Mario Chiariello

c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.

The cargo receptor NCOA4 mediates autophagic ferritin degradation. Here we show that NCOA4 deficiency in a knockout mouse model causes iron accumulation in the liver and spleen, increased levels of transferrin saturation, serum ferritin, and liver hepcidin, and decreased levels of duodenal ferroportin. Despite signs of iron overload, NCOA4-null mice had mild microcytic hypochromic anemia. Under an iron-deprived diet (2-3 mg/kg), mice failed to release iron from ferritin storage and developed severe microcytic hypochromic anemia and ineffective erythropoiesis associated with increased erythropoietin levels. When fed an iron-enriched diet (2 g/kg), mice died prematurely and showed signs of liver damage. Ferritin accumulated in primary embryonic fibroblasts from NCOA4-null mice consequent to impaired autophagic targeting. Adoptive expression of the NCOA4 COOH terminus (aa 239-614) restored this function. In conclusion, NCOA4 prevents iron accumulation and ensures efficient erythropoiesis, playing a central role in balancing iron levels in vivo.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic. Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

E-cadherins are surface adhesion molecules localized at the level of adherens junctions, which play a major role in cell adhesiveness by mediating calcium-dependent homophylic interactions at sites of cell-cell contacts.Recently, E-cadherins have been also implicated in a number of biological processes, including cell growth and differentiation, cell recognition, and sorting during developmental morphogenesis, as well as in aggregationdependent cell survival.As phosphatidylinositol (PI) 3-kinase and Akt play a critical role in survival pathways in response to both growth factors and extracellular stimuli, these observations prompted us to explore whether E-cadherins could affect intracellular molecules regulating the activity of the PI 3-kinase/Akt signaling cascade.Using Madin-Darby canine kidney cells as a model system, we show here that engagement of E-cadherins in homophylic calcium-dependent cell-cell interactions results in a rapid PI 3-kinase-dependent activation of Akt and the subsequent translocation of Akt to the nucleus.Moreover, we demonstrate that the activation of PI 3-kinase in response to cell-cell contact formation involves the phosphorylation of PI 3-kinase in tyrosine residues, and the concomitant recruitment of PI 3-kinase to E-cadherin-containing protein complexes.These findings indicate that E-cadherins can initiate outside-in signal transducing pathways that regulate the activity of PI 3-kinase and Akt, thus providing a novel molecular mechanism whereby the interaction among neighboring cells and their adhesion status may ultimately control the fate of epithelial cells.

The expression of the c-jun proto-oncogene is rapidly induced in response to mitogens acting on a large variety of cell surface receptors. The resulting functional activity of c-Jun proteins appears to be critical for cell proliferation. Recently, we have shown that a large family of G protein-coupled receptors (GPCRs), represented by the m1 muscarinic receptor, can initiate intracellular signaling cascades that result in the activation of mitogen-activated protein kinases (MAPK) and c-Jun NH2-terminal kinases (JNK) and that the activation of JNK but not of MAPK correlated with a remarkable increase in the expression of c-jun mRNA. Subsequently, however, we obtained evidence that GPCRs can potently stimulate the activity of the c-jun promoter through MEF2 transcription factors, which do not act downstream from JNK. In view of these observations, we set out to investigate further the nature of the signaling pathway linking GPCRs to the c-jun promoter. Utilizing NIH 3T3 cells, we found that GPCRs can activate the c-jun promoter in a JNK-independent manner. Additionally, we demonstrated that these GPCRs can elevate the activity of novel members of the MAPK family, including ERK5, p38α, p38γ, and p38δ, and that the activation of certain kinases acting downstream from MEK5 (ERK5) and MKK6 (p38α and p38γ) is necessary to fully activate the c-jun promoter. Moreover, in addition to JNK, ERK5, p38α, and p38γ were found to stimulate the c-jun promoter by acting on distinct responsive elements. Taken together, these results suggest that the pathway linking GPCRs to the c-junpromoter involves the integration of numerous signals transduced by a highly complex network of MAPK, rather than resulting from the stimulation of a single linear protein kinase cascade. Furthermore, our findings suggest that each signaling pathway affects one or more regulatory elements on the c-jun promoter and that the transcriptional response most likely results from the temporal integration of each of these biochemical routes.

The expression of human muscarinic acetylcholine receptors (mAChRs) in NIH 3T3 cells has been used as a model for studying proliferative signaling through G protein-coupled receptors. In this biological system, the m1 class of mAChRs can effectively transduce mitogenic signals (Stephens, E. V., Kalinec, G., Brann, M. R., and Gutkind, J. S.(1993) Oncogene 8, 19-26) and induce [Medline] malignant transformation if persistently activated (Gutkind, J. S., Novotny, E. A., Brann, M. R., and Robbins, K. C.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4703-4708). Moreover, available evidence suggests that the m1-signaling pathway converges at the level of p21ras with that emerging from tyrosine kinase receptors (Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S.(1994) Nature 369, 418-420). To explore nuclear events involved in growth regulation by G protein-coupled receptors in this setting, we compared the effect of platelet-derived growth factor (PDGF) and the cholinergic agonist, carbachol, on the expression of mRNA for members of the jun and fos family of nuclear proto-oncogenes. We found that activation of m1 receptors by carbachol induces the expression of a distinct set of nuclear transcription factors. In particular, carbachol caused a much greater induction of c-jun mRNA and AP-1 activity. These responses did not correlate with protein kinase C stimulation nor with the activation of mitogen-activated protein (MAP) kinases. Recently, it has been shown that a novel family of kinases structurally related to MAP kinases, stress-activated protein kinases, or Jun kinases (JNKs), phosphorylate in vivo the amino-terminal transactivating domain of the c-Jun protein, thereby increasing its transcriptional activity. In view of our results, this observation prompted us to ask whether m1 and PDGF can differentially activate JNKs. Here, we show that m1 mAChRs can induce a remarkable increase in JNK activity, which was temporally distinct from that of MAP kinase and was entirely protein kinase C independent. In contrast, PDGF failed to activate JNK in these cells, although it stimulated MAP kinase to an extent even greater than that for carbachol. These findings demonstrate that G protein-coupled receptors can signal through pathways leading to the activation of JNK, thus diverging at this level with those signaling routes utilized by tyrosine kinase receptors. The expression of human muscarinic acetylcholine receptors (mAChRs) in NIH 3T3 cells has been used as a model for studying proliferative signaling through G protein-coupled receptors. In this biological system, the m1 class of mAChRs can effectively transduce mitogenic signals (Stephens, E. V., Kalinec, G., Brann, M. R., and Gutkind, J. S.(1993) Oncogene 8, 19-26) and induce [Medline] malignant transformation if persistently activated (Gutkind, J. S., Novotny, E. A., Brann, M. R., and Robbins, K. C.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4703-4708). Moreover, available evidence suggests that the m1-signaling pathway converges at the level of p21ras with that emerging from tyrosine kinase receptors (Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S.(1994) Nature 369, 418-420). To explore nuclear events involved in growth regulation by G protein-coupled receptors in this setting, we compared the effect of platelet-derived growth factor (PDGF) and the cholinergic agonist, carbachol, on the expression of mRNA for members of the jun and fos family of nuclear proto-oncogenes. We found that activation of m1 receptors by carbachol induces the expression of a distinct set of nuclear transcription factors. In particular, carbachol caused a much greater induction of c-jun mRNA and AP-1 activity. These responses did not correlate with protein kinase C stimulation nor with the activation of mitogen-activated protein (MAP) kinases. Recently, it has been shown that a novel family of kinases structurally related to MAP kinases, stress-activated protein kinases, or Jun kinases (JNKs), phosphorylate in vivo the amino-terminal transactivating domain of the c-Jun protein, thereby increasing its transcriptional activity. In view of our results, this observation prompted us to ask whether m1 and PDGF can differentially activate JNKs. Here, we show that m1 mAChRs can induce a remarkable increase in JNK activity, which was temporally distinct from that of MAP kinase and was entirely protein kinase C independent. In contrast, PDGF failed to activate JNK in these cells, although it stimulated MAP kinase to an extent even greater than that for carbachol. These findings demonstrate that G protein-coupled receptors can signal through pathways leading to the activation of JNK, thus diverging at this level with those signaling routes utilized by tyrosine kinase receptors.

The serine/threonine kinase Cot is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family implicated in cellular transformation. Enhanced expression of this protein has been shown to activate both the MAPK and the c-Jun N-terminal kinase (JNK) pathways and to stimulate the nuclear factor of activated T cells and NF-κB-dependent transcription. However, the nature of the normal functions of the Cot protein and the molecular mechanisms responsible for its oncogenic potential are still largely unknown. Here, we show that overexpression of the cot proto-oncogene is sufficient to stimulate the expression of c-jun and that, in turn, the activity of c-Jun is required for Cot-induced transformation. These observations prompted us to explore the molecular events by which Cot regulates c-jun expression. We found that Cot potently stimulates the activity of the c-jun promoter utilizing JNK-dependent and -independent pathways, the latter involving two novel members of the MAPK family, p38γ (ERK6) and ERK5. Molecularly, this activity was found to be dependent on the ability of Cot to activate, in vivo, members of each class of the MAPK kinase superfamily, including MEK, SEK, MKK6, and MEK5. Furthermore, the use of dominant interfering molecules revealed that Cot requires JNK, p38s, and ERK5 to stimulate the c-jun promoter fully and to induce neoplastic transformation. These findings indicate that Cot represents the first example of a serine/threonine kinase acting simultaneously on all known MAPK cascades. Moreover, these observations strongly suggest that the transforming ability of Cot results from the coordinated activation of these pathways, which ultimately converge on the regulation of the expression and activity of the product of the c-junproto-oncogene.

RhoA regulates the actin cytoskeleton and the expression of genes associated with cell proliferation. This includes c-fos and c-jun, which are members of the AP1 family of transcription factors that play a key role in normal and aberrant cell growth. Whereas RhoA stimulates the c-fos SRE by a recently elucidated mechanism that is dependent on actin treadmilling, how RhoA regulates c-jun is still poorly understood. We found that RhoA stimulates c-jun expression through ROCK, but independently from the ability of ROCK to promote actin polymerization. Instead, we found that ROCK activates JNK, which then phosphorylates c-Jun and ATF2 when bound to the c-jun promoter. Thus, ROCK represents a point of signal divergence downstream from RhoA, as it promotes actin reorganization and the consequent expression from the c-fos SRE, while a parallel pathway connects ROCK to JNK, thereby stimulating c-jun expression. Ultimately, these pathways converge in the nucleus to regulate AP1 activity.

Non-small cell lung cancer (NSCLC) accounts for ∼80% of all lung cancers. Although some advances in lung cancer therapy have been made, patient survival is still quite poor. Two microRNAs, miR-221 and miR-222, upregulated by the MET proto-oncogene, have been already described to enhance cell survival and to induce TNF-related apoptosis-inducing ligand (TRAIL) resistance in NSCLC cell lines, through the downregulation of p27kip1, PTEN and TIMP3. Here, we further investigated this pathway and showed that miR-130a, expressed at low level in lung cancer cell lines, by targeting MET was able to reduce TRAIL resistance in NSCLC cells through the c-Jun-mediated downregulation of miR-221 and miR-222. Moreover, we found that miR-130a reduced migratory capacity of NSCLC. A better understanding of MET-miR-221 and 222 axis regulation in drug resistance is the key in developing new strategies in NSCLC therapy.

In the past decade, we have observed exciting advances in lung cancer therapy, including the development of targeted therapies. However, additional strategies for early detection and tumor-based therapy are still essential in improving patient outcomes. EGF receptor (EGFR) and MET (the receptor tyrosine kinase for hepatocyte growth factors) are cell-surface tyrosine kinase receptors that have been implicated in diverse cellular processes and as regulators of several microRNAs (miRNAs), thus contributing to tumor progression. Here, we demonstrate a biological link between EGFR, MET, and the miRNA cluster 23a ~ 27a ~ 24-2. We show that miR-27a regulates MET, EGFR, and Sprouty2 in lung cancer. In addition, we identify both direct and indirect mechanisms by which miR-27a can regulate both MET and EGFR. Thus, we propose a mechanism for MET and EGFR axis regulation that may lead to the development of therapeutics in lung cancer.

Polymeric nanoparticles (PNPs) may efficiently deliver in vivo therapeutics to tumors when conjugated to specific targeting agents. Gint4.T aptamer specifically recognizes platelet-derived growth factor receptor β and can cross the blood-brain barrier (BBB). We synthesized Gint4.T-conjugated PNPs able of high uptake into U87MG glioblastoma (GBM) cells and with astonishing EC50 value (38 pM) when loaded with a PI3K-mTOR inhibitor. We also demonstrated in vivo BBB passage and tumor accumulation in a GBM orthotopic model.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved catabolic process necessary for normal recycling of cellular constituents and for appropriate response to cellular stress. Although several genes belonging to the core molecular machinery involved in autophagosome formation have been discovered, relatively little is known about the nature of signaling networks controlling autophagy upon intracellular or extracellular stimuli. We discovered ATG8-like proteins (MAP1LC3B, GABARAP and GABARAPL1) as novel interactors of MAPK15/ERK8, a MAP kinase involved in cell proliferation and transformation. Based on the role of these proteins in the autophagic process, we demonstrated that MAPK15 is indeed localized to autophagic compartments and increased, in a kinase-dependent fashion, ATG8-like proteins lipidation, autophagosome formation and SQSTM1 degradation, while decreasing LC3B inhibitory phosphorylation. Interestingly, we also identified a conserved LC3-interacting region (LIR) in MAPK15 responsible for its interaction with ATG8-like proteins, for its localization to autophagic structures and, consequently, for stimulation of the formation of these compartments. Furthermore, we reveal that MAPK15 activity was induced in response to serum and amino-acid starvation and that this stimulus, in turn, required endogenous MAPK15 expression to induce the autophagic process. Altogether, these results suggested a new function for MAPK15 as a regulator of autophagy, acting through interaction with ATG8 family proteins. Also, based on the key role of this process in several human diseases, these results supported the use of this MAP kinase as a potential novel therapeutic target.

Small GTP-binding proteins of the Rho-family, Rho, Rac, and Cdc42, have been traditionally linked to the regulation of the cellular actin-based cytoskeleton. Rac and Cdc42 can also control the activity of JNK, thus acting in a molecular pathway transmitting extracellular signals to the nucleus. Interestingly, Rho can also regulate gene expression, albeit by a not fully understood mechanism. Here, we found that activated RhoA can stimulate c-jun expression and the activity of the c-jun promoter. As the complexity of the signaling pathways controlling the expression of c-jun has begun to be unraveled, this finding provided a unique opportunity to elucidate the biochemical routes whereby RhoA regulates nuclear events. We found that RhoA can initiate a linear kinase cascade leading to the activation of ERK6 (p38 gamma), a recently identified member of the p38 family of MAPKs. Furthermore, we present evidence that RhoA, PKN, MKK3/MKK6, and ERK6 (p38 gamma) are components of a novel signal transduction pathway involved in the regulation of gene expression and cellular transformation.

Rab proteins are small GTPases involved in the regulation of membrane traffic. Rab5a has been shown to regulate transport in the early endocytic pathway. Here we report the isolation of cDNA clones encoding two highly related isoforms, Rab5b and Rab5c. The two proteins share with Rab5a all the structural features required for regulation of endocytosis. Rab5b and Rab5c colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion between early endosomes in vitro and increase the rate of endocytosis when overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the regulation of endocytosis in eukaryotic cells.

The RET proto-oncogene encodes a functional receptor tyrosine kinase (Ret) for the Glial cell line Derived Neurotrophic Factor (GDNF). RET is involved in several neoplastic and non-neoplastic human diseases. Oncogenic activation of RET is detected in human papillary thyroid tumours and in multiple endocrine neoplasia type 2 syndromes. Inactivating mutations of RET have been associated to the congenital megacolon, i.e. Hirschprung's disease. In order to identify pathways that are relevant for Ret signalling to the nucleus, we have investigated its ability to induce the c-Jun NH2-terminal protein kinases (JNK). Here we show that triggering the endogenous Ret, expressed in PC12 cells, induces JNK activity; moreover, Ret is able to activate JNK either when transiently transfected in COS-1 cells or when stably expressed in NIH3T3 fibroblasts or in PC Cl 3 epithelial thyroid cells. JNK activation is dependent on the Ret kinase function, as a kinase-deficient RET mutant, associated with Hirschsprung's disease, fails to activate JNK. The pathway leading to the activation of JNK by RET is clearly divergent from that leading to the activation of ERK: substitution of the tyrosine 1062 of Ret, the Shc binding site, for phenylalanine abrogates ERK but not JNK activation. Experiments conducted with dominant negative mutants or with negative regulators demonstrate that JNK activation by Ret is mediated by Rho/Rac related small GTPases and, particularly, by Cdc42.

Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones.Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.By performing small RNA sequencing on A375 melanoma cells and a vemurafenibresistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib.We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features.miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2.www.impactjournals.com/oncotarget/

Mitochondria are the major source of reactive oxygen species (ROS), whose aberrant production by dysfunctional mitochondria leads to oxidative stress, thus contributing to aging as well as neurodegenerative disorders and cancer. Cells efficiently eliminate damaged mitochondria through a selective type of autophagy, named mitophagy. Here, we demonstrate the involvement of the atypical MAP kinase family member MAPK15 in cellular senescence, by preserving mitochondrial quality, thanks to its ability to control mitophagy and, therefore, prevent oxidative stress. We indeed demonstrate that reduced MAPK15 expression strongly decreases mitochondrial respiration and ATP production, while increasing mitochondrial ROS levels. We show that MAPK15 controls the mitophagic process by stimulating ULK1-dependent PRKN Ser108 phosphorylation and inducing the recruitment of damaged mitochondria to autophagosomal and lysosomal compartments, thus leading to a reduction of their mass, but also by participating in the reorganization of the mitochondrial network that usually anticipates their disposal. Consequently, MAPK15-dependent mitophagy protects cells from accumulating nuclear DNA damage due to mitochondrial ROS and, consequently, from senescence deriving from this chronic DNA insult. Indeed, we ultimately demonstrate that MAPK15 protects primary human airway epithelial cells from senescence, establishing a new specific role for MAPK15 in controlling mitochondrial fitness by efficient disposal of old and damaged organelles and suggesting this kinase as a new potential therapeutic target in diverse age-associated human diseases.

Glioblastoma multiforme (GBM) is the most aggressive primary tumor of the central nervous system and the diagnosis is often dismal. GBM pharmacological treatment is strongly limited by its intracranial location beyond the blood-brain barrier (BBB). While Temozolomide (TMZ) exhibits the best clinical performance, still less than 20% crosses the BBB, therefore requiring administration of very high doses with resulting unnecessary systemic side effects. Here, we aimed at designing new negative temperature-responsive gel formulations able to locally release TMZ beyond the BBB. The biocompatibility of a chitosan-β-glycerophosphate-based thermogel (THG)-containing mesoporous SiO2 nanoparticles (THG@SiO2) or polycaprolactone microparticles (THG@PCL) was ascertained in vitro and in vivo by cell counting and histological examination. Next, we loaded TMZ into such matrices (THG@SiO2-TMZ and THG@PCL-TMZ) and tested their therapeutic potential both in vitro and in vivo, in a glioblastoma resection and recurrence mouse model based on orthotopic growth of human cancer cells. The two newly designed anticancer formulations, consisting in TMZ-silica (SiO2@TMZ) dispersed in the thermogel matrix (THG@SiO2-TMZ) and TMZ, spray-dried on PLC and incorporated into the thermogel (THG@PCL-TMZ), induced cell death in vitro. When applied intracranially to a resected U87-MG-Red-FLuc human GBM model, THG@SiO2-TMZ and THG@PCL-TMZ caused a significant reduction in the growth of tumor recurrences, when compared to untreated controls. THG@SiO2-TMZ and THG@PCL-TMZ are therefore new promising gel-based local therapy candidates for the treatment of GBM.

Abstract Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4−/− mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12–induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12–induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38α and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38α/STAT4 pathway as an important mediator of IL-12 actions.

