African-American Prostate Cancer Cell Research Articles

Abstract A045: Whole transcriptome analysis of paired African American prostate cancer cell lines

Abstract Recent investigations on patient race have begun to characterize biological differences in prostate cancer, particularly among patients of African ancestry. Despite clinical trends continuously demonstrating this health disparity, very few African American cell line models exist for downstream molecular investigations, and even fewer have undergone proper characterization of their expression profiles. Here, we characterize the transcriptome of the non-malignant RC-77 N/E and malignant RC-77 T/E prostate epithelial cell lines previously established from prostate specimens from the same African American patient with early-stage primary prostate cancer. Total RNA sequencing was performed on the paired cell lines using Illumina NextSeq 2000 P2 to build transcriptomic profiles that have previously been undetermined. Following data analysis and normalization, we identified 369 genes with significant upregulation in the RC-77 T/E cell line in comparison to its normal counterpart. Among the top 15 upregulated genes, half of them were previously characterized in prostate cancer but not in the context of patient race. We further compared the transcriptome of RC-77 T/E to that of the African American metastatic MDA-PCa-2b. Finally, we analyzed differential gene expression by race among prostate cancer patients in The Cancer Genome Atlas (TCGA) to correlate our transcriptomics findings on the cell line level with that on the population level. These findings may allow for more targeted assessments of genes that are dependent on race as molecular tools are established to narrow the health disparities gap in prostate cancer. Citation Format: Kristi Y. Lee, Erica L. Beatson, Cindy H. Chau, Douglas K. Price, William D. Figg. Whole transcriptome analysis of paired African American prostate cancer cell lines [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A045.

Molecular Insight into Drug Resistance Mechanism Conferred by Aberrant PIK3CD Splice Variant in African American Prostate Cancer.

Targeting PI3Kδ has emerged as a promising therapy for hematologic and non-hematologic malignancies. Previously, we identified an oncogenic splice variant, PIK3CD-S, conferring Idelalisib resistance in African American (AA) prostate cancer (PCa). In the current study, we employed a comprehensive analysis combining molecular biology, biochemistry, histology, in silico simulation, and in vitro functional assays to investigate the PIK3CD-S expression profiles in PCa samples and to elucidate the drug resistance mechanism mediated by PI3Kδ-S (encoded by PIK3CD-S). The immunohistochemistry, RT-PCR, and Western blot assays first confirmed that PI3Kδ-S is highly expressed in AA PCa. Compared with PCa expressing the full-length PI3Kδ-L, PCa expressing PI3Kδ-S exhibits enhanced drug resistance properties, including a higher cell viability, more antiapoptotic and invasive capacities, and constitutively activated PI3K/AKT signaling, in the presence of PI3Kδ/PI3K inhibitors (Idelalisib, Seletalisib, Wortmannin, and Dactolisib). Molecular docking, ATP-competitive assays, and PI3 kinase assays have further indicated a drastically reduced affinity of PI3Kδ inhibitors with PI3Kδ-S vs. PI3Kδ-L, attributed to the lack of core binding residues in the PI3Kδ-S catalytic domain. Additionally, SRSF2 has been identified as a critical splicing factor mediating exon 20 skipping in PIK3CD pre-mRNA. The inhibition of the SRSF2 activity by SRPIN340 successfully sensitizes AA PCa cells to PI3Kδ inhibitors, suggesting a novel therapeutic option for Idelalisib-resistant tumors.

Abstract 3619: Vitamin D receptor function in African American prostate cancer is disrupted by reduced BAZ1A/SMARCA5

Abstract The biological drivers of prostate cancer (PCa) health disparities remain enigmatic. There are genomic differences, for example in the incidence of TMPRSS2 and ERG gene fusions which differ between European American (EA) and African American (AA) PCa. Accumulating epidemiological evidence suggests loss of vitamin D receptor VDR signaling contributes to AA PCa. Therefore, we investigated the VDR signaling in AA and EA prostate models. VDR protein levels were comparable between AA (RC43N, RC43T, RC77N, RC77T) and EA cells (HPr1AR, LNCaP) but RIME analyses identified significant AA-EA differences in VDR binding of coactivators (e.g. NCOA5), corepressors (e.g. HDAC2), and components of SWI/SNF and circadian rhythm complexes. ATAC-Seq identified 1α,25(OH)2D3-stimulated gain in nucleosome free (NF) regions was greatest in RC43N (non-malignant AA cells) and enriched for circadian rhythm transcription motifs (e.g. CLOCK). In RC43T AA PCa cells 1α,25(OH)2D3 led to a loss of NF free regions. Similarly, VDR ChIP-Seq revealed the cistrome was greater in AA cell models, and was dynamically altered in RC43N cells by 1α,25(OH)2D3 and overlapped with the SWI/SNF component SMARCA4, but 1α,25(OH)2D3 led to reduced VDR binding in RC43T cells. The 1α,25(OH)2D3-regulated transcriptome was largest in RC43N and RC43T but GSEA terms were frequently and oppositely enriched, for example Reactome S phase genes were negatively enriched in RC43N, but positively enriched in RC43T. Cistrome-transcriptome integration identified the most significant relationships between VDR binding and the magnitude of 1α,25(OH)2D3-regulated genes in RC43N. Global coregulator analyses identified significantly reduced BAZ1A/SMARCA5 in AA PCa in TCGA and a published cohort of ~1500 AA/EA tumors. Restoring BAZ1A expression in cells resulted in significantly enhanced 1α,25(OH)2D3-regulated transcriptome in RC43N and RC43T cells, and hypergeometric tests revealed the significant enrichment of direct VDR target genes. The footprint of altered AA VDR signaling was evident in three previously established clinical cohorts. Serum miRNA that predicted AA progression from HGPIN to PCa were enriched for 1α,25(OH)2D3-regulated VDR targets; AA tumors from a vitaminD3-treated chemotherapy trail were enriched for VDR-bound genes; and, tumors from men with low serum 25(OH)D3 levels were significantly enriched for VDR target genes identified in AA but not EA PCa. Together, these data suggest VDR transcriptional control is most potent and dynamic in AA prostate cells, and is primed to govern inflammatory and circadian pathways. Reduced BAZ1A/SMARCA5 expression and/or reduced environmental-regulated serum vitamin D3 levels most acutely suppress these actions in AA PCa. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes that contributes to PCa health disparities. Citation Format: Shahid Hussain, Manjunath Siddappa, Sajad A. Wani, Hedieh Jafari, Jaimie S. Gray, Hsu-Chang Wu, Hancong Tang, Mark D. Long, Isra Elhussin, Balasubramanyam Karanam, Honghe Wang, Rebecca Morgan, Gary Hardiman, Isaacson B. Adelani, Rotimi Oladapo, Adam R. Murphy, James R. Marshall, Chanita Highes Halbert, Lara E. Sucheston-Campbell, Clayton Yates, Moray J. Campbell. Vitamin D receptor function in African American prostate cancer is disrupted by reduced BAZ1A/SMARCA5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3619.

