Search

Effective clinical trials are meant to provide the safest and fastest way to find new treatments to improve health. The FDA affirms that because people may respond differently to treatments, it is imperative to test drugs and medical products in a variety of populations ( https://www.fda.gov/patients/clinical-trials-what-patients-need-know/basics-about-clinical-trials ). Unfortunately, clinical trial enrollment in the US remains largely homogeneous, with the majority of participants being non-Hispanic white men. Despite efforts to increase diversity in recruitment for clinical trials, enrollment of racial/ethnic minorities in this nation has decreased over the past two decades. 1

Vitamin D is a steroid hormone that confers anti-proliferative and anti-inflammatory properties in prostate cells. Vitamin D deficiency is associated with advanced prostate cancer (PCa) stage, grade, and increased mortality. Since AA men are more likely to be vitamin D deficient compared to EA men, elucidating the pleiotropic effects of vitamin D may provide additional drug targets to mitigate disparate outcomes for AA men with PCa. Total RNA sequencing (RNAseq) was performed on an AA prostate cell line, RC-77N/E, derived from non-malignant epithelial tissue obtained from a PCa patient. We compared untreated RC-77N/E cells with RC-77N/E cells treated with 10 nM of vitamin D3 metabolite, 1α,25(OH)2D3 at 24h. Bioinformatic analysis, public cohort data mining, and RT-qPCR was performed to filter and validate selected statistically significant differentially expressed genes from RNAseq analysis. Our comparison of 1α,25(OH)2D3 treated versus untreated control RC-77N/E cell replicates revealed, 1601 significantly differentially expressed genes (DEGs) (FDR p < 0.05). Ingenuity Pathway Analysis software was used to conduct pathway enrichment analysis, which predicted repression of signaling pathways involved in actin cytoskeleton organization, epithelial-mesenchymal transitioning, adherens junction pathway, and RhoA pathway signaling with high enrichment z-score and statistically significant p-value. We additionally report predicted repression of PCa cell viability, invasion, cell proliferation and organismal death, for disease and function. We prioritized differentially expressed genes by identifying genes with 1) vitamin D response elements (VDREs) and 2) predicted poor prognosis, by performing overall survival analyses using The Cancer Genome Atlas (TCGA) Prostate Adenocarcinoma (PRAD) Cohort. We identified ANLN (Anillin) and ECT2 (Epithelial Cell Transforming 2), which were further validated using an AA tumor/benign contralateral matched clinical cohort, and a public cohort study using a non-malignant European prostate cell line, RWPE1. Our data suggest 1α,25(OH)2D3 significantly downregulates ANLN and ECT2 (FDR p < 0.05) in the RC-77N/E cell line, where survival analyses in the TCGA PRAD cohort show low expression of both ANLN and ECT2 are associated with significantly better overall survival outcome (p < 0.05). In our clinical cohort validations, both ANLN and ECT2 also show significantly decreased expression in non-malignant compared to tumor (p < 0.05). In conclusion, our study provides more insight of vitamin D3 regulation of PCa biomarkers and their potential roles in AA prostate cancer prevention, adjuvant treatment, as well as improving cancer survivor outcome Citation Format: Jabril R. Johnson, Rachel N. Martini, Yate Yuan-Ching, Leanne Woods-Burnham, Mya Walker, Greisha L. Ortiz-Hernandez, Dorothy Galloway, Melissa B. Davis, Sean K. Kimbro, Adam B. Murphy, Rick A. Kittles. 1,25-dihydroxyvitamin D3-mediated suppression of genes associated with cell cycle regulation and actin organization in a non-malignant African American prostate cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3509.

