Metformin-induced TRAIL Upregulation Promotes Apoptosis in Triple Negative Breast Cancer and Non-small Cell Lung Cancer Cells

Background: Triple negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) are highly aggressive types of cancer with limited therapeutic options. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity and is well tolerated in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised in part by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Methods: Cell proliferation (MTS) and colony formation assays were performed to determine the anti-proliferative/anti-survival effects of metformin, a common drug for type II diabetes, on TNBC and NSCLC cells. A Live/Dead imaging assay and specific apoptotic ELISA analyzed cells undergoing apoptosis. Western blot analyses were used to examine protein expression and cleavage. A recombinant TRAIL-R2-Fc chimera protein was applied to block TRAIL binding to its receptors. Lentiviral vector containing shRNAs was used to specifically knockdown TRAIL expression. A tumor xenograft model was established by inoculation of H460 cells into nude mice. The tumor-bearing mice were treated with metformin to assess the drug’s antitumor activity. Immunohistochemistry was carried out to study the effects of metformin on tumor cell proliferation and induction of apoptosis and TRAIL in vivo. Results: Metformin upregulated TRAIL protein, but not mRNA expression, which correlated with increased apoptosis in TNBC and NSCLC cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-induced TRAIL was secreted into conditioned medium (CM) and functional, since the CM potently promoted apoptosis in MDA-MB-231 cells, which was effectively blocked by a recombinant TRAIL-R2-Fc chimera protein. Inhibition of TRAIL function by blockade of its binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth; it also elicited apoptosis and upregulated TRAIL expression in vivo. Conclusions: TRAIL upregulation and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.