Castration-resistant Prostate Cancer Bone Research Articles

Abstract 4618: PIM kinases drive cell adhesion mediated drug resistance in prostate cancer

Abstract Introduction: Castration-resistant prostate cancer (CRPC) is an incurable and lethal progression of the disease that does not respond to any treatment options. Cell adhesion to the extracellular matrix (ECM) activates survival pathways, allowing cancer cells to evade anti-cancer therapy, a phenomenon known as cell adhesion-mediated drug resistance. Bone, which is the primary site of prostate cancer metastasis, is known to be enriched in laminin, a ubiquitous ECM protein. In addition, laminin binding integrins, α6β1 and α3β1, are the major integrins expressed in CRPC, suggesting that laminin-dependent adhesion provides a survival advantage. Based on these findings, activation of the laminin-binding integrin, α6β1, is an intriguing factor mediating resistance to therapy in CRPC. PIM1 is an oncogenic Ser/Thr kinase that is elevated in CRPC and is known to promote resistance to therapy. In contrast, to many pro-survival kinases, PIM kinases do not possess any obvious regulatory domains. Current evidence suggests that PIM1 is constitutively active when it is expressed, so PIM1 expression level directly correlates with its catalytic activity. Despite this fact, little is known about the internal and external mechanisms that dictate PIM1 protein levels and spatial activation in cancer cells. Here, we identify PIM1 as a key survival signal that contributes to cell adhesion-mediated drug resistance. Experimental procedures: Supporting the importance of this signaling axis in human tumors, multiplex IHC of a TMA containing prostate adenocarcinoma and CRPC bone metastases to show that integrin α6 and PIM1 are coexpressed and spatially correlated in primary and metastatic prostate cancer. To examine the spatiotemporal dynamics of PIM1 during cell adhesion, We developed a kinase activity reporter (PIMKAR) to monitor PIM catalytic activity in live cells. Because we observed PIM was highly localized to the membrane in patient tumors, we tagged PIMKAR with a myristylation sequence to specifically measure PIM activity at the membrane vs cytosol. Immunofluorescence and biochemical techniques were used to demonstrate the effect of PIM1 on mitophagy and oxidative stress. Conclusions: We demonstrate that activation of integrin α6β1 downstream of cell adhesion stabilizes PIM1 by blocking its proteasomal degradation. The resulting upregulation of PIM1 serves to reduce oxidative stress through complementary mechanisms reducing mitochondrial fragmentation and increasing the removal of damaged mitochondria by increasing mitophagy. Mechanistically, PIM1 upregulates BNIP3 to enhance mitophagy and thereby reduce oxidative stress in the cell. Together, these results expand our understanding of the mechanisms regulating PIM1 activation and provide evidence for the use of PIM inhibitors in bone mCRPC and other cancers where laminin is a major component of the ECM Citation Format: Caitlyn Flores, Shailender Chauhan, Cynthia Miranti, Anne Cress, Gregory Rogers, Noel Warfel. PIM kinases drive cell adhesion mediated drug resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4618.

Abstract 2701: Multivariate transcriptome analysis identifies networks and key drivers of metastatic castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most cancer-related cause of death in men. Most PCa are characterized as slow-growing or indolent, though some patients are at a high risk of for recurrent and eventually metastatic disease. The most lethal form of PCa is metastatic castration-resistant prostate cancer (mCRPC) that has progressed to the bone. The molecular mechanism underlying PCa progression to bone has not been fully elucidated. Here, we identify gene networks associated with CRPC bone metastasis and survival risk. Networks were investigated by using a novel weighted gene network co-expression analysis (WGCNA) method and examining overrepresentation of upstream regulators and signaling pathways within co-expressed transcriptome modules across clinically annotated transcriptomes from mCRPC patients (N=60). Functional Enrichment analysis was used, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analysis, to identify biological functions of related hub genes overrepresented in our module of interest. WGCNA identified that cyan module was significantly associated with castration-resistant prostate cancer (CRPC) bone metastasis. KEGG and GO analysis results revealed genes in blue module were mainly related to collagen formation, cell signaling peptides, and bone regulation processes, including alkaline phosphatase, osteoblast formation, and endochondral ossification. Genes positively correlated with bone metastasis exhibited the following biological pathways: PI3K-Akt signaling, ECM-receptor interaction, and protein digestion and absorption pathways. This study provides novel insights into the biological pathways associated with CPRC metastasis to the bone. The module associated with bone metastasis and overall survival represent both known and novel pathways. Citation Format: Lawrence P. McKinney, Rajesh Singh, I. King Jordan, Sooryanarayana Varambally, Martin G. Sanda, Eric B. Dammer, James W. Lillard. Multivariate transcriptome analysis identifies networks and key drivers of metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2701.

