Promote Prostate Cancer Cell Survival Research Articles

LncRNA SNHG4 promotes prostate cancer cell survival and resistance to enzalutamide through a let-7a/RREB1 positive feedback loop and a ceRNA network

BackgroundProstate cancer threatens the health of men over sixty years old, and its incidence ranks first among all urinary tumors among men. Enzalutamide remains the first-line drug for castration-resistant prostate cancer, however, tumors inevitably become resistant to enzalutamide. Hence, it is of great importance to investigate the mechanisms that induce enzalutamide resistance in prostate cancer cells.MethodsBioinformatic analyzing approaches were used to identified the over-expressed genes in prostate cancer tumor tissues from three GEO datasets. qRT-PCR, western blotting and immunochemistry/In situ hybridization staining assays were performed to assess the expression of SNHG4, RRM2, TK1, AURKA, EZH2 and RREB1. Cell cycle was measured by flow cytometry. CCK-8, plate colony formation and EdU assays were performed to assess the cell proliferation. Senescence-associated β-Gal assay was used to detect the cell senescence level. γ-H2AX staining assay was performed to assess the DNA damages of PCa cells. Luciferase reporter assay and RNA immunoprecipitation assay were performed to verify the RNA-RNA interactions. Chromatin immunoprecipitation assay was performed to assess the bindings between protein and genomic DNA.ResultsWe found that RRM2 and NUSAP1 are highly expressed in PCa tumors and significantly correlated with poor clinical outcomes in PCa patients. Bioinformatic analysis as well as experimental validation suggested that SNHG4 regulates RRM2 expression via a let-7 miRNA-mediated ceRNA network. In addition, SNHG4 or RRM2 knockdown significantly induced cell cycle arrest and cell senescence, and inhibited DNA damage repair and cell proliferation, and the effects can be partially reversed by let-7a knockdown or RRM2 reoverexpression. In vitro and in vivo experiments showed that SNHG4 overexpression markedly enhanced cell resistance to enzalutamide. RREB1 was demonstrated to transcriptionally regulate SNHG4, and RREB1 was also validated to be a target of let-7a and thereby regulated by the SNHG4/let-7a feedback loop.ConclusionOur study uncovered a novel molecular mechanism of lncRNA SNHG4 in driving prostate cancer progression and enzalutamide resistance, revealing the critical roles and therapeutic potential of RREB1, SNHG4, RRM2 and let-7 miRNAs in anticancer therapy.

Biomarker analysis of phase (Ph) IB trial of radium-223 (Rad) and niraparib (Nira) in patients (Pts) with metastatic castrate-resistant prostate cancer (mCRPC) (NiraRad).

5036 Background: Rad is an alpha particle emitter that causes DNA double strand breaks and has been FDA-approved for use in mCRPC pts with bone metastases. PARP-1 activity critically supports androgen receptor (AR) activity in mCRPC and potentiates AR-dependent DNA damage response pathways that promote prostate cancer cell survival. Nira is a potent and selective PARP-1/2 inhibitor that has shown single agent clinical activity in mCRPC. We previously reported the safety of targeting the PARP-1/AR axis with Nira in combination with Rad. Herein we describe the results of an exploratory biomarker analysis. Methods: The primary objective of NiraRad is to determine the optimum Ph II dose of Nira plus Rad (55 kBq/kg of body weight IV every 4 weeks (wks) x 6) in pts with and without prior chemotherapy (docetaxel). Pts were enrolled to one of three dose levels of Nira (100, 200, or 300 mg PO daily). All cohorts were combined for exploratory biomarker analysis using Nanostring PanCancer Driver and Immune Pathways panels and the nSolver Advanced analysis module was performed on blood obtained from 23 pts at baseline, cycle (C) 1 day (D) 15, and C3D15. A favorable response was defined as any PSA reduction at week 12 or treatment (tx) duration > 18 wks, the median time on tx in the cohort of pts analyzed. A threshold of > 2 fold (X) differentially expressed genes was used. Results: Of the 23 pts with biomarker data, 7 (30%) experienced PSA reductions and 11 (48%) received tx for > 18 wks, 6 of which also had PSA reductions. Exploratory analysis revealed that the PI3K/Ras, MAPK, and transcriptional misregulation pathways were differentially regulated in pts who had favorable responses. The top downregulated gene, PAX5, which has been shown to promote prostate cancer growth, was decreased at C1D15 (2.7X, p < 0.01) and C3D15 (4.8X, p < 0.001) in pts with tx duration > 18 wks and at baseline in pts who had PSA reductions (3.1X, p < 0.05). Immune pathways analysis suggested downregulation of immunosuppressive B-cell (plasma cell) and upregulation of NK and T cell pathways in pts with tx duration > 18 wks. Conclusions: Previously Nira and Rad have been shown to have acceptable tolerability in mCRPC pts. This exploratory analysis suggests potential response biomarkers that warrant further investigation. Managed by: the Prostate Cancer Clinical Trials Consortium; Funded by: Janssen Pharmaceuticals and Bayer Healthcare Pharmaceuticals, Inc. Clinical trial information: NCT03076203.

