Andreana Holowatyj

Integrated genomic and functional analyses of histone demethylases identify oncogenic KDM2A isoform in breast cancer.

Histone lysine demethylases (KDMs) comprise a large class of enzymes that catalyze site‐specific demethylation of lysine residues on histones and other proteins. They play critical roles in controlling transcription, chromatin architecture, and cellular differentiation. However, the genomic landscape and clinical significance of KDMs in breast cancer remain poorly characterized. Here, we conducted a meta‐analysis of 24 KDMs in breast cancer and identified associations among recurrent copy number alterations, gene expression, breast cancer subtypes, and clinical outcome. Two KDMs, KDM2A and KDM5B, had the highest frequency of genetic amplification and overexpression. Furthermore, among the 24 KDM genes, KDM2A had the highest correlation between copy number and mRNA expression, and high mRNA levels of KDM2A were significantly associated with shorter survival of breast cancer patients. KDM2A has two isoforms: the long isoform is comprised of a JmjC domain, CXXC‐zinc finger, PHD zinc finger, F‐box, and the AMN1 protein domain; whereas the short isoform of KDM2A lacks the N‐terminal JmjC domain but contains all other motifs. Detailed characterization of KDM2A in breast cancer revealed that the short isoform of KDM2A is more abundant than the long isoform at DNA, mRNA, and protein levels in a subset of breast cancers. Furthermore, our data indicate that the short isoform of KDM2A has oncogenic potential and functions as an oncogenic isoform in a subset of breast cancers. Taken together, our findings suggest that amplification and overexpression of the KDM2A short isoform is critical in breast cancer progression.

Racial/Ethnic Disparities in Survival Among Patients With Young-Onset Colorectal Cancer

PURPOSE Racial disparities in colorectal cancer (CRC) persist, despite overall reductions in morbidity and mortality. In addition, incidence is rising among individuals younger than 50 years of age. We compared the survival of young-onset CRC among non-Hispanic black (NHB), non-Hispanic white (NHW), and Hispanic individuals. PATIENTS AND METHODS Using the National Cancer Institutes Surveillance, Epidemiology, and End Results program data, we identified individuals between the ages of 20 and 49 years, diagnosed with CRC between 2000 and 2009. Survival rates and Cox proportional hazards models were used to compare stage-specific 5-year survival among NHBs, NHWs, and Hispanics. RESULTS We identified 28,145 patients with young-onset CRC (19,497 NHW; 4,384 NHB; 4,264 Hispanic) during the 10-year study period. Overall survival at 5 years after CRC diagnosis was 54.9% among NHB, 68.1% among NHW, and 62.9% among Hispanic individuals (P < .001). NHB individuals had a significantly higher hazard of cancer-specific death compared with NHWs after adjusting for age, sex, race, stage, county-level poverty, and treatment history in cases of colon (hazard ratio [HR], 1.35; 95% CI 1.26 to 1.45) and rectum/rectosigmoid junction (HR, 1.51; 95% CI, 1.37 to 1.68) cancers, whereas there was no significant difference in survival between NHWs and Hispanics. The greatest racial disparities in cancer-specific survival were observed among NHB and NHW patients diagnosed with stage II cancers of the colon (HR, 1.69; 95% CI, 1.33 to 2.14) and stage III cancers of the rectum (HR, 1.98; 95% CI, 1.63 to 2.40). CONCLUSION Survival after CRC diagnosis at a young age is significantly worse among NHBs compared with NHWs, even among patients with early-stage disease. Further study is needed to determine whether differences in tumor biology and/or treatment are associated with racial disparities in outcomes, which would have implications for CRC treatment and prevention.

HER2 status and disparities in luminal breast cancers

National Comprehensive Care Network guidelines for adjuvant treatment of invasive breast cancer are based on HER2 and hormone receptor (HR) status, where HR+ disease encompasses all estrogen receptor (ER)+ and/or progesterone receptor (PR)+ tumors. We sought to explore clinical and demographic differences among patients with HR+ breast cancer subtypes, and the role of HER2 status, age, race/ethnicity, and socioeconomic status (SES) in disease risk. We evaluated breast cancer subtype distribution, defined by HR and HER2 status, using patient clinical, demographic, and socioeconomic characteristics. Differences in HR categories by demographic and tumor characteristics were examined using chi‐squared tests. Multinomial logistic regression was used to estimate odds ratios (OR) and 95% confidence intervals (CI) to quantify associations between breast cancer HR status and demographic factors. We found that differences in HR+ (ER−/PR+ vs. ER+/PR− or ER+/PR+) tumor biology are likely clinically significant and may play a role in breast cancer, regardless of HER2 status. While clinical and patient characteristics differed within each luminal subtype, we found disparities in SES only among Luminal A (HR+/HER2−) tumors. Among HR+/HER2− cases, we observed that ER−/PR+ patients tend to live in areas of higher poverty (OR = 1.20, 95% CI = 1.03–1.40) and are 70% more likely to be aged 50 years or older. However, this pattern was not found in women with Luminal B (HR+/HER2+) disease (Poverty OR = 0.98, 95% CI = 0.76–1.27; Age OR = 1.01, 95% CI = 0.81–1.26). Racial/ethnic disparities among non‐Hispanic black and Hispanic women persisted across HR+/HER2− cases compared to non‐Hispanic white women. Our findings suggest that while race/ethnicity and SES are correlated, each plays an independent role in contributing to disease among Luminal A tumors. Further study is needed to investigate how tumor biology, race/ethnicity, and socioeconomic disparities among HR+/HER2− cases may contribute to poorer patient prognosis.

