PTEN-deficient Prostate Cancer Research Articles

Abstract 4699: Preclinical activity of the AKT inhibitor AZD5363 in PTEN-deficient mouse models of prostate cancer

Abstract Increased phosphoinositide 3-kinase (PI3K) pathway activity is commonly observed in human prostate cancer and constitutes a potential target for therapeutic intervention. AZD5363 is a potent pan-AKT kinase inhibitor that is currently being investigated in phase 1 and 2 clinical trials in solid cancers including prostate. AZD5363 has demonstrated strong anti-tumor activity against human prostate cancer cells in vitro and in vivo. Genetically engineered mouse models mimic the genetic and biological evolution of human prostate cancer and address deficiencies encountered with xenograft models. In the present study, we examined in vivo target validation, pharmacodynamics, and antitumor responses of AZD5363 monotherapy in a PTEN-deficient mouse model of prostate cancer, and show the survival benefit in an advanced prostate cancer model driven by PTEN/P53 inactivation. Downstream targets of AKT were inhibited in a dose-dependent manner and activity of AZD5363 at was observed up to 8 hours after a single dose of 100 mg/kg. Tumor cell proliferation and the induction of apoptosis paralleled the activity of AZD5363. Compared to vehicle treated mice, chronic administration of AZD5363 at 100 mg/kg B.I.D. 5 times per week for 4 weeks inhibited growth of castration-naïve and castration-resistant prostate cancers by 15.9% ±4.3 and 20.2% ±4.4, respectively. Inhibition of S6 phosphorylation was effectively maintained the after chronic administration of AZD5363 and was accompanied by decreased proliferation rates and increase in apoptosis. Furthermore, in a long-term survival study of PTEN/P53 double knockout mice, treatment with AZD5363 significantly improved survival rates from 24 to 37 days, P<0.011. In conclusion, our findings indicate that AZD5363 is effective against autochthonous models of mouse prostate cancer and support further investigation of the AKT inhibitor for the treatment of PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yuji Hatanaka, Yurie Kura, Emiko Fukushima, Naomi Ando, Barry R. Davies, Yutaka Yamamoto, Takashi Oki, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Preclinical activity of the AKT inhibitor AZD5363 in PTEN-deficient mouse models of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2015-4699

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19ARF as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF–Mdm2–p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14ARF expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Identification of Androgen Receptor Splice Variants in the Pten Deficient Murine Prostate Cancer Model.

Androgen receptor (AR) variants are associated with resistance to anti androgen therapy both in human prostate cancer cell lines and clinical samples. These observations support the hypothesis that AR isoform accumulation is a consequence of selective therapeutic pressure on the full length AR. The Pten deficient prostate cancer model proceeds with well-defined kinetics including progression to castration resistant prostate cancer (CRPC). While surgical castration and enzalutamide treatments yield an initial therapeutic response, Pten-/-epithelia continue to proliferate yielding locally invasive primary tumor pathology. That most epithelium remains AR positive, but ligand independent, suggests the presence of oncogenic AR variants. To address this hypothesis, we have used a panel of recently described Pten-/- tumor cell lines derived from both from hormone intact (E4, E8) and castrated Pten mutants (cE1, cE2) followed by RACE PCR to identify and characterize three novel truncated, amino terminus containing AR variants (mAR-Va, b, c). Variants appear not only conserved throughout progression but are correlated with nearly complete loss of full length AR (AR-FL) at castrate androgen levels. The overexpression of variants leads to enhanced transcriptional activity of AR while knock down studies show reduced transcriptional output. Collectively, the identification of truncated AR variants in the conditional PTEN deletion model supports a role for maintaining the CRPC phenotype and provides further therapeutic applications of this preclinical model.

KLF5 inhibits angiogenesis in PTEN-deficient prostate cancer by attenuating AKT activation and subsequent HIF1α accumulation.

