SMAD4酪氨酸磷酸化调控细胞功能的分子机制

Tissue homeostasis and development depends on normal functions of all cell types in the body. Proliferation of normal cells requires the availability of appropriate growth signals. Transforming growth factor-beta (TGF-beta) inhibits the growth of most epithelial and hematopoietic cells, and thus controls a wide variety of developmental processes, including cell and tissue differentiation, morphogenetic processes and embryonic organization. The early physiologic changes during tumor development include sustained activation of mitogenic signals and the loss of sensitivity to TGF-beta. As a result, tumor cells proliferate in an uncontrolled fashion and are often resistant to the antiproliferative response to TGF-beta. In line with this, inactivating mutations and deletions of the central TGF-beta signal transducer Smad4 are common in gastrointestinal and pancreatic cancers, leading to loss of TGF-beta responses. Yet, genetic alterations in the TGF-beta pathways are rare in certain cancers such as lymphoma, lung cancer and neuroblastoma. This suggests an existence of alternative mechanisms underlying TGF-beta resistance. Thus, our long-term goal is to understand the molecular basis of how antiproliferative growth factors such as TGF-beta regulate cell growth and differentiation, and the underlying mechanisms through which alterations in antiproliferative signaling pathways lead to deregulation of growth control in human diseases. Our short-term strategy is to elucidate the mechanism underlying how Smad4 tyrosine phosphorylation affects TGF-beta growth suppressing functions. In preliminary studies, we have for the first time found that Smad4 binds to protein tyrosine kinase ALK and becomes phosphorylated by ALK. Herein we propose to determine a physiological function of Smad4 tyrosine phosphorylation in inhibiting TGF-beta antiproliferative actions and in promoting tumorigenesis. Our proposed studies will not only provide further understanding of the molecular basis underlying TGF-beta signaling in the normal tissues, gain novel insights into the mechanism of TGF-beta resistance in cancer, but also have strong clinical implications in developing new diagnostic tools and therapies for the prevention and treatment of human diseases.

细胞的正常功能直接关系着器官动态平衡和个体发育，功能异常则导致疾病产生。TGF-beta信号是抑制细胞增殖的主要机制，它的失活则会导致细胞增殖失调。作为TGF-beta信号的关键效应蛋白，SMAD4在消化系统肿瘤中发生突变或缺失，导致了肿瘤细胞增殖不受TGF-beta信号的抑制。在多数其它肿瘤中，SMAD4的遗传变异却不常见，而且这些肿瘤细胞仍然能够逃逸TGF-beta的抑制，但是具体的逃逸机制并不清楚。本实验室首次发现了激酶ALK对SMAD4进行酪氨酸磷酸化，长期以来本领域认为SMAD蛋白主要受丝氨酸/苏氨酸磷酸化的调控。进一步工作表明，酪氨酸磷酸化抑制了SMAD4对TGF-beta靶基因的转录调控，从而阻断了TGF-beta对细胞增殖的抑制。本项目将深入研究SMAD4酪氨酸磷酸化如何调控细胞增殖的分子机制，它将为肿瘤细胞增殖失调提供新的理论依据，以及为临床中肿瘤检测与防治提供新的思路。

TGF-beta在癌症发生早期具有重要抑癌作用，逃逸TGF-beta信号是人类癌症的重要标志之一。SMAD4作为TGF-beta信号转导的核心蛋白，在胰腺癌和消化道肿瘤中存在高频率的突变或者缺失。但是，在很多其它类型的癌症中却极少出现SMAD4或者其它TGF-beta信号相关基因的变异，如淋巴瘤、肺癌、神经母细胞瘤等，这表明还存在导致TGF-beta信号失活的新机制。阐明这类TGF-beta信号失活的新机制对于了解肿瘤发生发展及其精准治疗具有重大意义。我们的前期工作发现激酶ALK对SMAD4存在酪氨酸磷酸化修饰，从而抑制SMAD4对TGF-beta靶基因的转录调控，阻断TGF-beta对细胞增殖的抑制作用。因此，在本项目中我们继续深入研究SMAD4酪氨酸磷酸化调控TGF-beta信号通路以及肿瘤细胞增殖失调的分子机制。我们的研究结果证实肿瘤细胞中激活型ALK能够直接对SMAD4的Tyr95进行磷酸化修饰，使SMAD4丧失结合DNA以及调控基因表达的能力，导致TGF-beta的抑癌功能丧失。而ALK的激酶抑制剂能够恢复肿瘤细胞对TGF-beta信号的响应。我们还发现在淋巴瘤中，ALK阳性与SMAD4酪氨酸磷酸化呈现强烈的正相关，SMAD4磷酸化可以作为ALK阳性肿瘤的新型诊断标记。由于TGF-beta信号能够调节机体的许多生理和病理反应（包括免疫抑制、器官纤维化及肿瘤转移），该研究也提示了ALK抑制剂的潜在副作用。因此，本项的开展不仅揭示了肿瘤细胞沉默TGF-beta抑癌作用的新机制，而且为ALK癌症治疗中ALK和TGF-beta靶向药联合治疗的可行性提供了理论依据。