Abstract D114: VIitamin D signaling of immune-related genes in diverse prostate cancer cancer cell lines

Abstract BACKGROUND: African American (AA) men are disproportionately affected by both prostate cancer (PCa) and vitamin D deficiency compared with European American (EA) men. Vitamin D deficiency is linked to increased PCa aggressiveness and mortality. Inflammation also plays an important role in PCa pathogenesis and progression, and expression of immune-related in genes in PCa tissues differs significantly between AAs and EAs. While evidence linking vitamin D and immune response to PCa remains scarce, previous studies demonstrated that vitamin D has anti-inflammatory effects in prostate tissue and mediates immune-related gene expression. Therefore, we hypothesize that vitamin D deficiency in AA men may alter the expression of immune-related genes associated with prostatic inflammation and PCa progression. METHODS: This study examined the effects of vitamin Don the expression of vitamin D- and immune-related genes. Weexposed a racially diverse panel of PCa cell lines—MDA-PCa-2b (AA), RC77T/E (AA), PC3 (EA), 22Rv1 (EA), and DU145 (EA)—to various concentrations (2.5 nM, 10 nM, 50 nM) of 25-hydroxyvitamin D (25(OH)D) and 1,25 dihydroxyvitamin D (1,25(OH)2D) for 24 hours, and quantified transcript levels and protein expression of VDR, RXRa, RXRb, LRP2, CYP27B1, GDF15, GFRAL, TNFa, IL6, IL8, and IGFBP3 with qPCR and immunoblotting. RESULTS:All cell lines expressed basal levels of VDR, RXRa, and RXRbconfirming the function of vitamin D signaling within PCa cells. Of the downstream immune-related genes tested, we observed a trend demonstrating that vitamin D treatment induced the greatest upregulation in either transcript and/or protein levels among the AA PCa cell lines RC77T/E and/or MDA-PCa-2b. In particular, LRP2, GDF15, TNFa, IL6, IL8, and IGFBP3 significantly increased in AA PCa cell lines exposed to either 25(OH)D or 1,25(OH)2D. CONCLUSIONS: These findings provide insight into a potential differential regulation of immune-related pathways by vitamin D in AA men compared to EA men with PCa. Further studies are warranted to better define the immune profile in prostatic tissue in response to vitamin D deficiency in AA men, and the downstream inflammatory effects on the microenvironment and tumor aggressiveness. Citation Format: Madhavi Bathina, Leanne Woods-Burnham, Mya S Walker, K Sean Kimbro, Rick A Kittles. VIitamin D signaling of immune-related genes in diverse prostate cancer cancer cell lines [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D114.

MicroRNA-214 targets PTK6 to inhibit tumorigenic potential and increase drug sensitivity of prostate cancer cells

Prostate cancer is the most commonly diagnosed cancer in men with African American men disproportionally suffering from the burden of this disease. Biomarkers that could discriminate indolent from aggressive and drug resistance disease are lacking. MicroRNAs are small non-coding RNAs that affect numerous physiological and pathological processes, including cancer development and have been suggested as biomarkers and therapeutic targets. In the present study, we investigated the role of miR-214 on prostate cancer cell survival/migration/invasion, cell cycle regulation, and apoptosis. miR-214 was differentially expressed between Caucasian and African American prostate cancer cells. Importantly, miR-214 overexpression in prostate cancer cells induced apoptosis, inhibiting cell proliferation and colony forming ability. miR-214 expression in prostate cancer cells also inhibited cell migration and 3D spheroid invasion. Mechanistically, miR-214 inhibited prostate cancer cell proliferation by targeting protein tyrosine kinase 6 (PTK6). Restoration of PTK6 expression attenuated the inhibitory effect of miR-214 on cell proliferation. Moreover, simultaneous inhibition of PTK6 by ibrutinib and miR-214 significantly reduced cell proliferation/survival. Our data indicates that miR-214 could act as a tumor suppressor in prostate cancer and could potentially be utilized as a biomarker and therapeutic target.

