Inhibition of CREB1/CTL4 Pathway Diminishes the Viability, and Restrains the Proliferation, Migration and Invasion of Breast Cancer Cells by Improving Aberrant Choline Metabolism

Abstract

Background: Abnormal Choline (Cho) metabolism is a hallmark of breast cancer (BC). Since the key role of choline transporter-like protein 4 (CTL4) in Cho transport and acetylcholine (ACh) biosynthesis has been reported, this study aimed to investigate additional role and mechanisms of CTL4 in BC. Methods: CTL4 expressions in BC and cAMP-responsive element (CRE)-binding protein-1 (CREB1)-binding motif were analyzed by bioinformatic analysis. After BC cells were transfected with short hairpin RNA against CTL4 (shCTL4) or CREB1-specific small interfering RNA (siCREB1), the CTL4 and CREB1 expressions in BC cells were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and the levels of markers relevant to Cho metabolism were also studied. The binding of CREB1 to the CTL4 promoter was verified by chromatin immunoprecipitation assay. After BC cells were transfected with CTL4 overexpression plasmid or siCREB1, BC cell viability, proliferation, invasion, and migration were assessed by cell counting kit-8, 5ʹ-ethynyl-2ʹ-deoxyuridine (EdU) and Transwell assays. Furthermore, CTL4 protein expression and ACh content in BC cells were determined by western blot assay and high-performance liquid chromatography (HPLC) with enzyme-coupled electrochemical assay. Results: CTL4 was highly expressed in BC and its knockdown diminished cell viability, restrained proliferation, migratory and invasion capabilities, reduced ACh content, and down-regulated the level of markers relevant to Cho metabolism in BC cells (p < 0.01). CREB1 could bind to the CTL4 promoter in BC cells. CREB1 silencing decreased the viability, proliferation, migration, invasiveness, ACh content, and the expression levels of CTL4 and Cho metabolism-relevant markers in BC cells (p < 0.001). Moreover, these aforementioned effects were reversed by CTL4 overexpression (p < 0.001). Conclusions: Inhibition of CREB1-CTL4 cascade attenuated aberrant Cho metabolism, thus restraining the malignant progression of BC cells.