The cysteine-rich angiogenic inducer 61 (CYR61) is a member of the cellular communication network (CCN) protein family that can be induced by growth factors. CYR61-specific functions are dependent on cellular context. In prostate cancer (PCa), CYR61 contributes to cell growth and survival through its interactions with extracellular ligands, such as integrins avb3 and a6b4, in the extracellular matrix space. Interestingly, CYR61 contains an insulin-like growth factor-binding protein domain suggesting that it may interact with insulin-like growth factor-I (IGF-1). High serum levels of IGF-1 are directly linked to PCa development and recently have been studied as a predictor of metastatic disease. Given the important roles that IGF-1 and CYR61 play in PCa and their potential interaction, it is critical to investigate their molecular interplay. This study is designated to specifically determine and characterize the molecular interaction between CYR61 and IGF-1 in metastatic PCa and to determine if this interaction contributes to aggressive tumor properties (e.g., proliferation, tumorsphere formation). Using immunoblotting, we demonstrated that CYR61 is upregulated in the metastatic cell line PC3 and downregulated in the docetaxel-resistant cell line PC3-DR. Furthermore, optimized knockdown of CYR61 using siRNA significantly reduced PC3 cell proliferation, viability, and prostasphere formation. To examine the underlying mechanisms associated with IGF-1 signaling, we assessed the activation of either the PI3/Akt and MAPK pathways in PC3 cells with CYR61 silencing. CYR61 siRNA-mediated knockdown decreased activation of the PI3/Akt pathway but did not affect the MAPK pathway. We also initiated studies to determine the co-localization of CYR61 and IGF-1 using immunofluorescence microscopy (IF). Based on the initial IF observations, we will proceed to examine if their interaction is primarily intra- or extra-cellular. For intracellular interactions, we will co-immunoprecipitate CYR61 with IGF-1 and for extracellular interactions, we will use the AVidity-based EXtracellular Interaction Screen (AVEXIS) system. Our goal is to establish the contribution of CYR61-IGF1 protein-protein interaction to PCa cell proliferation. Specifically, the correlation between PCa aggressiveness and circulating levels of CYR61 and IGF-1 is poorly studied. Their combined role may explain a proportion of aggressive PCa. Citation Format: Greisha L. Ortiz-Hernandez, Mya Walker, Leanne Woods-Burnham, Rick A. Kittles, Carlos A. Casiano, Susan L. Neuhausen. CYR61, a member of the CCN protein family, regulates IGF-1 in metastatic prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3949.

African American (AA) men are much more likely to be diagnosed with prostate cancer (PCa) and die from the disease than any other race/ethnicity in the United States. The pursuit of therapeutic strategies that incorporate tailored medicine for AA men remains a paramount goal. Metastatic PCa initially responds to suppression of androgen signaling through standard-of-care androgen deprivation therapy. However, relapse is inevitable, and treatment options are limited. For this reason, inhibiting androgen-independent signaling pathways that promote metastasis —such as human epidermal growth factor receptor 2 (HER2) signaling— is a promising area of investigation. HER2 overexpression in PCa tumors correlates with worse prognosis and treatment-resistance, but HER2 overexpression has not been evaluated specifically in AA men. We hypothesize that HER2 overexpression correlates with West African genetic ancestry (WAA) in AA PCa patients and worsens clinical features, treatment response, and survival outcomes. In this study, we report that HER2/ERBB2 is upregulated in the majority of PCa tumors in the Oncomine database. We quantified HER2/ERBB2 with RNAseq analysis of prostate tissue samples collected from AA patients (n=36) and observed a moderate correlation with WAA determined by genotyping using a validated set of Ancestry Informative Markers. Using immunohistochemistry, we also observed detectable HER2 protein expression in primary prostate tumors of AA patients (n=10). Our observation that 30% of AA primary tumor tissue samples were HER2-positive is remarkable, especially as previous studies have reported that HER2 overexpression is directly associated with metastasis as opposed to early, localized tumors. Our findings may underscore a basal/increased predisposition for HER2 overexpression in the AA population. We also detected HER2/ERBB2 mRNA expression using qPCR in a racially diverse panel of commonly used PCa cell lines: AA (MDA-PCa-2b and RC-77T/E) and non-Hispanic white (PC3, DU145, 22Rv1). To determine the effect of HER2 blockade on cell viability, we treated cells with 20 nM of anti-HER2 drug Trastuzumab for 72 hours and performed CellTiter-Glo™ assays. The only cell lines with significantly reduced viability were the AA cell lines. Furthermore, the RC-77T/E cell line which had the highest expression of HER2/ERBB2 also had the most significant reduction in cell viability. This finding suggests that AAs may be more responsive to HER2-targeting drugs, and that this responsiveness may be increased in PCa patients with higher levels of HER2 overexpression. It is plausible that HER2 overexpression may occur at earlier disease stages in AA men than previously reported and could be utilized to characterize aggressive PCa tumors eligible for HER2-targeting inhibitors, thus providing an early and effective therapeutic intervention that may improve outcomes in this population and reduce this appreciable health disparity. Citation Format: Nicole Ursula Mavingire, Mya Walker, Jabril R. Johnson, Isaiah J. Sailors, Greisha L. Ortiz-Hernandez, Rachel N. Martini, Melissa B. Davis, Justin Tran, Frank A. Myers, Sean Kimbro, Zhirong Yin, Tanya B. Dorff, Rick A. Kittles, Leanne Woods-Burnham. HER2 expression in African American men with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1955.

