Experimental gene therapy employing DUSP6/MKP-3 for pancreatic cancer

12q21和12q22-q23.1位点杂合性缺失在原发性胰腺癌中较为常见，位于12q22位点的DUSP6/MKP-3基因在绝大多数胰腺癌细胞系中缺失表达。DUSP6/MKP-3蛋白是一种双特异性磷酸酶，可使ERK的活性形式去磷酸化，形成一个反馈回路来控制ERK的活性。KRAS2的功能获得性突变发生在绝大多数胰腺癌细胞中，DUSP6/MKP-3的表达缺失可能协同促进ERK的组成性激活和不受控制的细胞生长。为了研究反馈通路的缺失及其对胰腺癌细胞生长的影响，我们采用免疫组织化学方法研究了DUSP6/MKP-3在原发性胰腺癌组织中的表达；在轻度和重度发育异常原位癌细胞中表达上调，而在浸润性癌，尤其是低分化型中表达下调。腺病毒介导的将DUSP6/MKP-3重新引入培养的胰腺癌细胞，诱导重组DUSP6/MKP-3和磷酸化ERK还原蛋白以剂量依赖性的方式表达，并导致细胞生长受到抑制。此外，流式细胞术和免疫细胞化学分析显示，外源表达DUSP6/MKP-3可诱导细胞凋亡。在一项实验基因治疗的人胰脏细胞肿瘤在裸鼠显示短暂的肿瘤生长迟缓。然而，为了获得更有效的治疗效果，应改变剂量和给药方式。这些结果表明DUSP6具有明显的肿瘤抑制作用，提示DUSP6是12q22位点的强候选肿瘤抑制基因，是人胰腺癌的治疗靶点之一。

The loss of heterozygosity at 12q21 and 12q22-q23.1 is frequently found in primary pancreatic cancers ard DUSP6/MKP-3 gene residing in this region at 12q22 lost its expression in the great majority of pancreatic cancer cell lines. The DUSP6/MKP-3 protein is a dual spcificity phosphatase that dephosphorylates the active form of ERK, making a feed back loop to control ERK activity. Gain-of-function mutations of KRAS2 occur ih the great majority of pancreatic cancer cells, and loss of expression of DUSP6/MKP-3 may synergistically promote constitutive activation of ERK and uncontrolled cell growth. To study loss of the feed back pathway and its impact to pancreatic cancer cell growth, the expression of DUSP6/MKP-3 in primary pancreatic cancer tissues was investigated immunohistochemically ; upregulation in mildly as well as severely dysplasticlin situ carcinoma cells and downregulation in invasive carcinoma, especially that of the poorly differentiated type were found. Adenovirus-mediated reintroduction of DUSP6/MKP-3 into cultured pancreatic cancer cells induced strong expression of recombinant DUSP6/MKP-3 and reductin of phosphorylated ERK in a dose-dependent manner based on the multiplicity of infection and resulted in suppression of cell growth. Moreover, analyses by flow cytometry and immunocytochemistry revealed that the exogenous expression of DUSP6/MKP-3 induced apoptosis. In an experimental gene therapy for xenographted human pancreatic cell tumors in nude mice revealed transient retardation of tumor growth. However, dose and administrative ways should be altered for more efficient therapy result. These results shovv that DUSP6 exerts apparent tumor-suppressive effects, and suggest that DUSP6 is a strong candidate tumor suppressor gene at 12q22 locus and one of therapeutic targets of human pancreatic cancer.