PLK1在APC突变相关结直肠癌发生中的作用及机制

Mutation and inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene is an early event in the development of colorectal cancer. Inactivation of APC are not only responsible for one form of inherited colorectal cancer, familial adenomatous polyposis (FAP), but also play the initiation and promoting roles in the majority of sporadic colorectal cancers. ApcMin/+ mice aremodel of FAP and are heterozygous for an Apc truncation mutation, which serves as a classical genetic model for intestinal carcinogenesis. We found that Polo-like kinase 1 (PLK1) was upregulated in both intestinal adenoma formed in APC-Min mice and human colorectal cancer samples. More importantly, reduction in PLK1 levels in APC-Min mice through intercrossing APCMin/+ mice with PLK1+/- mice significantly abrogated the Min (multiple intestinal neoplasia) phenotype in these mice. PLK1 downregulation not only decreased the number of intestinal adenoma, but also reduced the expression of several proliferation related markers, suggesting that PLK1 is critical for Apc-dependent intestinal tumorigenesis via enhancing cell proliferation in mice. PLK1 is a member of the highly conserved serine/threonine protein kinase family. It is a key regulator of cell division and is also a central player in maintaining genome stability during DNA replication and in modulating the DNA damage response. PLK1 is frequently up-regulated in the vast majority of human tumors. Specific small molecule inhibitors against PLK1 display prominent antitumor efficacy with minimal side effects in animal models and in clinical trials. Based on aforementioned observations, we hypothesize that upregulation of PLK1 mediated by APC mutation contributes to the intestinal tumorigenesis through reinforcing malignant proliferation, and targeted suppression of PLK1 may inhibit the intestinal tumor formation mediated by APC mutation. To test this hypothesis, we propose following specific aims: 1) By employing conditional PLK1 knockin mouse and APC-Min mouse model, we will define the role of PLK1 in intestinal carcinogenesis, especially mediated by APC mutation; 2) We will examine whether PLK1 is a potent target for early prevention of intestinal tumorigenesis using APC-Min mouse model; 3) We will clarify molecular mechanisms underlying PLK1 upregulation in APC mutated cells; 4) We will analyze and determine the correlation between APC mutation and PLK1 overexpression, as well as related molecular aberrations in human colorectal tumor samples. This proposed study will unveil a novel mechanism of APC inactivation in intestinal tumor formation. In addition, our animal work will shed light on the role of PLK1 in APC mutation-related intestinal tumorigenesis. The information gained from this study will also provide a promising molecular target for early prevention of colorectal cancer, as well as potential molecular markers for colorectal cancer screening.

抑癌基因APC的突变失活在结直肠癌发生中起主要的启动和促进作用。我们前期研究发现PLK1在携带APC 基因种系突变的结直肠癌小鼠模型APC-Min中表达上调，下调PLK1表达可显著抑制其肠道腺瘤的发生，提示PLK1过表达在APC突变所致结直肠癌中发挥重要作用。本项目将利用APC-Min和PLK1基因敲入小鼠深入分析PLK1在APC突变相关结直肠癌发生中的作用；利用动物模型研究PLK1抑制剂对结直肠癌的预防作用；同时，结合体外研究揭示PLK1在APC突变细胞中表达上调的分子机制；从体内、体外及组织标本三个层面证实我们的假说"APC突变导致PLK1表达上调进而增强细胞的增殖活性促进结直肠癌的发生，抑制PLK1活性可能阻断APC突变所致结直肠癌的发生"，从而揭示PLK1在APC突变相关结直肠癌发生中的作用及PLK1表达上调的分子机制，为结直肠癌早期筛查提供新的分子标志并为其预防提供新的分子靶标。

抑癌基因APC（adenomatous polyposis coli）的突变失活在结直肠癌的发生中起主要的启动和促进作用，但相关分子机制尚未阐明。本项目研究发现蛋白激酶PLK1（polo-like kinase 1）的mRNA和蛋白表达水平在携带APC 基因种系突变的结直肠癌小鼠模型APCMin/+的肠道腺瘤组织中明显升高。利用APCMin/+小鼠、PLK1低表达（PLK1+/-）及PLK1过表达（PLK1fl/+）小鼠进行体内研究证实，APC突变导致的PLK1表达上调可通过增强细胞的恶性增殖能力促进结直肠癌的发生。分子水平的研究结果显示，APC突变的结肠上皮细胞中转录因子SP1和beta-catenin/TCF7L2均可转录激活PLK1的表达。APC突变可使细胞中SP1蛋白的稳定性增强，该作用可能与GSK-3beta对SP1磷酸化作用减弱，进而导致SP1不能被E3连接酶beta-TrCP识别并进行泛素化修饰有关。此外，我们研究发现SP1可以调控beta-catenin的转录，但beta-catenin不参与SP1的转录调控。这些结果提示SP1不仅可以直接转录激活PLK1的表达，而且也可通过激活beta-catenin的转录间接上调PLK1 的表达水平。值得注意的是，PLK1 的mRNA水平在结直肠腺瘤及早期腺癌组织中即有显著升高，提示PLK1可能作为结直肠癌筛查和早期诊断的候选分子标志。更为重要的是，PLK1特异性的小分子抑制剂BI6727可以显著抑制ApcMin/+小鼠肠道腺瘤的发生，提示PLK1是一个预防或早期干预结直肠癌发生的潜在分子靶点。综上所述，本研究揭示了PLK1在APC突变相关结直肠癌发生中的作用及其表达上调的分子机制，为结直肠癌的筛查和早期诊断提供了新的候选分子标志，并且为预防和早期干预结直肠上皮细胞癌变提供了新的潜在分子靶点。