African American Cell Lines Research Articles

Abstract 7060: Mitochondrial metabolism and racial disparity of bladder cancer

Abstract Bladder cancer mortality is significantly higher in African American patients compared to their European American counterparts. This inferior survival of African American cancer patients is driven at least in part by distinctive intrinsic tumor biology, more specifically altered metabolic activity. However, underlying molecular mechanism for the African American bladder cancer mortality are largely unexplored. Our pioneering metabolomic findings demonstrate alterations in metabolic pathways between African American and European American bladder cancer, specifically those involved in driving energy metabolism. Along these line, we observed increased electron transport chain activity and ATP production in African American bladder cancer could be a result of distinctive rewiring of energy metabolism compared to their European American counterparts. We further performed gene set enrichment analysis by mapping metabolites to genes and observed that oxidative phosphorylation pathway was uniquely enriched in African American tumors compared to European American tumors. In addition, from in vitro assays, mitochondrial complex protein and activity were altered in African American bladder cancer. To determine the role of glutamine mediated increased electron transport chain activity and ATP production in African American bladder cancer, we evaluated the metabolic flux using 13C labeled tracers in both African American and European American bladder cancer cell lines. Metabolic flux analysis revealed that African American cell lines are more glutamine dependent and that producing increased tricarboxylic acid intermediates. Overall, our data indicate that elevated mitochondrial metabolism and oxidative phosphorylation driven by enhanced glutaminolysis may facilitate African American bladder cancer progression. These findings provide the rationale to use mitochondrial-specific inhibitors to target at least a subset of African American bladder cancer patients. Citation Format: Karthik Reddy Kami Reddy, Jun Hyoung Park, Roni J. Bollag, Martha K. Terris, Seth P. Lerner, Minhaj Siddiqui, Yair Lotan, Jianjun Gao, Benny Abraham Kaipparettu, Nagireddy Putluri. Mitochondrial metabolism and racial disparity of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7060.

Abstract 2247: Cellular senescence as a potential mechanism underlying differences in chemoresistance between lung cancer cell lines derived from African-American and Caucasian patients

Abstract Lung cancer remains the leading cause of cancer-related deaths world-wide. Statistical data reveals significant difference in both lung cancer incidence and death rate between African-American (AA) and Caucasian (CA) populations in the United States. Although these disparities can be partially attributed to socio-economical reasons, the biological mechanisms underlying them are poorly understood. In this study, we compared the chemoresistance of cell lines derived from tumor tissues obtained from AA patients (H23, H292) versus CA patients (A549, H460). The cell lines were treated with FDA-approved anti-cancer drugs as well as with cardenolide analogues previously reported by our group to have anti-cancer properties. Irrespectively of drug class, cellular senescence appeared to develop more abundantly in cells derived from AA patients. However, AA-derived cell lines manifested higher drug resistance in comparison to CA-derived ones, especially to cardenolide analogues. Recovery experiments in drug-free media revealed faster and more efficient re-growth after long-term (2 weeks) drug treatment in AA-derived cell lines. Taken together, the data provides a potential role for cellular senescence as mechanism of the differential chemoresistance observed between AA and CA lung cancer cell lines, revealing new potential targets for anti-cancer therapy. Citation Format: Nina Semenova, Camille C. Robertson, Juan S. Yakisich, Robyn R. Ayscue, Anand K. Iyer, Neelam Azad. Cellular senescence as a potential mechanism underlying differences in chemoresistance between lung cancer cell lines derived from African-American and Caucasian patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2247.

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.

Abstract PR006: The racial disparity-linked gene CRYβB2 alters cell adhesion and tumor microenvironment to promote proliferation and invasion in triple-negative breast cancer

Abstract Background: Clinical and epidemiological data showed that African American (AA) women suffer higher incidence and mortality rates due to triple-negative breast cancer (TNBC) compared to European American (EA) women. These disparate disease outcomes are attributed to multiple factors including socioeconomic status, access to health care, and earlier disease onset. However, a growing body of evidence also shows the existence of different gene expression signatures and tumor microenvironments between these patient racial groups. We and others identified CRYβB2 as a cancer racial disparity gene that has higher expression in AA patients with breast, colon, and prostate cancers. We previously demonstrated, via in vitro and in vivo studies, that CRYβB2 promotes proliferation, metastasis, and IL-6 signaling. The objective of the current research is to identify the cellular and molecular mechanisms downstream of CRYβB2 leading to its tumor-promoting phenotypes. Methods: CRISPR/Cas9 and lentiviral knock-in gene editing tools were used to create TNBC cell lines with CRYβB2 knockout and overexpression respectively. The functional consequences of CRYβB2 were assessed in vitro using colony formation, tumor spheroid invasion, and collagenase activity assays. Additionally, tumor spheroids were grown and RNA sequencing (RNA-seq) was performed to assess the effect of CRYβB2 on gene expression. Results: Herein, functional assays showed that CRYβB2 promotes proliferation and invasion in vitro in both AA and EA cell lines. RNA-seq and Gene Ontology enrichment analyses revealed that CRYβB2 alters key pathways such as cell adhesion and extracellular matrix (ECM) organization. RT-qPCR validation also showed that CRYβB2 inhibits the expression of a specific class of adhesion molecules known as protocadherins (PCDHs). Downregulation of PCDHs has been shown to enhance tumor progression and is a predictive marker for low survival in many cancer types. Indeed, data mining of publicly available data shows that three of the identified PCDHs (PCDHGB5, PCDHGB6 and PCDHGB7) have significantly lower expression levels in breast cancer compared to normal tissue and their low expression leads to low overall survival. Furthermore, RNA-seq data showed that CRYβB2 promotes the expression of multiple matrix metalloproteinase (MMP) genes. This was functionally validated using collagenase activity assay where CRYβB2 enhanced collagen degradation in vitro. Conclusions: Our findings provide a mechanistic explanation for the role of CRYβB2 in driving disparate clinical outcome among AA and EA TNBC patients. We demonstrate a paradigm where high CRYβB2 expression promotes tumor progression by suppressing cell adhesion molecules and increasing ECM degradation to enhance tumor growth, invasion, and metastasis. Disclaimer: This work was prepared while Jodie Fleming was employed at NCCU. The opinions expressed in this article are the author's own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. Citation Format: Amr A. Waly, London Harper, Jodie M. Fleming, Lindsey M. Costantini. The racial disparity-linked gene CRYβB2 alters cell adhesion and tumor microenvironment to promote proliferation and invasion in triple-negative breast cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PR006.

