Novel sequential therapy with metformin enhances the effects of cisplatin in testicular germ cell tumours via YAP1 signalling

BACKGROUND: Testicular germ cell tumours (TGCTs) are the most commonly diagnosed malignancy in young men. Although cisplatin has been shown to be effective to treat TGCT patients, long-term follow-up has shown that TGCT survivors who accepted cisplatin treatment suffered from a greater number of adverse reactions than patients who underwent orchiectomy alone. As metformin has shown an anticancer effect in various cancers, we investigated whether metformin could enhance the effects of cisplatin to treat TGCTs. METHODS: The anticancer effects of different treatment strategies consisting of metformin and cisplatin in TCam-2 and NTERA-2 cells were assessed in vitro and in vivo. First, we used a colony formation assay, CCK-8 and MTT assays to explore the viability of TGCT cells. Flow cytometry was used to assess the cell cycle and apoptosis of TGCTs. Then, Western blotting was used to detect the protein expression of TGCTs cells after different treatments. In addition, a xenograft model was used to investigate the effects of the different treatments on the proliferation of TGCT cells. Immunohistochemistry assays were performed to analyse the expression of related proteins in the tissues from the xenograft model. RESULTS: Metformin inhibited the proliferation of TCam-2 and NTERA-2 cells by arresting them in G1 phase, while metformin did not induce apoptosis in TGCT cells. Compared with cisplatin monotherapy, the CCK-8, MTT assay and colony formation assay showed that sequential treatment with metformin and cisplatin produced enhanced anticancer effects. Further study showed that metformin blocked the cells in G1 phase by inducing phosphorylated YAP1 and reducing the expression of cyclin D1, CDK6, CDK4 and RB, which enhanced the chemosensitivity of cisplatin and activated the expression of cleaved caspase 3 in TGCTs. CONCLUSIONS: Our study discovers the important role of YAP1 in TGCTs and reports a new treatment strategy that employs the sequential administration of metformin and cisplatin, which can reduce the required cisplatin dose and enhance the sensitivity of TGCT cells to cisplatin. Therefore, this sequential treatment strategy may facilitate the development of basic and clinical research for anticancer therapies to treat TGCTs.