Abstract 2368: SOX9-binding proteins regulate SOX9 activity to control the growth of triple-negative breast cancer cells

Abstract

Abstract Background: Triple-negative breast cancers (TNBCs) are the most aggressive types of breast cancer, which lack the expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). TNBCs have a very poor prognosis and have few targeted therapy options. Therefore, the development of new TNBC treatment strategies is an urgent and unmet clinical need. Our previous studies have demonstrated that knockout of the SOX9 transcription factor decreased TNBC cell growth and tumor metastasis in vivo. Hypothesis: SOX9-binding proteins include upstream activators of this transcription factor which regulate SOX9 activity and control TNBC growth. Material and Methods: Immunoprecipitation (IP) in combination with mass spectrometry (IP-MS) analysis was used to identify SOX9 binding proteins, with support from MD Anderson’s proteomics core. The Mascot Score is a statistical score was used as the reference of targeted protein, and the data was summarized based on the Mascot Score. IP-Western Blotting analysis was used to confirm that the identified proteins bound SOX9 in TNBC cells. RNA-Seq analysis was used to identify SOX9 regulated genes in MDA MBA-231 and MDA MB-468 cells after SOX9 knockdown or in MCF-7 after SOX9 overexpression, with support from MD Anderson’s genomic biostatistics core. We then treated TNBC cells with or without siRNAs of these identified proteins to evaluate their effect on SOX9-regulated gene expression and TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. SOX9 transcriptional activity was measured using a reporter gene activity assay, and qRT-PCR 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 immunoprecipitation in combination with mass spectrometry (IP-MS) analysis, we identified multiple SOX9 binding proteins. Among them, proteins that demonstrated high Mascot Scores include Valosin-Containing Protein (VCP), protein S100A10 (S100A10); Caveolin 1 (CAV1). Knockdown of each of these SOX9-binding proteins (VCP, S100A10, or CAV1) decreased TNBC cell growth in vitro. Using mRNA-Seq analysis, we next identified 38 genes down-regulated by SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Using these genes as markers of SOX9 transcriptional activity, all three of these SOX9-binding proteins regulate SOX9 activity. Thus, VCP, CAV1, and S100A10 all are potential upstream proteins of SOX9 that can be targeted to inhibit TNBC growth. Conclusion: Our results demonstrate that VCP, S100A10, and CAV1, are SOX9-binding proteins that regulate SOX9 activity and control TNBC cell growth. Implications: These proteins represent potential targets for the treatment of TNBCs. Citation Format: Yanxia Ma, David H. Hawke, Ganiraju Manyam, Wenyi Wang, Abhijit Mazumdar, Powel Brown. SOX9-binding proteins regulate SOX9 activity to control the growth of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2368.