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A20 Promotes Melanoma Progression Via the Activation of Akt Pathway

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A20 Promotes Melanoma Progression Via the Activation of Akt Pathway Ma et al. Cell Death and Disease (2020) 11:794 https://doi.org/10.1038/s41419-020-03001-y Cell Death & Disease ARTICLE Open Access A20 promotes melanoma progression via the activation of Akt pathway Jinyuan Ma1, Huina Wang1,SenGuo1, Xiuli Yi1, Tao Zhao1,YuLiu1,QiongShi1,TianwenGao1,ChunyingLi1 and Weinan Guo1 Abstract Melanoma is the most life-threatening skin cancer with increasing incidence around the world. Although recent advances in targeted therapy and immunotherapy have brought revolutionary progress of the treatment outcome, the survival of patients with advanced melanoma remains unoptimistic, and metastatic melanoma is still an incurable disease. Therefore, to further understand the mechanism underlying melanoma pathogenesis could be helpful for developing novel therapeutic strategy. A20 is a crucial ubiquitin-editing enzyme implicated immunity regulation, inflammatory responses and cancer pathogenesis. Herein, we report that A20 played an oncogenic role in melanoma. We first found that the expression of A20 was significantly up-regulated in melanoma cell lines. Then, we showed that knockdown of A20 suppressed melanoma cell proliferation in vitro and melanoma growth in vivo through the regulation of cell-cycle progression. Moreover, A20 could potentiate the invasive and migratory capacities of melanoma cell in vitro and melanoma metastasis in vivo by promoting epithelial–mesenchymal transition (EMT). Mechanistically, we found that Akt activation mediated the oncogenic effect of A20 on melanoma development, with the involvement of glycolysis. What’s more, the up-regulation of A20 conferred the acquired resistance to Vemurafenib in BRAF-mutant melanoma. Taken together, we demonstrated that up-regulated A20 promoted melanoma progression via the activation of Akt pathway, and that A20 could be exploited as a potential therapeutic target for 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; melanoma treatment. Introduction pathogenesis and treatment resistance could be helpful Melanoma is the most lethal skin cancer that is the for melanoma patients. malignant transformation from epidermal melanocytes. In Inflammation-related signals, particularly pro- 2018, there are estimated 96,480 new cases of melanoma inflammatory cytokines like tumor necrosis factor α emerging and over 7230 deaths among them in the United (TNFα), have been proved essential for maintaining cell States1. Despite the progressive elucidation of melanoma survival in melanoma4. A20, which is encoded by Tumor pathogenesis and the revolutionary advances of targeted Necrosis Factor α- Induced Protein 3(TNFAIP3), was therapy and immunotherapy2, the prognosis for patients initially identified as a primary responsive gene induced remains unoptimistic, especially for those with metastasis by TNF-α5. It was a crucial negative regulator of immu- and resistance to current therapies3. Therefore, to further nity and inflammatory response via the suppression on investigate the mechanism underlying melanoma NF-κB6. Of note, the loss-of-function of A20 could trigger inflammatory response and contribute to the onset and development of many autoimmune diseases6. Apart from Correspondence: Chunying Li ([email protected])or the role in immunity, A20 is also highly related to tumor Weinan Guo ([email protected]) biology. Up-regulated A20 acted as an oncogene in breast 1Department of Dermatology, Xijing Hospital, Fourth Military Medical – ’ cancer to facilitate epithelial mesenchymal transition and University, No 127 of West Changle Road, 710032 Xi an, Shaanxi, China 7 These authors contributed equally: Jinyuan Ma, Huina Wang, Sen Guo tumor metastasis via the ubiquitination of Snail1 . Edited by A. Peschiaroli © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Ma et al. Cell Death and Disease (2020) 11:794 Page 2 of 12 However, the loss of A20 contributed to the progression melanocyte (HPM). Through qRT-PCR analysis, we found 8 of MYD88L265P-driven non-Hodgkin lymphoma .In that the transcriptional levels of A20 were generally addition, the down-regulation of A20 by its upstream increased in melanoma cells compared with melanocytes regulator miR-19a promoted the development of colitis- (Fig. 1a). In addition, the protein levels of A20 