Targeting the fibroblast growth factor pathway in molecular subtypes of castration-resistant prostate cancer.

BACKGROUND: Androgen receptor (AR) pathway inhibition remains the cornerstone for prostate cancer therapies. However, castration-resistant prostate cancer (CRPC) tumors can resist AR signaling inhibitors through AR amplification and AR splice variants in AR-positive CRPC (ARPC), and conversion to AR-null phenotypes, such as double-negative prostate cancer (DNPC) and small cell or neuroendocrine prostate cancer (SCNPC). We have shown previously that DNPC can bypass AR-dependence through fibroblast growth factor receptor (FGFR) signaling. However, the role of the FGFR pathway in other CRPC phenotypes has not been elucidated. METHODS: RNA-Seq analysis was conducted on patient metastases, LuCaP patient-derived xenograft (PDX) models, and CRPC cell lines. Cell lines (C4-2B, VCaP, and 22Rv1) and ex vivo LuCaP PDX tumor cells were treated with enzalutamide (ENZA) and FGFR inhibitors (FGFRi) alone or in combination and sensitivity was determined using cell viability assays. In vivo efficacy of FGFRi in ARPC, DNPC, and SCNPC were evaluated using PDX models. RESULTS: RNA-Seq analysis of FGFR signaling in metastatic specimens, LuCaP PDX models, and CRPC cell lines revealed significant FGF pathway activation in AR-low PC (ARLPC), DNPC, and SCNPC tumors. In vitro/ex vivo analysis of erdafitinib and CH5183284 demonstrated robust and moderate growth suppression of ARPC, respectively. In vivo studies using four ARPC PDX models showed that combination ENZA and CH5183284 significantly suppressed tumor growth. Additional in vivo studies using four ARPC PDX models revealed that erdafitinib monotherapy was as effective as ENZA in suppressing tumor growth, and there was limited combination benefit. Furthermore, two of three DNPC models and two of four SCNPC models responded to CH5183284 monotherapy, suggesting FGFRi responses were model dependent. RNA-Seq and gene set enrichment analysis of end-of-study ARPC tumors treated with FGFRi displayed decreased expression of E2F and MYC target genes and suppressed G2M checkpoint genes, whereas end-of-study SCNPC tumors had heterogeneous transcriptional responses. CONCLUSIONS: Although FGFRi treatments suppressed tumor growth across CRPC phenotypes, our analyses did not identify a single pathway or biomarker that would identify tumor response to FGFRi. This is very likely due to the array of FGFR1-4 expression and tumor phenotypes present in CRPC. Nevertheless, our data nominate the FGFR pathway as a clinically actionable target that promotes tumor growth in diverse phenotypes of treatment-refractory metastatic CRPC.

LuCaP Prostate Cancer Patient-Derived Xenografts Reflect the Molecular Heterogeneity of Advanced Disease an--d Serve as Models for Evaluating Cancer Therapeutics.

BACKGROUND: Metastatic prostate cancer is a common and lethal disease for which there are no therapies that produce cures or long-term durable remissions. Clinically relevant preclinical models are needed to increase our understanding of biology of this malignancy and to evaluate new agents that might provide effective treatment. Our objective was to establish and characterize patient-derived xenografts (PDXs) from advanced prostate cancer (PC) for investigation of biology and evaluation of new treatment modalities. METHODS: Samples of advanced PC obtained from primary prostate cancer obtained at surgery or from metastases collected at time of death were implanted into immunocompromised mice to establish PDXs. Established PDXs were propagated in vivo. Genomic, transcriptomic, and STR profiles were generated. Responses to androgen deprivation and docetaxel in vivo were characterized. RESULTS: We established multiple PDXs (LuCaP series), which represent the major genomic and phenotypic features of the disease in humans, including amplification of androgen receptor, PTEN deletion, TP53 deletion and mutation, RB1 loss, TMPRSS2-ERG rearrangements, SPOP mutation, hypermutation due to MSH2/MSH6 genomic aberrations, and BRCA2 loss. The PDX models also exhibit variation in intra-tumoral androgen levels. Our in vivo results show heterogeneity of response to androgen deprivation and docetaxel, standard therapies for advanced PC, similar to the responses of patients to these treatments. CONCLUSIONS: The LuCaP PDX series reflects the diverse molecular composition of human castration-resistant PC and allows for hypothesis-driven cause-and-effect studies of mechanisms underlying treatment response and resistance. Prostate 77: 654-671, 2017. © 2017 The Authors. The Prostate Published by Wiley Periodicals, Inc.

