IBP通过激活Rho GTPase促进结直肠癌细胞EMT的分子机制研究

In spite of rapid advances in our understanding of the molecular genetics of colorectal cancer (CRC), the burden of disease remains high and the outlook for patients with advanced cancer is still poor. Since this tumor type is particular, where the switch to the malignant phenotype is critical for outcome, it is of paramount importance to elucidate the molecular biology that underlies the metastatic evolution of this disease. It has been shown that the Epithelial Mesenchymal Transition (EMT) provides a powerful paradigm for investigating both genetic and epigenetic aspects of late stage tumor progression. Guaninenucleotide exchanging factors (GEFs) play critical roles in EMT of Colorectal cancer cell via activation Rho GTPases. For Rho GTPases serve as molecular switches are involved in EMT regulation of colorectal cancer cell. IRF-4 binding protein (IBP), a novel GEF, which is expressed in both central and peripheral lymphoid tissues, plays an important role in the maintenance of immune homeostasis. We firstly reported that IBP expression was elevated in colorectal cancer. IBP expression was correlated with the grades of differentiation of the tumors and TNM stage. And our previous studies showed that IBP over expression affects actin cytoskeleton, increase the activity of Rac1 and promote cell invasion in colorectal cancer. In summary, we presume that IBP could regulate EMT and promote cell invasion and metastasis in human colorectal cancer via Rho GTPase pathway. And we focus on the role of IBP in EMT process in human colorectal cancer. In addition, since over 80% of human cancers arise from the epithelium as carcinomas, the benefits of EMT research will not be restricted to CRC, and should impact the bulk of solid tumors. Insights to the biochemical framework by which EMT promotes invasion and metastasis will contribute to the development of novel diagnostic and therapeutic strategies for this insidious killer.

GEF通过激活Rho GTPase在结直肠癌细胞EMT过程中发挥重要作用。IBP是具有GEF活性的信号分子，在免疫系统中具有重要的功能和作用。我们首次发现IBP在结直肠癌细胞中异常高表达，而在正常肠上皮和腺瘤中不表达，IBP表达与肿瘤分期分级密切相关；前期研究证实IBP过表达能够提高结肠癌细胞中Rac1活性、增强肿瘤细胞侵袭力、影响细胞骨架。据此我们推测：IBP通过激活Rho GTPase调控结直肠癌细胞EMT进而促进肿瘤细胞侵袭与转移。本项目拟采用结肠癌细胞株、临床组织标本观察IBP与EMT标志分子表达间的联系；采用IP、流式细胞分析、间接免疫荧光、荷瘤小鼠等观察IBP与Rho GTPase相互作用及其对肿瘤细胞EMT和骨架的影响；采用免疫印迹、IHC等探讨IBP通过激活Rho GTPase促进肿瘤细胞EMT的信号通路及机制，为结直肠癌侵袭与转移的分子机制研究提供新的线索和思路。

IBP在免疫系统中具有重要的功能,我们首次报道了IBP与乳腺肿瘤细胞恶性行为密切相关，本研究对乳腺癌临床标本中的IBP表达模式做进一步的分析，通过一系列体内体外实验研究IBP调控乳腺癌细胞EMT的分子机制。主要研究内容和结果1.乳腺癌组织芯片和乳腺癌细胞株分析。1) 将109例乳腺癌原发灶和淋巴结转移灶肿瘤组织做IBP免疫组化染色，通过对原发灶和转移灶进行组间比较，发现转移灶中IBP表达强度明显高于原发灶。2) 预后分析发现IBP表达强度与乳腺癌患者生存率相关。3)在转移性较强的乳腺癌细胞中IBP表达水平高；在转移性相对低的乳腺癌细胞中IBP表达水平低，正常人乳腺上皮细胞中未检测到IBP的表达。同时发现IBP在结肠癌细胞中高表达。2.IBP调控乳腺癌细胞EMT及其分子机制1) IBP调节乳腺癌细胞EMT相关分子的表达水平。2) IBP改变乳腺癌细胞的EMT表型。3) EGF诱导的EMT过程中IBP表达水平显著升高。4) EGF活化乳腺癌细胞中EGFR信号通路，通过免疫印迹对EGFR酪氨酸磷酸化位点1068、1173，ERK1/2 和 p38MAPK 的磷酸化水平进行观察发现：IBP影响EGF诱导的EGFR及其下游信号分子的活化。5) IBP影响EMT相关转录因子Snail、Slug、ZEB1和ZEB2的水平。6) IBP促进乳腺癌细胞迁移和侵袭； IBP促进乳腺癌细胞中金属蛋白酶的表达。7) IBP调节细胞骨架重排，IBP促进伪足的形成，IBP抑制会引起乳腺癌细胞骨架排列紊乱。8)乳腺癌细胞中IBP通过其GEF活性调控Rac1, RhoA和Cdc42的活化。3.IBP影响乳腺癌细胞成瘤和转移能力。1) IBP对乳腺癌细胞成瘤能力的影响，抑制IBP能够降低乳腺癌细胞的皮下成瘤能力。2) IBP对乳腺癌细胞体内转移能力的影响，抑制IBP能够降低乳腺癌细胞的体内转移能力。综上所述，本课题IBP与乳腺肿瘤细胞的恶性行为密切相关，IBP高水平表达提示患者预后不良；利用一系列体内外实验证明IBP通过活化Rac1, RhoA和Cdc42信号调节乳腺癌细胞EMT进而促进其转移。本研究说明IBP作为一个EMT正向调节因子参与乳腺肿瘤发生、发展和转移的调控，将为乳腺癌的诊断和治疗提供新的靶点及思路。