Abstract 1565: A novel therapeutic target in lethal prostate cancer in African American males

Abstract Background: The largest US cancer health disparity exists in prostate cancer (PC), with African American (AA) men having a 1.6-fold increased risk of developing PC; younger age and more advanced stage at diagnosis; increased risk for recurrence; and a 2.5-fold increased mortality rate relative to Caucasian American (CA) men. In addition to important socioeconomic and environmental factors, this disparity is linked to incompletely understood genetic and other intrinsic biological factors. Consequently, there is an unmet need to identify actionable targets in AA PC in order to develop more effective therapeutics and, ultimately, improve clinical outcomes for AA PC patients. Rationale/Hypothesis: The p160 Steroid Receptor Coactivator (SRC) family (SRC-1, SRC-2, and SRC-3), are master regulators of transcriptional activity. They cooperate with several oncogenic transcription factors to mediate their gene expression programs, thus representing a critical connecting signaling node in PC. We have hypothesized that the p160 SRCs underlie the higher aggressiveness of AA PC and proposed that the inhibition of the p160 SRCs can target the oncogenic transcriptional programs of AA PC, suppress AA PC cell proliferation, survival, and metastasis, and improve outcomes for AA PC patients. Data from the three p160 SRC knockout mouse models (Src1, Src2, or Src3 knockout mice) show no negative effect on mouse lifespan, suggesting the possibility of a therapeutic window. Experimental approach: We utilized state-of-the-art in vitro assays and multi-Omics datasets to examine these hypotheses and shed light onto the role of the p160 SRCs in the intrinsic biological aggressiveness of AA PC. Recently, our BCM group synthesized an innovative panel of small molecule inhibitors (SMIs) of the p160 SRCs. We examined the efficacy of our novel p160 SRC SMIs against AA PC cell lines and patient-derived xenografts (PDXs). Results: The p160 SRCs exhibit increased activity in AA PC compared to CA PC. They are important regulators of the transcriptional programs that drive AA PC cell proliferation, metabolism, invasion, and metastasis. Importantly, our novel p160 SRC SMIs potently suppress multiple oncogenic signaling programs in AA PC cells and inhibit cell proliferation. They also potently suppress key AA PC driver transcription factors, including AR, SREBP1, and MNX1. The latter two are - so far - undruggable oncogenic transcription factors that our extended BCM group recently discovered to be specifically overexpressed in AA PC. Furthermore, our lead SMI is well tolerated in mice and can inhibit PC cell proliferation and tumor growth in vivo. Conclusions: We propose pharmacologic inhibition of the p160 SRC as an innovative treatment opportunity that will target several signaling pathways simultaneously and improve clinical outcomes in AA PC. Citation Format: Salma Kaochar, Michael Ittmann, Matthew Robertson, Jin Wang, Darlene Skapura, Christel Davis, Erik Ehli, Matthew Freedman, Cristian Coarfa, Bert O'Malley, Nicholas Mitsiades. A novel therapeutic target in lethal prostate cancer in African American males [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 1565.

Abstract 3561: MicroRNA-214 inhibits prostate cancer cell proliferation, migration, invasion and increases drug sensitivity by targeting PTK6

Abstract MicroRNAs (miRNAs) are involved in numerous physiological and pathological processes and play a critical role in cancer progression and the role miRNA-214 (miR-214) in prostate cancer remains elusive. In the present study, we investigated the effect of expression of miR-214 on cell survival/migration/invasion, cell cycle regulation, and apoptosis. Differential expression of miR-214 was found between Caucasian, African American prostate cancer cells compared with normal prostate epithelial cells. Expression of miR-214 in prostate cancer cells inhibited cell proliferation and colony forming capability, and induced apoptosis. miR-214 also inhibited cell migration and 3D spheroid invasion in prostate cancer cells. Interestingly, miR-214 targets protein tyrosine kinase 6 (PTK6) and reduced PTK6 expression. The restoration of PTK6 expression significantly blocked the inhibitory effect of miR-214 on cell proliferation. Moreover, inactivation of PKT6 by ibrutinib and expression of miR-214 significantly enhanced the cell death in prostate cancer cells. Collectively the data suggest that miR-214 functions as a tumor suppressor in prostate cancer, and may become a potential therapeutic target in prostate cancer. Citation Format: Patrice Cagle, Suryakant Niture, Anvesha Srivastava, Malathi Ramalinga, Leslimar Rios-Colon, Uchechukwu Chimeh, Deepak Kumar. MicroRNA-214 inhibits prostate cancer cell proliferation, migration, invasion and increases drug sensitivity by targeting PTK6 [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 3561.

Abstract 1331: Receptor tyrosine kinases are differentially phosphorylated in metastatic castration resistant prostate cancer

