MDA PCa 2b Research Articles

Abstract B004: Dealing with prostate cancer racial disparity: Galeterone and next-generation galeterone analog VNPP433-3β potently inhibits proliferation of prostate cancer cells from African American patient by degrading AR and Mnk1/2

Abstract Prostate cancer incidence and mortality rates are remarkably higher in African-American (AA) men as compared to their European-American (EA) counterparts even after factoring in socio economic status. The disproportionate incidence of prostate cancer and associated mortality in African men is currently becoming more evident as Several studies have reported significant variations in genetic, molecular, epigenetic, microRNA, and cell signaling pathways in patients of Caucasian and African race. As androgen is implicated in prostate cancer progression and about 90% of the cases are Androgen-dependent, blocking androgen action is an effective strategy in treating prostate cancer. Previously, we reported that galeterone and VNPP433-3β, a next generation galeterone analog potently inhibit metastatic castration resistant prostate cancer of Caucasian origin in vitro and in vivo by degrading AR/AR-V7 and Mnk1/2. Since the prostate cancer patients of African descent express significant levels of AR and Mnk1/2, we investigated the efficacy of galeterone and VNPP433-3β in treating prostate cancer of African men. Cell proliferation assay using the cell lines MDA-PCa 2b and RC77 T/E derived from AA prostate cancer patient demonstrates that galeterone and VNPP433-3β can effectively inhibit prostate cancer of African descent at very low concentration. These cell lines are Androgen-sensitive and further biochemical studies reveal that galeterone and VNPP433-3β degrade AR and Mnk1/2 in RC77 T/E and MDA-PCa 2b cell lines, thereby inhibiting its proliferation. Galeterone and VNPP433-3β significantly decrease the half-life of AR in these cell lines by increasing the rate of ubiquitination and proteasomal degradation of AR. Therefore, galeterone and VNPP433-3β hold significant promise for the clinical management of prostate cancer in patients of African descent Citation Format: Elizabeth Thomas, Retheesh Sulochana Thankan, Puranik Purushottachar, Vincent C.O. Njar. Dealing with prostate cancer racial disparity: Galeterone and next-generation galeterone analog VNPP433-3β potently inhibits proliferation of prostate cancer cells from African American patient by degrading AR and Mnk1/2 [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B004.

Tumor suppressive miR-99b-5p as an epigenomic regulator mediating mTOR/AR/SMARCD1 signaling axis in aggressive prostate cancer.

African American (AA) men exhibited 2.3-fold higher PCa incidence and 1.7-fold higher PCa mortality rates when compared to the European American (EA) men. Besides the socioeconomic factors, emerging evidence has highlighted that biological risk factors may play critical roles in the AA PCa disparities. Previously, we have shown that downregulated miR-99b-5p and upregulated mTOR cooperatively promotes the AA PCa aggressiveness and drug resistance. In this study, we aimed to explore the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis in AA PCa aggressiveness. The analyses used in the study included immunofluorescence, western blot, in-vitro functional assays (TUNEL, colony forming, and MTT), and chromatin immunoprecipitation (ChIP)-qPCR assays in 2D and/or 3D culture model of EA PCa and AA PCa cell lines. Specifically, the immunofluorescence staining, and western blot analysis has revealed that nuclear mTOR, AR, and SMARCD1 were highly expressed in AA PCa (MDA PCa 2b) compared to EA PCa (LNCaP) cell line. Western blot analysis further revealed that miR-99b-5p inhibited protein levels of mTOR, AR/AR-V7 and SMARCD1 in cytoplasm and nuclei of EA and AA PCa. The in-vitro functional (MTT, TUNEL, and clonogenic) assays have demonstrated that miR-99b-5p effectively inhibited cell proliferation/survival and induced cell apoptosis in EA and AA PCa cells. Moreover, combination of miR-99b-5p and enzalutamide (Enz) synergistically enhances the cytotoxicity against aggressive AA PCa and castration-resistant prostate cancer (CRPC). mTOR ChIP-qPCR assays further demonstrated that miR-99b-5p or miR-99b-5p/Enz significantly reduces the recruitment of mTOR to the genes involved in the metabolic reprogramming in CRPC. Taken together, miR-99b-5p may function as an epigenomic driver to modulate the mTOR/AR/SMARCD1 signaling axis in AA PCa and resistant CRPC.

Induction of Reactive Bone Stromal Fibroblasts in 3D Models of Prostate Cancer Bone Metastases.

A dynamic interplay between prostate cancer (PCa) cells and reactive bone stroma modulates the growth of metastases within the bone microenvironment. Of the stromal cells, metastasis-associated fibroblasts (MAFs) are known to contribute but are the least studied cell type in PCa tumour progression. It is the aim of the current study to establish a biologically relevant 3D in vitro model that mimics the cellular and molecular profiles of MAFs found in vivo. Using 3D in vitro cell culture models, the bone-derived fibroblast cell line, HS-5, was treated with conditioned media from metastatic-derived PCa cell lines, PC3 and MDA-PCa 2b, or mouse-derived fibroblasts 3T3. Two corresponding reactive cell lines were propagated: HS5-PC3 and HS5-MDA, and evaluated for alterations in morphology, phenotype, cellular behaviour, plus protein and genomic profiles. HS5-PC3 and HS5-MDA displayed distinct alterations in expression levels of N-Cadherin, non-functional E-Cadherin, alpha-smooth muscle actin (α-SMA), Tenascin C, and vimentin, along with transforming growth factor receptor expression (TGF β R1 and R2), consistent with subpopulations of MAFs reported in vivo. Transcriptomic analysis revealed a reversion of HS5-PC3 towards a metastatic phenotype with an upregulation in pathways known to regulate cancer invasion, proliferation, and angiogenesis. The exploitation of these engineered 3D models could help further unravel the novel biology regulating metastatic growth and the role fibroblasts play in the colonisation process.

