Castration-resistant Tumors Research Articles

Mechanisms of Lysophosphatidic Acid-Mediated Lymphangiogenesis in Prostate Cancer.

Prostate cancer (PCa) is the most common noncutaneous cancer in men worldwide. One of its major treatments is androgen deprivation therapy, but PCa frequently relapses as aggressive castration resistant local tumors and distal metastases. Hence, the development of novel agents or treatment modalities for advanced PCa is crucial. Many tumors, including PCa, first metastasize to regional lymph nodes via lymphatic vessels. Recent findings demonstrate that the bioactive lipid lysophosphatidic acid (LPA) promotes PCa progression by regulating vascular endothelial growth factor-C (VEGF-C), a critical mediator of tumor lymphangiogenesis and lymphatic metastasis. Many of the underlying molecular mechanisms of the LPA–VEGF-C axis have been described, revealing potential biomarkers and therapeutic targets that may aid in the diagnosis and treatment of advanced PCa. Herein, we review the literature that illustrates a functional role for LPA signaling in PCa progression. These discoveries may be especially applicable to anti-lymphangiogenic strategies for the prevention and therapy of metastatic PCa.

The Role of Castration-Resistant Bmi1+Sox2+ Cells in Driving Recurrence in Prostate Cancer.

Recurrence following androgen-deprivation therapy is associated with adverse clinical outcomes in prostate cancer, but the cellular origins and molecular mechanisms underlying this process are poorly defined. We previously identified a population of castration-resistant luminal progenitor cells expressing Bmi1 in the normal mouse prostate that can serve as a cancer cell-of-origin. Here, we investigate the potential of Bmi1-expressing tumor cells that survive castration to initiate recurrence in vivo. We employed lineage retracing in Bmi1-CreER; R26R-confetti; Ptenf/f transgenic mice to mark and follow the fate of emerging recurrent tumor clones after castration. A tissue recombination strategy was used to rescue transgenic mouse prostates by regeneration as grafts in immunodeficient hosts. We also used a small molecule Bmi1 inhibitor, PTC-209, to directly test the role of Bmi1 in recurrence. Transgenic prostate tumors (n = 17) regressed upon castration but uniformly recurred within 3 months. Residual regressed tumor lesions exhibited a transient luminal-to-basal phenotypic switch and marked cellular heterogeneity. Additionally, in these lesions, a subpopulation of Bmi1-expressing castration-resistant tumor cells overexpressed the stem cell reprogramming factor Sox2 (mean [SD] = 41.1 [3.8]%, n = 10, P < .001). Bmi1+Sox2+ cells were quiescent (BrdU+Bmi1+Sox2+ at 3.4 [1.5]% vs BrdU+Bmi1+Sox2- at 18.8 [3.4]%, n = 10, P = .009), consistent with a cancer stem cell phenotype. By lineage retracing, we established that recurrence emerges from the Bmi1+ tumor cells in regressed tumors. Furthermore, treatment with the small molecule Bmi1 inhibitor PTC-209 reduced Bmi1+Sox2+ cells (6.1 [1.4]% PTC-209 vs 38.8 [2.3]% vehicle, n = 10, P < .001) and potently suppressed recurrence (retraced clone size = 2.6 [0.5] PTC-209 vs 15.7 [5.9] vehicle, n = 12, P = .04). These results illustrate the utility of lineage retracing to define the cellular origins of recurrent prostate cancer and identify Bmi1+Sox2+ cells as a source of recurrence that could be targeted therapeutically.

ANO7 is associated with aggressive prostate cancer.

Prostate cancer is one of the most common and heritable human cancers. Our aim was to find germline biomarkers that can predict disease outcome. We previously detected predisposing signals at 2q37, the location of the prostate specific ANO7 gene. To investigate, in detail, the associations between the ANO7 gene and PrCa risk and disease aggressiveness, ANO7 was sequenced in castration resistant tumors together with samples from unselected PrCa patients and unaffected males. Two pathogenic variants were discovered and genotyped in 1769 patients and 1711 unaffected males. Expression of ANO7 vs. PrCa aggressiveness was investigated. Different databases along with Swedish and Norwegian cohorts were used for validation. Case–control and aggressive vs. nonaggressive association analyses were performed against risk and/or cancer aggressiveness. The ANO7 mRNA level and patient survival were analyzed using expression data from databases. Variant rs77559646 showed both risk (OR 1.40; p = 0.009, 95% CI 1.09–1.78) and association with aggressive PrCa (Genotype test p = 0.04). It was found to be an eQTL for ANO7 (Linear model p‐values for Finnish patients p = 0.009; Camcap prostate tumor p = 2.53E‐06; Stockholm prostate tumor cohort p = 1.53E‐13). rs148609049 was not associated with risk, but was related to shorter survival (HR 1.56; 95% CI 1.03–2.36). High ANO7 expression was independently linked to poor survival (HR 18.4; 95% CI 1.43–237). ANO7 genotypes correlate with expression and biochemical relapse, suggesting that ANO7 is a potential PrCa susceptibility gene and that its elevated expression correlates with disease severity and outcome.

Heterogeneous cancer-associated fibroblast population potentiates neuroendocrine differentiation and castrate resistance in a CD105-dependent manner.

