Therapeutic Target In Advanced Prostate Cancer Research Articles

The β-Secretase 1 Enzyme as a Novel Therapeutic Target for Prostate Cancer.

Recent studies have demonstrated the association of APP and Aβ with cancer, suggesting that BACE1 may play an important role in carcinogenesis. In the present study, we assessed BACE1's usefulness as a therapeutic target in prostate cancer (PCa). BACE1 expression was observed in human PCa tissue samples, patient-derived xenografts (PDX), human PCa xenograft tissue in nude mice, and transgenic adenocarcinoma of the mouse prostate (TRAMP) tissues by immunohistochemistry (IHC) analysis. Additionally, the downstream product of BACE1 activity, i.e., Aβ1-42 expression, was also observed in these PCa tissues by IHC as well as by PET imaging in TRAMP mice. Furthermore, BACE1 gene expression and activity was confirmed in several established PCa cell lines (LNCaP, C4-2B-enzalutamide sensitive [S], C4-2B-enzalutamide resistant [R], 22Rv1-S, 22Rv1-R, PC3, DU145, and TRAMP-C1) by real-time PCR and fluorometric assay, respectively. Treatment with a pharmacological inhibitor of BACE1 (MK-8931) strongly reduced the proliferation of PCa cells in in vitro and in vivo models, analyzed by multiple assays (MTT, clonogenic, and trypan blue exclusion assays and IHC). Cell cycle analyses revealed an increase in the sub-G1 population and a significant modulation in other cell cycle stages (G1/S/G2/M) following MK-8931 treatment. Most importantly, in vivo administration of MK-8931 intraperitoneal (30 mg/kg) strongly inhibited TRAMP-C1 allograft growth in immunocompetent C57BL/6 mice (approximately 81% decrease, p = 0.019). Furthermore, analysis of tumor tissue using the prostate cancer-specific pathway array revealed the alteration of several genes involved in PCa growth and progression including Forkhead O1 (FOXO1). All together, these findings suggest BACE1 as a novel therapeutic target in advanced PCa.

Abstract CT159: CYCLONE 1: abemaciclib in men with heavily pretreated metastatic castration-resistant prostate cancer (mCRPC)

Abstract Background: Treatment options for patients (pts) with heavily pretreated mCRPC are limited. Augmenting endocrine therapy by targeting CDK4/6 with abemaciclib has dramatically improved outcomes in HR+ breast cancer. Similar to ER, AR signaling activates CDK4 & 6 and abemaciclib showed preclinical anti-tumor activity in prostate cancer (PC). This signal seeking single arm Phase 2 study evaluated the safety and clinical activity of abemaciclib monotherapy in pts with heavily pretreated poor prognosis mCRPC who progressed after both a novel hormonal agent (NHA) and two taxanes. Methods: Eligible pts had progressive mCRPC, measurable disease by RECIST1.1, and previously received ≥1 NHA, docetaxel, and cabazitaxel for metastatic PC. Abemaciclib was administered at 200 mg BID on a continuous 28-day dosing schedule. The primary endpoint was investigator-assessed objective response rate without concurrent bone progression (ORR). A target ORR of ≥12.5% was deemed suitable to support further evaluation of abemaciclib monotherapy in this refractory mCRPC setting. Key secondary endpoints included disease control rate (DCR), time to PSA progression, radiographic progression-free survival (rPFS), overall survival (OS), and safety. Results: Forty-four pts (median age 68 yrs; 75% ECOG PS 1, median PSA of 96 ng/mL) were enrolled. In total, 46.5% had visceral metastasis, including 27.9% with liver metastases. Median time from development of mCRPC to study entry was 2.1 yrs and the median number of prior systemic regimens for mCRPC was 3. Median number of treatment cycles was 3 (1-13). Five pts (11.8%) had soft tissue response. The ORR without concurrent bone progression was 6.8%. Eighteen pts (40.9%) had stable disease, of which 6 (13.6%) lasted ≥6 months (mos); DCR was 47.7%. Median rPFS was 2.7 mos (95% CI 1.9, 3.7); 6-mos rPFS rate was 24.9% (95% CI 12.4, 39.5); median time to PSA progression was 6.5 mos. Median OS was 7.6 mos (95% CI 5.6, NE). Any grade (G) TRAEs experienced by ≥50% of pts were diarrhea (79.5%), decreased appetite (52.3%) and fatigue (50%); majority were G1/2. G3 TRAEs in ≥ 5% of pts were neutropenia (22.7%), anemia (6.8%), fatigue (6.8%) and diarrhea (6.8%). There were no G4 or G5 TRAEs. Discontinuation rate due to AE was 13.6%. Conclusions: Abemaciclib demonstrated modest but objective single-agent clinical activity in pts with very heavily pretreated progressive mCRPC. The safety profile of abemaciclib was consistent with the experience in breast cancer. Although the primary endpoint was not formally met, the single agent activity observed in this late line mCRPC setting validates CDK4/6 as a therapeutic target in advanced PC. The CYCLONE 2 and CYCLONE 3 studies are currently evaluating abemaciclib in combination with abiraterone/prednisone to leverage synergy of combined CDK4/6 and hormonal inhibition in first line mCRPC and mHSPC settings, respectively. Citation Format: Neeraj Agarwal, Daniel Castellano Guana, Teresa A. Gordoa, Jose A. Arija, Emeline Colomba, Gwenaelle Gravis, Loic Mourey, Stephanie Oudard, Aude Flechon, Macarena Gonzalez, Pablo Maroto-Rey, Michael Schweizer, Enrique Gallardo, Karim Nacerddine, Adams K. Appiach, Arjun Balar, Erica Johnston, José M. Piulats. CYCLONE 1: abemaciclib in men with heavily pretreated metastatic castration-resistant prostate cancer (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT159.