Glioblastoma Multiforme (GBM) is the most common and aggressive human brain tumor, associated with very poor survival despite surgery, radiotherapy and chemotherapy.The epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor β (PDGFRβ) are hallmarks in GBM with driving roles in tumor progression. In approximately half of the tumors with amplified EGFR, the EGFRvIII truncated extracellular mutant is detected. EGFRvIII does not bind ligands, is highly oncogenic and its expression confers resistance to EGFR tyrosine kinase inhibitors (TKIs). It has been demonstrated that EGFRvIII-dependent cancers may escape targeted therapy by developing dependence on PDGFRβ signaling, thus providing a strong rationale for combination therapy aimed at blocking both EGFRvIII and PDGFRβsignaling.We have recently generated two nuclease resistant RNA aptamers, CL4 and Gint4.T, as high affinity ligands and inhibitors of the human wild-type EGFR (EGFRwt) and PDGFRβ, respectively.Herein, by different approaches, we demonstrate that CL4 aptamer binds to the EGFRvIII mutant even though it lacks most of the extracellular domain. As a consequence of binding, the aptamer inhibits EGFRvIII autophosphorylation and downstream signaling pathways, thus affecting migration, invasion and proliferation of EGFRvIII-expressing GBM cell lines.Further, we show that targeting EGFRvIII by CL4, as well as by EGFR-TKIs, erlotinib and gefitinib, causes upregulation of PDGFRβ. Importantly, CL4 and gefitinib cooperate with the anti-PDGFRβ Gint4.T aptamer in inhibiting cell proliferation.The proposed aptamer-based strategy could have impact on targeted molecular cancer therapies and may result in progresses against GBMs.

CCDC6 gene product is a pro-apoptotic protein substrate of ATM, whose loss or inactivation enhances tumour progression. In primary tumours, the impaired function of CCDC6 protein has been ascribed to CCDC6 rearrangements and to somatic mutations in several neoplasia. Recently, low levels of CCDC6 protein, in NSCLC, have been correlated with tumor prognosis. However, the mechanisms responsible for the variable levels of CCDC6 in primary tumors have not been described yet.We show that CCDC6 turnover is regulated in a cell cycle dependent manner. CCDC6 undergoes a cyclic variation in the phosphorylated status and in protein levels that peak at G2 and decrease in mitosis. The reduced stability of CCDC6 in the M phase is dependent on mitotic kinases and on degron motifs that are present in CCDC6 and direct the recruitment of CCDC6 to the FBXW7 E3 Ubl. The de-ubiquitinase enzyme USP7 appears responsible of the fine tuning of the CCDC6 stability, affecting cells behaviour and drug response.Thus, we propose that the amount of CCDC6 protein in primary tumors, as reported in lung, may depend on the impairment of the CCDC6 turnover due to altered protein-protein interaction and post-translational modifications and may be critical in optimizing personalized therapy.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

The regulation of gene expression by cell surface receptors often involves the stimulation of signaling pathways including one or more members of the MAPK superfamily of serine-threonine kinases. Upon their activation in the cytosol, MAPKs can translocate to the nucleus and affect the activity of a variety of transcription factors. Recently, it has been observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming G protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation of c-jun expression through the activation of MEF2 transcription factors. How cell surface receptors, including GPCRs, stimulate ERK5 is still poorly understood. In this study, we have used transiently transfected COS-7 cells to begin delineating the biochemical route linking GPCRs to ERK5. We show that receptors that can couple to the Gq and G12/13 families of heterotrimeric G proteins, m1 and thrombin receptors, respectively, but not those coupled to Gi, such as m2 receptors, are able to regulate the activity of ERK5. To investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric system by which Gαq- and Gα13-mediated signaling pathways can be conditionally activated upon ligand stimulation. Using this system, as well as the expression of activated forms of G protein subunits, we show that the Gαq and Gα12/13 families of heterotrimeric G proteins, but not the Gαi, Gαs, and βγ subunits, are able to regulate ERK5. Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves a novel signaling pathway, which is distinct from those regulated by Ras and Rho GTPases. The regulation of gene expression by cell surface receptors often involves the stimulation of signaling pathways including one or more members of the MAPK superfamily of serine-threonine kinases. Upon their activation in the cytosol, MAPKs can translocate to the nucleus and affect the activity of a variety of transcription factors. Recently, it has been observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming G protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation of c-jun expression through the activation of MEF2 transcription factors. How cell surface receptors, including GPCRs, stimulate ERK5 is still poorly understood. In this study, we have used transiently transfected COS-7 cells to begin delineating the biochemical route linking GPCRs to ERK5. We show that receptors that can couple to the Gq and G12/13 families of heterotrimeric G proteins, m1 and thrombin receptors, respectively, but not those coupled to Gi, such as m2 receptors, are able to regulate the activity of ERK5. To investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric system by which Gαq- and Gα13-mediated signaling pathways can be conditionally activated upon ligand stimulation. Using this system, as well as the expression of activated forms of G protein subunits, we show that the Gαq and Gα12/13 families of heterotrimeric G proteins, but not the Gαi, Gαs, and βγ subunits, are able to regulate ERK5. Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves a novel signaling pathway, which is distinct from those regulated by Ras and Rho GTPases. mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun N-terminal kinase GTP-binding proteins mitogen-activated protein kinase/extracellular signal-regulated kinase kinase G protein-coupled receptors hemagglutinin Cdc42, Rac interactive binding domain chloramphenicol acetyltransferase serum response element glutathione S-transferase green fluorescent protein protease activated receptor 1 Mitogen-activated protein kinases (MAPKs)1 are serine-threonine protein kinases that play a central role in the transduction of environmental stimuli to the nucleus, thereby regulating the expression of genes involved in a variety of cellular processes, including cell proliferation, differentiation, programmed cell death, and neoplastic transformation (1.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4287) Google Scholar, 2.Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1409) Google Scholar). To date, MAPKs have been classified into at least six subfamilies: p44mapk and p42mapk, also called extracellular signal-regulated kinases (ERKs) 1 and 2, respectively (referred in here as MAPK); c-Jun N-terminal kinases (JNKs), also termed stress-activated protein kinases; p38 MAPKs; ERK5, also known as big MAPK 1; and the recently identified ERK7 (3.Abe M.K. Kuo W.L. Hershenson M.B. Rosner M.R. Mol. Cell. Biol. 1999; 19: 1301-1312Crossref PubMed Scopus (117) Google Scholar), and MOK (4.Miyata Y. Akashi M. Nishida E. Genes Cells. 1999; 4: 299-309Crossref PubMed Scopus (59) Google Scholar) (see Ref. 5.Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar for a review). These kinases are activated by a wide variety of extracellular stimuli such as growth factors, hormones, antigens, and cytokines and can also be stimulated in response to a diverse array of cellular stresses, such as UV irradiation, oxidative stress, and heat and osmotic shock (2.Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1409) Google Scholar). Many cell surface receptors can effectively stimulate MAPK cascades to signal to the nucleus, including the large family of receptors that transduce signals through the activation of heterotrimeric GTP-binding proteins (G proteins) (6.Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). For example, receptors coupled to Gαi proteins can potently stimulate MAPK (7.Moolenaar W.H. Kranenburg O. Postma F.R. Zondag G.C. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar), and that appears to be mediated primarily through the release of βγ subunits (8.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (781) Google Scholar) and the activation of a complex biochemical route involving phosphatidylinositol 3-kinases (9.Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (630) Google Scholar) and several nonreceptor and receptor tyrosine kinases (see Refs. 6.Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar and 10.Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (615) Google Scholar for reviews). In turn, activation of these tyrosine kinases leads to the phosphorylation of an adaptor protein, Shc, and the recruitment of the Grb2·Sos complex to the plasma membrane thus stimulating the exchange of GDP for GTP on Ras GTPases. This promotes the activation of a kinase cascade including Raf and MEK, which culminates with the activation of MAPK (1.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4287) Google Scholar, 11.Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3252) Google Scholar). Signals generated by the activation of G protein-coupled receptors (GPCRs) can also be transmitted through Gαi and Gαs, which can stimulate MAPK by regulating the small GTPase Rap1 (12.Mochizuki N. Ohba Y. Kiyokawa E. Kurata T. Murakami T. Ozaki T. Kitabatake A. Nagashima K. Matsuda M. Nature. 1999; 400: 891-894Crossref PubMed Scopus (194) Google Scholar, 13.Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 14.de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1657) Google Scholar), and by Gαq, which can activate Pyk2 and Src (15.Dikic I. Tokiwa G. Lev S. Courtneidge S.A. Schlessinger J. Nature. 1996; 383: 547-550Crossref PubMed Scopus (883) Google Scholar, 16.Igishi T. Gutkind J.S. Biochem. Biophys. Res. Commun. 1998; 244: 5-10Crossref PubMed Scopus (65) Google Scholar), and can stimulate Raf through protein kinase C (17.Kolch W. Heidecker G. Kochs G. Hummel R. Vahidi H. Mischak H. Finkenzeller G. Marme D. Rapp U.R. Nature. 1993; 364: 249-252Crossref PubMed Scopus (1182) Google Scholar). Similarly, JNK and p38 MAPKs have been shown to be activated by ligands acting on GPCRs, by the release of βγ dimers (18.Coso O.A. Teramoto H. Simonds W.F. Gutkind J.S. J. Biol. Chem. 1996; 271: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar) and through the Gαq and Gα12/13 classes of G proteins (19.Yamauchi J. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 1997; 272: 27771-27777Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 20.Prasad M.V. Dermott J.M. Heasley L.E. Johnson G.L. Dhanasekaran N. J. Biol. Chem. 1995; 270: 18655-18659Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). However, signaling pathways from GPCRs to these MAPKs are still largely unknown, although they appear to involve the activation of the small GTPases Rac1 and Cdc42 by βγ dimers and RhoA and Rac1 by members of the Gα12/13 class of G proteins (18.Coso O.A. Teramoto H. Simonds W.F. Gutkind J.S. J. Biol. Chem. 1996; 271: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar,21.Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1576) Google Scholar, 22.Nagao M. Kaziro Y. Itoh H. Oncogene. 1999; 18: 4425-4434Crossref PubMed Scopus (66) Google Scholar, 23.Collins L.R. Minden A. Karin M. Brown J.H. J. Biol. Chem. 1996; 271: 17349-17353Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Recently, we have observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming GPCRs and provided evidence that ERK5 participates in the regulation of c-junexpression by GPCRs through the activation of members of the MEF2 class of transcription factors (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar). ERK5 exhibits an extended C-terminal tail, which is absent in other types of MAPKs, suggesting that the regulation and function of this kinase might be different from that of other MAPKs (25.Lee J.D. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1995; 213: 715-724Crossref PubMed Scopus (294) Google Scholar, 26.Zhou G. Bao Z.Q. Dixon J.E. J. Biol. Chem. 1995; 270: 12665-12669Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar). How cell surface receptors, including GPCRs, stimulate ERK5 is still unknown. In this study, we have used transfected and endogenously expressed GPCRs and the co-expression of GPCRs with chimeric G protein α subunits to begin delineating the biochemical route linking GPCRs to ERK5. We show that the Gαq and Gα12/13 families of heterotrimeric G proteins α subunits, but not the Gαi, Gαs, or βγ subunits, are able to regulate ERK5 activity. Furthermore, we obtained evidence that the stimulation of the ERK5 cascade by GPCRs involves a novel pathway, which is distinct from those regulated by Ras and Rho GTPases. PAR1, kindly provided by Dr. L.F. Brass, was subcloned into the pCEFL vector as an EcoRI fragment. DNA encoding a G13/Gi chimera, in which 5 amino acids at the C terminus of Gα13 were replaced by the corresponding sequence of Gαi2, was prepared by polymerase chain reaction amplification using pcDNA3 HA-Gα13 (27.Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar) as a template, and the resulting DNA was subcloned into the pCEFL HA vector (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar) as aBglII-EcoRI fragment. Sequences of mutagenic oligonucleotides will be made available upon request. Plasmids expressing epitope-tagged ERK5, MAPK, and JNK, pCEFL HA-ERK5, pcDNA3 HA-MAPK, and pcDNA3 HA-JNK, respectively, as well as expression plasmids for constitutively activated forms of Ras, Rho, Rac1, Cdc42, Gαq, Gαi2, Gαs, Gα12, Gα13, β and γ subunits of G proteins, dominant negative mutants of Ras and Rho, the CRIB (Cdc42, Rac interactive binding) domain of PAK (PAK-N), m1 and m2 muscarinic receptors, a Gal4 fusion protein containing the transactivating domain of MEF2C and dominant negative and active mutants of MEK5, MEK5AA, and MEK5DD, respectively, were described previously (21.Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1576) Google Scholar, 24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar, 27.Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 28.Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). A DNA plasmid encoding a Gq/Gi chimeric protein, in which 5 amino acids at the C terminus of Gαq were replaced by the corresponding sequence of Gαi2, was a gift from Dr. B. Conklin (29.Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (626) Google Scholar). Reporter plasmids that express the chloramphenicol acetyltransferase (CAT) gene under the control of the mutant form of the serum response element (SRE) from the c-fos promoter, lacking the ternary complex factor binding site (SREmutL) as well as an expression vector for the C3 toxin were kindly provided by Dr. R. Treisman (30.Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1211) Google Scholar). COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Cells were transfected using LipofectAMINE PlusTM reagent (Life Technologies, Inc.) according to the manufacturer's protocol. In each experiment, the total amount of DNA was adjusted to 3–10 μg/plate with a plasmid for green fluorescent protein. The ERK5 kinase activity in cells transfected with an expression plasmid for HA-ERK5 was measured as described previously (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar), using 3 μg of GST·MEF2C fusion protein containing the transactivating domain of MEF2C as a substrate. MAPK and JNK activities in cells transfected with an epitope-tagged MAPK (HA-ERK2, referred in here as HA-MAPK) or JNK (HA-JNK) were determined as described previously (21.Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1576) Google Scholar), using myelin basic protein (Sigma) or bacterially expressed GST·ATF2(96) fusion protein as a substrate, respectively. The expression level of HA-ERK5, HA-MAPK, and HA-JNK in lysates from transfected cells was assessed by Western blot analysis after SDS-polyacrylamide gel electrophoresis with the specific antibody against HA (HA.11; Berkeley Antibody Company). The transactivating activity of MEF2C and the SRE activity were determined as described previously (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar, 27.Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Briefly, for MEF2C, COS-7 cells plated in a 24-well plate were transfected with different expression plasmids together with 2 ng of pCDNAIII-Gal4-MEF2C, a plasmid expressing a Gal4 fusion protein containing the transactivation domain of MEF2C (amino acids 161–350) as well as 50 ng of pGal4-Luc and 10 ng of pRL-null (Promega). To measure the SRE activity, COS-7 cells were transfected with the indicated plasmids together with 0.1 μg of pCDNAIII-β-galactosidase, a plasmid expressing the enzyme β-galactosidase, and 0.1 μg of pSREmutL, the reporter plasmid expressing a CAT gene under the control of the mutant SRE lacking a ternary complex factor binding site. After transfection, cells were cultured for ∼24 h in serum-free Dulbecco's modified Eagle's medium, then stimulated with the indicated ligands for an additional 6 h, and lysed using reporter lysis buffer (Promega). Luciferase activities in cell extracts were determined using a dual luciferase assay system (Promega). CAT activity was assayed in the cell extracts by incubation at 37 °C for 1 h in the presence of 0.25 μCi of [14C]chloramphenicol (100 mCi/mmol) (ICN) and 200 μg/ml butyryl-CoA (Sigma) in 0.25 m Tris-HCl, pH 7.4. Labeled butyrylated products were extracted using a mixture of xylenes and 2,6,10,14-tetramethyl-pentadecane (ratio 1:2), and radioactivity was counted. β−Galactosidase activity present in each sample was assayed by a colorimetric method and was used to normalize for transfection efficiency. ERK5 has been recently found to participate in the regulation of the c-jun promoter by transforming GPCRs (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar). To begin exploring the nature of the pathway linking these cell surface receptors to ERK5, we first investigated which classes of GPCRs are able to stimulate ERK5 kinase activity. For these experiments, COS-7 cells were transiently transfected with expression plasmids for a HA-tagged form of ERK5, and its kinase activity was measured by anin vitro kinase assay using MEF2C fused to GST as a substrate. As shown in Fig.1 A, stimulation by carbachol, a cholinergic agonist, of transfected Gαq-coupled m1 muscarinic receptors (31.Berstein G. Blank J.L. Smrcka A.V. Higashijima T. Sternweis P.C. Exton J.H. Ross E.M. J. Biol. Chem. 1992; 267: 8081-8088Abstract Full Text PDF PubMed Google Scholar) potently activated ERK5. Similarly, stimulation with a tyrosine kinase receptor agonist, epidermal growth factor, also enhanced ERK5 kinase activity, as recently reported (32.Kato Y. Tapping R.I. Huang S. Watson M.H. Ulevitch R.J. Lee J.D. Nature. 1998; 395: 713-716Crossref PubMed Scopus (368) Google Scholar,33.Kamakura S. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 26563-26571Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). However, the stimulation of m2 muscarinic receptors, which are typical Gi-coupled receptors (34.Peralta E.G. Ashkenazi A. Winslow J.W. Smith D.H. Ramachandran J. Capon D.J. EMBO J. 1987; 6: 3923-3929Crossref PubMed Scopus (694) Google Scholar), had no effect on ERK5 activity, although it potently activated MAPK, which served as an internal control (8.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (781) Google Scholar). ERK5 activity was also stimulated by exposure to thrombin, which acts on endogenously expressed GPCRs, and this effect was slightly enhanced by overexpression of its cognate receptors, PAR1 (Fig. 1 A). Kinetics of ERK5 activation mediated by m1 and thrombin receptors were very similar, and responses were evident within 5 min after agonist addition and reached a maximal level around 10 min (Fig. 1 B). As m1 and m2 muscarinic receptors couple to Gαq and Gαi types of heterotrimeric G proteins, respectively, and thrombin receptors can stimulate both the Gαq and Gαi as well as the Gα12/13 families of G proteins (35.Grand R.J. Turnell A.S. Grabham P.W. Biochem. J. 1996; 313: 353-368Crossref PubMed Scopus (326) Google Scholar, 36.Aragay A.M. Collins L.R. Post G.R. Watson A.J. Feramisco J.R. Brown J.H. Simon M.I. J. Biol. Chem. 1995; 270: 20073-20077Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 37.Majumdar M. Seasholtz T.M. Buckmaster C. Toksoz D. Brown J.H. J. Biol. Chem. 1999; 274: 26815-26821Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38.Gohla A. Offermanns S. Wilkie T.M. Schultz G. J. Biol. Chem. 1999; 274: 17901-17907Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), these findings suggest that receptors coupled to Gαq and, possibly Gα12/13, may harbor the ability to transduce a signal to ERK5, whereas Gi-coupled receptors do not. MEF2C is a physiological substrate for ERK5 (39.Kato Y. Kravchenko V.V. Tapping R.I. Han J. Ulevitch R.J. Lee J.D. EMBO J. 1997; 16: 7054-7066Crossref PubMed Scopus (504) Google Scholar). Thus, we next asked whether the ability to enhance the in vitrophosphorylating activity of ERK5 by GPCRs resulted in enhanced transcriptional activity of MEF2 proteins in vivo. For these experiments, we fused the transactivation domain of MEF2C to the DNA binding domain of Gal4 and tested the ability to induce the expression from a pGal4-Luc reporter plasmid, as described previously (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar). As shown in Fig. 2 A, expression from the Gal4-driven luciferase reporter was induced by the stimulation of m1 and thrombin receptors, but not m2 receptors, which is consistent with their abilities to stimulate ERK5 kinase activity. Furthermore, transfection of a DNA plasmid for MEK5AA, which acts as a dominant negative mutant of MEK5 (24.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (192) Google Scholar), completely blocked the increased transcriptional activity of MEF2C elicited by thrombin and partially inhibited m1 mediated-transcriptional activation (Fig. 2 B). As a control, the activation of SRE mediated by m1 and thrombin receptors was unaffected by co-expression of MEK5AA (Fig.2 B). These findings suggested that the MEK5-ERK5 kinase pathway is functionally activated by GPCRs and that this kinase cascade is involved in the activation of MEF2C by m1 and thrombin receptors. To investigate which classes of G proteins mediate ERK5 activation induced by GPCRs, we examined the effects of activated forms of Gα subunits as well as overexpression of βγ subunits of heterotrimeric G proteins on ERK5 kinase activity. As shown in Fig.3 A, expression of Gα12QL and Gα13QL could induce ERK5 activation, whereas ERK5 kinase activity was not altered by the expression of GαqQL, Gαi2QL, GαsQL, and the βγ subunit, thus suggesting the involvement of the Gα12/13 family of G proteins in GPCR-mediated signaling to ERK5. Regarding Gαi, these observations are in line with the failure of m2 muscarinic receptors, which transduce signals through Gαi and βγ subunits, to activate ERK5. In addition, ERK5 activation mediated by m1 and thrombin receptors was found to be insensitive to pertussis toxin, which ADP ribosylates and inactivates Gαi and Gαo (data not shown), together indicating that pertussis toxin-sensitive G proteins cannot signal to ERK5 and that they do not mediate the activation of ERK5 by thrombin and m1 receptors. Stimulation of m1 receptors, that couple to Gαq, can activate ERK5. However, expression of an activated form of Gαq could not enhance the ERK5 kinase activity. Interestingly, this is highly reminiscent to that observed for the MAPK pathway (8.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (781) Google Scholar, 40.Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar). Indeed, several lines of evidence suggest the ability of Gαq to stimulate the MAPK pathway, but expression of activated Gαq could not induce MAPK activation (8.Crespo P. Xu N. Simonds W.F. Gutkind J.S. Nature. 1994; 369: 418-420Crossref PubMed Scopus (781) Google Scholar, 40.Faure M. Voyno-Yasenetskaya T.A. Bourne H.R. J. Biol. Chem. 1994; 269: 7851-7854Abstract Full Text PDF PubMed Google Scholar), and it even prevents the further stimulation of MAPK by a variety of stimuli (41.Qian N.X. Winitz S. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4077-4081Crossref PubMed Scopus (59) Google Scholar). This discrepancy may be ascribed to the desensitization of downstream signaling pathways, such as that two days after the transfection of expression plasmids the activity of MAPK and/or ERK5 may no longer be demonstrable. To solve this problem, we took advantage of the finding that a Gq/Gichimera, where a C-terminal region of Gαq is replaced by the corresponding region of Gαi, can be stimulated by Gi-coupled receptors and is able to transmit Gq-mediated signaling pathways (29.Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (626) Google Scholar). Thus, upon coexpression of this Gq/Gi chimera together with a Gi-coupled receptor, such as m2 receptors, on and off Gq-mediated signaling can now be controlled by agonist addition. Moreover, the treatment with pertussis toxin can now make it possible to specifically activate Gq-mediated signals, because Gαi is inhibited by pertussis toxin and no longer has the ability to stimulate downstream signaling pathways. The potential usefulness of this system prompted us to design also a similar G13/Gi chimera in which the C-terminal 5 amino acids of Gα13 were replaced by the corresponding sequences of Gαi. Expression of this protein was confirmed by Western blot analysis (data not shown). The functional activity of these chimeras was assessed by examining their ability to stimulate the transcriptional activation of an SRE-containing reporter plasmid, as both Gαq and Gα12/13 classes of G proteins are known to activate a SRE, but not the stimulation of Gαi (42.Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). As previously reported, the stimulation of m2 by carbachol did not activate the SRE (42.Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), although transcription from the SRE was potently increased by m1 receptor stimulation (Fig.3 B). However, the exposure to carbachol of cells expressing both m2 receptors and either a Gq/Gi chimera or a G13/Gi chimera significantly induced SRE activation, whereas the expression of either a Gq/Gi chimera or a G13/Gi chimera alone did not affect the SRE activity (Fig. 3 B). These results indicated that these Gq/Gi and G13/Gichimeras can be stimulated by m2 receptors and are capable of transmitting Gq- and G13-mediated signaling pathways, respectively. We then used this system to investigate whether Gαq and Gα13 have the ability to stimulate ERK5. As shown in Fig.3 C, stimulation with carbachol induced a limited activation of ERK5 in cells expressing m2 receptors. However, in cells expressing either the Gq/Gi or G13/Gi chimera, m2 receptor stimulation significantly activated ERK5 (Fig. 3 C). These results indicated that both the Gαq and Gα12/13classes of heterotrimeric G proteins are able to signal to ERK5 and thus might participate in ERK5 activation by GPCRs. Gi- and Gq-coupled receptors can stimulate the Ras-MAPK pathway effectively (6.Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar). However, we found that Gi-coupled m2 receptors fail to stimulate ERK5, suggesting that the pathway linking GPCRs to ERK5 is different from that which communicates these receptors to MAPK. Indeed, we observed that activated Ras causes only a very limited increase in the enzymatic activity of ERK5, although it potently stimulates MAPK (Fig.4). On the other hand, activation of the JNK pathway is believed to be mediated by Rac and Cdc42, two members of the Rho family of GTPases (21.Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1576) Google Scholar). However, whereas expression of activated Rac and Cdc42 strongly enhanced the kinase activity of JNK, these GTPases failed to stimulate ERK5 (Fig. 4). Similarly, expression of an activated form of Rho, which stimulates the SRE-driven reporter plasmid potently (30.Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1211) Google Scholar), also failed to enhance the enzymatic activity of ERK5. Furthermore, although some minor variations in the activity of ERK5 can be observed upon expression of these GTPases, an activated form of MEK5, MEK5DD, consistently induced ERK5 activation under these experimental conditions (Fig. 4). Thus, activation of Ras, Rho, Rac, and Cdc42 may not be sufficient to stimulate the ERK5 pathway. However, it is still possible that these small GTPases are required for GPCR-mediated ERK5 activation. To address this possibility, we used the expression of dominant interfering molecules for each of these GTPases. As shown in Fig. 5 A, the activation of ERK5 mediated by m1 and thrombin receptors was not affected by the expression of a dominant negative mutant of Ras, RasN17, although this inhibitory molecule effectively inhibited MAPK activation when induced by m1 stimulation (Fig. 5 B) and by thrombin (data not shown), but not by phorbol esters, as previously reported (43.Seo B. Choy E.W. Maudsley S. Miller W.E. Wilson B.A. Luttrell L.M. J. Biol. Chem. 2000; 275: 2239-2245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 44.Ueda Y. Hirai S. Osada S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). Thus, together these data suggest that Ras is unlikely to play a prominent role in ERK5 activation by GPCRs. For Rac and Cdc42, we used the overexpression of a molecule containing the CRIB domain of PAK fused to GST, which can specifically bind the GTP-bound forms of Rac and Cdc42 thereby inhibiting these GTPases (28.Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 45.Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1451) Google Scholar, 46.Osada S. Izawa M. Koyama T. Hirai S. Ohno S. FEBS Lett. 1997; 404: 227-233Crossref PubMed Scopus (27) Google Scholar). Indeed, expression of the CRIB domain of PAK (PAK-N) significantly inhibited JNK activation evoked by the expression of activated forms of Rac and Cdc42 and by m1 stimulation but had only a limited effect on the activation of JNK by anisomycin, which served as a control for specificity (Fig. 5 C). However, PAK-N did not affect the abilities of m1 and thrombin receptors to activate ERK5 (Fig.5 A). Together, these results indicated that the small GTPases Ras, Rac, and Cdc42 are not involved in the signaling from GPCRs to ERK5.Figure 5Signaling from G protein-coupled receptors to ERK5 does not require Ras- and Rho-related GTPases. A–C, COS-7 cells were cotransfected with expression plasmid for HA-ERK5 (A), HA-MAPK (B), or HA-JNK (C) as well as with expression vectors carrying cDNAs for GFP, m1, and PAR1 receptors or the activated mutant of Rac1 (RacQL) and Cdc42 (Cdc42QL), together with plasmids encoding inhibitory molecules (RasN17, RhoN19, and PAK-N). Cells were stimulated with or without 100 μm carbachol (Cch), 5 units/ml thrombin (Thr), 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA), or 10 μg/ml anisomycin as indicated. Kinase reactions were performed using anti-HA immunoprecipitates from the corresponding cellular lysates. Labeled substrates are indicated. Data shown are from a representative experiment for each assay. Western blot (WB) analysis was performed with anti-HA antibodies using total cell lysates (HA-ERK5 and HA-MAPK) or anti-HA immunoprecipitates (HA-JNK). D, COS-7 cells were cotransfected with pSREmutL and pCMV-β-galactosidase plasmid DNAs as well as with expression vectors carrying cDNAs for GFP, m1 and PAR1 receptors, and the activated mutant of Cdc42 (Cdc42QL), with or without expression plasmids for C3 toxin, as indicated. The next day, the cells were stimulated with vehicle, 100 μm carbachol (Cch) or 5 units/ml thrombin (Thr) for 6 h. Cells were processed as described under “Experimental Procedures.” The data represent CAT activity normalized by the β-galactosidase activity present in each cellular lysate, expressed as fold induction with respect to control cells, and are the mean ± S.E. of triplicate samples from a typical experiment. Similar results were obtained in three separate experiments. E, COS-7 cells were transfected with expression vectors carrying DNA for HA-ERK5 and GFP, m1 or PAR1 receptors, with or without expression plasmid for C3 toxin, and stimulated with vehicle, 100 μm carbachol (Cch), 5 units/ml thrombin (Thr), or 100 ng/ml epidermal growth factor (EGF) for 10 min. Kinase reactions were performed in anti-HA immunoprecipitates from the corresponding cellular lysates. Data represent the mean ± S.E. of three independent experiments, expressed as fold increase with respect to unstimulated cells. Autoradiograms correspond to representative experiments. Western blot (WB) analysis was performed in the corresponding cellular lysates and immunodetected with the antibody to HA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Interestingly, both Gαq and Gα12/13 classes of G proteins, but not Gαi, have been shown to activate Rho-dependent signaling pathways (42.Mao J. Yuan H. Xie W. Simon M.I. Wu D. J. Biol. Chem. 1998; 273: 27118-27123Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), and recent studies suggested that Rho-specific exchange factors such as p115-RhoGEF and PDZ-RhoGEF could be directly activated by the Gα12/13family of G proteins (27.Fukuhara S. Murga C. Zohar M. Igishi T. Gutkind J.S. J. Biol. Chem. 1999; 274: 5868-5879Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 47.Hart M.J. Jiang X. Kozasa T. Roscoe W. Singer W.D. Gilman A.G. Sternweis P.C. Bollag G. Science. 1998; 280: 2112-2114Crossref PubMed Scopus (679) Google Scholar). Together, these findings suggested the possibility that Rho may participate in signaling to ERK5. However, an activated form of Rho did not enhance the kinase activity of ERK5 (see above, Fig. 4) and that of any other member of the MAPK superfamily in this cellular setting. Nonetheless, these observations cannot rule out the possibility that Rho stimulates certain MAPKs, which might not be revealed by the expression of its activated mutants for reasons such as those described for Gαq. Thus, to explore further the possibility that Gq- and G12/13-coupled receptors uses Rho to stimulate ERK5, we used as a more definitive approach the expression ofClostridium botulinum C3 exoenzyme, which specifically ADP ribosylates Rho thus preventing its activation (48.Aktories K. Hall A. Trends Pharmacol. Sci. 1989; 10: 415-418Abstract Full Text PDF PubMed Scopus (126) Google Scholar). As shown in Fig.5 D, a C3 toxin inhibited the activation of SRE, a typical Rho-dependent response (30.Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1211) Google Scholar), by m1 and thrombin receptors, although the Cdc42-mediated activation of SRE was unaffected and served as a control. However, this toxin did not change the abilities of m1 and thrombin receptors to activate ERK5 (Fig. 5 E), strongly suggesting that the activation of ERK5 by GPCRs is independent of Rho. In conclusion, the present study demonstrates that the ERK5 pathway can be functionally activated by the stimulation of GPCRs depending on their coupling specificity and that the Gq and G12/13 families of heterotrimeric G proteins can mediate this effect. As ERK5 regulates the activity of a growing number of nuclear transcription factors, these findings may help explain the distinct ability of Gq- and G12/13-coupled receptors to promote the expression of growth related genes. Furthermore, our observations raise the possibility of the existence of a novel signaling pathway whereby GPCRs enhance the activity of ERK5. Although the precise nature of this biochemical route is still unknown, we provide evidence that the pathway linking GPCRs to the MEK5-ERK5 kinase cascade is distinct from those utilized by these cell surface receptors to stimulate MAPK, JNK, and Rho GTPases.

