Development of a Novel GREB1 Monoclonal Antibody and Applications in Breast Cancer Research.

Abstract

It has been well established that GREB1 mRNA expression is induced by estrogen in breast cancers and that GREB1 is critically involved in the estrogen-induced growth of breast cancer. To date, all studies of GREB1 have focused on mRNA levels using PCR and array technologies with very little known about GREB1 protein expression in normal and breast cancer cells. The lack of a specific antibody to GREB1 has inhibited protein-based investigations in target cells. Our collaborative research group has generated a novel monoclonal GREB1 antibody (GREB1ab) for research use in Western blotting as well as immunohistochemical (IHC) applications.Our team created a hybridoma expressing a murine monoclonal antibody against a 119 amino acid peptide specific to human GREB1. By Western Blot Analysis, GREB1ab detects a 216 kD protein corresponding to GREB1a in ER+ breast cancer cells expressing GREB1 as well as cells transfected with a GREB1a expression plasmid. GREB1ab specificity was verified using ICI 182,780, an estrogen receptor antagonist to prevent GREB1 induction, as well as silencing siRNA targeting GREB1 mRNA expression. GREB1 protein expression was reduced in MCF-7 cells treated with estrogen plus ICI 182,780 compared with that of estrogen treatment. There was no detectable GREB1 protein expression when GREB1 mRNA is silenced by siRNA at 48 hours. In a time course study, inhibition of GREB1 protein occurred as early as 24 hours and lasted up to 72 hours. Thus, by Western Blot Analysis, the monoclonal GREB1ab is specific for GREB1 and can be employed to detect changes in GREB1 expression in breast cancer cell lines under various experimental conditions.GREB1ab was validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). IHC staining with GREB1ab revealed GREB1 was strongly expressed in the ERα-positive breast cancer cell line, MCF-7, with weak GREB1 expression in ERα-negative MDA-231 cells. A panel of breast cancer cell lines was screened for endogenous expression of GREB1 protein in a TMA format using IHC. As expected, ER-positive cell lines (n=5) were observed to express GREB1 while ER-negative cell lines (n=11) did not express detectable levels. Using breast cancer tissue whole sections, IHC with the GREB1ab indicated protein expression in ERα positive breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERα negative breast cancer tissue.The monoclonal GREB1ab is specific for GREB1 protein. This antibody will serve as a tool for investigations focused on the expression, distribution and function of GREB1 in normal breast and breast cancer tissues. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2126.