BackgroundKLF5 is a basic transcriptional factor that regulates multiple physiopathological processes. Our recent study showed that deletion of Klf5 in mouse prostate promotes tumorigenesis initiated by the deletion of Pten. While molecular characterization of Klf5-null tumors suggested that angiogenesis was partially responsible for tumor promotion, the precise function and mechanism of KLF5 deletion in prostate tumor angiogenesis remain unclear.ResultsApplying histological staining to Pten-null mouse prostates, we observed that deletion of Klf5 significantly increased the number of microvessels, accompanied by the upregulation of multiple angiogenesis-related genes based on microarray analysis with MetaCore software. In human umbilical vein endothelial cells (HuVECs), tube formation and migration, both of which are indicators of angiogenic activities, were decreased by conditioned media from PC-3 and DU 145 human prostate cancer cells with KLF5 overexpression, but increased by media from cells with KLF5 knockdown. HIF1α, a key angiogenesis inducer, was upregulated by KLF5 loss at the protein but not the mRNA level in both mouse tissues and human cell lines, as determined by immunohistochemical staining, real-time RT-PCR and Western blotting. Consistently, KLF5 loss also upregulated VEGF and PDGF, two pro-angiogenic mediators of HIF1α function, as analyzed by immunohistochemical staining in mouse tissues and ELISA in conditioned media. Mechanistically, AKT activity, which caused the accumulation of HIF1α, was increased by KLF5 knockout or knockdown but decreased by KLF5 overexpression. PI3K/AKT inhibitors consistently abolished the effects of KLF5 knockdown on angiogenic activity, HIF1α accumulation, and VEGF and PDGF expression.ConclusionKLF5 loss enhances tumor angiogenesis by attenuating PI3K/AKT signaling and subsequent accumulation of HIF1α in PTEN deficient prostate tumors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0365-6) contains supplementary material, which is available to authorized users.

Differential requirements for β-catenin in murine prostate cancer originating from basal versus luminal cells.

A driver mutation occurring in different cells of origin may impact cancer progression differently. Previously, we demonstrated higher invasiveness in Pten-deficient prostate cancer (CaP) arising from basal cells compared to that arising from luminal cells in mice. Here, we show higher expression of epithelial-mesenchymal transition (EMT)-inducing transcription factors and stem/progenitor properties in basal-derived CaP compared to luminal-derived CaP. We further explore the requirement for β-catenin in basal and luminal prostate cells during CaP progression. Genetic ablation and pharmacological inhibition of β-catenin specifically suppress basal-derived CaP progression through reduction of stemness and cell proliferation and increased γH2Ax-associated apoptosis. Lineage tracing revealed that loss of β-catenin in basal cells impairs basal-to-luminal differentiation; conversely, β-catenin loss is dispensable for luminal-derived CaP progression. Our findings suggest that β-catenin is required for basal-derived normal luminal cells and cancer cells, but not for luminal derivatives. Although the cellular origin of CaP in patients cannot be easily determined at present, the results imply that β-catenin inhibition is a potential therapeutic option for a subset of patients with basal-derived CaP.

Abstract A49: Cabozantinib attenuates CXCL8-driven macrophage-dependent migration and invasion of PTEN-deficient prostate cancer