Addition of PSMA ADC to enzalutamide therapy significantly improves survival in in vivo model of castration resistant prostate cancer.

BACKGROUND: Despite multiple new therapies available to patients with advanced castration-resistant prostate cancer (CRPC), the overall survival benefit still remains relatively short. Therefore, it is important to investigate additional treatment options that could achieve greater efficacy. Because of tumor heterogeneity and the development of resistance to treatment with single agents, combination therapies using existing drugs with new agents can potentially broaden individual therapeutic windows and achieve improved efficacy and safety profiles. The objective of the current studies was to evaluate the efficacy of combination of enzalutamide (ENZ) with prostate specific membrane antigen antibody drug conjugate (PSMA ADC) to inhibit CRPC patient-derived xenografts (PDX) in a preclinical setting. METHODS: Subcutaneous LuCaP 96CR prostate cancer PDX bearing mice were treated with a single dose of PSMA ADC (2.0 mg/kg) or 5 days a week ENZ (50 mg/kg) as monotherapy or with a combination of these two agents. The effects of the PSMA ADC+ENZ combination were compared to PSMA ADC alone, ENZ alone, and placebo control. IHC analyses were performed to determine PSMA, AR, ARV7, and GR expression and effects on proliferation. RESULTS: All treatments inhibited tumor progression but with different efficacy. At 6 weeks, in the control and ENZ groups all tumors were progressing, while in the PSMA ADC group only 5/11 were progressing, two remained unchanged and four tumors had decreased tumor volume. Moreover, all animals in the PSMA ADC+ENZ group had smaller tumors at week 6 when compared to their size at enrollment (week 0). A 14-week followup showed that all three treatments resulted in significant survival benefits but the combination effects were the most pronounced resulting in PSMA ADC+ENZ versus ENZ HR = 0.093 (P = 0.0045) and PSMA ADC+ENZ versus PSMA ADC HR = 0.051 (P = <0.0001) with no deaths observed in the combination group. CONCLUSIONS: Our results clearly indicate that the combination of PSMA ADC+ENZ possesses strong antitumor activity and significantly improves survival over ENZ monotherapy using the LuCaP 96CR PDX model. These results provide a strong rationale for clinical testing of PSMA ADC in combination with ENZ and/or other androgen-directed treatment strategies.

Efficacy studies of an antibody-drug conjugate PSMA-ADC in patient-derived prostate cancer xenografts.

BACKGROUND: It is timely and important to develop new treatment modalities for advanced prostate cancer, because even the newly FDA approved treatments, despite providing significant survival benefits, do not constitute cure of this disease. Antibody drug conjugates (ADCs) represent a promising approach to cancer therapy. Prostate-specific membrane antigen (PSMA) is expressed in advanced prostate cancer and targeting this protein is used for imaging of advanced prostate cancer as well as development of targeting strategies. The objective of our studies was to evaluate the efficacy of PSMA ADC against a series of patient-derived prostate cancer xenografts (LuCaP 58, LuCaP 77, LuCaP 96CR, and LuCaP 105) with different characteristics, including varying levels of PSMA expression and responses to androgen suppression. METHODS: Mice bearing subcutaneous LuCaP prostate cancer-derived xenografts received PSMA antibody monomethyl auristatin E (MMAE) drug conjugate (PSMA ADC) in which the antibody and MMAE are linked via a protease-cleavable linker. PSMA ADC dose ranged from 1 to 6 mg/kg. Unmodified PSMA mAb + free MMAE at the amount equivalent to those contained in 6 mg/kg PSMA ADC was used as control. All treatments were administered once a week via tail-vein injections and repeated four times once a week and tumor responses were monitored for 10 weeks. IHC analyses were performed to determine PSMA and AR expression and effects on proliferation. RESULTS: Treatment responses varied widely across the tumor models, from complete tumor regressions in LuCaP 96CR to largely unimpeded tumor progression of LuCaP 58, which had the lowest baseline level of PSMA expression. Intermediate antitumor effects were seen for LuCaP 77 and LuCaP 105 tumors, despite their having similar basal expression of PSMA as LuCaP 96CR. Interestingly, we detected substantial differences in responses even within the same model, indicating that PSMA expression is not the only factor involved in treatment outcomes. CONCLUSIONS: Our results show high efficacy of PSMA ADC in advanced prostate cancer but also considerable variability in effects despite PSMA expression. Further studies to identify tumor characteristics that are predictive of treatment response are ongoing.

