Francesca Belleudi

Dendritic cells (DCs) sense the microenvironment through several types of receptors recognizing pathogen-associated molecular patterns. In particular, C-type lectins, expressed by distinct subsets of DCs, recognize and internalize specific carbohydrate antigen in a Ca(2+) -dependent manner. Targeting of these receptors is becoming an efficient strategy of delivering antigens in DC-based anticancer immunotherapy. Here we investigated the role of the macrophage galactose type C-lectin receptor (MGL), expressed by immature DCs (iDCs), as a molecular target for α-N-acetylgalactosamine (GalNAc or Tn)-carrying tumor-associated antigens to improve DC performance. MGL expressed by ex vivo-generated iDCs from healthy donors was engaged by a 60-mer MUC1(9Tn) -glycopeptide as a Tn-carrying tumor-associated antigen, and an anti-MGL antibody, as a specific MGL binder. We demonstrated that MGL engagement induced homotrimers and homodimers, triggering the phosphorylation of extracellular signal-regulated kinase 1,2 (ERK1,2) and nuclear factor-κB activation. Analysis of DC phenotype and function demonstrated that MGL engagement improved DC performance as antigen-presenting cells, promoting the upregulation of maturation markers, a decrease in phagocytosis, an enhancement of motility, and most importantly an increase in antigen-specific CD8(+) T-cell activation. These results demonstrate that the targeting of MGL receptor on human DCs has an adjuvant effect and that this strategy can be used to design novel anticancer vaccines.

Treatment of advanced melanoma has been improved with the advent of the BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been identified to be responsible for the development of resistance, either MEK-dependent or MEK-independent. In order to overcome resistance due to reactivation of MEK signaling, MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It has been recently reported that ErbB3, a member of the EGFR receptor family, may be involved in the establishment of drug resistance.Three melanoma cell lines were tested: LOX IMVI (BRAF V600E), MST-L (BRAF V600R) and WM266 (BRAF V600D). Phosphorylation of Receptor Tyrosine Kinases (RTKs) was assessed by an RTK array. Western blot analysis was performed on total protein extracts using anti-ErbB3, anti-AKT and anti-ERK 1/2 antibodies. The expression of neuregulin after vemurafenib treatment was assessed by Real Time PCR and Western blotting. The growth inhibitory effects of vemurafenib, GSK1120212b and/or anti-ErbB3 mAbs were evaluated by in vitro colony formation assays.In the present study we demonstrate that ErbB3 is the main RTK undergoing rapidly hyperphosphorylation upon either treatment with a BRAF inhibitor or with a MEK inhibitor in a panel of melanoma cell lines harboring a variety of V600BRAF mutations and that this results in a strong activation of phospho-AKT. Importantly, ErbB3 activation is fully abrogated by the simultaneous use of anti-ErbB3 monoclonal antibodies, which are also shown to potently synergize with BRAF inhibitors in the inactivation of both AKT and ERK pathways and in the inhibition of melanoma cell growth. We show that upregulation of phospho-ErbB3 is due to an autocrine loop involving increased transcription and production of neuregulin by melanoma cells.On the basis of these results, we propose that initial co-treatment with BRAF and/or MEK inhibitors and anti-ErbB3 antibodies should be pursued as a strategy to reduce the ErbB3-dependent feedback survival mechanism and enhance duration of clinical response.

The FGFRs are receptor tyrosine kinases expressed by tissue-specific alternative splicing in epithelial IIIb or mesenchymal IIIc isoforms. Deregulation of FGF/FGFR signaling unbalances the epithelial-stromal homeostasis and may lead to cancer development. In the epithelial-context, while FGFR2b/KGFR acts as tumor suppressor, FGFR2c appears to play an oncogenic role. Based on our recent observation that the switching of FGFR2b versus FGFR2c induces EMT, here we investigated the biological outcome of the ectopic expression of FGFR2c in normal human keratinocytes. Morphological analysis showed that, differently from FGFR2b overexpression, the forced expression and activation of FGFR2c drive the epithelial cells to acquire a mesenchymal-like shape and actin reorganization. Moreover, the appearance of invasiveness and anchorage-independent growth ability in FGFR2c transfected keratinocytes was consistent with the potential tumorigenic role proposed for this receptor variant. Biochemical and molecular approaches revealed that the observed phenotypic changes were accompanied by modulation of EMT biomarkers and indicated the involvement of EMT transcription factors and miRs. Finally, the analysis of the expression pattern of discriminating markers strongly suggested that activation of FGFR2c triggers a process corresponding to the initiation of the pathological type III EMT, but not to the more physiological type II EMT occurring during FGFR2b-mediated wound healing.

Eps15 is a substrate for the tyrosine kinase of the epidermal growth factor receptor (EGFR) and is characterized by the presence of a novel protein:protein interaction domain, the EH domain. Eps15 also stably binds the clathrin adaptor protein complex AP-2. Previous work demonstrated an essential role for eps15 in receptor-mediated endocytosis. In this study we show that, upon activation of the EGFR kinase, eps15 undergoes dramatic relocalization consisting of 1) initial relocalization to the plasma membrane and 2) subsequent colocalization with the EGFR in various intracellular compartments of the endocytic pathway, with the notable exclusion of coated vesicles. Relocalization of eps15 is independent of its binding to the EGFR or of binding of the receptor to AP-2. Furthermore, eps15 appears to undergo tyrosine phosphorylation both at the plasma membrane and in a nocodazole-sensitive compartment, suggesting sustained phosphorylation in endocytic compartments. Our results are consistent with a model in which eps15 undergoes cycles of association:dissociation with membranes and suggest multiple roles for this protein in the endocytic pathway.

eps15 and eps1SR are substrates of the epidermal growth factor (EGF) receptor kinase that are characterized by the presence of a protein:protein interaction domain, the EH domain, and by their ability to bind to the clathrin adaptor protein complex adaptor protein 2. Indirect evidence suggests that eps15 and eps15R are involved in endocytosis. Here we show that microinjection of antibodies against eps15 and eps15R inhibits internalization of EGF and transferrin. In addition, fragments of eps15 (encompassing its EH domains or the COOH-terminal region that binds to adaptor protein 2) inhibit EGF internalization or endocytosis of Sindbis virus. These results demonstrate that eps15 and eps15R are essential components of the endocytic machinery.

The ErbB-4 receptors are unique in the EGFR/ErbB family for the ability to associate with WW domain-containing proteins. To identify new ligands of the cytoplasmic tail of ErbB-4, we panned a brain cDNA phage library with ErbB-4 peptides containing sequence motifs corresponding to putative docking sites for class-I WW domains. This approach led to identification of AIP4/Itch, a member of the Nedd4-like family of E3 ubiquitin protein ligases, as a protein that specifically interacts with and ubiquitinates ErbB-4 in vivo. Interaction with the ErbB-4 receptors occurs via the WW domains of AIP4/Itch. Functional analyses demonstrate that AIP4/Itch is recruited to the ErbB-4 receptor to promote its polyubiquitination and degradation, thereby regulating stability of the receptor and access of receptor intracellular domains to the nuclear compartment. These findings expand our understanding of the mechanisms contributing to the integrity of the ErbB signaling network and mechanistically link the cellular ubiquitination pathway of AIP4/Itch to the ErbB-4 receptor.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) deregulates epithelial homeostasis through the modulation of receptor tyrosine kinases and their signaling. Accordingly, the fibroblast growth factor receptor 2b (FGFR2b/KGFR), epithelial splicing transcript variant of the FGFR2, is down-modulated by the viral protein expression, leading to impairment of keratinocyte differentiation. Here, we report that, in cell models of transfected human keratinocytes as well as in cervical epithelial cells containing episomal HPV16, the down-regulation of FGFR2b induced by 16E5 is associated with the aberrant expression of the mesenchymal FGFR2c isoform as a consequence of splicing switch: in fact, quantitative RT-PCR analysis showed that this molecular event is transcriptionally regulated by the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) and is able to produce effects synergistic with those caused by TGFβ treatment. Immunofluorescence analysis revealed that this altered FGFR2 splicing leads to changes in the specificity for the ligands FGFs and in the cellular response, triggering epithelial-mesenchymal transition (EMT). Through 16E5 or FGFR2 silencing as well as inhibition of FGFR2 activity we demonstrated the direct role of the viral protein in the receptor isoform switching and EMT, suggesting that these early molecular events during HPV infection might represent additional mechanisms driving cervical transformation and tumor progression.

Human T-cell leukemia virus type 1 encodes a number of "accessory" proteins of unclear function; one of these proteins, p13^II, is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13^II through (i) determination of its submitochondrial localization and sequences required to alter mitochondrial morphology and (ii) an assessment of the biophysical and biological properties of synthetic peptides spanning residues 9–41 (p13^9–41), which include the amphipathic mitochondrial-targeting sequence of the protein. p13^9–41 folded into an α helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13^II accumulates in the inner mitochondrial membrane. p13^9–41 induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na⁺, K⁺) and released Ca²⁺ from Ca²⁺-preloaded mitochondria. These effects as well as the ability of full-length p13^II to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p13^9–41 were insensitive to cyclosporin A, suggesting that full-length p13^II might alter mitochondrial permeability through a permeability transition pore-independent mechanism, thus distinguishing it from the mitochondrial proteins Vpr and X of human immunodeficiency virus type 1 and hepatitis B virus, respectively.

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high‐affinity‐specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin‐coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c‐Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.

Infection with high-risk human papillomavirus (HR-HPV) genotypes, mainly HPV16 and HPV18, is a major risk factor for cervical cancer and responsible for its progression. While the transforming role of the HPV E6 and E7 proteins is more characterized, the molecular mechanisms of the oncogenic activity of the E5 product are still only partially understood, but appear to involve deregulation of growth factor receptor expression. Since the signaling of the transforming growth factor beta (TGFbeta) is known to play crucial roles in the epithelial carcinogenesis, aim of this study was to investigate if HPV16 E5 would modulate the TGF-BRII expression and TGFbeta/Smad signaling.The HPV16 E5 mRNA expression pattern was variable in low-grade squamous intraepithelial lesions (LSIL), while homogeneously reduced in high-grade lesions (HSIL). Parallel analysis of TGFBRII mRNA showed that the receptor transcript levels were also variable in LSILs and inversely related to those of the viral protein. In vitro quantitation of the TGFBRII mRNA and protein in human keratinocytes expressing 16E5 in a dose-dependent and time-dependent manner showed a progressive down-modulation of the receptor. Phosphorylation of Smad2 and nuclear translocation of Smad4 were also decreased in E5-expressing cells stimulated with TGFbeta1.Taken together our results indicate that HPV16 E5 expression is able to attenuate the TGFbeta1/Smad signaling and propose that this loss of signal transduction, leading to destabilization of the epithelial homeostasis at very early stages of viral infection, may represent a crucial mechanism of promotion of the HPV-mediated cervical carcinogenesis.

Autophagy plays key roles during host defense against pathogens, but viruses have evolved strategies to block the process or to exploit it for replication and successful infection.The E5 oncoprotein of human papillomavirus type 16 (HPV16 E5) perturbs epithelial homeostasis down-regulating the expression of the keratinocyte growth factor receptor (KGFR/FGFR2b), whose signaling induces autophagy.Here we investigated the possible effects of 16E5 on autophagy in human keratinocytes expressing the viral protein.The 16E5 presence strongly inhibited the autophagic process, while forced expression and activation of KGFR counteracted this effect, demonstrating that the viral protein and the receptor exert opposite and interplaying roles not only on epithelial differentiation, but also in the control of autophagy.In W12 cells, silencing of the 16E5 gene in the context of the viral full length genome confirmed its role on autophagy inhibition.Finally, molecular approaches showed that the viral protein interferes with the transcriptional regulation of autophagy also through the impairment of p53 function, indicating that 16E5 uses parallel mechanisms for autophagy impairment.Overall our results further support the hypothesis that a transcriptional crosstalk among 16E5 and KGFR might be the crucial molecular driver of epithelial deregulation during early steps of HPV infection and transformation.