Abstract C058: Deregulated microRNA-mRNA reciprocal pairs as novel precision biomarkers and therapeutic targets for African American prostate cancer

Abstract Prostate cancer (PCa) is the most frequently diagnosed cancer and second leading cause of cancer death among American men. Notably, African American (AA) men exhibit 1.6-fold higher incidence rate and 2.4-fold higher mortality rate when compared to European American (EA) men. Besides socioeconomic factors, accumulating genomic data suggest that intrinsic genetic risk elements also play critical roles in promoting PCa disparities. MicroRNAs (miRNAs) are involved in epigenetic mechanisms regulating mRNA expression in various human diseases, including cancers. In this study, we have applied a systems biology approach (by integrating mRNA and miRNA profiling data, target prediction, and pathway analysis) to identify critical signaling networks differentially regulated between AA and EA PCa. Using miRNA-drive algorithm mirPath V.3 coupled with mRNA mapping, and global test algorithm coupled with miRNA mapping, we identified several cancer pathways, such as ERBB, mTOR, HIF, and VEGF signaling, that are significantly regulated by AA-depleted/enriched miRNAs and mRNAs. Specifically, reciprocal miRNA-mRNA pairs miR-34a-5p/HIF1A, miR-34a-5p/PIK3CB, miR-34a-5p/IGFBP2, and miR-99b-5p/MTOR (downregulated/upregulated in AA vs. EA PCa), and miR-96-5p/MAPKAPK2 (upregulated/downregulated in AA vs. EA PCa) were identified as core miRNA/mRNA regulators in activation of mTOR and VEGF signaling in AA PCa. RT-qPCR, Western blotting, and immunohistochemistry assays in AA and EA PCa cell lines and patient samples have confirmed that these miRNAs and mRNAs are differentially expressed in AA and EA PCa. Furthermore, molecular targeting miRNA/mRNA pairings using miR-34a-5p, miR-99b-5p mimics and/or miR-96-5p antagomir consequently reduced cell proliferation and enhanced docetaxel-induced cytotoxicity in AA PCa cells. In summary, these results suggest that deregulated miRNA/mRNA reciprocal pairs play critical roles in promoting cancer aggressiveness and chemoresistance in AA PCa. Further development of these miRNA/mRNA pairings as precision diagnostic/prognostic biomarkers and novel therapeutic targets may pave a new path for PCa diagnosis and treatment. Citation Format: Himali Gujrati, Siyoung Ha, Bi-Dar Wang. Deregulated microRNA-mRNA reciprocal pairs as novel precision biomarkers and therapeutic targets for African American prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C058.

Abstract PO-126: Identification of distinct mRNA & microRNA signatures and mRNA-miRNA pairs associated with inter-ethnic differences in prostate cancer aggressiveness

