CTCF磷酸化参与乳腺癌细胞增殖转移的机制研究

CTCF(CCCTC-binding factor), a potential tumor supressor, is involved in the regulation of tumor cell proliferation and apoptosis. As a multifunctional protein, CTCF associated with tumor supressor gene such as p16, p53 and regulated their transcription by harboring an open chromatin configuration. The activity of CTCF is regulated by multiple posttranslational modifications, including poly(ADP-ribosyl)ation, sumoylation and phosphorylation. However, little is known about the CTCF phosphorylation and its relationship with tumor cell proliferation. Erk (Extracellular signal-regulated kinase) signal pathway plays an important role in the process of tumor cell proliferation and excessive activation of Erk was found in many human cancers such as breast cancer. We first predict that CTCF contains potential Erk phosphorylation sites by bioinformatics. Furthermore, we found that Erk can interact with and phosphorylate CTCF using immunofluorescence and IP assay. In this subject, we will study the phosphorylation of CTCF by active Erk and the influence of CTCF phosphorylation on its downstream target genes and tumor proliferation and transformation in breast cancer cells at the molecular, cellular and animal levels, in order to find new signaling pathways in regulation of cell proliferation and migration, and provide a new target for the diagnosis and treatment of tumors.

CTCF(CCCTC-binding factor)参与调节肿瘤细胞的增殖和凋亡，可结合于p16，p53等抑癌基因上，阻止异染色质对这些基因的抑制，调控转录。CTCF的活性受到多种翻译后修饰的调节，目前对CTCF磷酸化及其与肿瘤细胞增殖转移的关系所知甚少。胞外信号调节激酶(extracellular signal-regulated kinase, Erk)信号通路在肿瘤细胞增殖过程中起重要作用，许多人类癌症如乳腺癌中都发现Erk的过度激活。通过生物信息学预测到CTCF含有潜在Erk磷酸化位点，免疫荧光、IP等方法进一步证实，我们发现ERK可以和CTCF相互结合并使其磷酸化。本课题以乳腺癌为研究对象，Erk对CTCF蛋白的磷酸化为切入点，在分子、细胞和动物水平上研究CTCF磷酸化对CTCF下游靶基因及肿瘤增殖转化的影响，以期发现调控细胞增殖迁移新的信号通路，为肿瘤的诊断和治疗提供新的靶点。

CCCTC结合因子（CTCF）是一种重要的表观遗传调节因子，参与生长，增殖，分化和凋亡等多种细胞过程。CTCF可结合于p16，p53等抑癌基因上，阻止异染色质对这些基因的抑制，调控转录。已有研究报道在乳腺癌组织中可检测到CTCF的表达缺失或错义突变。然而，CTCF在乳腺癌中发挥的确切作用尚未完全阐明，参与哪些信号通路仍未知。本研究检测了CTCF在不同侵袭性的乳腺癌细胞系，乳腺癌患者组织和血清中的表达，并进一步探讨了CTCF在乳腺癌细胞中的生物学功能及其机制。研究结果表明，人乳腺癌细胞和组织中的CTCF表达显著低于正常乳腺细胞和组织，且相较于低侵袭性的乳腺癌细胞，高侵袭性的乳腺癌细胞中CTCF的表达更低。CTCF的表达与乳腺癌临床分期（P = 0.043）和病理分化（P = 0.029）显著相关。此外，CTCF过表达能抑制乳腺癌细胞增殖，迁移和侵袭，而CTCF敲低则可促进乳腺癌细胞的增殖，迁移和侵袭。最后，通过转录组学分析和进一步的实验验证，我们发现在乳腺癌细胞MDA-MB231中过表达CTCF可以减少NF-κB（p65）磷酸化，抑制NF-κB活性及其靶基因TNFAIP3（参与抑制凋亡），EGR1和GADD45a（细胞增殖相关）的表达。本研究提供了CTCF在乳腺癌发展中的肿瘤抑制作用的新见解，并表明CTCF / NF-κB通路是乳腺癌治疗的潜在靶点。