Glauber C. Brito

Abstract Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a “functional genomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ∼16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types. Significance: This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. Cancer Discovery; 2(2); 172–89. © 2011 AACR. This article is highlighted in the In This Issue feature, p. 95.

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

A large fraction of transcripts are expressed antisense to introns of known genes in the human genome. Here we show the construction and use of a cDNA microarray platform enriched in intronic transcripts to assess their biological relevance in pathological conditions. To validate the approach, prostate cancer was used as a model, and 27 patient tumor samples with Gleason scores ranging from 5 to 10 were analyzed. We find that a considerably higher fraction (6.6%, [23/346]) of intronic transcripts are significantly correlated (P< or =0.001) to the degree of prostate tumor differentiation (Gleason score) when compared to transcripts from unannotated genomic regions (1%, [6/539]) or from exons of known genes (2%, [27/1369]). Among the top twelve transcripts most correlated to tumor differentiation, six are antisense intronic messages as shown by orientation-specific RT-PCR or Northern blot analysis with strand-specific riboprobe. Orientation-specific real-time RT-PCR with six tumor samples, confirmed the correlation (P=0.024) between the low/high degrees of tumor differentiation and antisense intronic RASSF1 transcript levels. The need to use intron arrays to reveal the transcriptome profile of antisense intronic RNA in cancer has clearly emerged.

