Profiling phagosome proteins identifies PD-L1 as a fungal-binding receptor.

Phagocytosis is the process by which myeloid phagocytes bind to and internalize potentially dangerous microorganisms1. During phagocytosis, innate immune receptors and associated signalling proteins are localized to the maturing phagosome compartment, forming an immune information processing hub brimming with microorganism-sensing features2-8. Here we developed proximity labelling of phagosomal contents (PhagoPL) to identify proteins localizing to phagosomes containing model yeast and bacteria. By comparing the protein composition of phagosomes containing evolutionarily and biochemically distinct microorganisms, we unexpectedly identified programmed death-ligand 1 (PD-L1) as a protein that specifically enriches in phagosomes containing yeast. We found that PD-L1 directly binds to yeast upon processing in phagosomes. By surface display library screening, we identified the ribosomal protein Rpl20b as a fungal protein ligand for PD-L1. Using an auxin-inducible depletion system, we found that detection of Rpl20b by macrophages cross-regulates production of distinct cytokines including interleukin-10 (IL-10) induced by the activation of other innate immune receptors. Thus, this study establishes PhagoPL as a useful approach to quantifying the collection of proteins enriched in phagosomes during host-microorganism interactions, exemplified by identifying PD-L1 as a receptor that binds to fungi.

ONECUT2 acts as a lineage plasticity driver in adenocarcinoma as well as neuroendocrine variants of prostate cancer.

Androgen receptor- (AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 gene targets include the glucocorticoid receptor (GR; NR3C1) and the NE splicing factor SRRM4, which are key drivers of lineage plasticity. Thus, OC2, despite its previously described NEPC driver function, can indirectly activate a portion of the AR cistrome through epigenetic activation of GR. Mechanisms by which OC2 regulates gene expression include promoter binding, enhancement of genome-wide chromatin accessibility, and super-enhancer reprogramming. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC and support enhanced efforts to therapeutically target OC2 as a means of suppressing treatment-resistant disease.

ONECUT2 regulates RANKL-dependent enterocyte and microfold cell differentiation in the small intestine; a multi-omics study.

Microfold (M) cells reside in the intestinal epithelium of Peyer's patches (PP). Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an enterocyte and M cell precursor population. We used our newly developed computational tool SCEPIA to link precursor cell-specific gene expression to transcription factor motif activity in cis-regulatory elements, uncovering high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling axis to support enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating cell fate balance in the PP, and it provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium.

Targeting Key Players of Neuroendocrine Differentiation in Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer (PC) that commonly emerges through a transdifferentiation process from prostate adenocarcinoma and evades conventional therapies. Extensive molecular research has revealed factors that drive lineage plasticity, uncovering novel therapeutic targets to be explored. A diverse array of targeting agents is currently under evaluation in pre-clinical and clinical studies with promising results in suppressing or reversing the neuroendocrine phenotype and inhibiting tumor growth and metastasis. This new knowledge has the potential to contribute to the development of novel therapeutic approaches that may enhance the clinical management and prognosis of this lethal disease. In the present review, we discuss molecular players involved in the neuroendocrine phenotype, and we explore therapeutic strategies that are currently under investigation for NEPC.

Actionable Driver Events in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) stands out as the most aggressive form of lung cancer, characterized by an extremely high proliferation rate and a very poor prognosis, with a 5-year survival rate that falls below 7%. Approximately two-thirds of patients receive their diagnosis when the disease has already reached a metastatic or extensive stage, leaving chemotherapy as the remaining first-line treatment option. Other than the recent advances in immunotherapy, which have shown moderate results, SCLC patients cannot yet benefit from any approved targeted therapy, meaning that this cancer remains treated as a uniform entity, disregarding intra- or inter-tumoral heterogeneity. Continuous efforts and technological improvements have enabled the identification of new potential targets that could be used to implement novel therapeutic strategies. In this review, we provide an overview of the most recent approaches for SCLC treatment, providing an extensive compilation of the targeted therapies that are currently under clinical evaluation and inhibitor molecules with promising results in vitro and in vivo.

