Lenalidomide arrests cell cycle and modulates PD1-dependent downstream mTOR intracellular signals in melanoma cells

Despite numerous efforts to define the best therapeutic strategies in advanced melanoma, the response of many patients remains heterogeneous and of short duration. Lenalidomide, an immunomodulating drug, has shown anti-inflammatory, antiangiogenic and anticancer properties in haematological disorders; however, few preclinical data support the rationale for using this drug in melanoma patients. In this study, we investigate lenalidomide's potential role in melanoma by focusing on the in-vitro drug's antiproliferative activity. The antiproliferative action of lenalidomide was evaluated on two melanoma cell lines by MTT assay, cell cycle and apoptosis assay. P21 protein levels were evaluated with droplet digital PCR (ddPCR) and western blot analysis while his interaction with specific cyclin-dependent kinase (CDK) was assessed by immunoprecipitation test. The biological effect and molecular mechanisms of programmed cell death-1 (PD-1) in the regulation of proliferation were evaluated using ddPCR, flow cytometry, western blot and small interfering RNA transfection. We observed that lenalidomide exerts a cytostatic effect in melanoma cell lines by inducing cell cycle arrest in the G0-G1 phase through p21 upregulation and modulation of CDK complexes. Furthermore, we found that lenalidomide has an antiproliferative action through the downregulation of melanoma-PD1 expression and consequently the alteration of intracellular signaling of mammalian target of rapamycin/S6. The present study aims to provide new insights into the role of lenalidomide in melanoma and suggesting to potentially translating these findings into a clinical setting to use immunomodulatory derivatives for blocking the pro-tumorigenic activity of the melanoma through the PD-1/PD-L1 axis.