Transcriptome analysis identifies networks and key drivers in tumor microenvironment for metastatic castration-resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most cancer-related cause of death in men. The most lethal form of PCa is metastatic castration-resistant prostate cancer (CRPC) that has progressed to the bone. The molecular mechanism underlying PCa progression to bone has not been fully elucidated. Here, we identify gene networks and characterize the cell types that contribute to the heterogeneity and complexity of CRPC associated with bone metastasis. Networks were investigated by using a novel weighted gene network co-expression analysis (WGCNA) method and examining overrepresentation of upstream regulators and signaling pathways within co-expressed transcriptome modules across clinically annotated transcriptomes from mCRPC patients (N=60). Functional Enrichment analysis was used, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analysis to identify biological functions of related hub genes overrepresented in our module of interest. WGCNA identified significant modules that are associated with CRPC bone metastasis. KEGG and GO analysis results revealed genes in these modules were mainly related to collagen formation, cell signaling peptides, and bone regulation processes. Genes positively correlated with bone metastasis exhibited the following biological pathways: PI3K-Akt signaling, ECM-receptor interaction, and protein digestion and absorption pathways. This study provides novel insights into the biological pathways associated with CRPC metastasis to the bone and its tumor immune microenvironment (TIME) niche. The modules associated with bone metastasis and overall survival represent both known and novel pathways. Supported by NIH/NCI Grant: MSM/Tuskegee U/UAB Comp. Cancer Center Partnership Name & Number of Grant Award: NCI - U54CA118638

Fibroblast Growth Factor Receptor 1 Drives the Metastatic Progression of Prostate Cancer

BackgroundNo curative therapy is currently available for metastatic prostate cancer (PCa). The diverse mechanisms of progression include fibroblast growth factor (FGF) axis activation. ObjectiveTo investigate the molecular and clinical implications of fibroblast growth factor receptor 1 (FGFR1) and its isoforms (α/β) in the pathogenesis of PCa bone metastases. Design, setting, and participantsIn silico, in vitro, and in vivo preclinical approaches were used. RNA-sequencing and immunohistochemical (IHC) studies in human samples were conducted. Outcome measurements and statistical analysisIn mice, bone metastases (chi-square/Fisher’s test) and survival (Mantel-Cox) were assessed. In human samples, FGFR1 and ladinin 1 (LAD1) analysis associated with PCa progression were evaluated (IHC studies, Fisher’s test). Results and limitationsFGFR1 isoform expression varied among PCa subtypes. Intracardiac injection of mice with FGFR1-expressing PC3 cells reduced mouse survival (α, p < 0.0001; β, p = 0.032) and increased the incidence of bone metastases (α, p < 0.0001; β, p = 0.02). Accordingly, IHC studies of human castration-resistant PCa (CRPC) bone metastases revealed significant enrichment of FGFR1 expression compared with treatment-naïve, nonmetastatic primary tumors (p = 0.0007). Expression of anchoring filament protein LAD1 increased in FGFR1-expressing PC3 cells and was enriched in human CRPC bone metastases (p = 0.005). ConclusionsFGFR1 expression induces bone metastases experimentally and is significantly enriched in human CRPC bone metastases, supporting its prometastatic effect in PCa. LAD1 expression, found in the prometastatic PCa cells expressing FGFR1, was also enriched in CRPC bone metastases. Our studies support and provide a roadmap for the development of FGFR blockade for advanced PCa. Patient summaryWe studied the role of fibroblast growth factor receptor 1 (FGFR1) in prostate cancer (PCa) progression. We found that PCa cells with high FGFR1 expression increase metastases and that FGFR1 expression is increased in human PCa bone metastases, and identified genes that could participate in the metastases induced by FGFR1. These studies will help pinpoint PCa patients who use fibroblast growth factor to progress and will benefit by the inhibition of this pathway.

MP33-03 DORMANT CASTRATION-RESISTANT PROSTATE CANCER ORGANOIDS WITH HYBRID BASAL-LUMINAL CELLS AND LOSS OF SARS-COV-2 HOST FACTORS EMERGED UNDER ANDROGEN DEPRIVATION

