CGRRF1 as a novel biomarker of tissue response to metformin in the context of obesity

Abstract

Objective: Obesity-associated hyperestrogenism and hyperinsulinemia contribute significantly to the pathogenesis of endometrial cancer. We recently demonstrated that metformin, a drug long used for treatment of type 2 diabetes, attenuates both insulin- and estrogen-mediated proliferative signaling in the obese rat endometrium. In this study, we sought to identify tissue biomarkers that may prove clinically useful to predict tissue response for both prevention and therapeutic studies. We identified CGRRF1 (cell growth regulator with ring finger domain 1) as a novel metformin-responsive gene and characterized its possible role in endometrial cancer prevention.

Methods: CGRRF1 mRNA expression was evaluated by RT-qPCR in the endometrium of obese and lean rats, and also in normal and malignant human endometrium. CGRRF1 levels were genetically manipulated in endometrial cancer cells, and its effects on proliferation and apoptosis were evaluated by MTT and Western blot.

Results: CGRRF1 is significantly induced by metformin treatment in the obese rat endometrium. In vitro studies demonstrate that overexpression of CGRRF1 inhibits endometrial cancer cell proliferation. Analysis of human endometrial tumors reveals that CGRRF1 expression is significantly lower in hyperplasia, Grade 1, Grade 2, Grade 3, MMMT, and UPSC endometrial tumors compared to normal human endometrium (p<0.05), suggesting that loss of CGRRF1 is associated with the presence of disease.

Conclusion: CGRRF1 represents a novel, reproducible tissue marker of metformin response in the obese endometrium. Furthermore, our preliminary data suggests that up-regulation of CGRRF1 expression may prove clinically useful in the prevention or treatment of endometrial cancer.

Keywords: Estrogen; Insulin resistance; Metformin; Obesity.

Figure 1. Metformin induces CGRRF1 gene expression in vitro and in vivo

(A) CGRRF1 expression in Zucker rat (n=4-6) endometrium was evaluated by RT-PCR assay. * p<0.05 Lean E2 vs. Lean Control; # p<0.001 Obese E2 vs. Obese Control; $ p<0.05 Obese E2 vs. Lean E2; ^ p<0.05 Obese E2+Metformin vs. Obese E2. (B) Direct effects of metformin on CGRRF1 expression in ECC-1 cells were measured by RT-qPCR. * p<0.05 control vs. metformin.

Figure 2. CGRRF1 increases the sensitivity of Ishikawa cells to metformin therapy and modulated endometrial cell proliferation

(A) Ishikawa cells were transfected with a pCMV6/CGRRF1 expression construct (Cg) or control vector (Co) and stable clones were isolated. Cells were then treated with 10 mM metformin for 48 hours, and cell proliferation evaluated by MTT. * p<0.001 control vs. metformin; # p<0.001, Ishikawa (Co) vs. Ishikawa (Cg). (B) ECC-1 cells were transiently transfected with either control or CGRRF1 siRNA. 24 hours following transfection, MTT assays were performed as a measure of cell proliferation. A reduction in CGRRF1 expression is associated with an increased proliferative rate. * p<0.05 control vs. CGRRF1 siRNA.

Figure 3. CGRRF1 prevents cell cycle progression

Stable clones isolated from Ishikawa cells transfected with a pCMV6/CGRRF1 expression construct (Cg) or control vector (Co) were treated with metformin (10mM) for 48 hours, and then processed for cell cycle analysis. Data shown is one representative experiment from three replicated experiments.

Figure 4. Overexpressing CGRRF1 modulates endometrial cancer proliferation

(A) Ishikawa cells were transfected with a pCMV6/CGRRF1 expression construct (Cg) or control vector (Co) and stable clones established. CGRRF1 mRNA level was evaluated by RT-qPCR. (B) Phosphorylation of ribosomal S6 protein (pS6p), a downstream marker of proliferation, was compared in Ishikawa cells transfected with pCMV6 or pCMV6-CGRRF1 by Western blot analysis. CGRRF1 expression inhibits phosphorylation of ribosomal S6 protein (indicating reduced proliferation).

Figure 5. P53 induces CGRRF1 expression

(A) CGRRF1 protein levels were evaluated in HCT116 p53+/+ cells and HCT116 p53-/- cells by Western blot analysis. (B, C) The effect of p53 on CGRRF1 expression was evaluated by RT-qPCR assay. Expression of p53 was evaluated in ECC-1 cells treated with doxorubin (to induce p53) (B) or ECC-1cells treated with p53 siRNA (to silence p53) (C). CGRRF1 expression is significantly induced in doxorubicin-treated ECC-1 cells, and inhibited in p53 siRNA treated cells.

Figure 6. CGRRF1 expression in normal endometrium, endometrial hyperplasia and endometrial tumors

CGRRF1 expression in normal, hyperplasia human endometrium, or endometrial cancer tissue was evaluated by RT-qPCR assay. CGRRF1 was significantly decreased in all cases of early and late endometrial disease as compared with normal endometrium (* p<0.01).