Department of Burns and Plastic Surgery Unit, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, P.R. China.
Department of Burns and Plastic Surgery Unit, West China Hospital of Sichuan University, Chengdu, Sichuan 610041, P.R. China.
Oncol Rep. 2018 Jun;39(6):2855-2864. doi: 10.3892/or.2018.6387. Epub 2018 Apr 23.
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.
节点信号在胚胎发生过程中具有关键作用，并且对于维持细胞干性是必要的。然而，其在黑色素瘤中的上调与恶性潜能呈正相关，包括癌症进展、转移和复发，最近的一份报告揭示了其在促进黑色素瘤自我更新能力中的作用。我们的研究旨在通过调节节点来探讨缺氧暴露（黑色素瘤中化疗耐药的主要原因之一）对黑色素瘤癌干细胞样细胞（CSC）生理过程的影响。通过在无血清培养基（SFM）中培养来富集 A375 CSC，并通过半定量 Western blot 分析节点及其相关蛋白的表达水平。使用慢病毒包装的节点编码序列或短发夹 RNA（shRNA）。缺氧暴露后，检测葡萄糖摄取、ATP 产生和 O2 消耗的影响，评估节点是否有助于增殖、侵袭、集落形成、自我更新能力和化疗耐药性。我们证明了缺氧暴露诱导 A375 CSC 中节点的表达并激活 Smad2/3 途径。缺氧诱导的节点部分促进了达卡巴嗪耐药性，促进了侵袭和自我更新能力，但不促进增殖，这进一步通过节点敲低得到证实。用小分子抑制剂 SB431542 阻断节点信号活性部分逆转了节点诱导的耐药性。在 SB431542 预处理后，节点敲低进一步使 A375 CSC 对达卡巴嗪敏感，表明前节点参与了达卡巴嗪耐药性。引入突变的前节点诱导的耐药性进一步证实了前节点在这一过程中的作用。总之，我们的结果表明，缺氧暴露诱导的节点诱导 A375 CSC 产生恶性表型和化疗耐药性，前节点也有助于这些过程，表明节点可能是黑色素瘤的潜在治疗靶点。
