Defining oncogenic capacities of PAK7 mutations in human cancer

 描述（由申请人提供）：基因组测序工作已将PAK 7（p21激活激酶7）鉴定为潜在的重要癌症激酶。PAK 7在所有518种激酶中携带人类癌症驱动突变的概率排名第17位。人类癌症中的绝大多数PAK 7突变是错义的，因此暗示了作为致癌基因的作用。PAK 7突变发生在相当大比例的肺癌和胃肠道癌以及恶性黑色素瘤中。我们的初步数据表明，PAK 7中的癌症突变有效地刺激激酶活性。然而，PAK 7是一种最低限度表征的激酶，因此提供了解决以下目标的优点：（1）推定的PAK 7驱动突变是癌细胞中致癌能力所需的吗？(2)假定的PAK 7驱动突变足以促进上皮肿瘤发生吗？我们将通过基因组编辑严格解决这些目标，以避免与外源表达或基因敲减相关的混淆效应。首先，我们将纠正人类癌细胞系中的PAK 7错义突变。然后，我们将确定这些突变的校正是否恢复了这些细胞系所具有的任何转化能力。具体地说，我们将检测细胞增殖、凋亡、侵袭和致瘤性。 无胸腺小鼠其次，我们将把假定的PAK 7驱动突变引入相对分化良好的结肠Caco-2细胞和支气管16 HBE 140-细胞中。除了上述能力外，我们还将确定PAK 7癌症突变对正常上皮结构参数的影响;这是具有既定肿瘤抑制能力的分化上皮细胞的标志。具体来说，我们将测试癌症中天然存在的PAK 7突变是否会影响3D培养中的上皮细胞极性、细胞间粘附和管腔形成。总的来说，拟议的研究将提供一种严格的初始方法来定义PAK 7突变在人类癌症中的致癌潜力，并阐明PAK 7是否代表了扩大癌症治疗靶点的有希望的候选者。

 DESCRIPTION (provided by applicant): Genome sequencing efforts have identified PAK7 (p21-activated kinase 7) as a potentially important cancer kinase. PAK7 ranked 17 among all 518 kinases in probability of harboring driver mutations for human cancer. The vast majority of PAK7 mutations in human cancer are missense and thus suggestive of a role as an oncogene. PAK7 mutations occur in a significant proportion of lung and gastrointestinal carcinomas, as well as in malignant melanomas. Our preliminary data suggest that cancer mutations in PAK7 potently stimulate kinase activity. However, PAK7 is a minimally characterized kinase, thus providing merit to address the following aims: (1) Are putative PAK7 driver mutations required for oncogenic capacities in cancer cells? (2) Are putative PAK7 driver mutations sufficient to promote epithelial oncogenesis? We will rigorously address these aims by genome editing to avoid confounding effects associated with exogenous expression or knockdown of genes. First, we will correct PAK7 missense mutations in human cancer cell lines. We will then determine whether correction of these mutations reverts any transformed capacities that these cell lines possess. Specifically, we will assay cell proliferation, apoptosis, invasion, and tumorigenicity in athymic mice. Second, we will introduce putative PAK7 driver mutations into the relatively well-differentiated colonic Caco-2 cells and bronchial 16HBE14o- cells. In addition to the capacities listed above, we will determine effects of PAK7 cancer mutations on parameters of normal epithelial architecture; a hallmark of differentiated epithelial cells with established tumor suppressor capacities. Specifically, we will test whether naturally occurring PAK7 mutations in cancer affect epithelial cell polarity, cell-cell adhesion, and lumen formation in 3D culture. Collectively, the proposed studies will provide a stringent initial approach to define the oncogenic potential of PAK7 mutations in human cancer and elucidate whether PAK7 represents a promising candidate in expanding targets for cancer therapeutics.