DNA Methyltransferase 1 Targeting Using Guadecitabine Inhibits Prostate Cancer Growth by an Apoptosis-Independent Pathway.

Epigenetic alterations such as DNA methylation and histone modifications are implicated in repressing several tumor suppressor genes in prostate cancer progression. In this study, we determined the anti-prostate cancer effect of a small molecule drug guadecitabine (gDEC) that inhibits/depletes the DNA methylation writer DNA methyltransferase 1 (DNMT1). gDEC inhibited prostate cancer cell growth and proliferation in vitro without activating the apoptotic cascade. Molecular studies confirmed DNMT1 depletion and modulated epithelial-mesenchymal transition markers E-cadherin and β-catenin in several prostate cancer cell lines (LNCaP, 22Rv1, and MDA PCa 2b). gDEC treatment also significantly inhibited prostate tumor growth in vivo in mice (22Rv1, MDA PCa 2b, and PC-3 xenografts) without any observed toxicities. gDEC did not impact the expression of androgen receptor (AR) or AR-variant 7 (AR-V7) nor sensitize the prostate cancer cells to the anti-androgen enzalutamide in vitro. In further investigating the mechanism of cytoreduction by gDEC, a PCR array analyses of 84 chromatin modifying enzymes demonstrated upregulation of several lysine-specific methyltransferases (KMTs: KMT2A, KMT2C, KMT2E, KMT2H, KMT5A), confirmed by additional expression analyses in vitro and of harvested xenografts. Moreover, gDEC treatment increased global histone 3 lysine 4 mono-and di-methylation (H3K4me1 and H3K4me2). In sum, gDEC, in addition to directly depleting the corepressor DNMT1, upregulated KMT activating epigenetic enzymes, activating terminal epithelial program activation, and prostate cancer cell cycling exits independent of apoptosis.

MiR-92a-1-5p enriched prostate cancer extracellular vesicles regulate osteoclast function via MAPK1 and FoxO1

BackgroundWe have previously reported that extracellular vesicles (EVs) derived from osteoblastic, osteoclastic and mixed prostate cancer cells promote osteoclast differentiation and inhibit osteoblast differentiation via transferring miR-92a-1-5p. In the present study, we focused on engineering miR-92a-1-5p into EVs and determining any therapeutic roles and mechanisms of the engineered EVs.MethodsA stable prostate cancer cell line (MDA PCa 2b) overexpressing miR-92a-1-5p was constructed by lentivirus, and EVs were isolated by ultracentrifugation. The overexpression of miR-92a-1-5p in both cells and EVs was tested using qPCR. Osteoclast function was evaluated by Trap staining, mRNA expression of osteoclastic markers ctsk and trap, immunolabeling of CTSK and TRAP and microCT using either in vitro and in vivo assays. Target gene of miR-92a-1-5p was proved by a dual-luciferase reporter assay system. siRNAs were designed and used for transient expression in order to determine the role of downstream genes on osteoclast differentiation.ResultsStable overexpression cells of miRNA-92a-5p was associated with EVs upregulating this microRNA, as confirmed by qPCR. Further, miR-92a-1-5p enriched EVs promote osteoclast differentiation in vitro by reducing MAPK1 and FoxO1 expression, associated with increased osteoclast function as shown by TRAP staining and mRNA expression of osteoclast functional genes. siRNA targeting MAPK1 or FoxO1 resulted in similar increase in osteoclast function. In vivo, the miR-92a-1-5p enriched EVs given via i.v. injection promote osteolysis, which was associated with reduction of MAPK1 and FoxO1 expression in bone marrow.ConclusionThese experiments suggest that miR-92a-1-5p enriched EVs regulate osteoclast function via reduction of MAPK1 and FoxO1.Graphical

Transcripts of the Prostate Cancer-Associated Gene ANO7 Are Retained in the Nuclei of Prostatic Epithelial Cells.

Prostate cancer affects millions of men globally. The prostate cancer-associated gene ANO7 is downregulated in advanced prostate cancer, whereas benign tissue and low-grade cancer display varying expression levels. In this study, we assess the spatial correlation between ANO7 mRNA and protein using fluorescent in situ hybridization and immunohistochemistry for the detection of mRNA and protein in parallel sections of tissue microarrays prepared from radical prostatectomy samples. We show that ANO7 mRNA and protein expression correlate in prostate tissue. Furthermore, we show that ANO7 mRNA is enriched in the nuclei of the luminal cells at 89% in benign ducts and low-grade cancer, and at 78% in high-grade cancer. The nuclear enrichment of ANO7 mRNA was validated in prostate cancer cell lines 22Rv1 and MDA PCa 2b using droplet digital polymerase chain reaction (ddPCR) on RNA isolated from nuclear and cytoplasmic fractions of the cells. The nuclear enrichment of ANO7 mRNA was compared to the nuclearly-enriched lncRNA MALAT1, confirming the surprisingly high nuclear retention of ANO7 mRNA. ANO7 has been suggested to be used as a diagnostic marker and a target for immunotherapy, but a full comprehension of its role in prostate cancer progression is currently lacking. Our results contribute to a better understanding of the dynamics of ANO7 expression in prostatic tissue.