Abstract Background: The interaction between a tumor and the surrounding non-malignant stroma impacts on nearly every aspect of malignant progression. The heterogeneity of the tumor microenvironment (TME), however, makes mapping this interaction difficult. Each stromal population contributes to various functions and each tumor type has its own distinct pathway of interaction with different stromal populations. The majority of these interactions are mediated by soluble factors secreted into the TME. The loss of the tumor suppressor PTEN, an early event in prostate tumorigenesis, results in a subsequent increase in secretion of the pro-inflammatory CXCL8 chemokine into the TME (Maxwell et al 2013). The specific role of CXCL8 signaling within the prostate TME has not been fully classified and warrants further investigation. Methods: PTENnull PC3 cells were cultured in the absence and presence of human macrophage-representative THP-1 monocytes. Tumor-derived CXCL8 signaling was attenuated using (i) CXCL8 neutralizing antibodies, (ii) CXCR1 and CXCR2 receptor-targeting small peptide inhibitors (PEPducins), and (iii) a CXCL8-targeted shRNA method to generate stable PC3-120p cells with reduced CXCL8 secretion – a control line termed PC3-NTp was co-developed. PC3 cell motility and invasion was assessed by real-time transwell invasion through MatrigelTM and wound closure assays. Phospho-receptor tyrosine kinase (RTK) arrays were used to identify signaling pathways downstream of CXCL8 signaling which are relevant to prostate tumor cross-talk with infiltrating monocytes. Results: The presence of THP-1 cells more than doubled the rate of PC3 invasion through MatrigelTM (2.4-fold, p=0.0098) and increased wound closure after 6 h (50.63% v 25.65% for PC3 cells alone; p=0.0108). Macrophage-dependent enhancement of invasion and motility was attenuated following inhibition of CXCL8 signaling (i) by neutralizing antibody (90% decrease in normalized invasion and 43% decrease in wound closure, p<0.001 and p=0.03 respectively relative to IgG) and (ii) PEPducin inhibition of CXCR1 and CXCR2 (38% reduction in wound closure, p=0.0002) compared to non-targeting peptides. Low CXCL8-secreting PC3-120p cells were unable to initiate THP-1-potentiated motility and invasion, however, this response was rescued by addition of exogenous human CXCL8. Exogenous CXCL8 was shown to up-regulate secretion of RTK-activating cytokines and growth factors from THP-1 cells. Addition of the RTK inhibitor cabozantinib (XL184) but not crizotinib abrogated THP-1-dependent invasion and wound healing response of PTEN-deficient PC3 cells. Differential analysis of RTKs regulated by both cabozantinib and CXCL8 neutralizing antibody in co-culture identify c-Kit, RET, VEGFR1 and VEGFR2 as potential downstream regulators of CXCL8-dependent monocyte-enhanced PC3 invasion. Conclusions: Tumor associated macrophages (TAMs) are correlated with prostate cancer progression. Secretion of CXCL8 from PTEN-deficient prostate cancer cells activates macrophages, which then sustains cancer cell migration and invasion. Attenuating CXCL8 signaling within the TME and its downstream effects provides potential therapeutic targets to reduce metastasis in PTEN-deficient, CXCL8-high prostate cancer patients. Citation Format: Jessica L. Neisen, Nuala McCabe, Richard D. Kennedy, David JJ Waugh. Cabozantinib attenuates CXCL8-driven macrophage-dependent migration and invasion of PTEN-deficient prostate cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A49. doi:10.1158/1538-7445.CHTME14-A49

Abstract 1437: PTEN deficiency in prostate epithelial cells is associated with increased CAD phosphorylation and is inhibited by fisetin

Abstract Critical loss of phosphatase and tensin homolog (PTEN) beside increased insulin signaling often results in mTOR activation which is associated with progression of lethal prostate cancer (PCa). Recently mTOR was shown to control de novo pyrimidine synthesis via S6K which directly phosphorylates Ser1859 on CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydroorotase), the enzyme that catalyzes the first three steps of de novo pyrimidine synthesis. We assessed the CAD expression and phosprylation at Ser1859 in different in-vitro and in-vivo models of PCa and also tested the potential efficacy of fisetin, an agent which we previously established as a dual PI3K/AKT and mTOR inhibitor. We observed upregulation of CAD both at transcriptional and translational levels in PCa cell lines when compared with the non-tumorigenic prostate cells. However, a highly significant upregulation of CAD phosphorylation at Ser1859 was evident in PTEN deficient cells when compared with wild type PTEN cells. To confirm, we assessed the CAD phosphorylation at Ser1859 in PTEN knockout mice samples and observed significant increase in the CAD phosphorylation at Ser1859 but not in total CAD levels, suggesting high demands of de novo pyrimidine synthesis in PTEN deficient tumors. In another model of spontaneous PCa (TRAMP) an inverse association between CAD phosphorylation at Ser1859 and PTEN was observed. Based on the facts that PTEN deficient cells have high mTOR activity that is prerequisite for CAD phosphorylation we tested the efficacy of fisetin to inhibit CAD phosphorylation at Ser1859 in different PCa models. Fisetin significantly suppressed the CAD phosphorylation in tumorigenic PCa cells in-vitro. In-vivo efficay of fisetin for inhibition of CAD phosphorylation was also established in the xenograft study. However, decrease in abundance of CAD mRNA and protein in xenograft samples suggested fisetin additionally suppressed other mechanism(s) important for CAD expression. Next we determined the effect of fisetin to inhibit the growth factors (insulin/IGF-1/EGF) stimulated CAD oligomerization in PCa cells important for CAD activity. Fisetin significantly suppressed the growth factors induced CAD oligomerization as observed by electron microscopy and density gradient based western blotting. However, we did not find significant S-phase blockage by fisetin under similar conditions. Based on these data we suggest that PTEN deficient PCa epithelial tumors possess heightened de novo pyrimidine requirements, satisfied by growth factor stimulated and mTOR regulated CAD phosphorylation and oligomerization. Fisetin inhibits both CAD phosphorylation at Ser1859 and oligomerization and thereby limits pyrimidine sufficiency in PTEN deficient PCa cells. Our results add additional mechanistic insight in the fisetin mediated inhibition of PCa and suggest the use of fisetin in PTEN deficient PCa incidents. Citation Format: Mohammad Imran Khan, Vaqar Mustafa Adhami, Omar Mohammad Haidar, Bilal Bin Hafeez, Ajit Kumar Verma, Hasan Mukhtar. PTEN deficiency in prostate epithelial cells is associated with increased CAD phosphorylation and is inhibited by fisetin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1437. doi:10.1158/1538-7445.AM2014-1437

