Abstract P2-08-04: HMGA1 negatively regulates the expression of PRRX1 in triple negative breast cancer cells, which favors the progression to metastasis

Abstract

Abstract Background: Triple negative breast cancer (TNBC) presents a very aggressive behavior with a high rate of metastasis. Overexpression of HMGA1 has been reported in TNBC and has been associated with the induction of the Epithelial-Mesenchymal transition (EMT) and metastasis. Therefore, HMGA1 is considered a master regulator of tumor progression in TNBC. The objective of this work was to know which genes are directly or indirectly regulated by HMGA1 to better understand their participation in EMT and their role in aggressive TNBC. Methods: We performed the silencing of the HMGA1 gene using siRNA Silencer® Select Pre-designed (s6667 HMGA1, 4390849 GAPDH, 4390843 Negative control, all from Thermo Fisher, MA, USA) in two TNBC cell lines, HCC-1395 and MDA-MB-231, and we observed the effect of this gene inhibition by microarray global expression analysis using GeneChip Human Genome U133 Plus 2.0 (Affymetrix, CA, USA), comparing the conditions of inhibition versus their own control without inhibition. After the microarray data mining, results for the HMGA1 and PRRX1 genes were validated by qPCR using the Prime Time® Primers for HMGA1 (Hs.PT.58.38699366), PRRX1 (Hs.PT.58.2820749), and GAPDH as endogenous gene (Hs.PT.39a.22214836) with SybrGreen reagent (Roche, Basel, Switzerland). The level of expression of the HMGA1 and PRRX1 proteins was analyzed by Western blot in nuclear protein extracts of each cell line before and after gene silencing. Finally, we performed an in silico analysis using the "Gene 2 promoter" tool in the Genomatix platform to search the promoters and binding proteins of the PRRX1 gene. Results: The silencing of HMGA1 in a non-metastatic TNBC-cell line, HCC-1395, showed deregulation of genes associated with cell proliferation and angiogenesis. Meanwhile the silencing in a TNBC-metastatic cell line, MDA-MB-231, resulted in the deregulation of genes involved in the formation and organization of the cytoskeleton, including the overexpression of PRRX1. Validation of the expression changes of HMGA1 and PRRX1 by qPCR and Western blot was performed and HMGA1 was confirmed to negatively regulate the PRRX1 gene expression. Through in silico studies, we identified several binding sites of HMGA1 to the PRRX1 promoter. Conclusions: The subexpression of PRRX1 is necessary for EMT to occur. In this study, we present the interesting finding that HMGA1 regulates the subexpression of PRRX1, as supported by the experiments of transcriptional and translational expression presented in this work. To our knowledge, this is the first report describing a regulatory role of HMGA1 on PRRX1, which could explain the metastatic capacity of cancers that overexpress HMGA1. Key words: Epithelial-Mesenchymal Transition, Triple Negative Breast Cancer, Mesenchymal-Epithelial Transition, Metastasis. Citation Format: Aguayo-Millán CD, Santuario-Facio SK, Treviño-Alvarado V, Calvo-Anguiano G, Rojas-Martínez A, Camacho-Morales A, Ortíz-López R. HMGA1 negatively regulates the expression of PRRX1 in triple negative breast cancer cells, which favors the progression to metastasis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-08-04.