Histone lysine demethylases in Drosophila melanogaster

Epigenetic regulation of chromatin structure is a fundamental process for eukaryotes. Regulators include DNA methylation, microRNAs and chromatin modifications. Within the chromatin modifiers, one class of enzymes that can functionally bind and modify chromatin, through the removal of methyl marks, is the histone lysine demethylases. Here, we summarize the current findings of the 13 known histone lysine demethylases in Drosophila melanogaster, and discuss the critical role of these histone-modifying enzymes in the maintenance of genomic functions. Additionally, as histone demethylase dysregulation has been identified in cancer, we discuss the advantages for using Drosophila as a model system to study tumorigenesis.

Racial Differences in 21-Gene Recurrence Scores Among Patients With Hormone Receptor–Positive, Node-Negative Breast Cancer

Purpose The 21-gene recurrence score (RS) breast cancer assay is clinically used to quantify risk of 10-year distant recurrence by category (low, < 18; intermediate, 18 to 30; high, ≥ 31) for treatment management among women diagnosed with hormone receptor-positive, human epidermal growth factor receptor 2-negative, lymph node-negative breast cancer. Although non-Hispanic black (NHB) women have worse prognosis compared with non-Hispanic white (NHW) women, the equivalency of 21-gene RS across racial groups remains unknown. Patients and Methods Using the Metropolitan Detroit Cancer Surveillance System, we identified women who were diagnosed with hormone receptor-positive, human epidermal growth factor receptor 2-negative, lymph node-negative invasive breast cancer between 2010 and 2014. Multinomial logistic regression was used to quantify racial differences in 21-gene RS category. Results We identified 2,216 women (1,824 NHW and 392 NHB) with invasive breast cancer who met clinical guidelines for and underwent 21-gene RS testing. The mean RS was significantly higher in NHBs compared with NHWs (19.3 v 17.0, respectively; P = .0003), where NHBs were more likely to present with high-risk tumors compared with NHWs (14.8% v 8.3%, respectively; P = .0004). These differences were limited to patients younger than 65 years at diagnosis, among whom NHBs had significantly higher RS compared with NHWs (20 to 49 years: 23.6 v 17.3, respectively; P < .001 and 50 to 64 years: 19.6 v 17.4, respectively; P = .023). NHBs remained more likely to have high-risk tumors compared with NHWs after adjusting for age, clinical stage, tumor grade, and histology (odds ratio [OR], 1.75; 95% CI, 1.18 to 2.59). Conclusion NHBs who met clinical criteria for 21-gene RS testing had tumors with higher estimated risks of distant recurrence compared with NHWs. Further study is needed to elucidate whether differences in recurrence are observed for these women, which would have clinical implications for 21-gene RS calibration and treatment recommendations in NHB patients.

Novel immune cell subtypes linked to survival among African American women with triple-negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive disease that is twice as likely to be diagnosed in African American (AA) women compared to white women, with poor clinical outcomes. Tumor infiltrating lymphocytes (TILs) are associated with improved survival for TNBC, but the relevance of TILs and immune cell subtypes to survival in AA women with TNBC is unknown. We evaluated histopathologic TIL counts and molecular characteristics among 60 AA women diagnosed with TNBC with linkage to clinical outcomes using data from the Metropolitan Detroit Cancer Surveillance System. We utilized whole genome expression profiling of TN tumors and cell type deconvolution analysis to evaluate the underlying mechanisms and immune cell subtypes associated with survival patterns in the context of TILs. TILs were significantly associated with improved survival [1-10% Hazard Ratio (HR)=0.32, 95% Confidence Interval (CI) 0.12-0.90, p=0.031; >10% HR=0.18, 95% CI 0.05-0.67, 9.9×10−3]. 524 transcripts (326 coding, 198 non-coding) were associated with TIL levels, 34 of which were associated with both TILs and survival (p<0.05). While only naïve B cells were associated with survival when considering individual cell types [Median HR=2.43, 95% CI 1.07-5.55, p=0.035], increased naïve B cells, plasma cells, and activated NK cells, and decreased resting mast cells, M1 macrophages, and monocytes were associated with transcripts that predicted worse survival. These data provide evidence for novel roles for these immune cells types in TNBC, and further studies are needed to validate these findings and identify determinants of patterns of immune response in TNBC relevant to the AA population. Summary We found that increased naïve B cells, plasma cells, and activated natural killer cells, and decreased resting mast cells, M1 macrophages, and monocytes were associated with expression biomarkers of worse survival among African American women with triple negative breast cancer.