Abstract LB-44: Persistent androgen ablation promotes enhanced neuroendocrine features in Pten-deficient prostate cancer

Abstract Background: The development of potent androgens has increased survival of CRPC patients. Clinical data also suggests that the number of neuroendocrine (NE) CaPs frequently found in soft tissue metastasis has steadily increased over recent years and may be correlated with the use of potent androgen receptor (AR) inhibitors such as Enzalutamide or Aberaterone Acetate. These data suggest that NE cells may escape hormonal castration and that active inhibition of the AR may promote aggressive disease. Approach. To investigate this hypothesis, we have taken both genetic and pharmacological approaches to oppose AR signaling in Pten deficient CaP. Surgical castration of Pten-null mutant mice (Pb-Cre+;PtenL/L) led to both an increased EMT signature but also enhanced content of NE cells that were Synaptophysin+ and Pten-low/null. Chronic Enzalutamide treatment (4 wks., 10 mg/kg/d) promoted an increased percentage Synaptophysin+ cells, an observation that was potentiated when coinciding with surgical castration. Interestingly, the highest potentiation of NE cells was in the proximal anterior region of Pten-null mice. Consistent with our previous results, epithelial deletion of AR signaling in Pten-null epithelium (Pb-Cre+;PtenL/L;ArL/Y) did not prevent CaP progression under surgical castrate conditions. Moreover, compared to Pten-null mutants, Pten-null;Ar-null mutants showed an enhanced stem cell signature (p63+ cells, LSChigh subpopulation) but also marked increase content of NE cells upon both surgical castration and chronic Enzalutamide treatment (4-8 wks., 10 mg/kg/d). To consider whether total systemic ablation of the AR signaling axis could impact progression, resected Pten-null explants (“chuncks”) were isolated from Pb-Cre+;PtenL/L mutants and transplanted to the kidney capsule of Tfm;Rag1-null mice, immune incompetent hosts that are genetically null for Ar. Consistent with our hypothesis, Tfm;Rag1-null mutants showed more NE cells in explants than those transplanted to control AR intact control (Rag1-null) mice. Conclusion: Chronic blockade of the AR signaling axis results in a poorly differentiated phenotype characterized by increased stemness and enhanced NE phenotype. Citation Format: David J. Mulholland. Persistent androgen ablation promotes enhanced neuroendocrine features in Pten-deficient prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-44. doi:10.1158/1538-7445.AM2014-LB-44

Abstract 613: Combining PI3K and 5alpha-reductase inhibitors improves the treatment response in a mouse model of PTEN-deficient prostate cancer

Abstract Testosterone is converted to dihydrotestosterone (DHT) by the enzymes 5alpha-reductase types 1 and 2. Inhibition of both 5alpha-reducatase enzymes with dutasteride was effective in reducing the incidence of prostate cancer in high risk patients. This suggests that suppression of DHT with dutasteride may have a potential therapeutic effect on prostate cancer. In this study, we examined the antitumor effects of dutasteride in vivo using a preclinical model of prostate cancer that is based on the PSA-Cre mediated inactivation of PTEN. Our findings show that monotherapy with dutasteride significantly inhibited prostate tumor growth by approximately 25%. Furthermore, the combination of dutasteride with the mTOR inhibitor, everolimus, improved tumor growth suppression compared to monotherapy with either drug. Treatment with dutasteride had a minimal effect on tumor cell proliferation; however, it significantly increased the apoptotic rate in tumors. Interestingly, treatment with dutasteride resulted in increased activation of PI3K/mTOR signaling and a reduction of STAT3 pathway activation. Moreover, increased activation of MAPK, which occurs after treatment with mTOR inhibitors, was effectively reduced with the co-administration of dutasteride. The findings from our study suggest that inhibitors of 5alpha-reductase types 1 and 2 enzymes may have a potential therapeutic value in PTEN deficient prostate cancers, in particular when co-administered with PI3K inhibitors. Citation Format: Yurie Kura, Marco A. De Velasco, Naomi Ando, Emiko Fukushima, Yutaka Yamamoto, Yuji Hatanaka, Nobutaka Shimizu, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Combining PI3K and 5alpha-reductase inhibitors improves the treatment response in a mouse model of PTEN-deficient prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 613. doi:10.1158/1538-7445.AM2014-613