Abstract Metastatic castration resistant prostate cancer (mCRPC) is the leading cause of death among prostate cancer (PCa) patients. Patients of African American (AA) origin are particularly disproportionately affected as they are less responsive to current therapies, mainly androgen deprivation therapy (ADT) that target androgen receptor signalling, which is less expressed in AA patients. Receptor tyrosine kinases (RTKs) have been implicated in cancer development and progression and inhibition of their activities has been effective in inhibiting cancer progression in other cancers. However, targeting RTK signalling has not yielded satisfactory results in PCas. The objective of this study was to identify precise RTKs that may be targetable in mCRPCs. An in-vitro model for primary vs mCRPC was developed from AA PCa cell lines: RC77T and RC43T. Briefly, invasive RC77T and RC43T cells were isolated in a cell invasion chamber. The invasive sublines were gradually exposed to increasing concentration (up to 10µM) of MDV3100 over a nine-month period to develop castration resistance. The resulting invasive and castration resistant sublines: RCMI77-CR3 and RCMI43-CR3 were subsequently cultured and maintained in K-SFM containing 10µM of MDV3100. Human Receptor Tyrosine Kinase Phosphorylation Antibody Array and Co-Immunoprecipitation protocols were used to identify differences in RTK activation. TCGA patient data was analysed using R to identify correlation between RTK mRNA expression and PCa progressiveness. MTT assay was used to evaluate effect of RTK inhibitors and MDV3100 treatment on cell survival. The RC77T and RC43T cells were epithelial in appearance, while RCMI77-CR3 and RCMI43-CR3 cells tended to be mesenchymal. The RCMI77-CR3 and RCMI43-CR3 sublines were more resistance to MDV3100 compared to their primary PCa counterparts. Human RTK Array identified four RTKs: KIT, LYN, ITK and RYK to be hyperphosphorylated in RCMI77-CR3 and RCMI43-CR3 relative to RC77T and RC43T, while three RTKs including EphA3, EphB1 and ErbB3 were hypo-phosphorylated. Immunoprecipitation and western blot analysis revealed differential expression of phosphotyrosine kinases between RC77T and RCMI77-CR3 cells. Analysis of TCGA patient data showed, mRNA expression level of only LYN and RYK differed significantly (p<0.05) significantly with disease progression. Analysis of effect of tyrosine kinase inhibitors on cell proliferation showed RCMI77-CR3 to be more responsive to dasatinib and axitinib compared to RC77T. No similar differences in response was observed for the inhibitors; SU6656, IWR-1, ICG-001 and BMS. The current study suggests, activation of specific RTKs are involved survival and drug resistance in mCRPC cells. Inhibiting these specific RTK signalling pathways in segments of patients with specific RTK hyperactivation could significantly improve survival. Citation Format: Raven A. Williams, Joakin Mori, Hui-Xian Lin, Alahni Becks, Clayton Yates, Honghe Wang. Receptor tyrosine kinases are differentially phosphorylated in metastatic castration resistant 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 1331.

Abstract 2637: Novel targeted therapy-resistant FGFR3 splice variant increases oncogenic phenotype in African American prostate cancer

Abstract Introduction: In this study, we investigate the role of alternative splicing in the receptor tyrosine kinase family on prostate cancer health disparities in African American men. Background: Prostate cancer (PCa) is the most diagnosed cancer in men and the second leading cause of male-cancer related deaths in the U.S. Dramatic ethnic disparities have been observed in PCa patients, as African American (AA) men are 60% more likely to be diagnosed with PCa and have a 2.4 fold higher mortality rate compared to European American (EA) men. Increasing evidence suggests that, after accounting for epidemiological factors, a remaining component of this disparity is due to intrinsic genetic and biological factors. Interestingly, exon array data from our lab suggest that differential alternative splicing (dAS) may be occurring in AA PCa. We hypothesize that dAS involving exon 14 of FGFR3 is generating a shorter, more oncogenic variant in AA PCa, which is absent or weakly expressed in EA PCa. Differential splicing of FGFR3 in AA patients may be mechanisms contributing to AA PCa health disparities. Results: Cloning confirmed the presence of the FGFR3-L variant (containing exon 14) and the FGFR3-S variant (without exon 14) from EA and AA PCa cell lines, respectively. RNA-seq data analysis suggests decreased survival of PCa patients with high FGFR3-S/-L expression ratios. EA cell lines overexpressing FGFR3-S show increased small molecule inhibitor (SMI) TKI258-mediated resistance to proliferation, invasion, and apoptosis in vitro. Additionally, mice injected with cells overexpressing FGFR3-S have increased tumor growth, metastasis, and resistance to TKI258 treatment. Conclusions: We have identified an oncogene of interest, FGFR3, which undergoes exon skipping specific to AA PCa. We have successfully cloned this novel FGFR3 variant in cell lines and created stable cell lines overexpressing both short and long variants. In vitro and in vivo studies suggest FGFR3-S is more resistant to SMIs. Thus, dAS in FGFR3 may be one mechanism contributing to the increased aggressiveness of PCa in AA patients. Citation Format: Jacqueline Moy, Bi-Dar Wang, Norman H. Lee. Novel targeted therapy-resistant FGFR3 splice variant increases oncogenic phenotype in African American 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 2637.

Abstract 761: Up-regulation of miR-10a affect on prostate cancer racial disparity