Abstract Introduction: The incidence of aggressive prostate cancer (PCa) and mortality is disproportionately higher in men of African-American (AA) ancestry. Treatment options for these patients are docetaxel or cabazitaxel alone or in combination with bevacizumab, thalidomide, and prednisone or immunotherapy. However, these chemotherapeutics typically only improve survival slightly (3-4 months). Further, majority of patients develop metastatic PCa over time, resistant to conventional chemotherapy (metastatic castration resistance PCa or mCRPC). Therefore, an understanding of the molecular mechanisms underlying the ethnic differences in disease aggressiveness and progression in PCa is needed. Methods: In this study, we performed mRNA and miRNA expression (RNAseq) analysis on PCa cell lines representing different tumor types (aggressive androgen receptor (AR) nonresponsive AR- vs. non-aggressive AR+) and derived from patients with European American/EA (PC3, PC3M, DU145, DUTXR, 22RV1, LNCaP, VCaP, LaPC4, C4, C4-2B) and AA (MDA-Pca-2b, RC77, RC165, RC43) ancestry to identify differentially expressed genes (DEG) associated with tumor aggressiveness and ethnicity. Next, we validated the top DEG signatures using an AA vs EA patient cohort dataset as well as in silico validation using The Cancer Genome Atlas (TCGA) database. For each miRNA, functional analysis was performed using miRBase datasets and mRNA-miRNA pairs and binding sites were predicted by TargetScan. Finally, IPA (Ingenuity Pathway Analysis) was performed to identify key regulators and downstream effects on biological and disease processes based on the expression patterns of DEGs. Results: We identified distinct mRNA and miRNA expression signatures associated with aggressive vs. non-aggressive PCa of EA vs. AA origin. The top DEGs that were associated with patient survival (p< 0.0001), stratified by Gleason scores, were PLAU, TGFB1, SERPINE1, MET, TIPM1, ITGA3, SERPINB5, PLAUR, MMPs, CDKN1A, and IGF1. The transporter genes SLC25A, SLC16A, and ABCB6 were also identified as important markers of aggressiveness. Notably, PLAU, MCAM, MET, TIMP1 were top DEGs in AA vs EA cell lines while SERPINE1 and MCAM were DEGs in AA vs EA patient cohort. IPA identified the activation of the angiogenesis pathway as a crucial factor for cancer aggressiveness. Top predicted miRNA-mRNA pairs included SERPINE1-let7, PLAU- mir181 which potentially influence differential gene expression in late-stage cancers. Finally, our immunoblotting results confirmed the protein expression changes of top DEGs. Next, we plan to perform CRISPR-based gene editing to functionally validate the molecular (mRNA/miRNA) signatures. Conclusion: An -omics-based approach was used to identify genetic signatures that provide insights into the molecular basis of PCa aggressiveness between men of EA vs AA ancestry. We believe this strategy will aid in developing effective targeted ethnicity-specific personalized treatment schedules for aggressive forms of PCa. This promises to address the known health disparity that is observed in AA men. Citation Format: Taraswi Mitra Ghosh, Jason White, Suman Mazumder, Joshua Davis, Grace Hurley, Brian Cummings, Gary Piazza, Amit K Mitra, Clayton Yates, Robert Arnold. Identification of distinct mRNA & microRNA signatures and mRNA-miRNA pairs associated with inter-ethnic differences in prostate cancer aggressiveness [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-126.

Abstract PS7-86: Molecular determinants of racial disparity guide triple-therapy for triple negative breast cancer

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to White American (WA) women (5-year relative survival of only 14% for AA in comparison to 36% for WA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than WA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to WA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to WA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and WA TNBC cell lines to query the status self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in WA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to WA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to WA-TNBC cells. We observed that GLI1 and NICD co-localize and co-immunoprecipitate in AA-TNBC indicating a direct interaction between these two transcription factors. High expression of GLI1 and Notch1 correlated with poor recurrence free survival in TNBC patients. Analyses of clinical samples revealed higher levels of nuclear NICD and GLI1 in AA-TNBC in comparison to WA-TNBC. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC tumor growth in mice. Also, ex vivo analyses of tumor cells showed reduced migration and invasion potential as well as downregulation of CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI1 and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Our studies propose a ‘triple drug regimen’ for AA-TNBC tumors based on its molecular circuitry. Citation Format: Sumit Siddharth, Sheetal Parida, Nethaji Muniraj, Shawn M Hercules, Arumugam Nagalingam, Balázs Győrffy, Juliet Daniel, Dipali Sharma. Molecular determinants of racial disparity guide triple-therapy for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-86.

Epigenetic and Posttranscriptional Modulation of SOS1 Can Promote Breast Cancer Metastasis through Obesity-Activated c-Met Signaling in African-American Women.

Ethnicity is considered to be one of the major risk factors in certain subtypes of breast cancer. However, the mechanism of this racial disparity remains poorly understood. Here, we demonstrate that SOS1, a key regulator of Ras pathway, is highly expressed in African-American (AA) patients with breast cancer compared with Caucasian-American patients. Because of the higher obesity rate in AA women, increased levels of SOS1 facilitated signal transduction of the c-Met pathway, which was highly activated in AA patients with breast cancer via hepatocyte growth factor secreted from adipocytes. Elevated expression of SOS1 also enhanced cancer stemness through upregulation of PTTG1 and promoted M2 polarization of macrophages by CCL2 in metastatic sites. SOS1 was epigenetically regulated by a super-enhancer identified by H3K27ac in AA patients. Knockout of the super-enhancer by CRISPR in AA cell lines significantly reduced SOS1 expression. Furthermore, SOS1 was posttranscriptionally regulated by miR-483 whose expression is reduced in AA patients through histone trimethylation (H3K27me3) on its promoter. The natural compound, taxifolin, suppressed signaling transduction of SOS1 by blocking the interaction between SOS1 and Grb2, suggesting a potential utility of this compound as a therapeutic agent for AA patients with breast cancer. SIGNIFICANCE: These findings elucidate the signaling network of SOS1-mediated metastasis in African-American patients, from the epigenetic upregulation of SOS1 to the identification of taxifolin as a potential therapeutic strategy against SOS1-driven tumor progression.