Heterogeneous prostatic carcinoma associated fibroblasts (CAF) contribute to tumor progression and resistance to androgen signaling deprivation therapy (ADT). CAF subjected to extended passaging, compared to low passage CAF, were found to lose tumor expansion potential and heterogeneity. Cell surface endoglin (CD105), known to be expressed on proliferative endothelia and mesenchymal stem cells, was diminished in high passage CAF. RNA-sequencing revealed SFRP1 to be distinctly expressed by tumor-inductive CAF, which was further demonstrated to occur in a CD105-dependent manner. Moreover, ADT resulted in further expansion of the CD105+ fibroblastic population and downstream SFRP1 in 3-dimensional cultures and patient derived xenograft tissues. In patients, CD105+ fibroblasts were found to circumscribe epithelia with neuroendocrine differentiation. CAF-derived SFRP1, driven by CD105 signaling, was necessary and sufficient to induce prostate cancer neuroendocrine differentiation in a paracrine manner. A partially humanized CD105 neutralizing antibody, TRC105, inhibited fibroblastic SFRP1 expression and epithelial neuroendocrine differentiation. In a novel synthetic lethality paradigm, we found that simultaneously targeting the epithelia and its microenvironment with ADT and TRC105, respectively, reduced castrate resistant tumor progression, in a model where either ADT or TRC105 alone had little effect.

SRRM4 gene expression correlates with neuroendocrine prostate cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of castrate-resistant prostate cancer characterized by poor patient outcome. Whole transcriptome sequencing analyses identified a NEPC-specific RNA splicing program that is predominantly controlled by the SRRM4 gene, suggesting that SRRM4 drives NEPC development. However, whether SRRM4 expression in patients may aid pathologists in diagnosing NEPC and predicting patient survival remains to be determined. In this study, we have applied RNA in situ hybridization and immunohistochemistry assays to measure the expressions of SRRM4, NEPC markers (SYP, CD56, and CHGA), and adenocarcinoma (AdPC) markers (AR, PSA) in a series of tissue microarrays constructed from castrate-resistant prostate tumors, treatment-naïve tumors collected from radical prostatectomy, and tumors treated with neoadjuvant hormonal therapy (NHT) for 0-12 months. Three pathologists also independently evaluated tumor histology and NEPC marker status. Here, we report that SRRM4 in castrate-resistant tumors is highly expressed in NEPC, strongly correlated with SYP, CD56, and CHGA expressions (Pearson correlation r = 0.883, 0.675, and 0.881; P < 0.0001) and negatively correlated with AR and PSA expressions (Pearson correlation r = -0.544 and -0.310; P < 0.05). Overall survival is 12.3 months for patients with SRRM4 positive tumors, comparing to 23 months for patients with SRRM4 negative tumors. In treatment-naïve AdPC, low SRRM4 expression is detected in ∼16% tumor cores. It correlates with SYP and CHGA expressions, but not Gleason scores. AdPC treated with >7 month NHT has significantly higher SRRM4 expression. Based on these findings, we conclude that SRRM4 expression in castrate-resistant tumors is highly correlated with NEPC and poor patient survival. It may serve as a diagnosis and prognosis biomarker of NEPC.

Abstract B037: N-Myc driven cell plasticity in castrate-resistant prostate cancer

Abstract An emerging concept of anti-AR therapy resistance is the induction of epithelial plasticity in castration-resistant prostate adenocarcinoma (CRPC) cells that eventually evolve to a neuroendocrine phenotype (e.g., low to no AR signaling and expression of neuroendocrine markers). Neuroendocrine prostate cancer (NEPC) is clinically aggressive and carries a poor prognosis with an average survival of less than one year. We and others have identified and validated new therapeutic targets and drivers of cell transformation from CRPC to NEPC. These include induction of MYCN (encodes N-Myc) or RB1/TP53 loss of function. While N-Myc is a bona fide driver oncogene in several rare tumor types, the N-Myc-driven molecular reprogramming and acquisition of cell plasticity remains poorly understood. We have previously shown that intact N-Myc+/Pten-null genetically engineered mice (GEM) develop poorly differentiated, highly proliferative, invasive prostate cancer that is molecularly similar to human NEPC tumor (1). Similar morphologic features were observed in organoids derived from our GEM model. Flow analysis of these organoids revealed that N-Myc+/Pten-null GEM-derived organoids are comprised of a heterogeneous population of cells with luminal, basal, or features of both lineages compared to more homogeneous population of cells from Pten-null control organoids. Recently, we also found that N-Myc overexpression induced a faster recurrence in the context of castration in vivo, based on the GEM model and 22Rv1 isogenic xenografts. This is consistent with the significant N-Myc induced increased castrate-resistant tumor growth based on data from GEM organoids grown in vivo as allografts. In the GEM, we found that castration led to invasive prostate tumors that metastasize to multiple locations including the liver and that express the EMT marker vimentin and the NEPC marker neural cell adhesion molecule (NCAM1). Based on RNAseq data from 22Rv1 xenografts in castrated mice, we identified stem cell signatures including neural crest stem genes (GSEA q value = 7.0E-19) that were significantly enriched in the N-Myc upregulated genes including SOX2, SOX10, and SOX11, genes implicated in neural development and NEPC associated genes (e.g., NCAM1 and chromogranin A [CHGA]). N-Myc ChIP-Seq data confirmed N-Myc occupancy enriched at promoters of genes implicated in stem cell-associated pathways. RNAseq data from human NEPC organoid following MYCN depletion revealed similar N-Myc-associated gene expression changes. Altogether, these data led to a model in which N-Myc overexpression induces a castrate-resistant, lineage-plastic phenotype that would give rise to NEPC, consistent with the phenotype induced by RB1/TP53/PTEN loss [2-4].