LSD1 Promotes Prostate Cancer Cell Survival by Destabilizing FBXW7 at Post-Translational Level

Prostate cancer (PCa) is the most common cancer in men and the fifth leading cause of cancer death worldwide. Unfortunately, castration-resistant prostate cancer (CRPCa) is incurable with surgical treat and prone to drug resistance. Therefore, it is of great importance to find a new target for treatment. LSD1 is up-regulated in PCa and related with prognosis. The high-expression LSD1 has been shown to be a potential target for treatment and is widely studied for its demethylase-activity. However, its demethylation-independent function remains to be elusive in PCa. Recent study shows that LSD1 can destabilize cancer suppressor protein FBXW7 without demethylation-function. Hence, we hope to investigate the impact of non-canonical function of LSD1 on PCa cell survival. We over-expressed FBXW7 gene through plasmid vector in LNCaP and PC3 cell lines and the result shows that up-regulated FBXW7 can suppress the viability of PC cell through suppressing oncoproteins, such as c-MYC, NOTCH-1. After FBXW7 function experiment on PC cell, we knock-down LSD1 gene in the same kinds of cell lines. In western blot assay, we detected that down-regulation of LSD1 will cause the increasing of FBXW7 protein level and decreasing of its targeting oncoproteins. And mRNA level of FBXW7 did not change significantly after LSD1 knock-down, which means LSD1 may destabilize FBXW7 by protein-protein interactions. Moreover, exogenous wild type LSD1 and catalytically deficient mutant K661A both can abrogate previous effect of LSD1 knock-down. Consequently, LSD1 may promote PC cell survival by destabilizing FBXW7 without its demethylase-activity. Next, we compared two kinds inhibitors, and found that SP-2509 (Allosteric inhibitor) treatment suppress the cancer cell survival by blocking the LSD1–FBXW7 interaction, which is an effect that GSK-2879552 (catalytic inhibitor) cannot achieve. This work revealed a pivotal function of LSD1 in PCa, and indicated a new direction of LSD1 inhibitor research for PCa treatment.

KIFC1 overexpression promotes prostate cancer cell survival and proliferation in vitro by clustering of amplified centrosomes via interaction with Centrin 2

Mitotic kinesin KIFC1 plays critical roles in mitosis by regulating the spindle length, pole formation, and known for clustering extra centrosomes in cancer cells. Centrosome clustering is associated with the survival of cancer cells, but this phenomenon remains obscure in prostate cancer (PCa). The present study demonstrated that PCa cells showed centrosome amplification and clustering during interphase and mitosis, respectively. KIFC1 is highly expressed in PCa cells and tumor tissues of prostatic adenocarcinoma (PAC) patients. Up-regulation of KIFC1 facilitated the PCa cell survival in vitro by ensuring bipolar mitosis through clustering the multiple centrosomes, suggesting centrosome clustering could be a leading cause of prostate carcinogenesis. Conversely, the silencing of KIFC1 resulted in normal centrosome number or multipolar mitosis by inhibiting the clustering of amplified centrosomes in PCa cells. Besides, knockdown of KIFC1 by RNAi in PCa cells reduced cancer cell survival, and proliferation. KIFC1 interacted with centrosome structural protein Centrin 2 in clustering of amplified centrosomes in PCa cells to ensure the bipolar mitotic spindle formation. Knockdown of Centrin 2 in PCa cells inhibited the centrosome amplification and clustering. Moreover, up-regulated KIFC1 promotes PCa cell proliferation via progression of cell cycle possibly through aberrant activation of cyclin dependent kinase 1(Cdk1). Therefore, KIFC1 can be a prognostic marker and therapeutic target of PCa for inhibiting the cancer cell proliferation.