Abstract 282: MicroRNAs located at chromosome 8p21-22 region as novel factors underlying racial disparities in prostate cancer aggressiveness

Abstract The mechanistic basis of racial disparities in prostate cancer (PCa) aggressiveness between African American (AA) and Caucasian (CA) men are not yet fully understood. Further, molecular biomarkers to predict aggressive disease at its onset have been studied primarily in CAs, the relevance of these biomarkers to AAs are largely unknown. We propose a novel, paradigm-shifting hypothesis that expression of microRNA (miRNA) genes-miR-3622a and miR-383-located within frequently deleted chr8p21-22 region constitute important molecular factors contributing to racial disparities in PCa aggressiveness in AA vs CA men. Chromosome 8p deletions are a frequent alteration of PCa genome, with a common region of loss of heterozygosity at the chr8p21-22 locus. We previously showed that this region is associated with a set of tumor suppressive miRNAs (miR-3622a/b, -383, -4288) that are downregulated in PCa with important causal roles in tumor progression, recurrence and metastasis. Chr8p deletions increase significantly with tumor grade and are associated with poor prognosis. These deletions are well studied in CA men. However, there have been conflicting reports on frequency of chr8p deletions in AA men. In view of these conflicting studies and in light of our paradigm-shifting studies establishing the link between chr8p deletions and loss of miRNA genes in this region, we examined the expression of these miRNAs in AA vs CA clinical PCa tissues. We found that miR-3622a and miR-383 expression is significantly lower in AA men as compared to age, tumor stage and grade matched CAs. To examine if these miRNAs constitute causal factors contributing to PCa racial disparities, we overexpressed miR-3622a/miR-383/control miR in AA and CA PCa cell lines followed by functional assays. We found that these miRNAs exert more prominent effects on cellular proliferation and apoptosis in AA cell lines. To understand molecular mechanisms contributing to disparate functional effects of these miRNAs in AA vs CA cell lines, we examined potential miRNA target genes and found that miR-3622a regulates PCa stem cell marker, CD44 while miR-383 represses LEF1 and ZEB1 in a race-specific manner. Further, we determined the mechanistic basis of differential expression of miR-3622a in AAs vs CAs by examining copy number alterations (CNAs) and methylation at this locus using PCa clinical samples. Our analyses showed that while this locus is genomically altered at a lower frequency in AAs, significantly increased frequency of miRNA promoter hypermethylation was found in AAs as compared to CAs. In conclusion, our data suggests that miRNAs located in frequently deleted chr8p21-22 region constitute important causal factors contributing to racial disparities in PCa aggressiveness and clinical outcomes and are potential biomarkers to predict PCa aggressiveness and tumor recurrence in AA men. Citation Format: Divya Bhagirath, Nikhil Patel, Santu Ghosh, Roni Bollag, Sharanjot Saini. MicroRNAs located at chromosome 8p21-22 region as novel factors underlying racial disparities in prostate cancer aggressiveness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 282.

Abstract LB-222: Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to European American (EA) women (5-year relative survival of only 14% for AA in comparison to 36% for EA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than EA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to EA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in EA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. AA TNBC Tumor samples show higher expression of GLI1 and NICD in comparison to TNBC tumors from EA patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC growth and progression in mice; growth, proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Anirudh Saxena, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-222.

Abstract D116: Glucocorticoid receptor interacts with beta-catenin to promote stemness and therapy resistance in prostate cancer cells

Abstract Prostate cancer (PCa) is the most commonly diagnosed male cancer, with men of African ancestry showing a disproportionately high incidence and mortality. There is increasing evidence that glucocorticoid signaling through glucocorticoid receptor (GR) is amplified in African American men due to cumulative life stress, and that GR signaling is a key driver of resistance to androgen-targeted therapy, radiotherapy, and taxane chemotherapy in advanced PCa. Recent studies indicate that GR also promotes in PCa cells the formation of tumorspheres, a property of cancer stem cells (CSC); however, the mechanisms remain unclear. CSCs are intrinsically resistant to therapies due to their low-frequency, expression of transporters and efflux pumps, quiescent cell cycle and metabolic profile. Our previous studies demonstrated that docetaxel (DTX)-resistant PCa cells exhibit increased capacity for CSC-like properties, suggesting that these properties contribute to chemoresistance. Like GR, beta-catenin is overexpressed in metastatic and therapy-resistant tumors, and is considered a key regulator of cancer stemness and androgen-targeted therapy resistance. Thus, we hypothesized that GR may interact with beta-catenin in PCa cells to support stemness and chemoresistance. Using whole-cell and nuclear co-immunoprecipitation, we demonstrated the interaction between GR and beta-catenin in several DTX-sensitive and -resistant PCa cell line pairs. Pharmacological inhibition of GR using the selective inhibitor CORT-108297 with concomitant inhibition of beta-catenin using the small molecule inhibitor MSAB significantly enhanced DTX cytotoxicity in resistant PCa cells grown in both adherent and spheroid cultures. The BET bromodomain inhibitor JQ1, an antagonist of enhancer-mediated GR upregulation, also significantly reduced tumorsphere formation and stemness in DTX-resistant PCa cells. In order to gain insights into transcriptomic profiles activated by GR signaling that may contribute to stemness and therapy resistance, we performed RNA sequencing in a racially diverse panel of PCa cell lines (MDA-PCa-2b, VCaP, 22RV1, and PC3) treated with and without dexamethasone, a potent glucocorticoid. We observed that the African American cell line MDA-PCa-2b yielded the largest number of differentially regulated genes, perhaps due to the ability of dexamethasone to signal through both AR and GR in this particular cell line. RNAseq data is currently being mined and validated in cellular models treated with glucocorticoids or androgens, in the presence or absence of anti-GR or anti-androgen inhibitors, in order to identify GR-regulated genes. Our results offer novel insights into mechanisms by which GR signaling may influence tumorsphere formation and stemness, and suggest that combinatorial targeting of GR and beta-catenin could be a promising therapeutic strategy to overcome PCa therapy resistance and consequently reduce PCa mortality and its racial disparities. Citation Format: Shannalee R Martinez, Evelyn S Sanchez-Hernandez, Xin Chen, Alfonso M Duran, Charles H Wang, Carlos A Casiano. Glucocorticoid receptor interacts with beta-catenin to promote stemness and therapy resistance in prostate cancer cells [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 D116.

