Abstract P1-08-04: SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion

Background: SRY (Sex Determining Region Y)-related HMG-box (SOX) genes belong to a super-family of genes, which is characterized by a homologous sequence called the HMG-box residing on the Y-chromosome. There are 20 SOX genes present in humans and mice. We performed a siRNA screen of SOX transcription factors, and found that SOX9 was essential for breast cancer cell growth. The SOX9 protein recognizes the sequence CCTTGAG along with other members of the HMG-box class DNA-binding proteins and has been shown to be required for development, differentiation and lineage commitment. Moreover, SOX9 is expressed in adenocarcinomas, and is highly expressed in the most aggressive cancers. Our previous data shows SOX9 is highly expressed in “triple negative breast cancer” (TNBC) than in non-TNBC. Thus, we hypothesized that the SOX9 transcription factor acts as an essential molecule regulating TNBC growth and invasion. To test the hypothesis, we used SOX9-overexpressed, or SOX9-knockdown/knockout breast cancer cell models to determine whether SOX9 is necessary and/or sufficient to regulate TNBC cell proliferation, migration and invasion. Methods: We measured the cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell migration & invasion assays in ER-positive (MCF7 and ZR75-1) and ER-negative (MDA231 and MDA468) breast cancer cells. DOX-inducible SOX9-knockout cell lines were established in MDA231, MDA468, and LM2 cell lines using an inducible Cas9-CRISPR system. A SOX9 expressing lentivirus was used to overexpress SOX9, and siRNAs was used to knockdown SOXs in the different breast cancer cells. Protein and mRNA levels of SOX9 in TNBC, non-TNBC, immortalized human breast epithelial cell lines were examined by western blotting and qRT-PCR assay. Results: Knockdown of SOXs by siRNA caused decreased cell proliferation of MDA231 by ≥50% and of MDA468 by 30%-50% in siSOX4, siSOX6, siSOX9, siSOX10 and siSOX11 treatment groups (but not in siSOX8 and siSOX17 treatment groups). However, in MCF7 and T47D cell lines, treatment with siRNA to these SOX factors did not cause significant cell growth reduction. We demonstrated that SOX9 is more highly expressed in TNBC cells at both the mRNA and protein levels. Knockdown of SOX9 decreased cell migration and invasion of MDA231 to 25% and 50% respectively. The same effect also was observed in MDA468 cells, with approximately a 50% decline in migration and invasion. In SOX9-knockout MDA231, MDA468, and LM2 cells, cell proliferation, migration, and invasion were significantly reduced. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. We are now conducting in vivo studies to determine the effect of SOX9 on breast cancer cell metastasis. Conclusion: SOX9 is a critical regulator of TNBC cell proliferation, migration and invasion. These studies suggest that regulating SOX9 transcription factor and its signaling pathway will be a promising therapeutic strategy to treat TNBC and prevent metastasis. This work was supported by a Susan G. Komen Scientific Advisory Board Grant, SAB1600006 (PB), and a grant from the Breast Cancer Research Foundation 2015-2016 BCRF grant(PB), and by the Charles Cain Endowment (PB). Citation Format: Ma Y, Shepherd J, Mazumdar A, Zhao D, Bollu L, Hill J, Zhang Y, Brown P. SOX9 is a critical regulator of triple-negative breast cancer cell growth and invasion [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-04.