Rab GTPases play a fundamental role in the regulation of membrane traffic. Three different Rab5 isoforms have been reported but no differences in their function in endocytosis have been discovered. As the Rab5 isoforms show a conserved consensus site for Ser/Thr phosphorylation, we investigated whether this site was phosphorylated. Here, we report that the three Rab5 proteins are differentially recognized by different kinases. Rab5a is efficiently phosphorylated by extracellular‐regulated kinase 1 but not by extracellular‐regulated kinase 2, while cdc2 kinase preferentially phosphorylates Ser‐123 of Rab5b. These findings strongly suggest that phosphorylation could be important to differentially regulate the function of the Rab5 isoforms.

Rab proteins are small GTPases involved in the regulation of vesicular membrane traffic. Research done in the past years has demonstrated that some of these proteins are under the control of signal transduction pathways. Still, several recent papers point out to a new unexpected role for this family of Ras-related proteins, as potential regulators of intracellular signaling pathways. In particular, several evidence indicate that members of the Rab family of small GTPases, through their effectors, are key molecules participating to the regulation of numerous signal transduction pathways profoundly influencing cell proliferation, cell nutrition, innate immune response, fragmentation of compartments during mitosis and apoptosis. Even more surprisingly, direct involvement of Rab proteins in signaling to the nucleus has been demonstrated. This review will focus on aspects of Rab proteins function connected to signal transduction and, in particular, connections between membrane traffic and other cell pathways will be examined.

ERK8 (MAPK15) is a large MAP kinase already implicated in the regulation of the functions of different nuclear receptors and in cellular proliferation and transformation. Here, we identify ERRα as a novel ERK8-interacting protein. As a consequence of such interaction, ERK8 induces CRM1-dependent translocation of ERRα to the cytoplasm and inhibits its transcriptional activity. Also, we identify in ERK8 two LXXLL motifs, typical of agonist-bound nuclear receptor corepressors, as necessary features for this MAP kinase to interact with ERRα and to regulate its cellular localization and transcriptional activity. Ultimately, we demonstrate that ERK8 is able to counteract, in immortalized human mammary cells, ERRα activation induced by the EGF receptor pathway, often deregulated in breast cancer. Altogether, these results reveal a novel function for ERK8 as a bona fide ERRα corepressor, involved in control of its cellular localization by nuclear exclusion, and suggest a key role for this MAP kinase in the regulation of the biological activities of this nuclear receptor. ERK8 (MAPK15) is a large MAP kinase already implicated in the regulation of the functions of different nuclear receptors and in cellular proliferation and transformation. Here, we identify ERRα as a novel ERK8-interacting protein. As a consequence of such interaction, ERK8 induces CRM1-dependent translocation of ERRα to the cytoplasm and inhibits its transcriptional activity. Also, we identify in ERK8 two LXXLL motifs, typical of agonist-bound nuclear receptor corepressors, as necessary features for this MAP kinase to interact with ERRα and to regulate its cellular localization and transcriptional activity. Ultimately, we demonstrate that ERK8 is able to counteract, in immortalized human mammary cells, ERRα activation induced by the EGF receptor pathway, often deregulated in breast cancer. Altogether, these results reveal a novel function for ERK8 as a bona fide ERRα corepressor, involved in control of its cellular localization by nuclear exclusion, and suggest a key role for this MAP kinase in the regulation of the biological activities of this nuclear receptor.

A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel “druggable” link between BCR-ABL1 and human CML.

Aberrant activation of the Hedgehog (HH) signaling is a critical driver in tumorigenesis. The Smoothened (SMO) receptor is one of the major upstream transducers of the HH pathway and a target for the development of anticancer agents. The SMO inhibitor Vismodegib (GDC-0449/Erivedge) has been approved for treatment of basal cell carcinoma. However, the emergence of resistance during Vismodegib treatment and the occurrence of numerous side effects limit its use. Our group has recently discovered and developed novel and potent SMO inhibitors based on acylguanidine or acylthiourea scaffolds. Here, we show that the two acylguanidine analogs, compound (1) and its novel fluoride derivative (2), strongly reduce growth and self-renewal of melanoma cells, inhibiting the level of the HH signaling target GLI1 in a dose-dependent manner. Both compounds induce apoptosis and DNA damage through the ATR/CHK1 axis. Mechanistically, they prevent G2 to M cell cycle transition, and induce signs of mitotic aberrations ultimately leading to mitotic catastrophe. In a melanoma xenograft mouse model, systemic treatment with 1 produced a remarkable inhibition of tumor growth without body weight loss in mice. Our data highlight a novel route for cell death induction by SMO inhibitors and support their use in therapeutic approaches for melanoma and, possibly, other types of cancer with active HH signaling.

Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest.To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase.In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a "stress support" autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors.

Charcot-Marie-Tooth type 2B (CMT2B) disease is a dominant axonal peripheral neuropathy caused by 5 mutations in the RAB7A gene, a ubiquitously expressed GTPase controlling late endocytic trafficking. In neurons, RAB7A also controls neuronal-specific processes such as NTF (neurotrophin) trafficking and signaling, neurite outgrowth and neuronal migration. Given the involvement of macroautophagy/autophagy in several neurodegenerative diseases and considering that RAB7A is fundamental for autophagosome maturation, we investigated whether CMT2B-causing mutants affect the ability of this gene to regulate autophagy. In HeLa cells, we observed a reduced localization of all CMT2B-causing RAB7A mutants on autophagic compartments. Furthermore, compared to expression of RAB7AWT, expression of these mutants caused a reduced autophagic flux, similar to what happens in cells expressing the dominant negative RAB7AT22N mutant. Consistently, both basal and starvation-induced autophagy were strongly inhibited in skin fibroblasts from a CMT2B patient carrying the RAB7AV162M mutation, suggesting that alteration of the autophagic flux could be responsible for neurodegeneration.

Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene. Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene. A wide range of growth factors, cytokines, and mitogens is able to induce the expression of the c-myc proto-oncogene (1.Kelly K. Cochran B.H. Stiles C.D. Leder P. Cell. 1983; 35: 603-610Abstract Full Text PDF PubMed Scopus (1425) Google Scholar, 2.Roussel M.F. Cleveland J.L. Shurtleff S.A. Sherr C.J. Nature. 1991; 353: 361-363Crossref PubMed Scopus (138) Google Scholar). In turn, c-myc is necessary for cellular proliferation induced by different oncogenic tyrosine kinases (3.Barone M.V. Courtneidge S.A. Nature. 1995; 378: 509-512Crossref PubMed Scopus (283) Google Scholar). In normal cells as well as in tumors, the ability of c-myc to control cellular proliferation has been mostly correlated to changes in its mRNA levels through transcriptional and post-transcriptional mechanisms. In fact, most of the oncogenic alterations that target c-myc result in the increase of its messenger RNA and, in turn, of its protein (4.Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Crossref PubMed Scopus (1040) Google Scholar). Indeed, overexpression or gene amplification and translocations of c-myc are frequent causes of numerous solid and blood human tumors (5.Dang C.V. Resar L.M. Emison E. Kim S. Li Q. Prescott J.E. Wonsey D. Zeller K. Exp. Cell Res. 1999; 253: 63-77Crossref PubMed Scopus (319) Google Scholar). In line with its ability to promote cell cycle progression, in quiescent fibroblasts c-myc expression is virtually undetectable. However, upon stimulation with growth factors such as the platelet-derived growth factor (PDGF), 1The abbreviations used are: PDGFplatelet-derived growth factorHDACshistone deacetylasesHAhemagglutininSIDSin3-binding domainJNKc-Jun N-terminal kinaseAP-1activator protein 1MAPKmitogen-activated protein kinaseMEKKMAPK/extracellular signal-regulated kinase kinase kinaseErkextracellular signal-regulated kinaseChIPchromatin immunoprecipitation assayEMSAelectrophoretic mobility shift assayStat-3signal transducer and activator of transcription-3DBDDNA-binding domainLucluciferaseATFactivating transcription factorMLKmixed lineage kinase. its mRNA and then protein levels are rapidly induced until cells progress through the G1/S boundary of the cell cycle (6.Chiariello M. Marinissen M.J. Gutkind J.S. Nat. Cell Biol. 2001; 3: 580-586Crossref PubMed Scopus (113) Google Scholar, 7.Chiariello M. Gomez E. Gutkind J.S. Biochem. J. 2000; 349: 869-876Crossref PubMed Scopus (41) Google Scholar). Still, the mechanism by which growth factors promote the expression of c-myc is poorly understood. In this regard, we have recently described a Rac-dependent signaling pathway initiated by PDGF, controlling the expression of the c-myc proto-oncogene (6.Chiariello M. Marinissen M.J. Gutkind J.S. Nat. Cell Biol. 2001; 3: 580-586Crossref PubMed Scopus (113) Google Scholar). Nonetheless, the mechanism by which Rac mediates PDGF stimulation of the c-myc promoter has remained elusive. platelet-derived growth factor histone deacetylases hemagglutinin Sin3-binding domain c-Jun N-terminal kinase activator protein 1 mitogen-activated protein kinase MAPK/extracellular signal-regulated kinase kinase kinase extracellular signal-regulated kinase chromatin immunoprecipitation assay electrophoretic mobility shift assay signal transducer and activator of transcription-3 DNA-binding domain luciferase activating transcription factor mixed lineage kinase. In this study, we show that JNK is required for PDGF induction of c-myc expression. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the human, mouse, and even Drosophila c-myc promoter. Such element binds in vivo to members of the Jun family of transcription factors, c-Jun and JunD, as indicated by chromatin immunoprecipitation analysis. Finally, we show that through this element PDGF is able to control the activity of the c-myc promoter in an AP-1-dependent fashion, implying the existence of a novel signaling pathway linking the PDGF receptor through JNK and Jun proteins to nuclear events culminating in the expression of the c-myc proto-oncogene. Reagents—Human recombinant PDGF (Intergen) was used at a final concentration of 12.5 ng ml–1. The selective JNK inhibitor SP600125 (Biomol) was added to the cells 30 min before stimulation at the indicated concentrations. pcDNAIII/GS-Myc-V5 was purchased from Invitrogen. Myelin basic protein was purchased from Sigma. Expression vectors for Rac12V and the corresponding effector domain mutants Rac12V/33N, Rac12V/37L, and Rac12V/40H were kindly provided by C. J. Der (8.Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (385) Google Scholar). The bacterial expression vector pGEX-4T3 glutathione S-transferase-ATF2 and expression vectors for MEKK1 and MLK3 were described previously (9.Chiariello M. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2000; 20: 1747-1758Crossref PubMed Scopus (168) Google Scholar, 10.Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). pcDNAIII-Sis was generated by cloning the sis (PDGF) oncogene in the EcoRI and NotI restriction sites. The pmycAP-1 luciferase reporter vector was obtained by cloning two mouse AP-1 elements in the pGL3 reporter vector (Promega). PCR amplifications of the c-Fos and c-Jun cDNAs were cloned in the pCEFL AU5 and pCEFL AU1 expression vectors, respectively. The JunDBD-Sin3-binding domain of Mad (SID) expression vector was prepared cloning in pCEFL HA the DNA-binding domain of c-Jun and the Sin3-binding domain of Mad. The Gal4-driven luciferase reporter plasmid pGal4-Luc was constructed by inserting six copies of a Gal4-responsive element and a TATA oligonucleotide to replace the simian virus 40 minimal promoter in the pGL3 vector (Promega). The Gal4-VP16 expression vector was prepared cloning the transactivation domain of the VP16 transcription factor in-frame with the DNA-binding domain of Gal4 into the pCDNA III vector. Specific maps and restriction sites will be made available upon request. Cell Culture and Transfections—NIH 3T3 fibroblasts were maintained at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% (v/v) bovine calf serum (BioWhittaker), 2 mm l-glutamine, and penicillin-streptomycin (Invitrogen). NIH 3T3 cells were transfected by the LipofectAMINE Plus reagent or LipofectAMINE reagent (Invitrogen) in accordance with the manufacturer's instructions. Northern Blot Analysis—After 24 h of starvation, NIH 3T3 cells were washed with cold phosphate-buffered saline and total RNA was extracted by homogenization with TRIzol (Invitrogen) in accordance with manufacturer's specifications. Total RNA (10 μg) was fractionated in 2% formaldehyde-agarose gels, transferred to Hybond-XL nylon membranes (Amersham Biosciences), and hybridized with 32P-labeled DNA probes prepared with the Prime-a-Gene labeling system (Promega). As a probe, we used a 450-bp PstI DNA fragment from the human c-myc gene (pcDNAIII/GS-Myc-V5). The RNA membranes were pre-hybridized for >2 h in hybridization solution (ExpressHyb, Clontech) at 70 °C. The 32P-labeled probe was added to the blots and hybridized for another 16 h at 60 °C. The blots were washed twice for 30 min each in 2× SSC, 0.1% SDS at room temperature and then washed twice for 30 min each in 0.2× SSC, 0.1% SDS at 60 °C. Accuracy of RNA loading and transfer was confirmed by fluorescence under ultraviolet light after staining with ethidium bromide. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed using the chromatin immunoprecipitation assay kit (Upstate Biotechnology), in accordance with the manufacturer's instructions. Chromatin from NIH 3T3 cells has been fixed by directly adding formaldehyde (1% final) to the cell culture medium. Nuclear extracts have been isolated from the cells and then sonicated to obtain mechanical sharing of the fixed chromatin. Transcription factor-bound chromatin has been immunoprecipitated with specific antibodies, cross-linking has been reversed, and the isolated genomic DNA has been amplified by PCR using specific primers encompassing the murine c-myc promoter: forward AP66 (5′-ATACCTGTGACTATTCATTT-3′) and reverse AP67 (5′-GATGCTTCCTTGCCTAAGAC-3′). The PCR products were separated on a 2% agarose gel. Primers used as a control for the ChIP analysis amplify an unrelated DNA sequence located on murine chromosome 5: forward DE1 (5′-AGGACCCATCCAGATGGAGT-3′) and reverse DE2 (5′-CAATGCTCCAAATCCTGTCA-3′). Electrophoretic Mobility Shift Assays (EMSA)—Nuclear extracts were obtained from NIH 3T3 cells plated in 10-cm plates and grown to 70% confluency, starved overnight, and then stimulated with PDGF when needed. Cells were washed in cold phosphate-buffered saline and lysed in 400 μl of buffer A (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). After 15 min on ice, 25 μl of 10% Nonidet P-40 was added and vigorously vortexed for 10 s. Homogenates were centrifuged for 30 s. Nuclear pellets were resuspended in 50 μl of ice-cold hypotonic buffer C (20 mm HEPES, pH 7.9, 0.42 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride) and rocked at 4 °C for 15 min. Homogenates were centrifuged for 5 min, and the supernatants (nuclear extracts) were aliquoted and stored at –70 °C. After determining protein concentrations using Bio-Rad protein assay (Bio-Rad), 2 μg of proteins were incubated at room temperature with 1 μg of poly(dI-dC) and 0.1 μg of salmon sperm DNA in 20 μl of binding buffer (12 mm HEPES, pH 7.8, 60 mm KCl, 2 mm MgCl2, 0.12 mm EDTA, 0.3 mm dithiothreitol, 0.3 mm phenylmethylsulfonyl fluoride, 12% glycerol) for 15 min. Complementary synthetic oligonucleotides containing the AP-1-responsive element plus adjacent sequences from the mouse c-myc promoter (AP1F, 5′-ATACCTGTGACTCATTCATTT-3′, and AP1R, 5′-AAATGAATGAGTCACAGGTAT-3′) were obtained from MWG Biotech and labeled with [γ-32P]ATP using T4 polynucleotide kinase (Invitrogen). Labeled oligonucleotides were purified using G25 columns (Amersham Biosciences) and used as probes (20,000 cpm/reaction) and added to the reactions for an additional 15 min. Complexes were analyzed on non-denaturing (4.5%) polyacrylamide gels in TGE buffer (40 mm Tris, 270 mm glycine, 2 mm EDTA, pH 8.0) and run at 13 V/cm at 4 °C. For supershift assays, 1 μg of the indicated antisera was added to the binding reaction. Kinase Assays—Methods to evaluate the phosphorylating activity of MAPK and JNK by in vitro kinase assays have been described previously (11.Chiariello M. Gutkind J.S. Methods Enzymol. 2002; 345: 437-447Crossref PubMed Scopus (3) Google Scholar). Western Blot Analysis and Antibodies—Methods to evaluate the phosphorylating activity of MAPK and JNK by in vitro kinase assay have been described previously (11.Chiariello M. Gutkind J.S. Methods Enzymol. 2002; 345: 437-447Crossref PubMed Scopus (3) Google Scholar). Lysates of total cellular proteins were analyzed by protein immunoblotting after SDS-PAGE with specific rabbit antisera or mouse monoclonal antibodies. Immunocomplexes were detected by ECL Plus (Amersham Biosciences) by using goat antiserum against rabbit or mouse IgG coupled to horseradish peroxidase (Amersham Biosciences). EMSA, Western blots, immunoprecipitations, and ChIP analysis were performed using rabbit polyclonal antibodies against JNK1 (C-17), Erk2 (C-14), Rac1 (C-14), c-Jun (H-79), JunD (329), JunB (N-17), and ATF2 (C-19) (Santa Cruz Biotechnology); phospho-c-Jun (Ser-63) and phospho-c-Jun (Ser-73) (Cell Signaling Technology); mouse monoclonal antibodies against HA and AU5 epitopes (Berkley Antibody Company); and JNK1 (BD Biosciences). Reporter Gene Assays—For each well, cells were transfected by the LipofectAMINE reagent with different expression plasmids together with 50 ng of the indicated reporter plasmid and 10 ng of pRL-null (a plasmid expressing the enzyme Renilla luciferase from Renilla reniformis) as an internal control. In all of the cases, the total amount of plasmid DNA was adjusted with empty vector. After 16–20 h from transfection, firefly and Renilla luciferase activities present in cellular lysates from serum-starved cells were assayed using the Dual-luciferase reporter system (Promega) and light emission was quantified using the Lumat LB9507 luminometer (EG&G Berthold) as specified by the manufacturer. Rac Effector Domain Mutants Differentially Impair Endogenous c-myc Expression—To investigate the signaling pathways activated by Rac impinging on the regulation of c-myc expression, we used specific constitutively active Rac effector domain mutants that are differentially impaired in their downstream signaling activities (8.Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (385) Google Scholar, 12.Joyce D. Bouzahzah B. Fu M. Albanese C. D'Amico M. Steer J. Klein J.U. Lee R.J. Segall J.E. Westwick J.K. Der C.J. Pestell R.G. J. Biol. Chem. 1999; 274: 25245-25249Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Therefore, we compared the ability to stimulate c-myc expression of a constitutively active Rac12V mutant with that of Rac alleles harboring additional mutations in their effector domain (Rac12V/33N, Rac12V/37L, and Rac12V/40H). We first explored, by Northern blot analysis, the ability of Rac12V to induce c-myc expression using PDGF as a positive control. As expected, the activated Rac12V mutant significantly induced c-myc expression as evidenced by an increase in the level of c-myc mRNA (Fig. 1A). We next investigated the effect of the double mutants. As shown in Fig. 1B, both the Rac12V/37L- and Rac12V/40H-mutated proteins were ineffective in stimulating the expression of c-myc, whereas the Rac12V/33N protein was fully competent to induce the transcription of the c-myc proto-oncogene. Recent work has established an impairment of JNK activation as a consequence of the transfection of the Rac12V/37L and Rac12V/40H effector domain mutants (8.Westwick J.K. Lambert Q.T. Clark G.J. Symons M. Van Aelst L. Pestell R.G. Der C.J. Mol. Cell. Biol. 1997; 17: 1324-1335Crossref PubMed Scopus (385) Google Scholar). In line with these studies, the data in Fig. 1C show that both Rac12V/37L and Rac12V/40H not only were unable to stimulate c-myc expression but they were also incapable of stimulating the activity of JNK, thereby suggesting the involvement of this kinase in Rac-induced c-myc expression. Conversely, the Rac12V/33N-mutated protein activated JNK at a level similar to the positive control, Rac12V (Fig. 1C). All of the Rac mutants were expressed at comparable levels (Fig. 1D). Together, these results strongly suggest that the JNK pathway is involved in the regulation of c-myc expression. JNK Activity Is Necessary for PDGF Induction of c-myc Expression—The possibility that JNK participates in the regulation of c-myc expression prompted us to test whether the constitutive activation of this signal transduction pathway could stimulate the expression of the endogenous c-myc proto-oncogene. Therefore, we transfected NIH 3T3 cells with vectors expressing two upstream activators of the JNK cascade, the MEKK1 and MLK3 MAPK kinase kinases (10.Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 13.Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar). As shown in Fig. 2A, both proteins induced the transcription of the endogenous c-myc gene at levels comparable to the positive control Rac12V, indicating that JNK activation is sufficient to trigger the expression of c-myc. Although JNKs have been isolated and characterized as stress-activated kinases based on their strong response to environmental stresses and inflammation stimuli, different growth factors are also able to stimulate their activity (14.Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). Moreover, they have recently been involved in mediating the proliferative effects of some oncogenes, including the product of the bcr-abl oncogene (15.Hess P. Pihan G. Sawyers C.L. Flavell R.A. Davis R.J. Nat. Genet. 2002; 32: 201-205Crossref PubMed Scopus (142) Google Scholar). Based on our data, we next explored the participation of JNK in the regulation of c-myc expression induced by PDGF. We began exploring the ability of this mitogen to activate JNK. As shown in Fig. 2B, exposure of NIH 3T3 fibroblasts to PDGF induced the activation of JNK, which peaked 30 min after stimulation. As an approach to examine the involvement of JNK in PDGF-induced c-myc expression, we took advantage of the availability of a synthetic compound, SP600125, a reversible ATP-competitive inhibitor that blocks JNK without significantly affecting other related kinases (16.Bennett B.L. Sasaki D.T. Murray B.W. O'Leary E.C. Sakata S.T. Xu W. Leisten J.C. Motiwala A. Pierce S. Satoh Y. Bhagwat S.S. Manning A.M. Anderson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13681-13686Crossref PubMed Scopus (2244) Google Scholar). We first confirmed the ability of the drug to inhibit JNK-dependent pathways in our experimental model. Indeed, SP600125 abolished PDGF-induced phosphorylation of the endogenous c-Jun protein in a dose-dependent manner as scored by Western blot analysis using a mixture of anti-phospho-Ser-63 and phospho-Ser-73 c-Jun antibodies (Fig. 2C). Conversely, identical concentrations of the drug had no effect on PDGF-induced Erk1/2 activation (Fig. 2D) and on Erk-dependent c-Fos phosphorylation (data not shown), indicating the specificity of the SP600125 for the JNK pathway as compared with other highly related MAPK-signaling pathways. To test the involvement of JNK in PDGF-induced c-myc expression, we performed Northern blot analysis on NIH 3T3 cells pretreated with increasing concentrations of the JNK inhibitor and then stimulated with PDGF for 1 h. As a result, the drug strongly inhibited PDGF-induced c-myc expression, even at the lowest concentration of the drug (Fig. 2E). Remarkably, the kinetic of inhibition of c-myc expression was coincident with the results obtained for the inhibition of PDGF-induced JNK activation by SP600125 (Fig. 2C). Thus, the emerging picture from these data is that activation of the JNK pathway may regulate c-myc expression and, in turn, PDGF exploits JNK as a key molecule to promote c-myc expression, possibly through phosphorylation and activation of nuclear transcription factors. A Typical AP-1-responsive Element in the c-myc Promoter— Two principal promoters, P1 and P2, drive the transcription of the human c-myc gene (17.Spencer C.A. Groudine M. Adv. Cancer Res. 1991; 56: 1-48Crossref PubMed Google Scholar). Despite the extraordinary complexity in the regulation of c-myc expression, the rate of transcription from these two promoters is mainly governed by composite negative and positive regulatory elements comprised within a 2.3-kb domain located upstream of the promoters (18.Hay N. Bishop J.M. Levens D. Genes Dev. 1987; 1: 659-671Crossref PubMed Scopus (83) Google Scholar). Among these elements, E2F, Stat-3, NF-κB, and F cell receptor 4-binding sites have been identified (19.Kiuchi N. Nakajima K. Ichiba M. Fukada T. Narimatsu M. Mizuno K. Hibi M. Hirano T. J. Exp. Med. 1999; 189: 63-73Crossref PubMed Scopus (342) Google Scholar, 20.He T.C. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4092) Google Scholar, 21.Wong K.K. Zou X. Merrell K.T. Patel A.J. Marcu K.B. Chellappan S. Calame K. Mol. Cell. Biol. 1995; 15: 6535-6544Crossref PubMed Scopus (55) Google Scholar, 22.Ji L. Arcinas M. Boxer L.M. Mol. Cell. Biol. 1994; 14: 7967-7974Crossref PubMed Google Scholar). In search for additional responsive elements that could mediate the JNK-dependent regulation of the c-myc gene, we performed computer-assisted analysis of its promoter region by the TRANSFAC data base (23.Heinemeyer T. Wingender E. Reuter I. Hermjakob H. Kel A.E. Kel O.V. Ignatieva E.V. Ananko E.A. Podkolodnaya O.A. Kolpakov F.A. Podkolodny N.L. Kolchanov N.A. Nucleic Acids Res. 1998; 26: 362-367Crossref PubMed Scopus (1325) Google Scholar). Surprisingly, we could identify 1.3 kb upstream the human c-myc transcription start site, a TGAGTCA motif perfectly matching the canonical AP-1-responsive element (Fig. 3A) (24.Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar). Interestingly, a similar analysis found conserved responsive elements also in the promoters of murine and even Drosophila c-myc genes (Fig. 3A). The sequences of the respective responsive elements were highly related to each other (Fig. 3A, boxed nucleotides) as opposed to their immediate flanking regions, suggesting that a strong selective pressure was exerted to maintain these sites intact during evolution. Several short sequences similar to known response elements are frequently found in promoter regions of a variety of genes. However, the arrangement of these sites in relation to neighboring sequences often determines the functionality of the predicted binding site. Thus, we first studied the ability of an oligonucleotide containing the murine c-myc AP-1-responsive element plus adjacent sequences to form DNA/proteins complexes by means of EMSA. As shown in Fig. 3B, left panel, proteins from NIH 3T3 nuclear extracts recognized and strongly bound the c-myc AP-1-responsive element as evidenced by the presence of a shifted complex that was more prominent 4 h after PDGF addition as a consequence of the accumulation of AP-1 proteins in the stimulated NIH 3T3 cells (25.Lallemand D. Spyrou G. Yaniv M. Pfarr C.M. Oncogene. 1997; 14: 819-830Crossref PubMed Scopus (132) Google Scholar). The binding was specific, because it was efficiently competed by adding an excess of unlabeled c-myc AP-1 oligonucleotide (Fig. 3B, right panel). To further investigate the nature of the transcription factors bound to the described c-myc AP-1 element, we next performed supershift experiments by incubating the binding reactions in the presence of specific antibodies against Jun family members. These proteins have been in fact described as substrates of the JNK signaling pathway, and they could therefore possibly mediate the effect of this kinase on the c-myc promoter. As shown in Fig. 3C, both c-Jun and JunD antibodies strongly decreased the electrophoretic mobility of the complexes derived from NIH 3T3 cells stimulated 30 min with PDGF, whereas the JunB antibody had a much lower effect. As an additional control, no ATF2 was detected in the complexes (Fig. 3C), in line with the fact that Jun:ATF2 heterodimers bind more efficiently atypical 8-bp TGACGTCA sites (26.van Dam H. Huguier S. Kooistra K. Baguet J. Vial E. van der Eb A.J. Herrlich P. Angel P. Castellazzi M. Genes Dev. 1998; 12: 1227-1239Crossref PubMed Scopus (100) Google Scholar). Conversely, among Fos proteins, only Fra2 was detected as part of the complexes (data not shown). On the basis of the binding observed in vitro, we next examined by ChIP analysis whether members of the Jun family could actually bind in vivo the endogenous c-myc promoter. In NIH 3T3 cells, ChIP assays clearly demonstrated the binding of both c-Jun and JunD to the endogenous c-myc promoter 30 min after PDGF addition (Fig. 3D), coincidently with the time point at which PDGF induces maximal JNK stimulation (see Fig. 2B). Conversely, we did not observe any in vivo binding of JunB to the promoter (Fig. 3D). As expected (see above), we could not detect ATF2 bound to the c-myc promoter (Fig. 3D), whereas it was able to bind the c-jun promoter, which harbors an atypical 8-bp TGACATCA element (data not shown). The same analysis, performed on untreated quiescent NIH 3T3 cells, gave similar results (data not shown), confirming that Jun family members are pre-bound to their responsive elements (25.Lallemand D. Spyrou G. Yaniv M. Pfarr C.M. Oncogene. 1997; 14: 819-830Crossref PubMed Scopus (132) Google Scholar) and can be rapidly trans-activated by phosphorylation in response to external stimuli (27.Mechta-Grigoriou F. Gerald D. Yaniv M. Oncogene. 2001; 20: 2378-2389Crossref PubMed Scopus (279) Google Scholar). As an additional control, no amplification was observed from the same immunoprecipitates when using primers recognizing DNA sequences unrelated to the c-myc promoter (Fig. 3D, lower panel). At this point, it is important to notice that although ChIP analysis was not able to detect binding of JunB to the c-myc promoter, EMSA experiments showed a small amount of this protein bound to the c-myc AP-1-containing EMSA probes. We have attributed this apparent difference to the in vitro nature of the EMSA and its limitations to precisely recapitulate the binding of the transcription factors at the level of the endogenous promoters. At the same time, this situation underscores the importance of the results from the ChIP assay, showing in vivo binding of c-Jun and JunD to the promoter. Altogether, these results indicate that proteins of the AP-1 family, specifically c-Jun and JunD, are able to recognize and bind in vivo the AP-1 element present in the c-myc promoter, thus suggesting this element as a potential mediator of JNK-dependent regulation of c-myc expression induced by PDGF. The AP-1 Element Controls PDGF Stimulation of c-myc Expression—We next investigated whether the c-myc AP-1 element was able to mediate PDGF-induced stimulation of c-myc expression. The control of histone acetylation is a key step in the general regulation of cellular transcriptional events (28.Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2419) Google Scholar). In turn, a model has been recently proposed in which the transactivation potential of c-Jun and possibly that of its related proteins is constitutively repressed by a histone deacetylases (HDACs) containing complex, which physically interacts with c-Jun itself and can be released upon JNK-dependent phosphorylation of the protein (29.Weiss C. Schneider S. Wagner E.F. Zhang X. Seto E. Bohmann D. EMBO J. 2003; 22: 3686-3695Crossref PubMed Scopus (133) Google Scholar). Therefore, we reasoned that an artificial molecule specifically targeting HDACs to AP-1 elements could recapitulate HDAC-dependent negative regulation of AP-1-containing promoters but in a dominant repressive fashion (unable to be relieved by upstream stimuli). Therefore, we engineered a molecule in which the DBD of c-Jun has been fused to the SID. The resulting protein (JunDBD-SID) is able to bind Sin3 and, through this, recruit HDACs (30.Ayer D.E. Laherty C.D. Lawrence Q.A. Armstrong A.P. Eisenman R.N. Mol. Cell. Biol. 1996; 16: 5772-5781Crossref PubMed Scopus (152) Google Scholar). We expect this repressor to be able to specifically inhibit transcription by targeting through the c-Jun DBD, AP-1 elements that are present in the endogenous promoters. To control the specificity of the repressor, we first engineered a reporter plasmid carrying the luciferase gene expressed under the control of a tandemly repeated AP-1 element from the murine c-myc gene (pmycAP-1-Luc). Such a construct behaves as a typical AP-1 reporter, its activity being readily induced by the c-Jun and c-Fos members of the AP-1 family (Fig. 4A) and by upstream stimulators of the JNK pathway, MEKK1 and MLK3 (Fig. 4B) (10.Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 13.Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1012) Google Scholar). As expected, the activity of the pmycAP-1-Luc plasmid was also readily induced by PDGF, its effect being much stronger when the corresponding oncogene (Sis) was cotransfected with the reporter (Fig. 4C). We hypothesized that the expression of luciferase from this construct should be strongly inhibited by the JunDBD-SID repressor through specific targeting to the c-myc AP-1 and recruitment of HDACs. As expected, very low amounts of the JunDBD-SID repressor were sufficient to completely abolish the activity of the pmycAP-1-Luc reporter induced by sis, the oncogenic form of the PDGF-proto-oncogene (Fig. 4D), while not affecting the luciferase activity induced by a Gal4-VP16 (Fig. 4E) or a p53 molecule (data not shown) on their respective reporter vectors. Therefore, these data confirmed the effectiveness and specificity of the engineered protein and the dependence of its activity upon the presence of functional AP-1 elements. It is also important to notice that such experiments not only control the specificity of our approach but also contribute to establish that the c-myc AP-1 is a fully functional element that can be stimulated by PDGF and the JNK pathway. This finding further supports the hypothesis that JNK plays a key role in the regulation of c-myc expression, possibly induced by PDGF activation of its cognate receptors. Finally, to prove the ability of the AP-1 element to regulate the transcription of the c-myc promoter, we analyzed by Northern blot the RNAs produced by PDGF-treated cells expressing the JunDBD-SID protein. Strikingly, the AP-1 repressor clearly inhibited PDGF-induced accumulation of c-myc mRNA (Fig. 4F). Altogether, these findings strongly support the idea that the AP-1 sequence identified in the c-myc promoter is functional, being able to control the c-myc expression induced by PDGF through the recruitment of members of the AP-1 family of transcription factors. In all, these findings also contribute to understand some of the molecular mechanisms by which c-Jun acts as a positive regulator of the cell cycle (24.Shaulian E. Karin M. Oncogene. 2001; 20: 2390-2400Crossref PubMed Scopus (1394) Google Scholar, 27.Mechta-Grigoriou F. Gerald D. Yaniv M. Oncogene. 2001; 20: 2378-2389Crossref PubMed Scopus (279) Google Scholar) because only very few c-Jun targets involved in the control of the cell cycle have been yet identified. In fact, this study adds c-myc to the short list of prototypes genes, such as cyclin D1 and p53 (31.Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar, 32.Schreiber M. Kolbus A. Piu F. Szabowski A. Mohle-Steinlein U. Tian J. Karin M. Angel P. Wagner E.F. Genes Dev. 1999; 13: 607-619Crossref PubMed Scopus (469) Google Scholar), that are regulated by c-Jun and involved in cellular proliferation. Our finding also show that JunD is bound to the c-myc promoter and can regulate c-myc expression, which may help to explain the role of this protein as mediator of cellular survival (33.Weitzman J.B. Fiette L. Matsuo K. Yaniv M. Mol. Cell. 2000; 6: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 34.Lamb J.A. Ventura J.J. Hess P. Flavell R.A. Davis R.J. Mol Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). The ability of JNK to mediate a typical proliferative pathway such as the one connecting PDGF to c-myc induction contrasts with the pro-apoptotic effects attributed to JNK in response to different environmental stresses (14.Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). Nonetheless, JNK has been also implicated in mediating survival signals in response to integrins (35.Almeida E.A. Ilic D. Han Q. Hauck C.R. Jin F. Kawakatsu H. Schlaepfer D.D. Damsky C.H. J. Cell Biol. 2000; 149: 741-754Crossref PubMed Scopus (337) Google Scholar), tumor necrosis factor-α (34.Lamb J.A. Ventura J.J. Hess P. Flavell R.A. Davis R.J. Mol Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), and cellular oncogenes (15.Hess P. Pihan G. Sawyers C.L. Flavell R.A. Davis R.J. Nat. Genet. 2002; 32: 201-205Crossref PubMed Scopus (142) Google Scholar). Interestingly, recent evidence shows that JunD is able to act downstream of JNK as a sensor that transmit survival or apoptotic signals depending on the state of others transcription factors (34.Lamb J.A. Ventura J.J. Hess P. Flavell R.A. Davis R.J. Mol Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Two non-mutually exclusive mechanisms are usually taken into account to explain the possibility for JNK to mediate both apoptotic and survival signaling. The latter mechanism considers the possibility that cells may interpret a transient JNK activation as a survival signal as opposed to the apoptotic response elicited by a sustained JNK activity (36.Chen Y.R. Wang X. Templeton D. Davis R.J. Tan T.H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (856) Google Scholar). The latter mechanism considers the possibility that JNK signals for cellular survival depend on the cooperation with other pathways such as Akt and NF-κB (34.Lamb J.A. Ventura J.J. Hess P. Flavell R.A. Davis R.J. Mol Cell. 2003; 11: 1479-1489Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Indeed, both these mechanisms could operate in our PDGF-dependent system. In fact, we have shown a transient activation of JNK in response to PDGF (Fig. 2B) and PDGF itself is able to contemporarily stimulate multiple signaling pathways, including Akt and NF-κB (37.Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1670) Google Scholar). Nonetheless, the possibility remains that specific stimuli, activating JNK in the absence of pro-survival signals, may induce apoptosis through a c-myc-dependent pathway. As c-myc itself has been involved in both pro- and anti-apoptotic responses, the mechanism by which regulation of c-myc expression by JNK-c-Jun/JunD relates to these two opposite responses will warrant further investigation. Our previous results indicated that PDGF induces c-myc expression through the Src-dependent activation of the Vav2 exchange factor, acting on the small GTPase Rac (6.Chiariello M. Marinissen M.J. Gutkind J.S. Nat. Cell Biol. 2001; 3: 580-586Crossref PubMed Scopus (113) Google Scholar). By studying the downstream components of the Rac pathway, we now show that JNK and two AP-1 family members, c-Jun and JunD, are essential components of the signaling cascade that mediate PDGF stimulation of c-myc expression (Fig. 5), which significantly establishes a new functional connection between Jun proteins and the c-myc proto-oncogene. The proposed pathway also suggests a mechanism by which both JNK and Jun proteins might exert their proliferative or apoptotic potential through the expression of the c-myc proto-oncogene. Further work will be required to establish the contribution of the “JNK-Jun pathway” to the biological responses of tyrosine kinase receptors such as the PDGF receptors as well as other membrane receptors that use the c-Myc protein to signal cellular proliferation. We thank C. J. Der for reagents and M. Santoro, F. Carlomagno, M. Incoronato, and A. M. Musti for discussion and critical reading of the paper.