Abstract LB-378: Ankyrin repeat domain 30A as a novel molecular marker of columnar alterations in benign breast disease and subsequent breast cancer in African American women

An estimated 1.6 million breast biopsies are performed annually in the United States, and the vast majority are benign. Benign breast disease is an established breast cancer risk factor for both Caucasian and African American women. Yet little is still known about benign breast tissue from African American women, who suffer from disproportionately high-grade tumors and poorer breast cancer survival. To better characterize the risk of breast cancer among African American women with benign breast disease, we assessed benign breast disease pathology of African American women in our metropolitan Detroit cohort (n = 3,737). We classified benign breast lesions for pathological characteristics and used logistic regression to estimate subsequent breast cancer risk with histological characteristics. We observed that women whose biopsies showed columnar alterations, columnar cell lesions of the breast, were 1.78-fold more likely to develop an invasive breast cancer [odds ratio (OR) 1.78, 95% confidence interval (CI) 1.19-2.65] after adjusting for age at biopsy, biopsy year, and overall impression. Columnar alterations were not found to be significantly associated with risk of developing ductal carcinoma in situ (DCIS) among African American women [OR 1.53, 95% CI 0.83-2.82]. Next, to identify molecular markers associated with columnar alterations in benign breast lesions, we performed gene expression analyses on biopsy tissues from 27 African American women that subsequently developed an invasive breast cancer. Transcriptional profiling of these lesions identified differentially expressed genes significantly associated with columnar alterations, including ankyrin repeat domain 30A (ANKRD30A). ANKRD30A, also known as NY-BR-1, is a mammary tissue-specific differentiation antigen that is overexpressed in breast cancers. Increased expression of ANKRD30A in benign breast lesions with columnar alterations may be indicative of subsequent breast cancer development. Taken together, novel molecular markers, including ANKRD30A, and ethnicity can lead to better risk assessment and earlier diagnosis of breast cancer. Citation Format: Andreana N. Holowatyj, Rouba Ali-Fehmi, Julie J. Ruterbusch, Eman Abdulfatah, Vishakha Pardeshi, Woodlyne Roquiz, Baraa Alosh, Sudeshna Bandyopadhyay, Derek Radisky, Michele L. Cote. Ankyrin repeat domain 30A as a novel molecular marker of columnar alterations in benign breast disease and subsequent breast cancer in African American women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-378.

Abstract 98: Genetic alterations of KDM4 subfamily and therapeutic effect of novel demethylase inhibitor in breast cancer

The histone lysine demethylase KDM4 subfamily, comprised of four members (A, B, C, and D), play critical roles in controlling transcription, chromatin architecture and cellular differentiation. We previously demonstrated that KDM4C is significantly amplified and overexpressed in aggressive basal-like breast cancers and functions as a transforming oncogene. However, information regarding the genomic and transcriptomic alterations of the KDM4 subfamily in different subtypes of breast cancer remains largely incomplete. Here, we conducted a meta-analysis of KDM4A, B, C and D in breast cancer and identified associations among recurrent copy number alterations, gene expression and breast cancer subtypes. We demonstrated that KDM4A and D are also significantly overexpressed in basal-like breast cancer, whereas KDM4B overexpression is more dominant in estrogen-receptor-positive, luminal breast cancer. Next, we investigated the therapeutic potential of a novel histone demethylase inhibitor, NCDM-32B, in breast cancer. The treatment of basal breast cancer cell lines with NCDM-32B resulted in the decrease of cell viability and anchorage independent growth in soft agar. Furthermore, we found that NCDM-32B impaired several critical pathways that drive cellular proliferation and transformation in breast cancer. Our findings demonstrate genetic amplification and overexpression of the KDM4 demethylases in different subtypes of breast cancer. Furthermore, histone methylation is reversible and KDM4 demethylases are druggable targets. Thus, KDM4 inhibitors may serve as a novel therapeutic approach for a subset of aggressive breast cancer.