Recent advances in our understanding of racial disparities in prostate cancer (PCa) incidence and mortality that disproportionately affect African American (AA) men have provided important insights into the psychosocial, socioeconomic, environmental, and molecular contributors. There is, however, limited mechanistic knowledge of how the interplay between these determinants influences prostate tumor aggressiveness in AA men and other men of African ancestry. Growing evidence indicates that chronic psychosocial stress in AA populations leads to sustained glucocorticoid signaling through the glucocorticoid receptor (GR), with negative physiological and pathological consequences. Compelling evidence indicates that treatment of castration-resistant prostate cancer (CRPC) with anti-androgen therapy activates GR signaling. This enhanced GR signaling bypasses androgen receptor (AR) signaling and transcriptionally activates both AR-target genes and GR-target genes, resulting in increased prostate tumor resistance to anti-androgen therapy, chemotherapy, and radiotherapy. Given its enhanced signaling in AA men, GR—together with specific genetic drivers—may promote CRPC progression and exacerbate tumor aggressiveness in this population, potentially contributing to PCa mortality disparities. Ongoing and future CRPC clinical trials that combine standard of care therapies with GR modulators should assess racial differences in therapy response and clinical outcomes in order to improve PCa health disparities that continue to exist for AA men.

Responsible health disparities research requires a multifaceted approach to address genetic, biological, socioeconomic, and lifestyle contributors to disease. For researchers dedicated to the cause, thorough experimental standards at the bench, in the clinic, and within the community must prioritize adequate representation and cultural competency. Not everyone is up for the challenge.

Background: Given the scarcity of cell lines from underrepresented populations, it is imperative that genetic ancestry for these cell lines is characterized. Consequences of cell line mischaracterization include squandered resources and publication retractions. Methods: We calculated genetic ancestry proportions for 15 cell lines to assess the accuracy of previous race/ethnicity classification and determine previously unknown estimates. DNA was extracted from cell lines and genotyped for ancestry informative markers representing West African (WA), Native American (NA), and European (EUR) ancestry. Results: Of the cell lines tested, all previously classified as White/Caucasian were accurately described with mean EUR ancestry proportions of 97%. Cell lines previously classified as Black/African American were not always accurately described. For instance, the 22Rv1 prostate cancer cell line was recently found to carry mixed genetic ancestry using a much smaller panel of markers. However, our more comprehensive analysis determined the 22Rv1 cell line carries 99% EUR ancestry. Most notably, the E006AA-hT prostate cancer cell line, classified as African American, was found to carry 92% EUR ancestry. We also determined the MDA-MB-468 breast cancer cell line carries 23% NA ancestry, suggesting possible Afro-Hispanic/Latina ancestry. Conclusions: Our results suggest predominantly EUR ancestry for the White/Caucasian-designated cell lines, yet high variance in ancestry for the Black/African American–designated cell lines. In addition, we revealed an extreme misclassification of the E006AA-hT cell line. Impact: Genetic ancestry estimates offer more sophisticated characterization leading to better contextualization of findings. Ancestry estimates should be provided for all cell lines to avoid erroneous conclusions in disparities literature.

Glucocorticoid receptor (GR) is emerging as a key driver of prostate cancer (PCa) progression and therapy resistance in the absence of androgen receptor (AR) signaling. Acting as a bypass mechanism, GR activates AR-regulated genes, although GR-target genes contributing to PCa therapy resistance remain to be identified. Emerging evidence also shows that African American (AA) men, who disproportionately develop aggressive PCa, have hypersensitive GR signaling linked to cumulative stressful life events. Using racially diverse PCa cell lines (MDA-PCa-2b, 22Rv1, PC3, and DU145) we examined the effects of glucocorticoids on the expression of two stress oncoproteins associated with PCa therapy resistance, Clusterin (CLU) and Lens Epithelium-Derived Growth Factor p75 (LEDGF/p75). We observed that glucocorticoids upregulated LEDGF/p75 and CLU in PCa cells. Blockade of GR activation abolished this upregulation. We also detected increased GR transcript expression in AA PCa tissues, compared to European American (EA) tissues, using Oncomine microarray datasets. These results demonstrate that glucocorticoids upregulate the therapy resistance-associated oncoproteins LEDGF/p75 and CLU, and suggest that this effect may be enhanced in AA PCa. This study provides an initial framework for understanding the contribution of glucocorticoid signaling to PCa health disparities.

