Abstract B2-13: AU-rich expression in invasive ductal breast cancer: A systems biology approach in post-transcriptional regulation

Abstract

Abstract AU-Rich element (ARE)-dependent post-transcriptional regulation mechanisms are increasingly emerging as key regulators that are influencing our perception of aberrant gene expression in cancer. AREs are instability determinant sequences located in the 3' UTR of mRNAs and are fundamental to the maintenance of transient expression of gene products that regulate various cellular functions such as cell proliferation, apoptosis, inflammation and immunity. Here we used a systems biology approach to examine the patterns of ARE-containing gene expression that is perturbed in invasive ductal breast cancer (IDBC). Based on IDBC mRNA expression profiles obtained from The Cancer Genome Atlas (TCGA) database, we intersected 2396 over-expressed genes with the AU-rich element database (ARED) and found that 414 comprise IDBC upregulated ARE-containing genes. Functional classification using the gene ontology tool, DAVID, showed the highest enrichment of ARE-genes in IDBC in chromosome segregation (5-fold, p<0.001). AREs are recognized by RNA binding proteins such as tristetraprolin (TTP, ZFP36), that targets mRNAs for decay, and HuR (ELAVL1), an mRNA stabilizing protein. Abnormal expression patterns of both these proteins, i.e. TTP deficiency coupled with HuR overexpression, are found in many cancers leading to prolonged expression of factors contributing to the maintenance and progression of tumors. We examined the relationship between the IDBC ARE-genes, TTP and HuR expression and the TTP/HuR ratio by looking at their correlations (R-values) with each other. Results indicated that the upregulated ARE-genes have a greater tendency to correlate with low TTP and elevated HuR expression than non-ARE genes. Using clustering assessment, we derived an 11-gene cluster from the 414 that function in chromosome positioning and segregation and mitotic kinase activity. The genes are CDC6, CENPA, CENPA, KIF11, KIF18A, NCAPG, NEK2, NUF2, PBK, PRC1, and TOP2A and their mRNA expression correlates negatively with the TTP/HuR ratio, i.e. their mRNA expression increases as TTP is deficient and HuR is simultaneously elevated (r = -0.53 to -0.66). Examination of the mRNA expression patterns of the 11 genes in the invasive breast cancer cell line MDA-MB-231 compared to MCF-7 cells and normal-like mammary cell lines, MCF10A and MCF12A showed that all of these genes were increased in MDA-MB-231 cell line compared to MCF10A by at least 2-fold (p<0.001). Immunoprecipitation of TTP protein in MDA-MB-231 cells followed by examination of associated mRNAs showed that 7 of the 11 gene-products were significantly associated with TTP (greater than 2-fold, p<0.01) compared to the control suggesting post-transcriptional regulation by TTP. Moreover, reporter experiments confirmed the functionally of the AREs. Upon examining the METBRIC breast cancer database, a larger database, we found that not only do the 11 chromosomal segregation genes overexpress, they also tend to be upregulated simultaneously in the same patients indicating that they participate in common regulatory mechanisms. Furthermore, the 11-gene cluster also correlates negatively with the TTP/HuR ratio further alluding to TTP regulation. Kaplan Meier survival analysis demonstrated a higher likelihood (1.6 times) of death due to co-overexpression of the 11 genes compared with a lower-expressing cohort. The data presented here suggest novel regulatory mechanisms related to mitosis and cellular proliferation that might be targeted by the mRNA decay-promoting protein TTP. Moreover, TTP deficiency in IDBC may, at least in part, be responsible for the aberrantly prolonged expression of factors that actively participate in chromosomal segregation and in turn, lead to enhanced maintenance and progression of tumors. Citation Format: Norah A. Alsouhibani, Edward Hitti, Tala Bakheet, Khalid SA Khabar. AU-rich expression in invasive ductal breast cancer: A systems biology approach in post-transcriptional regulation. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B2-13.