Abstract PO-134: Differential effects of CXCR1 and CXCR2 receptors on prostate tumorigenesis

Abstract Chemokines and their receptors are important proteins that promote growth of various malignant tumors. In prostate cancer, some members of the CXC chemokine receptor have been shown to enhance angiogenesis, proliferation and metastasis. In this study we assess the roles of the interleukin-8 (IL-8/CXCL8) receptors CXCR1 and CXCR2 in prostate cancer development, using the African American MDA PCa 2b cell line. Our results show that overexpression of CXCR2 enhanced in-vitro cell proliferation, soft agar growth and in-vivo tumor xenograft in nude mice, whereas overexpression of CXCR1 inhibited cell proliferation and tumor growth, relative to control cells. Interestingly, cells overexpressing CXCR1 exhibited a more mesenchymal phenotype, characterized by reduced E-Cadherin but increased N-Cadherin and vimentin protein expression. CXCR1 overexpression also blocked AKT activation and signal transduction. CXCR2 overexpression, however, resulted in a neuroendocrine phenotype, characterized by reduced androgen (AR) receptor expression and altered chromogranin A expression. In conclusion, these results indicate that CXCR1 and CXCR2 overexpression may play alternate roles on prostate tumorigenesis. Citation Format: Timothy O. Adekoya, Nikia Smith, Parag Kothari, Ricardo M. Richardson. Differential effects of CXCR1 and CXCR2 receptors on prostate tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-134.

Abstract PO-134: Intracellular calcium regulates androgen receptor expression and basal-luminal features in prostate cancer from African American men

Abstract Background: Disparities in prostate cancer (PrCa) incidence and mortality between African American (AA) and European American (EA) men are partially mediated by underlying disease biology. The goal of this study was to determine how DNA methylation and transcriptomic changes drive PrCa health disparities in AA men. Methods: Transcriptomic alterations potentially mediated by DNA methylation were identified by Illumina arrays and RNA-sequencing in PrCa from 30 AA and 32 EA men. Androgen receptor (AR) protein expression was measured using tissues microarray of matched PrCa tissues from 95 EA and 92 AA men. In vitro, MDA PCa 2a (2a)/MDA PCa 2b (2b) cells derived from an AA man and LNCaP/LaPC-4 cells derived from EA men were stimulated with ionomycin, a calcium ionophore to measure intracellular calcium using fluorescent-based live imaging. Results: Unsupervised hierarchical clustering revealed DNA methylation clusters (Cluster A and Cluster B) with differential methylation of loci that regulate intracellular calcium levels including RYR2, TRPC6, and TRPA1. Increased methylation of calcium regulatory genes in Cluster A was associated with reduced disease-free time (DFT) (21.65 vs 46.71 months, p<0.05) only in AA men with PrCa. RYR2 (-0.122 vs -0.004, p=0.69), TRPC6 (0.006 vs -0.639, p=0.06) and TRPA1 (-0.070 vs -0.269, p<0.05) transcript levels were lower in Cluster A compared to Cluster B. These data suggest DNA methylation can regulate expression of calcium regulating genes. In vitro, we found AA PrCa derived 2b cells have reduced RNA levels of RYR2, TRPV6 and CALB1 compared to EA PrCa derived LNCaP cells. Reduced transcription can result in lower protein expression and thus activity of calcium regulatory genes. To test this, we stimulated cells with ionomycin and found a rapid increase in intracellular calcium in 2a/2b cells (within 60 seconds) compared to LNCaP/LAPC-4 cells (120-300 seconds). This suggests that decreased transcription correlates to reduced buffering capacity of calcium regulatory genes. Others have shown that increased intracellular calcium reduces AR protein levels. Therefore, we analyzed AR protein expression in a subset of tumors from Cluster A and B. AR levels were lower in adjacent non-tumor and tumor tissue in these overlapping samples. AR low PrCa are associated with basal like features and respond poorly to androgen deprivation therapy. Therefore, we analyzed the PAM50 basal/luminal gene sets and AR target genes and found differentially expression of these genes in AA PrCa derived 2b and EA PrCa derived LNCaP cells. Conclusions: Our study shows that AA men with PrCa have epigenetically dysregulated calcium signaling that is associated with worse DFT. Our ongoing work seeks to identify how these alterations regulate AR expression and thus basal/luminal features. Our long- term goal is to establish novel molecular subtypes using calcium-AR-basal/luminal features that can guide design of rationale therapies in a subset of AA men with PrCa. Citation Format: Swathi Ramakrishnan, Xuan Peng, Eduardo C. Gomez, Kristopher Attwood, Ivan V. Maly, Wilma A. Hoffmann, Wendy Huss, Gissou Azabdaftari, Li Yan, Jianmin Wang, Anna Woloszynska. Intracellular calcium regulates androgen receptor expression and basal-luminal features in prostate cancer from African American men [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-134.