TNFAIP8 drives metabolic reprogramming to promote prostate cancer cell proliferation

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a member of TIPE/TNFAIP8 family, has been involved in the development and progression of various human cancers. We hypothesized that TNFAIP8 promotes prostate cancer (PCa) progression via regulation of oxidative phosphorylation (OXPHOS) and glycolysis. Ectopic expression of TNFAIP8 increased PCa cell proliferation/migration/spheroid formation by enhancing cell metabolic activities. Mechanistically, TNFAIP8 activated the PI3K-AKT pathway and up-regulated PCa cell survival. TNFAIP8 was also found to regulate the expression of glucose metabolizing enzymes, enhancing glucose consumption, and endogenous ATP production. Treatment with a glycolysis inhibitor, 2-deoxyglucose (2-DG), reduced TNFAIP8 mediated glucose consumption, ATP production, spheroid formation, and PCa cell migration. By maintaining mitochondrial membrane potential, TNFAIP8 increased OXPHOS and glycolysis. Moreover, TNFAIP8 modulates the production of glycolytic metabolites in PCa cells. Collectively, our data suggest that TNFAIP8 exerts its oncogenic effects by enhancing glucose metabolism and by facilitating metabolic reprogramming in PCa cells. Therefore, TNFAIP8 may be a biomarker associated with prostate cancer and indicate a potential therapeutic target.

The ERG-Regulated LINC00920 Promotes Prostate Cancer Cell Survival via the 14-3-3ε-FOXO Pathway.

Numerous noncoding transcripts have been reported to correlate with cancer development and progression. Nevertheless, there remains a paucity of long noncoding RNAs (lncRNA) with well-elucidated functional roles. Here, we leverage the International Cancer Genome Consortium-Early Onset Prostate Cancer transcriptome and identify the previously uncharacterized lncRNA LINC00920 to be upregulated in prostate tumors. Phenotypic characterization of LINC00920 revealed its positive impact on cellular proliferation, colony formation, and migration. We demonstrate that LINC00920 transcription is directly activated by ERG, an oncogenic transcription factor overexpressed in 50% of prostate cancers. Chromatin isolation by RNA purification-mass spectrometry revealed the interaction of LINC00920 with the 14-3-3ε protein, leading to enhanced sequestration of tumor suppressive FOXO1. Altogether, our results provide a rationale on how ERG overexpression, partly by driving LINC00920 transcription, could confer survival advantage to prostate cancer cells and potentially prime PTEN-intact prostate cells for cellular transformation through FOXO inactivation. IMPLICATIONS: The study describes a novel lncRNA-mediated mechanism of regulating the FOXO signaling pathway and provides additional insight into the role of ERG in prostate cancer cells.

Human DECR1 is an androgen-repressed survival factor that regulates PUFA oxidation to protect prostate tumor cells from ferroptosis.

Fatty acid β-oxidation (FAO) is the main bioenergetic pathway in human prostate cancer (PCa) and a promising novel therapeutic vulnerability. Here we demonstrate therapeutic efficacy of targeting FAO in clinical prostate tumors cultured ex vivo, and identify DECR1, encoding the rate-limiting enzyme for oxidation of polyunsaturated fatty acids (PUFAs), as robustly overexpressed in PCa tissues and associated with shorter relapse-free survival. DECR1 is a negatively-regulated androgen receptor (AR) target gene and, therefore, may promote PCa cell survival and resistance to AR targeting therapeutics. DECR1 knockdown selectively inhibited β-oxidation of PUFAs, inhibited proliferation and migration of PCa cells, including treatment resistant lines, and suppressed tumor cell proliferation and metastasis in mouse xenograft models. Mechanistically, targeting of DECR1 caused cellular accumulation of PUFAs, enhanced mitochondrial oxidative stress and lipid peroxidation, and induced ferroptosis. These findings implicate PUFA oxidation via DECR1 as an unexplored facet of FAO that promotes survival of PCa cells.

