膜相关鸟苷酸激酶Dlg5通过Girdin/Tks5轴调节肝细胞癌侵袭性伪足形成的机制研究

Invasion and metastasis are important features of hepatocellular carcinoma (HCC) and greatly impact the prognosis of HCC patients. Recent studies have shown that tumor cell invasion is closely related to a special cell structure invadopodia. However, the mechanisms underlying the regulation and function of invadopodia in HCC invasion and metastasis remain unexplored. Our preliminary experiments indicated the potential role of Discs large homolog 5 (Dlg5) in HCC metastasis. Dlg5 expression was significantly lower in HCC tissues compared to neighboring non-tumor tissues, and was negatively correlated with the stage of HCC. Moreover, Dlg5 expression was significantly lower in high invasive HCC cell lines such as SK-Hep1 and MHCC97-H than in low invasive HCC cell lines such as HepG2 and MHCC97-L. In addition, knockdown of Dlg5 led to epithelial-mesenchymal transition of HepG2 cells, promoting the formation of invadopodia and the degradation of matrix. Evidence from our group and others suggests that Girders of actin filaments (Girdin), a bona fide metastasis-related protein, can interact with and activate tyrosine kinase substrate with five SH3 domains (Tks5), which is recognized as a “molecular switch” that directs invadopodium formation. Interestingly, a recent study reported that Dlg5 interacted with and inhibited the activity of Girdin, thereby suppressing the migration of prostate cancer cells. Therefore, we hypothesized that Dlg5 modulates Girdin/Tks5 pathway to inhibit the formation and function of invadopodia in HCC, finally impede HCC invasion and metastasis. To confirm our hypothesis, we will employ HCC specimens, HCC cell lines and xenografts in nude mice as in vitro and in vivo models. In clinical research, we will perform immunohistochemistry and qRT-PCR assays to detect the expression Dlg5 and Girdin in more than 60 pairs of primary HCC tissues and matched adjacent tissues. The relationship of Dlg5 and Girdin expression will be analyzed using Spearman method. The Cox proportional-hazards regression model will be established to analyze the relationship of Dlg5 expression and different clinic-pathological features including BCLC stage, and the influence of Dlg5 expression on the prognosis of HCC patients will be analyzed using Kaplan-Meier method. Then HCC cells will be transfected with different constructs, such as expression vectors or siRNA (or shRNA). The malignant phenotypes of HCC cells will be examined such as cell proliferation, migration and invasion as well as the formation and function of invadopodia. The growth and metastasis of HCC cells derived from xenografts will be examined in nude mice. Mechanistically, co-immunoprecipitation and immunofluorescence assay will be performed to confirm the interactions among Dlg5, Girdin, and Tks5. It is expected that these experiments will help elucidate the molecular mechanism by which Dlg5 inhibits the metastasis of HCC, and identify novel diagnostic biomarkers and therapeutic targets for HCC to improve the prognosis of HCC patients.

侵袭性伪足在肿瘤侵袭转移中起关键作用，但其形成的分子机制尚不完全清楚。研究表明促肿瘤侵袭分子Girdin（Girders of actin filaments）与酪氨酸激酶底物Tks5（Tyrosine kinase substrate with five SH3 domains）相互作用可促进侵袭性伪足形成；课题组发现敲低膜相关鸟苷酸激酶家族成员Dlg5（Discs large homolog 5）表达也能促进肝癌细胞侵袭性伪足形成，且肝癌组织普遍Dlg5表达降低。项目拟研究Dlg5是否通过Girdin/Tks5调控侵袭性伪足形成。拟采用已有肿瘤数据库、肝癌组织标本、肝癌细胞系及基因敲低和过表达技术，研究Dlg5如何调控Girdin和Tks5相互作用及活化，进而是否影响体外肝癌细胞侵袭性伪足的形成以及体内肝癌的生长和转移。预期研究结果对理解肝癌细胞侵袭和转移、发现新诊治靶标具有重要意义。

侵袭性伪足在肿瘤侵袭转移中起关键作用，但其形成的分子机制尚不完全清楚。研究表明促肿瘤侵袭分子Girdin（Girders of actin filaments）与酪氨酸激酶底物Tks5（Tyrosine kinase substrate with five SH3 domains）相互作用可促进侵袭性伪足形成；课题组发现敲低膜相关鸟苷酸激酶家族成员Dlg5（Discs large homolog 5）表达也能促进肝癌细胞侵袭性伪足形成，且肝癌组织普遍Dlg5表达降低。项目研究Dlg5是否通过Girdin/Tks5调控侵袭性伪足形成。采用已有肿瘤数据库、肝癌组织标本、肝癌细胞系及基因敲低和过表达技术，细胞培养、基因转染、Western blot、免疫共沉淀、免疫荧光共聚焦显微镜、MTT、集落形成、Transwell小室、裸鼠移植瘤等研究手段，研究Dlg5如何调控Girdin和Tks5相互作用及活化，进而是否影响体外肝癌细胞侵袭性伪足的形成以及体内肝癌的生长和转移。有以下主要发现/关键数据：（1）免疫组织化学：70%的非肿瘤肝组织检测到中强度的Dlg5表达；（2）生存曲线分析：低Dlg5表达与整体生存率差显著相关；（3）Western blot检测：Dlg5表达的表达与侵袭性呈负相关，TGF-β1可下调Dlg5表达，故可作为Dlg5／Girdin／Tks5通路生理性调节因子；（4）侵袭性伪足及基质胶实验：Dlg5下调促进细胞侵袭性伪足形成的作用，至少部分依赖于涉及Girdin与Tks5的分子机制；（5）Coip检测：Dlg5可抑制Girdin与Tks5相互结合，而这种结合有可能对Tks5磷酸化活化重要，从而促进侵袭性伪足产生；（6）裸鼠体内试验：过表达Dlg5可抑制SK-Hep1细胞裸鼠原位移植模型肝、肺转移瘤的数目和大小；（7）云南特色中草药蒲公英活性成分蒲公英甾醇（Taraxasterol）通过上调Hint1活性抑制肝细胞癌生长。本项目科学意义：（1）提出并验证了Dlg5抑制肝癌侵袭、转移新的分子机制，即Dlg5抑制Girdin/Akt相互结合，该抑制可能对Tks5磷酸化活化重要；（2）初步验证了蒲公英甾醇抗肝癌作用，并发现其新的靶点Hint1，有可能成为进展期/晚期肝癌病人治疗药物选择。