Mechanobiological evaluation of prostate cancer metastasis to bone using an in vitro prostate cancer testbed

Prostate cancer exhibits a propensity to metastasize to the bone, which often leads to fatality. Bone metastasis is characterized by complex biochemical, morphological, pathophysiological, and genetic changes to cancer cells as they colonize at bone sites. In this study, we report the evaluation of MDA PCa2b prostate cancer cells' nanomechanical properties during the mesenchymal-to-epithelial transition (MET) and during disease progression at the metastatic site. Bone-mimetic tissue-engineered 3D nanoclay scaffolds have been used to create in vitro metastatic site for prostate cancer. A significant softening of the prostate cancer cells during MET and further softening as disease progression occurs at metastasis is also reported. The significant reduction in elastic modulus of prostate cancer cells during MET was attributed to actin reorganization and depolymerization. This study provides input towards direct nanomechanical measurements to evaluate the time evolution of cells' mechanical behavior in tumors at bone metastasis site.

The MD Anderson Prostate Cancer Patient-derived Xenograft Series (MDA PCa PDX) Captures the Molecular Landscape of Prostate Cancer and Facilitates Marker-driven Therapy Development.

Advances in prostate cancer lag behind other tumor types partly due to the paucity of models reflecting key milestones in prostate cancer progression. Therefore, we develop clinically relevant prostate cancer models. Since 1996, we have generated clinically annotated patient-derived xenografts (PDXs; the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical prostate cancer. We studied two cell line-derived xenografts and the first 80 PDXs derived from 47 human prostate cancer donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (androgen receptor, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the speckle-type POZ protein-like (SPOPL) gene in PDXs derived from seven human donors of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of prostate cancer. SPOPL deletions are found in 7% of The Cancer Genome Atlas prostate cancers, which suggests that our cohort is a reliable platform for targeted drug development. The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of prostate cancers progressing under novel treatments and enables optimization of prostate cancer-specific, marker-driven therapy.

Abstract 3389: A multi-gene prostate cancer liquid biopsy with > 92% accuracy in diagnosis and assessment of disease status

Abstract Background: There are a paucity of blood-based biomarkers with clinical utility for prostate cancer (PCa). We examined a circulating-free mRNA (27-gene) prostate cancer signature to diagnose PCa. Clinical utility of the assay was then assessed in surgical patients. Methods: Gene identification and validation: Publicly available PCa transcriptome sets (n=5) (E-GEOD-46691, E-GEOD-46602, E-GEOD-62116, E-GEOD-62667, E-GEOD-72220: n=1,159 samples); PCA cell lines (n=7) gene expression in 22Rv1 and E006AA-hT (localized); VCaP; PC-3; LNCaP; DU145 and MDA PCa2b (all metastatic) from two normal prostate epithelial lines: PWR-1E and RWPE-1 and validated in PCa tumor tissue (n=50). Transcriptome analyses: tumor compared with normal blood-based transcriptomes using gene co-expression network enrichment, differential expression and functional enrichment analyses to identify candidate markers. Blood gene expression: PCa set #I: n=132, PCA set #II: n=50 (biochemical recurrence [BCR]), BPH: n=44, controls n=55. We then constructed an artificial intelligence PCa model using classification algorithm analyses. Scoring: normalized algorithmically analyzed gene expression (0 to 100), positive score ≥20. Matched tumor/ blood samples: n=50. PSA: BPH (n=44) and PCa (n=132). Clinical score assessment: Score utility was assessed in surgical cohorts: Surgical: (n=47), scores =pre-surgical and post: 1 week - 14 months. Statistics: Non-parametric (Kruskal-Wallis), Pearson-correlation, Fisher's and AUROC analyses (Mean±SEM). Results: Transcriptomic analysis identified a 27 gene blood signature for PCa. Expression levels were significantly elevated (p<0.001, 2.1-35.8-fold) in cell lines and PCA tumors. The matched tissue/blood correlation was r: 0.56 (p=0.0023). In PCa #I, levels were 47±2 (p<0.0001) compared to BPH (19±1) and controls (18±0.5); AUROC: 0.92 (BPH) and 0.94 (controls), with an accuracy of 85-88%, a sensitivity of 86% and specificities 82 & 93% respectively. For PSA the AUROC (PCa vs. BPH) was 0.51 (p=0.88). PSA was positive in 86% of BPH and was >10ng/ml in 30%. PSA was positive in 83% of PCa and >10ng/ml in 40% (Fisher's exact: p=0.28). PSA accuracy (>10ng/ml) was 48%. Levels in PCA #II (BCR) were 44±3. ProstaTest-was positive in 48 (96%). Surgical cohort (n=47): PCa Prostatest accuracy 100% pre-surgery. Resection significantly decreased levels (KW-statistic: 57.4, p<0.0001) from 52±1 to 23.5±2. Conclusion: The diagnostic accuracy of a PCa molecular blood-signature was 92%; significantly better than PSA (48%, p<0.0001). Surgical resection significantly (p<0.0001) decreased levels. Biochemical recurrence was accurately detected (96%). A multi-gene prostate cancer liquid biopsy seems likely to have clinical utility in the diagnosis and monitoring management of PCa. Citation Format: Kambiz Rahbar, Mark Kidd, Ignat Drozdov, Alexandra Kitz, Anna Malczewska, Pawel Rajwa, Christof Bernemann, Lisa Bodei, Irvin Modlin. A multi-gene prostate cancer liquid biopsy with > 92% accuracy in diagnosis and assessment of disease status [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3389.