Article10 July 2018Open Access Transparent process Phosphorylation switches Bax from promoting to inhibiting apoptosis thereby increasing drug resistance Justin Kale Justin Kale orcid.org/0000-0002-4915-0806 Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada Search for more papers by this author Ozgur Kutuk Ozgur Kutuk orcid.org/0000-0001-9854-7220 Department of Medical Genetics, Adana Medical and Research Center, Baskent University School of Medicine, Adana, Turkey Search for more papers by this author Glauber Costa Brito Glauber Costa Brito Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Dourados, Mato Grosso do Sul, Brazil Search for more papers by this author Tallulah S Andrews Tallulah S Andrews Wellcome Trust Sanger Institute, Cambridge, UK Search for more papers by this author Brian Leber Brian Leber Departments of Biochemistry and Biomedical Sciences, and Medicine, McMaster University, Hamilton, ON, Canada Search for more papers by this author Anthony Letai Corresponding Author Anthony Letai [email protected] orcid.org/0000-0002-1993-9013 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author David W Andrews Corresponding Author David W Andrews [email protected] orcid.org/0000-0002-9266-7157 Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada Departments of Biochemistry and Medical Biophysics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Justin Kale Justin Kale orcid.org/0000-0002-4915-0806 Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada Search for more papers by this author Ozgur Kutuk Ozgur Kutuk orcid.org/0000-0001-9854-7220 Department of Medical Genetics, Adana Medical and Research Center, Baskent University School of Medicine, Adana, Turkey Search for more papers by this author Glauber Costa Brito Glauber Costa Brito Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Dourados, Mato Grosso do Sul, Brazil Search for more papers by this author Tallulah S Andrews Tallulah S Andrews Wellcome Trust Sanger Institute, Cambridge, UK Search for more papers by this author Brian Leber Brian Leber Departments of Biochemistry and Biomedical Sciences, and Medicine, McMaster University, Hamilton, ON, Canada Search for more papers by this author Anthony Letai Corresponding Author Anthony Letai [email protected] orcid.org/0000-0002-1993-9013 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA Search for more papers by this author David W Andrews Corresponding Author David W Andrews [email protected] orcid.org/0000-0002-9266-7157 Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada Departments of Biochemistry and Medical Biophysics, University of Toronto, Toronto, ON, Canada Search for more papers by this author Author Information Justin Kale1,‡, Ozgur Kutuk2,‡, Glauber Costa Brito3, Tallulah S Andrews4, Brian Leber5, Anthony Letai *,6 and David W Andrews *,1,7 1Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada 2Department of Medical Genetics, Adana Medical and Research Center, Baskent University School of Medicine, Adana, Turkey 3Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Dourados, Mato Grosso do Sul, Brazil 4Wellcome Trust Sanger Institute, Cambridge, UK 5Departments of Biochemistry and Biomedical Sciences, and Medicine, McMaster University, Hamilton, ON, Canada 6Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 7Departments of Biochemistry and Medical Biophysics, University of Toronto, Toronto, ON, Canada ‡These authors contributed equally to this work *Corresponding author. Tel: +1 6176322348; E-mail: [email protected] *Corresponding author. Tel: +1 4164805120; E-mail: [email protected] EMBO Reports (2018)19:e45235https://doi.org/10.15252/embr.201745235 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Akt is a pro-survival kinase frequently activated in human cancers and is associated with more aggressive tumors that resist therapy. Here, we connect Akt pathway activation to reduced sensitivity to chemotherapy via Akt phosphorylation of Bax at residue S184, one of the pro-apoptotic Bcl-2 family proteins required for cells to undergo apoptosis. We show that phosphorylation by Akt converts the pro-apoptotic protein Bax into an anti-apoptotic protein. Mechanistically, we show that phosphorylation (i) enables Bax binding to pro-apoptotic BH3 proteins in solution, and (ii) prevents Bax inserting into mitochondria. Together, these alterations promote resistance to apoptotic stimuli by sequestering pro-apoptotic activator BH3 proteins. Bax phosphorylation correlates with cellular resistance to BH3 mimetics in primary ovarian cancer cells. Further, analysis of the TCGA database reveals that 98% of cancer patients with increased BAX levels also have an upregulated Akt pathway, compared to 47% of patients with unchanged or decreased BAX levels. These results suggest that in patients, increased phosphorylated anti-apoptotic Bax promotes resistance of cancer cells to inherent and drug-induced apoptosis. Synopsis Phosphorylation of Bax at S184 by Akt converts it from pro- to anti-apoptotic and confers resistance to drugs and pro-apoptotic signalling. In patients, Akt is increased in tumours with high Bax and phosphorylation correlates with drug resistance. Phosphorylation of Bax at residue S184 by Akt inhibits apoptosis in cancer cell lines. Phosphorylation converts pro-apoptotic Bax into an anti-apoptotic protein. Phosphomimetic Bax sequesters and thereby inactivates pro-apoptotic BH3-proteins. In patients, levels of Akt and Bax are correlated and Bax phosphorylation correlates with resistance to BH3-mimetics. Introduction Activation of the mitochondrial apoptosis pathway in cancer cells serves as an important route of cell death following treatment with chemotherapeutic agents. Alteration of this pathway can cause resistance to therapy in cancer cells. A decisive step for commitment to apoptosis is mitochondrial outer membrane permeabilization (MOMP) by the pro-apoptotic Bax and/or Bak proteins that release intermembrane space proteins including cytochrome c into cytosol 1, 2. Membrane permeabilization by Bax and Bak is provoked by activator proteins including the BH3 proteins Bim and Bid. Pro-survival Bcl-2 proteins (Bcl-2, Bcl-XL, Mcl-1, Bfl-1, and Bcl-W) inhibit MOMP by sequestering either activator BH3 proteins or Bax and Bak 3, 4. Other so-called sensitizer BH3 proteins, including Bad, Noxa, and Bik, cannot activate Bax or Bak, but rather exert a pro-death function by competing for the BH3 binding sites of pro-survival proteins 2, 5. Differences in the affinities of the interactions, expression levels, and post-translational modifications of these proteins together determine the fate of the cell. Measurement of MOMP upon incubating BH3 domain-derived peptides with mitochondria and identifying differential response patterns was successfully translated into an assay called BH3 profiling 6, 7. By interpreting the pattern of mitochondrial sensitivity to BH3 peptides of different affinities for anti-apoptotic proteins, BH3 profiling can be used to identify dependence on individual anti-apoptotic Bcl-2 proteins for survival and sensitivity to inhibitors. Certain BH3 domain peptides, including those from Bid and Bim, interact with all known anti-apoptotic proteins. Mitochondrial sensitivity to these peptides can be interpreted as a measure of how close a cell is to the threshold of apoptosis, or how “primed” a cell is for death 6, 8. The degree of priming predicts how sensitive the cell will be to toxic insults, and correlates with clinical response to chemotherapy 9. In cancer, particularly in breast cancer, upregulation of the Akt pathway is strongly associated with poor prognosis and resistance to therapy 10. PTEN (phosphatase and tensin homolog deleted on chromosome 10) functions as a lipid phosphatase to restrain Akt pathway activation by diminishing the phosphatidylinositol-3,4,5-biphosphate (PIP3) cellular pool through hydrolysis of 3-phosphate on PIP3 to generate phosphatidylinositol-4,5-biphosphate (PIP2). PI3Ks phosphorylate PIP2 to regenerate PIP3 which promotes Akt recruitment to plasma membrane through binding its pleckstrin-homology (PH) domain. Following recruitment to the plasma membrane by PIP3, Akt is phosphorylated by PDK1 at T308 and by mTORC2 at S473 which leads to its activation 11. Hence, inactivation or loss of PTEN results in increased accumulation of PIP3 and constitutively active Akt signaling which promotes cell growth and survival. The Akt pathway regulates fundamental processes in cells, including survival, cell cycle progression, and metabolism. Upregulation of the Akt signaling pathway is commonly detected in a wide spectrum of human cancers. Several mechanisms including genomic amplification of Akt or growth factor receptors, PTEN deletion or mutations, or activating mutations in pathway genes can activate Akt in cancer cells. Importantly, Akt blocks pro-death signaling upstream of MOMP 12. However, it is still unclear how pro-survival Akt signaling makes the critical connection to the Bcl-2 family that controls the mitochondrial apoptosis pathway. Some suggest an indirect effect, for instance, via transcriptional control of pro-apoptotic Bcl-2 family proteins via the FOXO family of transcriptional regulators 11. Akt could also play a more direct role since it can phosphorylate the pro-apoptotic BH3 protein Bad. However, Bad is