Recent advances in prostate cancer research: large-scale genomic analyses reveal novel driver mutations and DNA repair defects.

Prostate cancer (PCa) is a disease of mutated and misregulated genes. However, primary prostate tumors have relatively few mutations, and only three genes ( ERG, PTEN, and SPOP) are recurrently mutated in more than 10% of primary tumors. On the other hand, metastatic castration-resistant tumors have more mutations, but, with the exception of the androgen receptor gene ( AR), no single gene is altered in more than half of tumors. Structural genomic rearrangements are common, including ERG fusions, copy gains involving the MYC locus, and copy losses containing PTEN. Overall, instead of being associated with a single dominant driver event, prostate tumors display various combinations of modifications in oncogenes and tumor suppressors. This review takes a broad look at the recent advances in PCa research, including understanding the genetic alterations that drive the disease and how specific mutations can sensitize tumors to potential therapies. We begin with an overview of the genomic landscape of primary and metastatic PCa, enabled by recent large-scale sequencing efforts. Advances in three-dimensional cell culture techniques and mouse models for PCa are also discussed, and particular emphasis is placed on the benefits of patient-derived xenograft models. We also review research into understanding how ETS fusions (in particular, TMPRSS2-ERG) and SPOP mutations contribute to tumor initiation. Next, we examine the recent findings on the prevalence of germline DNA repair mutations in about 12% of patients with metastatic disease and their potential benefit from the use of poly(ADP-ribose) polymerase (PARP) inhibitors and immune modulation. Lastly, we discuss the recent increased prevalence of AR-negative tumors (neuroendocrine and double-negative) and the current state of immunotherapy in PCa. AR remains the primary clinical target for PCa therapies; however, it does not act alone, and better understanding of supporting mutations may help guide the development of novel therapeutic strategies.

Abstract 2868: Preclinical evaluation of the multi tyrosine kinase inhibitor TAS-115 in genetically engineered mouse models of prostate cancer

Abstract The tumor microenvironment (TME) is comprised of various cell types that can contribute to malignancy by promoting cancer cell proliferation, immune evasion, angiogenesis and metastasis. Here we use genetically engineered mouse models of prostate cancer to show that TAS-115, a multi-kinase inhibitor that targets c-MET, VEGF-R, and CSF1-R signal pathways, suppresses prostate cancer growth by targeting the TME. Conditional Pten-knockout (Pten-KO) and Pten/Trp53-double knockout (Pten/P53-DKO) mice were used to evaluate the pharmacodynamic activity of TAS-115 and efficacy studies were carried in 16-week-old Pten/P53-DKO mice. Four weeks of treatment with TAS-115 significantly suppressed prostate tumor growth in by 22.6%, P=0.041. Epithelial cancer cell proliferation in was reduced by 28.6%, (P=0.072) in TAS-115-treated mice, however, no changes were seen in the induction of apoptosis. Immunohistochemical (IHC) analysis revealed potent but focal inhibition of ERK phosphorylation in both epithelial and stromal cells. A tendency for reduced phosphorylation of AKT and STAT3 were also noted in mice receiving TAS-115. IHC analysis also revealed strong inhibition of CSF1-R phosphorylation in tumor infiltrating immune cells of mice treated with TAS-115. We also examined the effects of TAS-115 in a castration-resistant Pten/P53-DKO prostate tumor model. In this model, we did not note statistically significant reductions in tumor burden, however, 2 of 6 (33.3%) mice treated with TAS-115 had noticeably reduced tumor burden (&amp;gt;20% reduction relative to median overall tumor burden levels). No significant changes were observed in overall tumor proliferation, apoptotic rates or in AKT and ERK phosphorylation levels in the tumors of TAS-115-treated mice however, a significant reduction of STAT3 phosphorylation staining index (-39.3, P=0.0424) was observed, the prostates tumors of mice receiving TAS-115. Similar to castration-naïve treated tumors, CSF1-R phosphorylation was strongly inhibited in infiltrating immune cells of castration-resistant prostate tumors from TAS-115-treated mice. Unsupervised clustering from a qRT-PCR-based focused panel of immune-responsive genes showed greater consistency and a higher magnitude of response in TAS-115-treated mice in the castration-resistant tumor model. Further analysis of microvessel density showed a 20% reduction in tumor neovascularization in both intervention models. The direct antitumor effect of TAS-115 on cancer cells was examined in vitro using mouse prostate cancer cell lines. In these studies, TAS-115 showed moderate growth inhibition. Overall our studies show that TAS-115 is capable of suppressing prostate tumor growth by acting primarily on the TME and provide evidence to support further investigation of TME modulation using small molecule multi-kinase inhibitors. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sato, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Preclinical evaluation of the multi tyrosine kinase inhibitor TAS-115 in genetically engineered mouse models of prostate cancer [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 2868.