Inhibition of CCL2 signaling in combination with docetaxel treatment has profound inhibitory effects on prostate cancer growth in bone.

The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.

Concurrent Targeting of HDAC and PI3K to Overcome Phenotypic Heterogeneity of Castration-resistant and Neuroendocrine Prostate Cancers.

Castration-resistant prostate cancer (CRPC) consists of multiple phenotypic subtypes including androgen receptor (AR)-active prostate cancer (ARPC) and neuroendocrine prostate cancer (NEPC). Tumor cells with these phenotypes can coexist between metastases within a patient and within an individual tumor. Treatments that are effective across CRPC subtypes are currently lacking. Histone deacetylation is crucial for the regulation of chromatin structure and maintenance of cancer cell state and activation of the PI3K/AKT/mTOR signaling cascade is a tumor growth-promoting pathway. We therefore investigated combined targeting of histone deacetylase (HDAC) and PI3K using a rationally designed dual inhibitor, fimepinostat, in CRPC subtypes in vitro and in vivo. Dual HDAC1/2 and PI3K/AKT pathway inhibition by fimepinostat led to robust tumor growth inhibition in both ARPC and NEPC models including cell line- and patient-derived xenografts. HDAC1/2 inhibition combined with PI3K/AKT inhibition was more effective than targeting each pathway alone, producing growth inhibitory effects through cell-cycle inhibition and apoptosis. Molecular profiling revealed on-target effects of combined HDAC1/2 and PI3K/AKT inhibition independent of tumor phenotype. Fimepinostat therapy was also associated with the suppression of lineage transcription factors including AR in ARPC and Achaete-scute homolog 1 (ASCL1) in NEPC. Together, these results indicate that fimepinostat represents a novel therapeutic that may be effective against both ARPC and NEPC through CRPC subtype-dependent and -independent mechanisms. SIGNIFICANCE: CRPC is a heterogeneous disease constituting multiple phenotypic subtypes that often co-occur within tumors or across metastases in patients. Existing targeted therapies for CRPC do not take this into account. Here we show that fimepinostat, a dual HDAC1/2 and PI3K/AKT inhibitor investigated clinically in other cancer types but not prostate cancer, may overcome this heterogeneity by effectively inhibiting both ARPC and NEPC subtypes of CRPC.

Enhancer profiling identifies epigenetic markers of endocrine resistance and reveals therapeutic options for metastatic castration-resistant prostate cancer patients.

Androgen Receptor (AR) signaling inhibitors, including enzalutamide, are treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC), but resistance inevitably develops. Using metastatic samples from a prospective phase II clinical trial, we epigenetically profiled enhancer/promoter activities with H3K27ac chromatin immunoprecipitation followed by sequencing, before and after AR-targeted therapy. We identified a distinct subset of H3K27ac-differentially marked regions that associated with treatment responsiveness. These data were successfully validated in mCRPC patient-derived xenograft models (PDX). In silico analyses revealed HDAC3 as a critical factor that can drive resistance to hormonal interventions, which we validated in vitro . Using cell lines and mCRPC PDX tumors in vitro , we identified drug-drug synergy between enzalutamide and the pan-HDAC inhibitor vorinostat, providing therapeutic proof-of-concept. These findings demonstrate rationale for new therapeutic strategies using a combination of AR and HDAC inhibitors to improve patient outcome in advanced stages of mCRPC.

Response to supraphysiological testosterone is predicted by a distinct androgen receptor cistrome.