BoxCar and shotgun proteomic analyses reveal molecular networks regulated by UBR5 in prostate cancer.

Prostate cancer (PC) is a major health and economic problem in industrialized countries, yet our understanding of the molecular mechanisms of PC progression and drug response remains limited. Accumulating evidence showed that certain E3 ubiquitin ligases such as SIAH2, RNF7, and SPOP play important roles in PC development and progression. However, the roles and mechanisms of other E3s in PC progression remain largely unexplored. Through an integration analysis of clinical genomic and transcriptomic profiles of PC tumors, this study identified UBR5 as a top PC-relevant E3 ubiquitin ligase whose expression levels are strongly associated with PC progression and aggressiveness. BoxCar and shotgun proteomic analyses of control and UBR5-knockdown PC3 cells complementarily identified 75 UBR5-regulated proteins. Bioinformatic analysis suggested that the 75 proteins form four molecular networks centered around FANCD2, PAF1, YY1, and LAMB3 via direct protein-protein interactions. Experimental analyses demonstrated that UBR5 associates with and downregulates two key DNA damage repair proteins (XRCC3 and FANCD2) and confers PC cell sensitivity to olaparib, a PARP inhibitor in clinical use for cancer therapy. This study represents the first application of BoxCar in PC research, provides new insights into the molecular functions of UBR5 in PC, and suggests that PC patients with UBR5-high tumors may potentially benefit from PARP inhibitor treatment.

Antioxidant functions of DHHC3 suppress anti-cancer drug activities.

Ablation of protein acyltransferase DHHC3 selectively enhanced the anti-cancer cell activities of several chemotherapeutic agents, but not kinase inhibitors. To understand why this occurs, we used comparative mass spectrometry-based palmitoyl-proteomic analysis of breast and prostate cancer cell lines, ± DHHC3 ablation, to obtain the first comprehensive lists of candidate protein substrates palmitoylated by DHHC3. Putative substrates included 22-28 antioxidant/redox-regulatory proteins, thus predicting that DHHC3 should have antioxidant functions. Consistent with this, DHHC3 ablation elevated oxidative stress. Furthermore, DHHC3 ablation, together with chemotherapeutic drug treatment, (a) elevated oxidative stress, with a greater than additive effect, and (b) enhanced the anti-growth effects of the chemotherapeutic agents. These results suggest that DHHC3 ablation enhances chemotherapeutic drug potency by disabling the antioxidant protections that contribute to drug resistance. Affirming this concept, DHHC3 ablation synergized with another anti-cancer drug, PARP inhibitor PJ-34, to decrease cell proliferation and increase oxidative stress. Hence, DHHC3 targeting can be a useful strategy for selectively enhancing potency of oxidative stress-inducing anti-cancer drugs. Also, comprehensive identification of DHHC3 substrates provides insight into other DHHC3 functions, relevant to in vivo tumor growth modulation.

Chromosomal instability in untreated primary prostate cancer as an indicator of metastatic potential.

BACKGROUND: Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. METHODS: PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). RESULTS: The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene "metastasis" signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. CONCLUSIONS: Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis.

Protein S-acylation (also called palmitoylation) is a common post-translational modification whose deregulation plays a key role in the pathogenesis of many diseases. Acyl-biotinyl exchange (ABE), a widely used method for the enrichment of S-acylated proteins, has the potential of capturing the entire S-acylproteome in any type of biological sample. Here, we showed that current ABE methods suffer from a high background arising from the coisolation of non-S-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2'-dithiodipyridine prior to the biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free proteomics, 2 895 high-confidence candidate S-acylated proteins (including 1 591 known S-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largest S-acylproteome data set identified in a single study. Immunoblotting analysis confirmed the S-acylation of five known and five novel prostate cancer-related S-acylated proteins in LNCaP cells and suggested that their S-acylation levels were about 0.6-1.8%. In summary, the LB-ABE method largely eliminates the coisolation of non-S-acylated proteins and enables deep S-acylproteomic analysis. It is expected to facilitate a much more comprehensive and accurate quantification of S-acylproteomes than previous ABE methods.

