Patel B R, Tall G G
Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA.
Oncogenesis. 2016 Jun 27;5(6):e236. doi: 10.1038/oncsis.2016.45.
The heterotrimeric G protein α subunit oncogenes GNAQ or GNA11 carry Q209X or R183X activating mutations and are present with ~90% frequency in human uveal melanomas. Forced expression of GNAQ/11(Q209L) in melanocytes is sufficient to drive metastatic melanoma in immune-compromised mice. No known drugs directly target these oncogenic G proteins. Ric-8A is the molecular chaperone that selectively folds Gαq/i/13 subunits. Targeting Ric-8A serves as a rational, yet unexplored approach to reduce the functional abundance of oncogenic Gαq/11 in order to blunt cancer signaling. Here, using mouse melanocyte cell graft tumorigenesis models, we determined that Ric-8A genetic ablation attenuated the abundance and melanoma-driving potential of Gαq-Q209L. A new conditional Ric-8A(Flox/Flox); Rosa-CreER(+/)(-) mouse strain was derived and used as a tissue source to culture an immortalized, tamoxifen-inducible Ric-8A knockout melanocyte cell line that required 12-O-tetradecanoylphorbol-13-acetate (TPA, phorbol ester) for growth. The cell line failed to grow tumors when grafted into immune-compromised mice regardless of Ric-8A expression. Stable expression of human GNAQ(Q209L), but not GNAQ(WT) in the cell line promoted TPA-independent cell proliferation, and upon cell grafting in mice, the initiation and robust growth of darkly-pigmented melanoma tumors. Deletion of Ric-8A in GNAQ(Q209L) cells restored TPA-dependent growth, reduced Gαq-Q209L below detectable levels and completely mitigated tumorigenesis from primary or secondary cell line grafts. Interestingly, TPA treatment of cultured GNAQ(Q209L) cells or host animals grafted with GNAQ(Q209L) cells also sharply reduced Gαq-Q209L abundance and tumorigenic capacity. Finally, tumorigenesis initiated from GNAQ(Q209L) cell grafts, followed by host mouse systemic tamoxifen treatment to delete Ric-8A in the grafted cells completely abrogated GNAQ(Q209L)-driven tumor progression unless a stable human RIC-8A transgene was used to rescue the floxed Ric-8A alleles. Our work defines two new rational targets that may be developed as potential uveal melanoma therapies through reduction of Gαq/11-Q209L oncoprotein abundance: (1) Ric-8A inhibition and (2) phorbol ester treatment.
异三聚体G蛋白α亚基癌基因GNAQ或GNA11携带Q209X或R183X激活突变，在人类葡萄膜黑色素瘤中的出现频率约为90%。在黑色素细胞中强制表达GNAQ/11(Q209L)足以在免疫缺陷小鼠中驱动转移性黑色素瘤。尚无已知药物直接靶向这些致癌G蛋白。Ric-8A是选择性折叠Gαq/i/13亚基的分子伴侣。靶向Ric-8A是一种合理但尚未探索的方法，可降低致癌Gαq/11的功能丰度，从而抑制癌症信号传导。在此，我们使用小鼠黑色素细胞移植瘤发生模型确定，Ric-8A基因敲除减弱了Gαq-Q209L的丰度和驱动黑色素瘤的潜力。我们构建了一种新的条件性Ric-8A(Flox/Flox); Rosa-CreER(+/)(-)小鼠品系，并将其用作组织来源，以培养一种永生化的、他莫昔芬诱导的Ric-8A敲除黑色素细胞系，该细胞系生长需要十四烷酰佛波醇乙酯（TPA，佛波酯）。无论Ric-8A的表达情况如何，将该细胞系移植到免疫缺陷小鼠中均无法形成肿瘤。在该细胞系中稳定表达人GNAQ(Q209L)而非GNAQ(WT)可促进不依赖TPA的细胞增殖，并且在将细胞移植到小鼠体内后，可引发深色色素沉着的黑色素瘤肿瘤的起始和强劲生长。在GNAQ(Q209L)细胞中删除Ric-8A可恢复依赖TPA的生长，将Gαq-Q209L降低到检测不到的水平，并完全减轻原发性或继发性细胞系移植的致瘤性。有趣的是，用TPA处理培养的GNAQ(Q209L)细胞或移植了GNAQ(Q209L)细胞的宿主动物，也会大幅降低Gαq-Q209L的丰度和致瘤能力。最后，由GNAQ(Q209L)细胞移植引发肿瘤发生，随后对宿主小鼠进行全身他莫昔芬处理以删除移植细胞中的Ric-8A，可完全消除GNAQ(Q209L)驱动的肿瘤进展，除非使用稳定的人RIC-8A转基因来挽救floxed Ric-8A等位基因。我们的研究确定了两个新的合理靶点，通过降低Gαq/11-Q209L癌蛋白丰度，有可能开发为潜在的葡萄膜黑色素瘤治疗方法：(1)抑制Ric-8A和(2)佛波酯治疗。