The product of the hereditary breast cancer susceptibility gene BRCA1 is a multifunctional protein involved in the maintenance of genomic integrity, in transcriptional coactivation, and in the control of cell growth. BRCA1-deficient cells manifest chromosomal instability. During mitosis, BRCA1 is known to interact with gamma-tubulin in the centrosomes, key elements of the mitotic spindle. Using confocal microscopy and immunogold electron microscopy, we investigated the distribution of endogenous BRCA1 relative to mitotic spindle markers in breast cancer cells. By confocal analysis, BRCA1 and beta-tubulin colocalized to microtubules of the mitotic spindle and to the centrosomes. Immunogold electron microscopy confirmed these results and further revealed that BRCA1 and alpha-tubulin codistributed to the walls of the centrioles and to pericentriolar fibers at centrosomes. During chromatid segregation, codistribution was also detected along individual spindle microtubules and at sites of insertion of microtubules on chromosomes. At cytokinesis, BRCA1 and alpha-tubulin codistributed to the midbody. Coimmunoprecipitation supported the association of full-length BRCA1 with alpha- and beta-tubulin. These results are consistent with an involvement of BRCA1 in the dynamics of the mitotic spindle and in the segregation of duplicated chromosomes.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which acts specifically on epithelial cells, regulating their proliferation and differentiation. KGF elicits its activity through binding to and activation of KGF receptor, a splicing transcript variant of fibroblast growth factor receptor 2 (FGFR2). Here we analyzed the pathway of internalization of KGF and its receptor using several approaches, including the utilization in immunofluorescence and in immunoelectron microscopy of a functional KGF-HFc chimeric protein as a specific tool to follow the endocytosis of the growth factor and of its receptor. Western blot analysis with anti-FGFR2 and anti-phosphotyrosine antibodies, as well as parallel double immunofluorescence and confocal analysis of NIH3T3 KGFR transfectants treated with KGF at 4 degrees C, followed by incubations at 37 degrees C for different time points, showed that KGF induced endocytosis of tyrosine activated KGFRs. The use of KGF-HFc in immunofluorescence and in immunogold electron microscopy on KGFR transfectants, A253 epithelial tumor cells and human cultured keratinocytes allowed us to follow the early steps of KGF internalization and revealed that this process occurred through clathrin-coated pits. A quantitative ELISA assay confirmed that KGF-HFc binding on the cell surface rapidly decreased because of internalization. Our results demonstrate that KGF is internalized by receptor-mediated endocytosis and illustrate the involvement of clathrin-coated pits in this process.

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of 16E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with 16E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the fibroblast growth factor receptor substrate-2α tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5'-Bromo-deoxyuridine incorporation assay demonstrated that expression of 16E5 induces a decrease in the growth response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that 16E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation.

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

Membrane and actin cytoskeleton dynamics during phagocytosis can be triggered and amplified by the signal transduction of receptor tyrosine kinases. The epidermal keratinocytes appear to use the phagocytic mechanism of uptake to ingest melanosomes released by the melanocytes and play a pivotal role in the transfer process. We have previously demonstrated that the keratinocyte growth factor KGF/FGF7 promotes the melanosome uptake through activation of its receptor tyrosine kinase FGFR2b/KGFR. The aim of the present study was to investigate the contribution of KGFR expression, activation, and signaling in regulating the phagocytic process and the melanosome transfer. Phagocytosis was analyzed in vitro using fluorescent latex beads on human keratinocytes induced to differentiate. Melanosome transfer was investigated in keratinocyte-melanocyte cocultures. KGFR depletion by small interfering RNA microinjection and overexpression by transfection of wild type or defective mutant KGFR were performed to demonstrate the direct effect of the receptor on phagocytosis and melanosome transfer. Colocalization of the phagocytosed beads with the internalized receptors in phagolysosomes was analyzed by optical sectioning and 3-dimensional reconstruction. KGFR ligands triggered phagocytosis and melanosome transfer in differentiated keratinocytes, and receptor kinase activity and signaling were required for these effects, suggesting that FGFR2b/KGFR expression/activity and PLCγ signaling pathway play crucial roles in phagocytosis.

Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

Autophagy is a degradative pathway through which cells overcome stressful conditions and rapidly change their phenotype during differentiation. Despite its protective role, when exacerbated, autophagy may lead to cell death. Several growth factors involved in cell survival and in preventing differentiation are able to inhibit autophagy. Here we investigated the autophagic role of FGF7/KGF, an important player in epithelial cell protection and differentiation. Biochemical and quantitative fluorescence approaches showed that FGF7 and its signaling induce autophagy in human keratinocytes and the use of specific inhibitors indicated that this effect is independent of the PI3K-AKT-MTOR pathway. The selective block of autophagosome-to-lysosome fusion clarified that FGF7 induces autophagy stimulating autophagosome formation. However, quantitative fluorescence approaches also indicated that, upon a prolonged autophagic stimulus, FGF7 is able to accelerate autophagosome turnover. Moreover, in differentiating keratinocytes, the use of the autophagic inhibitor 3-MA as well as the depletion of BECN1 and ATG5, 2 essential regulators of the process, counteracted the FGF7-induced increase of the differentiation marker KRT1/K1, suggesting that autophagy is required for the FGF7-mediated early differentiation. These results provide the first evidence of a role of FGF7 in the regulation of sequential steps of the autophagic process and strengthen the hypothesis of a direct interplay between autophagy and differentiation. On the other hand, the ability of FGF7 to accelerate autophagosome turnover, preventing their dangerous accumulation, is consistent with the well-established protective role played by the growth factor in epithelial cells.

The E5 oncogenic protein of the human papillomavirus type 16 (HPV16 E5) cooperates in epithelial transformation perturbing the behaviour of differentiating suprabasal cells. Among the receptor tyrosine kinases deregulated by 16E5 expression, the key paracrine mediator of epithelial homeostasis keratinocyte growth factor receptor (KGFR/FGFR2b) is altered in its signaling and endocytic traffic in undifferentiated keratinocytes expressing 16E5 and it would represent a major target of the viral protein in differentiated cells. With the aim to specifically address the possible interplay of 16E5 with KGFR/FGFR2b in cells already committed to differentiation, we took advantage of an in vitro model for forced overexpression or depletion of KGFR in E5 expressing human keratinocytes under synchronous waves of differentiation. Quantitative RT-PCR, biochemical and immunofluorescence analysis showed that KGFR down-modulation is responsible for a E5-mediated decrease of the early differentiation marker K1 and that the receptor re-expression as well as triggering of its kinase activity and signaling are able to efficiently counteract the impairment of differentiation, providing a further demonstration of the tumor-suppressive role of KGFR in the new unexplored context of HPV16 E5-mediated carcinogenesis. In addition, KGFR induced a ligand-dependent decrease of p63 through a miR-203 independent mechanism and this effect was blocked by inhibition of the PI3K/Akt signaling, which is the main pathway involved in KGFR-dependent keratinocyte differentiation, suggesting that alterations of the KGFR/p63 crosstalk are responsible for the impairment of keratinocyte differentiation induced by 16E5 and that the opposite tumor-suppressive action of KGFR and oncogenic role of E5 might both involve p63.

Tumor cells release extracellular microvesicles (MVs) in the microenvironment to deliver biological signals to neighbouring cells as well as to cells in distant tissues. Tumor-derived MVs appear to play contradictory role promoting both immunosuppression and tumor growth and both evoking tumor specific immune response. Recent evidences indicate that tumor-derived MVs can positively impact Dendritic Cells (DCs) immunogenicity by reprogramming DC antigen processing machinery and intracellular signaling pathways, thus promoting anti-tumor response. DCs are considered pivot cells of the immune system due to their exclusive ability to coordinate the innate and acquired immune responses, cross-present exogenous antigens and prime naïve T cells. DCs are required for the induction and maintenance of long-lasting anti-tumor immunity and their exploitation has been extensively investigated for the design of anti-tumor vaccines. However, the clinical grade culture conditions that are required to generate DCs for therapeutic use can strongly affect their functions. Here, we investigated the immunomodulatory impact of MVs carrying the MUC1 tumor glycoantigen (MVsMUC1) as immunogen formulation on clinical grade DCs grown in X-VIVO 15 (X-DCs). Results indicated that X-DCs displayed reduced performance of the antigen processing machinery in term of diminished phagocytosis and acidification of the phagosomal compartment suggesting an altered immunogenicity of clinical grade DCs. Pulsing DCs with MVsMUC1 restored phagosomal alkalinization, triggering ROS increase. This was not observed when a soluble MUC1 protein was employed (rMUC1). Concurrently, MVsMUC1 internalization by X-DCs allowed MUC1 cross-processing. Most importantly, MVsMUC1 pulsed DCs activated IFNγ response mediated by MUC1 specific CD8+ T cells. These results strongly support the employment of tumor-derived MVs as immunogen platforms for the implementation of DC-based vaccines.

Ligation of the high-affinity receptor for IgE (Fc epsilonRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, Fc epsilonRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of Fc epsilonRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating Fc epsilonRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the Fc epsilonRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of Fc epsilonRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.

ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 °C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles. ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 °C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles. The low density lipoprotein receptor (LDLR) 2The abbreviations used are: LDLR, low density lipoprotein receptor; FH, familial hypercholesterolemia; ARH, autosomal recessive hypercholesterolemia; TRITC, tetramethylrhodamine isothiocyanate; siRNA, short interfering RNA; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate. plays a pivotal role in the regulation of cholesterol metabolism (1.Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4551) Google Scholar). LDLR is a ubiquitous cell surface glycoprotein of 839 amino acids that is able to bind low density lipoprotein (LDL), the major cholesterol transport vehicle in the plasma. The cellular and molecular biology of LDLR has been revealed through studies on familial hypercholesterolemia (FH) (1.Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4551) Google Scholar). FH is inherited as a co-dominant trait (2.Goldstein J.L. Hobbs H.H. Brown M.S. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Diseases. 8th Ed. McGraw Hill, New York2001: 2863-2913Google Scholar), and affected subjects show a markedly impaired LDLR function because of mutations in the LDLR gene (2.Goldstein J.L. Hobbs H.H. Brown M.S. Scriver C. Beaudet A. Sly W. Valle D. The Metabolic and Molecular Bases of Inherited Diseases. 8th Ed. McGraw Hill, New York2001: 2863-2913Google Scholar). As a consequence, they present a decreased removal rate of circulating LDL and a dramatic increase in plasma cholesterol levels. Early studies on normal and FH fibroblasts demonstrated that LDLRs, shortly after being synthesized, appear on the cell surface where they gather in coated pits (1.Brown M.S. Goldstein J.L. Science. 1986; 232: 34-47Crossref PubMed Scopus (4551) Google Scholar). These are specialized regions of cell membranes that are lined on the cytoplasmic surface by a protein called clathrin (3.Goldstein J.L. Anderson R.G. Brown M.S. Nature. 1979; 279: 679-685Crossref PubMed Scopus (1365) Google Scholar). After formation of the LDL-LDLR complex, the coated pits invaginate to form coated endocytic vesicles. Very quickly, the clathrin coat dissociates and multiple endocytic vesicles fuse to create endosomes (4.Pearse B.M. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1255-1259Crossref PubMed Scopus (469) Google Scholar, 5.Anderson R.G. Brown M.S. Goldstein J.L. Cell. 1977; 10: 351-364Abstract Full Text PDF PubMed Scopus (417) Google Scholar, 6.Carpentier J.L. Gorden P. Goldstein J.L. Anderson R.G. Brown M.S. Orci L. Exp. Cell Res. 1979; 121: 135-142Crossref PubMed Scopus (25) Google Scholar). At the acid pH in endosomes, the LDL dissociates from the receptor, which returns to the surface to initiate another cycle of endocytosis (7.Brown M.S. Anderson R.G. Goldstein J.L. Cell. 1983; 32: 663-667Abstract Full Text PDF PubMed Scopus (498) Google Scholar, 8.Rudenko G. Henry L. Henderson K. Ichtchenko K. Brown M.S. Goldstein J.L. Deisenhofer J. Science. 2002; 298: 2353-2358Crossref PubMed Scopus (399) Google Scholar). Recently, the identification of the molecular defect responsible for a recessive form of hypercholesterolemia that clinically resembles FH provided new insights into LDLR physiology. This disorder, called Autosomal Recessive Hypercholesterolemia (ARH), is caused by mutations in the putative adaptor protein ARH (9.Garcia C.K. Wilund K. Arca M. Zuliani G. Fellin R. Maioli M. Calandra S. Bertolini S. Cossu F. Grishin N. Barnes R. Cohen J.C. Hobbs H.H. Science. 2001; 292: 1394-1398Crossref PubMed Scopus (490) Google Scholar). In vivo studies demonstrated that ARH patients show a markedly reduced hepatic uptake of LDL similar to that found in homozygous FH (10.Zuliani G. Arca M. Signore A. Bader G. Fazio S. Chianelli M. Bellosta S. Campagna F. Montali A. Maioli M. Pacifico A. Ricci G. Fellin R. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 802-809Crossref PubMed Scopus (86) Google Scholar). ARH protein contains an ∼130-residue phosphotyrosine-binding domain evolutionarily related to other adaptor proteins. Adaptor proteins containing phosphotyrosine-binding domains bind the conserved sequence motif NPXY located in the cytoplasmic domain of various cell surface receptors and mediate several cellular functions, including receptor trafficking and endocytosis. The LDLR cytoplasmic tail contains a single NPXY motif that is required for clustering and endocytosis of the receptor in fibroblasts. Point mutations in this highly conserved LDLR sequence eliminate binding of ARH to LDLR in vitro. The phosphotyrosine-binding sequence also binds inositol phospholipids, which may anchor the protein to the plasma membrane (11.Mishra S.K. Watkins S.C. Traub L.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16099-16104Crossref PubMed Scopus (148) Google Scholar). The C-terminal portion of ARH protein contains a canonical clathrin box sequence (LLDLE in the human sequence) that binds the heavy chain of clathrin. ARH protein also has a highly conserved 27-amino acid sequence that binds the β2 adaptin subunit of AP-2, which is a structural component of the clathrin-coated pits. On the basis of these data it has been proposed that ARH may function as a specific LDLR adaptor protein. Elucidation of the specific role of ARH in the LDLR endocytosis has been hampered by the fact that cultured skin fibroblasts from ARH patients do not show major defects in LDL uptake and degradation (12.Arca M. Zuliani G. Wilund K. Campagna F. Fellin R. Bertolini S. Calandra S. Ricci G. Glorioso N. Maioli M. Pintus P. Carru C. Cossu F. Cohen J. Hobbs H.H. Lancet. 2002; 359: 841-847Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Therefore, the majority of studies so far available have been carried out on lymphocytes from ARH patients, in which the LDLR function is significantly impaired (13.Eden E.R. Patel D.D. Sun X.M. Burden J.J. Themis M. Edwards M. Lee P. Neuwirth C. Naoumova R.P. Soutar A.K. J. Clin. Investig. 2002; 110: 1695-1702Crossref PubMed Scopus (68) Google Scholar, 14.He G. Gupta S. Yi M. Michaely P. Hobbs H.H. Cohen J.C. J. Biol. Chem. 2002; 277: 44044-44049Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). It has been observed that the distribution of the immunodetectable LDLR is significantly altered in these cells and that it mostly resides on the plasma membrane. Moreover, although LDL degradation appears to be markedly reduced in lymphocytes, cell surface LDL binding is increased. This strongly indicates that ARH protein may be involved in the internalization of the LDL-LDLR complex (13.Eden E.R. Patel D.D. Sun X.M. Burden J.J. Themis M. Edwards M. Lee P. Neuwirth C. Naoumova R.P. Soutar A.K. J. Clin. Investig. 2002; 110: 1695-1702Crossref PubMed Scopus (68) Google Scholar). How ARH works in hepatocytes is less known. It has been hypothesized that ARH may direct LDLR to the sinusoidal membrane. Experiments performed in ARH-deficient mice demonstrated that this was not the case. In arh -/- mouse livers, LDLR appears to be normally sorted to the sinusoidal surface (15.Jones C. Hammer R.E. Li W.P. Cohen J.C. Hobbs H.H. Herz J. J. Biol. Chem. 2003; 278: 29024-29030Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). ARH might play a role in LDLR clustering, either by transporting the receptor to the coated pits or simply by anchoring the receptor in the pits (16.Cohen J.C. Kimmel M. Polanski A. Hobbs H.H. Curr. Opin. Lipidol. 2003; 14: 121-127Crossref PubMed Scopus (51) Google Scholar). It must be noted, however, that LDLRs are dispersed on the plasma membrane of hepatocytes but clustered in coated pits on fibroblasts (17.Pathak R.K. Yokode M. Hammer R.E. Hofmann S.L. Brown M.S. Goldstein J.L. Anderson R.G. J. Cell Biol. 1990; 111: 347-359Crossref PubMed Scopus (85) Google Scholar). Therefore, it is unlikely that an inappropriate anchorage of LDLR in coated pits is the major cause of LDLR malfunction in arh-/- hepatocytes. Moreover, immunoprecipitation experiments demonstrated that, at the steady state, the majority of ARH protein appears in fractions from which the LDLR receptor is absent (15.Jones C. Hammer R.E. Li W.P. Cohen J.C. Hobbs H.H. Herz J. J. Biol. Chem. 2003; 278: 29024-29030Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), clearly indicating that ARH is not constitutively associated with the LDLR-clathrin complex. An alternative possibility is that the ARH protein is directly involved in the endocytic internalization of LDLR. We further investigated this possibility in the human hepatocyte cell line HepG2. To evaluate the consequence of the absence of ARH protein on LDLR trafficking we employed short interfering RNA (siRNA) methodology. This has been demonstrated to be the most powerful way to selectively reduce the intracellular concentration of proteins (18.Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8256) Google Scholar, 19.Hinrichsen L. Harborth J. Andrees L. Weber K. Ungewickell E.J. J. Biol. Chem. 2003; 278: 45160-45170Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). The siRNA-transfected cells were examined by immunofluorescence, Western blotting, and functional endocytosis assay. Our results indicate that ARH is not constitutively associated with LDLR at the plasma membrane; instead, ARH is mainly recruited to the membrane after LDL binding, thus facilitating the endocytosis of the LDL-LDLR complex. Collectively, our data provide evidence that the ARH protein is an important component of the endocytic machinery of LDLR in hepatocytes. Reagents—Enhanced chemiluminescence (ECL) Western blotting detection reagents, Hybond ECL nitrocellulose membrane, and Protein G/Protein A-Sepharose 4 Fast-Flow beads were from Amersham Biosciences. Protease inhibitor mixture tablets with and without EDTA were purchased from Roche Diagnostics. Laemmli sample buffer and phosphate-buffered saline with Tween 20 (PBST, pH 7.4) were obtained from Sigma. Transfections were done with a TransMessenger transfection kit from Qiagen GmbH (Hilden, Germany). A Bio-Rad protein assay kit was purchased from Bio-Rad Diagnostics. Antibodies—Rabbit polyclonal antibodies against Rab-4 (D-20) and anti-lysosome-associated membrane glycoprotein (LAMP-1) (H-228), anti-early endosomal antigen 1 (EEA1), goat polyclonal antibodies against β-adaptin (N-19), Dab2 (C-20), and clathrin heavy chain (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against ARH and LDLR were a kind gift from Dr. H. H. Hobbs (University of Texas Southwestern Medical Center, Dallas, TX). Mouse monoclonal antibody against LDLR (Ab-1) was purchased from Oncogene Research Products (Boston, MA). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were from Amersham Biosciences. Horseradish peroxidase-conjugated goat antibody and rabbit polyclonal antibody against actin were from Sigma. Cell Culture—Human HepG2 cells (obtained from ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum plus antibiotics (Sigma). To reach different levels of confluence, HepG2 cells were plated on round glass coverslips coated with 2% gelatin (Sigma) at a density of 1 × 105 cells. To ensure that bile canaliculus-like regions were formed de novo during the culture period, cells were vigorously separated before plating. Cells were allowed to grow for 72 h to obtain confluent polarized cells. After this culture period, HepG2 cells develop bile canaliculus-like structures (BC) located between adjacent cells at the apical pole of the cell (20.van Uzendoom I.S.C. Zegers M.M. Kok J.W. Hoekstra D. J. Cell Biol. 1997; 137: 347-357Crossref PubMed Scopus (75) Google Scholar). The plasma membrane of BC has many microvilli that contain high concentrations of F-actin (21.Lian W.N. Tsai J.W. Yu P.M. Wu T.W. Yang S.C. Chau Y.P. Lin C.H. Hepatology. 1999; 30: 748-760Crossref PubMed Scopus (22) Google Scholar). Sucrose Gradients—HepG2 cells were cultured as described above. Sucrose gradient fractions were obtained according to Stockinger et al. (22.Stockinger W. Sailler B. Strasser V. Recheis B. Fashing D. Kahr L. Schneider W.J. Nimpf J. EMBO J. 2002; 16: 4259-4267Crossref Scopus (106) Google Scholar). Briefly, confluent dishes were serum starved for 16 h, kept on ice, and suspended in 300 μl of buffer (3 mm imidazole, pH 7.4, 1μm EDTA, Sigma protease inhibitor mixture) containing 8.5% sucrose. Cells were disrupted by 20 strokes in a Dounce homogenizer, and the efficiency was monitored by microscopy (Olympus Italia, Segrate, Milan, Italy). Nuclei were removed by 10 min of centrifugation at 1000 × g, and the supernatant was loaded on top of 4 ml of a 10–40% continuous sucrose gradient and spun for 16 h in a Beckman ultracentrifuge (swinging bucket rotor, SW60 Ti) at 40,000 rpm. Fractions (200 μl) were collected from the bottom of the tube by puncturing with an 18-gauge needle and analyzed by Western blotting using anti-ARH polyclonal antibody and anti-LDLR monoclonal antibody (Ab-1) as well as anti-LAMP-1, anti-EEA1, or anti-Rab-4 polyclonal antibodies. Short Interfering RNA—21-nucleotide RNA duplexes with symmetric 2-nucleotide 3′(2′-deoxy) thymidine overhangs (corresponding to the ARH gene nucleotides 88–112 relative to the start codon) were purchased from Xeragon (Zurich, Switzerland). RNA sequences were: sense, 5′-GCUGCCUGAGAACUGGACAdTdT-3′; antisense, 5′-UGUCCAGUUCUCAGGCAGCdTdT-3′. Selected sequences were submitted to BLAST searches against the human genome sequence to ensure that only the desired mRNA was targeted. The silencing efficiency of selected siRNAs was first tested in cell culture experiments. The experimental conditions were as follows: to obtain 50–80% confluence in 24 h, about 2 × 105 cells/well were seeded in 12-well plates on the day before transfection. On the day of transfection, the cells were washed with PBS and then each well received 600 μl of RPMI 1640 growth medium containing 200 μl of buffer EC-R, 1.6 μg of siRNA, 3.2 μl of Enhancer R, and 8 μl of TransMessenger prepared following the TransMessenger kit directions. After 3 h of incubation at 37 °C, cells were washed and normal growth medium (1 ml) was added. After 24 and 48 h, Western blot analysis of ARH was carried out. siRNA-treated cells were trypsin digested in 12-well plates at 37 °C until the cells detached. The cells were washed twice with PBS and then lysed in lysis buffer (1% Triton, 50 mm Tris, pH 8.0, 2 mm CaCl2, 80 mm NaCl) supplemented with protease inhibitors. Total protein content of cell lysates was determined by Bio-Rad protein assay. Lysate volumes containing comparable amounts of total proteins were used for Western blotting. Western Blotting—Lysate samples were boiled for 5 min in Laemmli sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue, and 0.125 m Tris-HCl, pH 6.8) and separated by SDS-PAGE on 10% running gels. The proteins were transferred to Hybond ECL nitrocellulose membranes that had been blocked for 1 h at 37 °C with PBST containing 5% dry milk and 5% calf serum. The membranes were then incubated for 1 h at 37°C with primary antibodies, followed by horseradish peroxidase-conjugated secondary antibody and detection by ECL. Co-immunoprecipitation—HepG2 cells (5 × 106 cells) were seeded in 100-mm dishes and grown for 2 days to reach confluence. Cells were serum starved for 16 h and kept on ice for 30 min. Purified LDL (50–100 μg) was added, and the cells were left for 1 h at 4 °C and then incubated at 37 °C for an additional 2, 3, 5, or 10 min before lysis. Cells were scraped with 1 ml of incubation buffer (5 mm Tris, pH 7.5, 100 mm NaCl, 2 mm CaCl2, 2 mm MgCl2) in the presence of protease inhibitors without EDTA and lysed by sonication for 5 s at room temperature. As a control, HepG2 cells were serum starved for 16 h and immediately lysed; in addition, one 100-mm dish was serum starved for 16 h, incubated at 4 °C for 1 h with purified LDL, and lysed. The amounts of protein in the lysates were estimated by spectrophotometry, and concentrations were normalized by addition of buffer. Lysates (an estimated 500 μg of protein) were incubated with 25 μl of Protein G/Protein A-Sepharose beads in 25 μl of incubation buffer for 1 h. The beads were removed by centrifugation, and the supernatants were incubated with anti-Rab-4, anti-clathrin, and anti-β-adaptin at 4 °C overnight. The antibody complexes were captured by addition of 25 μl of Protein G/Protein A-Sepharose beads in 25 μl of incubation buffer and a 1-h incubation at 4 °C in a rotating shaker. Bead-bound immune complexes were washed twice with incubation buffer, collected by centrifugation, resuspended in 30 μl of Laemmli sample buffer, and visualized by SDS-PAGE and Western blotting. Microinjection—siRNA (100 nm in 1 ml of PBS buffer containing dextran-FITC at 1 mg/ml) or buffer with dextran-FITC alone as a control were microinjected into the cytoplasm of HepG2 cells to induce RNA interference and consequent ARH silencing (23.Heinonen J.E. Smith C.I. Nore B.F. FEBS Lett. 2002; 527: 274-278Crossref PubMed Scopus (63) Google Scholar). Microinjection was performed using an Eppendorf microinjector (Eppendorf, Hamburg, Germany) and an inverted microscope (Zeiss, Oberkochen, Germany). Injection pressure was set at 30–80 hPa and the injection time at 0.3–0.5 s. Microinjected cells were left for 8 h at 37°C, serum starved for 16 h, and then treated with the fluorescent conjugate DiI-LDL (Molecular Probes, Eugene, OR) as below. Functional Assay of LDLR—The function of LDLR was tested by using Dil-LDL or anti-LDLR C7 monoclonal antibody. For DiI-LDL treatment, confluent polarized HepG2 cells were serum starved for 16 h, washed with medium, treated with medium containing 5 μg/ml DiI-LDL or anti-LDLR C7 antibody for 5 min at 37 °C to induce LDLR internalization, and immediately fixed with 4% p-formaldehyde in PBS for 30 min. Alternatively, cells serum starved for 16 h were washed with cold medium, incubated with medium containing 5 μg/ml DiI-LDL for 1 h at 4 °C, and either immediately fixed or washed with prewarmed medium and incubated at 37 °C for an additional 2, 5, and 10 min before fixation. Immunofluorescence Microscopy—For conventional immunofluorescence, cells grown on coverslips were fixed with 4% p-formaldehyde in PBS for 30 min at 25 °C and permeabilized with 0.1% Triton X-100 for 5 min. In double immunofluorescence experiments, cells were incubated with anti-ARH polyclonal antibody (1:50 in PBS) and anti-LDLR monoclonal antibody (1:100 in PBS). The primary antibodies were visualized using FITC-conjugated goat anti-rabbit IgG (1:300 in PBS; Cappel Research Products, Durham, NC), Texas Red-conjugated goat anti-rabbit IgG (1:100 in PBS; Jackson Immunoresearch Laboratories Inc., West Grove, PA), or Texas Red-conjugated goat anti-mouse IgG (1:100 in PBS; Jackson Immunoresearch Laboratories). F-actin in microvilli of bile canaliculus was stained with TRITC-conjugated phalloidin (TRITC-Ph) (1:50 in PBS; Sigma) for 45 min at 25 °C. Fluorescent images were recorded and analyzed using a cooled CCD color digital camera SPOT-2 (Diagnostic Instruments Inc., Milan, Italy) and FISH 2000/H1 software (Delta Sistemi, Rome, Italy). Colocalization of the fluorescence signals was evaluated using a Zeiss confocal laser scan microscope (Zeiss, Oberkochen, Germany). To prevent cross-talk between the two signals, the multitrack function was used. Quantitative analysis of the DiI-LDL internalization in uninjected or injected cells was performed evaluating five different areas of each slide randomly taken from three different experiments; results are expressed as percentage of cells presenting internalized DiI-LDL. The Intracellular Localization of ARH Protein Is Related to Cell Polarity—To analyze ARH expression and distribution in human hepatocytes, we investigated the intracellular localization of ARH protein and the possible colocalization with LDLR in HepG2 cells, a hepatoma cell line that grows to subconfluent monolayers in 12 h or to confluent polarized monolayers in 72 h. As previously described for primary cultured fibroblasts and the HeLa epithelial cell line (11.Mishra S.K. Watkins S.C. Traub L.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16099-16104Crossref PubMed Scopus (148) Google Scholar), we found that in non-confluent, non-polarized HepG2 cells the ARH signal appears punctate and homogenously dispersed throughout the cytoplasm (Fig. 1A). In confluent HepG2 monolayers, the cells are polarized and form distinct bile canaliculus-like structures that contain a well organized actin cytoskeleton and can be intensely stained with TRITC-phalloidin (21.Lian W.N. Tsai J.W. Yu P.M. Wu T.W. Yang S.C. Chau Y.P. Lin C.H. Hepatology. 1999; 30: 748-760Crossref PubMed Scopus (22) Google Scholar). The ARH signal in these cells is also punctate but prevalently distributed in the basolateral regions, and to a lesser extent evident in the proximity of the apical poles (Fig. 1B, arrow). To compare the localization of ARH and LDLR, we performed double immunofluorescence experiments in confluent, polarized cells using anti-ARH polyclonal antibody and anti-LDLR monoclonal antibody. In this experiment, ARH appeared to be colocalized with the receptor in the basolateral area of the cells (Fig. 1C). Activation of LDLR Promotes ARH Recruitment to Plasma Membrane—We then investigated the ARH and LDLR localization after the up-regulation of LDLR expression by serum starvation for 16 h. Although these conditions increase the presence of receptors on the cell surface, they did not modify the ARH and/or LDLR distribution or induce a colocalization of ARH with LDLRs on the plasma membrane (Fig. 2A). To determine whether ARH distribution is affected by binding of LDL to LDLR, we serum starved HepG2 cells for 16 h and then treated them with DiI-LDL for 5 min at 37 °C before fixation. Double immunofluorescence with anti-ARH polyclonal antibodies showed that ARH and DiI-LDL colocalized at the plasma membrane (Fig. 2B), suggesting that ARH could be partially recruited to the plasma membrane after LDL binding to the LDLR. To further explore whether the ARH recruitment might be the direct result of ligand binding to the receptor or due to lipid transfer, we performed additional experiments in which the LDLR pathway was activated by addition of anti-LDLR C7, an antibody directed against the extracellular portion of the LDLR. Anti-LDLR C7 is known to induce internalization and recycling of LDLR similarly to LDL particles (24.Beisiegel U. Schneider W.J. Goldstein J.L. Andersonm R.G. Brown M.S. J. Biol. Chem. 1981; 256: 11923-11931Abstract Full Text PDF PubMed Google Scholar). Double immunofluorescence with anti-ARH polyclonal antibodies showed that ARH protein partially colocalizes with LDLR-anti-LDLR complexes (Fig. 2C). As noted in the Introduction, LDLR tends to cluster in clathrin-coated pits on the cell surface under defined conditions. Low temperature permits LDL binding to LDLR on the plasma membrane but inhibits the clustering in clathrin-coated pits of LDL-LDLR complexes. Subsequent warming to 37 °C induces a synchronous wave of endocytosis, with a massive clustering of LDL-LDLR complexes in the clathrin-coated pits. To discriminate whether the recruitment of ARH to the cell surface was dependent on the binding of LDL to LDLR or on the LDL-LDLR clustering in the clathrin-coated pits, we serum starved HepG2 cells for 16 h as above, treated them with DiI-LDL for 1 h at 4°C, and then warmed the cells to 37 °C for different times. Double immunofluorescence and subsequent confocal analysis showed that, after treatment at low temperature, ARH colocalized only weakly with DiI-LDL distributed on the plasma membrane (Fig. 3). In fact, colocalization of ARH with fluorescent LDL at the level of the plasma membrane appeared maximal after 2 min at 37 °C (Fig. 3) and rapidly decreased, although it was still evident after 5 min (Fig. 3) when LDL appeared also in intracellular endocytic dots. These results indicate that ARH could be involved in the early steps of LDLR endocytosis. Very weak ARH/LDL colocalization after treatment with the ligand at low temperature and the maximal ARH/LDL overlap at very early time points of warming to 37 °C strongly suggest that ARH protein could play a role in the recruitment of LDL-LDLR complexes in clathrin-coated pits (Fig. 3). We also attempted to analyze the relative distribution of ARH and the LDLR within the various endocytic compartments in cells before and after stimulation of LDLR pathway by adding LDL. Analytical centrifugation was performed to fractionate vesicles from HepG2 cells on a continuous sucrose gradient (Fig. 4). LAMP-1, EEA1, and Rab-4 antibodies were also used in Western blot analyses to determine the position of endocytic structures. As previously reported (15.Jones C. Hammer R.E. Li W.P. Cohen J.C. Hobbs H.H. Herz J. J. Biol. Chem. 2003; 278: 29024-29030Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), in control untreated cells the majority of ARH co-sedimented with vesicles containing EEA1 and Rab-4 (Fig. 4A). Only a small percentage of ARH was present in the same fractions as the LDLR (Fig. 4A). Conversely, 2 min after LDL addition, ARH distribution showed a marked shift toward higher density fractions where LDLR was mainly localized (Fig. 4B). These findings are consistent with ARH being recruited to LDLR-containing early endocytic vesicles when LDLR internalization is stimulated. To provide a more direct confirmation of immunofluorescence as well as subcellular fractionation experiments, we evaluated the association of ARH with LDL-LDLR complexes in clathrin-coated pits at different times of incubation with LDL (Fig. 5). Cell-free lysates were immunoprecipitated using the anti-clathrin monoclonal antibody followed by Protein G/Protein A-Sepharose beads. Western blot analysis using the anti-ARH antibody revealed a positive band in samples treated with LDL for 2 min. The band was still evident after 5 min of treatment and declined thereafter. As expected, no association was found in control untreated cells. These findings further indicate that ARH could be partially recruited to the plasma membrane during early steps of LDLR-mediated endocytosis. Silencing of ARH Is Able to Block LDL-LDLR Endocytosis—To determine the involvement of ARH in binding of the LDL-LDLR complex to the clathrin-coated pits, we inhibited the expression of the ARH protein in the human hepatic