INTRODUCTION AND OBJECTIVE: Androgen deprivation therapy (ADT) can help maintain remission in advanced prostate cancer (PCa) patients with bone metastases, however, growth and metastatic spread often recur. To address the need for more predictive pre-clinical research platforms and to identify new targets and therapies for bone metastatic castration-resistant prostate cancer (CRPC). METHODS: We used patient-derived xenograft (PDX) tumors from bone metastatic prostate cancer patients to establish threedimensional (3D) organoids and investigated their response to ADT by either withholding di-hydro-testosterone (no DHT) or treating with enzalutamide. Cyst/spheroid quantitation, immunohistopathology, cell viability assay, qRT-PCR, RNA sequencing, gene set enrichment analysis (GSEA) and live cell cycle tracking using Fucci2BL were performed in PDXs: PCSD1, PCSD13 and PCSD25 and compared to PCa Cell lines: P, DU145 and LNCaP. RESULTS: ADT resulted in CRPC spheroids with CK5D CK8D cells, up-regulated stem-cell transcription factors, steroidogenic and neurogenic pathways and down-regulated AR-target genes, interferon, cell cycle, cell division and circadian pacemaker pathways. Enzalutamide-treated spheroids transitioned to G0 and AR protein was decreased but not AR mRNA. Moreover, ADT decreased both ACE2 and TMPRSS2, host cell viral entry factors for the severe acute respiratory syndrome (SARS) SARS-CoV-2. CONCLUSIONS: In organoids, or mini-tumors, established from prostate cancer bone metastasis PDXs, a novel type of dormant ADT-resistant cell with specific gene changes emerged which may be targeted in order to eradicate dormant metastases before they can progress. This study identified a new dormant CRPC basal-luminal hybrid prostate cancer cell and gene signature which may be therapeutically targeted to eradicate dormant CRPC bone metastases in order to prevent disease recurrence. ADT also reduced the cell factors required for SARS-CoV-2 or its variants to infect its host cells and thus may reduce COVID-19 disease severity. The PDX organoid models can be used to screen for therapies that target the dormant CRPC cells and that reduce ACE2 and TMPRSS2 expression to suppress viral load of SARS-CoV-2 and its variants.

A Novel Salicylanilide Derivative Induces Autophagy Cell Death in Castration-Resistant Prostate Cancer via ER Stress-Activated PERK Signaling Pathway.

Metastatic castration-resistant prostate cancer (CRPC) is currently incurable. Cancer growth and progression is intimately affected by its interaction with host microenvironment. Cotargeting of the stroma and prostate cancer is therefore an emerging therapeutic strategy for metastatic CRPC. Cancer-induced osteoclastogenesis is known to contribute to CRPC bone metastasis. This study is to extend pharmacologic value of our synthesized LCC03, a derivative of 5-(2',4'-difluorophenyl)-salicylanilide that has previously testified for its osteoclastogenesis activity, by exploring its additional cytotoxic properties and underlying mechanism in CRPC cells. LCC03 was chemically synthesized and examined for cell growth inhibition in a serial of CRPC cell lines. We demonstrated that LCC03 dose-dependently suppressed proliferation and retarded cell-cycle progression in CRPC cells. The classical autophagy features, including autophagosome formation and LC3-II conversion, were dramatically shown in LCC03-treated CRPC cells, and it was associated with the suppressed AKT/mTOR signaling pathways, a major negative regulator of autophagy. Moreover, an expanded morphology of the endoplasmic reticulum (ER), increased expression of the ER stress markers GRP78 and PERK, and eIF2α phosphorylation were observed. Blockage of autophagy and PERK pathways using small molecule inhibitors or shRNA knockdown reversed LCC03-induced autophagy and cell death, thus indicating that the PERK-eIF2α pathway contributed to the LCC03-induced autophagy. Furthermore, treatment of tumor-bearing mice with intraperitoneal administered LCC03 suppressed the growth of CRPC xenografts in mouse bone without systemic toxicity. The dual action of 5-(2',4'-difluorophenyl)-salicylanilide on targeting both the osteoclasts and the tumor cells strongly indicates that LCC03 is a promising anticancer candidate for preventing and treating metastatic CRPC.

Radium-223 mechanism of action: implications for use in treatment combinations.

The targeted alpha therapy radium-223 (223Ra) can prolong survival in men with castration-resistant prostate cancer (CRPC) who have symptomatic bone metastases and no known visceral metastases. Preclinical studies demonstrate that 223Ra preferentially incorporates into newly formed bone matrix within osteoblastic metastatic lesions. The emitted high-energy alpha particles induce DNA double-strand breaks that might be irreparable and lead to cell death in nearby exposed tumour cells, osteoblasts and osteoclasts. Consequently, tumour growth and abnormal bone formation are inhibited by these direct effects and by the disruption of positive-feedback loops between tumour cells and the bone microenvironment. 223Ra might also modulate immune responses within the bone. The clinical utility of 223Ra has encouraged the development of other anticancer targeted alpha therapies. A thorough understanding of the mechanism of action could inform the design of new combinatorial treatment strategies that might be more efficacious than monotherapy. On the basis of the current mechanistic knowledge and potential clinical benefits, combination therapies of 223Ra with microtubule-stabilizing cytotoxic drugs and agents targeting the androgen receptor axis, immune checkpoint receptors or DNA damage response proteins are being explored in patients with CRPC and metastatic bone disease.

