Abstract P1-06-02: Identifying hypermethylated tumor suppressor genes in breast cancer with an in vivo total genome knockdown screen

Abstract

Abstract Changes in gene expression are required for the progression of breast cancer; and DNA methylation, which silences tumor suppressor genes, is often responsible for these changes. Several de-methylating/DNA synthesis inhibiting drugs (such as decitabine) are potential breast cancer therapies; however, for their application to be successful, we need to identify which patients would most benefit from these treatments. To accomplish this, the critical genes, which must be silenced by hypermethylation in order for breast cancer to progress, need to be identified. For this purpose, we have performed an in vivo total genome knockdown screen. A genome-wide lentiviral-based shRNA screen was performed with breast cancer cell line MDA-MB-231 tumors implanted in the mammary fat pads of female NOD/SCID mice, with or without decitabine treatment (50mg/kg). The resulting tumors were harvested and shRNA sequences retrieved and hybridized to a Decode microarray, allowing for the identification of shRNA sequences that were enriched or depleted under decitabine treatment. The enriched shRNA sequences likely target genes of two categories: 1) they are methylated in the tumor and when expressed have tumor suppressive function, or 2) they are required for decitabine-mediated DNA synthesis inhibition and apoptosis. To determine if the shRNA-targeted genes identified in the screen fall within category 1 or 2, further analysis was done using GEO datasets for methylation in breast cancer cell lines, primary tumors, and normal breast tissues. In replicates of 6 mice, 111 shRNA sequences were enriched more than 2-fold in 5/6 mice. Of the 111 genes, 29 genes showed significant enrichment (p<0.05) in the decitabine-treated tumors. Using the GEO datasets, 19 of those significantly enriched genes fit a tumor suppressor methylation profile and merit further investigation of their methylation status in the MDA-MB-231 cell line. The remaining enriched genes may play a role in DNA synthesis inhibition and apoptosis- this will be confirmed with in vitro apoptosis assays. DAVID analyses did not reveal any functional clustering of the genes of interest, but did implicate several pathways that involved a single potential hypermethylated tumor suppressor gene. We have identified several genes that are hypermethylated in breast cancer and are potentially involved in tumor progression. Confirmation experiments will reveal a list of genes that when found hypermethylated in patient tumors would identify candidate breast cancer patients who would benefit from decitabine treatment. Citation Format: Margaret Thomas, Krysta Coyle, Mohammad Sultan, Luzhe Pan, Dae-Gyun Ahn, Patrick Lee, Carman Giacomantonio, Paola Marcato. Identifying hypermethylated tumor suppressor genes in breast cancer with an in vivo total genome knockdown screen [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-02.