Abstract 5749: Interleukin-1 (IL-1) induces a treatment-resistant gene expression pattern in prostate cancer (PCa)

Abstract Inflammation promotes the development and progression of cancer, where inflammatory cytokines secreted by infiltrating immune cells and tumor cell autocrine signaling are usurped to promote tumor cell survival and disease progression. The interleukin-1 (IL-1) inflammatory cytokine is elevated in prostate cancer (PCa) patient tissue and serum, where it promotes PCa metastasis and bone colonization. Performing in vitro analysis on PCa cell lines and RNA sequencing, we found that IL-1 concomitantly upregulates many pro-survival and lineage proteins, and represses the hormone receptor, androgen receptor (AR). Despite AR loss, a subpopulation of PCa cells remain viable. Our observation could reflect a role for IL-1 in the de novo accumulation of AR- PCa subpopulations. Indeed, AR- PCa subpopulations, such as stem and neuroendocrine cells, are implicated in PCa resistance to AR-targeted therapies. Thus, we hypothesize that IL-1 may reprogram AR+ PCa cells into treatment-resistant AR- PCa cells that survive hormone receptor repression through the upregulation of pro-survival pathways. To begin to address this hypothesis, we performed RNA sequencing and pathway analysis to identify an IL-1-conferred expression pattern in the LNCaP AR+ PCa cell line that mimics the PC3 AR- PCa cell line. We identified canonical inflammatory and immune response pathways, including NFκB, IL-8, and iNOS signaling cascades, likely facilitating PCa cell survival in an inflammatory tumor microenvironment. We also identified novel IL-1-regulated proteins that are known to promote PCa cell survival, such as SQSTM1/p62, SOX9, and ELF3. Experiments are underway to determine the functional significance of our IL-1-conferred gene expression profile in cell line and PDX models. Our investigation will provide insight into the mechanistic function of IL-1 in PCa resistance to AR-targeted therapies Citation Format: Shayna Elizabeth Thomas-Jardin, Mohammed Kanchwala, Joan Jacob, Rachel Meade, Nagham Gahnim, Nikki Delk. Interleukin-1 (IL-1) induces a treatment-resistant gene expression pattern in prostate cancer (PCa) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5749.

TNFAIP8 promotes prostate cancer cell survival by inducing autophagy.

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is a TNF-α inducible anti-apoptotic protein with multiple roles in tumor growth and survival. Mechanisms of cell survival by TNFAIP8 remain elusive. We investigated the role of TNFAIP8 in the regulation of the cell cycle, autophagy, cell survival and neuroendocrine differentiation in prostate cancer cells. We showed that TNFAIP8 dysregulates cell-cycle-related proteins, in PC3 cells. Oncogenic cell survival, drug resistance and dysregulation of cell cycle-related proteins are often associated with autophagy. We demonstrated that TNFAIP8 induces autophagy by increasing expression of autophagy effectors such as LC3β I/II, Beclin1, 4EBP1, p62, and SIRT1. We also demonstrated that TNFAIP8 interacts with autophagy-related protein 3 (ATG3). TNFα treatment increased the expression of TNFAIP8, which was associated with increased autophagy and decreased apoptosis. We also observed an increase in expression of neuroendocrine differentiation markers, synaptophysin and chromogranin A, and drug resistance to anticancer drugs, docetaxel and doxorubicin, in cells transfected with TNFAIP8. Collectively, our findings reveal that by the creation of cellular autophagy events, TNFAIP8 promotes cell survival and drug resistance in prostate cancer cells.

Loss of scinderin decreased expression of epidermal growth factor receptor and promoted apoptosis of castration-resistant prostate cancer cells.

Most patients with prostate cancer will eventually develop the castration‐resistant form characterised by metastasis. Cytoskeleton constituents, including F‐actin, play important roles in maintaining epithelial integrity and their disruption is a major cause of cancer progression. We previously showed that scinderin (SCIN), an important regulator of F‐actin organisation, is highly expressed in poorly differentiated cancer tissues. This study aimed to explore the mechanism of its regulation of cell proliferation. We discovered that SCIN knockdown significantly downregulated epidermal growth factor receptor (EGFR) protein expression, and inhibited epidermal growth factor (EGF)‐mediated cell proliferation and activation of the downstream mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) signalling pathway. Silencing of SCIN promoted apoptosis in two cell lines (PC‐3 and DU145), inhibited B‐cell lymphoma‐extra‐large (Bcl‐xl) expression and activated caspase signalling. Furthermore, in vivo studies showed that SCIN deletion slowed tumour growth and decreased EGFR expression. Thus, we conclude that SCIN promotes prostate cancer cell survival by stabilising EGFR and MEK/ERK signalling.