A Suite of Activity-Based Probes To Dissect the KLK Activome in Drug-Resistant Prostate Cancer.

Kallikrein-related peptidases (KLKs) are a family of secreted serine proteases, which form a network (the KLK activome) with an important role in proteolysis and signaling. In prostate cancer (PCa), increased KLK activity promotes tumor growth and metastasis through multiple biochemical pathways, and specific quantification and tracking of changes in the KLK activome could contribute to validation of KLKs as potential drug targets. Herein we report a technology platform based on novel activity-based probes (ABPs) and inhibitors enabling simultaneous orthogonal analysis of KLK2, KLK3, and KLK14 activity in hormone-responsive PCa cell lines and tumor homogenates. Importantly, we identifed a significant decoupling of KLK activity and abundance and suggest that KLK proteolysis should be considered as an additional parameter, along with the PSA blood test, for accurate PCa diagnosis and monitoring. Using selective inhibitors and multiplexed fluorescent activity-based protein profiling (ABPP), we dissect the KLK activome in PCa cells and show that increased KLK14 activity leads to a migratory phenotype. Furthermore, using biotinylated ABPs, we show that active KLK molecules are secreted into the bone microenvironment by PCa cells following stimulation by osteoblasts suggesting KLK-mediated signaling mechanisms could contribute to PCa metastasis to bone. Together our findings show that ABPP is a powerful approach to dissect dysregulation of the KLK activome as a promising and previously underappreciated therapeutic target in advanced PCa.

IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.