Host- versus cell-dependent effects of β-Arrestin 1 expression in prostate tumorigenesis

Abstract Prostate cancer (PCa) constitutes a serious health challenge and remains one of the main causes of cancer-related death among men. The more aggressive form of the disease is androgen independent resulting in lack of response to androgen deprivation therapy. The scaffold proteins β-arrestin 1 and 2 (βarr1 and βarr2) which are known to mediate G protein-coupled receptors (GPCRs) desensitization and internalization, were also shown to modulate prostate tumorigenesis. βarr1 is overexpressed (~4 fold) in PCa cells relative to βarr2. In this study, we investigate the effect of β-arrestins expression in prostate tumorigenesis using in-vivo xenografts and autochthonous transgenic adenocarcinoma mouse prostate (TRAMP) model deficient in β-arrestins expression. Depletion of βarr1 in TRAMP mice (TRAMP/βarr1−/−) increased PCa growth and decreased overall survival, relative to TRAMP and TRAMP/βarr2−/− animals. Tumors from TRAMP/βarr1−/− mice displayed increase androgen receptor (AR), but decrease prostate specific antigen (PSA) expression. In contrast, depletion of βarr1 in MDA PCa 2b cells (MDA PCa 2b-βarr1−/−) decreased cell proliferation, wound closure and anchorage independent cell growth compared to wild type MDA PCa 2b and MDA PCa 2b-βarr2−/− cells. Nude mice xenografts of MDA PCa 2b-βarr1−/− cells also showed significant decrease in tumor growth. Interestingly, tumor lysates from MDA PCa 2b-βarr1−/− xenografts exhibited decrease AR and increase PSA expression, relative to control tumors. Altogether, the data indicate that βarr1 modulates AR/PSA signaling to regulate prostate cancer aggressiveness. However, the effect of βarr1 is context-sensitive depending on its expression in the host relative to the tumor cells.

Antitumor Activity of the IGF-1/IGF-2-Neutralizing Antibody Xentuzumab (BI 836845) in Combination with Enzalutamide in Prostate Cancer Models.

Androgen deprivation therapy and second-generation androgen receptor signaling inhibitors such as enzalutamide are standard treatments for advanced/metastatic prostate cancer. Unfortunately, most men develop resistance and relapse; signaling via insulin-like growth factor (IGF) has been implicated in castration-resistant prostate cancer. We evaluated the antitumor activity of xentuzumab (IGF ligand-neutralizing antibody), alone and in combination with enzalutamide, in prostate cancer cell lines (VCaP, DuCaP, MDA PCa 2b, LNCaP, and PC-3) using established in vitro assays, and in vivo, using LuCaP 96CR, a prostate cancer patient-derived xenograft (PDX) model. Xentuzumab + enzalutamide reduced the viability of phosphatase and tensin homolog (PTEN)-expressing VCaP, DuCaP, and MDA PCa 2b cells more than either single agent, and increased antiproliferative activity and apoptosis induction in VCaP. Xentuzumab or xentuzumab + enzalutamide inhibited IGF type 1 receptor and AKT serine/threonine kinase (AKT) phosphorylation in VCaP, DuCaP, and MDA PCa 2b cells; xentuzumab had no effect on AKT phosphorylation and proliferation in PTEN-null LNCaP or PC-3 cells. Knockdown of PTEN led to loss of antiproliferative activity of xentuzumab and reduced activity of xentuzumab + enzalutamide in VCaP cells. Xentuzumab + enzalutamide inhibited the growth of castration-resistant LuCaP 96CR PDX with acquired resistance to enzalutamide, and improved survival in vivo The data suggest that xentuzumab + enzalutamide combination therapy may overcome castration resistance and could be effective in patients who are resistant to enzalutamide alone. PTEN status as a biomarker of responsiveness to combination therapy needs further investigation.

Prostate Cancer Phenotype Influences Bone Mineralization at Metastasis: A Study Using an In Vitro Prostate Cancer Metastasis Testbed.

ABSTRACTIn this study, two types of prostate cancer cell lines, highly metastatic PC‐3 and low metastatic MDA PCa 2b (PCa) were cultured on bone mimetic scaffolds to recapitulate metastasis to bone. A unique in vitro 3D tumor model that uses a sequential culture (SC) of human mesenchymal stem cells followed by seeding with cancer cells after bone formation was initiated to study the phenotype‐specific interaction between prostate cancer cells and bone microenvironment. The PCa cells were observed to be less prolific and less metastatic, and to form multicellular tumoroids in the bone microenvironment, whereas PC‐3 cells were more prolific and were highly metastatic, and did not form multicellular tumoroids in the bone microenvironment. The metastatic process exhibited by these two prostate cancer cell lines showed a significant and different effect on bone mineralization and extracellular matrix formation. Excessive bone formation in the presence of PC‐3 and significant osteolysis in the presence of PCa were observed, which was also indicated by osteocalcin and MMP‐9 expression as measured by ELISA and qRT‐PCR. The field emission scanning electron microscopy images revealed that the structure of mineralized collagen in the presence of PC‐3 is different than the one observed in healthy bone. All experimental results indicated that both osteolytic and osteoblastic bone lesions can be recapitulated in our tumor testbed model and that different cancer phenotypes have a very different influence on bone at metastasis. The 3D in vitro model presented in this study provides an improved, reproducible, and controllable system that is a useful tool to elucidate osteotropism of prostate cancer cells. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Abstract 1047: Novel roles for manganese superoxide dismutase polymorphisms in prostate cancer