Prostate Cancer (PCa) is the number one diagnosed cancer among Black men (BM) in the United States. Diagnosed earlier, with more aggressive disease, BM are dying at higher rates than other race/ethnic groups. Family history of PCa and lack of access to annual screenings are known contributors to the development of lethal disease. In addition, over 70% of BM are vitamin D deficient (VDD), which promotes tumor aggressiveness, and generally have little knowledge of their family history of PCa. When detected at an early stage, BM have higher survival rates for PCa than other racial/ethnic group. As a result, the American Cancer Society recommends that men with a higher risk of developing PCa should receive information about the benefits and limitations of screening between 40 and 50 years of. Community-based screening (CBS) interventions have been shown to increase early detection and increase knowledge of PCa risk for BM). The purpose of this study is to better understand the feasibility of CBS interventions in reducing PCa disparities among BM. Methodology: Participants were recruited between 2018-2020 from CBS events in Southern California. The men completed a 48-item questionnaire prior to receiving a Prostate-Specific Antigen (PSA) test. Vitamin-D levels were also measured. The survey included questions on demographics, general health, cancer history, and family history of cancer. Inclusion criteria involved BM over the age of 40, without a family history of PCa, with PSA scores greater than 4 ng/ml (abnormal screening result), and Vitamin-D values less than 20 ng/ml (Vitamin D deficiency level). Survey data was analyzed using SPSS Version 25. Results: Our participants (n=497) included 68.8% (n=342) BM, 10% (n=50) White, 11.9% (n=59) Latino, 6.8% (n=34) Asian, and 2.4% (n=12) other. 62% of participants (n=269) have never been screened for PCa, and 74% (n=313) are between 40 and 88 years old. Of the Black population, 62% (n=163) never received a PSA screening, and 66% (n=175) did not have a family history of PCa. In addition, 36% (n=113) of the BM were VDD. Of the BM with an Abnormal Screening Result (ASR) (n=32), 44% (n=14) have never been screened, and 53% (n=17) did not have a family history of PCa. In addition, 44% (n=14) of the participants with an ASR were VDD. Lastly, of the BM above the age of 40 with an ASR, and VDD, (n=14), 50% have never been screened and 57% did not have a family history of PCa. Discussion: This study aimed to identify the feasibility of a community based PCa screening intervention for BM in Southern California. As a result of our CBS program, we identified high risk men without a family history of PCa and were VDD. This program offers early detection and screening opportunities to further increase awareness of PCa risk factors for BM. Future research should include the impact of CBS interventions for BM at perceived lower risk of developing PCa. Citation Format: Dorothy Galloway, Dede Teteh, Leanne Woods-Burnham, Mya Walker, Rick A. Kittles. Connecting under-resourced populations: A community-based prostate cancer screening intervention [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-023.

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.

African American (AA)/Black men are more likely to develop aggressive prostate cancer (PCa), yet less likely to be screened despite guidelines espousing shared decision-making regarding PCa screening and prostate-specific antigen (PSA) testing. Given the documented racial disparities in PCa incidence and mortality, engaging interactions with physicians are especially important for AA/Black men. Thus, this study evaluated occurrence of physician–patient conversations among AA/Black men, and whether such conversations were associated with PCa knowledge. We also quantified the serum PSA values of participants who had, and had not, discussed testing with their physicians. Self-identified AA/Black men living in California and New York, ages 21–85, donated blood and completed a comprehensive sociodemographic and health survey ( n = 414). Less than half (45.2%) of participants had discussed PCa screening with their physicians. Multivariate analyses were used to assess whether physician–patient conversations predicted PCa knowledge after adjusting for key sociodemographic/economic and health-care variables. Increased PCa knowledge was correlated with younger age, higher income and education, and having discussed the pros and cons of PCa testing with a physician. Serum PSA values were measured by ELISA. Higher-than-normal PSA values were found in 38.5% of men who had discussed PCa screening with a physician and 29.1% who had not discussed PCa screening. Our results suggest that physician–AA/Black patient conversations regarding PCa risk need improvement. Encouraging more effective communication between physicians and AA/Black men concerning PCa screening and PSA testing has the potential to reduce PCa health disparities.

Patients with metastatic castration-resistant prostate cancer (mCRPC) develop resistance to conventional therapies including docetaxel (DTX). Identifying molecular pathways underlying DTX resistance is critical for developing novel combinatorial therapies to prevent or reverse this resistance. To identify transcriptomic signatures associated with acquisition of chemoresistance we profiled gene expression in DTX-sensitive and -resistant mCRPC cells using RNA sequencing (RNA-seq). PC3 and DU145 cells were selected for DTX resistance and this phenotype was validated by immunoblotting using DTX resistance markers (e.g. clusterin, ABCB1/P-gp, and LEDGF/p75). Overlapping genes differentially regulated in the DTX-sensitive and -resistant cells were ranked by Gene Set Enrichment Analysis (GSEA) and validated to correlate transcript with protein expression. GSEA revealed that genes associated with cancer stem cells (CSC) (e.g., NES, TSPAN8, DPPP, DNAJC12, and MYC) were highly ranked and comprised 70% of the top 25 genes differentially upregulated in the DTX-resistant cells. Established markers of epithelial-to-mesenchymal transition (EMT) and CSCs were used to evaluate the stemness of adherent DTX-resistant cells (2D cultures) and tumorspheres (3D cultures). Increased formation and frequency of cells expressing CSC markers were detected in DTX-resistant cells. DU145-DR cells showed a 2-fold increase in tumorsphere formation and increased DTX resistance compared to DU145-DR 2D cultures. These results demonstrate the induction of a transcriptomic program associated with stemness in mCRPC cells selected for DTX resistance, and strengthen the emerging body of evidence implicating CSCs in this process. In addition, they provide additional candidate genes and molecular pathways for potential therapeutic targeting to overcome DTX resistance.