The androgen receptor (AR) is a master transcription factor that regulates prostate cancer (PC) development and progression. Inhibition of AR signaling by androgen deprivation is the first-line therapy with initial efficacy for advanced and recurrent PC. Paradoxically, supraphysiological levels of testosterone (SPT) also inhibit PC progression. However, as with any therapy, not all patients show a therapeutic benefit, and responses differ widely in magnitude and duration. In this study, we evaluated whether differences in the AR cistrome before treatment can distinguish between SPT-responding (R) and -nonresponding (NR) tumors. We provide the first preclinical evidence to our knowledge that SPT-R tumors exhibit a distinct AR cistrome when compared with SPT-NR tumors, indicating a differential biological role of the AR. We applied an integrated analysis of ChIP-Seq and RNA-Seq to the pretreatment tumors and identified an SPT-R signature that distinguishes R and NR tumors. Because transcriptomes of SPT-treated clinical specimens are not available, we interrogated available castration-resistant PC (CRPC) transcriptomes and showed that the SPT-R signature is associated with improved survival and has the potential to identify patients who would respond to SPT. These findings provide an opportunity to identify the subset of patients with CRPC who would benefit from SPT therapy.

Cabozantinib can block growth of neuroendocrine prostate cancer patient-derived xenografts by disrupting tumor vasculature.

With the advent of potent second-line anti-androgen therapy, we and others have observed an increased incidence of androgen receptor (AR)-null small cell or neuroendocrine prostate cancer (SCNPC) in metastatic castration-resistant prostate cancer (mCRPC). Our study was designed to determine the effect of cabozantinib, a multi-targeted tyrosine kinase inhibitor that inhibits VEGFR2, MET and RET on SCNPC. Transcriptome analysis of the University of Washington rapid autopsy and SU2C mCRPC datasets revealed upregulated MET and RET expression in SCNPCs relative to adenocarcinomas. Additionally, increased MET expression correlated with attenuated AR expression and activity. In vitro treatment of SCNPC patient-derived xenograft (PDX) cells with the MET inhibitor AMG-337 had no impact on cell viability in LuCaP 93 (MET+/RET+) and LuCaP 173.1 (MET-/RET-), whereas cabozantinib decreased cell viability of LuCaP 93, but not LuCaP 173.1. Notably, MET+/RET+ LuCaP 93 and MET-/RET- LuCaP 173.1 tumor volumes were significantly decreased with cabozantinib treatment in vivo, and this activity was independent of MET or RET expression in LuCaP 173.1. Tissue analysis indicated that cabozantinib did not inhibit tumor cell proliferation (Ki67), but significantly decreased microvessel density (CD31) and increased hypoxic stress and glycolysis (HK2) in LuCaP 93 and LuCaP 173.1 tumors. RNA-Seq and gene set enrichment analysis revealed that hypoxia and glycolysis pathways were increased in cabozantinib-treated tumors relative to control tumors. Our data suggest that the most likely mechanism of cabozantinib-mediated tumor growth suppression in SCNPC PDX models is through disruption of the tumor vasculature. Thus, cabozantinib may represent a potential therapy for patients with metastatic disease in tumor phenotypes that have a significant dependence on the tumor vasculature for survival and proliferation.

RNA Splicing Factors SRRM3 and SRRM4 Distinguish Molecular Phenotypes of Castration-Resistant Neuroendocrine Prostate Cancer.

Neuroendocrine (NE) differentiation in metastatic castration-resistant prostate cancer (mCRPC) is an increasingly common clinical feature arising from cellular plasticity. We recently characterized two mCRPC phenotypes with NE features: androgen receptor (AR)-positive NE-positive amphicrine prostate cancer (AMPC) and AR-negative small cell or neuroendocrine prostate cancer (SCNPC). Here, we interrogated the regulation of RE1-silencing transcription factor (REST), a transcriptional repressor of neuronal genes, and elucidated molecular programs driving AMPC and SCNPC biology. Analysis of prostate cancer cell lines, mCRPC specimens, and LuCaP patient-derived xenograft models detected alternative splicing of REST to REST4 and attenuated REST repressor activity in AMPC and SCNPC. The REST locus was also hypermethylated and REST expression was reduced in SCNPC. While serine/arginine repetitive matrix protein 4 (SRRM4) was previously implicated in alternative splicing of REST in mCRPC, we detected SRRM3 expression in REST4-positive, SRRM4-negative AMPC, and SCNPC. In CRPC cell lines, SRRM3 induced alternative splicing of REST to REST4 and exacerbated the expression of REST-repressed genes. Furthermore, SRRM3 and SRRM4 expression defined molecular subsets of AMPC and SCNPC across species and tumor types. Two AMPC phenotypes and three SCNPC phenotypes were characterized, denoted either by REST attenuation and ASCL1 activity or by progressive activation of neuronal transcription factor programs, respectively. These results nominate SRRM3 as the principal REST splicing factor expressed in early NE differentiation and provide a framework to molecularly classify diverse NE phenotypes in mCRPC. SIGNIFICANCE: This study identifies SRRM3 as a key inducer of cellular plasticity in prostate cancer with neuroendocrine features and delineates distinct neuroendocrine phenotypes to inform therapeutic development and precision medicine applications.

