African American Prostate Cancer Cell Lines Research Articles

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.

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 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 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.

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 primary androgen-responsive African-American prostate cancer cell line, E006AA.

Establishment of human prostate cancer cell lines is essential to advance our understanding of complex processes associated with the initiation and progression of the disease. In the present study, we report the establishment of a primary African-American prostate cancer cell line (E006AA) as well as its associated stromal cells (S006AA). E006AA cell line was established as a spontaneously immortalized cells from a patient with a clinically localized prostate cancer. Extensive characterization of the cells was accomplished using androgen-dependent growth and sensitivity assays, Western analyses, RT-PCR/real-time PCR, cytogenetic analyses, and tumorigenicity in nude mice. E006AA cell line shows androgen-dependent growth, expresses PSA and the androgen receptor (AR) with 26 CAG repeats in exon 1 of AR. Cytogenetic analyses revealed a hypertriploid karyotype with additional numerical gains in chromosomes 5, 6, 8, 10, 17, 20, 21 and a marker chromosome of unknown origin as well as structural abnormalities in chromosomes 4, 5, 8, 9, 11-14, 18, and 20. This cell line is not tumorigenic in nude mice. S006AA cell line, isolated from the same tumor specimen, also expresses AR and shows the morphological characteristics of smooth muscle cells of prostatic stroma. These cell lines are the first available primary epithelial and stromal cells derived from an African-American patient with organ-confined prostate cancer and in conjunction with other established cell lines, could provide an in vitro model system to investigate early transforming events in prostate cancer.