Abstract 1303: PRTN3, RGCC and SLCO4C1 are critical SOX9-regulated genes that control TNBC growth
Authors: Ma Yanxia , Tahaney William M. , Qian Jing , Hill Jamal , Zhan Yun , Mazumdar Abhijit , Brown Powel H. 
Published: September 2020

Background: Breast cancer is the most common cancer in women, and about 12% U.S. women will develop invasive breast cancer over the lifetime. Triple-negative breast cancers (TNBCs) which lack expression of the estrogen and progesterone receptors and the HER2 protein.have a poor prognosis and are typically treated with chemotherapy. Our previous studies have demonstrated that SOX9 is essential for survival and metastasis of TNBCs via suppressing apoptosis and promoting epidermal to mesenchymal transition (EMT).

Hypothesis: SOX9-regulated proteins control TNBC growth, and represent promising targets for the treatment of these potentially lethal breast cancers.

Material and Methods: Several breast cancer datasets were used to analyze the association between overall survival and SOX9 expression. A doxycycline-inducible Cas9-CRISPR knockout or siRNA knockdown system was used to inhibit SOX9 in cells. RNA-Seq analysis was used to identify SOX9 downstream targets in TNBC cells (MDA MBA-232 and MDA MB-468 with SOX9 knockdown by siRNA transfection) or non-TNBC cells (MCF-7 with SOX9 overexpression by transfection of SOX9-expression plasmid), with support from MD Anderson's genomic biostatistics core. After identification of SOX9 downstream genes, we treated TNBC cells with or without siRNAs of these identified genes to evaluate their effect on 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. Significant genes are defined by using a false discovery rate (FDR) cut-off of 0.01 and log2 fold change of 2. Significant common genes are defined by using a FDR cut-off of 0.05 and log2 fold change of 1 among the cell lines. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student's t-test unless otherwise indicated.

Results: TNBCs and late stage breast cancers showed higher SOX9 expression compared to non-TNBCs and early stage breast cancers. Using SOX9 inhibited cells, we demonstrated that SOX9 is critical for TNBC cell growth in vitro and in vivo. Using mRNA-seq analysis, we identified downstream genes regulated by SOX9 in breast cancer. 340 genes and 843 genes were suppressed in MDA MB-231 and MDA MB-468 respectively upon SOX9 knockdown, while 250 genes were upregulated in MCF-7 cells upon SOX9 overexpression. Between MDA MB-231 and MDA MB-468 cell line, there were 301 common genes were down-regulated upon SOX9 knockdown. 38 genes were down-regulated upon SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Among them, the top 10 genes that show largest difference between TNBCs and MCF-7 cells are MIOX, LAMB2P1, SLCO4C1, RGCC, PRTN3, LINC00173, UCN, LOC100129, VPREB3 and CTF1 genes. We then investigated the effect of these SOX9 downstream targets on cell growth in vitro. Loss of RGCC, PRTN3, and SLCO4C1 reduced cell growth in MDA MBA-231, and MDA MB-468, while having minimal effect on the growth of MCF-7 cells.

Conclusion: Our results demostrate that the SOX9-regulated genes RGCC, PRTN3, and SLC04C1 are required for TNBC growth. These genes are critical regulators of TNBC growth and are potential novel targets for the treatment of these aggressive breast cancers.

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

Citation Format: Yanxia Ma, William M. Tahaney, Jing Qian, Jamal Hill, Yun Zhan, Abhijit Mazumdar, Powel H. Brown. PRTN3, RGCC and SLCO4C1 are critical SOX9-regulated genes that control TNBC growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1303.