The expression of osteoclastogenesis-associated factors and osteoblast response to osteolytic prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) has a propensity to metastasize to bone. Tumor cells replace bone marrow and can elicit an osteoblastic, osteolytic, or mixed bone response. Our objective was to elucidate the mechanisms and key factors involved in promoting osteoclastogenesis in PCa bone metastasis. METHODS: We cultured osteoblast-like MC3T3-E1 cells with conditioned medium (CM) from PC-3 and C4-2B cells. MC3T3-E1 mineralization decreased in the presence of PC-3 CM, whereas C4-2B CM had no effect on mineralization. Using oligo arrays and validating by real-time PCR, we observed a decrease in the expression of mineralization-associated genes in MC3T3-E1 cells grown in the presence of PC-3 CM. In addition, PC-3 CM induced the expression of osteoclastogenesis-associated genes IGFBP-5, IL-6, MCP-1, and RANKL while decreasing OPG expression in MC3T3-E1 cells. Furthermore, CM from MC3T3-E1 cells cultured in the presence of PC-3 CM, in association with soluble RANKL, increased osteoclastogenesis in RAW 264.7 cells. Investigation of PCa metastases and xenografts by immunohistochemistry revealed that the osteoclastic factor IL-6 was expressed in the majority of PCa bone metastases and to a lesser extent in PCa soft tissue metastases. In vitro it was determined that soluble IL-6R (sIL-6R) was necessary for IL-6 to inhibit mineralization in MC3T3-E1 cells. RESULTS: PC-3 cells inhibit osteoblast activity and induce osteoblasts to produce osteoclastic factors that promote osteoclastogenesis, and one of these factors, IL-6, is highly expressed in PCa bone metastases. CONCLUSIONS: IL-6 may have an important role in promoting osteoclastogenesis in PCa bone metastasis through its' interaction with sIL-6R.

Durable Response of Enzalutamide-resistant Prostate Cancer to Supraphysiological Testosterone Is Associated with a Multifaceted Growth Suppression and Impaired DNA Damage Response Transcriptomic Program in Patient-derived Xenografts.

BACKGROUND: Androgen deprivation therapy improves the survival of castration-resistant prostate cancer (CRPC) patients, yet ultimately fails with debilitating side effects. Supraphysiological testosterone (SPT)-based therapy produces clinical responses with improved quality of life in a subset of patients. Currently, no information defines a durable response to SPT. OBJECTIVE: To identify key molecular phenotypes underlying SPT response to improve patient selection and guide combination treatment to achieve a durable response. DESIGN, SETTING, AND PARTICIPANTS: A patient-derived xenograft (PDX) preclinical trial was performed with 13 CRPC PDXs to identify molecular features associated with SPT response. Comprehensive intratumoral androgen, tumor growth, and integrated transcriptomic and protein analyses were performed in three PDXs resistant to the newer androgen receptor (AR) pathway inhibitor enzalutamide (ENZ) to define SPT response and resistance. INTERVENTION: Testosterone cypionate. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: SPT efficacy was evaluated by PDX growth, prostate-specific antigen (PSA) change, and survival. Intratumoral androgens were analyzed using mass spectrometry. Global transcriptome analysis was performed using RNA sequencing, and confirmed by quantitative real-time polymerase chain reaction and immunohistochemistry. Log-rank and Mann-Whitney tests were used for survival and molecular analyses, respectively. RESULTS AND LIMITATIONS: A durable SPT responder was identified, presenting robust repressions of ARv7 and E2F transcriptional outputs, and a DNA damage response (DDR) transcriptomic program that were altogether restored upon SPT resistance in the transient responder. ENZ rechallenge of SPT-relapsed PDXs resulted in PSA decreases but tumor progression. CONCLUSIONS: SPT produces a durable response in AR-pathway inhibitor ENZ CRPC that is associated with sustained suppression of ARv7 and E2F transcriptional outputs, and the DDR transcriptome, highlighting the potential of combination treatments that maintain suppression of these programs to drive a durable response to SPT. PATIENT SUMMARY: Patients with ENZ-resistant prostate cancer have very limited treatment options. Supraphysiological testosterone presents a prominent option for improved quality of life and a potential durable response in patients with sustained suppression on ARv7/E2F transcriptional outputs and DNA repair program.

