Nek2A负反馈调节Hedgehog信号通路影响结肠癌细胞增殖的分子机制

Hedgehog (Hh) signaling pathway is found to be associated with development and multiple cancers. As the downstream transcriptional factor, Glis are regulated by many proteins, in which Suppressor of fused (Sufu) negatively regulates Hh signaling through combining with Glis. However, the mechanism remains poorly understood. To study the biological roles of Sufu gene, we performed a GAL4-based yeast two-hybrid screening using a human universal cDNA library. With human Sufu as bait, Nek2A is identified as a Sufu-binding protein, which was also confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. Functional study revealed that Nek2A increased the stability of Sufu. And GANT61, a Hh inhibitor, decreased expression of Nek2A mRNA in treated cells. Taken together, in our preliminary experiments, Sufu/Nek2A combination and a feedback regulatory loop between Nek2A and Hh were found but we need more evidence to verify. To further clarify the effects of Nek2A on Sufu stabilization, we plan to study the degradation pathways of Sufu regulated by Nek2A, including ubiquitin/proteasome system, phosphorylation, etc. We also want to find the biological significance of Nek2A/Hh feedback loop in proliferation of cancer, by using human cancer cells and tissues from surgery of colon cancer. Our project aims to illustrate new mechanisms of Hh signaling in proliferation of colon cancer through functional research of Sufu and its related proteins.

Hedgehog（Hh）信号通路在机体发育和肿瘤发生中发挥着重要作用，胞内组分Suppressor of Fused（Sufu）蛋白通过结合转录因子Gli，负调控Hh信号通路，但其介导的信号转导机制仍有待明晰。我们最近发现Nek2A与Sufu相互作用，并且Nek2A增加Sufu蛋白的稳定性，同时还发现Nek2A表达受Hh信号通路调控，提示Nek2A负反馈调节Hh信号通路。为了进一步明确Nek2A负反馈调节Hh信号通路的机制及其对结肠癌细胞增殖的影响，本研究拟通过生化学、细胞生物学等多学科手段探明Nek2A稳定Sufu的途径，并从细胞和整体水平研究Nek2A的负反馈调节作用对结肠癌增殖的影响，为阐明Hh信号通路在结肠癌中的作用机制提供新思路，为结肠癌的治疗提供新的分子靶点。

Hedgehog（Hh）信号通路与多种肿瘤的发生发展密切相关。作为下游的效应因子，Gli受到许多蛋白的调控，其中Sufu能通过结合Gli负向调节Hh信号通路。然而，人们对Sufu的调节机制仍然知之甚少。为了研究Sufu的调控机制，我们首先通过酵母双杂交法发现了Sufu蛋白的相互作用蛋白—Nek2A。接下来我们在真核细胞及原核生物中通过蛋白免疫共沉淀、GST pull down及蛋白片段验证了蛋白的相互作用及作用位点。进一步地，Nek2A促进内源性和外源性Sufu的稳定，这种稳定作用是剂量和时间依赖的。通过激酶突变体的实验证实，Nek2A对Sufu的稳定作用有赖于Nek2A激酶活性。接下来的磷酸化质谱分析进一步揭示Nek2A通过磷酸化Sufu蛋白的T225和S352两个位点稳定Sufu。最后，我们还发现Nek2A对Sufu在纤毛的定位功能具有调节作用。接下来，在我们的进一步研究中，发现Nek2A稳定Sufu蛋白的另一分子机制，即Nek2A阻遏Sufu的泛素/蛋白酶体降解途径，并发现了Nek2A负向调节Hh信号靶基因以及Gli2转录活性。另一方面，GANT61处理后的Hh信号抑制作用导致Nek2A基因转录下调，而Hh激动剂促进Nek2A的转录上调。染色质免疫共沉淀实验发现了Gli1/2与Nek2A启动子区的直接结合。因此，我们揭示了Nek2A负反馈调节Hh的新机制，达到了预期的研究目标。在探索性的研究中，我们发现微管相关蛋白TPX2受Hh信号通路调控，是新的Hh信号通路靶基因。通过该项目的研究，我们发现并验证了新的Sufu调控机制，即与Nek2A相互作用，并在Nek2A的去泛素化及磷酸化作用下，Sufu蛋白的稳定性增强，从而抑制Hh信号功能，而Nek2A又是Gli的靶基因，受Hh信号调控，形成负反馈调节环。这一发现为临床Hh抑制剂的耐药提供了理论依据和联合用药靶点。