The Hypertension Related Gene G-Protein Coupled Receptor Kinase 4 Contributes to Breast Cancer Proliferation

Abstract

Purpose: Clinical studies have shown that breast cancer risk is increased in hypertensive women. The underlying molecular mechanism remains undetermined. The current study tests our hypothesis that G protein coupled receptor kinase 4 (GRK4) is a molecule that links hypertension and breast cancer. GRK4 plays an important role in regulation of renal sodium excretion. Sustained activation of GRK4 as in the circumstances of single nucleotide polymorphism (SNPs) causes hypertension. Expression of GRK4 in the kidney is regulated by cMyc, an oncogene that is amplified in breast cancer. Methods: Western analysis was used to evaluate GRK4 protein expression in seven breast cancer cell lines. GRK4 gene single nucleotide polymorphism in breast cancer cell lines and in breast cancer cDNA arrays were determined using TaqMan Genotyping qPRC. The function of GRK4 was evaluated in MCF-7 cells with cMyc knock-down and GRK4 re-expression and in MDA-MB-468 cells expressing inducible GRK4 shRNA lentivirus constructs. Nuclei counting and 5-Bromo-2’-deoxy-uridine (BrdU) labeling were used to evaluate cell growth and proliferation. Results: Genotyping of GRK4 SNPs in breast cancer cDNA arrays (n = 94) revealed that the frequency of carrying two hypertension related SNPs A142 V or R65 L is threefold higher in breast cancer patients than in healthy people ( P = 7.53E-11). GRK4 protein is expressed in seven breast cancer cell lines but not the benign mammary epithelial cell line, MCF-10A. Three hypertension related SNPs in the GRK4 gene were identified in the breast cancer cell lines. Except for BT20, all other breast cancer lines have 1-3 GRK4 SNPs of which A142 V occurs in all 6 lines. MDA-MB-468 cells contain homozygous A142 V and R65 L SNPs. Knocking down cMyc in MCF-7 cells significantly reduced the growth rate, which was rescued by re-expression of GRK4. We then generated three stable GRK4 knock-down MDA-MB-468 lines using inducible lentiviral shRNA vectors. Doxycycline (Dox) induced GRK4 silencing significantly reduced GRK4 mRNA and protein levels, growth rates, and proliferation. As a marker of cell proliferation, the percentage of BrdU-labeled cells decreased from 45 ± 3% in the cells without Dox to 32 ± 5% in the cells treated with 0.1 µg/mL Dox. Conclusions: GRK4 acts as an independent proliferation promotor in breast cancer. Our results suggest that targeted inhibition of GRK4 could be a new therapy for both hypertension and breast cancer.