Cholinergic receptor muscarinic 3 (CHRM3) contributes to breast cancer tumorigenesis through angiogenesis regulation

Abstract

Objectives: Cholinergic muscarinic receptors are widely distributed in different human organs with various biological functionals such as synaptic transmission. Accumulating evidence revealed the overexpression of these receptors especially cholinergic receptor muscarinic 3 (CHRM3) with multiple roles in cancer behaviors such as proliferation, apoptosis, angiogenic phenotypes and even epithelial-mesenchymal transition (EMT) through various signaling pathways. However, its roles in breast cancer and angiogenesis still remain elusive. Methods: Gene expression and patient survival data were from the TCGA and the GTEx projects database. Cells were transfected with scrambled shRNA or CHRM3 shRNA to knockdown gene expression. pcDNA1.3 empty expression vector or CHRM3 expressing vector was used for ectopic expression in the cells. Cell proliferation was examined in different treatment groups by MTT assay and further subjected to cell cycle and apoptosis assay. Expression levels of CHRM3 in paired breast cancer tissues and different cell lines as well as angiogenesis-related markers in cells with different treatments were investigated by qRT-PCR. Results: CHRM3 expressions were significantly upregulated in triple negative breast cancer (TNBC) subtype, but not other subtypes of breast cancer, and was consistent with the data in TCGA database. Further investigation in different breast cancer cells revealed that CHRM3 expressions were higher in MDA-MB-231 and MDA-MB-468, which are both TNBC cell lines. After treatment with CHRM3-specific inhibitor, 4-DAMP, cell viability was significantly decreased in both MDA-MB-231 and MDA-MB-468 cells. G1-phase arrest and decreased S-phase cell populations and early apoptosis were increased upon 4-DAMP. Moreover, angiogenic markers such as ANG1 and VEGFD were significantly decreased upon CHRM3 knockdown in breast cancer cells or human umbilical vein endothelial cells (HUVEC). Gene correlation analysis from TCGA database indicated a significant positive correlation between CHRM3 and ANG1. Furthermore, CHRM3 shRNA or 4-DAMP treatment inhibited cell proliferation in both MDA-MB-231 and HUVEC. Conclusions: We found that CHRM3 is upregulated in TNBC tissues and cell lines. CHRM3 confers tumor oncogenic roles, and for the first time, we identified a potential relationship between CHRM3 and angiogenesis in breast cancer. Further experiments are warranted to explore the underlying mechanism of CHRM3 in regulating angiogenesis.