Abstract B084: Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes

Abstract Background: We previously showed that Nrdp1, an E3 ubiquitin ligase, is transcriptionally regulated by the androgen receptor (AR) in prostate cancer (CaP) cells and that Nrdp1 can post-translationally regulate ErbB3 (an EGFR family member) levels via ubiquitination and subsequent proteasomal degradation. Increased levels of ErbB3 are associated with worse CaP patient outcome. The goal of the current study was to determine whether dysregulation of the AR-Nrdp1-ErbB3 axis contributes to prostate cancer health disparities for African American (AA) men, and to identify the underlying mechanisms involved. Methods: Expression levels and localization of AR, Nrdp1, and ErbB3 were assessed in a total of 208 CaP patient samples (50 African American (AA) and 158 Caucasian (CA) CaP patients) and two cell lines (LNCaP (CA) and MDAPCa2b (AA)). Expression levels and localization of these molecules were determined by immunohistochemistry (IHC) and subcellular fractionation and western blot. A combination of knockdown (siRNA), forced overexpression (Nrdp1-FLAG construct), and treatment with enzalutamide or synthetic androgen (R1881) experiments were employed to investigate the relationship between AR, Nrdp1, and ErbB3 expression levels and localization and to identify differences in AA and CA cell lines. Immunoprecipitation allowed for investigation of the mechanisms which facilitate nuclear transport of Nrdp1. Results: A statistically significant negative association between cytoplasmic levels of AR (AR C) and nuclear Nrdp1 (Nrdp1 N) exists in both Caucasian (CA) and African American (AA) prostate cancer (CaP) patients, but this association was stronger in AA patients compared to CA patients (Spearman correlation coefficient of -0.62 for AA patients, and -0.36 for CA patients). Increased nuclear Nrdp1 expression levels were associated with increased 5-year survival rates, and reduced nuclear Nrdp1 expression levels predicted biochemical recurrence (AUC 0.63). Dysregulation of the AR-Nrdp1-ErbB3 axis in AA CaP cells was also observed in cell line studies; AA CaP cells expressed significantly lower levels of both total and nuclear Nrdp1 compared to their CA CaP counterparts. Manipulation of AR expression levels and localization via knockdown and inhibition with enzalutamide had a lesser impact on Nrdp1 expression levels and localization in AA versus CA CaP cells. Immunoprecipitation studies demonstrated that Nrdp1 can bind to AR in CA CaP cells but not AA CaP cells. Conclusions: Our combined data indicate that dysregulation of the AR-Nrdp1-ErbB3 axis occurs to a larger extent in AA than CA CaP cells and that this can contribute to worse patient outcomes. Our data also indicate that lack of binding between AR and Nrdp1 in AA CaP cells may account for the lower levels of nuclear Nrdp1 observed in AA CaP cells. Further understanding of the mechanisms which regulate Nrdp1 levels and localization may allow for the development of treatments which help abrogate cancer health disparities. Note: This abstract was not presented at the conference. Citation Format: Anhao Sam, Elizabeth Browning, Shawna Evans, Thomas Steele, Paramita Ghosh, Ruth Vinall. Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes [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 B084.

Abstract A070: Myosin heavy chain 9 (MYH9) is a novel interacting partner of kinesin family member C1 (KIFC1) in African American TNBC: Implications for racial disparity