Abstract 4697: Immune cell infiltration and its clinical correlations in paired castration-naive and castration-resistant prostate cancers

Abstract Ineffectiveness of immune checkpoint inhibitors in patients with castration- resistant prostate cancer (CRPC) suggests that CRPC may harbor a profound immunosuppressive tumor microenvironment. However, immune tumor microenvironment of CRPC has never been well investigated due to limited availability of castration-resistant tumor tissue. To characterize the changes in immune tumor microenvironment during CRPC progression and identify disease stage which is potentially favorable for immunotherapy, we studied the densities of tumor-infiltrating immune cells by using the immunohistochemical method and manual counting in paired castration-naive (CN) and castration-resistant (CR) tumors. We identified 11 metastatic prostate cancer patients who had received palliative transurethral resection of prostate both before androgen deprivation therapy (ADT) and at the time when castration resistance emerged from the hospital database during 2006.1~2017.3. We also used prostatic tissue from 5 patients with benign prostate hyperplasia as the normal prostate control. The median age at diagnosis of PC patients was 74.7 (range: 62-85) year-old. The numbers of tumors with Gleason score 7, 8, 9 and 10 were 1, 1, 6, and 2, respectively. The median PSA level at diagnosis was 96.6 (range: 3.3-847.7) ng/ml. The median overall survival after initiation of ADT was 48.8 months. The median density of CD4+ T cells (no./mm2) was 182.3 (45.0-363.5) for normal prostate, 160.9 (range: 12.6-240.6) for CNPC and 69.4 (range: 31-499.6) for CRPC (non-statistically significant; n.s.). The median density of CD8+ T cells (no./mm2) was 12.5 (range: 32.5-220.7) for normal prostate, 65.7 (range: 3.7-195.6) for CNPC and 53.8 (range: 4.4-190.4) for CRPC (n.s.). The median density of CD20+ B cells (no./mm2) was 132.1 (range: 54.6-514.4) for normal prostate, 112.2 (range: 3.7-298.9) for CNPC and 47.6 (range: 7.0-137.3) for CRPC (n.s.). The median density of CD68+ macrophages (no./mm2) was 31 (range: 0.4-248.7) for normal prostate, 77.5 (range: 3.7-403.7) for CNPC and 31.7 (range: 2.2-180.8) for CRPC (n.s.). The tumor-infiltrating immune cell densities in CNPC did not correlate with PSA level, Gleason score or ADT response. Only high B cell density in CNPC was prognostic for overall survival (median: 23.9 months for patients with high tumor-infiltrating B cell density vs. 64 months for patients with low tumor-infiltrating B cell density; P=0.0463). In conclusion, immune tumor microenvironment of CNPC and CRPC is very similar and is not different from normal prostate. It suggests that immune checkpoint inhibitors may not be effective against CNPC unless combining with other treatment, such as ADT or radiotherapy, which can reprogram the tumor microenvironment toward more immunosupportive. Citation Format: Ying-Chun Shen, Chia-Tung Shun, Ching-Ping Yeh, Jhe-Cyuang Kuo, Yu-Chieh Tsai, Chung-Hsin Chen, Chao-Yuan Huang, Yeong-Shiau Pu. Immune cell infiltration and its clinical correlations in paired castration-naive and castration-resistant prostate cancers [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 4697.

Abstract 5538: Arv7 validation in castrate resistant prostate tumors utilizing Rnascope in situ hybridization assay

Abstract Background Androgen receptor splice variant 7 (ARV7) is a ligand-independent transcription factor associated with resistance to androgen deprivation therapy. There are efforts in development to monitor ARV7 expression during the treatment of patients with prostate cancer. Methods We evaluated ARV7 expression in primary and metastatic prostate adenocarcinomas tissues utilizing automated RNAScope (Advanced Cell Diagnostics) ISH assay, Immunohistochemistry (IHC) and quantitative PCR (qPCR). The novel RNAscope ISH assay was used for direct chromogenic visualization of RNA transcripts and to confirm positivity as an orthogonal approach to validate IHC and quantitative qPCR assay findings. We screened a total of 44 CRPC FFPE tumor samples by IHC and a subset of 28 (excluding controls) with the ISH and qPCR assays. Samples for qPCR were enriched for tumor by macrodissection. RNAScope Scoring Criteria was used to score ISH expression and raw Ct values were normalized to the RPL19 housekeeping gene for qPCR. Thresholds for each assay was established by comparing the findings with the other 2 assays. The data was calculated as ARV7 (+) or ARV7 (-) based on thresholds and compared to the other 2 assays to establish assay performance statistics. Results The ISH screening of 28 CRPC tumors resulted in 16 tumors with some level of positivity and approximately 10 tumors with significant positivity. Of the 44 CRPC tumors stained by IHC, 16 tumors showed various levels of ARV7 nuclear positivity and 8 tumors with strong positivity. Conclusions The detection of ARV7 was achieved in three independent assays. The correlation of three orthogonal data sets were used to identify positive and negative tissues. ISH has the potential for greater sensitivity compared to traditional IHC. The development of automated RNAscope ISH assay allows for more samples to run in a standardized manner with minimal inter-operator variably and requires less hands on-time gives consistently reproducible results. Citation Format: Tanesha Cash-Mason, Jackson Wong, Martinez Martinez, Michael Sharp, Jayaprakash Karkera, John D. Alvarez, Gerald C. Chu, Shibu Thomas, Weiman Li, S. Ken Tian. Arv7 validation in castrate resistant prostate tumors utilizing Rnascope in situ hybridization assay [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 5538.