Abstract 3138: IL-6/Stat3 signaling is an indispensable modulator of oncogene-induced cellular senescence

Abstract IL-6/Stat3 signaling plays a role as a proto-oncogene in various tumors including prostate cancer, where IL-6 has been explored as therapeutic target. Deficiency of Pten, one of the most commonly mutated genes in cancer, directly triggers a senescence program critically depending on the p19Arf-p53 fail-safe pathway. We identified a novel and unexpected tumor suppressive role of IL-6/Stat3 signaling in a Pten-deficient murine prostate cancer model. Our data provide evidence that activation of IL-6/Stat3 signaling axis is implicated in oncogene-induced senescence (OIS). Strikingly, genetic ablation of IL-6 strongly promoted prostate cancer initiation, progression and metastasis in Pten-deficient mice. Mechanistically, IL6/Stat3 signaling regulates p19Arf-p53-induced senescence by modulating expression of the ubiquitin ligase MDM2 in pre-neoplastic cells. Critically, in prostate cancer patients low expression of Stat3 or p14Arf, the human homologue of p19Arf, correlates with poor survival. Therefore, the IL-6/Stat3 signaling axis may represent a novel molecular target for senescence-based cancer therapies. Citation Format: Jan Pencik, Michaela Schlederer, Melanie Hassler, Wolfgang Gruber, Fritz Aberger, Richard Kennedy, Stephen Walker, Stephan Rose-John, Valeria Poli, Robert Eferl, Harald Esterbauer, Osman Aksoy, Merima Herac, Peter Mazal, Andrea Haitel, Martin Susani, Richard Moriggl, Zoran Culig, Lukas Kenner. IL-6/Stat3 signaling is an indispensable modulator of oncogene-induced cellular senescence. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3138. doi:10.1158/1538-7445.AM2014-3138

Abstract 675: Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer

Abstract Inhibition of the androgen receptor (AR) results in activation of the PI-3K/mTOR pathway in PTEN deficient prostate cancer cells by reciprocal feedback inhibition (Carver BS, 2011). Enzalutamide or PI-3K/mTor inhibition result in feedback activation and combined inhibition abrogated this effect. We explored AR inhibition in CRPC cell lines with low, intermediate and high AR expression in the context of PI-3K/mTor inhibition to identify biomarkers of response and resistance. We utilized no/low (DU145, PC3), intermediate (LNCaP) and high (22Rv1,VCap) AR expressing prostate cancer (PC) cell lines to evaluate anti-tumor activity with 4 pan-PI3K/mTOR inhibitors (GDC-0980, GDC-0941, LY294002, PF-04691502), enzalutamide and abiraterone as single agents or various in combinations at clinically relevant doses. Cytotoxic activity of enzalutamide was more potent than abiraterone (IC50's was < 1µM and > 10µM) in LNCaP cells, however, the opposite was seen in DU145 and PC-3 cell lines. High AR expressing cells (22Rv1, VCap) were more sensitive to AR inhibition. PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 had IC50's in the range 50-500nM while LY294002 had an IC50 16-24 µM except for DU145. This cell line had increased IC50 values comparatively for PI-3K/mTor SMIs GDC-0980, GDC-0941 or PF-04691502 (1.5-3.75 µM) and LY29002 (48.5 µM). The combination index (CI) isobologram method indicated significant synergism for PI-3K/mTOR SMIs plus enzalutamide or abiraterone in all PC cell lines irrespective of AR status. Combination treatments were associated with increased apoptosis (Annexin-V staining and PARP-cleavage assay) in LNCaP and VCaP > 22Rv1 > DU145 and PC3. Cell cycle analysis by flow cytometry demonstrated that enzalutamide induces G1 arrest in LNCaP and DU145 cells and G2/M arrest in PC3 cells. PI-3K inhibition induces S arrest in LNCaP and DU145 and G2/M arrest in PC3 cells. The combination of enzalutamide plus PI-3K inhibition enhanced S arrest in LNCaP while in DU145 and PC3, G0/G1 arrest was prominent. PI-3K SMI plus enzalutamide completely inhibited pAkt308/473, pS6 and p4EBP1 in LNCaP cells, pAkt473 in PC3 cells and pS6 and p4EBP1 in 22Rv1 cells. Enzalutamide alone or in combination with PI-3K inhibition also suppressed AR expression in 22Rv1 cells. A 22Rv1 mouse xenograft model is evaluating safety and efficacy of the combination. This is a novel therapeutic strategy for CRPC patients to be evaluated in a clinical trial. Citation Format: Daruka Mahadevan, Carla Morales, Laurence S. Cooke, Bradley Somer, Wenqing Qi. Dual inhibition of the androgen receptor and PI-3K/mTor pathways has significant antitumor activity in castrate-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 675. doi:10.1158/1538-7445.AM2014-675