Discovery of a novel long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth

BackgroundVirtually all patients with metastatic prostate cancer (PCa) will relapse and develop lethal castration-resistant prostate cancer (CRPC). Long noncoding RNAs (lncRNAs) are emerging as critical regulatory elements of many cellular biological processes, and may serve as therapeutic targets for combating PCa progression. Here, we have discovered in a high-throughput RNAi screen a novel lncRNA in PCa, and assessed the oncogenic effects of this lncRNA.MethodsRapid amplification of cDNA ends and sequencing was utilized to identify a previously unannotated lncRNA lying within exon six and the 3’UTR of the lymphocyte-specific protein tyrosine kinase (LCK) gene. The levels of HULLK in the presence or absence of hormone and/or enzalutamide or coregulator inhibitors were measured by quantitative PCR (qPCR). The determination of HULLK transcription and localization were characterized by strand-specific qPCR and cellular fractionation followed by qPCR, respectively. The correlation between HULLK expression and prostate cancer Gleason score was analyzed by droplet digital PCR. CyQuant assays were conducted to evaluate the effects of knocking down HULLK with shRNAs or overexpressing HULLK on cell growth.ResultsIn this study, a previously unannotated lncRNA lying within exon six and 3’UTR of the LCK gene was dramatically upregulated by androgen in a dose-dependent manner, and the anti-androgen enzalutamide completely blocked this hormone-induced increase. Therefore, we labeled this lncRNA “HULLK” for Hormone-Upregulated lncRNA within LCK. Binding sites for two AR coregulators p300 and Brd4 reside near the HULLK transcriptional start site (TSS), and inhibitors of these coregulators downregulated HULLK. HULLK is transcribed from the sense strand of DNA, and predominantly localizes to the cytoplasm. HULLK transcripts are not only expressed in prostate cancer cell lines, but also prostate cancer patient tissue. Remarkably, there was a significant positive correlation between HULLK expression and high-grade PCa in multiple cohorts. shRNAs targeting HULLK significantly decreased PCa cell growth. Moreover, cells overexpressing HULLK were hypersensitive to androgen stimulation.ConclusionsHULLK is a novel lncRNA situated within the LCK gene that may serve as an oncogene in PCa. Our data enhances our understanding of lncRNA biology and may assist in the development of additional biomarkers or more effective therapeutic targets for advanced PCa.

Abstract B086: Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in PCa patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a tumor-promoting phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behavior of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of YKL-40 (Chitinase-3-like 1; CHI3L1) when compared with PCa-N patients and healthy controls (HC), classified as individuals with no known history of cancer. The only described receptor for YKL-40, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in ARCaPm cells; accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPm. In addition, siRNA directed against this receptor in ARCaPm cells caused a significant reduction in the invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: We show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of YKL-40, and YKL-40 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca Meza, David Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine Martins, Timothy R. Crother, Cory M. Hogaboam, Sungyong You, Neil Bhowmick, Edwin Posadas. Monocytes-produced Chitinase-3-like 1 is a driver of metastatic behavior in advanced prostate cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B086.

Circulating monocytes from prostate cancer patients promote invasion and motility of epithelial cells.

BackgroundRecruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. We tested the hypothesis that circulating blood monocytes from advanced prostate cancer (PCa) patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor‐derived signals.MethodsBlood monocytes from advanced and stable PCa patients were cultured, and the conditioned media (CM) were collected and analyzed using standard invasion and wound closure assays to measure effects on invasion and motility of PCa tumor cells. We then identified the proteome profile of these monocytes using proteome array and ELISA.ResultsConditioned media from circulating monocytes in patients with metastatic prostate cancer (PCa‐M) increased invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte‐derived CM from metastatic castration‐resistant (mCRPC) patients presented high levels of chitinase‐3‐like 1 (CHI3L1, YKL‐40) when compared to patients with stable disease (PCa‐N) and healthy control individuals (HC). The only described receptor for CHI3L1, interleukin‐13 receptor α2 (IL‐13Rα2), was significantly up‐regulated in the human metastatic PCa cell line, ARCaPM. Accordingly, we observed that the activation of IL‐13Rα2 from PCa‐M CM increased the invasiveness of ARCaPM cells while siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa‐M patients.ConclusionsThus, we show that circulating monocytes from metastatic PCa patients exert a tumor‐promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa.