Abstract 5152: Mechanism and therapeutic potential of the histone demethylase GASC1 in castration-resistant prostate cancer

The histone lysine demethylase, GASC1 (Gene Amplified in Squamous Cell Carcinoma 1) is an epigenetic regulatory protein both highly expressed during mammalian embryogenesis and overexpressed in several types of cancer, including aggressive Castration Resistant Prostate Cancer (CRPC). GASC1 mainly catalyzes demethylation of tri- and di-methylated forms of histone H3 lysine 9 (H3K9me3/me2) epigenetic repressive marks, regulating gene expression and chromatin architecture. This research aims to elucidate the fundamental mechanism by which GASC1 dysregulation contributes to CRPC progression, as well as to evaluate its potential as a therapeutic target against aggressive prostate cancer. Previously, our lab and others have shown that GASC1 interacts with the androgen receptor (AR) and functions as a co-activator of AR-induced transcription in prostate cancer. We have used an shRNA approach to determine whether GASC1 knockdown affects the proliferation and transformation of CRPC cells. We found that knocking down GASC1 inhibits the growth and colony formation of C4-2B and CWR22Rv1 cells in vitro. Knockdown also reduced transcript levels of classical and CRPC-specific AR target genes in either the presence or absence of the AR ligand. As a result, we are evaluating the therapeutic potential of novel and highly specific lysine demethylase inhibitors towards abating GASC1 function in prostate cancer cells in vitro. We have recently determined that one of these novel inhibitors is highly effective in modulating the survival and proliferation of several prostate cancer cell lines including CRPC lines with IC50 values, all well under 1 uM. Together, our data demonstrates that GASC1 is a therapeutically relevant target in controlling and preventing the emergence of prostate cancers, with particular application against aggressive CRPC subtypes. Citation Format: Roselyne M. Labbe, Qin Ye, Andreana Holowatyj, Lihong Zhang, Zeng Quan Yang. Mechanism and therapeutic potential of the histone demethylase GASC1 in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5152. doi:10.1158/1538-7445.AM2014-5152

Abstract 5154: Targeting the histone demethylase KDM4 subfamily as a potential therapeutic strategy in breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Epigenetic alterations, including histone modifications, play fundamental roles in breast cancer initiation and progression. We originally identified and cloned the GASC1 (gene amplified in squamous cell carcinoma 1, also known as KDM4C) gene from an amplified region at 9p24 in esophageal cancer cells; and recently demonstrated that KDM4C/GASC1 is amplified and over-expressed in breast cancer, particularly in the aggressive basal subtype. The KDM4C/GASC1 protein belongs to the KDM4 family of histone demethylases, and although KDM4 family members have a high degree of homology, they may play different roles in various types of breast cancer. The goal of this study is to analyze genomic anomalies and expression levels of KDM4 demethylases in breast cancer, and elucidate the fundamental role and mechanism of their dysregulation in promoting breast tumorigenesis. We conducted a large-scale meta-analysis of KDM4 demethylase expression across multiple available gene expression studies in breast cancer. Next, we examined KDM4 expression in a panel of non-tumorigenic and cancerous breast epithelial cell lines using quantitative RT-PCR and Western blot assays. We also assessed global methylation (H3K4, H3K9, H3K27 and H3K36) levels by Western blot in a panel of breast cancer cell lines. Finally, we tested a novel KDM4 inhibitor in breast cancer. We found that the KDM4 members show different expression patterns in subtypes of breast cancer. GASC1/KDM4C expression is high in estrogen receptor (ER)-negative, basal type breast cancers. In contrast, KDM4B expression is significantly higher in ER-positive luminal-type breast cancers. Expression levels of homologs KDM4A and D are not significantly different between ER-+/- breast cancers. Our findings suggest that H3 global methylation levels vary among different breast cancer cell lines. Furthermore, we demonstrated that inhibition of KDM4 with a novel small molecule inhibitor increased H3K9 methylation levels and slowed KDM4-overexpressed breast cancer cell growth in vitro. In summary, our data indicate that the KDM4 histone demethylase family may contribute to the dysregulation of histone methylation status differently in breast cancer subtypes. Moreover, breast cancer cell lines with defined histone methylation levels will provide a useful model for investigating biological and functional roles of KDM4 histone demethylases, and for developing novel anticancer epi-drugs in breast cancer. Citation Format: Andreana Holowatyj, Qin Ye, Lihong Zhang, Jack Wu, Zeng-Quan Yang. Targeting the histone demethylase KDM4 subfamily as a potential therapeutic strategy in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5154. doi:10.1158/1538-7445.AM2014-5154