Human antinuclear autoantibodies (ANAs) targeting the dense fine speckled (DFS) nuclear protein DFS70, commonly known as lens epithelium derived growth factor p75 (LEDGFp75), present a clinical puzzle since their significance remains elusive. While their frequencies are low in ANA-positive autoimmune rheumatic diseases, they are relatively elevated in clinical laboratory referrals, diverse inflammatory conditions, and ‘apparently’ healthy individuals. We reported previously that DFS70/LEDGFp75 is an autoantigen in prostate cancer that closely interacts with another 70 kD DFS nuclear protein, methyl CpG binding protein 2 (MeCP2). This led us to investigate if anti-DFS sera exclusively target DFS70/LEDGFp75 or also recognize MeCP2. Using several complementary autoantibody detection platforms and cellular/molecular approaches we evaluated 65 human sera producing anti-DFS autoantibodies. Our results show that these antibodies are highly specific for DFS70/LEDGFp75 and do not target MeCP2. Establishing the specificity of anti-DFS autoantibodies has implications for increasing our understanding of their biological significance and clinical utility.

Background Understanding how biological factors contribute to prostate cancer (PCa) health disparities requires mechanistic functional analysis of specific genes or pathways in pre-clinical cellular and animal models of this malignancy. The 22Rv1 human prostatic carcinoma cell line was originally derived from the parental CWR22R cell line. Although 22Rv1 has been well characterized and used in numerous mechanistic studies, no racial identifier has ever been disclosed for this cell line. In accordance with the need for racial diversity in cancer biospecimens and recent guidelines by the NIH on authentication of key biological resources, we sought to determine the ancestry of 22RV1 and authenticate previously reported racial identifications for four other PCa cell lines. Methods We used 29 established Ancestry Informative Marker (AIM) single nucleotide polymorphisms (SNPs) to conduct DNA ancestry analysis and assign ancestral proportions to a panel of five PCa cell lines that included 22Rv1, PC3, DU145, MDA-PCa-2b, and RC-77T/E. Results We found that 22Rv1 carries mixed genetic ancestry. The main ancestry proportions for this cell line were 0.41 West African (AFR) and 0.42 European (EUR). In addition, we verified the previously reported racial identifications for PC3 (0.73 EUR), DU145 (0.63 EUR), MDA-PCa-2b (0.73 AFR), and RC-77T/E (0.74 AFR) cell lines. Conclusions Considering the mortality disparities associated with PCa, which disproportionately affect African American men, there remains a burden on the scientific community to diversify the availability of biospecimens, including cell lines, for mechanistic studies on potential biological mediators of these disparities. This study is beneficial by identifying another PCa cell line that carries substantial AFR ancestry. This finding may also open the door to new perspectives on previously published studies using this cell line.

There is no cure for metastatic prostate cancer (PCa), the most commonly diagnosed cancer and third leading cause of cancer mortality among U.S. men. Patients with advanced PCa often develop resistance to conventional therapies resulting in metastatic castration-resistant PCa (mCRPC). Docetaxel (DTX) chemotherapy is the standard-of-care for mCRPC, followed by secondary chemotherapy with cabazitaxel (CBZ), yet chemoresistance and death inevitably occurs. While pharmacological agents targeting single survival pathways have shown promise at the bench side, clinical trials have failed. This is mostly due to the presence of redundant pathways utilized by PCa tumor cells to maintain chemoresistance. Therefore, we are interested in further defining molecular pathways associated with DTX-resistance in order to identify novel therapeutic targets to overcome this resistance. We hypothesized that RNA sequencing (RNAseq) analysis comparing the transcriptomes of chemosensitive and chemoresistant mCRPC cells will reveal potential targets for combinatorial therapies. RNAseq analysis of DTX-sensitive and -resistant PC3 and DU145 cells revealed a number of differentially regulated genes that were either unique to each cell line or common to both. We found that dipeptidyl peptidase 4 (DPP4), fatty acid binding protein 5 (FABP5), nestin (NES), and tetraspanin 8 (TSPAN8) were among the top most robustly and significantly upregulated genes in both DTX-resistant PC3 and DU145 cell lines. In-house qPCR studies validated the RNAseq results for these genes, and Western blotting analysis showed increased expression of these proteins in DTX-resistant cells, which correlated with increased mRNA expression. Interestingly, NES, TSPAN8, DPP4 are genes associated with development of the cancer stem-cell (CSC) phenotype. Other genes associated with CSCs were also upregulated in the DTX-resistant cells, suggesting that in vitro selection of mCRPC cells for DTX resistance may enrich for CSCs. This novel finding is consistent with recent studies implicating CSCs in PCa chemoresistance. Further analysis and validation of our RNAseq data will increase our understanding of DTX-resistance mechanisms in PCa cells, including the yet undefined functional role of CSC-related genes. These studies also reveal potential therapeutic targets to circumvent DTX resistance in PCa. Citation Format: Christina K. Cajigas-Du Ross, Leanne Woods-Burnham, Joshua Ramirez, Xin Chen, Charles Wang, Carlos A. Casiano. RNA sequencing analysis of taxane-resistant prostate cancer cells reveals potential candidate genes for therapeutic targeting [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 5902.