Abstract Purpose: Prostate cancer (PC) is the second most diagnosed cancer in men. African-American (AfA) men have higher incidence and twice the PC mortality rates compared to Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-10a in prostate cancer racial disparity. Methods: We used PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) to examine miR-10a expression levels. Total RNA was extracted from total 138 human prostate cancer clinical samples (AfA: n=75, CaA: n=63) and cell lines. Gene expression levels were determined by quantitative real-time PCR (qPCR). A miRNA target database (miRWalk that integrates mainly three databases, miRDB, TargetScan and miRTarBase) was used for miR-10a target prediction. Cell proliferation and apoptosis were monitored after miR-10a knockdown by using anti-miR-10a. Results: miR-10a was significantly up-regulated in AfA prostate cancer cell line, E006AA-hT compared to CaA cells. Utilizing SFVAMC and NDRI patient cohorts, we confirmed that miR-10a expression was linked to a racial difference between AfA/CaA PCa patients. Knockdown of miR-10a showed decreased growth and apoptosis, though the effect was less in CaA compared to AfA cells. We found NCOR2 which is frequently deleted in castration- resistant prostate cancer patient specimens and tumor-suppressor PTEN as miR-10a targets that overlapped in multiple miRNA target prediction databases. Conclusion: These results demonstrate that miR-10a may be a central regulator of crucial events that contribute to racial differences in prostate cancer. Regulation of miR-10a expression may be a new therapeutic strategy for AfA prostate cancer patients. Citation Format: Yutaka Hashimoto, Marisa Shiina, Yuichiro Tanaka, Pritha Dasgupta, Priyanka Kulkarni, Taku Kato, Ryan K. Wong, Varahram Shahryari, Shigekatsu Maekawa, Soichiro Yamamura, Divya Bhagiratha, Sharanjot Saini, Guoren Deng, Laura Tabatabai, Shahana Majid, Rajvir Dahiya, Rajvir Dahiya. Up-regulation of miR-10a affect on prostate cancer racial disparity [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 761.

Abstract 2437: Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia

Abstract Hypoxia in prostate cancer (PCa) selects for aggressive clones and causes treatment failure, metastatic progression and mortality. Despite the key role of hypoxia in prostate carcinogenesis, little is known about the specific effect of hypoxia in African Americans, a race that faces a serious PCa-related health disparity with the highest incidence and mortality rate. We hypothesize that metabolic adaptation to hypoxia is one mechanism that contributes to PCa aggressiveness in African Americans. Therefore, in the present work we focused on the metabolic adaptations, both in vitro and in vivo, contributing to proliferation of African American PCa cells under hypoxic conditions. PCa cells from men of African American ancestry (E006AA-hT, MDA PCa 2b, WFCB17 and 22Rv1) showed proliferation and significantly higher clonogenicity under hypoxia (1% O2) compared to normoxia. In contrast, Caucasian PCa cells (LNCaP and PC3) showed significant growth inhibition under similar hypoxic condition. The proliferation under hypoxia in African American PCa cells was associated with higher beta-oxidation of lipids as measured by malate response in Oroboros Oxygraph system. In addition, [18F]-fluoro-4-thia-oleate ([18F]-FTO) uptake in African American PCa cells under hypoxia was more than 2-fold higher (p <0.001) compared to normoxic cells, confirming higher beta-oxidation in hypoxic PCa cells. Furthermore, African American PCa cells showed more than 2-fold (p <0.001) higher uptake of [11C]-acetate under hypoxia, suggesting increased fatty acid synthesis during hypoxia in African American PCa cells. This suggests that African American PCa cells may increase fatty acid synthesis under hypoxia to provide metabolic fuel. Fatty acid oxidation is associated with mitochondrial metabolism; accordingly, we observed higher oxidative phosphorylation, complex II activity and expression under hypoxia in African American PCa cells. Importantly, treatment with complex II inhibitor Atpenin A5 (1-2 microM) strongly reduced the clonogenicity (51%-79%, p <0.0001) of African American PCa cells under hypoxia. In vivo, mPET/CT imaging using [18F]-FTO and [18F]-fluoromisonidazole as PET tracers showed greater tumor uptake of both tracers in 22Rv1 tumors in male athymic nude mice, suggesting that hypoxia and fatty acid oxidation are associated with African American PCa growth. Furthermore, analyses of 22Rv1 tumor tissues by confocal microscopy showed higher level of carnitine palmitoyltransferase 1 (a key regulator of β-oxidation) and succinate dehydrogenase B (a subunit of complex II) in hypoxic areas (pimonidazole+) of the tumor. Taken together, these data demonstrate that proliferation of African American PCa cells under hypoxia is supported by high lipid beta-oxidation, lipid synthesis and complex II activity, providing a novel mechanism to promote disease aggressiveness. Citation Format: Gati Panigrahi, Prakash Praharaj, Kiran Sai, Gargi Mahapatra, Taylor Peak, Sierra Patterson, Hakeem Oufkir, Anthony Molina, Steven Kridel, Gagan Deep. Novel metabolic adaptations support proliferation of African American prostate cancer cells under hypoxia [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 2437.

Hypoxia-induced exosome secretion promotes survival of African-American and Caucasian prostate cancer cells

African American men in the United States have higher mortality due to prostate cancer (PCa) compared to other races. One reason for this disparity is the lack of in-depth understanding of the PCa biology in African Americans. For example, hypoxia in prostate tumor microenvironment is associated with adverse prognosis; still, no hypoxia-related studies have been reported in African Americans. Here, we compared African-American and Caucasian PCa cells for exosome secretion under normoxic (21% O2) and hypoxic (1% O2) conditions. All cell lines showed higher exosome secretion under hypoxia but it was clearly more prominent in African-American PCa cells. Further, under hypoxia, Rab5 (a biomarker for early endosome) was clustered in perinuclear region; and CD63 (a biomarker for exosomes and multivesicular endosomes) showed greater co-localization with actin cytoskeleton especially in African American PCa cells. Importantly, exosome biogenesis inhibitors GW4869 (10–20 µM) or DMA (10–20 µg/ml) significantly decreased cell viability and clonogenicity in PCa cells. Interestingly, we also observed higher level of lactic acid loaded in exosomes secreted under hypoxia. Overall, under chronic hypoxia, PCa cells secrete more exosomes as a survival mechanism to remove metabolic waste.

