Abstract 3475: Suppression of inflammatory signaling by HOXA5 via modulation of the NF-KB pathway in breast cancer

Abstract

Abstract Background and Aims: HOXA5 is a tumor suppressor in breast cancer (BC) and transcriptionally regulates E-cadherin, CD24, progesterone receptor and p53. The goal of this study was to establish whether the loss of HOXA5 cooperates with common tumor suppressor genes and oncogenes in isogenic untransformed breast epithelial MCF10A sublines to induce tumorigenesis and to determine the mechanism thereof. 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 were used. Sanger sequencing was used to confirm genotype. Immunoblotting and qPCR was used to analyze gene expression. All-trans Retinal- ATAL (Sigma #R2500) (1 μM for 7 days) was used. For xenograft studies, 2 X 106 cells in 80% growth factor reduced Matrigel and 20% PBS were implanted subcutaneously. Agilent Human v2, 4x 44K Expression, two-color expression array was used (Biological replicates, n=3). Differentially expressed probes were sorted by p value (<0.05) and fold change (1.5 or greater). Results: Stable knock down (KD) of HOXA5 using shRNAs in the V777L DKI cell line (HER2 V777L and PIK3CA E545K) caused epithelial to mesenchymal transition (EMT): spindle-like morphology with attendant alterations in N-cadherin, Slug, Vimentin, E-cadherin, increased invasion and migration. Overexpression of HOXA5 in the p53 R248W cell line resulted in an opposing phenotype. ATAL treatment of p53 R248W restored expression of HOXA5 and its targets. V777L DKI-HOXA5 knock down cells formed pre-invasive outgrowths upon subcutaneous injection in NSG mice. Expression array and ingenuity pathway analysis showed IL-22 signaling as a top differentially regulated pathway and TNF as top upstream regulator. Validation in multiple BC cell lines by qPCR and western blot showed that inflammatory transcripts IL-6, IL-8, IL1β, PTX-3 and COX-2 were up-regulated upon HOXA5 KD and reduced upon overexpression of HOXA5 or ATAL treatment. Treatment with NF-κB inhibitor BAY11-7085 partially abrogated the effect of HOXA5 KD, suggesting a NF-κB mediated regulation. Reduced p65 nuclear localization and increased IκB-α expression was observed upon HOXA5 overexpression. KD of HOXA5 increased NF-κB-mediated luciferase reporter activity. Conclusions: KD of HOXA5 expression in the partially transformed V777L DKI cell line results in EMT, and transformation from normal ductal structures to preneoplastic nodules when implanted in NSG mice. Expression array of KD cells showed that NF-κB regulated inflammatory transcripts were perturbed. HOXA5 was found to suppress NF-κB mediated transcription, thus reducing levels of inflammatory cytokines and chemokines. Citation Format: Priya Pai, Guannan Wang, Diana Ráez Rodríguez, Wayne Yu, Saraswati Sukumar. Suppression of inflammatory signaling by HOXA5 via modulation of the NF-KB pathway in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3475.