Prostate cancer (PCa) is more aggressive in African American (AA) men compared to European American (EA) men, resulting in increased incidence and mortality disparities. There is a critical need to identify biological determinants contributing to these disparities. Glucocorticoids—a type of stress hormone—have recently been implicated as biological factors driving PCa progression and response to therapy. The underlying mechanism involves endogenous glucocorticoid binding to its receptor (GR), prompting GR to transcriptionally activate genes that are typically androgen-regulated and that promote tumor aggressiveness and therapy resistance. However, a clinical dilemma exists as glucocorticoids, necessary in the palliative care of PCa patients, are now emerging as accelerators of disease progression. In addition, AA men have chronically elevated levels of glucocorticoids linked to stressful life events, and are also hypersensitive to glucocorticoid exposure. We examined the effects of glucocorticoids on the activation of the stress oncoproteins LEDGF/p75 (Lens Epithelium Derived Growth Factor of 75kd) and CLU (Clusterin), linked by our group and others to prostate tumor aggressivenes and taxane resistance. Our goal is to establish the interplay between glucocorticoids and the expression of these proteins in the context of PCa health disparities. We hypothesized that LEDGF/p75 and CLU may be induced by glucocorticoids through GR in PCa cells. Treatment of a racially diverse panel PCa cell lines (MDA-PCa-2b [AA], 22Rv1 [AA/EA], PC3 [EA], and DU145 [EA]) with 10nM cortisol or dexamethasone for up to 48h significantly upregulated both transcript and protein levels of LEDGF/p75 and CLU. This upregulation was more robust in the AA cell lines. Depletion of GR with siRNAs abrogated dexamethasone-induced upregulation of LEDGF/p75 and CLU. Pharmacological inhibition of GR with mifepristone, in the presence of dexamethasone, also abrogated the induction of these proteins in a cell type-dependent manner. We also observed that dexamethasone exposure increased cell migration, a characteristic of tumor aggressiveness. Furthermore, using an ELISA platform we detected increased serum levels of LEDGF/p75 in AA PCa patients, as well as increased levels of CLU in non-PCa AA men, compared to EA patients and controls. These findings show that glucocorticoids induce the expression of oncoproteins associated with PCa aggressiveness and therapy resistance, and this upregulation may occur more robustly in AA men, priming them to develop highly aggressive PCa tumors. Citation Format: Leanne Woods-Burnham, Christina K. Cajigas-Du Ross, Anamika Basu, Evelyn S. Sanchez, Arthur Love, Carlos A. Casiano. Glucocorticoids trigger GR-mediated expression of the chemoresistance-associated oncoproteins LEDGF/p75 and Clusterin: Implications for prostate cancer health disparities [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 5043.

Prostate cancer (PCa) is the most commonly diagnosed male cancer and the second leading cause of cancer deaths in U.S. men. Late-stage PCa is characterized by metastasis followed by castration resistant PCa (mCRPC), for which there is no cure. Docetaxel (DTX) is the first-line chemotherapeutic drug for late-stage PCa, and resistance to this drug is inevitable. Our group has previously reported the elevated expression of the stress oncoprotein lens epithelium-derived growth factor (LEDGF/p75) in PCa cells and tissues, and this upregulation promotes resistance to DTX-induced non-apoptotic lysosomal cell death. Another stress oncoprotein, clusterin (CLU), is also upregulated in DTX-resistant PCa, and protects cells from DTX-induced mitochondrial-triggered apoptosis. In this study we observed that targeting LEDGF/75 with siRNAs partially resensitized DTX-resistant (DR) mCRPC cell lines to DTX treatment in clonogenic assays. Similarly, other groups have shown that knockdown of CLU in mCRPC-DR cells resensitizes them to treatment with taxanes. However, recent clinical trials targeting CLU have not been effective, suggesting that blocking this protein alone is insufficient to overcome DTX resistance. We hypothesized that simultaneous knockdown or inhibition of both LEDGF/p75 and CLU could be more effective in resensitizing chemoresisant PCa cells to taxane therapy than targeting each protein alone. Because of LEDGF/p75’s role as a stress transcription co-activator, we first sought to determine if this protein contributes to CLU expression by performing siRNA-mediated knockdown of LEDGF/p75 on the taxane-sensitive and -resistant mCRPC cell lines PC3 and DU145. Western blotting revealed that CLU protein expression was not downregulated by LEDGF/p75 knockdown, compared to treatment with scrambled siRNA control. Similarly, CLU knockdown in these cell lines did not downregulate LEDGF/p75. These results indicated that CLU expression is independent of LEDGF/p75, consistent with previous observations that these two proteins promote chemoresistance via distinct, possibly redundant, mechanisms. We then performed siRNA-mediated knockdown of LEDGF/p75 and CLU, separately or together, in PC3 DR and DU145 DR cells, in the presence and absence of varying concentrations of DTX. MTT and clonogenic assays were used to determine cell death viability and clonogenicity, respectively. Simultaneous knockdown of LEDGF/p75 and CLU, in combination with DTX treatment, resulted in increased sensitization to DTX than single knockdown of each protein. These findings suggest that co-targeting LEDGF/p75 and CLU could be an effective strategy for ameliorating chemoresistance in mCRPC, and set the foundation for combinatorial therapies targeting multiple stress pathways linked to taxane resistance together with taxane chemotherapy for treatment of late-stage PCa. Citation Format: Christina K. Cajigas-Du Ross, Leslimar Rios-Colon, Leanne Woods-Burnham, Carlos A. Casiano. Co-targeting the stress survival proteins LEDGF/p75 and clusterin to resensitize chemoresistant prostate cancer cells to docetaxel [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 4167. doi:10.1158/1538-7445.AM2017-4167