Cell migration is a physiological process that requires endocytic trafficking and polarization of adhesion molecules and receptor tyrosine kinases (RTKs) to the leading edge. Many growth factors are able to induce motility by binding to specific RTK on target cells. Among them, keratinocyte growth factor (KGF or FGF7) and fibroblast growth factor 10 (FGF10), members of the FGF family, are motogenic for keratinocytes, and exert their action by binding to the keratinocyte growth factor receptor (KGFR), a splicing variant of FGFR2, exclusively expressed on epithelial cells. Here we analyzed the possible role of cortactin, an F-actin binding protein which is tyrosine phosphorylated by Src and is involved in KGFR-mediated cell migration, in the KGFR endocytosis and polarization to the leading edge of migrating cells upon ligand-induced stimulation. Biochemical phosphorylation study revealed that both KGF and FGF10 were able to induce tyrosine phosphorylation of Src and in turn of cortactin, as demonstrated by using the specific pharmacological Src-inhibitor SU6656, although FGF10 effect was delayed with respect to that promoted by KGF. Immunofluorescence analysis demonstrated the polarized localization of KGFR upon ligand stimulation to the leading edge of migrating keratinocytes, process that was regulated by Src. Moreover, we showed that the colocalization of cortactin with KGFR at the plasma membrane protrusions and on early endosomes after KGF and FGF10 treatment was Src-dependent. Further, by using a RNA interference approach through microinjection, we showed that cortactin is required for KGFR endocytosis and that the clathrin-dependent internalization of the receptor is a critical event for its polarization. Finally, KGFR expression and polarization enhanced cell migration in a scratch assay. Our results indicate that both Src and cortactin play a key role in the KGFR endocytosis and polarization at the leading edge of migrating keratinocytes, supporting the crucial involvement of RTK trafficking in cell motility.

