Abstract 2411: CEACAM1 is a Critical SOX9-regulated gene that suppresses TNBC growth

Abstract

Abstract Background: Triple-negative breast cancers (TNBCs), which lack expression of the estrogen and progesterone receptors and the HER2 protein, are typically treated with chemotherapy and have a poor prognosis. Our previous studies have demonstrated that the SOX9 transcription factor is highly expressed in TNBC and is essential for survival and metastasis of these aggressive breast cancers. Hypothesis: CEACAM1 is a SOX9 regulated gene that regulates TNBC growth and metastasis. Material and Methods: RNA-Seq analysis was used to identify SOX9 downstream targets in MDA MB-231, MDA MB-468 or MCF-7 cells, with support from MD Anderson's genomic biostatistics core. Cells were treated with or without siRNAs or overexpression plasmids to knock down or over-express target genes. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. CHIP assay was used to examine direct binding of SOX9 protein on promoter of CEACAM1 gene. Several breast cancer datasets were used to analyze CEACAM1 expression in TNBCs and non-TNBCs, and the association between SOX9 and CEACAM1 expression. 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: Upon SOX9 knockdown, 340 genes and 843 genes levels were significantly changed in MDA MB-231 and MDA MB-468 respectively. Upon SOX9 overexpression, 250 genes were regulated in MCF-7 cells. There were 38 common genes regulated by SOX9 in MDA MBA-231, MDA MB-468 cells and MCF-7 cells. Among 38 common genes, 5 genes showed positive correlation with SOX9 level (MIOX, LAMB2P1, SLCO4C1, RGCC, and PRTN3 genes). The most highly up-regulated genes upon SOX9 knockdown in TNBC cells were CEACAM1, PRSS35, CALB1, and SLC6A19. We then investigated how these SOX9 downstream genes affected TNBC cell growth in vitro. Knockdown of RGCC, PRTN3, or SLCO4C1 by siRNA reduced cell growth in TNBC cells, but had minimal effect on the growth of MCF-7 cells. Loss of CEACAM1 promoted the growth of MDA MBA-231 and MDA MB-468 cells, while overexpression of CEACAM1 decreased cell growth in TNBC cells. Bioinformatic analysis of the Curtis dataset demonstrated that TNBCs had lower CEACAM1 expression as compared to non-TNBCs. CEACAM1 was inversely correlated with SOX9 expression in the Curtis and TCGA breast cancer datasets. CHIP assays were then performed that showed that the CEACAM1 gene promoter is bound by the SOX9 transcript factor in MDA MD-231 cells. Conclusion: Our results demonstrate that CEACAM1 is a SOX9-regulated gene and is a suppressor of TNBC growth. Grant Support: These studies were supported by a Breast Cancer Research Foundation (BCRF) grant (PB). Citation Format: Yanxia Ma, William M. Tahaney, Ganiraju C. Manyam, Wenyi Wang, Abhijit Mazumdar, Powel H. Brown. CEACAM1 is a Critical SOX9-regulated gene that suppresses TNBC growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2411.