Therapeutic effects of human monoclonal PSMA antibody-mediated TRIM24 siRNA delivery in PSMA-positive castration-resistant prostate cancer.

Background and Aims: Prostate specific membrane antigen (PSMA) is specifically expressed on prostate epithelial cells and markedly overexpressed in almost all prostate cancers. TRIM24 is also up-regulated from localized prostate cancer to metastatic castration-resistant prostate cancer (CRPC). Because of the high relevance of TRIM24 for cancer development and the universal expression of PSMA in CPRC, we investigated the efficacy of human monoclonal PSMA antibody (PSMAb)-based platform for the targeted TRIM24 siRNA delivery and its therapeutic efficacy in CRPC in vivo and in vitro.Methods: The therapeutic complexes were constructed by conjugating PSMAb and sulfo-SMCC-protamine, and encapsulating TRIM24 siRNA. Flow cytometry, immunofluorescence, and fluorescence imaging were performed to detect the receptor-binding, internalization, and targeted delivery of PSMAb-sulfo-SMCC-protamine (PSP)-FAM-siRNA complex (PSPS) in vitro and in vivo. CCK-8, plate-colony formation, apoptosis, cell cycle, and Transwell assays were performed to evaluate the therapeutic potential of the PSP-TRIM24 siRNA complex in vitro, whereas the in vivo therapeutic efficacy was monitored by small animal imaging, radiography, and micro CT.Results: We confirmed that PSP could efficiently protect siRNA from enzymatic digestion, enable targeted delivery of siRNA, and internalize and release siRNA into PSMA-positive (PSMA+) prostate cancer cells in vitro and in vivo. Silencing TRIM24 expression by the PSP-TRIM24 siRNA complex could dramatically suppress proliferation, colony-formation, and invasion of PSMA+ CRPC cells in vitro, and inhibit tumor growth of PSMA+ CRPC xenografts and bone loss in PSMA+ CRPC bone metastasis model without obvious toxicity at therapeutic doses in vivo.Conclusion: PSMAb mediated TRIM24 siRNA delivery platform could significantly inhibit cell proliferation, colony-formation, and invasion in PSMA+ CRPC in vitro and suppressed tumor growth and bone loss in PSMA+ CRPC xenograft and bone metastasis model.

Identification of an oncogenic network with prognostic and therapeutic value in prostate cancer.

Identifying critical pathways governing disease progression is essential for accurate prognosis and effective therapy. We developed a broadly applicable and novel systems‐level gene discovery strategy. This approach focused on constitutively active androgen receptor (AR) splice variant‐driven pathways as representative of an intractable mechanism of prostate cancer (PC) therapeutic resistance. We performed a meta‐analysis of human prostate samples using weighted gene co‐expression network analysis combined with experimental AR variant transcriptome analyses. An AR variant‐driven gene module that is upregulated during human PC progression was identified. We filtered this module by identifying genes that functionally interacted with AR variants using a high‐throughput synthetic genetic array screen in Schizosaccharomyces pombe. This strategy identified seven AR variant‐regulated genes that also enhance AR activity and drive cancer progression. Expression of the seven genes predicted poor disease‐free survival in large independent PC patient cohorts. Pharmacologic inhibition of interacting members of the gene set potently and synergistically decreased PC cell proliferation. This unbiased and novel gene discovery strategy identified a clinically relevant, oncogenic, interacting gene hub with strong prognostic and therapeutic potential in PC.

Abstract 1162: Castration-resistant prostate cancer bone metastasis model to assess new therapeutics