Abstract Prostate Cancer is the most common non-cutaneous cancer and second leading cause of cancer related death in American men. Epithelial Mesenchymal Transition (EMT), a key event in prostate cancer metastasis, is a process in which polarized epithelial cells undergo biochemical changes, allowing them to assume mesenchymal morphologies, enhancing invasiveness and migration. Reactive Oxygen Species (ROS) are chemically reactive molecules of cellular metabolism that induce oxidative stress, and increase levels of SNAI1 (Snail) transcription factor while also promoting EMT. In Phase II clinical trials, patients were treated with natural product, Muscadine Grape Skin Extract (MSKE), and patients with SOD2 Ala/Ala single nucleotide polymorphism (SNP) responded better than those with Val/Val SNP. Ala/Ala SNP is associated with higher dismutase activity [superoxide (O2-) to hydrogen peroxide (H2O2)] than Val/Val. We hypothesize that SOD2 Ala/Ala genotype is associated with increased H2O2 and EMT, potentially antagonized by MSKE in prostate cancer cells. Prostate cancer cell lines were analyzed for SOD2 SNPs by pyrosequencing. Site-directed mutagenesis was conducted to create the Ala/Ala and Val/Val SNP vectors. LNCaP prostate cancer cells were transiently and subsequently stably transfected with SOD2 Val/Val cDNA, as well as empty vector (Neo) control, and verified through western blot analysis. ROS activity was evaluated for baseline ROS activity. Our results showed that most prostate cancer cell lines were heterozygous for SOD2 SNP (Ala/Val), although several had extra copies, for example, LNCaP had Val/Val/Ala, suggesting multiple copy numbers. Only metastatic MDA PCa 2a and MDA PCa 2b contained Ala/Ala homozygous SNP. LNCaP cells transiently and stably transfected with Val/Val showed partial induction of EMT, concomitant with higher levels of Snail and Vimentin, as compared to LNCaP Neo control. Furthermore, data also suggested that transient transfection marginally affected ROS activity while stable transfection caused significant changes in ROS activity compared to LNCaP Neo cells. We have begun to stably transfect Ala/Ala cDNA into LNCaP cells and are interested to see the differences in ROS activity and EMT induction between the two clone cell types (LNCaP Ala/Ala and Val/Val). As previous data suggests, we believe that Ala/Ala will show a higher expression of SOD2 and mesenchymal markers Snail and Vimentin, demonstrating full induction of EMT. In future we will test ROS activity, EMT marker expression in response to treatment of LNCaP Ala/Ala and Val/Val cells with MSKE. These studies may uncover the differential functions and response to treatment of SOD2 SNPs. Acknowledgements: These studies were supported by the NIH/NIMHD/RCI Grant #5G12MD007590-31, NIH/NIGMS/RISE Grant #5R25GM060414, and https://www.thecommunityfoundation.org/ Citation Format: Janae D. Sweeney, Valerie Odero-Marah, Channing Paller. Novel roles for manganese superoxide dismutase polymorphisms in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1047.

Abstract 5305: Reexpression of LSAMP, a gene frequently deleted in African American prostate cancers, suppresses tumor growth and β-catenin activity

Abstract Introduction: African American (AA) men have the highest prostate cancer incidence and mortality rates in the US. The biological contribution to this disparity, however, is not well understood. LSAMP inactivation has been implicated in several cancers, and was recently identified in prostate cancer. A higher frequency of LSAMP inactivation has been observed in AA prostate cancer, and this aberration has been associated with a significantly greater risk of disease progression. In the characterization of LSAMP in prostate cancer cells lines, we found the copy number to be variable and the expression to be low or undetectable. LNCaP, MDA PCa 2B, and DU 145 prostate cancer cell lines were stably transduced to express LSAMP in an inducible or constitutive manner. LSAMP expression in these cell lines resulted in reduced cell proliferation, and induced a reversion to indolent cell-cell, and cell-extra-cellular-matrix adhesion characteristics, consistent with its tumor suppressive role. LSAMP expression also resulted in the down-regulation of receptor tyrosine kinases EPHA3, FGFR2, and FGFR4, and reduced activation of their downstream ERK and AKT pathways. Several Integrins were also up-regulated upon LSAMP expression. Additionally, β-catenin localization was altered, suggesting a potential reduction in transcriptional activity. We assessed the tumor suppressive function of LSAMP further, using in vitro assays and in vivo mouse models. Methods: LSAMP expressing and control DU 145 cells were used to investigate the tumor suppressive function of LSAMP in vivo. Athymic nude mice were injected either subcutaneously, to determine effect of LSAMP expression on prostate tumor growth rates, or intravenously, to determine effect of LSAMP expression on tumor formation. We performed the TOPflash/FOPflash luciferase reporter assay to determine whether LSAMP expression modulates transcriptional activity of β-catenin in vitro. Results: LSAMP expression resulted in a significant inhibition of tumor growth in the subcutaneous xenograft model. In the intravenous xenograft model, LSAMP expression resulted in a reduced incidence of distant metastases. Consistent with the negative modulation of signal transduction, and β-catenin localization previously observed, LSAMP expression resulted in a reduction of β-catenin transcriptional activity in vitro. Conclusion: These studies provide in vivo evidence of the suppressive function of LSAMP in prostate tumors, corroborating previous in vitro and clinical findings. LSAMP expression reduced tumor growth rates, and incidence of distant metastases. LSAMP expression also reduced β-catenin transcriptional activity in vitro. These findings provide further support for a biological mechanism underlying the aggressive prostate cancer phenotype observed with LSAMP inactivation. Citation Format: Kevin Babcock, Taduru Sreenath, Charles P. Xavier, Inger L. Rosner, Shiv Srivastava, Albert Dobi, Shyh-Han Tan. Reexpression of LSAMP, a gene frequently deleted in African American prostate cancers, suppresses tumor growth and β-catenin activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5305.

