Metformin Exhibits Anticancer Effects Through Inhibition Of The Sonic Hedgehog Signaling Pathway In Breast Cancer

[Research background and purpose]Metformin is an oral biguanide agent widely used for the treatment of type 2 diabetes mellitus. Recently, several studies have indicated that metformin could lower the risk of developing several cancers, furthermore, some in vitro studies and animal experiments showed that metformin can inhibit cancer cell proliferation and tumor growth in animal models. However, the mechanisms underlying this effect remain incompletely understood. Here, we investigate the effectiveness of metformin in inhibiting the proliferation, migration, invasion, and sternness of breast cancer cells, and reveal a role for the Sonic hedgehog pathway (Shh) in mediating these effects. Moreover, we further explore an association between the Shh signaling pathway and AMPK.[Methods]The expression of Shh, Smo, Ptc and Gli1 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot in MDA-MB-231, MCF-7 and BT549 cells treated with metformin. Cells were exposed to rhShh (1μg/mL), metformin (3mM), or a combination of the two, we investigated the effect of Shh signaling on the proliferation, migration, invasion, and sternness in breast cancer cells treated with metformin using cell proliferation assays, wound scratch migration assay, invasion assays, mammosphere culture, and flow cytometry. MDA-MB-231 cells expressing green fluorescent protein (GFP) were injected subcutaneously into the abdominal mammary fat pads of BALB/c-nu mice, treatment was initiated with intra-tumoral injections of rhShh alone, oral gavage of metformin alone, or a combination of both drugs. In vivo imaging system (IVST) were used to detect the size of tumor, tumor growth was compared between groups. MDA-MB-231 cells were transfected with siCtr or siAMPK (siAMPK#2) in the presence of rhShh and metformin, Gli1 protein levels were analyzed by western blot; sternness was analyzed by mammosphere culture and flow cytometry.[Results]1. Metformin decreased Shh, Smo, Ptc, and Gli1 expression in breast cancer cells, levels of mRNA and protein expressions decreased in a dose- and time-dependent manner following metformin treatment.2. Metformin inhibits rhShh-induced proliferation in breast cancer cells. The influence of rhShh, metformin, and rhShh combined with metformin on the proliferation of MCF-7 and MDA-MB-231 breast cancer cells was assessed with the MTT assay and colony-forming assay. Treatment with rhShh increased the proliferation of these cells; metformin treatment significantly decreased the growth of cells, while metformin inhibited rhShh-induced proliferation (**,##P<0.01). The colony-forming assay showed that incubation with metformin resulted in a significant decrease in both the number and size of colonies compared with the control group, what is more, metformin inhibited the increase in the number and size of colonies induced by rhShh when compared with rhShh treatment alone (**,##P<0.01).3. Metformin inhibits rhShh-induced tumor growth in vivo. An analysis of bioluminescent imaging data indicated that the rhShh treatment group generated significantly larger tumors than the control group; metformin inhibited rhShh-induced tumor growth (**,##P<0.01). Immunohistochemistry of the tumors showed that Glil expression was higher in the rhShh treatment group, but less Glil expression was observed in sections from the combination treatment than in those of the rhShh treatment group.4. Metformin suppresses rhShh-induced MDA-MB-231 cells migration and invasion. The wound scratch migration assay showed that rhShh-treated MDA-MB-231 cells displayed a higher rate of migration; in contrast, metformin treatment resulted in a significant reduction in cellular migration compared with the control group; and the combination treatment revealed that metformin remarkably inhibited rhShh-induced wound gap closure (**,##P<0.01). The matrigel invasion assay showed that the number of rhShh-treated cells that migrated across both the matrigel and the insert was three times higher than that of the control group; meanwhile, cells subjected to combined treatment had a dramatically reduced invasive capacity compared with those treated with rhShh alone (**,##P<0.01).5, Metformin inhibits rhShh-induced BCSCs sternness. The mammosphere culture assay indicated that MDA-MB-231 cells treated with rhShh produced more and larger spheres compared with control group; metformin treatment significantly reduced the number and size of these spheres compared with control cells, and also inhibited the rhShh-induced development of more and larger spheres (**,##P<0.01). The population of CD44+/CD24-/low cells was measured using flow cytometry. MDA-MB-231 cells exposed to rhShh had a higher proportion of CD44+/CD24-/low cells in the population; metformin treatment induced a statistically significant reduction of CD44+/CD24-/;low cells in the population; and the combination treatment showed that metformin remarkably inhibited the increase in the proportion of CD44+/CD24-/low cells induced by rhShh (**,##P<0.01).6. Western blot revealed a significant increase in the Gli1 expression following rhShh and metformin combination treatment of MDA-MB-231 cells transfected with siAMPK#2 compared with those transfected with siCtr or untransfected.7.1n the presence of rhShh and metformin, siAMPK#2-transfected cells produced more and larger spheres compared with both siCtr-transfected cells and untransfected cells(&&P<0.01). Meanwhile, in the presence of rhShh and metformin, siAMPK#2-transfected cells exhibited a significantly higher proportion of CD44+/CD24-/loW stem-like cells compared with both siCtr-transfected cells and untransfected cells (&&P<0.01).[Conclusion]Our findings identified that metformin exhibited anticancer effects through inhibition of the Shh signaling pathway in breast cancer. The downregulation of Shh signaling by metformin inhibited proliferation of cancer cells, impaired cellular migration and invasion, and reduced BCSC survival and self-renewal capacity; in addition, metformin inhibited tumor growth in vivo. Furthermore, the metformin-mediated inhibition of the Shh signaling pathway was dependent on AMPK.