Abstract 1201: Establishment and characterization of cell lines derived from a murine model of PTEN-deficient prostate cancer

Abstract Inactivation of the tumor suppressor PTEN is a common event in human prostate cancer that leads to increased activation of the PI3K pathway. Crosstalk between the PI3K signaling cascade and other cancer regulatory pathways contributes to the growth and survival of cancer cells. To better understand the mechanisms involved in the transformation process and to establish an in vivo screening system for novel treatment strategies, we previously developed a genetically engineered mouse model of prostate cancer that is based on the conditional inactivation of PTEN. In this model, inactivation of PTEN results in the stage-specific development of prostate cancer that recapitulates most features of the human disease including the development of castration-resistant prostate cancer (CRPC). The purpose of our study was to establish and characterize a panel of cell lines derived from this model in order to study the disease mechanisms at the cellular level and to establish a cell-based assay system to complement our in vivo program for pre-clinical screening of novel anticancer drugs. A panel of 7 cell lines derived from castration-naïve prostate tumors and 6 cell lines derived from CRPC were established. Cells lines were characterized for morphological, growth, differentiation, and molecular profiles in vitro as well as for their tumorigenic potential in vivo. The cell lines were also tested against a panel of cytotoxic and molecular targeting drugs and compared to the sensitivity of human prostate cell lines. Overall, our comprehensive analysis summarizes the functionality and potential application as cell-based system for developing treatment strategies against prostate cancer. Citation Format: Kazuhiro Yoshikawa, Marco A. De Velasco, Yurie Kura, Naomi Ando, Emiko Fukushima, Yuji Hatanaka, Yutaka Yamamoto, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuto Nishio, Hirotsugu Uemura. Establishment and characterization of cell lines derived from a murine model of PTEN-deficient prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1201. doi:10.1158/1538-7445.AM2014-1201

Down-modulation of Bcl-2 sensitizes PTEN-mutated prostate cancer cells to starvation and taxanes.

The critical role of PTEN in regulating the PI3K/Akt/mTOR signaling pathway raises the possibility that targeting downstream effectors of the PI3K pathway, such as Bcl-2, might be an effective anti-proliferative strategy for PTEN-deficient prostate cancer cells. Four prostate cancer cell lines (LNCaP, PC3, DU145, 22Rv1) were assayed for their levels of total Akt and Ser473 phosphorylated Akt (p-Akt) by Western Blotting; their growth rates and sensitivity to different doses of paclitaxel were determined by cell counts after Trypan Blue dye exclusion assay. Cells were subjected to different combinations of starvation (growth factors and/or aminoacids withdrawal), paclitaxel treatment and Bcl-2 silencing by siRNA. Cell viability was evaluated by Trypan Blue dye exclusion assay, Propidium Iodide (PI) and Annexin-V/PI staining. We assessed the sensitivity of different prostate cancer cell lines to starvation and we observed a differential response correlated to the levels of Akt activation. The four prostate cancer cell lines also showed different sensitivity to taxol treatments; LNCaP and 22Rv1 cells were more resistant to paclitaxel than DU145 and PC3 cells. Combining taxol with growth factors and aminoacids deprivation leaded to a more than additive reduction of cell viability compared to single treatments in PTEN-mutant LNCaP cells. Down-modulation of anti-apoptotic Bcl-2 protein by siRNA sensitized LNCaP cells to taxanes and starvation induced cell death. Silencing Bcl-2 in PTEN-mutated prostate cancer cells enhances the apoptotic effects of combined starvation and taxol treatments, indicating that inhibition of Bcl-2 may be of significant value in PTEN-mutant tumor therapy.