Abstract 4285: p66Shc/ROS enhances the progression of androgen-sensitive towards castration-resistant prostate cancer cells

Abstract Purpose and Background: Prostate cancer (PCa) is the second leading cause of cancer-related death in United States men. Androgen deprivation therapy is the standard-of-care treatment for metastatic PCa; most patients eventually relapse and develop castration-resistant (CR) tumors that currently have no effective treatment. Thus, a useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies for CR PCa. In this study, we established a PCa cell progressive model in three independent cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells, recapitulating clinical PCa progression. Experimental Methods: AS and AI human prostate adenocarcinoma cell lines LNCaP, MDA PCa2b and VCaP were utilized in this study to demonstrate the molecular and signaling alterations seen in CR PCa. Stable p66Shc cDNA transfected subclones were established from LNCaP-AS cells. The tumorigenicity of AS and AI cells as well as AS PCa cells treated with H2O2 and NAC were evaluated via trypan blue exclusion, transwell migration, clonogenic assays and xenograft mouse models. The signaling profiles including phosphoprotein microarray and immunoblotting were conducted. Results: AI PCa cells have enhanced tumorigenicity, recapitulating the clinical CR phenotype, including AR expression, proliferation and tumorigenicity under androgen-deprived conditions. Further, AI cells exhibit increased ROS levels as well as enhanced signaling of proliferation and survival pathways. We further identified oxidase p66Shc as one of the potential sources of the ROS-mediated phenotypic and cell signaling alterations in AI PCa cells. LNCaP-AI cells and p66Shc subclones have a greater oxidative environment compared to LNCaP-AS cells. Increased ROS via H2O2 enhanced AS cell growth and migration, which was counteracted by antioxidant NAC. Treatment of LNCaP-AS cells with H2O2 resulted in a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Further, the ROS-driven alterations of p66Shc subclone cell signaling can be mitigated via p66Shc knockdown or inactive p66Shc mutant. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules. Molecular profiling showed that alterations of signaling in LNCaP-AI cells and p66Shc subclones attributed to p66Shc/ROS. Conclusions: We report the establishment of a PCa cell progression model in three commonly used PCa cell lines that replicates clinical PCa progression from the AS to the AI/CR phenotype. Altogether, the data shows ROS produced by p66Shc promotes PCa tumorigenicity and progression to the CR phenotype. Further characterization of the PCa progressive model will aid in the understanding of advanced PCa progression to help in treatment of this lethal disease. Citation Format: Dannah Miller, Matthew Ingersoll, Arpita Chatterjee, Rebecca Oberley-Deegan, Ming-Fong Lin. p66Shc/ROS enhances the progression of androgen-sensitive towards castration-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4285.

Abstract 1311A: CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target

Abstract Background: Previously we demonstrated that CYP3A5 positively regulates androgen receptor (AR) signaling and growth of prostate cancer (PC) cells. CYP3A5 inhibition decreases nuclear AR localization and downregulates AR downstream signaling (e.g. PSA and TMPRSS2), resulting in decreased PC cell growth both with and without dihydrotestosterone (DHT) induction. Our current work focusses on the effect of CYP3A5 modulation on AR signaling and therapeutic resistance, mostly in African Americans (AA) widely carrying (73%) wild type CYP3A5 (*1/*1). AAs and have higher incidence of PC and double the mortality rate compared to Non-Hispanic White Americans (NHWA) carrying mutated CY3A5 gene (*3/3, 95%) and expressing truncated non-functional CYP3A5 protein. Methods: Immunoprecipitation was used to identify binding partners of AR. q-RT-PCR based profiler assays was used to detect changes in AR downstream signaling and effect on genes responsible for therapeutic resistance after CYP3A5 inhibition. Effect of CYP3A5 on AR nuclear translocation and induction of synthetic lethality in combination with PARP inhibitors were evaluated using cell fractionation and immunocytochemistry studies. Results: Co-immunoprecipitation of flag-tagged CYP3A5 transfected LNCaP cell extracts showed that CYP3A5 was in a complex with HSP90, AR, HSP70 and HSP40, also confirmed with reverse immunoprecipitation using AR/ HSP90 antibodies and agarose A/G beads. AR regulated genes (SCL45A3, FKBP5, MYC, ELL2, and MT2A) were downregulated with CYP3A5 siRNA treatment in MDA PCa 2b cells (*1/*3, AA origin) in a qPCR-based profiler assay with high fold difference and a P value of ≤0.005. PC patients are often prescribed medications for comorbidities, many of which are CYP3A modulators. Our results indicate that CYP inhibitors (amiodarone, ritonavir, fluoxetine etc.) decrease AR nuclear translocation and inducers (phenytoin, rifampicin, pioglitazone and hyperforin) increase AR nuclear translocation and activation. CYP3A5 siRNA treatment downregulates genes involved in cancer drug therapeutic resistance (TOP2A, BRCA1/2, CCNE1, CDK2/4, DHFR, MVP, MYC, RARB and HPRT1, P ≤ 0.005 ). Loss of TOP2A, BRCA2 and BRCA1is known to impair homologous recombination (HR). In combination with topoisomerase inhibitor etoposide, CYP3A5 siRNA in PC cells increases number of gamma-H2AX foci indicating synthetic lethality. Conclusion: A-Developing CYP3A5-AR-HSP90 interface as a target may provide a novel strategy to block PC growth. B-CYP3A5 modulation of AR signaling can affect the efficacy of androgen deprivation therapy specifically AA patients carrying wild type CYP3A5. C-Since CYP3A5 inhibition, downregulates DNA damage repair genes and impairs HR, CYP3A5 inhibition in combination with PARP inhibitors can be a novel strategy to induce synthetic lethality in PC patients. Citation Format: Priyatham Gorjala, Oscar B. Goodman, Ranjana Mitra. CYP3A5 regulation of androgen receptor signaling: Relevance in African American prostate cancer patients and its development as novel target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1311A.