Abstract Women of African descent in the United States are twice as likely to develop triple-negative breast cancer (TNBC), compared to women of European descent. African American (AA) women with TNBC present a much more aggressive disease course relative to their European American (EA) counterparts. Thus, there is an urgent need to evaluate race-specific biomarkers and their molecular regulation to improve survival outcome in African American (AA)patients. Kinesin family member C1 (KIFC1), a minus-end-directed microtubule motor protein is involved in centrosomal clustering and chromosomal instability. KIFC1 is highly overexpressed (fold change=1.50, p=0.000392) in TNBC relative to non-TNBC. We previously showed that ablation of KIFC1 impaired proliferation and migration of AA TNBC cells to a greater extent than European American (EA) equivalent. Our published data also showed that the weighted index (WI) of nuclear KIFC1 (nKIFC1) is higher in AA TNBC (WI: 154.66 vs. 133.74, AA and EA, respectively, p = 0.036) and nKIFC1 is an independent poor prognosis biomarker exclusively for AA TNBC patients. While the centrosome clustering role of KIFC1 during mitosis is well-established, its predominant nuclear localization in interphase may underlie its previously unrecognized race-specific association. We and others have shown that nKIFC1 immunoprecipitates with various histones, helicases, and actinins. Immunoprecipitation (IP) of KIFC1 from nuclear fractions of TNBC cell lines followed by mass-spectrometry analysis revealed myosin heavy chain 9 (MYH9), a tumor suppressor protein that stabilizes p53 and promotes its nuclear retention, as the top nKIFC1 binding partner in AA TNBC cell lines (MDA-MB-468 and HCC1806). However, we observed a rather low affinity (rank 5th) or no binding of MYH9 with nKIFC1 in EA TNBC cell lines (MDA-MB-436, HCC1937). Our IP-MS data suggest that a) the relative abundance of MYH9 in nKIFC1 immunoprecipitates is higher in the AA TNBC cell lines and b) MYH9 expression is higher in AA TNBC compared to EA TNBC cells. We hypothesize that nKIFC1 impairs the tumor suppressor function of MYH9 more effectively in AA compared to EA TNBC cells. Intriguingly, KIFC1 immunoprecipitates also showed the presence of beta, alpha-, and gamma actin along with several dead box helicases such as DDX5, DDX17, exclusively in AA TNBC cell lines, suggesting that a combination therapy regimen of KIFC1 inhibitor (SR31527, CW069, and AZ82) and taxane/anthracycline/platinating agent might be more effective in AA than in EA TNBC cells. RNA seq analyses to identify other KIFC1partners using KIFC1 knock out using CRISPR-CAS9 approach in AA and EA TNBC cell lines are currently underway. Citation Format: Chakravarthy Garlapati, Shriya Joshi, Luciane Cavalli, Uma Krishnamurti, Shobhna Kapoor, Ritu Aneja. Myosin heavy chain 9 (MYH9) is a novel interacting partner of kinesin family member C1 (KIFC1) in African American TNBC: Implications for racial disparity [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 A070.

Abstract B083: Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes

Abstract Background: We previously showed that Nrdp1, an E3 ubiquitin ligase, is transcriptionally regulated by the androgen receptor (AR) in prostate cancer (CaP) cells and that Nrdp1 can post-translationally regulate ErbB3 (an EGFR family member) levels via ubiquitination and subsequent proteasomal degradation. Increased levels of ErbB3 are associated with worse CaP patient outcome. The goal of the current study was to determine whether dysregulation of the AR-Nrdp1-ErbB3 axis contributes to prostate cancer health disparities for African American (AA) men, and to identify the underlying mechanisms involved. Methods: Expression levels and localization of AR, Nrdp1, and ErbB3 were assessed in a total of 208 CaP patient samples (50 African American (AA) and 158 Caucasian (CA) CaP patients) and two cell lines (LNCaP (CA) and MDAPCa2b (AA)). Expression levels and localization of these molecules were determined by immunohistochemistry (IHC) and subcellular fractionation and western blot. A combination of knockdown (siRNA), forced overexpression (Nrdp1-FLAG construct), and treatment with enzalutamide or synthetic androgen (R1881) experiments were employed to investigate the relationship between AR, Nrdp1, and ErbB3 expression levels and localization and to identify differences in AA and CA cell lines. Immunoprecipitation allowed for investigation of the mechanisms which facilitate nuclear transport of Nrdp1. Results: A statistically significant negative association between cytoplasmic levels of AR (AR C) and nuclear Nrdp1 (Nrdp1 N) exists in both Caucasian (CA) and African American (AA) prostate cancer (CaP) patients, but this association was stronger in AA patients compared to CA patients (Spearman correlation coefficient of -0.62 for AA patients, and -0.36 for CA patients). Increased nuclear Nrdp1 expression levels were associated with increased 5-year survival rates, and reduced nuclear Nrdp1 expression levels predicted biochemical recurrence (AUC 0.63). Dysregulation of the AR-Nrdp1-ErbB3 axis in AA CaP cells was also observed in cell line studies; AA CaP cells expressed significantly lower levels of both total and nuclear Nrdp1 compared to their CA CaP counterparts. Manipulation of AR expression levels and localization via knockdown and inhibition with enzalutamide had a lesser impact on Nrdp1 expression levels and localization in AA versus CA CaP cells. Immunoprecipitation studies demonstrated that Nrdp1 can bind to AR in CA CaP cells but not AA CaP cells. Conclusions: Our combined data indicate that dysregulation of the AR-Nrdp1-ErbB3 axis occurs to a larger extent in AA than CA CaP cells and that this can contribute to worse patient outcomes. Our data also indicate that lack of binding between AR and Nrdp1 in AA CaP cells may account for the lower levels of nuclear Nrdp1 observed in AA CaP cells. Further understanding of the mechanisms which regulate Nrdp1 levels and localization may allow for the development of treatments which help abrogate cancer health disparities. Citation Format: Anhao Sam, Shawna Evans, Elizabeth Browning, Sheryl Krig, Neelu Batra, Thomas Steele, Salma Siddqui, Paramita Ghosh, Ruth Vinall. Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes [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 B083.

