High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer

<div><p>Objectives</p><p>STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin.</p><p>Methods</p><p>In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the <i>in vivo</i> efficacy of metformin.</p><p>Results</p><p>Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. <i>In vitro</i>, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. <i>In vivo</i>, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins.</p><p>Conclusions</p><p>These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.</p></div

Xenograft endometrial tumor weight and expression of STAT3 in mice treated with metformin.

<p>A xenograft study was done in which nude mice were injected with 1 x 10⁶ Ishikawa endometrial cancer cells subcutaneously in the right flank. After tumors were at least 3–5 mm in diameter, treatment with control, metformin 100 mg/kg, or metformin 200 mg/kg was started. Mice were sacrificed after 4 weeks of treatment. <b>A</b>. Tumor weight (in grams) from the mice with the 3 largest tumors in each group. <b>B</b>. Average body weight (g) of the mice in each group at conclusion of the study. <b>C</b>. Western blot results of STAT3 and associated proteins after treatment with control, 100 mg/kg, or 200 mg/kg of metformin.</p

Expression of apoptosis and cell proliferation-related proteins in metformin-treated grade 1 endometrial cancer cells overexpressing STAT3.

<p>Ishikawa endometrial cancer cells were transfected with a STAT3-overexpressing plasmid. <b>A</b>. Western blot confirming overexpression of pSTAT3 ser727 and total STAT3. <b>B</b>. Western blot of proteins involved in apoptosis or cell proliferation in control or STAT3-overexpressing Ishikawa cells treated with control or 20 mM metformin in high-glucose medium for 48h. (C: control, TR: transfection reagent only, OE: transfected with STAT3-overexpressing plasmid, Ctrl: control, Met: metformin).</p

Metformin inhibits STAT3, its regulatory proteins and upregulated apoptosis-related proteins, in grade 1 endometrial cancer cells.

<p><b>A</b>. Western blot of STAT3 and its regulatory proteins in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h in high-glucose conditions. <b>B</b>. qPCR of STAT3 and some of its regulatory genes in Ishikawa cells after treatment with control, 10 mM, or 20 mM metformin for 48h. Groups significantly different than control (p<0.05) are indicated with an asterisk (*). <b>C</b>. Metformin did not affects pSTAT3 and STAT3 in normal glucose conditions.</p

Metformin inhibits grade 1 endometrial cancer cell proliferation, survival, migration, and induces apoptosis.

<p><b>A</b>. Sulforhodamine B (SRB) assay measured Ishikawa cell proliferation with increasing concentrations of metformin after 24h or 48h, in high-glucose media. <b>B</b>. Proportion of Ishikawa cells surviving (compared to control) after treatment with 10 mM or 20 mM of metformin. <b>C</b>. Cell migration assay, with Ishikawa cells subjected to control, 10 mM, or 20 mM metformin for 24h. Control (0h) is also shown. <b>D</b>. Quantification of % wound closure; the 10 mM and 20 mM treatment groups were each significantly different than control (p<0.05; noted with asterisk [*]). <b>E</b>. Western blot of apoptosis-related protein expression in Ishikawa cells after treatment with control, 10 mM, or 20 mM for 48h.</p

Degradation of pSTAT3 ser727 in grade 1 endometrial cancer cells treated with meformin.

<p>After Ishikawa cells were treated with control, 10 mM, or 20 mM metformin in high glucose media for 48h, samples were subjected to ubiquitin assay. The ubiquitinated proteins were subjected to immunoblot for pSTAT3 Ser727 and blotted with ubiquitin antibody. A ubiquitination smear of pSTAT3 Ser727 is seen in the metformin treated ishikawa cells under proteasomal inhibition using MG-132.</p

Relative expression of STAT3 in grade 1 endometrial cancer cells with respect to glucose concentration.

<p><b>A & B</b>. Ishikawa cells were cultured in low, normal, or high glucose media for 24 hours. Relative expression of STAT3 and its regulatory genes were calculated by normalizing the values of normal glucose to 1. <b>C</b>. STAT3-associated microRNA was then evaluated with qPCR. Groups significantly different than control (p<0.05) are indicated with an asterisk (*).</p

Associations between SNPs in 9p22.2 with ovarian cancer risk for <i>BRCA1</i> and <i>BRCA2</i> mutation carriers.

<p>In each plot, the purple diamond corresponds to the strongest associated SNP and the colour code indicates the linkage disequilibrium with respect to this variant. Horizontal lines indicate the -log₁₀ p-value such that the SNPs above the line are the potential causal ones. This set was defined based on a likelihood ratio for a particular SNP as being less or equal than 100, relative to the most likely variant and r²>0.1. (A) <i>BRCA1</i> mutation carriers, (B) <i>BRCA2</i> mutation carriers.</p

Pairwise correlations (r²) between selected SNPs.

<p>SNPs correspond to: rs10124837, the strongest associated in <i>BRCA1</i>; rs62543583, the strongest associated in <i>BRCA2</i> mutation carriers; rs7046326, the strongest associated in <i>BRCA1/2</i> meta-analysis; rs3814113, was the strongest associated variant in the initial GWAS analysis.</p

Genomic features surrounding the 9p22.2 locus.

<p>Illustration of the genomic region (chr9:16,839,835–16,924,468) encompassing peaks (shaded areas) containing candidate causal variants associated with ovarian cancer risk in <i>BRCA1</i> and <i>BRCA2</i> mutation carriers. Epigenomic data from Coetzee et al., (2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158801#pone.0158801.ref020" target="_blank">20</a>] representing potential regulatory elements in ovarian cells (iOSE4 and iOSE11) and fallopian tube (FTSEC33) cells derived from formaldehyde assisted identification of regulatory elements sequencing (FAIRE-seq) and histone modification ChIP-seq are shown as black bars. Variants which overlap one of these features are coloured red. Data from the ENCODE project including histone modification ChIP-seq for three modifications (H3K4me1, H3K4me3, and H3K27ac) are shown as coloured histograms, as well as DNaseI hypersensitive site mapping and transcription factor ChIP-seq. The positions of all common SNPs from dbSNP build 142 are shown in the lowest track.</p