Abstract 3347: SOX9 is a critical regulator of triple-negative breast cancer growth and invasion
Authors: Ma Yanxia , Shepherd Jonathan , Zhao Dekuang , Bollu Lakshmi , Hill Jamal , Zhang Yun , Mazumdar Abhijit , Brown Powel H. 
Published: August 2018

Background: Triple-negative breast cancers (TNBCs) have the worst prognosis of all breast cancers, and have few available therapies other than non-specific and toxic chemotherapy. To identify novel targets for TNBCs, we investigated expression levels of transcription factors (TFs) in TNBCs compared to those in Non-TNBCs. Our previous analyses identified the TFs highly expressed in TNBCs. Of these, six SRY (Sex Determining Region Y)-related HMG-box (SOX) TFs were highly expressed in TNBCs as compared to non-TNBCs. SOX genes belong to a superfamily of gene. There are approximately 20 SOX genes in humans and mice.

Hypothesis: SOX TFs are important regulators of TNBC cell growth and metastasis.

Material and Methods: We measured breast cancer cell growth using an automated cell counting assay. Cell migration and invasion were detected by transwell assays in non-TNBC (MCF7 and ZR75-1) and TNBC (MDA231 and MDA468) cells. DOX-inducible SOX9-knockout (KO) 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 were used to knockdown (KD) SOXs in the different breast cancer cells. Cell cycle phase and apoptosis were detected using flow cytometric analysis. Protein and mRNA levels of SOX9 in cell lines were examined by western blotting and qRT-PCR assays. SOX9 RNA expression data were obtained from the Oncomine Database. The Curtis dataset was used to analyze survival according to SOX9 expression using Kaplan-Meier survival curves and the statistical significance was determined using the log-rank test.

Results: We performed a screen using specific siRNA targeting the 6 SOX transcription factors (SOX4, 6, 8, 9, 10 and 11) that are high-expressed in TNBC, and measured the effect of loss on SOX expression on TNBC cell growth and invasion. SOX4, 6, 8, 9, and 10-KD using siRNA caused decreased cell proliferation (by 30% or more) of MDA231 and MDA468 TNBC cells, but not of MCF7 and T47D cell lines (non-TNBC). SOX11 or 17-KD had no effect on breast cancer growth. SOX9-KD and SOX9-KO decreased cell migration and invasion of MDA231 and MDA468 cells. Reduced expression of SOX9 also inhibited the in vivo growth and metastasis of MDA MB-231 and LM2 cells in mice. In contrast, overexpression of SOX9 in MCF7 and ZR75-1 cells increased cell migration and invasion. Our studies also demonstrated that loss of SOX9 induced a G1 to S cell cycle arrest and apoptosis. In addition, high expression of SOX9 was correlated with worse overall survival and 5-year disease-free survival in patients with TNBC breast cancer.

Conclusion: Our results demonstrate that the SOX9 acts as an essential molecule regulating TNBC growth and invasion. In the future, it may be possible to target SOX9 and its downstream genes to treat TNBC and prevent its metastasis.

Grant Support: These studies were supported by a Susan G. Komen for the Cure Promise Grant (PB).

Citation Format: Yanxia Ma, Jonathan Shepherd, Dekuang Zhao, Lakshmi Bollu, Jamal Hill, Yun Zhang, Abhijit Mazumdar, Powel H. Brown. SOX9 is a critical regulator of triple-negative breast cancer growth and invasion [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 3347.