Abstract The role of androgens in the regulation of growth of primary prostate cancer is well established but less recognized in the context of advanced bone metastatic disease, which is the cause of high morbidity in patients. At advanced stage, prostate cancer cells typically lose their dependence on endogenous androgens leading to metastatic castration resistant prostate cancer (mCRPC). The aim of the study was to establish a novel mCRPC model resembling clinical aspects of the bone metastatic disease, which could be used for the evaluation of efficacy of new therapies. To obtain a model for castration resistant growth, 5-6 weeks old NOD.Scid male mice were divided into three study groups. Mice in two study groups were castrated either before inoculation of the cancer cells or at four weeks after inoculation when the tumors were already growing. The mice in one study group were left intact. All mice received an intratibial injection of 2x106 VCaP human prostate cancer cells originally derived from vertebral metastatic site. Tumor growth was followed biweekly for 16 weeks by PSA measurements and X-ray imaging of tumor induced bone changes (lesions). At endpoint, androgen dependent organs were weighed and tumor-bearing tibias were subjected to histological evaluation. At endpoint, a tumor take of 60%, 40% and 50% was observed in the intact mice and in the mice castrated one week before and four weeks after VCaP cell inoculation, respectively. Tumor take was assessed by PSA, X-ray and histology at endpoint. The PSA levels were higher in the intact mice and in the mice castrated at four weeks compared to the mice castrated before cancer cell inoculation. The PSA levels became detectable between 6-8 weeks after cancer cell inoculation in the intact mice and in the mice castrated at four weeks. In the mice castrated before cancer cell inoculation the PSA levels started to increase very late at 14-16 weeks. VCaP cells induced mainly new bone formation, an osteoblastic bone reaction typical for prostate cancer patients. In castrated mice, fewer bone lesions were observed compared to intact mice. The bone lesion areas quantified from X-ray images were larger in the intact mice compared to the mice castrated before cancer cell inoculation. As expected, the weight of androgen dependent organs was lower in the castrated mice compared to the intact mice. In conclusion, a model mimicking important clinical aspects of castration resistant prostate cancer bone metastases was established. Tumor take and growth rates indicated that early phases of tumor development into the bone are androgen dependent whereas tumor growth at later stage relies on the bone microenvironment. The results highlight the significance of the tumor microenvironment in establishing clinically relevant preclinical models for drug development. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jussi M. Halleen, Jenni Bernoulli, Pascale Lejeune. Castration-resistant prostate cancer bone metastasis model to assess new therapeutics [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 1162.

Hematologic Toxicity From Radium-223 Therapy for Bone Metastases in Castration-Resistant Prostate Cancer: Risk Factors and Practical Considerations.

Radium-223 dichloride is an α-emitting radiopharmaceutical that localizes to bone matrix and is approved for the treatment of patients with metastatic castration-resistant prostate cancer (CRPC) and symptomatic bone metastases. The cumulative impact of Ra-223 and other therapeutic agents for metastatic CRPC on myelosuppression in bone marrow is unknown. The phase 3 randomized, double-blind, placebo-controlled ALSYMPCA trial of Ra-223 in patients with CRPC and symptomatic bone metastases demonstrated a significant improvement in overall survival. Of the 571 patients subsequently followed for 3 years, few in either the Ra-223 or placebo arm experienced hematologic adverse events. Little evidence shows secondary malignancies associated with Ra-223 treatment; only 2 cases of secondary leukemia after Ra-223 treatment were found in the literature. The goals of this review were to summarize safety and efficacy results from clinical trials and institutional safety data pertaining to hematologic adverse events occurring with Ra-223, and to discuss practical management issues.

AB065. EP4 antagonist suppresses bone metastasis in prostate cancer

BackgroundBone metastasis is one of the most common complications in clinical intractable castration-resistant prostate cancer (CRPC) and advanced prostate cancer period. The invasion ability of cancer cells was thought to have important relationship with the potential of cancer metastasis. EP4 receptor is overexpressed in several cancers, and enhanced EP4 receptor signaling has been previously shown to correlate with the invasion of several different cancer types. In present study, we found that EP4 antagonist can in vitro suppress the invasion phenotype abilities of CRPC PC3 cells, LNCaP cells with EP4 overexpressed which have the tendency to castration-resistance and in vivo suppress the CRPC bone metastasis animal models.MethodsRNA interference was used to stably transfected EP4 to LNCaP with the pcDNA3.1-EP4, so as to be the LNCaP with vector control (LNCaP-mock) and LNCaP with EP4 overexpressed (LNCaP/EP4+) as described in our previous article. Effect on cell proliferation was determined firstly by the MTT assay. Transwell invasion assay was used to in vitro examine the metastasis abilities of PC3 cells, LNCaP cells, LNCaP/EP4+ and all after EP4 antagonist affected. Cells with ONO-AE3-208 at the final concentration of 0.1, 1 and 10 µmol/L were administrated to be the experimental group. To observe the effect of EP4 antagonist on the bone metastasis of prostate cancer in vivo, PC3/Luc cells were inoculated into the left cardiac ventricle of 5-week-old male nude mice. The mice developed osteolytic lesions with a mean endpoint at 52±7 days. To assess whether EP4 antagonist suppresses bone metastasis, ONO-AE3-208 (10 mg/kg/d) was given to the animals 5 days a week i.p. which the same volume of DDW was given as the control. To detect PC3 cell dissemination, bioluminescent imaging (BLI) was applied using a cooled CCD camera. We evaluated the efficacy of ONO-AE3-208 by measuring the photon counts of the metastatic lesions in the mandible and both hip joints in a blinded manner.ResultsThe invasion assay showed that ONO-AE3-208 can inhibit the invasive abilities of both PC3 and LNCaP/EP4+ in a dose-dependent manner (P<0.01). In animal experiment, metastatic bone lesions in the control group progressed while the photon emission was significantly suppressed in the ONO-AE3-208 treatment group during the observation period from day 28 every 7 days (P<0.01).ConclusionsWe demonstrated that EP4 antagonist can in vitro inhibit the invasive abilities of CRPC PC3 and LNCaP/EP4+ cells. The administration of it can also suppress the bone metastasis of CRPC in vivo.