Prostate cancer (PCa) presents the greatest US cancer health disparity in terms of incidence and mortality, disproportionately affecting African American (AA) men. The biological characteristics of prostate tumors are exaggerated in AA men compared to European American (EA) men at time of diagnosis. The mechanisms underlying increased PCa aggressiveness in AA men are not fully understood; therefore, there is a critical need to identify biological determinants contributing to PCa mortality disparities. Glucocorticoids—a type of stress hormone—have been implicated as driving factors in PCa progression. The underlying mechanism involves endogenous glucocorticoid (cortisol) binding to its glucocorticoid receptor (GR) and activating genes that promote tumor aggressiveness and therapy resistance. A clinical dilemma exists as glucocorticoids, important in the palliative care of PCa patients, are now emerging as accelerators of disease progression and shortened survival. Chronically elevated levels of cortisol have been documented in AA men compared to EA men. This unhealthy cortisol production has been linked to stressful life events, which occur more frequently in the AA population. In addition, AA men are more sensitive to glucocorticoid exposure than EA men. These observations support the hypothesis that chronic stress leading to elevated cortisol in AA men may contribute to PCa health disparities. This study focused on examining the effects of glucocorticoid agonists and antagonists on the expression of stress oncoproteins, particularly LEDGF/p75 (Lens epithelial-derived growth factor of 75 kd) and CLU (clusterin), which are upregulated in PCa tumors and promote features of tumor aggressiveness, including resistance to clinically relevant taxanes used for advanced PCa treatment. We exposed a racially diverse panel of PCa cell lines, MDA-PCa-2b, 22Rv1, PC3, and DU145, to glucocorticoids (cortisol and dexamethasone), and observed by immunoblotting a time and dose-dependent upregulation of GR, LEDGF/p75 and CLU, particularly and more robustly in AA cells. Treatment of cells with mifepristone, a GR antagonist, attenuated this glucocorticoid-induced upregulation. In addition, siRNA- and shRNA-knockdown of LEDGF/p75 in PC3 cells resulted in decreased GR expression, suggesting a functional interplay between these two proteins in PCa cells. Interestingly, we also observed, using ELISA, increased circulation levels of LEDGF/p75 in AA PCa patients compared to EA patients and non-PCa controls, suggesting that this stress oncoprotein might play a more prominent role in AA PCa patients. These findings imply that activation of GR-signaling by glucocorticoids contributes to the activation of stress survival pathways associated with PCa aggressiveness and chemoresistance. These results add to the emerging contribution of GR signaling in PCa, particularly in the context of PCa health disparities. Citation Format: Leanne Woods-Burnham, Arthur Love, Christina K. Cajigas-Du Ross, Laura Stiel, Evelyn S. Sanchez, Kwame Amponsah, Susanne Montgomery, Colwick Wilson, Carlos A. Casiano. Glucocorticoid-mediated upregulation of stress oncoproteins: implications for prostate cancer health disparities [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 5285. doi:10.1158/1538-7445.AM2017-5285

