Correlation of GREB1 mRNA with protein expression in breast cancer: validation of a novel GREB1 monoclonal antibody

Abstract

Studies of gene regulated by estrogen in breast cancer 1 (GREB1) have focused on mRNA levels with limited evidence about GREB1 protein expression in normal and breast cancer cells. A monoclonal antibody that recognizes GREB1 protein in breast tissues could be applied to correlate protein expression with established mRNA expression data. A hybridoma expressing a murine monoclonal antibody targeting a 119 amino acid peptide specific to human GREB1 was generated. The novel monoclonal GREB1 antibody (GREB1ab) was validated for use in Western blotting as well as immunohistochemical (IHC) applications. GREB1ab detects a 216 kDa protein corresponding to GREB1 in estrogen receptor alpha (ERalpha+) breast cancer cells as well as ERalpha- breast cancer cells transduced with a GREB1 expression vector. GREB1ab specificity was verified using an ERalpha antagonist to prevent GREB1 induction as well as a silencing siRNA targeting GREB1 mRNA. GREB1ab was further validated for detection of GREB1 by IHC in breast cancer cell lines and breast tissue microarrays (TMA). ERalpha+ cell lines were observed to express GREB1 while ERalpha- cell lines did not express detectable levels of the protein. Using breast cancer tissue whole sections, IHC with the GREB1ab identified protein expression in ERalpha+ breast cancer tissue as well as normal breast tissue, with little GREB1 expression in ERalpha- breast cancer tissue. Furthermore, these data indicate that GREB1 mRNA expression correlates well with protein expression. The novel 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.

The Orphan Nuclear Receptor LRH-1 and ERα Activate GREB1 Expression to Induce Breast Cancer Cell Proliferation

“…The role of GREB1 in cell proliferation was demonstrated with the suppression of GREB1 with siRNA, causing a significant reduction in cell proliferation [26]. Recently a monoclonal antibody for GREB1 was created, allowing the detection of a 216 kDa protein whose expression positively correlated to ERα expression in breast cancer cell lines and tumor samples, as well as to GREB1 mRNA transcript levels [31]. While expression of GREB1 protein appears predominantly nuclear, some cytoplasmic staining was also observed.…”

“…While expression of GREB1 protein appears predominantly nuclear, some cytoplasmic staining was also observed. GREB1 protein was expressed in tumor epithelial cells and CAFs [31]. As commercially available antibodies report variations in size of detected band in western blots and variability in cell localisation, the current study focussed on transcript expression analysis.…”

GREB1 (growth regulation by estrogen in breast cancer 1)

“…Its regulation is not only correlated with the presence of a subset of nuclear receptors such as ERα (Deschênes et al, 2007;Hnatyszyn et al, 2010;Lin et al, 2004;Pellegrini et al, 2012), androgen receptor (AR) (Rae et al, 2006) and Liver receptor Homolog 1 (LRH-1) (Chand et al, 2012) but also depends on their activation. In breast cancers, GREB1 protein is detected in ERalpha+ but not ERalpha-negative breast tumour tissue (Hnatyszyn et al, 2010). In addition GREB1, regulated by androgens is expressed in proliferating prostatic tissue and prostate cancer (Rae et al, 2006).…”

“…GREB1 protein expression is predominantly nuclear with some cytoplasmic appearance (Hnatyszyn et al, 2010).…”

“…Subsequently, GREB-1 is now a well characterised estrogen responsive gene used to identify ERα activity (Cai et al, 2011;Chand et al, 2012;Gupta et al, 2012;Liu et al, 2012;Rae et al, 2005;Sun et al, 2007;Woodfield et al, 2010). Hnatyszyn, et al developed a novel GREB-1 antibody which was used to detect GREB-1 protein expression in ERα +ve breast cancer cells and tissue (Hnatyszyn et al, 2010). This positive correlation of GREB1 expression with ERα expression is validated in clinical cohorts (Ghosh et al, 2000).…”