Vulnerabilities of PTEN–TP53-Deficient Prostate Cancers to Compound PARP–PI3K Inhibition

Prostate cancer is the most prevalent cancer in males, and treatment options are limited for advanced forms of the disease. Loss of the PTEN and TP53 tumor suppressor genes is commonly observed in prostate cancer, whereas their compound loss is often observed in advanced prostate cancer. Here, we show that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the prosurvival PI3K-AKT signaling pathway limits the efficacy of a PARP single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human prostate cancer cell lines and in a Pten/Trp53-deficient mouse model of advanced prostate cancer. Our findings, therefore, identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for PTEN-deficient prostate cancer. The paucity of therapeutic options in advanced prostate cancer displays an urgent need for the preclinical assessment of novel therapeutic strategies. We identified differential therapeutic vulnerabilities that emerge upon the loss of both PTEN and p53, and observed that combined inhibition of PARP and PI3K provides increased efficacy in hormone-insensitive advanced prostate cancer.

PTEN-deficient tumors depend on AKT2 for maintenance and survival.

Loss of PTEN is a common event in many cancers and leads to hyperactivation of the PI3K-AKT signaling pathway. The mechanisms by which AKT isoforms mediate signaling to phenotypes associated with PTEN inactivation in cancer have not been defined. Here, we show that AKT2 is exclusively required for PTEN-deficient prostate tumor spheroid maintenance, whereas AKT1 is dispensable. shRNA silencing of AKT2 but not AKT1 promotes regression of prostate cancer xenografts. Mechanistically, we show that AKT2 silencing upregulates p21 and the proapoptotic protein BAX and downregulates the insulin-like growth factor receptor-1. We also show that p21 is an effector of AKT2 in mediating prostate tumor maintenance. Moreover, AKT2 is also exclusively required for the maintenance and survival of other PTEN-deficient solid tumors, including breast cancer and glioblastoma. These findings identify a specific function for AKT2 in mediating survival of PTEN-deficient tumors and provide a rationale for developing therapeutics targeting AKT2. Depletion of AKT2, but not AKT1, induces potent tumor regression in PTEN-deficient prostate cancer xenografts, concomitant with upregulation of p21, which may serve as a potential biomarker for screening AKT2 activity in clinical samples. The specific role of AKT2 in tumor maintenance provides a rationale for the development of isoform-specific inhibitors for patients with PTEN-deficient cancers.

Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells.

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

Safety/efficacy of MK-8669 (ridaforolimus) plus MK-2206 (AKT inhibitor) in patients with advanced breast cancer with low RAS signature and PTEN deficient prostate cancer.

2509 Background: The PI3K/AKT/mTOR signaling pathway is aberrantly activated in a variety of cancers. The combination of ridaforolimus (mTOR inhibitor) and MK-2206 may lead to blockade of the PI3K ...

A novel PI3K inhibitor displays potent preclinical activity against an androgen-independent and PTEN-deficient prostate cancer model established from the cell line PC3

Recent studies demonstrated that targeting the phosphatidylinositide 3-kinase (PI3K)/AKT signaling pathway is a major strategy for the treatment of androgen-independent prostate cancer. In the present study, we developed an analog BENC-511 from a recently reported PI3K inhibitor S14161 by structural optimization. Using PC3 and DU145 as the model cell lines, we found PTEN-deficient PC3 cells were more sensitive than PTEN-expressing DU145 ones in terms of cell proliferation, apoptosis, and caspase-3 activation. These findings were consistent with the inhibition on PI3K/AKT signals. BENC-511 preferably suppressed AKT activation in PC3 over DU145 cells. Notably, PTEN restoration attenuated BENC-511 induced apoptosis. Moreover, BENC-511 displayed great therapeutic efficacy in a PC3-derived prostate cancer model in nude mice. With an oral dosage of 50mg/kg, BENC-511 decreased tumor growth more than 50% in 27 days, which was accompanied with PARP cleavage, but did not show overt toxicity. This study lays a solid rationale for the development of BENC-511 as a drug for the treatment of PTEN-deficient and androgen-independent prostate cancers.

Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear.NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB–TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6–NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB–TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

P300 Acetyltransferase Regulates Androgen Receptor Degradation and PTEN-Deficient Prostate Tumorigenesis

Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion-induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNA interference-mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these findings, in PTEN-deficient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss.