Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer.

Abstract

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is an important susceptibility locus for nicotine addiction and lung cancer. Depletion of CHRNA5 has been associated with reduced cell viability, increased apoptosis and alterations in cellular motility in different cancers yet not in breast cancer. Herein we first showed the expression of CHRNA5 was variable and positively correlated with the fraction of total genomic alterations in breast cancer cell lines and tumors indicating its potential role in DNA damage response (DDR). Next, we demonstrated that silencing of CHRNA5 expression in MCF7 breast cancer cell line by RNAi affected expression of genes involved in cytoskeleton, TP53 signaling, DNA synthesis and repair, cell cycle, and apoptosis. The transcription profile of CHRNA5 depleted MCF7 cells showed a significant positive correlation with that of A549 lung cancer cell line while exhibiting a negative association with the CHRNA5 co-expression profile obtained from Cancer Cell Line Encylopedia (CCLE). Moreover, it exhibited high similarities with published MCF7 expression profiles obtained from exposure to TP53 inducer nutlin-3a and topoisomerase inhibitors. We then demonstrated that CHRNA5 siRNA treatment reduced cell viability and DNA synthesis indicating G1 arrest while it significantly increased apoptotic sub-G1 cell population. Accordingly, we observed lower levels of phosphorylated RB (Ser807/811) and an increased BAX/BCL2 ratio in RNAi treated MCF7 cells. We also showed that CHRNA5 RNAi transcriptome correlated negatively with DDR relevant gene expression profile in breast cancer gene expression datasets while the coexposure to topoisomerase inhibitors in the presence of CHRNA5 RNAi enhanced chemosensitivity potentially due to reduced DDR. CHRNA5 RNAi consistently lowered total CHEK1 mRNA and protein levels as well as phosphorylated CHEK1 (Ser345) in MCF7 cells. We also detected a significant positive correlation between the expression levels of CHRNA5 and CHEK1 in CCLE, TCGA and METABRIC breast cancer datasets. Our study suggests CHRNA5 RNAi is associated with cell cycle inhibition, apoptosis as well as reduced DDR and increased drug sensitivity in breast cancer yet future studies are warranted since dose- and cell line-specific differences exist in response to CHRNA5 depletion. Gene expression microarray data can be accessed from GEO database under the accession number GSE89333.