dispensable for apoptosis induced by several mechanisms 13, 14, suggesting that a more central Bcl-2 family protein such as Bax might also be controlled by AKT 15, 16. However, reports on the function of phosphorylated Bax are inconsistent—one suggests that S184 phosphorylation activates Bax 17, while others suggest that S184-phosphorylated Bax is inhibited 15, 16, 18, 19. Here, we show that Akt directly phosphorylates Bax and can localize to mitochondria. Unexpectedly, phosphorylation switches the function of Bax from pro- to anti-apoptotic, thereby impeding mitochondrial priming for apoptosis. Mechanistically, we show (i) that phosphorylation of Bax blocks its insertion into membranes upstream of the oligomerization essential for its pro-apoptotic membrane permeabilization function, and (ii) that after phosphorylation, Bax acts as a dominant negative by binding to and sequestering activator BH3 proteins thereby inhibiting BH3-protein-mediated apoptosis. Consistent with a role for this switch in Bax function in human disease, we find that in cancer patients with elevated Bax, Bax expression is positively associated with an increase in expression of genes in the Akt pathway. Our data suggest that in cancers with upregulated Akt pathway signaling, one means of inhibiting apoptosis is to select for increased levels of Bax which is then phosphorylated switching on its anti-apoptotic function. Given the established role of Bax as a critical effector of MOMP downstream of many pro-apoptotic signals, phosphorylation of Bax is likely a key mediator of the anti-apoptotic effect exerted by upregulated Akt in cancer. Results A post-translational modification alters mitochondrial priming in ABT-737-resistant cell lines BH3 profiling with Bad can predict cellular sensitivity to Bcl-2 antagonism by ABT-737 6, 8, 20. To extend this observation, we tested this correlation in a new cancer cell line panel. As shown in Fig 1A, we found that mitochondria from MDA-MB-435, MDA-MB-468, SF539, and 768-O cells were at least as sensitive to Bad as MCF-7, T47-D, or ZR-75-1 cell lines. Surprisingly, mitochondrial sensitivity was not predictive of cellular responses as only MDA-MB-435 cells were sensitive to ABT-737 (EC50, 32 nM), and other cells were relatively insensitive to ABT-737 with EC50 values at micromolar levels (Fig 1B). Figure 1. A post-translational modification alters priming of mitochondria from cancer cell lines Mitochondria isolated from the indicated cell lines were incubated with Bad peptide and MOMP measured as % cytochrome c release determined by ELISA. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Cells were treated with indicated concentrations of ABT-737 for 48 h. ABT-737 EC50 values of cancer cell lines were determined by using MTT assay (mean ± SEM, n = 4 experimental replicates). Mitochondria isolated from the indicated cell lines were incubated with the indicated BH3 peptides in the presence or absence of phosphatase inhibitor cocktail PhosSTOP in all buffers. MOMP was measured as % cytochrome c release determined by ELISA. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. One-way ANOVA was used followed by post hoc t-tests with Bonferroni correction for multiple comparisons (ns = not significant). MDA-MB-468 (Bim ****P < 0.0001; Bad **P = 0.0059; BMF ***P = 0.0002). ZR-75-1 (Bid ***P = 0.0003; Bim ****P < 0.0001; Bad **P = 0.0041; Puma ***P = 0.0007). Download figure Download PowerPoint One possible explanation for the deviation from sensitivity predicted by BH3 profiling is that post-translational modifications (PTMs) are present in cells that are not present in our in vitro system. Since the primary post-translational modification of Bcl-2 family proteins is phosphorylation 21, 22, we tested whether including phosphatase inhibitors in mitochondrial isolation buffer and during BH3 profiling in experimental buffer would modulate the priming profile (Fig 1C). We found that phosphatase inhibitors significantly reduced sensitivity to BH3 peptides in ABT-737-resistant cells (MDA-MB-468 and ZR-75-1) but had no significant effect on BH3 peptide response in ABT-737-sensitive cells (MDA-MB-435 and MCF-7). Taken together, our results suggest that a kinase is unpriming mitochondria selectively in the ABT-737-resistant cell lines. For this reason, we used a phosphatase inhibitor cocktail in all subsequent BH3 profiling experiments. Bax is phosphorylated in ABT-737-resistant cell lines Resistance to the BH3 mimetic ABT-199 is reportedly overcome by inhibition of the Akt pathway 23. Furthermore, since Bax can be phosphorylated by Akt at residue S184 and inhibited 15, 16, we examined the phosphorylation status of Bax in ABT-737-resistant cells. Immunoprecipitating Bax and blotting for phosphoserine suggest Bax is phosphorylated, while blotting with a phospho-Bax S184 antibody demonstrated that residue S184 is phosphorylated in ABT-737-resistant MDA-MB-468 cells, but not in ABT-737-sensitive MDA-MB-435 cells (Figs 2A and EV1A). Figure 2. Bax is phosphorylated in ABT-737-resistant cell lines Top two panels: Phosphorylation of Bax was evaluated using lysates from MDA-MB-435 and MDA-MB-468 cells by Western blotting with the indicated antibodies (IB) after immunoprecipitation with the indicated antibodies (IP). Lower two panels: 5–10% of the total lysates (input) were probed for Bax (Bax ∆21 antibody) and actin as expression and loading controls, respectively, by Western blotting with the indicated antibodies (IB). Phosphorylation of Bax was evaluated using lysates from GFP-Bax- and GFP-Bax S184A-expressing MDA-MB-468 cells by blotting with the antibodies indicated at the right after precipitation with the antibodies indicated below the panels. Lysates from untransfected cells (MDA-MB-468 WT) and IgG IP are used as negative controls. Lower panels: 5–10% of the total lysates were probed for GFP and actin as expression and loading controls, respectively. Asterisk (*) indicates a cross-reacting band. Bax delta-21 antibody was used for IB of Bax. MDA-MB-468 cells were transfected with plasmids encoding GFP-Bax, GFP-Bax S184A, or GFP-Bax S184E, and the localization of Bax was evaluated by confocal microscopy (upper panel). Cells were co-stained with MitoTracker Red CMXRos (mitochondria) and DAPI (nucleus). Whole-image Pearson's correlation coefficients between MitoTracker and GFP for the single images shown were 0.644, 0.607, and 0.206 for GFP-Bax, GFP-Bax S184A, and GFP-Bax S184E, respectively (see Appendix Fig S1). Scale bars: 20 μm. MDA-MB-468 cells that were either untransfected or expressing GFP, GFP-Bax, GFP-Bax S184A, or GFP-Bax S184E were treated with ABT-737 (100 nM, 48 h), and apoptosis was evaluated by Annexin V staining followed by fluorescence-activated cell sorting (FACS). Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. One-way ANOVA was used followed by post hoc t-tests with Bonferroni correction (****P < 0.001, ns = not significant). Data information: See also Fig EV1. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Related to Fig 2: Bax is phosphorylated on residue S184 Top two panels: Phosphorylation of Bax S184 was evaluated using lysates from MDA-MB-435 and MDA-MB-468 cells by Western blotting with the indicated antibodies (IB) after immunoprecipitation with the indicated antibodies (IP). Lower two panels: 5–10% of the total lysates (input) were probed for Bax and actin as expression and loading controls, respectively, by Western blotting with the indicated antibodies (IB). Top two panels: Phosphorylation of Bax S184 was evaluated using lysates from GFP-Bax- and GFP-Bax S184A-expressing MDA-MB-468 cells by blotting with the antibodies indicated at the right after precipitation with the antibodies indicate below the panels. Lysates from untransfected cells (MDA-MB-468 WT) and IgG IP are used as negative controls. Lower panels: 5–10% of the total lysates were probed for GFP and actin as expression and loading controls, respectively. Localization of GFP-Bax constructs in MDA-MB-468 cells. MDA-MB-468 cells transiently expressing GFP-Bax S184A, GFP-Bax, or GFP-Bax S184E were lysed and separated into cytosolic (C), mitochondrial (M), or nuclear fractions (N) and then immunoblotted for GFP. GAPDH and CoxIV were immunoblotted for cytosolic and mitochondrial marker proteins, respectively. Download figure Download PowerPoint Bax can be phosphorylated at multiple residues 21. To further confirm the specific phosphorylation of Bax on residue S184 in the ABT-737-resistant MDA-MB-468 cells, we created a mutant Bax (S184A) that would prevent Bax phosphorylation at residue S184. In ABT-737-resistant MDA-MB-468 cells, GFP immunoprecipitation revealed that GFP-Bax was phosphorylated at residue S184, whereas GFP-Bax S184A was not (Figs 2B and EV1B). These data support S184 as a primary phosphorylation site on Bax in the ABT-737-resistant cells and corroborate other studies that also observed Bax S184 phosphorylation by Akt 15, 16, 19, 24. While Bax is localized both in the cytosol and at mitochondria of many cells, migration to the mitochondrion is essential for Bax pro-apoptotic function. We evaluated the subcellular localization of GFP-tagged WT Bax and the S184A (phosphorylation-resistant) and S184E (phosphomimetic) mutants in ABT-737-resistant MDA-MB-468 cells. GFP-Bax was localized diffusely throughout the cell and also