were also associated colon cancer7,9. These reports indicate that up-regulated in melanoma cells (Fig. 1b). To further while A20 is a potential therapeutic target for cancer, its confirm the expression status of A20, we performed effect is tumor type-specific. For melanoma, a previous immunofluorescence staining on the tails of B6. Cg-Tg study revealed that A20 mediated the effect of IL-17RC on (KRT14-Kitl*) 4XTG2Bjl/J mice, which express mutant the progression of xenograft tumor of B16 melanoma mouse Kitl cDNA and possess abundant melanocytes in cells10. In addition, the suppression of A20 in myeloid- the epidermis, as well as the xenograft tumors of B16 and derived suppressor cells or dendritic cells could potentiate B16F10 mouse melanoma cells implanted in C57BL/6 the anti-tumor immunity and restrain the progression of mice. The intensity of A20 was markedly increased in B16 implanted melanoma in mice11,12. These reports have and B16F10 cells compared with mouse melanocytes (Fig. partially elucidated the biological role of A20 in mela- 1c). Moreover, we turned to Talantov data set (GSE3189) noma, focusing on anti-tumor immunity. However, the and found that the mRNA level of A20 was significantly expression status of A20 in human melanoma cell and its increased in human melanoma specimens compared to influence on melanoma pathogenesis, especially on the benign melanocytic nevus tissues (Fig. 1d). Taken toge- malignant behavior of melanoma cell, yet to be fully ther, the results from in vitro and in vivo demonstrated elucidated. the up-regulation of A20 in melanoma. Akt is a serine-threonine kinase partially activated through the phosphorylation of Thr-308 and fully acti- A20 promotes melanoma growth by regulating cell-cycle vated upon Ser-473 phosphorylation13. Notably, Akt is progression constitutively activated in up to 70% of human melano- We then explored the biological function of A20 in mas and plays an important role in melanoma patho- melanoma. Lentiviral transfection was employed to obtain genesis14. Increased phosphorylation of Akt was reported stable knockdown of A20 in both A2058 and A375 cell to be highly correlated with the clinical stages and the lines, and the knockdown efficiency was confirmed (Fig. thickness of melanomas, as well as the prognosis of 2a). The knockdown of A20 led to impaired cell pro- patients15. Moreover, Akt could simultaneously stabilize liferation and colony formation in melanoma (Fig. 2b, c). cells with extensive mitochondrial DNA mutation and Forwardly, we employed xenograft tumor model to promote the expression of the ROS-generating enzyme explore whether increased A20 expression contributed to NOX4 to increase angiogenesis and superoxide genera- melanoma growth in vivo. A2058 melanoma cells with or tion, fostering more aggressive tumor behavior in mela- without the knockdown of A20 were subcutaneously noma16. The oncogenic effect of Akt could be possibly injected into nude mice. 5 weeks later, the tumor volume attributed to its multiple biological functions, among and tumor weight were markedly decreased in the A20- which the activation of glycolysis might provide sufficient knockdown group compared to the control (Fig. 2d–f). energy and intermediate metabolite for the rapid pro- Further, we investigated the mechanism underlying the liferation of tumor cells17. However, the upstream reg- pro-proliferative role of A20. Previously, several resear- ulatory mechanism of Akt activation and the concomitant ches have revealed the regulatory role of A20 in various glycolysis in melanoma remains elusive. modalities of cell death including cell apoptosis, necrop- – In the present study, we first found that A20 expression tosis, and pyroptosis18 20. Therefore, to figure out whe- was remarkably up-regulated in melanoma cells. Subse- ther A20 was involved in the regulation of cell death, we quently, the function of A20 in melanoma growth and adopted positive controls of melanoma cell apoptosis, metastasis was examined both in vitro and in vivo. Fur- necroptosis, and pyroptosis induced by TS (TNF-α and thermore, the mechanism underlying the oncogenic role SM-164), TSZ (TNF-α, SM-164 and Z-VAD-FMK), and of A20 was investigated, involving the regulation of Akt FC (FeSO4 and CCCP) respectively as described pre- – and glycolysis. Ultimately, we examined the role of A20 in viously21 23. TS and TSZ could effectively trigger cell the acquired resistance
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