Abstract 5208: Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients

Abstract Purpose: The identification, functional status, distribution, and interactions of immune cells in prostate cancer (PCa) patients have yet to be fully characterized. Understanding these details could provide key insights as to how promising current and developing immunotherapies might be directed toward PCa. Recruited myeloid cells are known to promote cancer initiation, malignant progression, metastasis, and resistance to therapy in the tumor niche. In the present study, we tested the hypothesis that circulating blood monocytes from advanced PCa patients exhibit a protumor phenotype and directly influence the tumor microenvironment in response to tumor-derived signals. Experimental Design: Monocytes from metastatic (PCa-M) and nonmetastatic (PCa-N) patient blood samples were isolated, cultured, and conditioned media (CM) was obtained. To evaluate the role of monocytes in metastatic behaviors of PCa cells, in vitro invasion (via Matrigel) and motility assays (Incucyte® Live Cell Analysis System) were performed using monocyte-CM. To identify the candidate PCa-M monocyte-secreted protein(s) that might be implicated in tumor progression, we analyzed the CM using Proteome Profiler Analysis (R&D). Data were validated using ELISA and qPCR. Results: Herein, we report that CM from circulating monocytes in patients with PCa/mCRPC (metastatic castration-resistant PCa) increased motility and invasion of epithelial PCa cells in vitro. Proteome Profiler Analysis revealed that monocyte-derived CM from metastatic PCa/mCRPC patients presented high levels of Chitinase-3-like 1 (CHI3L1, YKL-40) when compared with PCa-N patients and healthy-control individuals (HC). The only described receptor for CHI3L1, interleukin-13 receptor α2 (IL-13Rα2), was significantly upregulated in the human metastatic prostate cancer cell line, ARCaPM. Accordingly, we observed that the activation of IL-13Rα2 from PCa-M CM increased the invasiveness of ARCaPM cells, and siRNA directed against this receptor significantly reduced invasiveness of these cells in the presence of CM from PCa-M patients. Conclusions: Thus, we show that circulating monocytes from metastatic PCa/mCRPC patients exert a tumor-promoting role via the secretion of CHI3L1, and CHI3L1 demands further exploration as a possible therapeutic target in advanced PCa. Citation Format: Karen A. Cavassani, Rebecca J. Meza, David M. Habiel, Jie-Fu Chen, Alexander Montes, Manisha Tripathi, Gislaine A. Martins, Timothy R. Crother, Sungyong You, Cory M. Hogaboam, Neil Bhowmick, Edwin M. Posadas. Monocyte-produced Chitinase-3-like 1 is a driver of metastatic behavior in prostate cancer patients [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 5208.

ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer.

The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoid acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits coactivators SRC-1 and -3 to an AR-RORE to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR gene network. Lastly, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing but not AR-negative xenograft PCa models, and effectively sensitized CRPC tumors to enzalutamide, without overt toxicity in mice. Together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and a potential therapeutic target for advanced PCa.

Identification of the epigenetic reader CBX2 as a potential drug target in advanced prostate cancer

BackgroundWhile localized prostate cancer (PCa) can be effectively cured, metastatic disease inevitably progresses to a lethal state called castration-resistant prostate cancer (CRPC). Emerging evidence suggests that aberrant epigenetic repression by the polycomb group (PcG) complexes fuels PCa progression, providing novel therapeutic opportunities.ResultsIn the search for potential epigenetic drivers of CRPC, we analyzed the molecular profile of PcG members in patient-derived xenografts and clinical samples. Overall, our results identify the PcG protein and methyl-lysine reader CBX2 as a potential therapeutic target in advanced PCa. We report that CBX2 was recurrently up-regulated in metastatic CRPC and that elevated CBX2 expression was correlated with poor clinical outcome in PCa cohorts. Furthermore, CBX2 depletion abrogated cell viability and induced caspase 3-mediated apoptosis in metastatic PCa cell lines. Mechanistically explaining this phenotype, microarray analysis in CBX2-depleted cells revealed that CBX2 controls the expression of many key regulators of cell proliferation and metastasis.ConclusionsTaken together, this study provides the first evidence that CBX2 inhibition induces cancer cell death, positioning CBX2 as an attractive drug target in lethal CRPC.