Abstract 1338: Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2A and RAB signaling

Abstract Background and Objectives: African American men experience worse prostate cancer (PCa) outcomes compared with those of Caucasian men not only in incidence and mortality rates but also harbor higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, there may be biological basis for such phenomenon. Hypoxia in the tumor microenvironment is the key determinant of disease aggressiveness regulating primary growth, angiogenesis, metastasis and treatment outcomes. But role of hypoxia in the aggressiveness of PCa in African-American men has not been characterized yet. Here, we focused on hypoxia signaling, exosome biogenesis and characterization in African-American PCa cells compared to Caucasian PCa cells. Methods: We employed several techniques (cell viability assays, ultracentrifugation, nanoparticle tracking analyses [NTA], electron microscopy, and western blotting) to compare exosomes biogenesis and underlying molecular mechanisms in African-American and Caucasian PCa cells under normoxic and hypoxic conditions. Results: Compared to normoxic condition, African-American PCa cells (E006AA-hT and RC77T) either survived better or proliferated under hypoxia (1% O2), while growth of Caucasian PCa cells (PC3 and LNCaP) was strongly inhibited under hypoxia. NTA analyses showed that all PCa cells secreted more exosomes under hypoxia compared to normoxia, but more prominently in African-American PCa cells (E006AA-hT: 4.4 fold, RC77T: 35 fold, PC3: 3 fold and LNCaP: 2 fold). Exosomes secreted by E006AA-hT cells under hypoxia were loaded with higher amount of HSP70, HSP90, IL6 and Annexin II, and lower TGFβ. These exosomes also showed significantly higher MMP9 loading compared to E006AA-hT cell lysates. More importantly, under hypoxia, exosome biogenesis inhibition by GW4869 (20 µM) and 5-(N,N-dimethyl) amiloride (0.1-5 µg/ml) significantly reduced the E006AA-hT cell viability by 30% (p=0.0001) and 20-60% (p< 0.005 - 0.0001), respectively in 48 hrs. Further studies on hypoxia signaling revealed that E006AA-hT cells have significantly higher HIF2α and HIF1β but low HIF1α expression compared to LNCaP cells. Interestingly, HIF2α was present largely in nuclear fraction and its expression was similar under both normoxic and hypoxic conditions in E006AA-hT cells. Studies on the RAB and its effector proteins indicated that RabGAP5 expression was strongly increased under hypoxia in both E006AA-hT and LNCaP cells. Further, compared to LNCaP cells, E006AA-hT cells showed higher expression of Rab5 and Rabex5 but lower Rab4, and these molecules did not change significantly under hypoxia. Conclusion: African-American PCa cells have growth advantage under hypoxia mainly through higher exosome secretion; and higher expression/activity of HIF2α and RAB-signaling molecules could be associated with PCa aggressiveness in African-Americans. Citation Format: Gatikrushna Panigrahi, Taylor C. Peak, Sierra L. Patterson, Nicole Kasica, Ravi Singh, Ashok Hemal, Gagan Deep. Exosome secretion promotes proliferation of African American prostate cancer cells under hypoxia: Role of HIF2A and RAB signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1338. doi:10.1158/1538-7445.AM2017-1338

Abstract 2328: Race specific hyper-activation of CCR9-mediated survival signals and its impact on efficacy of docetaxel in prostate cancer

Abstract Current approaches to treat and manage prostate cancer (PCa) have failed to reduce racial disparity primarily due to undefined molecular mechanism. We have shown higher expression of chemokine receptor-9 in PCa cells and CCR9 expression was higher in PCa cells (MDA-PCa-2b) derived from African American (AA) patient compared to PCa cells derived form European American (EA) patients (LNCaP and PC3). Using antibody microarray we observed hyper activation of survival molecules and down-regulation of apoptotic molecules in AA PCa cells compared to EA PCa cell lines, following CCL25 stimulation. Comparative heat map analysis showed significant increase (~2-3 fold) in phosphorylation of pro-survival proteins in AA cells compared to EA cells treated with CCL25, which was further confirmed by western blot analysis. Furthermore, CCL25 treated PCa cells showed decreased expression of pro-apoptotic proteins (Bim, Bid, Bak), which was significantly abrogated by CCR9 shRNA. This effect was more pronounced in AA cells compared to EA cells. Efficacy of docetaxel (DTX) was higher after CCR9 blockade in presence of CCL25 compared to cells treated with CCL25 without blocking CCR9. Improvement in efficacy of DTX was due to inhibition of cell survival and activation of pro-apoptotic signals following CCR9 blockade. These observations suggest potential involvement of CCR9 mediated molecular pathways in disparity associated with outcome of PCa. Citation Format: Neeraj Kapur, Hina Mir, Guru Sonpavde, Shailesh Singh. Race specific hyper-activation of CCR9-mediated survival signals and its impact on efficacy of docetaxel in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2328. doi:10.1158/1538-7445.AM2017-2328

Abstract C42: Alternative splicing of FGFR3 as a mechanism for African American prostate cancer health disparities

