Abstract 717: Diagnosis of basal like breast cancer using DNA methylation markers at the promoters of long noncoding RNAs

Abstract

Abstract Background: Patients with basal-like breast cancer (BLBC) have poor overall survival and suffer a high rate of metastasis to the brain or lung within three to five years of initial presentation. Absence of a cure for advanced BLBC warrants early detection of BLBC, which might save more lives, especially those of women of African ancestry who are disproportionately affected by young onset BLBC. Aberrant DNA methylation is frequently observed in BLBC. DNA methylation is the most robust epigenetic marks and can be analyzed using clinical specimens including FFPE, tumor biopsies and liquid biopsies. Because expression of long non-coding RNAs (lncRNAs) is controlled temporally in response to neoplastic stimuli, we investigated the potential for lncRNA promoter methylation marks to be used for detection and prediction of BLBC development and progression in African Americans. Methods: To identify lncRNAs dysregulated in BLBC, we performed a Ribo-Zero RNA-seq or microarray analysis on breast tissues isolated from African American (n=63) and European American patients (n=14). Differentially methylated and hydroxymethylated regions of lncRNA genes were identified in African American samples (n=30) using our chemical methods to assay 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The Cancer Genome Atlas (TCGA) datasets were utilized to validate our results. To functionally annotate lncRNAs, we knocked down lncRNAs using LNA-antisense oligonucleotides (ASO) and performed Caspase 3/7 assays, flow cytometry, and qRT-PCR. We also utilized the CRISPR-Cas9 editing tools to knock out lncRNAs. Results: LncRNAs displayed distinct expression patterns between tumors and normal breast tissues. Out of hundreds of lncRNAs specifically expressed in BLBC tumors, we selected two lncRNAs, lnc19 and lnc98, which represent, respectively, a significantly increased and decreased lncRNA in BLBC tumors, compared to normal breast tissues and other subtype tumors. Lnc19 is highly up-regulated (a 39-fold increase) whereas lnc98 is dramatically down-regulated (a 59-fold decrease) in BLBC tumors. Methylation analysis showed significantly lower levels of promoter methylation for lnc19 and higher levels for lnc98 in BLBC tumors. A significant inverse correlation between methylation and expression of lnc19 was observed. Depletion of lnc19 resulted in rapid cell death of BLBC cells, with increased sensitivity to chemotherapy drugs. The data suggests that chemo-resistance of BLBC might be partly due to lnc19 overexpression, which is mediated through epigenetic control. Conclusion: We identified two epigenetically dysregulated lncRNAs in BLBC tumors, which contribute to chemo-resistance of these tumors. Our findings on the regulation of lncRNAs by cytosine methylation raise the possibility of new epigenetic biomarkers for diagnosis and prognosis of aggressive BLBC tumors. Citation Format: Yoo J. Han, Sonja M. Boatman, Jing Zhang, Albert C. Yeh, Yonglan Zheng, Olufunmilayo I. Olopade. Diagnosis of basal like breast cancer using DNA methylation markers at the promoters of long noncoding RNAs [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 717. doi:10.1158/1538-7445.AM2017-717