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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 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.

Despite numerous advances in cancer care, breast cancer remains one of the leading causes of cancer-related death among Canadian women. Previous work by our lab has identified aldehyde dehydrogenase 1A3 (ALDH1A3) as a breast cancer stem cell marker, and patients with high levels of ALDH1A3 in their tumors have worse outcomes. Furthermore, we found that via its retinoic acid (RA) production function, ALDH1A3 also contributes to breast tumor growth and metastasis in a murine xenograft model. RA induces transcription of many genes (directly and indirectly), initiates cell signaling and regulates many cellular processes involved in cell growth and differentiation. To identify the key RA-inducible genes which mediate breast cancer progression we performed a genome-wide lentiviral-based shRNA screen using MDA-MB-231 breast cancer cells xenotransplanted into NOD/SCID mice with or without RA treatment. From the in vivo screen, 50 genes were identified as being putatively involved in RA-mediated breast tumor growth. 14 genes have been confirmed in vitro as differentially expressed in the presence of RA. We have generated individual shRNA knockdown clones of the genes and are validating their involvement in RA-mediated breast cancer progression in vivo. Analysis of the genes identified by this screen revealed potential interactions between RA and Wnt signaling, implicating extensive cross-talk between these two pathways. Completion of this study will define the mechanism of RA-mediated breast cancer progression and potentially identify new therapeutic targets for breast cancer treatment. Citation Format: Krysta M. Coyle, Cheryl Dean, Lu-Zhe Pan, Dae-Gyun Ahn, Mohammad Sultan, Jayme Salsman, Graham Dellaire, Carman Giacomantonio, Patrick W. Lee, Paola Marcato. Identifying genes involved in retinoic-acid-mediated breast tumor progression by total-genome-knockdown screen. [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 3404. doi:10.1158/1538-7445.AM2014-3404