Identification of Therapeutic Vulnerabilities in Small-cell Neuroendocrine Prostate Cancer.

PURPOSE: Small-cell neuroendocrine prostate cancer (SCNPC) exhibits an aggressive clinical course and incidence rates seem to be increasing following resistance to potent androgen receptor (AR) antagonists. Currently, treatment options are limited and few model systems are available to identify new approaches for treatment. We sought to evaluate commonalities between SCNPC and other aggressive neuroendocrine carcinomas to identify therapeutic targets. EXPERIMENTAL DESIGN: We generated whole transcriptome RNA-sequencing data from AR-active prostate cancers (ARPCs) and SCNPCs from tumors collected at rapid autopsy and two other neuroendocrine carcinomas, Merkel cell carcinoma (MCC), and small-cell lung cancer. We performed cross-tumor comparisons to identify conserved patterns of expression of druggable targets. We tested inhibitors to highly upregulated drug targets in a panel of prostate cancer cell lines and in vivo patient-derived xenograft (PDX) models. RESULTS: We identified BCL2 as highly upregulated in SCNPC compared with ARPC. Inhibitors targeting BCL2 induced apoptotic cell death in SCNPC cell lines at nanomolar concentrations while ARPC cell lines were resistant. Treatment with the BCL2 inhibitor navitoclax leads to a reduction of growth of SCNPC PDX tumors in vivo, whereas ARPC PDX models were more resistant. We identified Wee1 as a second druggable target upregulated in SCNPC. Treatment with the combination of navitoclax and the Wee1 inhibitor AZD-1775 repressed the growth of SCNPC PDX resistant to single-agent BCL2 inhibitors. CONCLUSIONS: The combination of BCL2 and Wee1 inhibition presents a novel therapeutic strategy for the treatment of SCNPC.

Nemo-like kinase induces apoptosis and inhibits androgen receptor signaling in prostate cancer cells.

BACKGROUND: The mitogen-activated protein kinases (MAPKs) regulate cell growth, differentiation, and stress responses, and many critical signaling pathways are subject to cross-regulation by MAPK signaling. Previous studies have yielded evidence of cross-talk between the MAPK pathways and androgen receptor (AR) signaling, which plays a critical role in growth control of both normal prostate and prostate cancer (PCa). Objective of this study was to evaluate the expression of MAPK-like protein nemo-like kinase (NLK) in PCa and its effects on AR-mediated transcription. METHODS: Real-time PCR and IHC were used to evaluate levels of NLK in prostatic samples. Effects of over-expression of NLK on apoptosis and proliferation were determined using Western blot and flow cytometry. Effects on AR signaling were evaluated using over-expression and knockdown of NLK in PCa cells in combination with PCR, Western blotting and reporter assays. RESULTS: Our results show that the expression of NLK is decreased in PCa metastases in comparison to normal prostate epithelium and primary PCa. Our results also show that over-expression of NLK resulted in induction of apoptosis, which was more pronounced in AR-expressing LNCaP versus AR-negative PC-3 cells. Higher levels of NLK decreased levels of AR mRNA and protein as well as inhibited AR-mediated transcription. CONCLUSIONS: NLK expression is altered during PCa progression and it is involved in regulation of AR signaling in these cells. A deeper understanding of the roles of NLK in regulation of AR-mediated transcription and control of PCa progression may point the way to new modes of therapeutic intervention in this disease.

