Aberrant cell growth signaling in oral cancer

本研究旨在阐明口腔鳞状细胞癌中异常生长信号的详细机制。得到了以下结果。(1)我们首次在人类肿瘤组织中发现了322位密码子的CD域从Glu到Lys的ERK2突变，该突变伴随着电荷的改变和PAGE的快速迁移。类似的突变已经在Drosphila中被鉴定为七个标记物，据报道该突变蛋白与MKP结合较弱，MKP是一种用于使ERK失活的磷酸酶。除HSC6外，我们还对3个细胞株进行了EGFR扩增，发现经EGF处理后，EGFR在846位和1068位出现高水平的磷酸化，下调调控大部分被抑制。此外，我们还发现了一种未检测到mkp1/2被诱导的细胞系。口腔鳞状细胞癌生长信号的上述变化可能至少部分参与了ERK的结构性激活。(2)利用酵母2杂交系统分离了两个未知与ERK2相互作用的蛋白。NaFI通过EGF/ERK被磷酸化。EGF/ERK信号转导减弱。进一步的实验表明，该蛋白在M期出现超磷酸化(SP)，G2/M抑制剂处理诱导了SP物种的形成。此外，我们还发现NaFI的C末端1/3增强了诱导细胞凋亡的作用。(3)检测15例口腔鳞癌组织中ERK的磷酸化水平，并与临床病理资料进行比较。与组织分化程度有较好的相关性，与TNM分期无相关性。(4)口腔鳞状细胞癌LINSS细胞中35个耐药基因的RNA表达水平与依托泊苷的敏感性无关，而依托泊苷调控的2个基因的表达水平随依托泊苷敏感性的不同而不同。(5)在印度口腔鳞癌组织中发现5个抑癌基因的高甲基化，而在正常口腔鳞癌组织中未发现该基因的高甲基化，可作为口腔鳞癌的诊断标记物。

This study was undertook to elucidate detailed mechanisms of aberrant growth signaling in oral squamous cell carcinoma (OSCC). The following results were obtained. (1) We, first in human cancer, found an ERK2 mutation within CD domain of codon 322 from Glu to Lys, which is accompanied with charge alteration and faster migration in PAGE. The similar mutation has been identifed in Drosphila as sevenmaker and the mutant protein reported to have weak binding to MKP, a phosphatase to inactivate ERK. In addition to HSC6, we examined 3 cell lines with EGFR amplification, and found that EGFR was phosphorylated at high levels for 846 and 1068 positions upon EGF treatment and the down regulation was mostly suppressed. Moreover, we found a cell line in which MKP1/2 was not detectably induced. The above changes in growth signaling of OSCC might have contributed at least in part to the constitutive activation of ERK. (2) By yeast 2 hybrid system we isolated two proteins that had not been known to interact with ERK2. Nafi was phosphorylated through EGF/ERK. signaling, while attenuated EGF/ERK signaling. Further experiments showed the protein to be super-phsphoaated (SP) in M phase and the treatment with G2/M inhibitors induced SP species. Besides, we found the C-terminal 1/3 of Nafi enhanced to induce apoptosis. (3) 15 tumor specimen of oral squamous cell carcinoma were examined for levels of phosphorylation of ERK which was related to clinicopathological data. There was good correlation to histopathological differentiation grade but not to TNM classification. (4) RNA expression levels of 35 drug-resitnat genes were not changed in OSCC cell linss irrespective of sensitivity to etoposide, but the expression of etoposide-regulated 2 genes was differentially expressed according to the sensitivity to etoposide. (5) Hypermethylation of 5 tumor-suppressor related genes was found in OSCCs of India but not in normal tissues, which could be used as diagnostic markers.