Abstract 616: Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced prostate cancer

Abstract Most of castration-refractory prostate cancers (CRPC) continue to express androgen receptor (AR) that maintains its activity at low levels or even in the absence of androgen, driving tumor cell proliferation. Androgen-depleting drugs and androgen antagonists have little or no effect against AR+ cancers that are fully androgen-independent (AI). On the other hand, experimental compounds that cause AR degradation prevent AR-mediated repression of tumor-promoting genes, a tumor-suppressive effect of AR. We report a novel druggable target for CRPC, which mediates the induction but not the repression of gene expression by AR and inhibits the growth of AI prostate cancer (PCa) cells. This target is two closely related transcription-regulating kinases CDK8/CDK19 that unlike some other members of the CDK family are not required for cell cycle progression but play critical roles in several transcriptional signaling pathways. CDK8 and CDK19 are highly expressed in AR+ PCa cells and elevated under the conditions of androgen deprivation; CDK19 is overexpressed in AR+ PCa cell lines and in metastatic clinical PCa. A novel selective CDK8/19 kinase inhibitor (Senexin B) blocks ligand-induced AR-mediated transcriptional activation in androgen-dependent (AD) PCa cells but does not affect the AR-mediated gene repression. The CDK8/19 inhibitor also inhibits ligand-independent AR activation and AI cell growth in AI-PCa cells resistant to the anti-androgenic drug enzalutamide, both in vitro and in a mouse xenograft model. We have also discovered that CDK8/19 inhibition decreases NFκB mediated transcriptional activation of a group of tumor-promoting chemokines, such as CXCL1, CXCL2 and IL8, suggesting that CDK8/19 may promote prostate cancer chemoresistance and metastasis through the NFκB pathway. The above results suggest that CDK8/19 is an especially attractive therapeutic target for advanced PCa, which controls both AR-driven cell proliferation and other oncogenic pathways. Citation Format: Mengqian Chen, Vimala Kaza, Jiaxin Liang, Martina McDermott, Igor Roninson. Transcription-regulating kinases CDK8 and CDK19 as novel therapeutic targets for advanced 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 616. doi:10.1158/1538-7445.AM2014-616

Silencing of LASS2/TMSG1 enhances invasion and metastasis capacity of prostate cancer cell

Homo sapiens longevity assurance homolog 2 of yeast LAG1 (LASS2), also known as tumor metastasis suppressor gene 1 (TMSG1), was firstly cloned by our laboratory in 1999. However, its antitumor molecular mechanisms are still unclear. LASS2/TMSG-1 could directly interact with the C subunit of Vacuolar H(+) ATPase (V-ATPase), which suggested that LASS2/TMSG1 might inhibit the invasion and metastasis through regulating the function of V-ATPase. In this study, we explored the effect of small hairpin RNA (shRNA) targeting LASS2/TMSG1 on the invasion and metastasis of human prostate carcinoma cell line PC-3M-2B4 with low metastatic potential and its functional interaction with V-ATPase. Silencing of LASS2/TMSG1 gene in PC-3M-2B4 cells increased V-ATPase activity, extracellular hydrogen ion concentration and in turn the activation of secreted MMP-2 and MMP-9, which coincided with enhancing cell proliferation, cell survival, and cell invasion in vitro, as well as acceleration of prostate cancer (PCA) growth and lymph node metastases in vivo. Thus we concluded that silencing of LASS2/TMSG1 enhances invasion and metastasis of PCA cell through increase of V-ATPase activity. These results establish LASS2/TMSG1 as a promising therapeutic target for advanced PCA.

Metabolomic Characterization of Human Prostate Cancer Bone Metastases Reveals Increased Levels of Cholesterol