Abstract 2342: TNFAIP8 promotes prostate cancer cell survival by modulating autophagy

Abstract TNF-α inducible protein (TNFAIP8) is an antiapoptotic protein with roles in tumor cell growth and survival. Mechanisms of cell survival by TNFAIP8 remains elusive. In the current study, we investigated the role and molecular mechanism related to TNFAIP8 in the modulation of cell/cycle, autophagy and drug resistance/cell survival in prostate cancer cells. Microarray data from PC3 prostate cancer cells ectopically expressing TNFAIP8 demonstrated modulation of cell cycle related genes such as CDKs, CDCs and PCNA. Immunoblotting data from overexpression of TNFAIP8 in PC3 cells shows upregulation of cell cycle proteins cyclin A, cyclin B1, Myt1 and Chk1. However, no change in cell cycle was found. Phosphorylation of Histone-S10, CDC2-Tyr15 and Wee1-S642] was also observed. Autophagy plays an important role in tumor cell survival. We evaluated the effects of TNFAIP8 in modulating autophagy. Overexpression of TNFAIP8 leads to induction of autophagy. TNFAIP8 positively modulates the expression/stabilization of autophagy markers and effectors such as LC3β I/II, Beclin1, 4EBP1, oncogene p62 and SIRT1. Knockdown of TNFAIP8 inhibits autophagy induced by nutrient starvation in PC3 prostate and MCF7 breast cancer cells. We also evaluated cell growth and survival in PC3 cells. We demonstrate that TNFAIP8 promotes cell growth and proliferation in PC3, LNCaP and C4-2 prostate cancer cells. Furthermore, we have found that TNFAIP8 increase resistance against anticancer drugs docetaxel and doxorubicin. These data collectively suggest that by the creation of cellular autophagy events TNFAIP8 promotes cell survival and drug resistance in prostate cancer cells. Citation Format: Suresh Niture, Malathi Ramalinga, Habib Kedir, Deepak Kumar. TNFAIP8 promotes prostate cancer cell survival by modulating autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2342. doi:10.1158/1538-7445.AM2017-2342

Lipid degradation promotes prostate cancer cell survival.

Prostate cancer is the most common male cancer and androgen receptor (AR) is the major driver of the disease. Here we show that Enoyl-CoA delta isomerase 2 (ECI2) is a novel AR-target that promotes prostate cancer cell survival. Increased ECI2 expression predicts mortality in prostate cancer patients (p = 0.0086). ECI2 encodes for an enzyme involved in lipid metabolism, and we use multiple metabolite profiling platforms and RNA-seq to show that inhibition of ECI2 expression leads to decreased glucose utilization, accumulation of fatty acids and down-regulation of cell cycle related genes. In normal cells, decrease in fatty acid degradation is compensated by increased consumption of glucose, and here we demonstrate that prostate cancer cells are not able to respond to decreased fatty acid degradation. Instead, prostate cancer cells activate incomplete autophagy, which is followed by activation of the cell death response. Finally, we identified a clinically approved compound, perhexiline, which inhibits fatty acid degradation, and replicates the major findings for ECI2 knockdown. This work shows that prostate cancer cells require lipid degradation for survival and identifies a small molecule inhibitor with therapeutic potential.

GSTP1 Loss results in accumulation of oxidative DNA base damage and promotes prostate cancer cell survival following exposure to protracted oxidative stress.

