Abstract 2618: SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes
Authors: Ma Yanxia , Bollu Lakshmi , Hill Jamal , Zhang Yun , Tahaney William M. , Mazumdar Abhijit , Brown Powel H. 
Published: August 2019

Background: There are few effective targeted therapies available for triple-negative breast cancers (TNBCs), the most aggressive form of breast cancer. A better understanding of the critical molecular regulators is necessary to develop effective targeted therapies for TNBCs. Our previous study focused on SOX9 transcription factor and determined that SOX9 is highly expressed in TNBCs which is required for TNBC cell survival and metastasis.

Hypothesis: SOX9 controls TNBC cell growth and metastasis via directly regulating cell apoptosis and epithelial-mesenchymal transition (EMT) genes.

Material and Methods: The breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. Cell growth was measured using an automated cell counting assay. Cell migration and invasion were detected by transwell migration and invasion assays. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. Xenograft mice model was established to evaluate the effect of SOX9 on TNBC cell growth in vivo. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. An EMT PCR array and the CHIP-seq enrichment analysis based on published SOX9-CHIP-seq datasets were performed to identify effect of SOX9 on downstream pathways. SOX9 binding peaks in gene promoter region were determined and confirmed by SOX9-CHIP-seq datasets and CHIP assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student’s t-test unless otherwise indicated.

Results: Using available gene profiling datasets, we found that high expression of SOX9 was correlated with worse overall survival in Boersma breast dataset, but not in other datasets. Late stage breast cancers showed higher SOX9 expression compared to early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell survival and invasion in vitro and in vivo. To identify the molecular mechanisms of these functions, we used CHIP-seq data and CHIP assays to demonstrate that SOX9 directly binds to the promoter of several extrinsic apoptosis-inducing factors (such as fadd and tnfrsf10b) and represses their expressions. Loss of SOX9 caused up-regulation of the expression of these genes, consistent with the induction of apoptosis. We then investigated the effect of SOX9 on EMT-regulating genes. Loss of SOX9 induced down-regulation of most EMT genes. Using CHIP assays, we demonstrated that SOX9 directly binds the promoters of several EMT genes (such as vim and ctnnb1) and increases the expression of these genes.

Conclusion: Our results demonstrate that by directly binding and regulating cell apoptosis and EMT gene expression, SOX9 controls TNBC survival and induces EMT resulting in aggressive and potentially lethal tumors.

Grant Support: These studies were supported by a Breast Cancer Research Fund (BCRF) grant (PB).

Citation Format: Yanxia Ma, Lakshmi Bollu, Jamal Hill, Yun Zhang, William M. Tahaney, Abhijit Mazumdar, Powel H. Brown. SOX9 controls TNBC growth and metastasis via regulating the expression of apoptosis and EMT genes [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 2618.