Quantitative proteomic analysis of prostate tissue specimens identifies deregulated protein complexes in primary prostate cancer.

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and a leading cause of mortality among males in developed countries. However, our understanding of the global changes of protein complexes within PCa tissue specimens remains very limited, although it has been well recognized that protein complexes carry out essentially all major processes in living organisms and that their deregulation drives the pathogenesis and progression of various diseases. METHODS: By coupling tandem mass tagging-synchronous precursor selection-mass spectrometry/mass spectrometry/mass spectrometry with differential expression and co-regulation analyses, the present study compared the differences between protein complexes in normal prostate, low-grade PCa, and high-grade PCa tissue specimens. RESULTS: Globally, a large downregulated putative protein-protein interaction (PPI) network was detected in both low-grade and high-grade PCa, yet a large upregulated putative PPI network was only detected in high-grade but not low-grade PCa, compared with normal controls. To identify specific protein complexes that are deregulated in PCa, quantified proteins were mapped to protein complexes in CORUM (v3.0), a high-quality collection of 4274 experimentally verified mammalian protein complexes. Differential expression and gene ontology (GO) enrichment analyses suggested that 13 integrin complexes involved in cell adhesion were significantly downregulated in both low- and high-grade PCa compared with normal prostate, and that four Prothymosin alpha (ProTα) complexes were significantly upregulated in high-grade PCa compared with normal prostate. Moreover, differential co-regulation and GO enrichment analyses indicated that the assembly levels of six protein complexes involved in RNA splicing were significantly increased in low-grade PCa, and those of four subcomplexes of mitochondrial complex I were significantly increased in high-grade PCa, compared with normal prostate. CONCLUSIONS: In summary, to the best of our knowledge, the study represents the first large-scale and quantitative, albeit indirect, comparison of individual protein complexes in human PCa tissue specimens. It may serve as a useful resource for better understanding the deregulation of protein complexes in primary PCa.

Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes.

Since their first description, extracellular vesicles (EVs) have been the topic of avid study in a variety of physiologic contexts and are now thought to play an important role in cancer. The state of knowledge on biogenesis, molecular content and horizontal communication of diverse types of cancer EVs has expanded considerably in recent years. As a consequence, a plethora of information about EV composition and molecular function has emerged, along with the notion that cancer cells rely on these particles to invade tissues and propagate oncogenic signals at distance. The number of in vivo studies, designed to achieve a deeper understanding of the extent to which EV biology can be applied to clinically relevant settings, is rapidly growing. This review summarizes recent studies on cancer-derived EV functions, with an overview about biogenesis and molecular cargo of exosomes, microvesicles and large oncosomes. We also discuss current challenges and emerging technologies that might improve EV detection in various biological systems. Further studies on the functional role of EVs in specific steps of cancer formation and progression will expand our understanding of the diversity of paracrine signaling mechanisms in malignant growth.

Evidence for Feedback Regulation Following Cholesterol Lowering Therapy in a Prostate Cancer Xenograft Model.

BACKGROUND: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts. METHODS: Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 105 LAPC-4 cells. Two weeks post-injection, mice were randomized to control, 11 mg/kg/day simvastatin, 30 mg/kg ezetimibe, or the combination and sacrificed 42 days post-randomization. We used a generalized linear model with the predictor variables of treatment, time, and treatment by time (i.e., interaction term) with tumor volume as the outcome variable. Total serum and tumor cholesterol were measured. Tumoral RNA was extracted and cDNA synthesized from 1 ug of total RNA for quantitative real-time PCR. RESULTS: Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors. CONCLUSIONS: Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate 77:446-457, 2017. © 2016 Wiley Periodicals, Inc.

High-throughput sequencing of two populations of extracellular vesicles provides an mRNA signature that can be detected in the circulation of breast cancer patients.

Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo.

Universal Solid-Phase Reversible Sample-Prep for Concurrent Proteome and N-Glycome Characterization.