Abstract B006: Development of a novel therapeutic splice-switching oligonucleotide targeting race-related androgen receptor signaling and aggressive prostate cancer

Abstract Background: Age-adjusted incidence and mortality rates for prostate cancer (PCa) among African American (AA) men are 1.6- and 2.4-fold greater, respectively, than among white men. The more aggressive characteristics of AA PCa account for a significant component of the PCa disparity, in addition to social determinants of health. Differences in androgenic activities in AA versus white populations and PCa patients have been observed and, clinically, AAs have a less complete response to androgen deprivation than whites do. One of the critical androgen receptor signaling targets in PCa is AR-V7, an androgen receptor variant that lacks the ligand-binding domain, is constitutively active, and associates with castration-resistant PCa, poorer clinical outcomes, and resistance to androgen ablation/androgen receptor inhibition therapies. This work addresses the urgent need to develop a novel therapeutic strategy capable of inhibiting AR-V7. Methods: We have designed and synthesized a novel chemically modified splice switching oligonucleotide (SSO) to correct aberrant splicing leading to production of AR-V7 as well as a control scrambled SSO. After transfection of PCa cell lines derived from AA and white patients with these SSOs, we have examined AR-V7 as well as androgen receptor RNA and protein levels using qPCR and Western blot analysis, respectively. Resulting alterations in proliferation have been assessed by monitoring cell growth using an Incucyte Live-Cell Imaging System and associated software. Results: Transfection of a panel of PCa cell lines derived from AA and white patients with AR-V7 SSO decreases AR-V7 RNA and protein in a dose-dependent manner while maintaining expression levels of full-length androgen receptor. Preliminary data suggest this biochemical response correlates with a biologically significant phenotype. AR-V7 SSO decreases proliferation in PCa cells, including proliferation in the presence of the androgen receptor inhibitor, enzalutamide, over and above the reduction seen in response to enzalutamide alone in both enzalutamide-sensitive and -resistant PCa cell lines. Additionally, transfection of PCa cells with SSOs designed to target nearby splicing enhancer sequences partially reduces AR-V7 RNA expression. Further studies to examine the effects of this SSO on transactivation activity, signaling and PCa cell biology, and the therapeutic efficacy of this SSO in AA and white PCa patient-derived primary cell lines and xenografts are under way. Conclusions: These studies suggest that a SSO can be developed to modulate androgen receptor signaling, inhibiting constitutive signaling and restoring ligand dependency, with interference of nearby splicing enhancer sequences contributing to its function. Such an SSO could represent a novel therapeutic strategy with the potential to reduce PCa disparities for AA men and improve outcomes for men of all races with aggressive disease driven by this mechanism. Citation Format: Bonnie L. LaCroix, Brendon M. Patierno, Bruce A. Sullenger, Daniel J. George, Steven R. Patierno, Jennifer A. Freedman. Development of a novel therapeutic splice-switching oligonucleotide targeting race-related androgen receptor signaling and aggressive prostate cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B006.

Abstract 780: High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians

Abstract Background: African-Americans have higher risk for developing prostate cancer and when diagnosed, the cancer is more aggressive and African-Americans are more than twice as likely to die of the disease compared to Caucasians. To clarify the mechanisms involved in this disparity, we analyzed the role of miR-155 in African-American and Caucasian prostate cancer tissues and cell lines. Materials and Methods: Human tissue samples from African-Americans and Caucasians clinical FFPE (Formaldehyde Fixed Paraffin Embedded) were used to analyzed miR-155 expression by Real Time PCR. We selected prostate cancer cell lines from Caucasians (DU-145, LNCaP, PC3), African-American (E006AA-hT) and normal epithelial cell line (PWR-1E) which express different levels of miR-155, to mimic the tissue samples and help identify the mechanisms related to racial disparity. We knocked down miR-155 in cells lines and performed cell viability assays using a CellTiter-Glo luminescent assay. For migration analysis, miR-155 knockdown cells were seeded in chambers and we measured the capacity of cell motility towards a chemo-attractant. To identify miR-155 mediated downstream regulators, three target prediction algorithms (miRDB, Targetscan and miRecords) were used. Results: We found miR-155 expression to be higher in African-American prostate cancer tissues compared to Caucasian patients with localized disease. Also, expression of miR-155 in African-American cell line, E006AA-hT, was significantly higher compared to Caucasian cell lines, DU-145, LNCaP and PC3. To determine the role of miR-155 in the differences between African-Americans and Caucasians, miR-155 was knocked down in E006AA-hT and DU-145 cells. miR-155 knockdown in E006AA-hT cells caused a decrease in cell viability compared with negative control, while there was no difference in DU-145 cells. We observed inhibition of migration E006AA-hT miR-155 knockdown cells but not on DU-145 miR-155 knockdown cells, suggesting that miR-155 knockdown have the more potent effect on African-American cell line. We analyzed three target prediction algorithms and found that four genes overlapped, SPI1, JARID2, ETS1 and CEBPB that may play a pivotal role in the treatment of aggressive African-American prostate cancers. Conclusion: We conclude that high levels of miR-155 is associated with prostate cancer aggressiveness in African-Americans. Citation Format: Marisa Shiina, Yutaka Hashimoto, Priyanka Kulkarni, Pritha Dasgupta, Varahram Shahryari, Guoren Deng, Divya Bhagirath, Laura Tabatabai, Sharanjot Saini, Shahana Majid, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya. High expression of miR-155 promotes prostate cancer aggressiveness in African-Americans compared to Caucasians [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 780.

