Abstract 4467: The role of HOXA5 in the malignant progression of engineered isogenic MCF10A sublines harboring common breast cancer mutations

Abstract

Abstract Background and Aims: HOXA5 behaves as a tumor suppressor gene in breast cancer and regulates E-cadherin, CD24, progesterone receptor and p53. Retinoic acid activates transcription of HOXA5 by binding to RAR elements in the HOXA5 locus. The goal of this study was to establish whether the loss of HOXA5 cooperates with loss or mutations in common tumor suppressor genes and oncogenes in isogenic untransformed breast epithelial MCF10A sublines to induce tumorigenesis. Materials and Methods: Isogenic MCF10A sublines with a single knock in mutation in p53 (R248W), or double knock in mutations in both HER2 (V777L) and PIK3CA (E545) generated by Cre recombinase mediated excision/insertion by the Park lab were used. Genomic DNA extraction followed by PCR, gel extraction and Sanger sequencing was used to confirm genotype. Western blotting and quantitative real time PCR was used to analyze gene expression. All-trans Retinal- ATAL (Sigma #R2500) (1 μM for 7 days) was used treat p53 R248W cell line. Colony formation assay was performed and assessed using 0.5% crystal violet staining. XTT assay was used for cell proliferation assays. Immunocytochemical images were taken. For xenograft studies, 2 X 106 cells in 80% growth factor reduced Matrigel and 20% PBS were implanted subcutaneously. Results: Stable knock down (KD) of HOXA5 using shRNAs in the V777L DKI cell line (HER2 V777L and PIK3CA E545K) resulted in a more spindle like, elongated morphology. Elevated levels of mesenchymal markers N-cadherin, Slug and Vimentin, and reduced levels of epithelial marker E-cadherin, at both the protein and RNA levels were seen. Ectopic overexpression of HOXA5 in the p53 R248W cell line caused reduced proliferation, colony formation and wound healing relative to vector transfected cells. ATAL treatment of p53 R248W restored expression of HOXA5, E-Cadherin, progesterone receptor and other HOXA5 targets. Subcutaneous injection of V777L DKI SCR cells into NSG mice resulted in small growths of ductal structures with large lumens surrounded by a single layer of cells, while V777L DKI-HOXA5 knock down cells formed structures with smaller lumens surrounded by multiple layers of cells, resembling hyperplastic outgrowths. Conclusions: Knock down of HOXA5 expression in the partially transformed V777L DKI cell line results in a more mesenchymal phenotype accompanied by an increase in EMT marker expression, and a transformation from normal ductal structures to hyperplastic nodules when implanted in NSG mice. Whether the hyperplastic nodules will progress to invasive cancer as the tumor grows is under study. Conversely, ectopic expression of HOXA5 in the p53 R248W cell line causes a reduction in functional properties associated with a transformed phenotype: proliferation, colony formation and wound healing. Citation Format: Priya Pai, Guannan Wang, Saraswati Sukumar. The role of HOXA5 in the malignant progression of engineered isogenic MCF10A sublines harboring common breast cancer mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4467.