Abstract Introduction: In this study, we investigate the role of differential expression of splicing factors and exon skipping in the receptor tyrosine kinase family on prostate cancer health disparities in African American men. Background: Prostate cancer (PCa) is the most diagnosed cancer in men and the second leading cause of male-cancer related deaths in the U.S. Dramatic ethnic disparities have been observed in PCa patients, as African American (AA) men are 60% more likely to be diagnosed with PCa and have a 2.4 fold higher mortality rate compared to European American (EA) men. Increasing evidence suggests that, after accounting for epidemiological factors, a remaining component of this disparity is due to intrinsic genetic and biological factors. Interestingly, recent exon array data from our lab suggest that differential expression of splicing factors (SFs) and differential alternative splicing may be occurring in AA PCa. We hypothesize that differential alternative splicing involving exon 14 of the FGFR3 gene is generating a shorter, more oncogenic variant in AA PCa, which is absent or weakly expressed in EA PCa. Differential splicing of FGFR3 and increased expression of SFs in AA patients may be mechanisms contributing to AA PCa health disparities. Results: Exon array data suggested FGFR3 as a candidate for differential alternative splicing. Exon profiling and RT-PCR validated enriched expression of a short variant of FGFR3 due to skipping of exon 14 in AA patient samples and cell lines. Cloning confirmed the presence of the FGFR3-L variant (containing exon 14) and the FGFR3-S variant (without exon 14) from an EA and AA PCa cell line, respectively. Enrichment of FGFR3-S resulted in increased cell proliferation in an AA cell line. RNA-seq data analysis suggests decreased survival of PCa patients with high FGFR3-S/-L expression ratios. Additionally, our exon array data predicted increased expression of seven SFs in AA patients. RT-PCR and IHC analysis validated increased expression in AA specimens. Knockdown of these SFs resulted in decreased invasion and FGFR3 splice switching in an AA cell line. Conclusions: We have identified an oncogene of interest, FGFR3, which undergoes exon skipping that is specific to AA PCa. In cell lines, this shorter isoform of FGFR3 leads to an increased oncogenic phenotype based on proliferation assays. We have shown that AA PCa patient specimens have increased expression of specific SFs compared to EA specimens and knockdown of SFs reduces AA PCa cell invasion and causes splice switching of FGFR3. Thus, differential expression of SFs and exon skipping in FGFR3 may be one mechanism contributing to the increased aggressiveness of PCa in AA patients. Citation Format: Jacqueline Olender, Bi-Dar Wang, Norman H. Lee. Alternative splicing of FGFR3 as a mechanism for African American prostate cancer health disparities. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr C42.

Mitochondrial dysfunction-mediated apoptosis resistance associates with defective heat shock protein response in African-American men with prostate cancer.

Background:African–American (AA) patients with prostate cancer (PCa) respond poorly to current therapy compared with Caucasian American (CA) PCa patients. Although underlying mechanisms are not defined, mitochondrial dysfunction is a key reason for this disparity.Methods:Cell death, cell cycle, and mitochondrial function/stress were analysed by flow cytometry or by Seahorse XF24 analyzer. Expression of cellular proteins was determined using immunoblotting and real-time PCR analyses. Cell survival/motility was evaluated by clonogenic, cell migration, and gelatin zymography assays.Results:Glycolytic pathway inhibitor dichloroacetate (DCA) inhibited cell proliferation in both AA PCa cells (AA cells) and CA PCa cells (CA cells). AA cells possess reduced endogenous reactive oxygen species, mitochondrial membrane potential (mtMP), and mitochondrial mass compared with CA cells. DCA upregulated mtMP in both cell types, whereas mitochondrial mass was significantly increased in CA cells. DCA enhanced taxol-induced cell death in CA cells while sensitising AA cells to doxorubicin. Reduced expression of heat shock proteins (HSPs) was observed in AA cells, whereas DCA induced expression of CHOP, C/EBP, HSP60, and HSP90 in CA cells. AA cells are more aggressive and metastatic than CA cells.Conclusions:Restoration of mitochondrial function may provide new option for reducing PCa health disparity among American men.

Cell line modeling to study biomarker panel in prostate cancer.

African-American men with prostate cancer (PCa) present with higher-grade and -stage tumors compared to Caucasians. While the disparity may result from multiple factors, a biological basis is often strongly suspected. Currently, few well-characterized experimental model systems are available to study the biological basis of racial disparity in PCa. We report a validated in vitro cell line model system that could be used for the purpose. We assembled a PCa cell line model that included currently available African-American PCa cell lines and LNCaP (androgen-dependent) and C4-2 (castration-resistant) Caucasian PCa cells. The utility of the cell lines in studying the biological basis of variance in a malignant phenotype was explored using a multiplex biomarker panel consisting of proteins that have been proven to play a role in the progression of PCa. The panel expression was evaluated by Western blot and RT-PCR in cell lines and validated in human PCa tissues by RT-PCR. As proof-of-principle to demonstrate the utility of our model in functional studies, we performed MTS viability assays and molecular studies. The dysregulation of the multiplex biomarker panel in primary African-American cell line (E006AA) was similar to metastatic Caucasian cell lines, which would suggest that the cell line model could be used to study an inherent aggressive phenotype in African-American men with PCa. We had previously demonstrated that Protein kinase D1 (PKD1) is a novel kinase that is down regulated in advanced prostate cancer. We established the functional relevance by over expressing PKD1, which resulted in decreased proliferation and epithelial mesenchymal transition (EMT) in PCa cells. Moreover, we established the feasibility of studying the expression of the multiplex biomarker panel in archived human PCa tissue from African-Americans and Caucasians as a prelude to future translational studies. We have characterized a novel in vitro cell line model that could be used to study the biological basis of disparity in PCa between African-Americans and Caucasians.