An exploratory analysis of alkaline phosphatase, lactate dehydrogenase, and prostate-specific antigen dynamics in the phase 3 ALSYMPCA trial with radium-223

BackgroundBaseline clinical variables are prognostic for overall survival (OS) in patients with castration-resistant prostate cancer (CRPC). Their prognostic and predictive value with agents targeting bone metastases, such as radium-223, is not established.Patients and methodsThe radium-223 ALSYMPCA trial enrolled patients with CRPC and symptomatic bone metastases. Prognostic potential of baseline variables was assessed using Cox models. Percentage changes in biomarker levels from baseline were evaluated during the trial period; changes from baseline to week 12 were evaluated for association with OS and surrogacy.ResultsEastern Cooperative Oncology Group performance status, total alkaline phosphatase (tALP), lactate dehydrogenase (LDH), and prostate-specific antigen (PSA) at baseline were associated with OS (P ≤ 0.0003) in the intent-to-treat population (radium-223, N = 614; placebo, N = 307). tALP declined from baseline within 4 weeks after beginning radium-223, by week 12 declining in 87% of radium-223 and 23% of placebo patients (P < 0.001). LDH declined in 51% and 34% (P = 0.003), whereas PSA declined in 27% and 14% (P = 0.160). Mean tALP change from baseline was 32.2% decrease with radium-223 and 37.2% increase with placebo. Radium-223 patients with tALP decline from baseline to week 12 (confirmed ≥3 weeks from week 12) had 55% lower risk of death (hazard ratio = 0.45; 95% CI 0.34–0.61) versus those with no confirmed tALP decline. Proportional treatment effect (PTE) values for tALP, LDH, and PSA changes from baseline at week 12 as OS surrogate markers were 0.34 (95% CI: 0–0.746), 0.07 (95% CI: 0–0.211), and 0 (95% CI: 0–0.082), respectively.ConclusionsSignificant tALP declines (versus placebo) occurred as early as 4 weeks after beginning radium-223 therapy. tALP or LDH declines at 12 weeks correlated with longer OS, but did not meet statistical surrogacy requirements. Dynamic changes in tALP and LDH during radium-223 treatments may be useful to monitor, but do not serve as surrogates for survival.

High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.

The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion. AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc.

Subgroups of Castration-resistant Prostate Cancer Bone Metastases Defined Through an Inverse Relationship Between Androgen Receptor Activity and Immune Response

BackgroundNovel therapies for men with castration-resistant prostate cancer (CRPC) are needed, particularly for cancers not driven by androgen receptor (AR) activation. ObjectivesTo identify molecular subgroups of PC bone metastases of relevance for therapy. Design, setting, and participantsFresh-frozen bone metastasis samples from men with CRPC (n=40), treatment-naïve PC (n=8), or other malignancies (n=12) were characterized using whole-genome expression profiling, multivariate principal component analysis (PCA), and functional enrichment analysis. Expression profiles were verified by reverse transcription–polymerase chain reaction (RT-PCR) in an extended set of bone metastases (n=77) and compared to levels in malignant and adjacent benign prostate tissue from patients with localized disease (n=12). Selected proteins were evaluated using immunohistochemistry. A cohort of PC patients (n=284) diagnosed at transurethral resection with long follow-up was used for prognostic evaluation. Results and limitationsThe majority of CRPC bone metastases (80%) was defined as AR-driven based on PCA analysis and high expression of the AR, AR co-regulators (FOXA1, HOXB13), and AR-regulated genes (KLK2, KLK3, NKX3.1, STEAP2, TMPRSS2); 20% were non–AR-driven. Functional enrichment analysis indicated high metabolic activity and low immune responses in AR-driven metastases. Accordingly, infiltration of CD3+ and CD68+ cells was lower in AR-driven than in non–AR-driven metastases, and tumor cell HLA class I ABC immunoreactivity was inversely correlated with nuclear AR immunoreactivity. RT-PCR analysis showed low MHC class I expression (HLA-A, TAP1, and PSMB9 mRNA) in PC bone metastases compared to benign and malignant prostate tissue and bone metastases of other origins. In primary PC, low HLA class I ABC immunoreactivity was associated with high Gleason score, bone metastasis, and short cancer-specific survival. Limitations include the limited number of patients studied and the single metastasis sample studied per patient. ConclusionsMost CRPC bone metastases show high AR and metabolic activities and low immune responses. A subgroup instead shows low AR and metabolic activities, but high immune responses. Targeted therapy for these groups should be explored. Patient summaryWe studied heterogeneities at a molecular level in bone metastasis samples obtained from men with castration-resistant prostate cancer. We found differences of possible importance for therapy selection in individual patients.