Epigenetic silencing of glutathione S-transferase π (GSTP1) is a hallmark of transformation from normal prostatic epithelium to adenocarcinoma of the prostate. The functional significance of this loss is incompletely understood. The present study explores the effects of restored GSTP1 expression on glutathione levels, accumulation of oxidative DNA damage, and prostate cancer cell survival following oxidative stress induced by protracted, low dose rate ionizing radiation (LDR). GSTP1 protein expression was stably restored in LNCaP prostate cancer cells. The effect of GSTP1 restoration on protracted LDR-induced oxidative DNA damage was measured by GC-MS quantitation of modified bases. Reduced and oxidized glutathione levels were measured in control and GSTP1 expressing populations. Clonogenic survival studies of GSTP1- transfected LNCaP cells after exposure to protracted LDR were performed. Global gene expression profiling and pathway analysis were performed. GSTP1 expressing cells accumulated less oxidized DNA base damage and exhibited decreased survival compared to control LNCaP-Neo cells following oxidative injury induced by protracted LDR. Restoration of GSTP1 expression resulted in changes in modified glutathione levels that correlated with GSTP1 protein levels in response to protracted LDR-induced oxidative stress. Survival differences were not attributable to depletion of cellular glutathione stores. Gene expression profiling and pathway analysis following GSTP1 restoration suggests this protein plays a key role in regulating prostate cancer cell survival. The ubiquitous epigenetic silencing of GSTP1 in prostate cancer results in enhanced survival and accumulation of potentially promutagenic DNA adducts following exposure of cells to protracted oxidative injury suggesting a protective, anti-neoplastic function of GSTP1. The present work provides mechanistic backing to the tumor suppressor function of GSTP1 and its role in prostate carcinogenesis.

OXER1, a G protein-coupled oxoeicosatetraenoid receptor, mediates the survival-promoting effects of arachidonate 5-lipoxygenase in prostate cancer cells

Inhibition of 5-Lox induces apoptosis in prostate cancer cells by inactivating PKCε which is prevented by 5-oxoETE, and activators of PKCε prevent 5-Lox inhibition-induced apoptosis, suggesting that 5-Lox metabolites exert survival signaling via PKCε. However, mechanisms by which 5-Lox metabolites activate PKCε are not understood yet. We found that prostate cancer cells express high levels of OXER1, a G protein-coupled 5-oxoETE receptor, which delivers signal by generating diacyl-glycerol through phospholipase C-beta. Interestingly, we found that U73122, an inhibitor of PLC-beta, interrupts the apoptosis-preventing effect of 5-oxoETE, and exogenous diacyl-glycerol effectively prevents 5-Lox inhibition-induced apoptosis, suggesting that 5-oxoETE signals via OXER1 to promote prostate cancer cell survival.

Abstract 3131: Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation.

Abstract Prostate cancer is the most common cancer in men in the United States and the second leading cause of cancer death. The androgen receptor (AR) is the central signaling pathway in prostate cancer. Because of this, androgen deprivation therapy (ADT), which involves reducing levels of androgen ligands or interfering with binding of ligands to AR, has been the major therapeutic focus for the past 70 years. However, in many patients there is no benefit from ADT, and all prostate cancers eventually progress. At progression on ADT, the AR is ubiquitously expressed. For this reason, we contend that AR-dependent mechanisms are critical for prostate cancer progression after ADT. Indeed, recent work demonstrates that the AR can function independently of ligands to promote prostate cancer cell survival. However, specific ligand-independent AR target genes that account for this effect were not well-characterized. We show here that c-Myc, which is a key mediator of ligand-independent prostate cancer progression, is a key ligand-independent AR target gene. Using microarray analysis, we found that c-Myc and AR expression levels strongly correlated with each other in tumors from patients with castration-resistant prostate cancer (CRPC) progressing despite ADT. We confirmed that AR directly regulates c-Myc transcription in a ligand-independent manner, that AR and c-Myc suppression reduces ligand-independent prostate cancer cell growth, and that ectopic expression of c-Myc attenuates the anti-tumor activity of AR suppression. Importantly, treatment with the bromodomain inhibitor JQ1 suppressed c-Myc function and suppressed ligand-independent prostate cancer cell survival. Our results define a new link between two critical proteins in prostate cancer - AR and c-Myc - and demonstrate the potential of AR and c-Myc-directed therapies to improve prostate cancer control. Citation Format: Lina Gao, Jacob Schwartzman, Angela Gibbs, Richard Kleinschmidt, Beth Wilmot, Daniel Bottomly, Ilsa Coleman, Peter Nelson, Shannon McWeeney, Joshi J. Alumkal. Androgen receptor promotes ligand-independent prostate cancer progression through c-Myc upregulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3131. doi:10.1158/1538-7445.AM2013-3131

