去泛素化酶USP15正反馈Hippo-YAP通路促胃癌细胞转移的分子机制

Gastric cancer is the fourth most commonly diagnosed cancer and the second major cause of cancer-related death in the world. Despite the declining incidence of GC, patients have poor prognosis due to the high rate of metastasis and postsurgical recurrence. The Hippo pathway is an emerging signaling pathway that plays important roles in development and disease. Recent studies show Hippo-YAP pathway plays crucial roles in gastric cancer cell metastasis, however, the underlying molecular mechanism of Hippo inactivation remain largely unknown. In our preliminary work, analysis of immunohistochemistry on gastric cancer tissues revealed significant overexpression of USP15 was associated with TNM staging and lymph node metastasis, and the high cytoplasm expression of USP15 predicted a poor overall surviral time of patients. Moreover, we found USP15 binded to YAP and deubiquitinated and stabilized YAP protein in BGC-823 cell line, and USP15 maybe a direct target gene of Hippo-YAP signaling pathway. Knockdown of endogenous USP15 significantly inhibited cell proliferation and invasion of gastric cancer cells. So, we hypothesized that USP15 promoted gastric cancer cell metastasis via a positive feedback loop involving Hippo-YAP pathway. Therefore, in the present project, we aim to use both in vitro and in vivo systems to verify the function of USP15 on gastric cancer cells. Moreover, we will use co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (Chip), GST pull-down assay and protein mass spectrometry to illuminate how USP15 deubiquitinates and stabilizes YAP, and whether USP15 is a direct target gene of YAP/TEAD complex, to establish this positive feedback regulation in gastric cancer cells. Finally, we aim to use the cancer genome atlas TCGA data and clinical samples to further investigate the clinical significant of this feedback in gastric patients. Those results may provide new biomarkers and therapeutic targets for the diagnosis and treatment of gastric cancer in the future.

转移是胃癌患者主要死因，Hippo-YAP通路在胃癌转移过程中发挥重要作用，然而其调控机制尚未阐明。我们前期研究发现：1）去泛素化酶USP15在胃癌组织中表达升高，其高表达提示患者易转移和预后不良；2）USP15可能去泛素化稳定YAP，还可能是Hippo通路靶基因；3）下调USP15的表达显著抑制胃癌细胞增殖和侵袭。据此，我们推测USP15正反馈Hippo-YAP通路促胃癌细胞转移。为此，我们将构建抑制或高表达USP15的细胞模型，利用细胞培养和裸鼠动物实验观察调节USP15对胃癌细胞转移的作用；通过免疫共沉淀、Chip和质谱等方法，探索USP15去泛素化稳定YAP蛋白的机制，证实USP15是Hippo通路新的靶基因，解析此正反馈级联的作用机制；采用TCGA数据结合胃癌组织样本，明确USP15潜在临床意义。本研究揭示了Hippo通路新的调控机制，为胃癌的早期诊断和靶向治疗提供新的治疗策略。

Hippo-Yes 相关蛋白 1 (YAP1) 通路在胃癌发生和转移中的重要性引起了相当多的研究关注；然而，YAP1 在胃癌 (GC) 中的调控网络尚不完全清楚。在本研究中，泛素特异性肽酶 49 (USP49) 被鉴定为 YAP1 的新型去泛素酶，敲低 USP49 可抑制胃癌细胞的增殖、转移、化疗耐药和腹膜转移。过表达USP49显示相反的生物学效应。此外，USP49 被 YAP1/TEAD4 复合物转录激活，与 YAP1 形成正反馈回路促进胃癌细胞的恶性进展。最后，我们收集了 482 名胃癌患者的组织样本和临床随访信息。结果表明，USP49在胃癌细胞中的表达较高，并且与YAP1及其靶基因结缔组织生长因子（CTGF）和富半胱氨酸蛋白61（CYR61）的表达呈正相关。生存分析和 Cox 回归分析表明，USP49 高表达与胃癌患者预后不良相关，是预后差的独立因素。此外，具有高 USP49 和 YAP1 表达的胃癌患者的总生存期极短。本研究结果表明USP49/YAP1正反馈回路的异常激活在胃癌的恶性进展中起关键作用，为胃癌提供了潜在的新的预后因素和治疗靶点。