钙蛋白酶CAPNS1负调控TAOK3/NF2激活Hippo-CAPNS1反馈环路促进结直肠癌增殖的机制研究

Malignant proliferation of colorectal cancer is difficult to overcome the clinical challenges, the mechanism continue to develop but still not fully revealed. CAPNS1, a member of the Calpain family, has the function of regulating calpain1 and calpain2, and its carcinogenic effect has been validated in gastrointestinal tumors. Hippo-YAP signaling pathway is a key tumor suppressor pathway affecting tumor progression. However, the relationship between these two has not been reported. Applicants found that CAPNS1 was overexpressed in colorectal cancer in the previous work. Through proteomic screening of colorectal cancer cells stably knocked down CAPNS1, two key activation genes of Hippo signaling pathway, TAOK3 and NF2, was significantly rescued. Further study found that knockdowning CAPNS1 can significantly activate Hippo signal pathway, at the same time in vivo and in vitro significantly inhibited the proliferation phenotype of colorectal cancer. CAPNS1 transcription level could be suppressed by overexpression TAOK3 or NF2.From further study, we found Hippo pathway target gene TEAD could binding to the promoter region of CAPNS1. This study intends to investigate the role of CAPNS1-Calpain1/2-TAOK3/NF2-Hippo-CAPNS1, a feedback loop regulation mechanism, in the proliferation of colorectal cancer from three levels of cells, animals and population.

结直肠癌的恶性增殖是临床上难以攻克的难题，其机制仍然未能完全揭示。CAPNS1具有正调控calpain1/2的功能，且有证据显示在消化道肿瘤中具有促癌效应；Hippo信号通路是影响肿瘤进展的关键抑癌通路，但上述二者关系未见报道。我们在前期工作中发现CAPNS1在结直肠癌中过表达，通过对稳定敲减CAPNS1的结直肠癌细胞进行全蛋白组学筛查发现，Hippo通路的关键激活基因TAOK3与NF2的表达显著提高，随后证实敲减CAPNS1可以显著地激活Hippo信号通路。在过表达TAOK3与NF2的细胞中，我们发现CAPNS1的表达也被显著抑制。进一步研究发现，Hippo靶基因TEAD可与CAPNS1启动子结合反馈激活CAPNS1转录。本研究拟从细胞、动物、人群三个层次上深入探讨CAPNS1-Calpain1/2-TAOK3/NF2-Hippo-CAPNS1这一反馈环路调节机制在结直肠癌增殖中的作用。

结直肠癌的恶性增殖是临床上难以攻克的难题，其机制仍然未能完全揭示。CAPNS1具有正调控calpain1/2的功能，且有证据显示在消化道肿瘤中具有促癌效应；Hippo信号通路是影响肿瘤进展的关键抑癌通路，但上述二者关系未见报道。在本研究中，我们通过蛋白组学筛查探索了CAPNS1可能影响的下游蛋白，其中包括Hippo信号通路的调节基因TAOK3与NF2。因此，我们认为Calpain家族分子具有调节Hippo信号通路的作用并通过一系列实验加以证实。首先，我们发现在结直肠癌组织中CAPNS1在转录层面和蛋白层面显著过表达，并仅在蛋白层面上调节TAOK3与NF2的表达，二者在转录层面无显著增高。进一步研究发现，在结直肠癌细胞中敲减CAPNS1将抑制结直肠癌的体内和体外增殖表型。在机制上我们证明了在结直肠癌中CAPNS1 调节的calpain 家族成员calpain1、calpain2 负调控TAOK3 和NF2 的表达，进而抑制Hippo 通路的激活，导致YAP 去磷酸化入核与TEAD 形成转录激活复合物反馈激活CAPNS1，维持CAPNS1 在结直肠癌中的高表达，从而促进结直肠癌的增殖。本项目首次发现了CAPNS1 是一个TAOK3 与NF2 共同的上游调控分子，将这两个以往认为相对独立调控下游Hippo 通路的分子联系在了一起。进一步地，我们还探索了癌细胞中Hippo 下游转录激活因子TEAD 激活CAPNS1 转录的反馈环路，拓展了对Hippo 信号通路与calpain 蛋白水解酶交互对话的新机制和新认识。