Epithelial mesenchymal-like transition occurs in a subset of cells in castration resistant prostate cancer bone metastases.

TGFβ is a known driver of epithelial-mesenchymal transition (EMT) which is associated with tumor aggressiveness and metastasis. However, EMT has not been fully explored in clinical specimens of castration-resistant prostate cancer (CRPC) metastases. To assess EMT in CRPC, gene expression analysis was performed on 149 visceral and bone metastases from 62 CRPC patients and immunohistochemical analysis was performed on 185 CRPC bone and visceral metastases from 42 CRPC patients. In addition, to assess the potential of metastases to seed further metastases the mitochondrial genome was sequenced at different metastatic sites in one patient. TGFβ was increased in bone versus visceral metastases. While primarily cytoplasmic; nuclear and cytoplasmic Twist were significantly higher in bone than in visceral metastases. Slug and Zeb1 were unchanged, with the exception of nuclear Zeb1 being significantly higher in visceral metastases. Importantly, nuclear Twist, Slug, and Zeb1 were only present in a subset of epithelial cells that had an EMT-like phenotype. Underscoring the relevance of EMT-like cells, mitochondrial sequencing revealed that metastases could seed additional metastases in the same patient. In conclusion, while TGFβ expression and EMT-associated protein expression is present in a considerable number of CRPC visceral and bone metastases, nuclear Twist, Slug, and Zeb1 localization and an EMT-like phenotype (elongated nuclei and cytoplasmic compartment) was only present in a small subset of CRPC bone metastases. Mitochondrial sequencing from different metastases in a CRPC patient provided evidence for the seeding of metastases from previously established metastases, highlighting the biological relevance of EMT-like behavior in CRPC metastases.

Relationship between serum response factor and androgen receptor in prostate cancer.

Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. Our data support SRF as a promising therapeutic target in combination with current treatments.

Whole transcriptome sequencing reveals extensive unspliced mRNA in metastatic castration-resistant prostate cancer.

Men with metastatic prostate cancer who are treated with androgen deprivation therapies (ADT) usually relapse within 2 to 3 years with disease that is termed castration-resistant prostate cancer (CRPC). To identify the mechanism that drives these advanced tumors, paired-end RNA-sequencing (RNA-seq) was performed on a panel of CRPC bone marrow biopsy specimens. From this genome-wide approach, mutations were found in a series of genes with prostate cancer relevance, including AR, NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1, and CDH1, and a novel SND1:BRAF gene fusion. Among the most highly expressed transcripts were 10 noncoding RNAs (ncRNAs), including MALAT1 and PABPC1, which are involved in RNA processing. Notably, a high percentage of sequence reads mapped to introns, which were determined to be the result of incomplete splicing at canonical splice junctions. Using quantitative PCR (qPCR), a series of genes (AR, KLK2, KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a greater proportion of unspliced RNA in CRPC specimens than in normal prostate epithelium, untreated primary prostate cancer, and cultured prostate cancer cells. This inefficient coupling of transcription and mRNA splicing suggests an overall increase in transcription or defect in splicing. Inefficient splicing in advanced prostate cancer provides a selective advantage through effects on microRNA networks but may render tumors vulnerable to agents that suppress rate-limiting steps in splicing.

Abstract 3808: Preclinical efficacy of the novel, oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) on castration resistant prostate cancer