Abstract 4000: Biological basis of aggressive phenotype in African-American prostate cancer cell line

Abstract Introduction: It is well established that African American men (AAM) with prostate cancer (PC) present more frequently with higher grade tumors compared to Caucasians. While the disparity is likely multifactorial, the biological basis is perhaps the strongest contender. Protein kinase D1 (PKD1) is a tumor and metastasis suppressor in PC. Down regulation of PKD1 was shown to increased cell proliferation, migration and metastasis in PC. Aim and experimental procedure: In order to address the fundamental question whether PC in AAM is inherently aggressive, we compared the proliferation, invasion and the expression of PKD1-centered signaling based 13-biomarker panel in primary and metastatic AAM cell lines (E006AA and MDApca2b) to benign (RWPE) and metastatic Caucasian cell lines (LNCaP and C4-2) using Western blot within different cell compartments. The results were further validated by RT-PCR and immunofluoroscent staining. Results: The proliferation, viability and invasion assays showed that E006AA cells proliferate and invade matrigel significantly more rapidly than metastatic LNCaP and MDAPca2b cells. The 13-biomarker panel analysis showed PKD1-centered dysregulaiton in AAM prostate cancer cell lines. Although E006AA is a primary PC cell line, PKD1 was down regulated similar to more aggressive C4-2 cells. Nuclear androgen receptor (AR), which shows active AR, was higher in LNCaP and MD2Pca2b; Vimentin showed higher expression in C4-2 and E006AA. Localization of E-Cadherin, N-Cadherin, T120 phosphorylated ß- Catenin, MMP-2 and S11 phosphorylated snail in MD2Pca2b was similar to LNCaP cells but different to C4-2 and E006AA. Conclusion: Our Data demonstrates that the dysregulaiton of PKD1-centered signaling occurs in primary AAM prostate cancer cell line E006AA is akin to other metastatic PC cell lines suggesting an inherently aggressive nature of PC in AAM. Acknowledgment: Authors would like to thank Dr. Shahriar Koochekpour (Roswell park institute, University at Buffalo,State University of NewYork) for his kind gift E006AA cell line. Citation Format: Bita Nickkholgh, Xiaolan Fang, Kenneth Gyabaah, K.C. Balaji. Biological basis of aggressive phenotype in African-American prostate cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4000. doi:10.1158/1538-7445.AM2014-4000

Establishment and characterization of a highly tumorigenic African American prostate cancer cell line, E006AA-hT.

Genuine racial differences in prostate cancer (PCa) biology have been considered among the potential reasons to explain PCa disparities. There is no animal model to represent all aspects of human PCa and, more specifically, to be used for PCa disparity research. The lack of a spontaneously transformed in vitro cell-based model system has been a significant impediment to investigating and understanding potential molecular mechanisms, and the hormonal, genetic, and epigenetic factors underlying the biological and clinical aggressiveness of PCa in African American (AA) men.In this study, we established and characterized the E006AA-hT cell line as a highly tumorigenic subline of the previously characterized primary AA-PCa cell line, E006AA. Extensive characterization of the E006AA-hT cell line was accomplished using cytodifferentiation and prostate-specific markers, spectral karyotyping, cell line authentication assays, cell proliferation and migration assays, and in vitro tumorigenesis assays.Spectral karyotyping of E006AA-hT showed a hypertriploid chromosome complement and shared cytogenetic changes similar to its parental cells such as diploid X, absence of Y-chromosomes, numerical gains in chromosomes 5,6,8,10,17,20,21, and marker chromosomes of unknown origin. In addition, E006AA-hT also presented numerous clonal and structural aberrations such as insertion, deletion, duplication, and translocations in chromosomes 1-5, 8, 9, 11, 13, 14, 17, and 18. The E006AA-hT cell line was shown to be highly tumorigenic and produced tumors at an accelerated growth rate in both athymic nude and triple-deficient SCID mice.Silencing the mutated androgen receptor (AR-599 Ser>Gly) did not affect proliferation (loss-of-function), but decreased migration (gain-of-function) in E006AA-hT and its parental cell type. These data support that AR-point mutations may lead simultaneously to different “loss-of-function” and “gain-of-function” phenotypes in PCa cells. E006AA-Par and its subline as the only available spontaneously transformed low- and highly-tumorigenic primary AA-PCa cell lines could be used for basic and translational research aimed in supporting prostate cancer disparity research.

Androgen Receptor-Target Genes in African American Prostate Cancer Disparities

The incidence and mortality rates of prostate cancer (PCa) are higher in African American (AA) compared to Caucasian American (CA) men. To elucidate the molecular mechanisms underlying PCa disparities, we employed an integrative approach combining gene expression profiling and pathway and promoter analyses to investigate differential transcriptomes and deregulated signaling pathways in AA versus CA cancers. A comparison of AA and CA PCa specimens identified 1,188 differentially expressed genes. Interestingly, these transcriptional differences were overrepresented in signaling pathways that converged on the androgen receptor (AR), suggesting that the AR may be a unifying oncogenic theme in AA PCa. Gene promoter analysis revealed that 382 out of 1,188 genes contained cis-acting AR-binding sequences. Chromatin immunoprecipitation confirmed STAT1, RHOA, ITGB5, MAPKAPK2, CSNK2A,1 and PIK3CB genes as novel AR targets in PCa disparities. Moreover, functional screens revealed that androgen-stimulated AR binding and upregulation of RHOA, ITGB5, and PIK3CB genes were associated with increased invasive activity of AA PCa cells, as siRNA-mediated knockdown of each gene caused a loss of androgen-stimulated invasion. In summation, our findings demonstrate that transcriptional changes have preferentially occurred in multiple signaling pathways converging (“transcriptional convergence”) on AR signaling, thereby contributing to AR-target gene activation and PCa aggressiveness in AAs.