Abstract 1775: Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer

Abstract Enzalutamide (ENZ) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and approved for the treatment of castration resistant prostate cancer (CRPC). Studies show increased survival in men with metastatic CRPC after chemotherapy when treated with ENZ. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance and resistance to AR inhibitors may lead to increase in N-cadherin. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR positive and negative prostate cancers. Co-targeting AR and N-cadherin with ENZ and mAb respectively show promise in addressing not only castration resistant tumor progression but also ENZ resistance. Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with ENZ. In vivo studies on promising candidates were performed using castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to ENZ, mAb alone, or in combination. At end-point, tumors were harvested and further analyzed. Final candidate mAbs were then selected and re-formatted into fully humanized N-cadherin monoclonal antibodies and further evaluated. A molecular model of the mouse chimeric antibodies were built based on structure of similar antibodies and the CDR loops were grafted to the human framework region and back-translated into nucleotide sequences. Codon-optimized DNA encoding the humanized variable domains connected by a 15 glycine-rich amino acid linker was synthesized by GeneArt and sub-cloned into expression vectors containing light and heavy chain constant regions. Clones were then screened and selected. Candidates were transiently expressed in 293 cells and supernatant were collected and purified for further analysis. These newly generated humanized mAbs engineered from the promising human anti-mouse N-cadherin antibody candidates exhibited similar outcomes to their chimeric counterparts, revealing potential in providing methods for diagnosis and treatment of advanced diseases. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Keyu Li, Joyce Yamashiro, Chun Wang, Anna Wu, Robert E. Reiter. Development of fully humanized N-cadherin monoclonal antibodies for treatment of castration resistant prostate cancer [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 1775.

Abstract 5787: Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models

Abstract Introduction: Therapeutic targeting the PI3K/AKT signaling pathway is an attractive strategy to combat solid tumors that exhibit hyper-AKT activity. Ipatasertib, a potent pan-AKT inhibitor, has exhibited significant inhibitory effects on tumor growth in preclinical studies, including models of Androgen Receptor (AR)-independent prostate cancer (PCa) that lack PTEN, a negative regulator of the PI3K/AKT pathway. It is currently being investigated in several ongoing clinical trials, including evaluating its efficacy in combination with abiraterone in castrate resistant PCa (CRPC) (NCT01485861). There is cross-talk between androgen signaling and PI3K/AKT pathway activation, however the optimal setting for AKT inhibition in PCa is not clear. Here we seek to determine the benefit of ipatasertib for androgen sensitive (AS) PCa alone or in combination with androgen deprivation (AD), as well as the benefit of ipatasertib alone or in combination with the antiandrogen enzalutamide in CRPC. Methods: In vitro studies utilized human androgen sensitive PCa cell lines LAPC, LAPC4shPTEN, and LNCAP, as well as two LNCaP-derived, castrate resistant cell lines. Cell viability was determined using CellTitre Glo assay and apoptosis by flow cytometry. Inhibitory effects of AKT signaling pathway was determined by western blot assays against AKT phosphorylation sites and downstream targets as well as detection for hormone receptor expression. In vivo studies utilized LuCaP 136 and LuCaP 136-derived castrate resistant (LuCaP 136CR) patient derived xenograft (PDX) models. Cells were injected into athymic mice and grouped into 4 arms. Intact mice bearing AS tumors were treated with vehicle, ipatasertib (100mg/Kg), AD, or ipatasertib + AD. Castrated mice bearing castrate-resistant tumors were treated with vehicle, ipatasertib, enzalutamide (10mg/Kg), or ipatasertib + enzalutamide. Tumor growth was determined twice/week via blinded caliper measurements. Results: Ipatasertib effectively inhibited AKT signaling, as indicated by significant decrease in phosphorylation of the downstream targets P70S6K and 4E-BP1. Our in vitro data indicate significant anti-proliferation effects of ipatasertib across all PCa models, both androgen sensitive and castrate resistant. Interestingly, this was irrespective of PTEN status. In addition, we observed a decrease in glucocorticoid receptor (GR) expression in enzalutamide resistant lines, although AR expression was not significantly altered. Preclinical animal studies showed significant response to tumor growth inhibition with ipatasertib monotherapy, which was enhanced with combination treatment. Conclusion: Ipatasertib is beneficial in models of androgen sensitive and castrate resistant PCa and can be effective in the treatment of PCa alone or in combination with current standard therapies targeting the androgen receptor. Citation Format: Remi M. Adelaiye-Ogala, Yen T. Nguyen, Jose A. Rodriguez-Nieves, Aian Neil Alilin, Supreet Agarwal, David Vanderweele. Ipatasertib impairs tumor growth in androgen sensitive and castrate resistant prostate cancer: Evidence in preclinical models [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 5787.

