Metformin Promotes Anti-tumor Biomarkers in Human Endometrial Cancer Cells

Abstract

Metformin (MET) is increasingly implicated in reducing the incidence of multiple cancer types in patients with diabetes. However, similar effects of MET in non-diabetic women with endometrial cancer (EC) remain unknown. In a pilot study, obese non-diabetic women diagnosed with type 1, grade 1/2 EC, and consenting to participate were randomly assigned to receive MET or no MET (control (CON)) during the pre-surgical window between diagnosis and hysterectomy. Endometrial tumors obtained at surgery (MET, n = 4; CON, n = 4) were analyzed for proliferation (Ki67), apoptosis (TUNEL), and nuclear expression of ERα, PGR, PTEN, and KLF9 proteins in tumor glandular epithelial (GE) and stromal (ST) cells. The percentages of immunopositive cells for PGR and for KLF9 in GE and for PTEN in ST were higher while those for ERα in GE but not ST were lower, in tumors of MET vs. CON patients. The numbers of Ki67-and TUNEL-positive cells in tumor GE and ST did not differ between groups. In human Ishikawa endometrial cancer cells, MET treatment (60 μM) decreased cell numbers and elicited distinct temporal changes in ESR1, KLF9, PGR, PGR-B, KLF4, DKK1, and other tumor biomarker mRNA levels. In the context of reduced KLF9 expression (by siRNA targeting), MET rapidly amplified PGR, PGR-B, and KLF4 transcript levels. Our findings suggest that MET acts directly in EC cells to modify steroid receptor expression and signaling network and may constitute a preventative strategy against EC in high-risk non-diabetic women.

Metformin regulation of progesterone receptor isoform‑B expression in human endometrial cancer cells is glucose‑dependent

“…The in vivo relevance of PGR signaling as a MET target is supported by our previous study showing the induction of total PGR protein levels in the endometrial tumors of non-diabetic patients with EC following short-term pre-surgical MET treatment (13). The present study reports the novel finding that changes in glucose conditions modify the effects of MET on PGR signaling.…”

“…The Ishikawa human endometrial epithelial carcinoma cell line (a gift from Dr Bruce Lessey, Greenville Health System; Prisma Health) was authenticated and propagated as previously described ( 13 ). Cells were initially grown in Minimal Essential Media (MEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and containing phenol red and 1% antibiotic-antimycotic solution (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified incubator (5% CO 2 /95% air) at 37°C.…”

“…MET-induced changes in total PGR and PGR-B transcript levels are dependent on glucose. We previously showed that MET inhibited the expression of the antitumor biomarker PGR in EC cells in vivo (protein) and in vitro (transcript) (13). Transcript levels of total PGR and its antiproliferative isoform PGR-B were evaluated in Ishikawa cells in response to 100 µM MET, following a 24-h pre-treatment with E2.…”

“…In a previous study, it was shown that the short-term treatment of non-diabetic and obese women with MET during the pre-surgical window between diagnosis and hysterectomy promoted the expression of antitumor biomarkers; this effect was recapitulated in Ishikawa human endometrial cancer cells treated with a physiological dose of MET under normal glucose conditions ( 13 ). While these results support the ability of MET to reduce EC risk and development, meta-analyses of current clinical data indicate conflicting MET outcomes, with some showing no effect ( 14 ) and others showing inhibitory effects ( 15 ).…”

Therapeutic effect of metformin in the treatment of endometrial cancer (Review)

“…Also, the results of an in vitro study using ECC-1 and Ishikawa cells demonstrated that metformin reduced cell proliferation and increased AMPK activation in a dose-dependent manner, and indicated that the mTOR signaling pathway inhibition contributed to these effects (48). Metformin has been found to be functionally similar to an mTOR inhibitor, and the PTEN signaling pathway, which is tightly associated with the carcinogenesis of EC, may also be a target of metformin (49). Experiments in mice demonstrated a 50% reduction in the weight of EC xenografts following treatment with metformin, mediated via inhibition of the LKB1-AMPK-mTOR signaling pathway (50).…”