Search for Molecular Targets by Proteome Analysis and Its Use to Taylored-Made Therapy against Oral Cancer

通过双向PAGE和MALDI-TOFMS分析，我们发现在口腔癌细胞系中维生素A特异性地诱导了主要组织相容性复合体I类链相关分子A(MICA)蛋白，它是大多数NK细胞、LAK细胞和细胞毒性T细胞上的激活受体NKG2D的配体。逆转录聚合酶链式反应(RT-PCR)和流式细胞仪(FACS)分析显示维生素A衍生物ATRA分别诱导口腔癌细胞MICA基因和蛋白的表达。我们采用直接测序和片段分析的方法分析了100例口腔鳞状细胞癌(OSCC)和103例非相关对照的MICA基因多态性(跨膜区GCT三联体重复序列多态性)。结果发现，口腔癌患者MICA5.1表型的比例为30%，对照组为15%(X^2=16.203，P值=0.00006)。有5.1个等位基因的患者NK活性低于其他等位基因。通过流式细胞仪分析，所有细胞系均检测到细胞表面MICA蛋白。据报道，肿瘤有逃避人体免疫系统的方法。作为其中的一个系统，血清中存在多种形式的云母(Smica)。用双抗体夹心ELISA法检测了45例口腔鳞癌患者血清中的SMICA。我们发现口腔鳞癌患者的SMICA水平显著高于对照组。另一方面，血清SMICA水平与口腔鳞癌患者的临床状态密切相关，可作为一种新的口腔鳞癌生物标志物。提示口腔鳞癌的易感性与MICA基因微卫星多态性密切相关，5.1等位基因可能是口腔鳞癌的危险因素，血清SMICA水平可作为口腔鳞癌诊断、治疗和预后的新的分子靶点/生物标志物。

By two-dimensional PAGE and MALDI-TOFMS analysis, we have found that a major histocompatibility complex class I chain-related molecule A (MICA) protein, the ligand of NKG2D which is activation receptor on the most NK cells, LAK cells, and cytotoxic T cells, was specifically induced by vitamin A in oral cancer cell lines. RT-PCR and FACS analysis revealed that vitamin A derivative such as ATRA induced mRNA and protein expression of MICA in oral cancer cell lines, respectively.We analysed MICA polymorphism (GCT triplet repeat polymorphism in the transmembrane domain) in 100 cases with Oral Squamous Cell Carcinoma(OSCC), and 103 randomly selected unrelated controls by direct sequencing and fragment analysis. Five distinct MICA polymorphisms were studied.As a result, we have found that the percentage of the patient who has MICA 5.1 phenotype among total oral cancer patients is 30%, but that of control is 15% (X^2=16.203,P value=0.00006). NK activity of the patient of 5.1 allele is lower than that of the others of other alleles. By flow-cytometric analysis, MICA protein on the cell surface was detected in all cell lines. It has been reported that tumors have ways of evading the body's immune system. As one of the system of these, there are soluble forms of MICA (sMICA) in the serum. We have examined sMICA in the serum of 45 OSCC patients by ELISA. We have found the sMICA levels in OSCC patients were significantly higher than those in controls. On the other hand, sMICA serum concentration strongly correlated with the clinical state of OSCC patients and might serve as a novel biomarker of OSCC.These results suggest that the susceptibility to OSCC is closely linked to the MICA microsatellite polymorphism and 5.1 allele might confer the risk of OSCC, and that serum levels of sMICA may serve as a novel molecular target/biomarker of OSCC for its diagnosis, therapy, and prognosis.