Abstract Exportin 1/ Chromosomal Maintenance Protein 1 (XPO1/CRM1) is a key nuclear export protein whose inhibition leads to the nuclear accumulation of tumor suppressor proteins such as p53, FOXO, PTEN, pRB and I-κB. Selinexor is an orally bioavailable XPO1 inhibitor that represents a novel class of small molecule compounds with potent activity against a wide variety of cancers. Here we report the activity of Selinexor against Castration resistant prostate cancer (CRPC). CRPC progression is mediated by activation of various adaptive Androgen Receptor (AR) signaling pathways. These pathways are currently being targeted by novel anti-androgen therapies such as abiraterone acetate and enzalutamide with favorable outcomes. However, resistance to anti-androgen therapy can be developed through deregulation of tumor suppressor pathways. XPO-1 is highly expressed in prostate cancer cells and therefore we tested the effects of Selinexor on CRPC cells and tumors models. Treatment of the C4-2B prostate cancer cell line with Selinexor in vitro significantly inhibited cell proliferation and resulted in nuclear accumulation of p53 and p21. In addition, Selinexor inhibited expression of the tumor-promoting gene Ubiquitin-Conjugating Enzyme E2C (UBE2C), which is activated by AR in these cells. Selinexor was also tested in tumor graft models of two patient tumors in castrated male mice. The MDA-PCa-133 tumor is an adenocarcinoma derived from a clinical CRPC bone metastasis. Subcutaneous MDA-PCa-133 tumors in castrated male mice express full-length AR and Prostate-Specific Antigen (PSA) but lack expression of p53. The MDA-PCa-144-13 tumor is a small cell carcinoma derived from a lethal variant of prostate cancer with anaplastic clinical phenotype and does not express AR or PSA but expresses a gain-of-function p53 mutant. Vehicle or Selinexor treatment (10 mg/kg p.o. QoDX3 M/W/F) of MDA-PCa-133 tumors for 34 days resulted in almost complete inhibition of tumor growth with a 30-fold reduction of PSA (p&amp;lt;0.001). Treatment of MDA-PCa-144-13 tumors for 22 days also resulted in significant reduction of tumor volume (&amp;gt;8 fold compared to vehicle only; p&amp;lt;0.0047). Immunohistochemical analysis of MDA-PCa-133 tumors showed that Selinexor induced nuclear accumulation of XPO1 cargos FOXO3a and p27 with concomitant reduction in the proliferation marker KI67. In conclusion, Selinexor demonstrated robust inhibition of CRPC tumor growth and activated multiple tumor suppressors in prostate cancer cells. Selinexor is now being evaluated in Phase 1 clinical studies in patients with advanced hematological and solid tumor cancers, and preliminary results show adequate tolerability with evidence of anti cancer activity. Future clinical studies in patients with CRPC are planned and will provide more information on the use of Selinexor as monotherapy and in combination with anti-androgen therapy of CRPC. Citation Format: Sankar Narayan Maity, Guanglin Wu, Jing-Fang Lu, Anh Hoang, Yosef Landesman, Dilara McCauley, Sharon Shacham, Michael G. Kauffman, Ana M. Aparicio, Eleni Efstathiou, John C. Araujo, Christopher J. Logothetis. Preclinical efficacy of the novel, oral Selective Inhibitor of Nuclear Export (SINE) Selinexor (KPT-330) on castration 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 3808. doi:10.1158/1538-7445.AM2014-3808

Abstract 4599: Negative feedback loop between serum response factor and androgen receptor in castrate-resistant prostate cancer

Abstract Despite initial response to androgen ablation, patients with advanced disease relapse to develop castrate-resistant prostate cancer (CRPC) which is difficult to treat. We previously identified Serum Response Factor (SRF) as an important transcription factor in an in vitro model of CRPC. Since CRPC is associated with androgen receptor (AR) hypersensitivity and previous studies linked SRF with AR, we investigated the relationship between SRF and AR. DHT stimulation increased SRF transcriptional activity in both parental LNCaP cells and their castration-resistant Abl subline. To investigate AR involvement in this process, AR expression was down-regulated using siRNA, resulting in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in the AR-negative PC346 DCC cells. However, when AR was over-expressed, SRF induction post-DHT was restored. To explore whether this was reciprocal, SRF was down-regulated by siRNA and AR transcriptional activity and protein expression were assessed. These experiments showed AR increased activity and expression following SRF knock-down, suggesting a negative feedback loop, which was confirmed by immunohistochemistry (IHC) in CRPC bone metastases (n=42), showing a negative association between SRF and AR positivity. This negative feedback was also supported by a computational model based on our experimental data. We next assessed cell proliferation following SRF inhibition and demonstrated that SRF inhibition by siRNA or CCG-1423 was as effective as AR inhibition in decreasing cell proliferation. Moreover concomitant inhibition of AR and SRF showed a synergistic effect. To confirm SRF relevance in clinical samples, we performed IHC staining for SRF on 94 primary tumours and 21 castration-resistant patients who received a transurethral resection of the prostate. This analysis showed that SRF nuclear staining was associated with castration-resistance with 95% of castration-resistant patients showing positive SRF staining versus only 50% of primary tumours. In addition SRF expression in bone metastases in a cohort of 38 patients who died of CRPC, was an independent predictor of survival from (a) prostate cancer diagnosis and (b) diagnosis with CRPC. Overall our data highlight the importance of SRF in CRPC and support SRF as a promising therapeutic target alone or in combination with current treatments. Citation Format: Maria Prencipe, Gillian O'Hurley, Amanda O'Neill, Dara Lundon, Lan K. Nguyen, Susie Boyce, Colm Morrissey, Helmut Klocker, Elaine W. Kay, William Gallagher, William Watson. Negative feedback loop between serum response factor and androgen receptor 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 4599. doi:10.1158/1538-7445.AM2014-4599