co-localized with mitochondria, while GFP-Bax S184A showed punctate, mostly mitochondrial localization (Fig 2C). In contrast, GFP-Bax S184E showed a diffuse cytoplasmic and nuclear pattern without mitochondrial localization (Fig 2C). These data were confirmed by subcellular fractionation, where GFP-Bax S184A is predominantly mitochondrial, GFP-Bax S184E is cytosolic and nuclear, and GFP-Bax levels are similar in all fractions (Fig EV1C). In transient transfection experiments, both GFP-Bax and GFP-Bax S184A were highly toxic to MDA-MB-468 cells. Spontaneous cell death was also increased in cells stably expressing these constructs (Fig 2D), whereas there was minimal basal cell death in GFP-Bax S184E-expressing cells. Consistent with these results, GFP-Bax- and GFP-Bax S184A-expressing MDA-MB-468 cells were significantly more sensitive to ABT-737 treatment. By contrast, cells expressing GFP-Bax S184E remained resistant to ABT-737 (Fig 2D). Akt phosphorylates Bax in ABT-737-resistant cells Since Bax can be phosphorylated at residue S184 by Akt, this pathway may mediate the resistance to ABT-737 we observed in cancer cells (Fig 1B). To test this hypothesis, we examined the effect of small molecule inhibitors of the Akt pathway on mitochondrial priming. As predicted, treatment of ABT-737-resistant MDA-MB-468 cells (Fig 3A, top left panel) or ZR-75-1 cells (Fig 3A, bottom left panel) with Akt pathway inhibitors significantly enhanced priming in the majority of cases as shown by increased response to BH3 peptides (Appendix Table S1). As expected, there was no significant difference in the priming of ABT-737-sensitive MDA-MB-435 cells (Fig 3A, top right panel) or MCF-7 cells (Fig 3A, bottom right panel) when treated with Akt pathway inhibitors (Appendix Table S1). Furthermore, Bax phosphorylation at S184 is substantially reduced when the ABT-737-resistant cell lines, MDA-MB-468 and ZR-75-1, are treated with AKT pathway inhibitors (Figs 3B and EV2A), directly linking Akt activation to Bax S184 phosphorylation. Figure 3. AKT phosphorylates and inhibits Bax, preventing mitochondrial cytochrome c release in response to BH3 peptides and proteins BH3-peptide-resistant (left panels; MDA-MB-468 and ZR-75-1) and BH3-peptide-sensitive (right panels; MDA-MB-435 and MCF-7) cells were treated with direct Akt inhibitors (MK-2206 [1 μM], A-443654 [0.4 μM]) or Akt pathway inhibitors (LY294002 [25 μM], deguelin [10 nM]) for 6 h, and BH3 profiles were analyzed. The phosphatase inhibitor cocktail PhosSTOP was used in all buffers for these experiments. Cytochrome c release was determined by ELISA. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Two-way ANOVA was conducted on the influence of two independent variables (BH3 peptide, drug) on cytochrome c release of isolated mitochondria for each cell line followed by post hoc t-tests with Bonferroni correction for multiple comparisons. Each drug treatment was statistically compared to DMSO control within each peptide treatment group. The percentage of P-values that were significant (DMSO control to each drug) was 65% for MDA-MB-468, 0% for MDA-MB-435, 95% for ZR-75-1, and 0% for MDA-MB-435. See Appendix Table S1 for P-values of each comparison. Top two panels: MDA-MB-468 and ZR-75-1 cells were treated with 1 μM MK-2206, 0.4 μM A-443654, 25 μM LY294002, or 10 nM deguelin where indicated. Phosphorylation of Bax was evaluated using lysates from the treated cells by Western blotting with the indicated antibodies (IB) after immunoprecipitation with the indicated antibodies (IP). Lower two panels: 5%–10% of the total lysates (input) were probed for Bax and actin as expression and loading controls, respectively, by Western blotting with the indicated antibodies (IB). Bax ∆21 antibody was used for both IP and IB of Bax. S100 fractions from untreated cells (MDA-MB-468) or from cells treated with direct Akt inhibitors (MK-2206 [1 μM], A-443654 [0.4 μM]) or Akt pathway inhibitors (LY294002 [25 μM], deguelin [10 nM]) were isolated. Akt was immunodepleted by sequential immunoprecipitation of untreated S100 fractions, and the efficiency of immunodepletion was tested by immunoblot analysis. IgG was used as a negative control for immunodepletion experiments (inset). Indicated S100 fractions were incubated with MDA-MB-435 mitochondrial preparations, and the resulting extent of priming was assessed by measuring the cytochrome c released from MDA-MB-435 mitochondria by Bid BH3 peptide (treatments with Bim, Bad, Puma, and Bmf are located in Fig EV2B). Cytochrome c release was determined by ELISA. The phosphatase inhibitor cocktail PhosSTOP was used in all buffers for these experiments. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Two-way ANOVA was conducted on the influence of two independent variables (BH3 peptide, conditions) on cytochrome c release of isolated mitochondria followed by post hoc t-tests with Bonferroni correction for multiple comparisons. Each condition was statistically compared to control (white bar) within each peptide treatment group. Shown here are P-values for treatment with Bid BH3 peptide (ns = not significant). MDA-MB-468 S100 (**P = 0.0031), A443654-pre-treated MDA-MB-468 S100 (*P = 0.0324), and IgG-pre-treated MDA-MB-468 S100 (*P = 0.0432). See Appendix Table S2 for P-values. MDA-MB-435 mitochondria were incubated with recombinant active Akt in kinase assay buffer containing ATP, and alteration of priming in MDA-MB-435 mitochondria was detected by using BH3 profiling. Cytochrome c release was determined by ELISA. The phosphatase inhibitor cocktail PhosSTOP was used in all buffers for these experiments. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Two-way ANOVA was conducted on the influence of two independent variables (BH3 peptide, treatment) on cytochrome c release. BH3 peptides included Bid, Bim, Bad, Puma, and Bmf. Treatment included Akt or buffer alone. Each drug treatment was statistically compared to control (white bar) within each peptide group using t-tests with Bonferroni correction for multiple comparisons (ns = not significant). Bid, Akt (*P = 0.0283); Bim, Akt (*P = 0.0482); Puma, Akt (*P = 0.0170); Bmf, Akt (**P = 0.0035). tBid (5 nM) and Bax (50 nM), with or without active Akt plus ATP, were incubated with mitochondria isolated from Bax−/− Bak−/− DKO MEFs for 1 h, and mitochondrial cytochrome c release was determined by ELISA. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. tBid, Bax, Akt, ATP, and the Akt inhibitor MK-2206, as indicated, were incubated with mitochondria isolated from Bax−/− Bak−/− DKO MEFs for 1 h, and mitochondrial cytochrome c release was determined by ELISA. The shaded and circled reagents were pre-incubated before the addition of the other indicated reagents. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. tBid (5 nM) and Bax S184A (50 nM), with or without active Akt plus ATP, were incubated with mitochondria isolated from Bax−/− Bak−/− DKO MEFs for 1 h, and mitochondrial cytochrome c release was determined by ELISA. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Data information: See also Fig EV2. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Related to Fig 3: Akt pathway inhibitors change the primed state of mitochondria, the localization of Akt, and the phosphorylation status of Bax S184 MDA-MB-468 and ZR-75-1 cells were treated with 1 μM MK-2206, 0.4 μM A-443654, 25 μM LY294002, or 10 nM deguelin where indicated. Phosphorylation of Bax S184 was evaluated using lysates from the treated cells by Western blotting with the indicated antibodies (IB) after immunoprecipitation with the indicated antibodies (IP). S100 fractions from untreated cells (MDA-MB-468) or from cells treated with direct Akt inhibitors (MK-2206 [1 μM], A-443654 [0.4 μM]) or Akt pathway inhibitors (LY294002 [25 μM], deguelin [10 nM]) were isolated. Akt was immunodepleted by sequential immunoprecipitation in untreated S100 fractions, and the efficiency of immunodepletion was tested by immunoblot analysis. IgG was used as a negative control for immunodepletion experiments. The indicated S100 fractions were incubated with MDA-MB-435 mitochondrial preparations, and change in priming was assessed by using BH3 profiling. Responses to various BH3 peptides are shown. The phosphatase inhibitor cocktail PhosSTOP was used in all buffers. Cytochrome c release was determined by ELISA. Bars indicate the mean of three independent experiments (n = 3). Symbols indicate the mean of at least two technical replicates for each independent experiment. Two-way ANOVA was conducted on the influence of two independent variables (BH3 peptide, treatment) on cytochrome c release of isolated mitochondria from ABT-737-sensitive MDA-MB-435 cells that were treated with the S100 fraction isolated from ABT-737-resistant MDA-MB-468 or ZR-75-1 cells. Each treatment was statistically compared to control within each peptide group using t-tests with Bonferroni correction for multiple comparisons. See Appendix Table S2 for P-values. ABT-737-resistant MDA-MB-468 and ZR-75-1 and ABT-737-sensitive MDA-MB-435 and MCF-7 cells were treated with direct Akt inhibitors (MK-2206 [1 μM], A-443654 [0.4 μM]) or Akt pathway inhibitors (LY294002 [25 μM], deguelin [10 nM]), and mitochondrial and cytosolic fractions were immunoblotted for Akt. CoxIV was probed as a mitochondrial marker protein, and

