Abstract P5-11-03: Knockdown of TMEM45A regulates malignant progression of triple-negative breast cancer by inhibiting TGF-β/Smad signaling pathway-mediated EMT

Abstract

Abstract Objectives: The incidence of breast cancer has jumped to first place in the global malignant tumor. As an essential component of the TMEM family, TMEM45A is abnormally highly expressed in some malignant tumors, and plays a role in promoting and regulating the occurrence and development of tumors. This study analyzed the expression of TMEM45A in breast cancer tissues and breast cancer cell lines, and explored the relationship with clinical subtypes. To examine the effects of TMEM45A on TNBC cell proliferation, migration, invasion and other biological functions, and to preliminarily clarify the molecular mechanism of TMEM45A regulating the epithelial-mesenchymal transition of TNBC cell lines. Methods: 1. Download and analyze the TCGA database, analyze the difference in the expression of TMEM45A in normal breast tissue and different subtypes of breast cancer tissue, and detect the protein expression level of TMEM45A in 8 pairs of cancer tissues and adjacent tissues by western blot experiment. 2. Detection of TMEM45A protein expression in normal breast epithelial cell line MCF10A and five breast cancer cell lines (MCF-7, MDA-MB-231, BT-549, MDA-MB-468, SK-BR-3) by western blot experiment. 3. Using small interfering RNA technology to down-regulate the protein expression levels of MDA-MB-231 and BT-549 TNBC cell lines with relatively high TMEM45A expression. The changes in cell proliferation ability were detected by CCK8 assay and clone formation assay, and the changes in cell migration ability and invasion ability were detected by wound healing assay and transwell assay, respectively. 4. After down-regulating TMEM45A in two TNBC cell lines, the expression changes of epithelial cell phenotype marker E-cadherin, mesenchymal phenotype marker N-cadherin, Vimentin, and critical molecules in TGF-β/Smad signal transduction pathway (TGFβ1, Smad2, Smad3, p-Smad2, p-Smad3) were detected by western blot experiment. Results: 1. Based on the TCGA database, the results showed that the expression level of TMEM45A mRNA in breast cancer was significantly higher than in normal breast tissue. The expression level of TMEM45A mRNA in any breast cancer subtype was higher than that in normal breast tissue, and it was the highest in the TNBC subtype. In the results of western blot experiments of clinical breast cancer specimens, the expression of TMEM45A in breast cancer tissues was significantly higher than that in the corresponding adjacent tissues, which further confirmed the reliability of the results of the TCGA database. 2. In normal breast epithelial cell line MCF10A and five breast cancer cell lines, MCF10A has the lowest expression level of TMEM45A protein. Among the five breast cancer cell lines, MDA-MB-231 had the highest expression of TMEM45A, followed by BT-549. 3. CCK8 assay, clone formation assay, wound healing assay and transwell assay showed that down-regulating TMEM45A protein levels in the two TNBC cell lines could significantly reduce cell proliferation, migration and invasion abilities. 4. After down-regulating TMEM45A protein levels in two TNBC cell lines, EMT-related indicators E-cadherin protein expression levels increased, N-cadherin and Vimentin protein expressions decreased, and the expression levels of essential protein molecules Smad2 and Smad3 in the TGF-β/Smad signal transduction pathway did not change significantly, while p-Smad2, p-Smad3 and TGF-β1 protein expressions decreased. Conclusions: TMEM45A is relatively highly expressed in breast cancer tissues and cell lines, especially in the subtype of TNBC. Down-regulation of TMEM45A expression can inhibit the proliferation, migration and invasion of TNBC cell lines. Mechanistically, TMEM45A may reverse EMT by inhibiting the activity of the TGF-β/Smad pathway. Citation Format: Yingkun Xu, Shengchun Liu. Knockdown of TMEM45A regulates malignant progression of triple-negative breast cancer by inhibiting TGF-β/Smad signaling pathway-mediated EMT [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-11-03.