Neuroendocrine‐derived peptides promote prostate cancer cell survival through activation of IGF‐1R signaling

Neuroendocrine (NE) cells promote the progression of prostate cancer to a castration-resistant state through the production of paracrine growth factors. We have demonstrated this principle using in vitro and in vivo proliferative endpoints; however, the contributions of NE-derived pro-survival factors and anti-apoptosis to this phenomenon have not been thoroughly investigated. Here, we utilized conditioned-medium (CM) from LNCaP cells, engineered to undergo NE differentiation, and examined its effects on PC3 and LNCaP cell survival. Statistically significant changes in clonogenic survival, Annexin V staining, PARP cleavage and trypan blue positivity of approximately twofold were observed in the presence of NE-derived CM relative to control-CM for both LNCaP and PC3 cells. These changes were partially abrogated by antagonists of the neuropeptides neurotensin, bombesin, and PTHrP. Selective inhibitors of IGF-1R, EGFR or Src caused significant and nearly complete blockade of prostate cancer cell survival due to NE secretions. Similar increases in cell survival were observed for LNCaP or PC3 cells treated with NE-derived medium in the presence of docetaxel. Increased phosphorylation of IGF-1R, following treatment with NE-derived medium, was accompanied by decreased protein tyrosine phosphatase, receptor type F (PTPRF) mRNA, and protein levels. Overexpression of PTPRF decreased cell survival, the amplitude and duration of IGF-1R phosphorylation, and enhanced PARP cleavage in the presence of NE-derived medium. These data support the hypothesis that NE-derived factors act upon prostate cancer cells to stimulate pro-survival signaling and describe a novel mechanism of cross-talk between NE-derived factors and IGF-1R, mediated in part by PTPRF.

Abstract 1241: In vivo analysis of the role of Lyn tyrosine kinase in castrate-resistant LNCaP xenograft tumor growth

Abstract Introduction: It is proposed that advanced Prostate Cancer (PCa) tumors develop mechanisms, which re-activate the androgen receptor (AR) axis via oncogenic pathways, in which tyrosine kinases play a crucial role. Genetic manipulation of several tyrosine kinases in vivo revealed that only a knockout of Lyn tyrosine Kinase, compromised prostate gland development suggesting that Lyn plays a critical role in the regulation of AR in normal prostate development. Our results demonstrated that Lyn kinase enhances AR transcriptional activity and promotes prostate cancer cell survival. Thus, we hypothesized that Lyn kinase overexpression accelerates castrate-resistant LNCaP tumor growth and serum PSA relapse to pre-castration levels. Method: LNCaP cells stably overexpressing Lyn kinase (LNCaPLyn) or Empty vector (LNCapEmpty) were generated using clonal selection. 1 × 106 LNCaPLyn and LNCaPEmpty were injected into the male nude mice. Serum PSA level and tumor volumes were followed weekly and mice were castrated when serum PSA values reached 50ng/ml. Results: Interestingly, a higher rate of tumor growth and PSA relapse was observed after casteration in mice bearing the LNCaPLyn compare to the LNCapEmpty-bearing mice. Total protein was extracted from the xenograft tumors and levels of AR, PSA, FKBP52, AKT, pAKT were analyzed. Results of Protein analysis further confirmed the role of Lyn kinase in prostate cancer cell survival and progression to castrate resistant stage. Conclusion: Together, these results suggest that Lyn plays a key role in CRPC development and that inhibiting Lyn expression could be considered as a potential therapeutic target for CRPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1241. doi:1538-7445.AM2012-1241

Human periprostatic adipose tissue promotes prostate cancer aggressiveness in vitro