We describe a novel solid-phase reversible sample-prep (SRS) platform that enables rapid sample preparation for concurrent proteome and N-glycome characterization for nearly all protein samples. SRS utilizes a uniquely functionalized, silica-based bead that has strong affinity toward proteins with minimal to no affinity for peptides and other small molecules. By leveraging this inherent size difference between proteins and peptides, SRS permits high-capacity binding of proteins, rapid removal of small molecules (detergents, metabolites, salts, peptides, etc.), extensive manipulation including enzymatic and chemical treatments on bead-bound proteins, and easy recovery of N-glycans and peptides. SRS was evaluated in a wide range of samples including glycoproteins, cell lysate, murine tissues, and human urine. SRS was also coupled to a quantitative strategy to investigate the differences between DU145 prostate cancer cells and its DIAPH3-silenced counterpart. Previous studies suggested that DIAPH3 silencing in DU145 induced transition to an amoeboid phenotype that correlated with tumor progression and metastasis. In this pilot study we identified distinct proteomic and N-glycomic alterations between them. A metastasis-associated tyrosine kinase receptor ephrin-type-A receptor (EPHA2) was highly up-regulated in DIAPH3-silenced cells, indicating a possible connection between EPHA2 and DIAPH3. Moreover, distinct alterations in the N-glycome were identified, suggesting cross-links between DIAPH3 and glycosyltransferase networks.

Urinary metabolite profiling combined with computational analysis predicts interstitial cystitis-associated candidate biomarkers.

Interstitial cystitis/painful bladder syndrome (IC) is a chronic syndrome of unknown etiology that presents with bladder pain, urinary frequency, and urgency. The lack of specific biomarkers and a poor understanding of underlying molecular mechanisms present challenges for disease diagnosis and therapy. The goals of this study were to identify noninvasive biomarker candidates for IC from urine specimens and to potentially gain new insight into disease mechanisms using a nuclear magnetic resonance (NMR)-based global metabolomics analysis of urine from female IC patients and controls. Principal component analysis (PCA) suggested that the urinary metabolome of IC and controls was clearly different, with 140 NMR peaks significantly altered in IC patients (FDR < 0.05) compared to that in controls. On the basis of strong correlation scores, fifteen metabolite peaks were nominated as the strongest signature of IC. Among those signals that were higher in the IC group, three peaks were annotated as tyramine, the pain-related neuromodulator. Two peaks were annotated as 2-oxoglutarate. Levels of tyramine and 2-oxoglutarate were significantly elevated in urine specimens of IC subjects. An independent analysis using mass spectrometry also showed significantly increased levels of tyramine and 2-oxoglutarate in IC patients compared to controls. Functional studies showed that 2-oxoglutarate, but not tyramine, retarded growth of normal bladder epithelial cells. These preliminary findings suggest that analysis of urine metabolites has promise in biomarker development in the context of IC.

Assess the expression of ubiquitin specific protease USP2a for bladder cancer diagnosis.

BACKGROUND: Given that a deubiquitinating enzyme, ubiquitin-specific protease 2a (USP2a), regulates ubiquitination, trafficking, and degradation of EGFR, which plays a critical role in bladder cancer, in this study, we aimed to quantify the USP2a gene expression, and to determine the possibility that USP2a can be used for bladder cancer diagnosis. METHODS: Using two independent cohorts (cohort 1, n = 339 in total; cohort 2, n = 140 in total) consisting of human bladder tissues from BC patients and normal controls, we analyzed the gene expression levels of USP2a. A quantitative real-time PCR amplification was performed using a Rotor Gene 6000 instrument to quantify the expression of USP2a mRNA. RESULTS: A comparison of 305 bladder cancers and 34 age-matched controls showed an 81.4% reduction in USP2a expression in bladder cancers as compared to normal bladder tissues (p < 0.001). In the independent cohort consisting of 140 BC tissues and matched adjacent normal bladder tissues, the levels of USP2a in the specimens of BC patients were reduced by 86.9% as compared to matched surrounding normal specimens from the same patients (p < 0.001). Furthermore, there was 36.3% reduction of USP2a gene expression in muscle invasive bladder cancer (MIBC, n = 121), compared to non muscle invasive bladder cancer (NMIBC, n = 184) (p = 0.004). Lastly, USP2a mRNA expression was significantly reduced in higher stages of MIBC patients (p = 0.024), but not in NMIBC patients. CONCLUSIONS: Our findings suggest that USP2a mRNA may be considered as a diagnostic marker candidate for bladder cancer, in particular, to stratify MIBC patients with a more invasive phenotype.

Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancer regulate the tumor microenvironment.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), including gefitinib, are the first-line treatment of choice for nonsmall cell lung cancer patients who harbor activating EGFR mutations, however, acquired resistance to EGFR-TKIs is inevitable. The main objective of this study was to identify informative protein signatures of extracellular vesicles (EV) derived from gefitinib-resistant nonsmall cell lung cancer cells using proteomics analysis. Nano-LC-MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) 664 EV proteins enriched in PC9R cells, which are resistant to gefitinib due to EGFR T790M mutation. Computational analyses suggested components of several signal transduction mechanisms including the AKT (also PKB, protein kinase B)/mTOR (mechanistic target of rapamycin) pathway are overrepresented in EV from PC9R cells. Treatment of recipient cells with EV harvested from PC9R cells increased phosphorylation of signaling molecules, and enhanced proliferation, invasion, and drug resistance to gefitinib-induced apoptosis. Dose- and time-dependent pharmaceutical inhibition of AKT/mTOR pathway overcame drug resistance of PC9R cells and those of H1975 exhibiting EGFR T790M mutation. Our findings provide new insight into an oncogenic EV protein signature regulating tumor microenvironment, and will aid in the development of novel diagnostic strategies for prediction and assessment of gefitinib resistance.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells.

BACKGROUND: Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. RESULTS: Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. CONCLUSIONS: These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

Loss of caveolin-1 in prostate cancer stroma correlates with reduced relapse-free survival and is functionally relevant to tumour progression.

Levels of caveolin-1 (Cav-1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav-1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav-1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav-1 in the stroma correlated with reduced relapse-free survival (p = 0.009), suggesting a role for stromal Cav-1 in inhibiting advanced disease. Silencing of Cav-1 by shRNA in WPMY-1 prostate fibroblasts resulted in up-regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5-fold increase in TGF-ß1 and γ-synuclein (SNCG) gene expression. Moreover, silencing of Cav-1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down-regulation of TGF-ß1 and SNCG, suggesting that loss of Cav-1 in the stroma can influence Akt-mediated signalling in the tumour microenvironment. Cav-1-depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav-1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up-regulation of TGF-ß1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma.

ONECUT2 is a druggable driver of luminal to basal breast cancer plasticity.

PURPOSE: Tumor heterogeneity complicates patient treatment and can be due to transitioning of cancer cells across phenotypic cell states. This process is associated with the acquisition of independence from an oncogenic driver, such as the estrogen receptor (ER) in breast cancer (BC), resulting in tumor progression, therapeutic failure and metastatic spread. The transcription factor ONECUT2 (OC2) has been shown to be a master regulator protein of metastatic castration-resistant prostate cancer (mCRPC) tumors that promotes lineage plasticity to a drug-resistant neuroendocrine (NEPC) phenotype. Here, we investigate the role of OC2 in the dynamic conversion between different molecular subtypes in BC. METHODS: We analyze OC2 expression and clinical significance in BC using public databases and immunohistochemical staining. In vitro, we perform RNA-Seq, RT-qPCR and western-blot after OC2 enforced expression. We also assess cellular effects of OC2 silencing and inhibition with a drug-like small molecule in vitro and in vivo. RESULTS: OC2 is highly expressed in a substantial subset of hormone receptor negative human BC tumors and tamoxifen-resistant models, and is associated with poor clinical outcome, lymph node metastasis and heightened clinical stage. OC2 inhibits ER expression and activity, suppresses a gene expression program associated with luminal differentiation and activates a basal-like state at the gene expression level. We also show that OC2 is required for cell growth and survival in metastatic BC models and that it can be targeted with a small molecule inhibitor providing a novel therapeutic strategy for patients with OC2 active tumors. CONCLUSIONS: The transcription factor OC2 is a driver of BC heterogeneity and a potential drug target in distinct cell states within the breast tumors.