Prostate cancer (PCa) presents the greatest US cancer health disparity in terms of incidence and mortality, disproportionately affecting African American (AA) men. The biological characteristics of prostate tumors are exaggerated in AA men compared to European American (EA) men at time of diagnosis, including higher PSA levels, higher Gleason scores, and advanced tumor stage. These factors influence the relatively poorer prognosis and worse overall survival rate observed in AA men. There is, however, a gap in our understanding of the mechanisms underlying increased PCa aggressiveness in AA men; therefore, there is a critical need to identify biological determinants contributing to PCa mortality disparities. Glucocorticoids—a type of steroid hormone produced in the human biological response to stress—have been implicated as driving factors in PCa progression. The underlying mechanism involves endogenous glucocorticoid (cortisol) binding to its glucocorticoid receptor (GR) and activating genes that promote tumor aggressiveness and therapy resistance. Results from phase 3 of clinical trial AFFIRM revealed that men receiving glucocorticoids to reduce the negative side effects of advanced PCa and treatment showed a significantly worse survival rate than those not receiving glucocorticoids. A clinical dilemma exists as glucocorticoids, important in the palliative care of PCa patients are now emerging as accelerators of disease progression and shortened survival. Chronically elevated levels of cortisol, which promote metabolic disorders, have been documented in AA men compared to EA men. This unhealthy cortisol production has been linked to stressful life events, which occur more frequently in the AA population. In addition, AAs are more sensitive to glucocorticoid exposure than EAs. These observations support the hypothesis that chronic stress leading to elevated cortisol in AA men may contribute to PCa health disparities. This study focused on examining the effects of glucocorticoid agonists and antagonists on the expression of stress oncoproteins, particularly LEDGF/p75 and clusterin, which are upregulated in PCa tumors and promote features of tumor aggressiveness, including resistance to clinically relevant taxanes used for advanced PCa treatment. We exposed a racially diverse panel of PCa cell lines, MDA-PCa-2b, 22Rv1, PC3, and DU145, to glucocorticoids (cortisol and dexamethasone), and observed by immunoblotting a time and dose-dependent upregulation of LEDGF/p75 and clusterin, particularly and more robustly in AA cells. Treatment of cells with mifepristone, a GR antagonist, attenuated this glucocorticoid-induced upregulation. In addition, shRNA-mediated depletion of LEDGF/p75 in PC3 cells resulted in decreased GR expression, suggesting a functional interplay between these two proteins in PCa cells. Interestingly, we also observed, using ELISA, increased circulation levels of LEDGF/p75 in AA PCa patients compared to EA patients and non-PCa controls, suggesting that this stress oncoprotein might play a more prominent role in AA PCa patients. These findings imply that activation of GR-signaling by glucocorticoids contributes to the activation of stress survival pathways associated with PCa aggressiveness. These results add to the emerging contribution of GR signaling in PCa, particularly in the context of PCa health disparities. Citation Format: Leanne Woods-Burnham, Arthur Love, Christina K. Cajigas-Du Ross, Anamika Basu, Laura Stiel, Susanne Montgomery, Colwick Wilson, Carlos A. Casiano. Glucocorticoid-mediated upregulation of stress oncoproteins: Implications for 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 C43.

African American (AA) men are more likely to develop and die from prostate cancer (PCa) yet less likely to receive information about screening from physicians. Although guidelines espouse shared decision-making regarding PCa screening and PSA testing, studies reveal lack of consistency. Several studies demonstrate that PCa knowledge strongly influences informed decisions, and that engaging interactions are especially important for AA men who are more likely to progress from early stage to aggressive end-stage PCa at an earlier age and faster rate than other populations. This study evaluated the frequency of physician-patient conversations among AA men and whether this translated into increased PCa knowledge and PSA testing. In this study, self-identified Black men ages 21-85 donated blood and completed a health survey (n=414) via Project C.H.A.N.G.E. (Changing Health in Adult Men with New and Great Experiences), a transdisciplinary cross-sectional study conducted at Loma Linda University. PCa knowledge following physician conversations in light of socio-economic and health care variables was evaluated using univariate, bivariate, and multiple regression analyses. PSA values were measured with ELISA. Our results revealed that 52.7% of participants had never discussed the pros and cons of PCa screening with their physicians. The likelihood of physician-patient conversations increased with age (p.001), and health insurance positively predicted these discussions (p.001). Multiple regression analysis indicated ethnicity and high school education as predictors of improved PCa knowledge (p.001). We found that 38.5% of men who had discussed PCa screening with a physician and 28.5% who had not discussed PCa screening had PSA values that could be considered high-risk for PCa diagnosis. These findings expose a remaining deficiency in physician compliance regarding national guidelines for shared decision-making conversations with high-risk men. This is problematic as a large group of AA men at evident risk for PCa have not ever discussed this disease with their physicians and may be harboring undetected PCa. Encouraging more effective interactions between AA men and their physicians concerning PCa screening and PSA testing has the potential to reduce PCa health disparities. Citation Format: Leanne Woods-Burnham, Laura Stiel, Colwick Wilson, Susanne Montgomery, Carlos A. Casiano. Physician consultations, prostate cancer knowledge, and PSA screening in African American men: Are the conversations effective? [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 A56.