HER3 belongs to the human epidermal growth factor receptor (HER) family which also includes HER1/EGFR/erbB1, HER2/erbB2, and HER4/erbB4. As a unique member of the HER family, HER3 lacks or has little intrinsic tyrosine kinase activity. It frequently co-expresses and forms heterodimers with other receptor tyrosine kinases (RTKs) in cancer cells to activate oncogenic signaling, especially the PI-3K/Akt pathway and Src kinase. Elevated expression of HER3 has been observed in a wide variety of human cancers and associates with a worse survival in cancer patients with solid tumors. Studies on the underlying mechanism implicate HER3 expression as a major cause of treatment failure in cancer therapy. Activation of HER3 signaling has also been shown to promote cancer metastasis. These data strongly support the notion that therapeutic inactivation of HER3 and/or its downstream signaling is required to overcome treatment resistance and improve the outcomes of cancer patients.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised 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. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL 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 but also elicited apoptosis and enhanced TRAIL expression in vivo . Collectively, we have demonstrated that upregulation of TRAIL 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.

Introduction: Triple-negative breast cancer (TNBC) does not respond to conventional targeted therapy, necessitating novel treatment options. Metformin possesses unique anti-proliferative and pro-apoptotic properties in TNBC cells. However, the molecular mechanism through which metformin may induce apoptosis is incompletely understood. In the current study, we aim to determine whether tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptotic signaling is involved in metformin-induced cytotoxicity on TNBC cells. Methods: TNBC cell lines HCC70, MDA-MB-468, BT549 and MDA-MB-231 were used in the study. A cell death detection ELISA kit was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes). Western blot analyses were carried out to assess the expression of TRAIL, TRAIL receptors DR5 and DR4, and apoptosis-related proteins (PARP, Caspase-8 and Caspase-3). Flow cytometric analyses were performed to define the expression of cell surface TRAIL receptors DR5 and DR4. Secreted TRAIL protein level in the conditioned medium (CM) of cell culture was measured by using a highly specific ELISA and western blot. MDA-MB-231 cells were treated with concentrated CM from metformin-treated cells to test the activity of secreted TRAIL in inducing cell apoptosis. A soluble recombinant TRAIL decoy receptor, which contains a normal extracellular domain of death receptor 5 (DR5) but a truncated intracellular domain and thus is unable to transduce death signals, was used to block TRAIL function. Infection with lentivirus containing specific shRNAs was performed to knockdown TRAIL expression. Results: Metformin induced apoptosis in a panel of TNBC cell lines in a dose-dependent manner, as evidenced by the increase of cytoplasmic histone-associated DNA fragments and the cleavage of PARP, caspase-8 and caspase-3. Interestingly, during the process of TNBC cells undergoing apoptosis, metformin elicited a dose-dependent upregulation of TRAIL protein levels (both cellular TRAIL and secreted TRAIL) without altering the expression of TRAIL receptors DR5 and DR4. The secreted TRAIL from concentrated CM of metformin-treated cells showed activity in inducing apoptosis of MDA-MB-231 cells. To determine if the induction of TRAIL was essential for metformin-induced cytotoxicity effects on TNBC cells, we examined whether inhibition of TRAIL function by a decoy receptor or specific knockdown of TRAIL expression with shRNAs would affect metformin-induced apoptosis. In all three cell lines (HCC70, MDA-MB-468, BT549) we tested, both the TRAIL decoy receptor and TRAIL-specific shRNAs significantly attenuated metformin-induced DNA fragmentation and cleavage of PARP, caspase-8, and caspase-3. Conclusion: We demonstrate that metformin promotes TNBC cells undergoing apoptosis via induction of TRAIL expression. Our data suggest that TRAIL-mediated apoptosis critically contributes to metformin's antitumor activity against TNBC. Keywords: Metformin, TRAIL, Apoptosis, triple-negative breast cancer Citation Format: Shuang Liu, Erik V Polsdofer, Lukun Zhou, Susan Edgerton, Ann Thor, Bolin Liu. Metformin exhibits cytotoxicity effects on triple-negative cancer cells via TRAIL-mediated apoptosis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-18.