Abstract 3468: Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression

Abstract Background and Aim: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. Mortality from TNBC is significantly higher in African American (AA) women compared to European American (EA) women (5-year relative survival of 14% for AA compared to 36% for EA). Irrespective of stage at diagnosis, AA-TNBC is aggressive with higher metastasis and poorer survival than EA-TNBC; hence, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall this study aims to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. AA-TNBC cells also exhibited higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population. To decipher the molecular mechanism underlying these functional differences, we analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC (Pearson coefficient = 0.308303) with a negative correlation in EA-TNBC (Pearson coefficient = -0.06695). Immunoblot analyses showed increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, NICD, Hes1 and FOXM1) in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC cells and their interaction was confirmed using co-immunoprecipitation assays. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and Carboplatin) effectively inhibited AA TNBC growth and progression in mice; proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ALDH1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression [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 3468.

Abstract 5800: Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer

Abstract Triple-negative breast cancer is a highly heterogeneous and aggressive subtype of breast cancer. Epigenetic drugs are continuing to emerge as promising therapies for triple-negative breast cancer (TNBC). These drugs, including the one used in this study, vorinostat, a histone deacetylase inhibitor (HDAC), are capable of multiple actions and are considered multifunctional drugs. Epigenetic mechanisms have been shown to play a role in the etiology of this cancer, through modulation of DNA methylation, histone modification or microRNAs. We have shown in this study that vorinostat and indole-3-carbinol (I3C) modulated the expression of microRNAs 221 and 222 differently in basal-like-2 (BL2), MSL, basal-like1 (BL1) and M subtypes of TNBC cells. Additional studies demonstrated that combination of tamoxifen and vorinostat significantly decreased expression of miRNA 221 in TNBC cell lines. MiRNAs are short noncoding RNAs that regulate the function of target genes post-transcriptionally. In addition, breast cancer microarrays further showed modulation of a number of other miRNAs that play a role in cell proliferation, drug resistance, cell invasion and metastasis. MicroRNAs are epigenetic targets for drug development. Using a global HDAC activity assay, it was also shown that subtypes of TNBC cells respond differently to vorinostat and I3C treatment, depending on their HDAC activity. HCC70, an African American cell line and a BL2 subtype, had the highest HDAC activity and its activity was significantly decreased by vorinostat treatment compared to BT549 (M), HCC 1806 (BL2), MCF7 (ER positive) and a basal nonmalignant cell line, MCF10A. Both vorinostat and I3C show great promise alone and in combination as effective agents in triple-negative breast cancer. Histone deacetylase inhibitors and dietary agents are emerging as therapy for triple-negative breast cancer and are currently in clinical trials. Citation Format: Beverly Lyn-Cook, Fatemeh NouriEmamzaden, Anfernee Hawkins, George Hammons, Beverly Word. Vorinostat and indole-3-carbinol modulate microRNAs and histone deacetylase activity in subtypes of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5800.

Abstract 5043: Glucocorticoids trigger GR-mediated expression of the chemoresistance-associated oncoproteins LEDGF/p75 and Clusterin: Implications for prostate cancer health disparities

Abstract 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.

Abstract A73: Role of microsatellite stability and mismatch DNA repair mechanism in the response to 5-fluorouacil treatment in African American colon cancer cell lines

Abstract Despite progress in closing the gap, health disparities still persist among African American (AA) colon cancer patients both in incidence and death rates as well as worse prognosis after treatment with 5-fluorouacil (5-FU) when compared to Caucasian American (CA) patients. In addition, AA colon cancer patients have higher incidence of mutations on the mismatch DNA-repair mechanism (MMR) than CA patients, and previous studies have shown a correlation between mutations in MMR and microsatellite instability (MSI) to resistance to 5-FU. Therefore, we aim to examine if differential MSI and MMR play a role in the efficacy of 5-FU treatment in AAs. Methods: We examined the microsatellite status and MMR mutations found in the tumors from AA colon cancer patients at our institution from 2010 to 2017 and utilized the AA tumor-derived colon cancer cell line SB-521, generated in Dr. Williams's laboratory, and the CA colon cancer cell line HT-29 for in vitro studies. In order to assess the response to 5-FU, we used the protein levels of the ribonucleotide reductase catalytic subunit M1 (RRM1) protein as a marker for drug resistance, the cleavage of caspase-3 for apoptosis initiation, c-Jun N-terminal kinases (JNK) phosphorylation as indicator of cellular stress, and histone 3 protein levels as reference of gene methylation changes. Results: Our retrospective data (N=83 patients) demonstrated that up to 20% of our colon cancer patients have MSI and up to 30% showed MMR mutations. As hypothesized, the protein levels of the chosen targets in the two cell lines exhibited differences after treatment with 5-FU. The AA cell line SB-521 appears to be more sensitive to the chemotherapeutic in terms of apoptosis, stress response, and drug resistance when compared to HT-29 cells. Conclusions: Altogether, our results illustrate the range of MSI, MMR, and protein patterns in AA tumors and cell line. The cell line SB-521 is a potential model to study 5-FU treatment for patients with MSI and MMR mutations as it has high MSI, does not express MLH1, has reduced MSH2, and overexpresses MSH6 (MMR proteins). Previous data suggest that lack of a functional MMR mechanism may play a role in the response to this chemotherapeutic. Further studies are needed to elucidate the differences in chemotherapy treatment responses between AAs and CAs and their role in colon cancer health disparities. Citation Format: Jenny E. Paredes, Ping Ji, Maria Munoz-Sagastibelza, Malhaar Agrawal, Jennie Williams, Laura Martello-Rooney. Role of microsatellite stability and mismatch DNA repair mechanism in the response to 5-fluorouacil treatment in African American colon cancer cell lines [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A73.