Molecular profiling stratifies diverse phenotypes of treatment-refractory metastatic castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease with diverse drivers of disease progression and mechanisms of therapeutic resistance. We conducted deep phenotypic characterization of CRPC metastases and patient-derived xenograft (PDX) lines using whole genome RNA sequencing, gene set enrichment analysis and immunohistochemistry. Our analyses revealed five mCRPC phenotypes based on the expression of well-characterized androgen receptor (AR) or neuroendocrine (NE) genes: (i) AR-high tumors (ARPC), (ii) AR-low tumors (ARLPC), (iii) amphicrine tumors composed of cells co-expressing AR and NE genes (AMPC), (iv) double-negative tumors (i.e. AR-/NE-; DNPC) and (v) tumors with small cell or NE gene expression without AR activity (SCNPC). RE1-silencing transcription factor (REST) activity, which suppresses NE gene expression, was lost in AMPC and SCNPC PDX models. However, knockdown of REST in cell lines revealed that attenuated REST activity drives the AMPC phenotype but is not sufficient for SCNPC conversion. We also identified a subtype of DNPC tumors with squamous differentiation and generated an encompassing 26-gene transcriptional signature that distinguished the five mCRPC phenotypes. Together, our data highlight the central role of AR and REST in classifying treatment-resistant mCRPC phenotypes. These molecular classifications could potentially guide future therapeutic studies and clinical trial design.

Host-derived RANKL is responsible for osteolysis in a C4-2 human prostate cancer xenograft model of experimental bone metastases.

BACKGROUND: C4-2 prostate cancer (CaP) cells grown in mouse tibiae cause a mixed osteoblastic/osteolytic response with increases in osteoclast numbers and bone resorption. Administration of osteoprotegerin (OPG) blocks these increases, indicating the critical role of RANKL in osteolysis in this model. The objective of our study was to investigate whether RANKL expressed by tumor cells (human origin) directly stimulates osteolysis associated with the growth of these cells in bone or whether the increased osteolysis is caused by RANKL expressed by the host environment cells (murine origin). The relative contribution of tumor-vs. host-derived RANKL has been difficult to establish, even with human xenografts, because murine and human RANKL are both capable of stimulating osteolysis in mice, and the RANKL inhibitors used to date (OPG and RANK-Fc) inhibit human and murine RANKL. METHODS: To address this question we used a neutralizing, antibody (huRANKL MAb), which specifically neutralizes the biological activities of human RANKL and thereby the contribution of C4-2 derived RANKL in this tibial injection model of experimental bone metastases. RESULTS: Administration of huRANKL MAb did not inhibit the osteolytic response of the bone to these cells, or affect the establishment and growth of the C4-2 tumors in this environment. CONCLUSION: In conclusion, our results suggest that in this model, murine RANKL and not the tumor-derived human RANKL is the mediator of the osteolytic reaction associated with C4-2 growth in bone. We hypothesize that C4-2 cells express other factor/s inducing host production of RANKL, thereby driving tumor-associated osteolysis.

Osteoprotegerin in prostate cancer bone metastasis.

Osteoprotegerin (OPG), a critical regulator of osteoclastogenesis, is expressed by prostate cancer cells, and OPG levels are increased in patients with prostate cancer bone metastases. The objective of this study was to investigate the effects of OPG overexpression on prostate cancer cells and prostate cancer/bone cell interactions in vitro and in vivo. OPG-transfected C4-2 cells expressed 8.0 ng OPG per mL per 10(6) cells, whereas no OPG was detected in the media of C4-2 cells transfected with a control plasmid. OPG overexpressed by C4-2 cells protected these cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and decreased osteoclast formation. Subcutaneous OPG-C4-2 and pcDNA-C4-2 tumors exhibited similar growth and take-rate characteristics. However, when grown in bone, tumor volume was decreased in OPG-C4-2 versus pcDNA-C4-2 (P=0.0017). OPG expressed by C4-2 cells caused increases in bone mineral density (P=0.0074) and percentage of trabecular bone volume (P=0.007), and decreases in numbers of osteoblasts and osteoclasts when compared with intratibial pcDNA-C4-2 tumors (P=0.003 and P=0.019, respectively). In summary, our data show that increased expression of OPG in C4-2 cells does not directly affect proliferation of prostate cancer cells but indirectly decreases growth of C4-2 tumors in the bone environment. Our data also show that OPG expressed by C4-2 cells inhibits bone lysis associated with C4-2 bone metastasis, which results in net increases in bone volume. We therefore hypothesize that OPG expressed in prostate cancer patient bone metastases may be at least partially responsible for the osteoblastic character of most prostate cancer bone lesions.

