Metformin downregulates the insulin/IGF-I signaling pathway and inhibits different uterine serous carcinoma (USC) cells proliferation and migration in p53-dependent or -independent manners

Abstract

Accumulating epidemiological evidence shows that obesity is associated with an increased risk of several types of adult cancers, including endometrial cancer. Chronic hyperinsulinemia, a typical hallmark of diabetes, is one of the leading factors responsible for the obesity-cancer connection. Numerous cellular and circulating factors are involved in the biochemical chain of events leading from hyperinsulinemia and insulin resistance to increased cancer risk and, eventually, tumor development. Metformin is an oral anti-diabetic drug of the biguanide family used for treatment of type 2 diabetes. Recently, metformin was shown to exhibit anti-proliferative effects in ovarian and Type I endometrial cancer, although the mechanisms responsible for this non-classical metformin action remain unclear. The insulin-like growth factors (IGFs) play a prominent role in cancer biology and their mechanisms of action are tightly interconnected with the insulin signaling pathways. Given the cross-talk between the insulin and IGF signaling pathways, the aim of this study was to examine the hypothesis that the anti-proliferative actions of metformin in uterine serous carcinoma (USC) are potentially mediated via suppression of the IGF-I receptor (IGF-IR) pathway. Our results show that metformin interacts with the IGF pathway, and induces apoptosis and inhibition of proliferation and migration of USC cell lines with both wild type and mutant p53. Taken together, our results suggest that metformin therapy could be a novel and attractive therapeutic approach for human USC, a highly aggressive variant of endometrial cancer.

Figure 1. Effect of metformin on IGF-I-mediated signal transduction and mTOR and Ampk signalling pathway in endometrial cancer cells.

A, Ishikawa, ECC-1, USPC-2 and USPC-1 cells were treated with metformin (10 mM) for 24 h (or left untreated) in the presence or absence of IGF-I (50 ng/ml) during the last 10 min of the incubation period. Whole cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with antibodies against pIGF-IR, TIGF-IR, IR, pAKT, TAKT, pERK1/2, TERK1/2 and actin, followed by incubation with an HRP-conjugated secondary antibody. The figure shows the results of a typical experiment, repeated three times with similar results. B, USPC-2 and USPC-1 cell lines were treated with metformin for 24 h (or left untreated) and/or IGF-I during the last 10 min of the incubation. Whole cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with antibodies against pmTOR, TmTOR, pAmpk, TAmpk, and p85. The figure shows the results of a typical experiment, repeated three times with similar results.

Figure 2. Regulation of IGF-IR and IR promoter activities and transcriptional activators by metformin in USC cells.

USPC-1 and USPC-2 cells were transiently transfected with an IGF-IR promoter-luciferase reporter plasmid, p(-476/+640)LUC (A), or an IR promoter-luciferase reporter construct (B), along with a ß-galactosidase expression plasmid. Promoter activities were expressed as luciferase values normalized for ⇓-galactosidase activity. Results are mean ± SEM (duplicates samples of three independent experiments). *, p<0.05 versus untreated cells; **, p<0.05 versus USPC-2 cells transfected with IGF-IR or IR promoter luciferase constructs. C, Western blot analysis of Sp1, pTen, and p53 in USPC-2 and USPC-1 cells treated with metformin (24 h) and/or IGF-I (10 min). Whole-cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Results are representative of three independent experiments.

Figure 3. Effect of metformin on apoptosis.

A, Western blot analysis of PARP1 in USPC-2 and USPC-1 cells. B, Western blot analysis of caspase 9 in USPC-2 and USPC-1 cells. C, Western blot analysis of caspase 3 in USPC-2 cells. Cells were treated with metformin for 24 h in the presence or absence of IGF-I. Whole-cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Results are representative of three independent experiments.

Figure 4. Effect of metformin on proliferation and cell cycle regulatory proteins in USC cells.

Cells were plated in 24-well plates at a density of 5×10⁴ cells/well for USPC-2 (A) and 3.6×10⁴ cells/well for USPC-1 (B). Cells were incubated in the absence (open bars) or presence (solid bars) of metformin, and proliferation was evaluated at 24, 48 and 72 h by MTT measurements. A value of 100% was given to the cell number at time 0. The bars represent the mean ± S.E.M. of three independent experiments, performed each in triplicate samples; *p<0.05 versus untreated cells. C, Western blot analysis of cyclin D1, p21, Ras, Rb and E2F1 in USPC-2 and USPC-1 cells treated with metformin for 24 h in the absence or presence of IGF-I. Whole-cell lysates (100 µg) were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Results are representative of three independent experiments.

Figure 5. Effect of metformin on cell migration.

Wounds were made on monolayers of USPC-2 (A) and USPC-1 (B) cells grown to 100% confluence. Cells were then incubated in serum-free media containing IGF-I (50 ng/ml), metformin (10 mM), or both, for 48, 72 and 96 h (USPC-2) and for 48 and 72 h (USPC-1). Treated or untreated (control) cells were photographed just after scratch (time 0), and after 48, 72 and 96 h. Results presented here are representative of triplicate independent samples of each cell line. The rate of migration was measured by quantifying the total distance that the cells (as indicated by rulers) moved from the edge of the scratch toward the centre of the scratch. A value of 100% was given to the wound area at time 0. The migration of IGF-I and/or metformin treated samples was compared to wound area at time 0.

Figure 6. Effect of metformin on GSK3ß and Foxo1 expression.

A, Western blot of pGSK3ß and GSK3ß in USPC-2 and USPC-1 cells treated with metformin for 24 h and/or IGF-I. The figure shows the results of a typical experiment repeated three times. B, Western blot analysis of Foxo1 on USPC-2 and USPC-1 cells treated for 24 h with metformin and/or IGF-I. The figure shows the results of a characteristic experiment, repeated three times with similar results.