Abstract 3348: The role of SOX2 in promoting resistance to AR-targeted therapies in prostate cancer

Abstract Prostate cancer is the most common type of cancer in men, with approximately 181,000 new cases diagnosed in 2016. Due to the central role of the androgen receptor (AR) in prostate development and more importantly prostate cancer cell survival and proliferation, strategies targeting AR have been the mainstay therapy for over 70 years. However, despite potent inhibition of AR pathway activation, many patients develop castration-resistant prostate cancer (CRPC). Second-line therapies, such as enzalutamide, has increased overall survival in CRPC, but resistance to these therapies inevitably emerges, suggesting that other pathways apart from AR signalling are contributing to the failure of treatments. We have previously demonstrated that SOX2 [SRY (sex determining region Y)-box 2] is an AR-repressed gene that is expressed in a large percentage of high Gleason grade prostate tumours, as well as in most metastases. Additionally, expression of SOX2 within a castration-sensitive cell line is sufficient to enable castration-resistant tumor formation in vivo, and enzalutamide resistance in vitro. In prostate cancer, SOX2 is not found with its normal stem cell co-factors, NANOG and OCT4, leading to the hypothesis that SOX2 is interacting with a novel co-factor in prostate cancer to regulate expression of genes promoting castration-resistance. A chromatin-immunoprecipitation experiment in a castration-resistant prostate cancer cell line was performed to determine if SOX2 binds the same genes and activates similar pathways in prostate cancer as in embryonic stem cells. Approximately half of the SOX2 bound genes in the CRPC cell line were shared with the embryonic stem cell line, and these genes are involved in signalling pathways and maintenance of stem cell pluripotency; the genes bound uniquely in the CRPC cell line are present in pathways involved with metabolic processes. To identify potential binding partners of SOX2, computational analysis of the sites bound by SOX2 in the promoter regions of target genes determined the FOXA1 motif is within close proximity to the SOX2 motif, and the physical interaction of these proteins was confirmed through co-immunoprecipitation. Further understanding of SOX2 target genes and the pathways that SOX2 activates in the presence of enzalutamide will be crucial to identify mechanisms of resistance and enable the development of novel therapies for castration-resistant prostate cancer. Citation Format: Larischa de Wet, Anthony Williams, Marc Gillard, Steve Kregel, Tzintzuni Garcia, Erin McAuley, Ryan Brown, Donald Vander Griend. The role of SOX2 in promoting resistance to AR-targeted therapies in prostate cancer [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 3348.

Abstract 4510: SHH and GATA interplay: A potential therapeutic target for prostate cancer

Abstract Background: Prostate cancer is the second leading cause of cancer deaths in males worldwide. Typically, prostate cancer cells depend on androgens, and many therapeutic strategies target receptors involved in androgen regulation. However, certain castration-resistant tumors of the prostate are resistant to androgen deprivation therapies. Moreover, advanced-stage prostate tumors can proliferate uncontrollably either due to androgens or due to alteration in other signaling pathways. For instance, the activity of the sonic hedgehog (SHH) pathway, which usually decreases during post-natal development, is increased during prostate cancer progression. Of interest, a recent study reported a novel role for GATA transcription factors as potential repressors of SHH signaling. Based on the above-mentioned findings, we hypothesize that the balance between GATA and SHH pathways is perturbed in prostate cancer leading to overactivation of SHH pathways and its downstream effector proteins. Methods: We screened human prostate cancer cell lines, DU145, 22Rv1, and PC3, as well as a human immortalized normal prostate epithelial cell line, RWPE-1, for baseline levels of expression of SHH downstream effector proteins via real-time PCR using specific primers. Subsequently, we treated the cell lines with bone morphogenetic protein 4 (BMP4)-conditioned media in order to induce the expression of GATA transcription factors, and then we measured the levels of expression for each of the SHH pathway effector proteins by real-time PCR. Results: We demonstrated that GATA can interfere with the expression of SHH and its effector proteins in the human prostate cancer cell lines. Upon treatment with BMP4, we noted that there was a decrease in expression of Gli1, Gli2, and Gli3, downstream effectors of SHH, suggesting an anti-proliferative response. Additionally, Patched1 expression was also attenuated after treatment with BMP4. Similarly, Sox9 expression was also abrogated. Conclusion: Our results demonstrate that GATA transcription factors can act as potential repressors of the SHH pathway in the context of prostate cancer in vitro by interfering with the expression of SHH downstream effector proteins. Citation Format: Rami Z. Morsi, Wassim Abou Kheir, Layal El-Khatib, Hiba Msheik, Georges Daoud. SHH and GATA interplay: A potential therapeutic target for prostate cancer [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 4510.

