Role of PDZ Domain-Containing 1 (PDZK1) in MCF-7 Breast Cancer Cells.

Abstract

Breast cancer is the most common cancer and second leading cause of cancer death in women in the U.S. Approximately 70% of the breast cancers are estrogen receptor (ER) positive at the time of presentation. Estrogen induced genes are either considered as markers for diagnosis or candidate oncogenes of breast cancer. PDZ domain-containing 1 (PDZK1) is expressed in ER positive primary breast cancers and PDZK1 expression is induced by estrogen in ER positive MCF-7 breast cancer cells. Chromosome 1 was reported to be involved in the anomalies of 50-60% of breast cancer. PDZK1 gene is localized in chromosome 1q21 and shows significant overexpression correlated to copy number increase in breast cancer cells. PDZK1 is involved in the maintenance of endothelial cell monolayer integrity, stabilization of scavenger receptor class B, type I (SR-BI) in the liver, and lipid metabolism. SR-BI is a physiological receptor for high density lipoprotein (HDL) and HDL was reported to increase MCF-7 breast cancer cell proliferation. However, the role of PDZK1, its signaling pathway after estrogen stimulation, and its interaction with SR-BI in breast cancer cells are not known. The objectives of this study were to determine whether 1) estrogen induced PDZK1 expression is mediated through ER, 2) PDZK1 binds to Src kinase, 3) PDZK1 expression affects cholesterol ester uptake from HDL by its interaction with SR-BI, and 4) PDZK1 binding proteins are induced by estrogen in the MCF-7 breast cancer cells. MCF-7 breast cancer cells were cultured with 1 nM estradiol-17β (E2). PDZK1 mRNA and protein expression was determined by real-time PCR and western blotting, respectively. Knockdown of ERα and ERβ was performed by specific siRNA. PDZK1 binding to Src kinase was determined by immunoprecipitation followed by western blotting. Uptake of cholesterol ester was determined by flowcytometer and by fluorescence microscope after incubating cells with fluorescent 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled HDL. Expression of PDZK1 binding proteins of ATP-binding cassette, subfamily C, member 2 (ABCC2) and PDZK1 interacting protein 1 (PDZK1-IP1) mRNAs was determined by real-time PCR. PDZK1 mRNA and protein expression was detectable after 12h and 24h of 1 nM E2 treatment, respectively. Knockdown of ERα by siRNA reduced E2 stimulated PDZK1 expression to the basal level. Knockdown of ERβ by siRNA reduced PDZK1 expression less than that of ERα. Immunoprecipitation by Src antibody showed that Src binds to PDZK1 and ERα. Fluorescence detected after incubation with DiI-HDL in the presence of E2 stimulation was reduced by PDZK1 or SR-BI siRNA. Though E2 induced PDZK1 expression, but it did not stimulate the expression of ABCC2 or PDZK1-IP1 mRNA. In conclusion, estrogen induced PDZK1 expression is mediated by ER in ER positive MCF-7 breast cancer cells. PDZK1 may bind phosphorylated Src after estrogen stimulated ER binds to Src, since PDZK1 protein was induced 24h after estrogen stimulation. This reaction may occur on the cell membrane. Estrogen treatment may enhance cholesterol ester uptake from circulating HDL by SR-BI and it may stimulate cell proliferation. Since PDZK1 binding proteins of ABCC2 and PDZK1-IP1 were not induced by estrogen and therefore may not participate as scaffolding proteins in the signaling of PDZK1. This pathway may be targeted as a new therapeutic intervention blocking the effect of estrogen on cell proliferation. (poster)