Abstract 14‐3‐3 is a family of highly conserved adapter proteins that is attracting much interest among medicinal chemists. Small‐molecule inhibitors of 14‐3‐3 protein–protein interactions (PPIs) are in high demand, both as tools to increase our understanding of 14‐3‐3 actions in human diseases and as leads to develop innovative therapeutic agents. Herein we present the discovery of novel 14‐3‐3 PPI inhibitors through a multidisciplinary strategy combining molecular modeling, organic synthesis, image‐based high‐content analysis of reporter cells, and in vitro assays using cancer cells. Notably, the two most active compounds promoted the translocation of c‐Abl and FOXO pro‐apoptotic factors into the nucleus and sensitized multidrug‐resistant cancer cells to apoptotic inducers such as doxorubicin and the pan‐Akt inhibitor GSK690693, thus becoming valuable lead candidates for further optimization. Our results emphasize the possible role of 14‐3‐3 PPI inhibitors in anticancer combination therapies.

Pyrazolo[3,4-d]pyrimidines are potent protein kinase inhibitors with promising antitumor activity but suboptimal aqueous solubility, consequently worth being further optimized. Herein, we present the one-pot two-step procedure for the synthesis of a set of pyrazolo[3,4-d]pyrimidine prodrugs (1a-8a and 9a-e) with higher aqueous solubility and enhanced pharmacokinetic and therapeutic properties. ADME studies demonstrated for the most promising prodrugs a better aqueous solubility, a favorable hydrolysis in human and murine serum, and an increased ability to cross cell membranes with respect to the parental drugs, explaining their better 24 h in vitro cytotoxicity against human glioblastoma U87 cell line. Finally, the 4-4a couple of drug/prodrug was also evaluated in vivo, revealing a profitable pharmacokinetic profile of the prodrug associated with a good efficacy. The application of the prodrug approach demonstrated to be a successful strategy for improving aqueous solubility of the parental drugs, determining a positive impact also in their biological efficacy.

Iron is essential for deoxyribonucleotides production and for enzymes containing an Fe-S cluster involved in DNA replication and repair. How iron bioavailability and DNA metabolism are coordinated remains poorly understood. NCOA4 protein mediates autophagic degradation of ferritin to maintain iron homeostasis and inhibits DNA replication origin activation via hindrance of the MCM2-7 DNA helicase. Here, we show that iron deficiency inhibits DNA replication, parallel to nuclear NCOA4 stabilization. In iron-depleted cells, NCOA4 knockdown leads to unscheduled DNA synthesis, with replication stress, genome instability, and cell death. In mice, NCOA4 genetic inactivation causes defective intestinal regeneration upon dextran sulfate sodium-mediated injury, with DNA damage, defective cell proliferation, and cell death; in intestinal organoids, this is fostered by iron depletion. In summary, we describe a NCOA4-dependent mechanism that coordinates iron bioavailability and DNA replication. This function prevents replication stress, maintains genome integrity, and sustains high rates of cell proliferation during tissue regeneration.

Rab proteins are small GTPases involved in the regulation of intracellular membrane traffic in mammalian cells. In order to find Rab-interacting proteins we performed a two-hybrid screening using a human brain cDNA library. Here we report the isolation of a full-length human cDNA clone coding for a protein of 185 amino acids. This protein interacts strongly with the Rab4b, Rab5a, and Rab5c proteins and weakly with Rab4a, Rab6, Rab7, Rab17, and Rab22 in the two-hybrid assay. Comparison with the Data Bank revealed that this clone represents the human homolog of the previously isolated rat Prenylated Rab Acceptor (rPRA1). Analysis of mRNA expression shows a single abundant mRNA of about 0.8 kb ubiquitously expressed. Western blot analysis of the overexpressed protein shows a band of the expected size equally distributed between cytosol and membranes.