Abstract 3737: Apalutamide (ARN-509) demonstrates therapeutic efficacy in genetically engineered mouse models of Pten-deficient prostate cancer

Abstract Apalutamide (ARN-509) is an oral nonsteroidal androgen receptor (AR) antagonist that is currently undergoing late-stage clinical development for the management of castration-resistant prostate cancer (CRPC). In this study, we use genetically engineered mouse prostate cancer (GEM-PCa) models to examine the preclinical activity of apalutamide in the setting of castration-naïve and castration-resistant disease and delve into the molecular mechanisms underlying CRPC progression. In an early-stage efficacy model of Pten-deficient prostate cancer, four weeks of treatment with apalutamide (30 mg/kg/d) significantly reduced tumor burden by 33.5% (P=0.002) in castration-naive mice but did not alter tumor burden in orchidectomized mice bearing castration-resistant tumors. Interim analyses from long-term survival studies, using models of advanced prostate cancer driven by the conditional inactivation of Pten and Trp53, have indicated improved median overall survival (OS) times in both castration-naïve (17 days, 95% CI 11.6-38.9 in control mice vs. 36 days, 95% CI 21.8-56.9 in apalutamide treated-mice, P=0.280) and CRPC settings (21 days, 95% CI 15.1-25.8 in control mice vs. 40 days, 95% CI 31.2-50.1 in apalutamide treated-mice, P=0.001). In the CRPC model, time to tumor progression was significantly longer for mice treated with apalutamide, median time 20 days 95% CI 9.8-20.6, in apalutamide treated-mice vs. 7 days, 95% CI 5.1-10.8, in control mice, P=0.029. Molecular characterization indicated increased PI3K-AKT signaling in response to treatment with apalutamide in castration-naïve prostate tumors. In vitro studies using four mouse prostate cancer cell lines revealed synergistic responses with apalutamide and AKT inhibition (GSK-690693), the mean combination index at 50% effective dose ranged from 0.585-0.791. In vivo efficacy studies of combination therapy in Pten-KO mice showed tumor burden reductions of 33.4%, 17.7% and 40.9% in apalutamide, GSK-690693 and combination treatments, respectively, P=0.002 in the castration-naïve setting, and reductions of 1.0%, 12.7% and 15.5% in apalutamide, GSK-690693 and combination treatments, respectively, P=0.101 in the CRPC setting. Studies to further evaluate the therapeutic benefit of apalutamide plus PI3K/AKT signal blockade in models of advanced prostate cancer are underway. In conclusion, our findings show that apalutamide is active in GEM-PCa models and support further investigation for developing rational treatment combinations for the management of advanced prostate cancer. Citation Format: Marco A. De Velasco, Masahiro Nozawa, Yurie Kura, Naomi Ando, Noriko Sato, Kazuhiro Yoshimura, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Apalutamide (ARN-509) demonstrates therapeutic efficacy in genetically engineered mouse models of Pten-deficient prostate cancer [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 3737.

Development of Neuroendocrine Prostate Cancers by the Ser/Arg Repetitive Matrix 4-Mediated RNA Splicing Network.

While the use of next-generation androgen receptor pathway inhibition (ARPI) therapy has significantly increased the survival of patients with metastatic prostate adenocarcinoma (AdPC), several groups have reported a treatment-resistant mechanism, whereby cancer cells can become androgen receptor (AR) indifferent and gain a neuroendocrine (NE)-like phenotype. This subtype of castration-resistant prostate cancer has been termed “treatment-induced castration-resistant neuroendocrine prostate cancer” (CRPC-NE). Recent reports indicate that the overall genomic landscapes of castration-resistant tumors with AdPC phenotypes and CRPC-NE are not significantly altered. However, CRPC-NE tumors have been found to contain a NE-specific pattern throughout their epigenome and splicing transcriptome, which are significantly modified. The molecular mechanisms by which CRPC-NE develops remain unclear, but several factors have been implicated in the progression of the disease. Recently, Ser/Arg repetitive matrix 4 (SRRM4), a neuronal-specific RNA splicing factor that is upregulated in CRPC-NE tumors, has been shown to establish a CRPC-NE-unique splicing transcriptome, to induce a NE-like morphology in AdPC cells, and, most importantly, to transform AdPC cells into CRPC-NE xenografts under ARPI. Moreover, the SRRM4-targeted splicing genes are highly enriched in various neuronal processes, suggesting their roles in facilitating a CRPC-NE program. This article will address the importance of SRRM4-mediated alternative RNA splicing in reprogramming translated proteins to facilitate NE differentiation, survival, and proliferation of cells to establish CRPC-NE tumors. In addition, we will discuss the potential roles of SRRM4 in conjunction with other known pathways and factors important for CRPC-NE development, such as the AR pathway, TP53 and RB1 genes, the FOXA family of proteins, and environmental factors. This study aims to explore the multifaceted functions of SRRM4 and SRRM4-mediated splicing in driving a CRPC-NE program as a coping mechanism for therapy resistance, as well as define future SRRM4-targeted therapeutic approaches for treating CRPC-NE or mitigating its development.