The FGFRs trigger divergent responses, such as proliferation and differentiation, and the cell type as well as the context-dependent signaling are crucial for the functional outcome. The FGFR2b/KGFR is expressed exclusively on epithelial cells and plays a key role in skin homeostasis. Here we analyzed in vitro the role of KGFR in the early differentiation of keratinocytes modulating its expression by KGFR cDNA transient transfection or KGFR siRNA microinjection and inducing a synchronous wave of differentiation in pre-confluent cells. Immunofluorescence, biochemical and molecular approaches demonstrated that KGFR overexpression increased the early differentiation marker keratin 1 at both transcriptional and translational levels, while receptor depletion reduced it. Ligand-dependent receptor activation and signaling were required for this differentiative effect. Overexpression of kinase negative KGFR mutant or Tyr769 KGFR signaling mutant, which is not able to recruit and activate PLC-γ, showed that the receptor kinase activity, but not its PLCγ-mediated signaling, is required for differentiation. Reduction of K1 expression, obtained by AKT inhibition, demonstrated that the PI3K/Akt signaling pathway is involved in the control of KGFR-mediated keratinocyte differentiation. This in vitro experimental model indicates that FGFR2b/KGFR expression represents a key event regulating keratinocyte early differentiation during the switch from undifferentiated to differentiating cells.

Abstract Tumor-associated glycoproteins are a group of antigens with high immunogenic interest: The glycoforms generated by the aberrant glycosylation are tumor-specific and the novel glycoepitopes exposed can be targets of tumor-specific immune responses. The MUC1 antigen is one of the most relevant tumor-associated glycoproteins. In cancer, MUC1 loses polarity and becomes overexpressed and hypoglycosylated. Changes in glycan moieties contribute to MUC1 immunogenicity and can modify the interactions of tumor cells with antigen-presenting cells such as dendritic cells that would affect the overall antitumor immune response. Here, we show that the form of the MUC1 antigen, i.e., soluble or as microvesicle cargo, influences MUC1 processing in dendritic cells. In fact, MUC1 carried by microvesicles translocates from the endolysosomal/HLA-II to the HLA-I compartment and is presented by dendritic cells to MUC1-specific CD8+ T cells stimulating IFN-γ responses, whereas the soluble MUC1 is retained in the endolysosomal/HLA-II compartment independently by the glycan moieties and by the modality of internalization (receptor-mediated or non–receptor mediated). MUC1 translocation to the HLA-I compartment is accompanied by deglycosylation that generates novel MUC1 glycoepitopes. Microvesicle-mediated transfer of tumor-associated glycoproteins to dendritic cells may be a relevant biologic mechanism in vivo contributing to define the type of immunogenicity elicited. Furthermore, these results have important implications for the design of glycoprotein-based immunogens for cancer immunotherapy. Cancer Immunol Res; 2(2); 177–86. ©2013 AACR.

Dendritic cells (DCs) are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs) enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS) accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco-2 cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.

UVB exposure of epidermal cells is known to trigger early and late molecular pathways dependent on receptor tyrosine kinases and reactive oxygen species (ROS). We have recently reported that UVB irradiation induces tyrosine phosphorylation, kinase activation, and internalization of the receptor for the keratinocyte growth factor (KGFR), a paracrine mediator of epithelial growth, differentiation, and survival. Here we analyzed in more detail the UVB-induced endocytic pathway of KGFR and the role of KGFR activation and internalization in regulating UVB-promoted apoptosis and cell cycle arrest. Immunogold electron microscopy and confocal analysis revealed that the UVB-induced endocytosis of KGFR occurs through clathrin-coated pits and that the internalized receptors are sorted to the degradative route and reach the lysosomal compartment with a timing similar to that induced by their ligand KGF. Treatment with the anti-oxidant N-acetylcysteine inhibited KGFR endocytosis, suggesting that the receptor internalization is mediated by the intracellular production of ROS. The ligand-independent KGFR endocytic pathway induced by UVB requires receptor kinase activity and tyrosine phosphorylation and involves transient receptor ubiquitination. Inhibition of KGFR activity reduces both the KGF-mediated proliferative response and the UVB-promoted apoptotic cell death, indicating a different effect of ligand-induced and UVB-induced KGFR triggering. In addition, receptor internalization leads to protection from apoptosis caused by UVB exposure. Finally, we compared directly the behavior of KGFR with that of the epidermal growth factor receptor (EGFR) upon UVB exposure. Surprisingly, biochemical and immunofluorescence analysis showed that EGFR, differently from KGFR, does not undergo UVB-induced tyrosine phosphorylation and internalization. Taken together, our results suggest a differential role of KGFR and EGFR in the response of epidermal cells to UVB possibly because KGFR endocytosis could be crucial for attenuation of survival signals in the suprabasal layers of human skin.

hMena and the epithelial specific isoform hMena(11a) are actin cytoskeleton regulatory proteins belonging to the Ena/VASP family. EGF treatment of breast cancer cell lines upregulates hMena/hMena(11a) expression and phosphorylates hMena(11a), suggesting cross-talk between the ErbB receptor family and hMena/hMena(11a) in breast cancer. The aim of this study was to determine whether the hMena/hMena(11a) overexpression cooperates with HER-2 signalling, thereby affecting the HER2 mitogenic activity in breast cancer. In a cohort of breast cancer tissue samples a significant correlation among hMena, HER2 overexpression, the proliferation index (high Ki67), and phosphorylated MAPK and AKT was found and among the molecular subtypes the highest frequency of hMena overexpressing tumors was found in the HER2 subtype. From a clinical viewpoint, concomitant overexpression of HER2 and hMena identifies a subgroup of breast cancer patients showing the worst prognosis, indicating that hMena overexpression adds prognostic information to HER2 overexpressing tumors. To identify a functional link between HER2 and hMena, we show here that HER2 transfection in MCF7 cells increased hMena/hMena(11a) expression and hMena(11a) phosphorylation. On the other hand, hMena/hMena(11a) knock-down reduced HER3, AKT and p44/42 MAPK phosphorylation and inhibited the EGF and NRG1-dependent HER2 phosphorylation and cell proliferation. Of functional significance, hMena/hMena(11a) knock-down reduced the mitogenic activity of EGF and NRG1. Collectively these data provide new insights into the relevance of hMena and hMena(11a) as downstream effectors of the ErbB receptor family which may represent a novel prognostic indicator in breast cancer progression, helping to stratify patients.

The altered isoform switching of the fibroblast growth factor receptor 2 (FGFR2) and aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells is involved in cancer progression. We have recently described that the ectopic expression of FGFR2c in normal human keratinocytes induces epithelial‐mesenchymal transition and leads to invasiveness and anchorage‐independent growth. Here, we extended our analysis to the effects of this FGFR2c forced expression on human keratinocyte differentiation and stratification. Our findings demonstrated that, differently from cells overexpressing the epithelial splicing variant FGFR2b, keratinocytes ectopically expressing FGFR2c are not able to form a monolayer and display decreased expression of early differentiation markers. This impaired ability to enter the differentiation program is related to the up‐modulation of the transcription factor ΔNp63. In addition, FGFR2c‐expressing keratinocytes undergo defective stratification and invasion of the collagen matrix in 3D organotypic cultures, further suggesting their tumorigenic potential. Taken together, our results support the hypothesis that the receptor switching and the consequent appearance of the mesenchymal FGFR2c variant in the epithelial context would drive early steps of carcinogenesis, unbalancing the p63/FGFR interplay, and altering the paracrine response to the microenvironment.

Fibroblast growth factor receptor (FGFR) signaling is a key modulator of cellular processes dysregulated in cancer. We recently found that the high expression of the mesenchymal FGFR2c variant in human pancreatic ductal adenocarcinoma (PDAC)-derived cells triggers the PKCε-mediated improvement of EMT and of MCL-1/SRC-dependent cell invasion. Since other membrane proteins can affect the receptor tyrosine kinase signaling, including transient receptor potential channels (TRPs), in this work, we investigated the role of TRPs in the FGFR2c/PKCε oncogenic axis. Our results highlighted that either the FGFR2c/PKCε axis shut-off obtained by shRNA or its sustained activation via ligand stimulation induces TRPA1 downregulation, suggesting a channel/receptor dependence. Indeed, biochemical molecular and immunofluorescence approaches demonstrated that the transient depletion of TRPA1 by siRNA was sufficient to attenuate FGFR2c downstream signaling pathways, as well as the consequent enhancement of EMT. Moreover, the biochemical check of MCL1/SRC signaling and the in vitro assay of cellular motility suggested that TRPA1 also contributes to the FGFR2c-induced enhancement of PDAC cell invasiveness. Finally, the use of a selective channel antagonist indicated that the contribution of TRPA1 to the FGFR2c oncogenic potential is independent of its pore function. Thus, TRPA1 could represent a putative candidate for future target therapies in PDAC.

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.

Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic.

The tumor suppressor epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) induces human keratinocyte early differentiation. Moreover, protein kinases C (PKCs) are known to regulate the differentiation program in several cellular contexts, including keratinocytes. Therefore, in this paper we propose to clarify if FGFR2b could play a role also in the late steps of keratinocyte differentiation and to assess if this receptor-induced process would sequentially involve PKCδ and PKCα isoforms. Immunofluorescence, biochemical, and molecular approaches, performed on 2D cultures or 3D organotypic rafts of human keratinocytes overexpressing FGFR2b by stable transduction, showed that receptor signaling induced the precocious onset and an accelerated progression of keratinocyte differentiation, indicating that FGFR2b is a crucial regulator of the entire program of keratinocyte differentiation. In addition, the use of specific inhibitors and gene silencing approaches through specific siRNA demonstrated that PKCδ controls the onset of FGFR2b-triggered differentiation, while PKCα plays a role restricted to the terminal stages of the process. Molecular analysis revealed that the two PKC isoforms sequentially act via induction of KLF4 and DLX3, two transcription factors linked by negative loops to p63, suggesting that p63 would represent the hub molecule at the crossroad of an intricate signaling network downstream FGFR2b, involving multiple PKC-induced transcription factors.

Abstract Background The epithelial isoform of the fibroblast growth factor receptor 2 (FGFR2b) controls the entire program of keratinocyte differentiation via the sequential involvement of protein kinase C (PKC) δ and PKCα. In contrast, the FGFR2 isoform switch and the aberrant expression of the mesenchymal FGFR2c isoform leads to impairment of differentiation, epithelial-mesenchymal transition (EMT) and tumorigenic features. Aim of our present study was to contribute in clarifying the complex network of signaling pathways involved in the FGFR2c-mediated oncogenic outcomes focusing on PKCε, which appears to be involved in the induction of EMT and tumorigenesis in several epithelial contexts. Methods Biochemical and molecular analysis, as well as in vitro invasion assays, combined with the use of specific small interfering RNA (siRNA), were performed in human keratinocytes stably expressing FGFR2c or FGFR2b isoforms. Results Our results showed that aberrant expression and signaling of FGFR2c, but not those of FGFR2b, in human keratinocytes induced a strong phosphorylation/activation of PKCε. The use of siRNA approach showed that PKCε is the hub signaling downstream FGFR2c responsible for the modulation of EMT markers and for the induction of the EMT-related transcription factors STAT3, Snail1 and FRA1, as well as for the acquisition of the invasive behavior. Moreover, experiments of depletion of ESRP1, responsible for FGFR2 splicing in epithelial cells, indicated that the activation of PKCε is the key molecular event triggered by FGFR2 isoform switch and underlying EMT induction. Conclusions Overall, our results point to the identification of the downstream PKC isoform responsible for the FGFR signaling deregulation occurring in epithelial tissues from the physiological oncosoppressive to the pathological oncogenic profile. Graphical abstract

Actinic keratosis (AK) is a preneoplastic skin disorder which can rapidly progress to cutaneous squamous cell carcinomas (SCCs). In light of our previous findings, indicating a possible oncogenic role of the mesenchymal isoform of FGFR2 (FGFR2c) aberrantly expressed in AK keratinocytes, we analyzed the possible tumor-promoting role of this receptor in the stromal AK counterpart in this work. Molecular analysis showed that, particularly in early AK lesions, FGFR2c dermal upregulation is accompanied by the downregulation of the cancer-associated fibroblasts (CAF) transcription repressor CSL, the upregulation of the CAF activator ULK3, and the consequent CAF gene induction. Immunofluorescence and molecular analysis, coupled with silencing approaches by siRNA, applied on primary cultures of KIN I-derived fibroblasts, indicated that FGFR2c upregulation contribute to CAF signature and the increased autophagy in response to FGF2. Magnetic bead-based multiplex assay, combined with FGFR2 signaling shut-off approaches, indicated that, especially in response to FGF2, IL-6 secretion could depend on FGFR2c high expression and signaling, suggesting the possible establishment of FGFR2c-dependent secretory autophagy, contributing to tumor-promoting factor release. Overall, our results identified FGFR2c as a signaling molecule involved in controlling precancerous/stromal cell oncogenic crosstalk, pointing to this receptor as a possible early molecular marker predictive for AK's rapid malignant progression.

The keratinocyte growth factor receptor or fibroblast growth factor receptor 2b (KGFR/FGFR2b) is activated by the specific interaction with the keratinocyte growth factor (KGF/FGF7), which targets the receptor to the degradative pathway, and the fibroblast growth factor 10 (FGF10/KGF2), which drives the receptor to the juxtanuclear recycling route. Hrs plays a key role in the regulation of the endocytic degradative transport of ubiquitinated receptor tyrosine kinases, but the direct involvement of this protein in the regulation of FGFR endocytosis has not been investigated yet. We investigated here the possible role of Hrs in the alternative endocytic pathways of KGFR. Quantitative immunofluorescence microscopy and biochemical analysis showed that both overexpression and siRNA interference of Hrs inhibit the KGF-triggered KGFR degradation, blocking receptor transport to lysosomes and causing its rapid reappearance at the plasma membrane. In contrast, the FGF10-induced KGFR targeting to the recycling compartment is not affected by Hrs overexpression or depletion. Coimmunoprecipitation approaches indicated that Hrs is recruited to KGFR only after KGF treatment, although it is not tyrosine phosphorylated by the ligand. In conclusion, Hrs regulates the KGFR degradative pathway, but not its juxtanuclear recycling transport. In addition, the results suggest that Hrs recruitment to the receptor, but not its ligand-induced phosphorylation, could be required for its function.

The molecular mechanisms and cellular pathways involved in cutaneous pigmentation, as well as the crucial role played by the epidermal keratinocytes in the process, are just starting to be elucidated. In fact, a number of recent studies from different authors including our group have pointed out that the uptake by keratinocytes of the melanosomes released by the melanocytes occurs through phagocytic ingestion and is regulated by the activity of some receptors, such as protease-activated receptor-2 (PAR-2) and keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGFR/FGFR2b), followed by actin cytoskeleton reorganization 1-6. Dermal fibroblasts are known to participate in this complex cellular interplay controlling pigmentation through the modulated secretion of growth factors 7, some of them acting directly on the melanocytes and stimulating the melanogenesis, such as stem cell factor and basic fibroblast growth factor 8, while others promoting the melanosome phagocytic uptake by the keratinocytes, as occurring in the case of keratinocyte growth factor/fibroblast growth factor 7 (KGF/FGF7): in this context, in fact, we have proposed that the paracrine growth factor KGF, released from dermal fibroblasts, promotes melanosome transfer through binding to and activation of its tyrosine kinase receptor KGFR, expressed on the keratinocytes, but not on melanocytes or fibroblasts: the receptor signalling recruits and activates phospholipase Cγ, an essential player of the phagocytic process 5. In mouse keratinocytes, KGFR stimulates melanosome uptake also through a signalling pathway involving integrin-linked kinase and RAS-related C3 botulinum toxin substrate 1 (Rac1) 9, suggesting the existence of a crosstalk between KGFR and integrins. In addition, the contribution of increased expression of KGF/FGF7 in hyperpigmented solar lentigo lesions has been demonstrated 10. Hypopigmentary disorders such as vitiligo and nevus depigmentosus (ND) are characterized by a local or diffuse altered skin pigmentation. In addition, a hypopigmented halo surrounding a central benign melanocytic nevus is the hallmark of the Sutton's nevus. Although the loss of melanocytes is considered the main factor leading to skin colour impairment in such disorders, an altered melanogenesis or a reduced melanosome transfer from melanocytes to keratinocytes is also involved. In fact, it has been proposed that the differential feature of the ND disorder, compared with vitiligo, is the presence of melanocytes with defective melanosome transfer 11, 12. Given the crucial role of the secreted KGF/FGF7 in the modulation of the melanosome uptake by keratinocytes 2, 4, 9 and taking advantage of our in vitro models of melanosome transfer 5, we first investigated here the efficiency of melanosome transfer in the above-mentioned hypopigmentation conditions as well as the ability of supernatants (SNs) collected from primary cultured human dermal fibroblasts, derived from the different lesional skin samples or from healthy donors as described in the Data S1, to stimulate the process. To this aim, the human melanoma cell line MST-L was cocultured with human HaCaT keratinocytes at a seeding ratio of 1:20, as previously described 2, 5, serum starved for 12 hrs and incubated for 6 hrs at 37°C with the SNs (undiluted or diluted 1:2 or 1:5) obtained from fibroblasts derived from normal skin (NHFs) or from a nevus depigmentosus lesion (ND HFs), from a vitiligo biopsy (vitiligo HFs) or from the hypopigmented regression area surrounding a Sutton's nevus (rSutton HFs). As positive control, stimulation of the melanosome transfer was induced treating the cocultures with KGF. Double immunofluorescence analysis was performed with anti-tyrosinase polyclonal antibodies, to visualize melanosomes, and anti-pancytokeratin monoclonal antibody, to identify the keratinocytes. Quantitation of tyrosinase fluorescence intensity in the cytosolic area of the keratinocytes, performed as described 5, showed a significant decrease of the tyrosinase-positive dots upon stimulation with lesional-derived SNs with respect to that observed under treatment with SN from NHFs (Fig. 1A, upper panels). To evaluate if the effects of the various SNs would be ascribed, at least in part, to the presence of KGFR/FGFR2b ligands released in the fibroblast culture medium, as previously demonstrated in previous papers from our group 13, 14, addition of the specific FGFR2 tyrosine kinase inhibitor SU5402 was also performed: significant inhibition of the melanosome uptake was found only when the inhibitor was added to the SN from NHFs or to the KGF-treated cultures (Fig. 1A, lower panels), suggesting a possible deficiency of paracrine KGFR ligands in the pathological lesions. Then, to assess if the reduction of melanosome transfer in response to SNs from lesional fibroblasts would be dependent on an altered expression of KGF, the growth factor mRNA transcript levels were analysed by real-time RT-PCR and normalized with respect to β-actin, showing a clear decrease of KGF mRNA expression in all groups of HFs derived from lesional skin compared with the control NHFs (Fig. 1B). In addition, ELISA test demonstrated that KGF protein levels were significantly decreased in SNs from all lesional HFs compared with NHFs (Fig. 1C). Interestingly, consistent with the mRNA expression data, the KGF released by vitiligo HFs was significantly reduced if compared with that secreted by both ND HFs and rSutton HFs (Fig. 1C). None of the SNs was cytotoxic for the cells at different times of treatment (6, 24 or 48 hrs) when assayed by MTT test (Fig. 1D). Thus, the loss of pigmentation in all the three hypopigmentary conditions could be explained, at least in part, by a reduced expression and secretion of KGF from dermal fibroblasts, which impair the melanosome uptake by the keratinocytes. To evaluate the contribution of the lesional keratinocytes on the inefficient melanosome transfer, we focused our attention on the above ND biopsy, because of the postulated defect of the organelle uptake in such disorder 11, 12. To dissect in vitro the process, we cocultured the MST-L melanocytes with primary keratinocytes derived from the ND (ND HKs) or from normal skin, at a seeding ratio of 1:40. Serum starvation and treatment with KGF in the presence or absence of SU5402 were performed as above. The quantitative double immunofluorescence revealed that the KGF-induced increase of the tyrosinase-positive dots in the cytoplasm of ND HKs was much lower compared with NHKs (Fig. 2A, middle panels). Brightfield and phase-contrast microscopy were used to unequivocally demonstrate the decreased melanosome transfer to the lesional keratinocytes (Fig. 2B). Again, the addition of SU5402 was able to abolish the KGF effect in both cocultures (Fig. 2A, lower panels), providing a further evidence of the involvement of KGFR activation and signalling in the process and suggesting a decreased receptor expression in the pathological condition. Therefore, with the aim to analyse the receptor expression, we quantified KGFR transcript levels by real-time RT-PCR and we found a decreased receptor mRNA expression in ND HKs compared with NHK control cells (Fig. 2C). Thus, at least in the ND disorder, low levels of KGFR might significantly contribute to the reduction of KGF-mediated melanosome transfer. Taken together, our results further support the key roles played, on the melanosome transfer in normal skin, by KGF/FGF7 released by dermal fibroblasts and by its receptor KGFR/FGFR2b expressed and activated on the epidermal keratinocytes (Fig. 2D, cartoon on the left) and suggest a deficient expression of both players (Fig. 2D, cartoon on the right) as an additional pathogenic mechanism involved in hypopigmentary disorders. This work was partially supported by grants from MIUR and from AIRC – Associazione Italiana per la Ricerca sul Cancro (IG 10272), Italy. The authors state no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 microM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.

Dendritic cell (DC)-based immunotherapy is an attractive approach to induce long lasting antitumor effector cells aiming to control cancer progression. DC targeting is a critical step in the design of DC vaccines in order to optimize delivery and processing of the antigen, and several receptors have been characterized for this purpose. In this study, we employed the FcγRs to target DCs both in vitro and in vivo. We designed a recombinant molecule (HER2-Fc) composed of the immunogenic sequence of the human tumor-associated antigen HER2 (aa 364-391) and the Fc domain of a human IgG(1). In a mouse model, HER2-Fc cDNA vaccination activated significant T cell-mediated immune responses towards HER2 peptide epitopes as detected by IFN-γ ELIspot and induced longer tumor latency as compared to Ctrl-Fc-vaccinated control mice. Human in vitro studies indicated that the recombinant HER2-Fc immunogen efficiently targeted human DCs through the FcγRs resulting in protein cross-processing and in the activation of autologous HER2-specific CD8(+) T cells from breast cancer patients.

Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

The paracrine networks of the human melanoma microenvironment are able to influence tumor growth and progression. Among the paracrine growth factors involved in skin homeostasis, the KGF/FGF7 secreted by dermal fibroblasts promotes the epidermal proliferation and differentiation as well as the release from keratinocytes of other paracrine mediators. To evaluate the possible role played by KGF in affecting the behavior of different subtypes of melanoma carrying activating mutations or overexpression of the SCF receptor c-KIT, we used human melanoma cell lines, characterized by different expression levels of c-KIT and opposing responsivity to SCF, and HaCaT keratinocytes. Quantitative real-time reverse transcription-polymerase chain reaction assay and ELISA test on KGF-treated keratinocytes showed enhanced expression and secretion of SCF in response to KGF and dependent on functional KGF receptor. Immunofluorescence microscopy and biochemical analysis showed, in one of the selected melanoma cell models, SCF-dependent c-KIT activation induced by stimulation with the culture supernatants collected from KGF-treated keratinocytes. In keratinocyte-melanoma cocultures stained for the Ki67 proliferation marker, incubation with KGF induced enhanced growth not only of the keratinocytes but also of the melanoma cells, which could be blocked by the c-KIT inhibitor imatinib, demonstrating the establishment of a KGF-induced paracrine signaling network owing to the coexpression of biologically active SCF released from keratinocytes and functional c-KIT on melanoma cells.

Signalling of the epithelial splicing variant of the fibroblast growth factor receptor 2 (FGFR2b) induces both autophagy and phagocytosis in human keratinocytes. Here, we investigated, in the cell model of HaCaT keratinocytes, whether the two processes might be related and the possible involvement of PLCγ signalling. Using fluorescence and electron microscopy, we demonstrated that the FGFR2b-induced phagocytosis and autophagy involve converging autophagosomal and phagosomal compartments. Moreover, the forced expression of FGFR2b signalling mutants and the use of specific inhibitors of FGFR2b substrates showed that the receptor-triggered autophagy requires PLCγ signalling, which in turn activates JNK1 via PKCδ. Finally, we found that in primary human keratinocytes derived from light or dark pigmented skin and expressing different levels of FGFR2b, the rate of phagocytosis and autophagy and the convergence of the two intracellular pathways are dependent on the level of receptor expression, suggesting that FGFR2b signalling would control in vivo the number of melanosomes in keratinocytes, determining skin pigmentation.

Fibroblast growth factor receptor 2b (FGFR2b) is a receptor tyrosine kinase expressed exclusively in epithelial cells. We previously demonstrated that FGFR2b induces autophagy and that this process is required for the triggering of FGFR2b-mediated early differentiation of keratinocytes. However, the molecular mechanisms regulating this interplay remain to be elucidated. Since we have also recently shown that Jun N-terminal protein kinase 1 (JNK1) signaling is involved in FGFR2b-induced autophagy and a possible role of the JNK pathway in epidermal differentiation has been suggested (though it is still debated), we investigated here the cross talk between FGFR2b-mediated autophagy and differentiation, focusing on the downstream JNK signaling. Biochemical, molecular, and immunofluorescence approaches in 2-dimensional (2-D) keratinocyte cultures and three-dimensional (3-D) organotypic skin equivalents confirmed that FGFR2b overexpression increased both autophagy and early differentiation. The use of FGFR2b substrate inhibitors and the silencing of JNK1 highlighted that this signaling is required not only for autophagy but also for the triggering of early differentiation. In contrast, the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway did not appear to be involved in the two processes, and AKT signaling, whose activation contributes to the FGFR2b-mediated onset of keratinocyte differentiation, was not required for the triggering of autophagy. Overall, our results point to JNK1 as a signaling hub that regulates the interplay between FGFR2b-induced autophagy and differentiation.

Abstract Signalling of the epithelial splicing variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, inhibition of autophagy as well as the induction of the epithelial‐mesenchymal transition (EMT). In light of the widely proposed negative loop linking autophagy and EMT in the early steps of carcinogenesis, here we investigated the possible involvement of FGFR2c aberrant expression and signalling in orchestrating this crosstalk in human keratinocytes. Biochemical, molecular, quantitative immunofluorescence analysis and in vitro invasion assays, coupled to the use of specific substrate inhibitors and transient or stable silencing approaches, showed that AKT/MTOR and PKCε are the two hub signalling pathways, downstream FGFR2c, intersecting with each other in the control of both the inhibition of autophagy and the induction of EMT and invasive behaviour. These results indicate that the expression of FGFR2c, possibly resulting from FGFR2 isoform switch, could represent a key upstream event responsible for the establishment of a negative interplay between autophagy and EMT, which contributes to the assessment of a pathological oncogenic profile in epithelial cells.

Actinic keratosis (AK) is the ultra violet (UV)-induced preneoplastic skin lesion clinically classified in low (KIN I), intermediate (KIN II), and high (KIN III) grade lesions. In this work we analyzed the expression of Fibroblast Growth Factor Receptors (FGFRs), as well as of keratinocyte differentiation and epithelial-to-mesenchymal transition (EMT)-related markers in differentially graded AK lesions, in order to identify specific expression profiles that could be predictive for direct progression of some KIN I lesions towards squamous cell carcinoma (SCC). Our molecular analysis showed that the keratinocyte differentiation markers keratin 1 (K1), desmoglein-1 (DSG1), and filaggrin (FIL) were progressively downregulated in KIN I, II, and III lesions, while the modulation of epithelial/mesenchymal markers and the induction of the transcription factors Snail1 and Zinc finger E-box-binding homeobox 1 (ZEB1) compatible with pathological EMT, even if observable, did not appear to correlate with AK progression. Concerning FGFRs, a modulation of epithelial isoform of FGFR2 (FGFR2b) and the mesenchymal FGFR2c isoform compatible with an FGFR2 isoform switch, as well as FGFR4 upregulation were observed starting from KIN I lesions, suggesting that they could be events involved in early steps of AK pathogenesis. In contrast, the increase of FGFR3c expression, mainly appreciable in KIN II and KIN III lesions, suggested a correlation with AK late progression. Interestingly, the strong modulation of FIL, Snail1, as well as of FGFR2c, FGFR4, and of their ligand FGF2, observed in some of the KIN I samples, may indicate that they could be molecular markers predictive for those low graded lesions destined to a direct progression to SCC. In conclusion, our data point on the identification of molecular markers predictive for AK rapid progression through the “differentiated” pathway. Our results also represent an important step that, in future, will help to clarify the molecular mechanisms underlying FGFR signaling deregulation in epithelial tissues during the switch from the pre-neoplastic to the oncogenic malignant phenotype.

Infection with human papillomavirus type 16 (HPV16) is one of the major risk factors for the development of cervical cancer. Our previous studies have demonstrated the involvement of the early oncoprotein E5 of HPV16 (16E5) in the altered isoform switch of fibroblast growth factor receptor 2 (FGFR2) and the consequent expression in human keratinocytes of the mesenchymal FGFR2c isoform, whose aberrant signaling leads to EMT, invasiveness, and dysregulated differentiation. Here, we aimed to establish the possible direct link between these pathological features or the appearance of FGFR2c and the expression of 16E5 in low-grade squamous intraepithelial lesions (LSILs). Molecular analysis showed that the FGFR2c expression displayed a statistically significant positive correlation with that of the viral oncoprotein, whereas the expression values of the epithelial FGR2b variant, as well as those of the differentiation markers keratin 10 (K10), loricrin (LOR) and involucrin (INV), were inversely linked to the 16E5 expression. In contrast, the expression of EMT-related transcription factors Snail1 and ZEB1 overlapped with that of 16E5, becoming a statistically significant positive correlation in the case of Snail2. Parallel analysis performed in human cervical LSIL-derived W12 cells, containing episomal HPV16, revealed that the depletion of 16E5 by siRNA was able to counteract these molecular events, proving to represent an effective strategy to identify the specific role of this viral oncoprotein in determining LSIL oncogenic and more aggressive profiles. Overall, coupling in vitro approaches to the molecular transcript analysis in ectocervical early lesions could significantly contribute to the characterization of specific gene expression profiles prognostic for those LSILs with a greater probability of direct neoplastic progression.

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin‐mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down‐modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin‐mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C‐terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor‐mediated endocytosis, the EGFR ligand‐induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand‐induced internalization. Taken together our results indicate that the clathrin‐mediated uptake of KGFR is not mediated by eps15.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribo-some-inactivating protein saporin (p-uPA-SapTRITC), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.—Ippoliti, R., Lendaro, E., Benedetti, P. A., Torrisi, M. R., Belleudi, F., Carpani, D., Soria, M. R., Fabbrini, M. S. Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism. FASEB J. 14, 1335–1344 (2000)

The pathogenic Nef protein of the human immunodeficiency virus type 1 (HIV-1) downregulates CD4 by inducing its endocytosis and by inhibiting the transport of the receptor to the cell membrane. By means of in vivo -selected mutations, we show that L37, P78 and E177 residues of Nef are required for its effect on CD4 internalization and recycling but dispensable for Nef-induced retention and degradation of intracellular CD4. Of note, the function of Nef on the anterograde transport of newly synthesized CD4 molecules is irrelevant in cells with a slow constitutive CD4 turnover such as T cell lines. Moreover, we show that a mutated CD4 that is unresponsive to Nef-mediated endocytosis, CD4LL 144 AA, is retained intracellularly and degraded by Nef like wild-type CD4. Thus, Nef's abilities to enhance endocytosis and induce intracellular retention of CD4 are mediated by separate protein surfaces and occur through distinct mechanisms.

Pancreatic ductal adenocarcinoma (PDAC) is a treatment-resistant malignancy characterized by a high malignant phenotype including acquired EMT signature and deregulated autophagy. Since we have previously described that the aberrant expression of the mesenchymal FGFR2c and the triggering of the downstream PKCε signaling are involved in epidermal carcinogenesis, the aim of this work has been to assess the contribution of these oncogenic events also in the pancreatic context. Biochemical, molecular and immunofluorescence approaches showed that FGFR2c expression impacts on PDAC cell responsiveness to FGF2 in terms of intracellular signaling activation, upregulation of EMT-related transcription factors and modulation of epithelial and mesenchymal markers compatible with the pathological EMT. Moreover, shut-off via specific protein depletion of PKCε signaling, activated by high expression of FGFR2c resulted in a reversion of EMT profile, as well as in a recovery of the autophagic process. The detailed biochemical analysis of the intracellular signaling indicated that PKCε, bypassing AKT and directly converging on ERK1/2, could be a signaling molecule downstream FGFR2c whose inhibition could be considered as possible effective therapeutic approach in counteracting aggressive phenotype in cancer.

Signaling of the epithelial splice variant of fibroblast growth factor receptor 2 (FGFR2b) triggers both differentiation and autophagy, while the aberrant expression of the mesenchymal FGFR2c isoform in epithelial cells induces impaired differentiation, epithelial mesenchymal transition (EMT) and tumorigenic features. Here we analyzed in the human keratinocyte cell line, as well as in primary cultured cells, the possible impact of FGFR2c forced expression on the autophagic process. Biochemical and quantitative immunofluorescence analysis, coupled to the use of autophagic flux sensors, specific substrate inhibitors or silencing approaches, showed that ectopic expression and the activation of FGFR2c inhibit the autophagosome formation and that AKT/MTOR is the downstream signaling mainly involved. Interestingly, the selective inhibition of AKT or MTOR substrates caused a reversion of the effects of FGFR2c on autophagy, which could also arise from the imbalance of the interplay between AKT/MTOR pathway and JNK1 signaling in favor of JNK1 activation, BCL-2 phosphorylation and possibly phagophore nucleation. Finally, silencing experiments of depletion of ESRP1, responsible for FGFR2 splicing and consequent FGFR2b expression, indicated that the switching from FGFR2b to FGFR2c isoform could represent the key event underlying the inhibition of the autophagic process in the epithelial context. Our results provide the first evidence of a negative impact of the out-of-context expression of FGFR2c on autophagy, suggesting a possible role of this receptor in the modulation of the recently proposed negative loop between autophagy and EMT during carcinogenesis.