The TNM Classification of Malignant Tumours (TNM) staging system is the primary means of determining a prognosis for gastric adenocarcinoma (GC). However, tumor behavior in the individual patient is unpredictable and in spite of treatment advances, a classification of 'advanced stage' still portends a poor prognosis. Thus, further insights from molecular analyses are needed for better prognostic stratification and determination of new therapeutic targets.A total of fifty-one fresh frozen tumor samples from patients with histopathologically confirmed diagnoses of GC, submitted to surgery with curative intent, were included in the study. Total RNA was extracted from an initial group of fifteen samples matched for known prognostic factors, categorized into two subgroups, according to patient overall survival: poor (<24 months) or favorable (at or above 24 months), and hybridized to Affymetrix Genechip human genome U133 plus 2.0 for genes associated with prognosis selection. Thirteen genes were selected for qPCR validation using those initial fifteen samples plus additional thirty-six samples.A total of 108 genes were associated with poor prognosis, independent of tumor staging. Using systems biology, we suggest that this panel reflects the dampening of immune/inflammatory response in the tumor microenvironment level and a shift to Th2/M2 activity. A gene trio (OLR1, CXCL11 and ADAMDEC1) was identified as an independent marker of prognosis, being the last two markers validated in an independent patient cohort.We determined a panel of three genes with prognostic value in gastric cancer, which should be further investigated. A gene expression profile suggestive of a dysfunctional inflammatory response was associated with unfavorable prognosis.

Abstract The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non‐neoplastic tissue were obtained from six patients. A custom‐built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down‐regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave‐one‐out patient cross‐validation. Among the down‐regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein‐coding genes. Lower levels of expression of SIN3B , TRIP3 , SYNJ2BP and NDE1 ( P &lt; 0.02), and of intronic transcripts derived from SND1 and ACTN4 loci ( P &lt; 0.05), were confirmed in clear cell RCC by Real‐time RT‐PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. © 2008 Wiley‐Liss, Inc.

Tail-anchored (TA) proteins are a unique class of functionally diverse membrane proteins defined by their single C-terminal membrane-spanning domain and their ability to insert post-translationally into specific organelles with an Ncytoplasm-Corganelle interior orientation. The molecular mechanisms by which TA proteins are sorted to the proper organelles are not well understood. Herein we present results indicating that a dibasic targeting motif (i.e., -R-R/K/H-X{X≠E}) identified previously in the C terminus of the mitochondrial isoform of the TA protein cytochrome b5, also exists in many other A. thaliana outer mitochondrial membrane (OMM)-TA proteins. This motif is conspicuously absent, however, in all but one of the TA protein subunits of the translocon at the outer membrane of mitochondria (TOM), suggesting that these two groups of proteins utilize distinct biogenetic pathways. Consistent with this premise, we show that the TA sequences of the dibasic-containing proteins are both necessary and sufficient for targeting to mitochondria, and are interchangeable, while the TA regions of TOM proteins lacking a dibasic motif are necessary, but not sufficient for localization, and cannot be functionally exchanged. We also present results from a comprehensive mutational analysis of the dibasic motif and surrounding sequences that not only greatly expands the functional definition and context-dependent properties of this targeting signal, but also led to the identification of other novel putative OMM-TA proteins. Collectively, these results provide important insight to the complexity of the targeting pathways involved in the biogenesis of OMM-TA proteins and help define a consensus targeting motif that is utilized by at least a subset of these proteins.

Cellular interaction networks can be used to analyze the effects on cell signaling and other functional consequences of perturbations to cellular physiology. Thus, several methods have been used to reconstitute interaction networks from multiple published datasets. However, the structure and performance of these networks depends on both the quality and the unbiased nature of the original data. Due to the inherent bias against membrane proteins in protein-protein interaction (PPI) data, interaction networks can be compromised particularly if they are to be used in conjunction with drug screening efforts, since most drug-targets are membrane proteins. To overcome the experimental bias against PPIs involving membrane-associated proteins we used a probabilistic approach based on a hypergeometric distribution followed by logistic regression to simultaneously optimize the weights of different sources of interaction data. The resulting less biased genome-scale network constructed for the budding yeast Saccharomyces cerevisiae revealed that the starvation pathway is a distinct subnetwork of autophagy and retrieved a more integrated network of unfolded protein response genes. We also observed that the centrality-lethality rule depends on the content of membrane proteins in networks. We show here that the bias against membrane proteins can and should be corrected in order to have a better representation of the interactions and topological properties of protein interaction networks.

Methicillin-resistant Staphylococcus aureus (MRSA) screening plays a great role in preventing infections in surgical patients. This study aims to evaluate clonality, virulence and resistance of MRSA in pre- and post-liver transplantation (LT) patients. Nasal and groin swabs of 190 patients were collected. PCR for virulence genes and staphylococcal cassette chromosome mec (SCCmec) types, microarray, PFGE, multilocus sequence typing and MIC were performed. MRSA carriers were detected in 20.5 % (39/190) of the patients. However, only three colonized patients developed infections post-LT. Sixty-nine MRSA isolates were identified, and the most frequent SCCmec type was type II (29/69; 42.0 %). Most isolates (57/69; 82.6 %) were susceptible to trimethoprim-sulfamethoxazole (TMP/SMX) and harboured the lukD, lukE, clf and fnbA genes as determined by PCR. Five sequence types (ST) were identified among nine clones; 36.2 % (25/69) isolates belonged to a predominant clone (ST105 and SCCmec type II) that was susceptible to TMP/SMX, mupirocin and chlorhexidine, which had 87.9 % similarity with the New York/Japan clone. The array showed virulence difference in isolates of the same clone and patients and that colonized isolates (pre-LT patients) were less virulent than those post-LT and those infected. Therefore, despite the high frequency of MRSA colonization, infection due to MRSA was uncommon in our LT unit. MRSA isolates presented great diversity. Isolates of the same clone expressed different virulence factors by array. Colonizing isolates pre-LT expressed less virulent factors than post-LT and infecting isolates.

Abstract Background Tail-anchored membrane proteins (TAMPs) differ from other integral membrane proteins, because they contain a single transmembrane domain at the extreme carboxyl-terminus and are therefore obliged to target to membranes post-translationally. Although 3–5% of all transmembrane proteins are predicted to be TAMPs only a small number are well characterized . Results To identify novel putative TAMPs across different species, we used TAMPfinder software to identify 859, 657 and 119 putative TAMPs in human ( Homo sapiens) , plant ( Arabidopsis thaliana) , and yeast ( Saccharomyces cerevisiae ), respectively. Bioinformatics analyses of these putative TAMP sequences suggest that the list is highly enriched for authentic TAMPs. To experimentally validate the software predictions several human and plant proteins identified by TAMPfinder that were previously uncharacterized were expressed in cells and visualized at subcellular membranes by fluorescence microscopy and further analyzed by carbonate extraction or by bimolecular fluorescence complementation. With the exception of the pro-apoptotic protein harakiri, which is, peripherally bound to the membrane this subset of novel proteins behave like genuine TAMPs. Comprehensive bioinformatics analysis of the generated TAMP datasets revealed previously unappreciated common and species-specific features such as the unusual size distribution of and the propensity of TAMP proteins to be part of larger complexes. Additionally, novel features of the amino acid sequences that anchor TAMPs to membranes were also revealed. Conclusions The findings in this study more than double the number of predicted annotated TAMPs and provide new insights into the common and species-specific features of TAMPs. Furthermore, the list of TAMPs and annotations provide a resource for further investigation.

In renal cell carcinoma (RCC) of the clear cell type, inactivity of the VHL gene induces overexpression of HIF1 α and its targets, the tyrosine kinase receptors, promoting RCC development and progression. The discovery of tyrosine kinase inhibitors (TKIs) changed the treatment of these tumors. Other molecular pathways involved in the TKI mechanisms of action have not been described in the literature. The aim of our study was to elucidate alternative mechanisms of action of sunitinib in tumor tissue after neoadjuvant treatment of RCC.The gene expression profile was accessed using microarray (Affymetrix Human Genome U133 Plus 2.0 platform) and frozen RCC tissues collected from 5 patients with locally advanced non-metastatic tumors who underwent nephrectomy after being treated with 2 cycles of neoadjuvant sunitinib. The results were compared with matched controls comprising 6 patients with no neoadjuvant intervention.There was underexpression of the majority of genes after sunitinib treatment. The lower expression levels of IGFBP1, CCL20, CXCL6 and FGB were confirmed by qRT-PCR in all cases. The downregulation of gene expression leads us to search for methylation as a mechanism of action of the TKI. IGFBP1 was shown to be methylated by methylation-sensitive high-resolution melting technique.The ultimate genetic effects of sunitinib may explain its actions as an antitumor drug that apparently suppresses the expression of important genes related to cell survival, adhesion, invasion and immunomodulation. The methylation of gene promoters was shown to be part of the mechanism of action of this class of drugs.

&lt;p&gt;XLS file - 51K&lt;/p&gt;

&lt;p&gt;XLS file - 75K&lt;/p&gt;

&lt;p&gt;XLS file - 15.2MB&lt;/p&gt;

&lt;p&gt;XLS file - 22K&lt;/p&gt;

&lt;p&gt;XLS file - 22K&lt;/p&gt;

&lt;p&gt;PDF file - 217K&lt;/p&gt;

&lt;p&gt;XLS file - 49K&lt;/p&gt;

&lt;p&gt;XLS file - 32K&lt;/p&gt;

&lt;p&gt;XLS file - 303K&lt;/p&gt;

&lt;p&gt;XLS file - 41K&lt;/p&gt;

&lt;p&gt;PDF file - 310K&lt;/p&gt;

&lt;p&gt;XLS file - 22K&lt;/p&gt;

&lt;p&gt;XLS file - 47K&lt;/p&gt;

&lt;p&gt;XLS file - 55K&lt;/p&gt;

&lt;p&gt;XLS file - 49K&lt;/p&gt;

&lt;p&gt;XLS file - 22K&lt;/p&gt;

&lt;p&gt;PDF file - 310K&lt;/p&gt;

&lt;p&gt;XLS file - 22K&lt;/p&gt;

&lt;p&gt;XLS file - 75K&lt;/p&gt;

&lt;p&gt;XLS file - 32K&lt;/p&gt;

&lt;p&gt;XLS file - 22K&lt;/p&gt;

&lt;p&gt;XLS file - 47K&lt;/p&gt;

&lt;p&gt;XLS file - 51K&lt;/p&gt;

&lt;p&gt;XLS file - 15.2MB&lt;/p&gt;

&lt;p&gt;XLS file - 55K&lt;/p&gt;

&lt;p&gt;XLS file - 303K&lt;/p&gt;

&lt;p&gt;PDF file - 217K&lt;/p&gt;

&lt;p&gt;XLS file - 41K&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a “functional genomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ∼16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. &lt;i&gt;Cancer Discovery; 2(2)&lt;/i&gt;; 172–89. &lt;i&gt;© 2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;This article is highlighted in the In This Issue feature, p. 95.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;Genomic analyses are yielding a host of new information on the multiple genetic abnormalities associated with specific types of cancer. A comprehensive description of cancer-associated genetic abnormalities can improve our ability to classify tumors into clinically relevant subgroups and, on occasion, identify mutant genes that drive the cancer phenotype (“drivers”). More often, though, the functional significance of cancer-associated mutations is difficult to discern. Genome-wide pooled short hairpin RNA (shRNA) screens enable global identification of the genes essential for cancer cell survival and proliferation, providing a “functional genomic” map of human cancer to complement genomic studies. Using a lentiviral shRNA library targeting ∼16,000 genes and a newly developed, dynamic scoring approach, we identified essential gene profiles in 72 breast, pancreatic, and ovarian cancer cell lines. Integrating our results with current and future genomic data should facilitate the systematic identification of drivers, unanticipated synthetic lethal relationships, and functional vulnerabilities of these tumor types.&lt;/p&gt;&lt;p&gt;&lt;b&gt;Significance:&lt;/b&gt; This study presents a resource of genome-scale, pooled shRNA screens for 72 breast, pancreatic, and ovarian cancer cell lines that will serve as a functional complement to genomics data, facilitate construction of essential gene profiles, help uncover synthetic lethal relationships, and identify uncharacterized genetic vulnerabilities in these tumor types. &lt;i&gt;Cancer Discovery; 2(2)&lt;/i&gt;; 172–89. &lt;i&gt;© 2011 AACR&lt;/i&gt;.&lt;/p&gt;&lt;p&gt;This article is highlighted in the In This Issue feature, p. 95.&lt;/p&gt;&lt;/div&gt;

Introduction - Epidemiologic studies indicate that air pollution affects lung health in prenatal and early childhood. However, the mechanisms involved remain unknown. Aims - Investigate the influence of gestational exposure to concentrated ambient particles (CAPs) derived from vehicular emissions on the expression of genes involved in lung development. Methods - Dams were continuously exposed to either filtered air (control group) or CAP (600µg/m 3 /day) from gestational day 5 (intrauterine exposure) until 14 days post conception (dpc), using a Harvard Ambient Particle Concentrator. At the end of the exposures, the total RNA was isolated from the lungs of fetuses at 14 dpc (n=5/group), using the RNeasy Mini kit (Qiagen). Gene expression profiles were investigated with AffymetrixGeneChip® Mouse 2.0 ST Arrays . Enrichment analysis was performed by Funcassociate 2.0. Results- Microarray data analysis revealed that 86 genes, related to embryogenic development, were differentially expressed in the fetal lung tissue of exposed group when compared to the control group. Fifty-two upregulated (e.g. Shh) and 34 downregulated (e.g. Hoxa5) genes. Notably, upregulated genes were involved in G-protein coupled receptor activity (GO:0004930, corrected p-value l0.001). Furthermore, downregulated genes were enriched in genes regulated by the polycomb repressor complex, which contains both EZH2 and Suz12 transcription factors. Conclusions - Exposure to particulate air pollution during intrauterine life affects the regulation of important genes involved in lung development. We speculate that these alterations in gene regulation could be related to abnormal lung development and function observed in previous studies.

Additional file 3: Table S2. TAMPfinder datasets for H. sapiens, A. thaliana, and S. cerevisiae.

Additional file 4: Table S3. Direct comparison of Kalbfleisch and TAMPfinder for potential tail-anchored proteins in H. sapiens using UniProt data of single-pass IV membrane proteins.

Additional file 1: Table S1. List of all proteins used for training and test set.

Additional file 10: Table S4. Performance of TAMPfinder program in comparison with Kalbfleisch et al. Both exclusive and common membership was analyzed in terms of GO annotation.