Clinical implications of PTEN loss in prostate cancer

Genomic aberrations of the PTEN tumour suppressor gene are among the most common in prostate cancer. Inactivation of PTEN by deletion or mutation is identified in ∼20% of primary prostate tumour samples at radical prostatectomy and in as many as 50% of castration-resistant tumours. Loss of phosphatase and tensin homologue (PTEN) function leads to activation of the PI3K-AKT (phosphoinositide 3-kinase-RAC-alpha serine/threonine-protein kinase) pathway and is strongly associated with adverse oncological outcomes, making PTEN a potentially useful genomic marker to distinguish indolent from aggressive disease in patients with clinically localized tumours. At the other end of the disease spectrum, therapeutic compounds targeting nodes in the PI3K-AKT-mTOR (mechanistic target of rapamycin) signalling pathway are being tested in clinical trials for patients with metastatic castration-resistant prostate cancer. Knowledge of PTEN status might be helpful to identify patients who are more likely to benefit from these therapies. To enable the use of PTEN status as a prognostic and predictive biomarker, analytically validated assays have been developed for reliable and reproducible detection of PTEN loss in tumour tissue and in blood liquid biopsies. The use of clinical-grade assays in tumour tissue has shown a robust correlation between loss of PTEN and its protein as well as a strong association between PTEN loss and adverse pathological features and oncological outcomes. In advanced disease, assessing PTEN status in liquid biopsies shows promise in predicting response to targeted therapy. Finally, studies have shown that PTEN might have additional functions that are independent of the PI3K-AKT pathway, including those affecting tumour growth through modulation of the immune response and tumour microenvironment.

Androgen receptor splice variants bind to constitutively open chromatin and promote abiraterone-resistant growth of prostate cancer.

Androgen receptor (AR) splice variants (ARVs) are implicated in development of castration-resistant prostate cancer (CRPC). Upregulation of ARVs often correlates with persistent AR activity after androgen deprivation therapy (ADT). However, the genomic and epigenomic characteristics of ARV-dependent cistrome and the disease relevance of ARV-mediated transcriptome remain elusive. Through integrated chromatin immunoprecipitation coupled sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analysis, we identified ARV-preferential-binding sites (ARV-PBS) and a set of genes preferentially transactivated by ARVs in CRPC cells. ARVs preferentially bind to enhancers located in nucleosome-depleted regions harboring the full AR-response element (AREfull), while full-length AR (ARFL)-PBS are enhancers resided in closed chromatin regions containing the composite FOXA1-nnnn-AREhalf motif. ARV-PBS exclusively overlapped with AR binding sites in castration-resistant (CR) tumors in patients and ARV-preferentially activated genes were up-regulated in abiraterone-resistant patient specimens. Expression of ARV-PBS target genes, such as oncogene RAP2A and cell cycle gene E2F7, were significantly associated with castration resistance, poor survival and tumor progression. We uncover distinct genomic and epigenomic features of ARV-PBS, highlighting that ARVs are useful tools to depict AR-regulated oncogenic genome and epigenome landscapes in prostate cancer. Our data also suggest that the ARV-preferentially activated transcriptional program could be targeted for effective treatment of CRPC.

Abstract A52: Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer

Abstract Introduction: Recent advances improved survival in men with metastatic castration-resistant prostate cancer (CRPC) after chemotherapy when treated with androgen ablation therapies, including the well-known androgen receptor (AR) antagonist enzalutamide. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next-generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR-positive and -negative prostate cancers. Here we report the further development of effective N-cadherin specific monoclonal antibodies against CRPC and their value in combination therapies. Method: Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen-dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with enzalutamide. Further, enzalutamide-resistant cell lines were also generated and evaluated. In vivo studies were performed using castration-resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to enzalutamide or mAb alone or in combination. When the tumors reached 100-200 mm3 in volume the mice were castrated and treated with 5-10 mg/kg enzalutamide and/or 5-10 mg/kg mAb candidates. Tumors were monitored for growth, local invasion, and metastasis. At endpoint, tumors were harvested and further analyzed. Promising mAb candidates, as well as new epitope targets, were then selected and reformatted into full humanized N-cadherin monoclonal antibodies and further analyzed. Results: Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. In vitro studies showed that combination treatment of N-cadherin positive and AR-positive cells reduced invasion and cell proliferation than either treatment alone. Certain enzalutamide-resistant cell lines generated showed an increase in N-cadherin expression. Such cells responded to N-cadherin antibodies in androgen-deprived conditions, suggesting a role for N-cadherin in a subset of enzalutamide resistance. In vivo studies using CRPC xenograft models expressing N-cadherin and AR showed inhibition of tumor growth when the animals were castrated and treated with a combination of enzalutamide and N-cadherin mAb than either treatment alone. Androgen-dependent xenograft models in castrated mice also significantly delayed castration-resistant tumor growth when treated in combination than either treatment alone. The newly generated humanized mAbs showed similar outcomes. Conclusion: Previous studies suggested that therapeutic targeting of N-cadherin with monoclonal antibodies have significant effect in inhibiting tumor progression. We have verified that humanized N-cadherin mAb exhibit significant efficacy, fostering our search for increasingly effective immunotherapy in the clinic. Further, the antibodies showed synergy in combination with enzalutamide, suggesting promise in potential combination therapies for CRPC. Other androgen receptor antagonists and other drugs targeting those potentially involved in downstream pathways may prove to have additional clinical benefit. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Joyce Yamashiro, Wang Chun, Della Z. Hu, Anna M. Wu, Robert E. Reiter. Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A52.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature