α-辅肌动蛋白4 (ACTN4) 通过稳定受体相互作用蛋白激酶1 (RIP1) 促进黑色素瘤的恶性进展

Melanoma in China appears as fast growth, high malignant degree, dismal prognosis, and it is becoming one of the most lethal tumor. At present, curative treatment of metastatic melanoma remains an unmet health problem around the world. Although BRAF, MEK, and combined BRAF/MEK inhibition with small molecule inhibitors have achieved unprecedented response rates in the treatment of metastatic melanoma, the benefits are often of limited duration. On the other hand, blocking antibodies against immune checkpoints (anti-CTLA4, anti-PD1, and anti-PD-L1) can result in long-lasting melanoma regression, but only a subset of metastatic melanoma patients benefit from these agents. Investigation of additional genes and signaling pathways that are deregulated in melanoma is therefore a high priority for identification of novel therapeutic targets..Alpha-actinin-4 (ACTN4), a member of a family of actin crosslinking proteins, is emerging as an important promoter of the pathogenesis of various types of cancers. The increased expression of ACTN4 is commonly associated with disease progression and poor prognosis of the patients. Indeed, we have found that ACTN4 is physically associated with receptor-interacting protein kinase 1 (RIP1) that we have recently shown to be an oncogenic driver in melanoma, and that this association is necessary for stabilization of RIP1 and subsequent activation of NF-κB. But how RIP1 itself is regulated in melanoma is to be illuminated. We have also found that ACTN4 is upregulated in melanoma cell lines and fresh melanoma isolates, which promotes proliferation and resistance to BRAF/MEK (MAPK) inhibitors in melanoma cells. .In this project, we propose these hypotheses: ACTN4 is a biomarker of melanoma progression and poor prognosis of the patients; ACTN4 promotes melanoma cell proliferation through RIP1-mediated activation of NF-κB; targeting ACTN4 inhibits melanoma growth and improves the therapeutic efficacy of MAPK inhibitors; and ACTN4 is essential for the expression of RIP1 in melanoma cells. .The specific aims of this project are: To clarify whether ACTN4 upregulation is associated with malignant progression of melanoma and is predictive of poor patient prognosis; To define the downstream effectors responsible for ACTN4-mediated melanoma cell proliferation; To determine the effect of targeting ACTN4 on melanoma growth and responses to MAPK inhibitors in preclinical melanoma models; To define the mechanism responsible for upregulation of ACTN4 and the mechanism responsible for ACTN4-mediated expression of RIP1 in melanoma cells..The results from this project will potentially identify ACTN4 as a biomarker predictive of melanoma progression and poor prognosis of the patients and a molecular target for therapeutic intervention in melanoma. Moreover, the results will potentially identify co-targeting ACTN4 and the MAPK pathway as a novel combined approach in the treatment of melanoma.

黑色素瘤在我国呈现为增长速度快、恶性程度高、预后极差，为最致命的肿瘤之一。前期发现，受体相互作用蛋白激酶1（RIP1）作为重要促癌因子，通过激活NF-κB信号通路促进黑色素瘤的发生发展，但RIP1自身受调控的机制有待进一步阐明。我们的质谱结果确定了α-辅肌动蛋白4（ACTN4）与RIP1结合，并调控RIP1蛋白稳定性；进而发现ACTN4在黑色素瘤表达上调，促进细胞增殖并对MAPK抑制剂有抵抗作用。因此我们推断ACTN4通过RIP1介导的NF-κB通路促进黑色素瘤的恶性进展。本课题拟建立ACTN4 shRNA knockdown和过表达的黑色素瘤细胞系，通过裸鼠成瘤实验，确定ACTN4可作为黑色素瘤病情及不良预后的指标；阐明ACTN4下游信号通路；探索以其为靶点，MAPK等抑制剂对黑色素瘤的疗效。本课题将较全面阐明ACTN4在黑色素瘤恶性进展中的具体机制，提供其可作为潜在治疗靶点的实验依据。

受体相互作用蛋白激酶1（receptor interacting protein 1, RIP1）作为重要促癌因子，是细胞内重要的压力感受器，能够介导细胞存活和死亡的信号转导通路。我们的研究发现，在体或离体的黑色素瘤细胞中RIP1普遍高表达，RIP1 knockdown能够降低细胞活力，而且与TNFα及MEK或PI3K的抑制剂联合应用能够诱导黑色素瘤细胞凋亡，表明RIP1在促进黑色素瘤存活中发挥作用；RIP1通过激活NF-κB信号通路促进黑色素瘤的发生发展。进一步研究表明，RIP1通过ERK-Snail1-CYLD-RIPK1-NF-κB信号轴介导黑色素瘤细胞对BRAFi的耐药，在此过程中，K63泛素化维持了RIP1的蛋白水平，从而发挥对细胞死亡信号的抵抗作用。但RIP1的受调机制仍不明确。我们通过免疫沉淀及后续的质谱鉴定，筛选到肌动蛋白交联蛋白α-辅肌动蛋白4（ACTN4）是RIP1的互作蛋白。我们建立了ACTN4 shRNA knockdown和过表达的黑色素瘤细胞系，通过体外、体内裸鼠成瘤实验，发现ACTN4在黑色素瘤表达上调，沉默ACTN4可抑制黑色素瘤细胞增殖，延缓黑色素瘤异种移植瘤的生长；而过表达ACTN4促进黑素细胞和黑素瘤细胞增殖，并促进黑素细胞非锚定生长及对MAPK抑制剂的抵抗作用。我们阐明了ACTN4作为稳定RIPK1所必需支架的一种新功能：ACTN4 分别通过其N端肌动蛋白结合结构域和C端类似CAM的结构域与RIPK1和细胞凋亡抑制蛋白1（cIAP1）结合，对促进RIPK1和cIAP1之间的结合、促进RIPK1的稳定至关重要，进而激活NF-κB通路，促进细胞增殖及MAPK耐药，且不依赖于其核转位。而且，ACTN4的表达被NF-κB转录激活。我们进一步通过RIP1、ACTN4 shRNA KD前后的蛋白组学分析及相应的KEGG分析、GSEA分析，获得了一系列ACTN4的下游信号通路，实验表明ACTN4可能在MAPK、Akt、NF-κB三条信号通路上游发挥分子开关的作用，并可控制MAPK信号通路向Akt信号通路的转换，从而调节黑色素瘤细胞的生长。总之，我们的研究发现ACTN4是一种致癌调控因子，能够驱动ACTN4- RIP1-NF-κB的正反馈信号轴，并且在多条促癌信号通路上游发挥分子开关的作用，可作为治疗黑色素瘤的潜在靶点。