BackgroundMetastasis to the bone is one clinically important features of prostate cancer (PCa). Current diagnostic methods cannot predict metastatic PCa at a curable stage of the disease. Identification of metabolic pathways involved in the growth of bone metastases therefore has the potential to improve PCa prognostication as well as therapy.Methodology/Principal FindingsMetabolomics was applied for the study of PCa bone metastases (n = 20) in comparison with corresponding normal bone (n = 14), and furthermore of malignant (n = 13) and benign (n = 17) prostate tissue and corresponding plasma samples obtained from patients with (n = 15) and without (n = 13) diagnosed metastases and from men with benign prostate disease (n = 30). This was done using gas chromatography-mass spectrometry for sample characterization, and chemometric bioinformatics for data analysis. Results were verified in a separate test set including metastatic and normal bone tissue from patients with other cancers (n = 7). Significant differences were found between PCa bone metastases, bone metastases of other cancers, and normal bone. Furthermore, we identified metabolites in primary tumor tissue and in plasma which were significantly associated with metastatic disease. Among the metabolites in PCa bone metastases especially cholesterol was noted. In a test set the mean cholesterol level in PCa bone metastases was 127.30 mg/g as compared to 81.06 and 35.85 mg/g in bone metastases of different origin and normal bone, respectively (P = 0.0002 and 0.001). Immunohistochemical staining of PCa bone metastases showed intense staining of the low density lipoprotein receptor and variable levels of the scavenger receptor class B type 1 and 3-hydroxy-3-methylglutaryl-coenzyme reductase in tumor epithelial cells, indicating possibilities for influx and de novo synthesis of cholesterol.Conclusions/SignificanceWe have identified metabolites associated with PCa metastasis and specifically identified high levels of cholesterol in PCa bone metastases. Based on our findings and the previous literature, this makes cholesterol a possible therapeutic target for advanced PCa.

Heat shock protein 27 confers resistance to androgen ablation and chemotherapy in prostate cancer cells through eIF4E

One strategy to improve therapies in advanced prostate cancer (PC) involves targeting genes that are activated by androgen withdrawal to delay the emergence of the androgen-independent (AI) phenotype. Heat shock protein 27 (Hsp27) expression becomes highly upregulated in PC cells after androgen withdrawal or chemotherapy, in which it functions as a cytoprotective chaperone to confer broad-spectrum treatment resistance. The purpose of this study is to elucidate anti-apoptotic pathways regulated by Hsp27 that are activated during PC progression. Using two-hybrid experiment, we found that Hsp27 was having a major role in the protein translational initiation process. Furthermore, using complementary DNA (cDNA) microarray analysis, 4E binding protein 1 was identified as being proportionately and highly regulated by Hsp27. These data led us to analyze the protein synthesis initiation pathway, which is a prerequisite for cell growth and proliferation. Using northern and western blot analysis, we found that Hsp27 downregulation decreased eukaryotic translation initiation factor 4E (eIF4E) expression at the protein, but not mRNA, level. The cytoprotection afforded by Hsp27 overexpression was attenuated by eIF4E knockdown using specific eIF4E short interfering RNA (siRNA). Co-immunoprecipitation and co-immunofluorescence confirmed that Hsp27 colocalizes and interacts directly with eIF4E. Hsp27-eIF4E interaction decreases eIF4E ubiquitination and proteasomal degradation. By chaperoning eIF4E, Hsp27 seems to protect the protein synthesis initiation process to enhance cell survival during cell stress induced by castration or chemotherapy. Forced overexpression of eIF4E induces resistance to androgen-withdrawal and paclitaxel treatment in the prostate LNCaP cells in vitro. These findings identify Hsp27 as a modulator of eIF4E and establish a potential mechanism for the eIF4E-regulated apoptosis after androgen ablation and chemotherapy. Targeting Hsp27-eIF4E interaction may serve as a therapeutic target in advanced PC.

A Novel Experimental Heme Oxygenase-1–Targeted Therapy for Hormone-Refractory Prostate Cancer