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr981, a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr981-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions. Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr981, a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr981-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions. MAP 3The abbreviations used are: MAP, mitogen-activated protein; MAPK, MAP kinase; MEN2, multiple endocrine neoplasias type 2; PTC, papillary thyroid carcinomas; HA, hemagglutinin; PDGF, platelet-derived growth factor; RT,; reverse transcriptase; qRT, quantitative real-time; SH, Src homology; Luc, luciferase; PP1, protein phosphatase-1. kinases are a family of proline-directed serine/threonine kinases that play a central role in signal transduction in all eukaryotic cells, from yeast to humans (1Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar). They coordinate signaling from a variety of extracellular and intracellular stimuli by acting as components of modular systems that involve other kinases and regulatory proteins. These stimuli induce specific phosphorylation on a conserved Thr-Xaa-Tyr motif present in all MAP kinases, thereby inducing their activation. Consequently, these proteins transmit the signals to a vast array of cellular regulatory proteins including protein kinases, transcription factors, cytoskeletal proteins, and other enzymes (1Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2292) Google Scholar). Erk8 is the last identified member of the MAP kinase family of proteins (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Expressed in humans at high levels in the brain, kidney, and lung, its activity can be modulated by serum and c-Src (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Although possessing a very typical MAP kinase domain, Erk8 also presents a peculiarly long C-terminal domain containing two putative SH3-binding sites (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Information concerning its upstream activators, downstream effectors, and cellular functions is still extremely limited. RET is a typical trans-membrane receptor tyrosine-kinase, essential for the development of the sympathetic, parasympathetic, and enteric nervous system and of the kidney (3Schuchardt A. D'Agati V. Larsson-Blomberg L. Costantini F. Pachnis V. Nature. 1994; 367: 380-383Crossref PubMed Scopus (1428) Google Scholar). In complex with four glycosylphosphatidylinositol-anchored coreceptors, GFR-α 1-4, the RET protein binds growth factors of the glial-derived neurotrophic factor family, mediating their intracellular signaling (4Airaksinen M.S. Saarma M. Nat. Rev. Neurosci. 2002; 3: 383-394Crossref PubMed Scopus (1462) Google Scholar). As for other receptor tyrosine-kinases, ligand interaction triggers autophosphorylation of different RET intracellular tyrosine residues that work as docking sites for several adaptor and effector signaling molecules (5Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (144) Google Scholar). Among such tyrosines, although Tyr981 is a binding site for c-Src, Tyr1062 has been shown to mediate the interactions with most of RET effectors and to be responsible for activation of the Ras/Erk, phosphatidylinositol 3-kinase/Akt, Jnk, p38, and Erk5 signaling pathways (6Kurokawa K. Kawai K. Hashimoto M. Ito Y. Takahashi M. J. Intern. Med. 2003; 253: 627-633Crossref PubMed Scopus (25) Google Scholar). Finally, Tyr1015 is a recognized docking site for phospholipase C γ (7Borrello M.G. Alberti L. Arighi E. Bongarzone I. Battistini C. Bardelli A. Pasini B. Piutti C. Rizzetti M.G. Mondellini P. Radice M.T. Pierotti M.A. Mol. Cell. Biol. 1996; 16: 2151-2163Crossref PubMed Google Scholar). Gain-of-function mutations of RET have been repeatedly described in several human tumors (8Pasini B. Ceccherini I. Romeo G. Trends Genet. 1996; 12: 138-144Abstract Full Text PDF PubMed Scopus (159) Google Scholar). RET germline point mutations are in fact responsible for the three clinical subtypes of the multiple endocrine neoplasias type 2 (MEN2) syndrome, MEN2A, MEN2B, and familial medullary thyroid carcinoma (5Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (144) Google Scholar). In addition, fusion of the intracellular kinase domain of RET with heterologous genes, caused by chromosomal inversions or translocations, generates the RET/PTC oncogenes, which represent the genetic hallmark of papillary thyroid carcinomas (PTC), accounting for more than 80-90% of all thyroid carcinomas (9Sherman S.I. Lancet. 2003; 361: 501-511Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). Among the at least 10 different RET/PTC rearrangements, RET/PTC1 and RET/PTC3, generated by the fusion with the H4 and RFG genes, respectively, are the most common types, accounting for more than 90% of all rearrangements (10Nikiforov Y.E. Endocr. Pathol. 2002; 13: 3-16Crossref PubMed Google Scholar). Antibodies—As primary antibodies, we used rabbit polyclonal antisera to Erk2 (C-14) (Santa Cruz Biotechnology), phospho-MAPK (p42/p44) (Cell Signaling), RET and phospho-RET (phospho-Tyr905) (11Carlomagno F. Guida T. Anaganti S. Vecchio G. Fusco A. Ryan A.J. Billaud M. Santoro M. Oncogene. 2004; 23: 6056-6063Crossref PubMed Scopus (211) Google Scholar), and mouse monoclonal antibodies to enhanced green fluorescent protein, AU5, and HA epitopes (Berkeley Antibody Company), Src (Ab-1; Oncogene Science), c-Abl (Pharmingen), and phospho-tyrosine, PY (Santa Cruz Biotechnology and Upstate Biotechnology). Expression Vectors—The expression vectors pCEFLP-SrcYF (constitutively active) and pCEFLP-SrcYF KM (dominant negative) were obtained by subcloning the corresponding cDNA obtained from pSM-SrcYF and pSM-SrcYF KM, kindly provided by H. Varmus (12Chiariello M. Marinissen M.J. Gutkind J.S. Nat. Cell Biol. 2001; 3: 580-586Crossref PubMed Scopus (113) Google Scholar). The HA-tagged form of Erk8 was generated by cloning the corresponding cDNA, kindly provided by M. Abe (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), in the pCEFL-HA vector. The HA-tagged forms of Erk8 deletion mutants (1-504; 1-486; 1-386; 1-373) were generated by cloning the corresponding cDNAs, obtained by PCR amplification, in the pCEFL-HA vector. The expression vector for the dominant negative Erk8 KR molecule was also provided by M. Abe (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). To generate the pCEFL-HA-Erk8δ expression vector, we amplified by PCR the corresponding cDNA using an “expressed sequence tag” obtained from ResGen (Clone ID 5742965). These sequence data have been submitted to the GenBank™ data base under accession number AY994058. pCDNA3-RET/PTC3 and pBABE-RET/MEN2B expression plasmid have been described previously (13Carlomagno F. De Vita G. Berlingieri M.T. de Franciscis V. Melillo R.M. Colantuoni V. Kraus M.H. Di Fiore P.P. Fusco A. Santoro M. EMBO J. 1996; 15: 2717-2725Crossref PubMed Scopus (119) Google Scholar, 14Melillo R.M. Santoro M. Ong S.H. Billaud M. Fusco A. Hadari Y.R. Schlessinger J. Lax I. Mol. Cell. Biol. 2001; 21: 4177-4187Crossref PubMed Scopus (112) Google Scholar). The RET/PTC3Y981, RET/PTC3Y1015, RET/PTC3Y1062, RET/PTC3 Kindead, and RET/PTC3V804 expression plasmid were generated by the QuikChange™ site-directed mutagenesis kit (Stratagene), using pCDNA3-PTC3 as a template. Expression vectors for the activated form of c-Abl (Abl Act) and Bcr/Abl p210 (Bcr/Abl) have been described previously (15Lobo V. Luquero C.I.A. Alvarez-Vallina L. Tipping A.J. Viniegra J.G. Losa J.H. Cobo C.P. Moya E.M.G. Cruz J.G. Melo J.V. Cajal S.R.Y. Sanchez-Prieto R. Biochem. J. 2005; 387: 231-238Crossref PubMed Scopus (22) Google Scholar, 16Sanchez-Prieto R. Sanchez-Arevalo V.J. Servitja J.M. Gutkind J.S. Oncogene. 2002; 21: 974-979Crossref PubMed Scopus (97) Google Scholar). The dominant negative c-Abl (Abl-KD) expression vector was obtained by mutating a critical lysine in the kinase domain of c-Abl, contained in the pCEFL-AU5 vector. The c-myc and c-jun promoter reporter plasmids, pMyc-Luc and pJLuc, respectively, and the pCDNAIII-β-galactosidase expression vector have been described previously (12Chiariello M. Marinissen M.J. Gutkind J.S. Nat. Cell Biol. 2001; 3: 580-586Crossref PubMed Scopus (113) Google Scholar, 17Chiariello M. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2000; 20: 1747-1758Crossref PubMed Scopus (168) Google Scholar). The identity and integrity of the different clones were confirmed by dideoxy DNA sequencing. For each plasmid, additional information will be provided upon request. Western Blot Analysis—Lysates of total cellular proteins or immunoprecipitates were analyzed by protein immunoblotting after SDS-PAGE with specific rabbit antisera or mouse monoclonal antibodies. Immunocomplexes were visualized by enhanced chemiluminescence detection (ECL or ECL Plus, Amersham Biosciences) with the use of goat antiserum to rabbit or mouse immunoglobulin G, coupled to horseradish peroxidase (Amersham Biosciences). Reagents—The PP1 inhibitor was purchased from BIOMOL. PDGF was purchased from Serologicals Corp. Cell Culture and Transfections—293T, Cal62, Kat4, and ARO cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, and 100 units/ml penicillin-streptomycin (Invitrogen). NIH 3T3 fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf bovine serum (BioWhittaker), 2 mm l-glutamine, and 100 units/ml penicillin-streptomycin (Invitrogen). Rat Pc cells were maintained in Coon's modified Ham's F12 medium supplemented with 5% calf serum and a mixture of six hormones, including 10 milliunits/ml thyrotropin, 10 nm hydrocortisone, 10 μg/ml insulin, 5 μg/ml apo-transferrin, 10 ng/ml somatostatin, and 10 ng/ml glycyl-histidyl-lysin (Sigma). 293T and NIH 3T3 cells were transfected by the Lipofectamine reagent (Invitrogen), whereas ARO cells were transfected by the Lipofectamine 2000 reagent (Invitrogen), respectively, in accordance with the manufacturer's instructions. For transfections, 200 ng of HA-Erk8 and HA-Erk8δ and 100 ng of SrcYF, Abl Act, Bcr/Abl, and of the different Ptc3 expression vectors were always used. Reporter Gene Assays—NIH 3T3 cells were transfected with different expression plasmids together with 100 ng of the pMyc Luc reporter plasmid. ARO cells were transfected with different expression plasmids together with 20 ng of the pJLuc reporter plasmid. After a 24-h incubation in serum-free medium, the cells were lysed using reporter lysis buffer (Promega). Luciferase activity present in cellular lysates was assayed using d-luciferin and ATP as substrates, and light emission was quantitated using the 20n/20n luminometer as specified by the manufacturer (Turner BioSystems). RNA Extraction, RT-PCR, and Quantitative Real-time PCR—Total RNA was isolated using the RNeasy kit (Qiagen) and subjected to on-column DNase digestion with the RNase-free DNase set (Qiagen) according to the manufacturer's instructions. The quality of RNA was verified by electrophoresis through 1% agarose gel and visualized with ethidium bromide. We synthesized random-primed first-strand cDNA in a 50-μl reaction volume starting from 2 μg of RNA using the Gene-Amp RNA PCR core kit (Applied Biosystems). Quantitative (real-time) reverse transcription-polymerase chain reactions (qRT-PCRs) were performed by using the SYBR Green PCR master mix (Applied Biosystems) in the iCycler apparatus (Bio-Rad). Amplification reactions (25-μl final reaction volume) contained 200 nm of each primer, 3 mm MgCl2, 300 μm deoxyribonucleoside triphosphates (dNTPs), 1× SYBR Green PCR buffer (Applied Biosystems), 0.1 units/μl AmpliTaq Gold DNA Polymerase (Applied Biosystems), 0.01 units/μl Amp Erase (Applied Biosystems), RNase-free water, and 2 μl of cDNA samples. Thermal cycling conditions were optimized standard conditions and are available upon request. To verify the absence of nonspecific products, we performed 80 cycles of melting (55 °C for 10 s). The melting curve confirmed that a single product was generated. Amplification was monitored by measuring the increase in fluorescence caused by the binding of SYBR Green to double-stranded DNA. Primers were synthesized by MWG Biotech. Nucleotide sequences of the primers used in this study were as follows: qErk8Forw, 5′-GGAGTTTGGGGACCATCC-3′, and qErk8Rev, 5′-GCGTTCAGGTCAGTGTCC-3′. Src in Vitro Kinase Assay—Cells were lysed in a buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1% (v/v) Nonidet P-40, 1 mm EDTA, 50 mm NaF, 20 mm sodium pyrophosphate, 1 mm sodium vanadate, 2 mm phenylmethylsulfonyl fluoride, and 0.2 mg/ml each aprotinin and leupeptin. Lysates were centrifuged at 10,000 × g for 30 min and immunoprecipitated by incubating 1 mg of total proteins with anti-Src antibodies for 60 min at 4 °C. Immunocomplexes were recovered by incubation with protein G-Sepharose beads (Amersham Biosciences) on a rotating platform at 4 °C for 60 min. After three washes with lysis buffer, the immunoprecipitates were washed with kinase buffer (20 mm Tris-HCl (pH = 7.0), 5 mm MgCl2) and resuspended in 30 μl of kinase buffer, 10 mCi of [γ-32P]ATP (>10,000 Ci/mmol, Amersham Biosciences), and 10 mm cold ATP. After 30 min of incubation at room temperature, the beads were washed twice with lysis buffer, and the reaction was terminated by adding an equal volume of SDS-gel loading buffer (62.5 mm Tris-HCl (pH = 6.8), 2% SDS, 5% glycerol, 0.7 m 2-mercaptoethanol, and 0.25% bromphenol blue). When indicated, 5 mg of acid-denatured enolase were added to the reaction (Roche Applied Science). The samples were electrophoresed on SDS-10% polyacrylamide gel. After the run, gels were incubated three times for 30 min in a fixing solution (20% methanol, 10% acetic acid), dried, and processed for auto-radiography with phosphor screens. Laser densitometry was used to quantitate Src kinase activity. Erk8 Is Activated by a RET-dependent Signaling Pathway—We performed an in silico analysis of Erk8 gene expression in mouse tissues through the public Mouse Gene Prediction Database resource (18Zhang W. Morris Q.D. Chang R. Shai O. Bakowski M.A. Mitsakakis N. Mohammad N. Robinson M.D. Zirngibl R. Somogyi E. Laurin N. Eftekharpour E. Sat E. Grigull J. Pan Q. Peng W.T. Krogan N. Greenblatt J. Fehlings M. van der Kooy D. Aubin J. Bruneau B.G. Rossant J. Blencowe B.J. Frey B.J. Hughes T.R. J. Biol. (Bronx N. Y.). 2004; 3: 21Google Scholar). Among other tissues, the mouse orthologue Erk8 gene was expressed at very high levels in the thyroid. To verify Erk8 expression also in human thyroid-derived cell lines, we next performed qRT-PCR in three human anaplastic thyroid carcinoma cell lines, ARO, Cal62, and Kat4. qRT-PCR evidenced high expression of Erk8 in the three human cell lines tested, whereas as a control, the human-specific primers did not detect any signal in a rat papillary thyroid carcinoma cell line, Pc (Fig. 1A). These data therefore suggest a role for Erk8 in signaling pathways involved in the homeostasis and/or pathology of the thyroid. As RET/PTC oncogenes are frequently involved in human papillary thyroid carcinomas (5Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (144) Google Scholar), we decided to investigate their ability to modulate Erk8 activation. In particular, we investigated the role of RET/PTC3, a chimeric oncogene generated by the fusion of RET with the RFG gene (Fig. 1B) (19Santoro M. Dathan N.A. Berlingieri M.T. Bongarzone I. Paulin C. Grieco M. Pierotti M.A. Vecchio G. Fusco A. Oncogene. 1994; 9: 509-516PubMed Google Scholar). As an approach to score Erk8 activation, we used an anti-phospho-MAPK (Erk2) antibody that recognizes phosphorylation in the conserved MAP kinase TEY motif. We performed Western blot analysis of 293T cells transfected with an HA epitope-tagged form of the Erk8 kinase, as described previously by Abe et al. (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), and then distinguished the transfected HA-Erk8 and the endogenous Erk2 by their different molecular masses, ∼60 and ∼45 kDa, respectively. As shown in Fig. 1C, RET/PTC3 overexpression readily induced Erk8 activation at a level comparable with an activated form of c-Src (Src YF), used as a positive control (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Of note, no signal in the ∼60-kDa range was detected in the absence of HA-Erk8 transfection (Fig. 1C), indicating that the anti-phospho-MAPK antisera specifically recognized the Erk8 protein. As an additional control for the activity of RET/PTC3 and Src YF, both proteins activated the Erk2 MAP kinase (Fig. 1C), also scored by anti-phospho-MAPK Western blot. As the RET/PTC3 oncogene is generated by the fusion of RET with the RFG gene, we next wanted to address the specific contribution of RET to the activation of the Erk8 protein. To this aim, we used a different RET-derived oncogene, RET/MEN2B, representing a specific point mutant of the intracellular RET domain (5Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (144) Google Scholar), therefore lacking the RFG component present in the RET/PTC3 chimeric protein. As shown in Fig. 1D, increasing amounts of the RET/MEN2B protein also induced Erk8 activation, therefore suggesting a specific role for the RET intracellular tyrosine kinase domain in the activation of this MAP kinase. Altogether, these results indicate that activated forms of the RET protooncogene stimulate Erk8 activity. A Kinase-defective Mutant for Erk8 Interferes with RET/PTC3 Signaling—The expression of the c-jun proto-oncogene is rapidly and transiently induced by different growth factors and cellular oncogenes (20Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (191) Google Scholar). Among them, an oncogenic rearrangement of the RET proto-oncogene is able to strongly induce c-jun expression (21Ishizaka Y. Takahashi M. Ushijima T. Sugimura T. Nagao M. Biochem. Biophys. Res. Commun. 1991; 179: 1331-1336Crossref PubMed Scopus (4) Google Scholar), therefore establishing this gene as part of the RET signaling pathway. To investigate whether the RET/PTC3 oncogene was able to stimulate the activity of the c-jun promoter, we took advantage of the availability of a reporter plasmid carrying the luciferase gene under the control of the c-jun promoter, pJLuc (17Chiariello M. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2000; 20: 1747-1758Crossref PubMed Scopus (168) Google Scholar, 20Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (191) Google Scholar). A variety of thyroid cell lines have been used as model systems to examine RET biological functions. Among them, human anaplastic thyroid carcinoma ARO cells have the advantage to be readily transfectable and to express endogenous Erk8 (Fig. 1A). Cotransfection of thyroid ARO cells with the pJLuc reporter plasmid and increasing concentrations of the RET/PTC3 cDNA revealed that this oncogene could strongly induce the activity of the c-jun promoter (Fig. 2A). To evaluate whether Erk8 activation is involved in RET/PTC3 signaling to the c-jun promoter, we next used a dominant negative, kinase defective (data not shown) Erk8 molecule, Erk8 KR. For these experiments, we therefore cotransfected RET/PTC3 with the c-jun reporter plasmid and increasing amounts of the Erk8 KR expression vector. As shown in Fig. 2B, the dominant negative Erk8 molecule caused a strong, although incomplete, inhibition of RET/PTC3-dependent c-jun promoter stimulation, suggesting the existence of both Erk8-dependent and Erk8-independent pathways linking RET/PTC3 to the expression of the c-jun proto-oncogene. Tyrosine 981 of RET/PTC3 Is Necessary for Erk8 Activation—Tyrosine phosphorylated residues in the kinase domain of RET, as well as of its derivative oncogenes, usually represent docking sites for adaptor proteins and enzymes that are able to propagate the signal to the intracellular environment (5Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (144) Google Scholar). We therefore used RET/PTC3 molecules in which different tyrosine phosphorylation sites have been inactivated by mutating them to phenylalanines to ascertain the dependence of RET/PTC3-induced Erk8 activation on the presence of these specific residues. Also, as these tyrosines have already been linked to the activation of different specific signaling pathways (5Santoro M. Melillo R.M. Carlomagno F. Vecchio G. Fusco A. Endocrinology. 2004; 145: 5448-5451Crossref PubMed Scopus (144) Google Scholar), this approach could grant us the possibility to suggest the participation of some of these effectors in the modulation of Erk8 activity. In particular, tyrosine 981 binds c-Src (22Encinas M. Crowder R.J. Milbrandt J. Johnson Jr., E.M. J. Biol. Chem. 2004; 279: 18262-18269Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), tyrosine 1015 is a docking site for phospholipase C γ (7Borrello M.G. Alberti L. Arighi E. Bongarzone I. Battistini C. Bardelli A. Pasini B. Piutti C. Rizzetti M.G. Mondellini P. Radice M.T. Pierotti M.A. Mol. Cell. Biol. 1996; 16: 2151-2163Crossref PubMed Google Scholar), and tyrosine 1062 is a multiple docking site that mediates most of the RET signaling pathways (6Kurokawa K. Kawai K. Hashimoto M. Ito Y. Takahashi M. J. Intern. Med. 2003; 253: 627-633Crossref PubMed Scopus (25) Google Scholar), including Erk2 activation (23Chiariello M. Visconti R. Carlomagno F. Melillo R.M. Bucci C. de Franciscis V. Fox G.M. Jing S. Coso O.A. Gutkind J.S. Fusco A. Santoro M. Oncogene. 1998; 16: 2435-2445Crossref PubMed Scopus (108) Google Scholar). 293T cells were transiently transfected with the HA-Erk8 molecule, together with RET/PTC3, RET/PTC3Y981, RET/PTC3Y1015, or RET/PTC3Y1062, respectively (numbers indicating RET/PTC3 tyrosine residues correspond to their position in the wild-type RET receptor). Surprisingly, based on the observation that tyrosine 1062 mediates most of the RET signaling pathways (6Kurokawa K. Kawai K. Hashimoto M. Ito Y. Takahashi M. J. Intern. Med. 2003; 253: 627-633Crossref PubMed Scopus (25) Google Scholar), the RET/PTC3Y1062 mutant activated Erk8 at an extent comparable with the RET/PTC3 molecule, whereas as expected (23Chiariello M. Visconti R. Carlomagno F. Melillo R.M. Bucci C. de Franciscis V. Fox G.M. Jing S. Coso O.A. Gutkind J.S. Fusco A. Santoro M. Oncogene. 1998; 16: 2435-2445Crossref PubMed Scopus (108) Google Scholar), this mutation strongly affected Erk2 activation (Fig. 3). The tyrosine 1015 mutation, involving a known binding site for phospholipase C γ (7Borrello M.G. Alberti L. Arighi E. Bongarzone I. Battistini C. Bardelli A. Pasini B. Piutti C. Rizzetti M.G. Mondellini P. Radice M.T. Pierotti M.A. Mol. Cell. Biol. 1996; 16: 2151-2163Crossref PubMed Google Scholar), also did not affect Erk8 activation by RET/PTC3 (Fig. 3). Conversely, tyrosine 981 mutation determined a dramatic reduction in RET/PTC3-dependent Erk8 activation, although such mutation proved irrelevant to Erk2 activation (Fig. 3). As a control, RET/PTC3 Kindead, a kinase-inactive form of RET/PTC3 containing a mutation in the ATP-binding catalytic lysine (Lys758), was unable to activate both Erk8 and Erk2 (Fig. 3). These results therefore imply tyrosine 981 of RET/PTC3 as a major site recognized by signaling molecules mediating RET/PTC3-dependent Erk8 activation. In addition, as tyrosine 981 has been previously recognized as a key residue for the binding of c-Src to RET (22Encinas M. Crowder R.J. Milbrandt J. Johnson Jr., E.M. J. Biol. Chem. 2004; 279: 18262-18269Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), they also suggest a role for c-Src in mediating RET/PTC3-initiated signals impinging on Erk8 activation. Src Activity Is Dispensable for RET/PTC3-dependent Erk8 Activation—Based on the above information and on the observation that c-Src activates Erk8 (2Abe M.K. Saelzler M.P. Espinosa R. II I Kahle K.T. Hershenson M.B. Le Beau M.M. Rosner M.R. J. Biol. Chem. 2002; 277: 16733-16743Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), we next sought to investigate whether c-Src was able to mediate RET/PTC3-dependent Erk8 activation. A classical approach to establish a role for Src kinases in cellular processes takes advantage of a pyrazolo-pyrimidine compound, PP1, which binds the ATP-binding pocket of these kinases, therefore blocking their enzymatic activity (24Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790) Google Scholar) and biological functions (12Chiariello M. Marinissen M.J. Gutkind J.S. Nat. Cell Biol. 2001; 3: 580-586Crossref PubMed Scopus (113) Google Scholar). Although PP1 has been described to affect RET kinase activity (25Carlomagno F. Vitagliano D. Guida T. Napolitano M. Vecchio G. Fusco A. Gazit A. Levitzki A. Santoro M. Cancer Res. 2002; 62: 1077-1082PubMed Google Scholar), a specific mutation in valine 804 in the RET kinase domain confers resistance (>50-fold increase of the IC50) to the compound (11Carlomagno F. Guida T. Anaganti S. Vecchio G. Fusco A. Ryan A.J. Billaud M. Santoro M. Oncogene. 2004; 23: 6056-6063Crossref PubMed Scopus (211) Google Scholar). We, therefore, introduced such mutation in the RET/PTC3 kinase domain (RET/PTC3V804), rendering its activity significantly resistant to PP1, as scored by RET/PTC3V804 autophosphorylation and activation of Erk2 (Fig. 4A). As expected, kinase activity of the parental RET/PTC3 molecule was completely abolished at comparable concentrations (compare the 5-10 μm PP1 lanes) as evidenced by both RET/PTC3 autophosphorylation and activation of Erk2 (Fig. 4B). Surprisingly, PP1 treatment of RET/PTC3V804-transfected cells only slightly affected Erk8 activity even at the highest doses tested (10 μm) (Fig. 4C) and after extensive times of treatment (up to 10 h of treatment, at 5 μm concentration) (Fig. 4D), thus excluding a role for c-Src and its related kinases (24Hanke J.H. Gardner J.P. Dow R.L. Changelian P.S. Brissette W.H. Weringer E.J. Pollok B.A. Connelly P.A. J. Biol. Chem. 1996; 271: 695-701Abstract Full Text Full Text PDF PubMed Scopus (1790)

The properties of adenyl-cyclase and of cAMP phosphodiesterase of normal thyroid and of a transplantable thyroid tumor of the rat have been compared. The adenyl-cyclase of the tumor and the normal thyroid were both stimulated by sodium fluoride. TSH significantly stimulates adenyl-cyclase of normal thyroid, but fails to stimulate the tumor enzyme. cAMP-phosphodiesterase was found in the 22,000 g supernatant of both tissues. In the presence of 5 × 10-3M theophylline the enzyme of both tissues was inhibited by 40%. TSH also fails to stimulate 1-14C glucose oxidation and phospholipid metabolism in tumor slices, whereas, DBcAMP significantly stimulates the metabolism of both. The inability of the tumor adenyl-cyclase to respond to TSH may be due to a structural alteration of the receptor site for TSH in the neoplastic cell. (Endocrinology90: 1483, 1972)

Macroautophagy/autophagy is one of the major responses to stress in eukaryotic cells and is implicated in several pathological conditions such as infections, neurodegenerative diseases and cancer. Interestingly, cancer cells take full advantage of autophagy both to support tumor growth in adverse microenvironments and to oppose damages induced by anti-neoplastic therapies. Importantly, different human oncogenes are able to modulate this survival mechanism to support the transformation process, ultimately leading to ‘autophagy addiction’. Still, oncogenic signaling events, impinging on the control of autophagy, are poorly characterized, limiting our possibilities to take advantage of these mechanisms for therapeutic purposes. Here, we screened a library of activated kinases for their ability to stimulate autophagy. By this approach, we identified novel potential regulators of the autophagic process and, among them, the IKBKE oncogene. Specifically, we demonstrate that this oncoprotein is able to stimulate autophagy when overexpressed, an event frequently found in breast tumors, and that its activity is strictly required for breast cancer cells to support the autophagic process. Interestingly, different oncogenic pathways typically involved in breast cancer, namely ERBB2 and PI3K-AKT-MTOR, also rely on IKBKE to control this process. Ultimately, we show that IKBKE-dependent autophagy is necessary for breast cancer cell proliferation, suggesting an important supporting role for this oncogene and autophagy in these tumors.Abbreviations: AAK1: AP2 associated kinase 1; AMPK: 5ʹ-prime-AMP-activated protein kinase; AKT1: AKT serine/threonine kinase 1; BAF: bafilomycin A1; CA: constitutively activated; CDK17: cyclin dependent kinase 17; CDK18: cyclin dependent kinase 18; CHUK: conserved helix-loop-helix ubiquitous kinase; EGF: epidermal growth factor; ERBB2: erb-b2 receptor tyrosine kinase 2; FGF: fibroblast growth factor; FM: full medium; GALK2: galactokinase 2; IKBKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKE: inhibitor of nuclear factor kappa B kinase subunit epsilon; IKK: IκB kinase complex; KD: kinase dead; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1: mitogen-activated protein kinase 1; MAPK15: mitogen-activated protein kinase 15; MTORC1: mammalian target of rapamycin kinase complex 1; myr: myristoylation/myristoylated; NFKBIA: NFKB inhibitor alpha; PDGF: platelet derived growth factor; PFKL: phosphofructokinase, liver type; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKCD: protein kinase C delta; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TNBC: triple-negative breast cancer; TSC2: TSC complex subunit 2; WB: western blot; WT: wild-type.

In the last few years, several pyrrolo-pyrimidine derivatives have been either approved by the US FDA and in other countries for the treatment of different diseases or are currently in phase I/II clinical trials. Herein we present the synthesis and the characterization of a novel series of pyrrolo[2,3-d]pyrimidines, compounds 8a-j, and their activity against Glioblastoma multiforme (GBM). Docking studies and MM-GBSA analysis revealed the ability of such compounds to efficiently interact with the ATP binding site of Src. Enzymatic assays against a mini-panel of kinases (Src, Fyn, EGFR, Kit, Flt3, Abl, AblT315I) have been performed, showing an unexpected selectivity of our pyrrolo[2,3-d]pyrimidines for Src. Finally, the derivatives were tested for their antiproliferative potency on U87 GBM cell line. Compound 8h showed a considerable cytotoxicity effect against U87 cell line with an IC50 value of 7.1 μM.

Abstract Pyrazolo[3,4- d ]pyrimidines are a class of compounds with a good activity against several cancer cell lines. Despite the promising anticancer activity, these molecules showed a poor aqueous solubility. This issue could threat the future development of pyrazolo[3,4- d ]pyrimidines as clinical drug candidates. With the aim of improving their solubility profile and consequently their pharmacokinetic properties, we have chosen four compounds (1–4) on the base of their anti-neuroblastoma activity and we have developed albumin nanoparticles and liposomes for the selected candidates. Albumin nanoparticles and liposomes were prepared and characterized regarding size and ζ-potential distribution, polidispersity index, entrapment efficiency and activity against SH-SY5Y human neuroblastoma cell line. The most promising nanosystem, namely LP-2, was chosen to perform further studies: confocal microscopy, stability and drug release in physiological conditions and biodistribution. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo[3,4- d ]pyrimidines in liposomes represent an effective method to overcome the poor water solubility.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats’ eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

Pyrazolo[3,4-d]pyrimidine derivatives 1-5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1-4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent.

In this paper a straightforward synthesis of a novel pyrazole derivative is reported. Prominent feature of this synthetic process is a 1,3-Dipolar Cycloaddition of a suitable nitrile imine with an activated α,β-unsaturated lactam to afford directly and regioselectively the corresponding ring-fused pyrazole. Having obtained the central core of the synthetic target, a double stepwise functionalization with a "side chain" characterized by a terminal cyclic aliphatic amine was carried out. This molecular structure was designed to interact strongly with typical biological residues, and indeed it showed potent anticancer capability: in vitro cytotoxicity test on five different cancer cell lines showed interesting IC50 values in the range of 15-60 μM for exposure time of 24-72 h, thus resulting comparable with commercially available and nowadays therapeutically exploited anticancer compounds, such as 5-FU and NVP-BEZ235.

Herein we report the synthesis of cellulose nanocrystals covalently bound to a model chemotherapeutic drug (DOXO) via a novel spacer arm, which acts both as linker and as selective releasing agent. The carbamate linkage present in the linker, shows stability in aqueous environments for a wide range of conditions and can only be hydrolyzed in the presence of cells, freeing the active drug, with unmodified chemotherapeutic properties.

Hybrid hydrogels made of chemically modified pectin, gelatin and xanthan gum have been formulated and processed through a double crosslinking step, aimed at wound healing applications.

Mitogen-activated protein (MAP) kinases, p42(MAPK) and p44(MAPK), are central components of growth-promoting signalling pathways. However, how stimulation of MAP kinases culminates in cell-cycle progression is still poorly understood. Here we show that mitogenic stimulation of NIH 3T3 cells causes a sustained activation of MAP kinases, which lasts until cells begin progressing through the G(1)/S boundary. Furthermore, we observed that disruption of the MAP-kinase pathway with a selective MEK (MAP kinase/extracellular-signal-regulated protein kinase kinase) inhibitor, PD98059, prevents the activation of cyclin-dependent kinase (Cdk) 2 and DNA synthesis, even when added during late G(1) phase, once the known mechanisms by which MAP kinase controls G(1) progression, accumulation of G(1) cyclins and degradation of Cdk inhibitors have already taken place. Moreover, we provide evidence indicating that MAP kinases control Cdk2 Thr-160 activating phosphorylation and function, possibly by regulating the activity of a Cdk-activating kinase, thus promoting the re-initiation of DNA synthesis. These findings suggest the existence of a novel mechanism whereby signal-transducing pathways converging on MAP kinases can affect the cell-cycle machinery and, ultimately, participate in cell-growth control.

Autophagy, a pathway for bulk protein degradation and removal of damaged organelles, represents one of the major responses of cells to stress, thereby exerting a strict control on their correct functioning. Consequently, this process has been involved in the pathogenesis and therapeutic responses of several human diseases. Mitogen-activated protein (MAP) kinase 15 (MAPK15) is an atypical member of the MAP kinase family that recently emerged as a key modulator of autophagy and, through this, of cell transformation. Still, no information is available about signaling pathways mediating the effect of MAPK15 on this process, nor is it known which phase of autophagosome biogenesis is affected by this MAP kinase. Here, we demonstrate that MAPK15 stimulated 5′-AMP–activated protein kinase–dependent activity of UNC-51-like kinase 1 (ULK1), the only protein kinase among the ATG-related proteins, toward downstream substrates and signaling intermediates. Importantly, MAPK15 directly interacted with the ULK1 complex and mediated ULK1 activation induced by starvation, a classical stimulus for the autophagic process. In turn, ULK1 and its highly homologous protein ULK2 are able to transduce MAPK15 signals stimulating early phases of autophagosomal biogenesis in a multikinase cascade that offers numerous potential targets for future therapeutic intervention in cancer and other autophagy-related human diseases. Autophagy, a pathway for bulk protein degradation and removal of damaged organelles, represents one of the major responses of cells to stress, thereby exerting a strict control on their correct functioning. Consequently, this process has been involved in the pathogenesis and therapeutic responses of several human diseases. Mitogen-activated protein (MAP) kinase 15 (MAPK15) is an atypical member of the MAP kinase family that recently emerged as a key modulator of autophagy and, through this, of cell transformation. Still, no information is available about signaling pathways mediating the effect of MAPK15 on this process, nor is it known which phase of autophagosome biogenesis is affected by this MAP kinase. Here, we demonstrate that MAPK15 stimulated 5′-AMP–activated protein kinase–dependent activity of UNC-51-like kinase 1 (ULK1), the only protein kinase among the ATG-related proteins, toward downstream substrates and signaling intermediates. Importantly, MAPK15 directly interacted with the ULK1 complex and mediated ULK1 activation induced by starvation, a classical stimulus for the autophagic process. In turn, ULK1 and its highly homologous protein ULK2 are able to transduce MAPK15 signals stimulating early phases of autophagosomal biogenesis in a multikinase cascade that offers numerous potential targets for future therapeutic intervention in cancer and other autophagy-related human diseases.

RAB7A is a small GTPase that controls the late endocytic pathway but also cell migration through RAC1 (Ras-related C3 botulinum toxin substrate 1) and vimentin. In fact, RAB7A regulates vimentin phosphorylation at different sites and vimentin assembly, and, in this study, we identified vimentin domains interacting with RAB7A. As several kinases could be responsible for vimentin phosphorylation, we investigated whether modulation of RAB7A expression affects the activity of these kinases. We discovered that RAB7A regulates AKT and PAK1, and we demonstrated that increased vimentin phosphorylation at Ser38 (Serine 38), observed upon RAB7A overexpression, is due to AKT activity. As AKT and PAK1 are key regulators of several cellular events, we investigated if RAB7A could have a role in these processes by modulating AKT and PAK1 activity. We found that RAB7A protein levels affected beta-catenin and caspase 9 expression. We also observed the downregulation of cofilin-1 and decreased matrix metalloproteinase 2 (MMP2) activity upon RAB7A silencing. Altogether these results demonstrate that RAB7A regulates AKT and PAK1 kinases, affecting their downstream effectors and the processes they regulate, suggesting that RAB7A could have a role in a number of cancer hallmarks.

Glioblastoma (GB) is a highly malignant primary brain tumor with limited treatment options and poor prognosis. Despite current treatment approaches, including surgical resection, radiation therapy, and chemotherapy with temozolomide (TMZ), GB remains mostly incurable due to its invasive growth pattern, limited drug penetration beyond the blood-brain barrier (BBB), and resistance to conventional therapies. One of the main challenges in GB treatment is effectively eliminating infiltrating cancer cells that remain in the brain parenchyma after primary tumor resection. We’ve reviewed the most recent challenges and surveyed the potential strategies aimed at enhancing local treatment outcomes.

A complex intracellular signaling network mediates the multiple biological activities of G-protein-coupled receptors (GPCRs). Among them, monomeric GTPases and a family of closely related proline-targeted serine-threonine kinases, collectively known as Mitogen-Activated Protein Kinases (MAPKs), appears to play central roles in orchestrating the proliferative responses to multiple mitogens that act on GPCRs. Upon GDP/GTP exchange, monomeric GTPases control the phosphorylation of conserved threonine and tyrosine residues in MAPKs by their immediate upstream kinases, increasing their enzymatic activity and inducing their translocation to the nucleus where they phosphorylate transcription factors, thereby regulating the expression of genes playing a key role in normal and aberrant cell growth. Recently, a number of GPCRs have been engineered to provide exclusive activation by synthetic drug-like compounds while becoming insensitive to endogenous ligands. These engineered receptors, named Receptors Activated Solely by Synthetic Ligands (RASSLs), promise better understanding of GPCRs signaling in vitro and in vivo, thus representing ideal tools to selectively modulate MAPK signaling routes controlling a wide range of biological functions, from proliferation to differentiation, migration, invasion, and cell survival or death by apoptosis.

Abstract A novel and straightforward synthesis of highly substituted isoquinoline‐5,8‐dione fused tricyclic pyrazoles is reported. The key step of the synthetic sequence is a regioselective, Ag 2 CO 3 promoted, 1,3‐dipolar cycloaddition of C ‐heteroaryl‐ N ‐aryl nitrilimines and substituted isoquinoline‐5,8‐diones. The broad functional group tolerability and mild reaction conditions were found to be suitable for the preparation of a small library of compounds. These scaffolds were designed to interact with multiple biological residues, and two of them, after brief synthetic elaborations, were analyzed by molecular docking studies as potential anticancer drugs. In vitro studies confirmed the potent anticancer effects, showing promising IC 50 values as low as 2.5 μ m against three different glioblastoma cell lines. Their cytotoxic activity was finally positively correlated to their ability to inhibit PI3K/mTOR kinases, which are responsible for the regulation of diverse cellular processes in human cancer cells.

Epiretinal membranes (ERMs) are sheets of tissue that pathologically develop in the vitreoretinal interface leading to progressive vision loss. They are formed by different cell types and by an exuberant deposition of extracellular matrix proteins. Recently, we reviewed ERMs' extracellular matrix components to better understand molecular dysfunctions that trigger and fuel the onset and development of this disease. The bioinformatics approach we applied delineated a comprehensive overview on this fibrocellular tissue and on critical proteins that could really impact ERM physiopathology. Our interactomic analysis proposed the hyaluronic-acid-receptor cluster of differentiation 44 (CD44) as a central regulator of ERM aberrant dynamics and progression. Interestingly, the interaction between CD44 and podoplanin (PDPN) was shown to promote directional migration in epithelial cells. PDPN is a glycoprotein overexpressed in various cancers and a growing body of evidence indicates its relevant function in several fibrotic and inflammatory pathologies. The binding of PDPN to partner proteins and/or its ligand results in the modulation of signaling pathways regulating proliferation, contractility, migration, epithelial-mesenchymal transition, and extracellular matrix remodeling, all processes that are vital in ERM formation. In this context, the understanding of the PDPN role can help to modulate signaling during fibrosis, hence opening a new line of therapy.

Abstract Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes ( OSGIN1 , RGS17 , SRXN1 , AKR1B10 ) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

Oxidation processes in mitochondria and different environmental insults contribute to unwarranted accumulation of reactive oxygen species (ROS). These, in turn, rapidly damage intracellular lipids, proteins, and DNA, ultimately causing aging and several human diseases. Cells have developed different and very effective systems to control ROS levels. Among these, removal of excessive amounts is guaranteed by upregulated expression of various antioxidant enzymes, through activation of the NF-E2-Related Factor 2 (NRF2) protein. Here, we show that Mitogen Activated Protein Kinase 15 (MAPK15) controls the transactivating potential of NRF2 and, in turn, the expression of its downstream target genes. Specifically, upon oxidative stress, MAPK15 is necessary to increase NRF2 expression and nuclear translocation, by inducing its activating phosphorylation, ultimately supporting transactivation of cytoprotective antioxidant genes. Lungs are continuously exposed to oxidative damages induced by environmental insults such as air pollutants and cigarette smoke. Interestingly, we demonstrate that MAPK15 is very effective in supporting NRF2-dependent antioxidant transcriptional response to cigarette smoke of epithelial lung cells. Oxidative damage induced by cigarette smoke indeed represents a leading cause of disability and death worldwide by contributing to the pathogenesis of different chronic respiratory diseases and lung cancer. Therefore, the development of novel therapeutic strategies able to modulate cellular responses to oxidative stress would be highly beneficial. Our data contribute to the necessary understanding of the molecular mechanisms behind such responses and identify new potentially actionable targets.

A novel highly biocompatible nanosystem containing Mg nanoparticles is reported, characterized and tested as a suitable and non-toxic tool for photothermal therapy.

14-3-3 are regulatory proteins that through protein-protein interactions (PPI) with numerous binding partners could be involved in several human diseases, including cancer, neurodegenerative disorders, and pathogens infections. Following our research interest in the development of 14-3-3 PPI inhibitors, here we exploited the privileged 4-aminoantipyrine scaffold in the design and synthesis of some derivatives endowed with antiproliferative activity against K-562 cells, and capable of binding to recombinant 14-3-3σ as evidenced by NMR spectroscopy. The binding mode was further explored by molecular modelling, while coupling confocal microscopy with intensitometric analysis showed that compound 1 was able to promote the nuclear translocation of c-Abl at low micromolar concentrations. Overall, 1 is chemically stable compared to parent 14-3-3 PPI inhibitors, and thus emerged as a confirmed hit for further development.

BV02 is a reference inhibitor of 14-3-3 protein-protein interactions, which is currently used as chemical biology tool to understand the role of 14-3-3 proteins in pathological contexts. Due to chemical instability in certain conditions, its bioactive form has remained unclear. Here, we use NMR spectroscopy to prove for the first time the direct interaction between the molecule and 14-3-3σ, and to depict its bioactive form, namely the phthalimide derivative 9. Our work provides molecular insights to the bioactive form of the 14-3-3 PPI inhibitor and facilitates further development as candidate therapeutic agent.

The 14-3-3/c-Abl protein–protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

ABSTRACT The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired autophagy and reduced TNFα production was demonstrated in HEK293 cells transfected with TLR3-L412F plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (P=0.038). An increased frequency of autoimmune disorders as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.

During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.

Histone deacetylases (HDACs) are key regulatory enzymes involved in the control of gene expression and their inhibition by specific drugs has been widely correlated to cell cycle arrest, terminal differentiation, and apoptosis. Here, we investigated whether HDAC activity was required for PDGF-dependent signal transduction and cellular proliferation. Exposure of PDGF-stimulated NIH3T3 fibroblasts to the HDAC inhibitor trichostatin A (TSA) potently repressed the expression of a group of genes correlated to PDGF-dependent cellular growth and pro-survival activity. Moreover, we show that TSA interfered with STAT3-dependent transcriptional activity induced by PDGF. Still, neither phosphorylation nor nuclear translocation and DNA-binding in vitro and in vivo of STAT3 were affected by using TSA to interfere with PDGF stimulation. Finally, TSA treatment resulted in the suppression of PDGF-dependent cellular proliferation without affecting cellular survival of NIH3T3 cells. Our data indicate that inhibition of HDAC activity antagonizes the mitogenic effect of PDGF, suggesting that these drugs may specifically act on the expression of STAT-dependent, PDGF-responsive genes.

Extracellular signal-regulated kinase 8 (ERK8) has been already implicated in cell transformation and in the protection of genomic integrity and, therefore, proposed as a novel potential therapeutic target for cancer. In the absence of a crystal structure, we developed a three-dimensional model for its kinase domain. To validate our model we applied a structure-based virtual screening protocol consisting of pharmacophore screening and molecular docking. Experimental characterization of the hit compounds confirmed that a high percentage of the identified scaffolds was able to inhibit ERK8. We also confirmed an ATP competitive mechanism of action for the two best-performing molecules. Ultimately, we identified an ERK8 drug-resistant "gatekeeper" mutant that corroborated the predicted molecular binding mode, confirming the reliability of the generated structure. We expect that our model will be a valuable tool for the development of specific ERK8 kinase inhibitors.

DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not been established yet, but a weakened immune system is certainly the main cause. We envisaged the possibility to screen a small library of compounds synthesized in our laboratory against opportunistic tumors mainly due to HIV infection like Burkitt’s Lymphoma. From cellular assays and gene expression analysis we identified two promising compounds. These derivatives have the dual action required inhibiting HIV replication in human TZM-bl cells infected with HIV-1 NL4.3 and showing cytotoxic activity on human colon HT-29 and breast adenocarcinoma MCF-7 cells. In addition, preclinical in vitro adsorption, distribution, metabolism, and excretion studies highlighted a satisfactory pharmacokinetic profile.

In medulloblastomas, genetic alterations resulting in over-activation and/or deregulation of proteins involved in Hedgehog (HH) signaling lead to cellular transformation, which can be prevented by inhibition of primary ciliogenesis. Here, we investigated the role of MAPK15 in HH signaling and, in turn, in HH-mediated cellular transformation. We first demonstrated, in NIH3T3 mouse fibroblasts, the ability of this kinase of controlling primary ciliogenesis and canonical HH signaling. Next, we took advantage of transformed human medulloblastoma cells belonging to the SHH-driven subtype, i.e., DAOY and ONS-76 cells, to ascertain the role for MAPK15 in HH-mediated cellular transformation. Specifically, medullo-spheres derived from these cells, an established in vitro model for evaluating progression and malignancy of putative tumor-initiating medulloblastoma cells, were used to demonstrate that MAPK15 regulates self-renewal of these cancer stem cell-like cells. Interestingly, by using the HH-related oncogenes SMO-M2 and GLI2-DN, we provided evidences that disruption of MAPK15 signaling inhibits oncogenic HH overactivation in a specific cilia-dependent fashion. Ultimately, we show that pharmacological inhibition of MAPK15 prevents cell proliferation of SHH-driven medulloblastoma cells, overall suggesting that oncogenic HH signaling can be counteracted by targeting the ciliary gene MAPK15, which could therefore be considered a promising target for innovative "smart" therapies in medulloblastomas.

Rab7 is a small GTP-ase localized on late endosomes, which regulates late endocytic membrane traffic in mammalian cells. Moreover it has been shown that this protein has a fundamental role in the cellular vacuolation induced by the cytotoxin VacA of Helicobacter pylori. We report here for the first time the isolation of a cDNA encoding human Rab7 from a placenta cDNA library. The open reading frame for human Rab7 encodes a protein of 207 amino acids which exhibits high homology with the mouse, rat, and dog counterparts. Northern blot analysis of total RNAs isolated from different cell lines with a cDNA probe containing the entire open reading frame revealed two mRNA transcripts of 2.5 and 1.8 kilobases. The isolation of human Rab7 cDNA will allow further characterization of its function in normal and pathological states.

To report the implementation of cardiovascular secondary prevention guidelines following a cardiovascular event in Italy.Data were collected from 878 consecutive patients, who had suffered a cardiovascular event requiring hospitalisation in the preceding 12-24 months and who presented at 49 outpatient clinics across Italy. Cardiovascular risk markers were assessed through clinical examination, interview and reviewing of patients' charts; in addition, we collected information on changes in prevalence of selected risk factors that occurred since the time of index event. At the time of evaluation, increased body mass index (BMI) was observed in 35% of patients, with 20% being obese; 26% had diabetes and 21% uncontrolled hypertension. Although 91% of patients were on statins, no measurement of low-density lipoprotein (LDL)-cholesterol was available in the previous 6 months in 27% of patients and 16% had no knowledge of any lipid parameter in the same period. In the remaining patients, LDL was <100 mg dl(-1) in 57% and <70 mg dl(-1) in 20% of them. From the time of index event to interview, prevalence of uncontrolled hypertension remained stable, from 24% to 21% of patients; according to the patients' self-reporting, smoking had declined from 32% to 13% of patients and physical inactivity from 43% to 33% of patients.This survey shows, in a large national cohort, a suboptimal implementation of lifestyle changes and inadequate lipid control in patients at high cardiovascular risk after a cardiovascular event. Reinforcement of patients and physicians, implementation and adherence to guidelines is needed to reduce the burden of cardiovascular disease.

Recently the use of phosphorescent heavy-metal complexes in bioimaging techniques has been a promising research field and has been attracted increasing interest.

Head and neck squamous cell carcinomas (HNSCC) are a diverse group of tumors with high morbidity and mortality that have remained mostly unchanged over the past decades. The epidermal growth factor receptor (EGFR) is often overexpressed and activated in these tumors and strongly contributes to their pathogenesis. Still, EGFR-targeted therapies such as monoclonal antibodies and kinase inhibitors have demonstrated only limited improvements in the clinical outcome of this disease. Here, we take advantage of the extraordinary affinity of EGF for its cognate receptor to specifically target magnetite-containing nanoparticles to HNSCC cells and mediate, in vitro, their cellular upload. On the basis of this, we show efficient accumulation, in vivo, of such nanoparticles in subcutaneous xenograft tumor tissues in sufficient amounts to be able to mediate visualization by magnetic resonance imaging. Overall, our EGF-coated nanosystem may warrant, in the near future, novel and very efficient theranostic approaches to HNSCC.

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds.

Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4−/− mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12–induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12–induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38α and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38α/STAT4 pathway as an important mediator of IL-12 actions.

In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA.A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers.The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis.Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein-protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.

A cDNA clone coding for the Rab7 protein was isolated from an NIH3T3 cell line (mouse fibroblasts) cDNA library. Sequence analysis shows high homology to the rat and dog cDNAs. Northern blot analysis showed the presence of two messenger RNA that differ at the 3' untranslated region.

Arginine-vasopressin (AVP) plays an important role in regulating water balance in humans. Its secretion is under control of several mechanisms, some of which are not completely understood. The purpose of the present study was to evaluate the effects of an acute oral salt load on AVP secretion in normal subjects. Six normal volunteers received 350 mEq of NaCl per os. Pulmonary capillary wedge pressure and right atrial pressure, plasma AVP, plasma sodium and potassium concentration, plasma osmolality, hematocrit, urinary sodium and potassium excretion, and urinary flow were measured at baseline and every 30 minutes for two hours after the salt load. Hemodynamics as well as urinary sodium and potassium excretion did not change over the study. Ninety minutes after the salt load, plasma AVP increased from the basal value of 6.0 +/- 0.9 pg per ml to 10.1 +/- 1.2 pg per ml (mean +/- SE, p less than 0.005) and a significant reduction in diuresis of about 50% was observed. However, plasma osmolality and plasma sodium concentration increased significantly only 120 min after the salt load, from the initial value of 277.7 +/- 2.2 mOsm per kg and 145.3 +/- 1.4 mEq per 1 (mean +/- SE) to 284.8 +/- 2.5 mOsm per kg and 148.7 +/- 1.5 mEq per 1, respectively (p less than 0.01). Ninety minutes after the salt load, no correlation was found between plasma osmolality and plasma AVP concentration, indicating that AVP secretion was independent of changes in systemic blood osmolality.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the effects of thyroid state on the mechanisms underlying rat heart mitochondrial capacity to remove H2O2 produced by an exogenous source. The removal rates were higher in the presence of respiratory substrates independently from thyroid state and were higher in hyperthyroid than in hypothyroid preparations. The thyroid state-linked changes in H2O2 removal rates, mirrored those in H2O2 release rates, showing that endogenous and exogenous H2O2 do not compete for the removing system. Mitochondrial content of coenzyme Q9 and Q10 was lower in hypothyroidism and higher in hyperthyroidism suggesting that the thyroid state-linked changes in the rates of H2O2 production are due to changes in the ubiquinone mitochondrial content. The rates of H2O2 removal in the presence of antioxidant enzyme inhibitors indicated that the contribution of each antioxidant is dependent on the thyroid state. This was supported by enzymatic activity measurements. Pharmacological inhibition also showed that the overall percentage contribution of the enzymatic processes, as well as that of non-enzymatic processes, is not affected by thyroid state. Cytochrome levels, inferred by light emission measurements, and western blot determination of cytochrome c, were lower in hypothyroid and higher in hyperthyroid preparations supporting the idea that the levels of reducing compounds were modified in opposite way by the changes in thyroid state. Further support was obtained showing that the whole antioxidant capacity, which provides an evaluation of capacity of the systems, different from cytochromes, assigned to H2O2 scavenging, was lower in hyperthyroid than in hypothyroid state.

Glioblastoma Multiforme (GBM) is one of the most lethal human cancer types, with a 5-year survival rate of approximately 5%. A key reason for this is usually considered its poor accessibility to systemically administered drugs that only limitedly overcome the Blood Brain Barrier, ultimately causing the likely dismal appearance of recurrences. Here, we comment on our successful use, in GBM preclinical models, of novel thermogel based drug-delivery platforms for loco-regional treatment of tumor recurrences after primary surgery. The innovation as well as the pitfalls of our processes are outlined and discussed with an eye towards potential advancements in the realm of personalized medicine applications.

Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

A remarkably diverse array of stimuli, including growth factors, vasoactive polypeptides, chemoattractants, neurotransmitters, hormones, phospholipids, photons, odorants, and taste ligands, can elicit biological responses by stimulating receptors that transmit signals through the activation of heterotrimeric G proteins. The family of G-protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors involved in signal transmission, and accounts for more than 1% of the total proteins encoded by the human genome. This large number of GPCRs, together with their ability to stimulate distinct Gα and βγ subunits and intracellular effector molecules, explains the remarkable complexity of the GPCR-G protein signal-transducing system.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Abstract High-grade glioblastoma multiforme (GBM) is the most aggressive and common of gliomas in human populations, accounting for 55% of primary brain tumors. The prognosis of GBM is very poor and most patients die of tumor recurrence. The epidermal growth factor receptor (EGFR) and the platelet-derived growth factor receptor β (PDGFRβ) are hallmarks in GBM since they influence multiple aspects of tumor biology including cell proliferation, migration, invasiveness and resistance to treatment. In approximately half of the tumors with amplified EGFR, the EGFRvIII truncated extracellular mutant is detected. EGFRvIII does not bind ligand, is highly oncogenic and appears to be relatively resistant to treatment with conventional anti-EGFR agents such as ligand blocking monoclonal antibodies or EGFR tyrosine kinase inhibitors (TKIs). Recently, it has been demonstrated that EGFRvIII-dependent cancers may escape targeted therapy by developing dependence on PDGFRβ signaling, thus providing a strong rationale for combination therapy aimed at blocking both EGFRvIII and PDGFRβ signaling. We have generated two nuclease resistant 2′F-Py RNA aptamers, CL4 and Gint4.T, as high affinity ligands and inhibitors of human wild-type EGFR (EGFRwt) and PDGFRβ, respectively. Thanks to their unique characteristics (low size, good target affinity, no immunogenicity, high stability) aptamers represent a new class of molecules with a great potential to rival monoclonal antibodies in both therapy and diagnosis. Herein, by different approaches we demonstrate that CL4 aptamer binds to the EGFRvIII mutant even though it lacks amino acids 6-273 in the extracellular domain. As a consequence of binding, the aptamer inhibits EGFRvIII activation by hampering receptor homodimerization and downstream STAT3 pathway, thus confirming the critical role of EGFRvIII dimerization for signaling. Further, we show that targeting EGFRvIII by CL4, as well as by erlotinib and gefitinib, causes upregulation of PDGFRβ as a compensatory response to support cancer cell survival. Importantly, CL4 and EGFRTKIs cooperate with the anti-PDGFRβ aptamer in inhibiting survival and proliferation of EGFRvIII-overexpressing glioblastoma cells. Given the paucity of selective inhibitors for receptor tyrosine kinases, this study could have impact in the fields of targeted molecular cancer therapeutics and may result in progress against GBM. Citation Format: Simona Camorani, Elvira Crescenzi, David Colecchia, Andrea Carpentieri, Angela Amoresano, Mario Chiariello, Laura Cerchia. Aptamer-mediated inhibition of EGFRvIII mutant in glioblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-022. doi:10.1158/1538-7445.AM2015-LB-022

Aberrant production of reactive oxygen species (ROS) from dysfunctional mitochondria leads to oxidative stress and DNA damage, which induces the cellular senescence stress response pathway. This, while exerting strong beneficial suppressive effects on the development of cancer, also contributes to aging and different age-related disorders. Mitophagy is a key mechanism to constantly eliminate old and damaged mitochondria, strongly contributing to keep low levels of intracellular ROS. Here, we discuss our recent findings showing the involvement of the atypical MAP kinase family member MAPK15 in controlling the mitophagic process, thereby preventing ROS accumulation, extensive DNA damage and activation of the cellular senescence phenotype.

Macroautophagy/autophagy is one of the major responses to stress in eukaryotic cells and is implicated in several pathological conditions such as infections, neurodegenerative diseases and cancer. Interestingly, cancer cells take full advantage of autophagy both to support tumor growth in adverse microenvironments and to oppose damages induced by anti-neoplastic therapies. Importantly, different human oncogenes are able to modulate this survival mechanism to support the transformation process, ultimately leading to ‘autophagy addiction’. Still, oncogenic signaling events, impinging on the control of autophagy, are poorly characterized, limiting our possibilities to take advantage of these mechanisms for therapeutic purposes. Here, we screened a library of activated kinases for their ability to stimulate autophagy. By this approach, we identified novel potential regulators of the autophagic process and, among them, the IKBKE oncogene. Specifically, we demonstrate that this oncoprotein is able to stimulate autophagy when overexpressed, an event frequently found in breast tumors, and that its activity is strictly required for breast cancer cells to support the autophagic process. Interestingly, different oncogenic pathways typically involved in breast cancer, namely ERBB2 and PI3K-AKT-MTOR, also rely on IKBKE to control this process. Ultimately, we show that IKBKE-dependent autophagy is necessary for breast cancer cell proliferation, suggesting an important supporting role for this oncogene and autophagy in these tumors. <b>Abbreviations:</b> AAK1: AP2 associated kinase 1; AMPK: 5ʹ-prime-AMP-activated protein kinase; AKT1: AKT serine/threonine kinase 1; BAF: bafilomycin A₁; CA: constitutively activated; CDK17: cyclin dependent kinase 17; CDK18: cyclin dependent kinase 18; CHUK: conserved helix-loop-helix ubiquitous kinase; EGF: epidermal growth factor; ERBB2: erb-b2 receptor tyrosine kinase 2; FGF: fibroblast growth factor; FM: full medium; GALK2: galactokinase 2; IKBKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKE: inhibitor of nuclear factor kappa B kinase subunit epsilon; IKK: IκB kinase complex; KD: kinase dead; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1: mitogen-activated protein kinase 1; MAPK15: mitogen-activated protein kinase 15; MTORC1: mammalian target of rapamycin kinase complex 1; myr: myristoylation/myristoylated; NFKBIA: NFKB inhibitor alpha; PDGF: platelet derived growth factor; PFKL: phosphofructokinase, liver type; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKCD: protein kinase C delta; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TNBC: triple-negative breast cancer; TSC2: TSC complex subunit 2; WB: western blot; WT: wild-type.

first_page settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing:    Column Width:    Background: Open AccessExtended Abstract Small Molecules as Potential Inhibitors of the 14-3-3/c-Abl Interaction for the Treatment of CML † by Leire Iralde-Lorente 1,*, Giusy Tassone 1, Ylenia Cau 1, Claire Munier 2, Lorenzo Franci 4, Cecilia Pozzi 1, Adriano Angelucci 3, Mario Chiariello 4, Matthew Perry 2, Stefano Mangani 1 and Maurizio Botta 1 1 Department of Biotechnology, Chemistry and Farmacy- Department of Excellence 2018-2022, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy 2 Medicinal Chemistry, Respiratory, Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, SE-43183 Gothenburg, Sweden 3 Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, 67100 L'Aquila, Italy 4 Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and University of Siena, Via Fiorentina 1, 53100 Siena, Italy * Author to whom correspondence should be addressed. † Presented at the 2nd Molecules Medicinal Chemistry Symposium (MMCS): Facing Novel Challenges in Drug Discovery, Barcelona, Spain, 15–17 May 2019. Proceedings 2019, 22(1), 17; https://doi.org/10.3390/proceedings2019022017 Published: 7 August 2019 (This article belongs to the Proceedings of The 2nd Molecules Medicinal Chemistry Symposium (MMCS): Facing Novel Challenges in Drug Discovery) Download Download PDF Versions Notes Excerpt Note: In lieu of an abstract, this is an excerpt from the first page. c-Abl is a tyrosine kinase implicated in the regulation of proliferation, adhesion, motility, andcell survival [...] Keywords: 14-3-3; Protein-protein interactions; c-Abl; chronic myeloid leukemia (CML) 14-3-3; Protein-protein interactions; c-Abl; chronic myeloid leukemia (CML) Share and Cite MDPI and ACS Style Iralde-Lorente, L.; Tassone, G.; Cau, Y.; Munier, C.; Franci, L.; Pozzi, C.; Angelucci, A.; Chiariello, M.; Perry, M.; Mangani, S.; Botta, M. Small Molecules as Potential Inhibitors of the 14-3-3/c-Abl Interaction for the Treatment of CML. Proceedings 2019, 22, 17. https://doi.org/10.3390/proceedings2019022017 AMA Style Iralde-Lorente L, Tassone G, Cau Y, Munier C, Franci L, Pozzi C, Angelucci A, Chiariello M, Perry M, Mangani S, Botta M. Small Molecules as Potential Inhibitors of the 14-3-3/c-Abl Interaction for the Treatment of CML. Proceedings. 2019; 22(1):17. https://doi.org/10.3390/proceedings2019022017 Chicago/Turabian Style Iralde-Lorente, Leire, Giusy Tassone, Ylenia Cau, Claire Munier, Lorenzo Franci, Cecilia Pozzi, Adriano Angelucci, Mario Chiariello, Matthew Perry, Stefano Mangani, and Maurizio Botta. 2019. "Small Molecules as Potential Inhibitors of the 14-3-3/c-Abl Interaction for the Treatment of CML" Proceedings 22, no. 1: 17. https://doi.org/10.3390/proceedings2019022017 Find Other Styles Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here. Article Metrics No No Article Access Statistics Multiple requests from the same IP address are counted as one view.

Accumulation and proliferation of vascular smooth muscle cells are associated with atherosclerosis and hypertension. Proliferation of smooth muscle cells constitutes a major pathological event responsible for long-term failure of coronary and peripheral arterial bypass graft as well as the development of restenosis after percutaneous transluminal coronary angioplasty (PTCA). The incidence of restenosis after PTCA has been reported to be as high as 40-45% within 3-6 months. Major advantages in recombinant deoxyribonucleic acid (DNA) technology and eukaryotic gene regulation allow to hypothesize gene therapy as a potential treatment for inherited and acquired diseases. Gene therapy is the introduction of genes into somatic cells to correct an inherited or acquired disorder through the synthesis of missing or defective protein. Although no disease has yet been treated by gene therapy, several gene transfer protocols have recently been undertaken. We have studied the expression of foreign DNA that has been introduced into smooth muscle cells after balloon carotid injury in a rat model of angioplasty. The effects of different degree of balloon injury on neointima formation and c-fos expression was also assessed. Our data demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of restenosis after PTCA.

With more than 1000 members, the family of G protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors. The vast majority of these receptors exhibit a common structural motif consisting of the presence of seven membrane-spanning regions (1) (Fig. 1) that, when activated, undergo a dramatic conformational change, resulting in the exposure of previously masked G-protein binding sites in the intracellular loops (2–4). This causes the exchange of GDP for GTP bound to the G-protein α subunit and the dissociation of Gα from the βγ heterodimers. Then GTP-bound G-protein α subunits and βγ complexes initiate intracellular signaling responses by activating a variety of effector molecules, including adenylyl cyclases, phosphodiesterases, phospholipases, ion channels, ion transporters, and several intracellular kinases (5–8).