Background A major issue in the management of cancer is the development of drug resistance. In metastatic melanoma bearing V600 mutations in the BRAF oncogene, all patients undergo disease relapse after combination therapy with BRAF and MEK inhibitors. Hence, understanding the mechanisms at the basis of development of resistance is fundamental to the discovery of new therapeutic approaches. In our group we have spent the last years to identify mechanisms of early adaptation of BRAF mutated melanoma to BRAF and or MEK inhibitors. We have recently shown that the ErbB3 receptor is involved in the activation of an early feedback survival loop upon cell exposure to BRAF and/or MEK inhibitors. Upregulation of pErbB3, due to enhanced production of its ligand neuregulin-1 (HRG), causes increased AKT phosphorylation and cell survival. Furthermore, we demonstrated that activation of the ErbB3/AKT axis is abrogated by cotreatment with anti-ErbB3 mAbs previously generated in our laboratory.

<p>Cellular distribution of HLAII-DR molecules in iDCs was visualized after 2 or 12 hours from cell harvesting and incubation at 37{degree sign}C (columns 1 and 2, respectively) by immunofluorescence staining by Apotome module employing the anti-HLA-DR MoAb L243 (red signal). Nuclei are visualized in blu DAPI staining. Magnification 60X; Bars: 10μm.</p>

<p>A, MUC1 glycoform expression of MUC1-DG75 transfectant detected by flow cytometry employing anti-MUC1 glycoform specific MoAbs. Only the ST-MUC1 glycoform, (MoAb MYE12), is expressed on the surface of transfectant cells. B, MUC1 distribution on MVs shed by MUC1-DG75 cells as detected by Immunogold staining in TEM using the anti-MUC1 MoAb Ma552. C, MUC1 detection in cell extracts and MVs of MUC1-DG75 and DG75 cell lines by Western Blot analysis employing the anti-MUC1 MoAb Ma552. The specific bands above 170kDa are present only in the MUC1-DG75 cell extract and MVs. D, ELISA of the MUC1 glycoprofile expressed by MUC1-DG75-MVs and by rST-MUC1. The ST-MUC1 is the unique MUC1 glycoepitope detected in both samples.</p>

<p>A, MUC1 glycoform expression of MUC1-DG75 transfectant detected by flow cytometry employing anti-MUC1 glycoform specific MoAbs. Only the ST-MUC1 glycoform, (MoAb MYE12), is expressed on the surface of transfectant cells. B, MUC1 distribution on MVs shed by MUC1-DG75 cells as detected by Immunogold staining in TEM using the anti-MUC1 MoAb Ma552. C, MUC1 detection in cell extracts and MVs of MUC1-DG75 and DG75 cell lines by Western Blot analysis employing the anti-MUC1 MoAb Ma552. The specific bands above 170kDa are present only in the MUC1-DG75 cell extract and MVs. D, ELISA of the MUC1 glycoprofile expressed by MUC1-DG75-MVs and by rST-MUC1. The ST-MUC1 is the unique MUC1 glycoepitope detected in both samples.</p>

<p>Cellular distribution of HLAII-DR molecules in iDCs was visualized after 2 or 12 hours from cell harvesting and incubation at 37{degree sign}C (columns 1 and 2, respectively) by immunofluorescence staining by Apotome module employing the anti-HLA-DR MoAb L243 (red signal). Nuclei are visualized in blu DAPI staining. Magnification 60X; Bars: 10microm.</p>

<div>Abstract<p>Tumor-associated glycoproteins are a group of antigens with high immunogenic interest: The glycoforms generated by the aberrant glycosylation are tumor-specific and the novel glycoepitopes exposed can be targets of tumor-specific immune responses. The MUC1 antigen is one of the most relevant tumor-associated glycoproteins. In cancer, MUC1 loses polarity and becomes overexpressed and hypoglycosylated. Changes in glycan moieties contribute to MUC1 immunogenicity and can modify the interactions of tumor cells with antigen-presenting cells such as dendritic cells that would affect the overall antitumor immune response. Here, we show that the form of the MUC1 antigen, i.e., soluble or as microvesicle cargo, influences MUC1 processing in dendritic cells. In fact, MUC1 carried by microvesicles translocates from the endolysosomal/HLA-II to the HLA-I compartment and is presented by dendritic cells to MUC1-specific CD8<sup>+</sup> T cells stimulating IFN-γ responses, whereas the soluble MUC1 is retained in the endolysosomal/HLA-II compartment independently by the glycan moieties and by the modality of internalization (receptor-mediated or non–receptor mediated). MUC1 translocation to the HLA-I compartment is accompanied by deglycosylation that generates novel MUC1 glycoepitopes. Microvesicle-mediated transfer of tumor-associated glycoproteins to dendritic cells may be a relevant biologic mechanism <i>in vivo</i> contributing to define the type of immunogenicity elicited. Furthermore, these results have important implications for the design of glycoprotein-based immunogens for cancer immunotherapy. <i>Cancer Immunol Res; 2(2); 177–86. ©2013 AACR</i>.</p></div>

<p>Cellular distribution of HLAII-DR molecules in iDCs was visualized after 2 or 12 hours from cell harvesting and incubation at 37{degree sign}C (columns 1 and 2, respectively) by immunofluorescence staining by Apotome module employing the anti-HLA-DR MoAb L243 (red signal). Nuclei are visualized in blu DAPI staining. Magnification 60X; Bars: 10μm.</p>

<p>A, MUC1 glycoform expression of MUC1-DG75 transfectant detected by flow cytometry employing anti-MUC1 glycoform specific MoAbs. Only the ST-MUC1 glycoform, (MoAb MYE12), is expressed on the surface of transfectant cells. B, MUC1 distribution on MVs shed by MUC1-DG75 cells as detected by Immunogold staining in TEM using the anti-MUC1 MoAb Ma552. C, MUC1 detection in cell extracts and MVs of MUC1-DG75 and DG75 cell lines by Western Blot analysis employing the anti-MUC1 MoAb Ma552. The specific bands above 170kDa are present only in the MUC1-DG75 cell extract and MVs. D, ELISA of the MUC1 glycoprofile expressed by MUC1-DG75-MVs and by rST-MUC1. The ST-MUC1 is the unique MUC1 glycoepitope detected in both samples.</p>

<p>A, MUC1 glycoform expression of MUC1-DG75 transfectant detected by flow cytometry employing anti-MUC1 glycoform specific MoAbs. Only the ST-MUC1 glycoform, (MoAb MYE12), is expressed on the surface of transfectant cells. B, MUC1 distribution on MVs shed by MUC1-DG75 cells as detected by Immunogold staining in TEM using the anti-MUC1 MoAb Ma552. C, MUC1 detection in cell extracts and MVs of MUC1-DG75 and DG75 cell lines by Western Blot analysis employing the anti-MUC1 MoAb Ma552. The specific bands above 170kDa are present only in the MUC1-DG75 cell extract and MVs. D, ELISA of the MUC1 glycoprofile expressed by MUC1-DG75-MVs and by rST-MUC1. The ST-MUC1 is the unique MUC1 glycoepitope detected in both samples.</p>

<p>Cellular distribution of HLAII-DR molecules in iDCs was visualized after 2 or 12 hours from cell harvesting and incubation at 37{degree sign}C (columns 1 and 2, respectively) by immunofluorescence staining by Apotome module employing the anti-HLA-DR MoAb L243 (red signal). Nuclei are visualized in blu DAPI staining. Magnification 60X; Bars: 10microm.</p>

<div>Abstract<p>Tumor-associated glycoproteins are a group of antigens with high immunogenic interest: The glycoforms generated by the aberrant glycosylation are tumor-specific and the novel glycoepitopes exposed can be targets of tumor-specific immune responses. The MUC1 antigen is one of the most relevant tumor-associated glycoproteins. In cancer, MUC1 loses polarity and becomes overexpressed and hypoglycosylated. Changes in glycan moieties contribute to MUC1 immunogenicity and can modify the interactions of tumor cells with antigen-presenting cells such as dendritic cells that would affect the overall antitumor immune response. Here, we show that the form of the MUC1 antigen, i.e., soluble or as microvesicle cargo, influences MUC1 processing in dendritic cells. In fact, MUC1 carried by microvesicles translocates from the endolysosomal/HLA-II to the HLA-I compartment and is presented by dendritic cells to MUC1-specific CD8<sup>+</sup> T cells stimulating IFN-γ responses, whereas the soluble MUC1 is retained in the endolysosomal/HLA-II compartment independently by the glycan moieties and by the modality of internalization (receptor-mediated or non–receptor mediated). MUC1 translocation to the HLA-I compartment is accompanied by deglycosylation that generates novel MUC1 glycoepitopes. Microvesicle-mediated transfer of tumor-associated glycoproteins to dendritic cells may be a relevant biologic mechanism <i>in vivo</i> contributing to define the type of immunogenicity elicited. Furthermore, these results have important implications for the design of glycoprotein-based immunogens for cancer immunotherapy. <i>Cancer Immunol Res; 2(2); 177–86. ©2013 AACR</i>.</p></div>

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy whose main characterizations are Kirsten Rat Sarcoma-activating mutations (KRAS) and a highly aggressive phenotype. Based on our recent findings demonstrating that the highly aberrant expression of the mesenchymal isoform of Fibroblast Growth Factor Receptor 2 (FGFR2c) in PDAC cells activates Protein-Kinase C Epsilon (PKCε), which in turn controls receptor-mediated epithelial to mesenchymal transition (EMT), here we investigated the involvement of these signaling events in the establishment of additional tumorigenic features. Using PDAC cell lines expressing divergent levels of the FGFR2c and stable protein depletion approaches by short hairpin RNA (shRNA), we found that FGFR2c expression and its PKCε downstream signaling are responsible for the invasive response to Fibroblast Growth Factor 2 (FGF2) and for anchorage-independent growth. In addition, in vitro clonogenic assays, coupled with the check of the amount of cleaved Poly Adenosine Diphosphate-Ribose Polymerase 1 (PARP1) by Western blot, highlighted the involvement of both FGFR2c and PKCε in cell viability. Finally, monitoring of Myeloid Cell Leukemia 1 (MCL-1) expression and Sarcoma kinase family (SRC) phosphorylation suggested that the FGFR2c/PKCε axis could control cell migration/invasion possibly via MCL-1/SRC-mediated reorganization of the actin cytoskeleton. Being PKCs RAS-independent substrates, the identification of PKCε as a hub molecule downstream FGFR2c at the crossroad of signaling networks governing the main malignant tumor hallmarks could represent an important advance towards innovative target therapies overcoming RAS.

Background Therapy of advanced melanoma has been improved with the advent of BRAF inhibitors. However, a limitation to such treatment is the occurrence of resistance. Several mechanisms have been implied in the development of resistance, which in most cases lead to downstream MEK reactivation. In order to overcome resistance MEK inhibitors are being clinically developed with promising results. However, also in this case resistance inevitably occurs. It is commonly believed that the establishment of resistance is facilitated by adaptive changes that take place in cancer cells shortly after exposure to kinase inhibitors. Our laboratory is interested in the identification of these early adaptive changes with the intent to discover additional targets for therapeutic intervention. Methods

Meeting abstracts The induction of an efficacious anti-tumor immune response (IR) requires the cross-processing and presentation of tumor antigen by Dendritic Cells (DCs). Block of endocytated tumor associated antigen (TAA) in the early compartments of the intracellular processing machinery shifts