Characterization of an Abiraterone Ultraresponsive Phenotype in Castration-Resistant Prostate Cancer Patient-Derived Xenografts.

Purpose: To identify the molecular signature associated with abiraterone acetate (AA) response and mechanisms underlying AA resistance in castration-resistant prostate cancer patient-derived xenografts (PDXs).Experimental Design: SCID mice bearing LuCaP 136CR, 77CR, 96CR, and 35CR PDXs were treated with AA. Tumor volume and prostate-specific antigen were monitored, and tumors were harvested 7 days after treatment or at end of study for gene expression and immunohistochemical studies.Results: Three phenotypic groups were observed based on AA response. An ultraresponsive phenotype was identified in LuCaP 136CR with significant inhibition of tumor progression and increased survival, intermediate responders LuCaP 77CR and LuCaP 96CR with a modest tumor inhibition and survival benefit, and LuCaP 35CR with minimal tumor inhibition and no survival benefit upon AA treatment. We identified a molecular signature of secreted proteins associated with the AA ultraresponsive phenotype. Upon resistance, AA ultraresponder LuCaP 136CR displayed reduced androgen receptor (AR) signaling and sustainably low nuclear glucocorticoid receptor (nGR) localization, accompanied by steroid metabolism alteration and epithelial-mesenchymal transition phenotype enrichment with increased expression of NF-κB-regulated genes; intermediate and minimal responders maintained sustained AR signaling and increased tumoral nGR localization.Conclusions: We identified a molecular signature of secreted proteins associated with AA ultraresponsiveness and sustained AR/GR signaling upon AA resistance in intermediate or minimal responders. These data will inform development of noninvasive biomarkers predicting AA response and suggest that further inhibition along the AR/GR signaling axis may be effective only in AA-resistant patients who are intermediate or minimal responders. These findings require verification in prospective clinical trials. Clin Cancer Res; 23(9); 2301-12. ©2016 AACR.

Androgen Receptor Pathway-Independent Prostate Cancer Is Sustained through FGF Signaling.

Androgen receptor (AR) signaling is a distinctive feature of prostate carcinoma (PC) and represents the major therapeutic target for treating metastatic prostate cancer (mPC). Though highly effective, AR antagonism can produce tumors that bypass a functional requirement for AR, often through neuroendocrine (NE) transdifferentiation. Through the molecular assessment of mPCs over two decades, we find a phenotypic shift has occurred in mPC with the emergence of an AR-null NE-null phenotype. These "double-negative" PCs are notable for elevated FGF and MAPK pathway activity, which can bypass AR dependence. Pharmacological inhibitors of MAPK or FGFR repressed the growth of double-negative PCs in vitro and in vivo. Our results indicate that FGF/MAPK blockade may be particularly efficacious against mPCs with an AR-null phenotype.

Administration of zoledronic acid enhances the effects of docetaxel on growth of prostate cancer in the bone environment.

BACKGROUND: After development of hormone-refractory metastatic disease, prostate cancer is incurable. The recent history of chemotherapy has shown that with difficult disease targets, combinatorial therapy frequently offers the best chance of a cure. In this study we have examined the effects of a combination of zoledronic acid (ZOL), a new-generation bisphosphonate, and docetaxel on LuCaP 23.1, a prostate cancer xenograft that stimulates the osteoblastic reaction when grown in the bone environment. METHODS: Intra-tibial injections of LuCaP 23.1 cells were used to generate tumors in the bone environment, and animals were treated with ZOL, docetaxel, or a combination of these. Effects on bone and tumor were evaluated by measurements of bone mineral density and histomorphometrical analysis. RESULTS: ZOL decreased proliferation of LuCaP 23.1 in the bone environment, while docetaxel at a dose that effectively inhibited growth of subcutaneous tumors did not show any effects in the bone environment. The combination of the drugs significantly inhibited the growth of LuCaP 23.1 tumors in the bone. CONCLUSION: In conclusion, the use of the osteolysis-inhibitory agent ZOL in combination with docetaxel inhibits growth of prostate tumors in bone and represents a potential treatment option.