Activator of G protein signaling 3 modulates prostate tumor development and progression

Prostate cancer (PCa) is a leading cause of cancer death among men, with greater prevalence of the disease among the African American population in the USA. Activator of G-protein signaling 3 (AGS3/G-protein signaling modulator 1) was shown to be overexpressed in prostate adenocarcinoma relative to the prostate gland. In this study, we investigated the correlation between AGS3 overexpression and PCa malignancy. Immunoblotting analysis and real-time quantitative-PCR showed increase in AGS3 expression in the metastatic cell lines LNCaP (~3-fold), MDA PCa 2b (~2-fold), DU 145 (~2-fold) and TRAMP-C1 (~20-fold) but not in PC3 (~1-fold), relative to control RWPE-1. Overexpression of AGS3 in PC3, LNCaP and MDA PCa 2b enhanced tumor growth. AGS3 contains seven tetratricopeptide repeats (TPR) and four G-protein regulatory (GPR) motifs. Overexpression of the TPR or the GPR motifs in PC3 cells had no effect in tumor growth. Depletion of AGS3 in the TRAMP-C1 cells (TRAMP-C1-AGS3-/-) decreased cell proliferation and delayed wound healing and tumor growth in both C57BL/6 (~3-fold) and nude mice xenografts, relative to control TRAMP-C1 cells. TRAMP-C1-AGS3-/- tumors also exhibited a marked increase (~5-fold) in both extracellular signal-regulated kinase (ERK) 1/2 and P38 mitogen-activated protein kinase (MAPK) activation, which correlated with a significant increase (~3-fold) in androgen receptor (AR) expression, relative to TRAMP-C1 xenografts. Interestingly, overexpression of AGS3 in TRAMP-C1-AGS3-/- cells inhibited ERK activation and AR overexpression as compared with control TRAMP-C1 cells. Taken together, the data indicate that the effect of AGS3 in prostate cancer development and progression is probably mediated via a MAPK/AR-dependent pathway.

Abstract B061: Alternative RNA splicing and prostate cancer aggressiveness

Abstract Prostate cancer (PCa) is the most common cancer in men in the United States. Aggressive pathology, metastasis, and drug resistance of cancer cells are the most important prognostic factors for patients with PCa. African Americans (AAs) suffer disproportionately from PCa, with higher aggressiveness, progression, and mortality. A recently published analysis involving our laboratory reported more than 2,500 alternatively RNA spliced (ARS) genes between AA and white PCa biopsy tissue specimens. Subsequent analysis revealed that, among the race-related ARS genes in PCa tissue, 19 are also ARS in breast, lung, and liver cancer. Several of the genes that are ARS across multiple tumor types are important for cancer aggressiveness, including CD44, FN1, and LMO7. We designed PCR primers to further explore the expression of LMO7 variants in preclinical models of white and AA PCa, and found that a LMO7 variant that skips exons 10, 11, and 12 is predominantly expressed in VCaP (white) and MDA PCA 2b (AA) PCa cell lines. In addition, we detected a unique race-related LMO7 splicing event that excludes exons 10 and 11, but preserves exon 12. This event is detected in MDA PCA 2b cells, but not in VCaP cells. Importantly, both of these LMO7 splicing events are in-frame and are not frameshifts leading to truncated or extended C-terminal proteins. These LMO7 variants translate to proteins of 1297 amino acids (missing 344 amino acids coded by skipped exons 10, 11, and 12) and 1287 amino acids (missing 334 amino acids coded by skipped exons 10 and 11), which differ from the full-length LMO7 variant that translates to a protein of 1631 amino acids. Interestingly, the enrichment of these variants in cancer cells suggests an important regulatory role for the amino acids encoded by the skipped exons. Further work to study the impact of LMO7 variants as well as other candidate RNA splice variants on PCa aggressiveness is ongoing, using gene overexpression, gene depletion, and splicing manipulation by siRNA or CRISPR-cas, followed by assessment of resulting alterations in growth, migration, invasion, and drug sensitivity. In summary, we have identified genes that undergo race-related ARS in multiple tumor types and novel ARS events that may functionally contribute to increased tumor aggressiveness, including that of PCa in AA men. Citation Format: Muthana Al Abo, Steven R. Patierno, Jennifer A. Freedman. Alternative RNA splicing and prostate cancer aggressiveness [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B061.