BackgroundObesity is associated with prostate cancer aggressiveness and mortality. The contribution of periprostatic adipose tissue, which is often infiltrated by malignant cells, to cancer progression is largely unknown. Thus, this study aimed to determine if periprostatic adipose tissue is linked with aggressive tumor biology in prostate cancer.MethodsSupernatants of whole adipose tissue (explants) or stromal vascular fraction (SVF) from paired fat samples of periprostatic (PP) and pre-peritoneal visceral (VIS) anatomic origin from different donors were prepared and analyzed for matrix metalloproteinases (MMPs) 2 and 9 activity. The effects of those conditioned media (CM) on growth and migration of hormone-refractory (PC-3) and hormone-sensitive (LNCaP) prostate cancer cells were measured.ResultsWe show here that PP adipose tissue of overweight men has higher MMP9 activity in comparison with normal subjects. The observed increased activities of both MMP2 and MMP9 in PP whole adipose tissue explants, likely reveal the contribution of adipocytes plus stromal-vascular fraction (SVF) as opposed to SVF alone. MMP2 activity was higher for PP when compared to VIS adipose tissue. When PC-3 cells were stimulated with CM from PP adipose tissue explants, increased proliferative and migratory capacities were observed, but not in the presence of SVF. Conversely, when LNCaP cells were stimulated with PP explants CM, we found enhanced motility despite the inhibition of proliferation, whereas CM derived from SVF increased both cell proliferation and motility. Explants culture and using adipose tissue of PP origin are most effective in promoting proliferation and migration of PC-3 cells, as respectively compared with SVF culture and using adipose tissue of VIS origin. In LNCaP cells, while explants CM cause increased migration compared to SVF, the use of PP adipose tissue to generate CM result in the increase of both cellular proliferation and migration.ConclusionsOur findings suggest that the PP depot has the potential to modulate extra-prostatic tumor cells' microenvironment through increased MMPs activity and to promote prostate cancer cell survival and migration. Adipocyte-derived factors likely have a relevant proliferative and motile role.

CaM kinase control of AKT and LNCaP cell survival

AKT and its substrate BAD have been shown to promote prostate cancer cell survival. Agonists, such as carbachol, and hormones that increase intracellular calcium concentration can activate AKT leading to cancer cell survival. The LNCaP prostate cancer cells express the carbachol-sensitive M(3) -subtype of G protein-coupled receptors that cause increases in intracellular calcium and activate the family of Ca(2+) /calmodulin-dependent protein kinases (CaM Ks). One type of CaM Kinase, CaM Kinase Kinase (CaM KK), phosphorylates several substrates including AKT on threonine 308. AKT phosphorylation and activation enhances cell survival through phosphorylation of BAD protein and the subsequent blockade of caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT and BAD in LNCaP prostate cancer cells and evaluate whether CaM KK may be mediating carbachol's activation of AKT and cell survival. Our results suggest that carbachol treatment of LNCaP cells promoted cell survival through CaM KK and its phosphorylation of AKT. The bacterial toxin anisomycin triggered caspase-3 activation in LNCaP cells that was blocked by carbachol in a CaM KK- and AKT-dependent manner. AKT and BAD phosphorylation were blocked by the selective CaM KK inhibitor, STO-609, as well as siRNA directed against CaM KK. BAD phosphorylation was also blocked by treating cells with the AKT inhibitor, AKT-X, as well as siRNA to AKT. Additionally, epinephrine promoted LNCaP cell survival through activation of AKT that was insensitive to STO-609. Taken together these data suggest a survival role for CaM KK operating through AKT and BAD in LNCaP prostate cancer cells.

Integration of Regulatory Networks by NKX3-1 Promotes Androgen-Dependent Prostate Cancer Survival

The NKX3-1 gene is a homeobox gene required for prostate tumor progression, but how it functions is unclear. Here, using chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) we showed that NKX3-1 colocalizes with the androgen receptor (AR) across the prostate cancer genome. We uncovered two distinct mechanisms by which NKX3-1 controls the AR transcriptional network in prostate cancer. First, NKX3-1 and AR directly regulate each other in a feed-forward regulatory loop. Second, NKX3-1 collaborates with AR and FoxA1 to mediate genes in advanced and recurrent prostate carcinoma. NKX3-1- and AR-coregulated genes include those found in the "protein trafficking" process, which integrates oncogenic signaling pathways. Moreover, we demonstrate that NKX3-1, AR, and FoxA1 promote prostate cancer cell survival by directly upregulating RAB3B, a member of the RAB GTPase family. Finally, we show that RAB3B is overexpressed in prostate cancer patients, suggesting that RAB3B together with AR, FoxA1, and NKX3-1 are important regulators of prostate cancer progression. Collectively, our work highlights a novel hierarchical transcriptional regulatory network between NKX3-1, AR, and the RAB GTPase signaling pathway that is critical for the genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.