Heme oxygenase-1 (HO-1), a member of the heat shock protein family, plays a key role as a sensor and regulator of oxidative stress. Herein, we identify HO-1 as a biomarker and potential therapeutic target for advanced prostate cancer (PCA). Immunohistochemical analysis of prostate tissue using a progression tissue microarray from patients with localized PCA and across several stages of disease progression revealed a significant elevation of HO-1 expression in cancer epithelial cells, but not in surrounding stromal cells, from hormone-refractory PCA (HRPCA) compared with hormone-responsive PCA and benign tissue. Silencing the ho-1 gene in HRPCA cells decreased the HO-1 activity, oxidative stress, and activation of the mitogen-activated protein kinase-extracellular signal-regulated kinase/p38 kinase. This coincided with reduced cell proliferation, cell survival, and cell invasion in vitro, as well as inhibition of prostate tumor growth and lymph node and lung metastases in vivo. The effect of ho-1 silencing on these oncogenic features was mimicked by exposure of cells to a novel selective small-molecule HO-1 inhibitor referred to as OB-24. OB-24 selectively inhibited HO-1 activity in PCA cells, which correlated with a reduction of protein carbonylation and reactive oxygen species formation. Moreover, OB-24 significantly inhibited cell proliferation in vitro and tumor growth and lymph node/lung metastases in vivo. A potent synergistic activity was observed when OB-24 was combined with Taxol. Together, these results establish HO-1 as a potential therapeutic target for advanced PCA.

Ligand-Independent Androgen Receptor Variants Derived from Splicing of Cryptic Exons Signify Hormone-Refractory Prostate Cancer

Suppression of androgen production and function provides palliation but not cure in men with prostate cancer (PCa). Therapeutic failure and progression to hormone-refractory PCa (HRPC) are often accompanied by molecular alterations involving the androgen receptor (AR). In this study, we report novel forms of AR alteration that are prevalent in HRPC. Through in silico sequence analysis and subsequent experimental validation studies, we uncovered seven AR variant transcripts lacking the reading frames for the ligand-binding domain due to splicing of "intronic" cryptic exons to the upstream exons encoding the AR DNA-binding domain. We focused on the two most abundantly expressed variants, AR-V1 and AR-V7, for more detailed analysis. AR-V1 and AR-V7 mRNA showed an average 20-fold higher expression in HRPC (n = 25) when compared with hormone-naive PCa (n = 82; P < 0.0001). Among the hormone-naive PCa, higher expression of AR-V7 predicted biochemical recurrence following surgical treatment (P = 0.012). Polyclonal antibodies specific to AR-V7 detected the AR-V7 protein frequently in HRPC specimens but rarely in hormone-naive PCa specimens. AR-V7 was localized in the nuclei of cultured PCa cells under androgen-depleted conditions, and constitutively active in driving the expression of canonical androgen-responsive genes, as revealed by both AR reporter assays and expression microarray analysis. These results suggest a novel mechanism for the development of HRPC that warrants further investigation. In addition, as expression markers for lethal PCa, these novel AR variants may be explored as potential biomarkers and therapeutic targets for advanced PCa.

Calcitonin promotes in vivo metastasis of prostate cancer cells by altering cell signaling, adhesion, and inflammatory pathways

Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer (PC). Although the significance of CT-CTR axis in PC cell growth, invasion, and epithelial to mesenchymal transition has been established, its role in tumor metastasis has not been examined. To examine the role of CT-CTR axis in tumor metastasis, we employed stable CT-CTR activated and silenced system of three PC cell lines, LNCaP cells that lack endogenous CT, PC-3 cells that lack endogenous CTR, and PC-3M cells that co-express CT and CTR. Enforced expression of CT in LNCaP cells and CTR in PC-3 cells increased their ability to form orthotopic tumors and distant metastases in multiple organs. By contrast, silencing of CT expression in PC-3M cells not only reduced their tumorigenicity, but also completely abrogated their metastatic potential. To investigate the effect of in vivo silencing of CT expression on tumor growth, we employed recombinant adeno-associated virus (rAAV) to deliver anti-CT ribozymes in preexisting tumors of nude mice and large probasin promoter (LPB)-Tag transgenic mice. rAAV-CT(-) treatment not only abrogated the growth of pre-implanted tumors in nude mice, but also significantly reduced the growth of spontaneous tumors in LPB-Tag mice. Analysis of CT upregulated and silenced PC-3M transcriptomes revealed 105 genes affected by the modulation of CT expression. These CT signature genes generated survival, adhesion, pro-inflammatory, and pro-metastatic pathways. Added together, these data indicate a pivotal role for CT-CTR axis in PC metastasis and may serve as a potential therapeutic target for advanced PC.