Nodal Induced by Hypoxia Exposure Contributes to Dacarbazine Resistance and the Maintenance of Stemness in Melanoma Cancer Stem‑like Cells

Nodal signaling has a critical role in the processes of embryogenesis and is necessary for maintaining cell stemness. However, its upregulation in melanoma is positively correlated with malignant potential, including cancer progression, metastasis and recurrence, and a recent report has revealed its role in promoting self‑renewal capacity in melanoma. Our study aimed to explore the effects of hypoxia exposure, which is one of the main causes of chemoresistance in melanoma, on the physiological processes of melanoma cancer stem‑like cells (CSCs) via regulating Nodal. A375 CSCs were enriched by culturing in serum‑free medium (SFM) and were analyzed for the expression levels of Nodal and its correlated proteins by semi‑quantitative western blotting. Lentiviral‑packaged Nodal coding sequence or short‑hairpin RNA (shRNA) was employed. After hypoxia exposure, the effects on glucose uptake, ATP production and O2 consumption were detected, and whether Nodal contributed to the proliferation, invasion, colony formation, self‑renewal capacity and chemoresistance was evaluated. We demonstrated that hypoxia exposure induced Nodal expression and activated the Smad2/3 pathway in A375 CSCs. Hypoxic‑induced Nodal partially promoted dacarbazine resistance, promoted invasion and self‑renewal capacity, but not proliferation which was further confirmed using Nodal knockdown. Blockage of Nodal signaling activity with the small‑molecule inhibitor SB431542 partially reversed Nodal‑induced chemoresistance. Nodal knockdown further sensitized A375 CSCs to dacarbazine after SB431542 pretreatment, indicating the involvement of proNodal in dacarbazine resistance. The introduction of mut‑proNodal induced chemoresistance further confirming the role of proNodal in this process. Taken together, our results demonstrated that Nodal induced by hypoxia exposure induced a malignant phenotype and chemoresistance in A375 CSCs, and proNodal also contributed to these processes, indicating that Nodal may be a potential therapeutic target for melanoma.