ASAP1激活Wnt通路促进喉鳞癌发生上皮间质转化的机制探究

Epithelial-mesenchymal transition (EMT) has a critical role in promoting the invasion and metastasis of tumor. We have established nude mice lung metastasis model of subcutaneous transplantation tumor using laryngeal squamous cancer cell line successfully, and found that ASAP1 expression raised most significantly in lung metastasis tumor tissues compared to that in subcutaneous transplantation tumor tissues, based on the gene expression analysis. Moreover, downregulation of ASAP1 in Hep-2 cells transfected with ShRNA-ASAP1 lentivirus resulted in downregulated β-catenin expression of the Wnt/β-catenin pathway compared to the control groups, which correlated to increase of E-cadherin, an epithelial marker. The goals of this research are to demonstrate the effect of ASAP1 through regulating Wnt/β-catenin pathway on the EMT and invasion of laryngeal squamous cancer, to explore the proteins interconnecting with ASAP1 using genes expression profile chip, co-immunoprecipitation technology and mass spectrometric analysis, and so on. Based on the above results, the mechanism of ASAP1 regulates EMT and promotes the invasion and metastasis of LSCC will be described. Moreover, we will detect ASAP1 expression of the plasma and tumor tissues of patients with LSCC, and evaluate its relationship with the tumor’s metastasis and patients’ prognosis. The project will improve the understanding of the invasion and metastasis of LSCC and provide the theoretical basis for molecules treatment targeting ASAP1.

上皮间质转化（EMT）和肿瘤细胞的侵袭转移能力密切相关。我们前期利用喉鳞癌细胞系建立裸鼠皮下移植瘤肺转移模型，取肺转移病灶及皮下瘤进行表达谱检测，发现差异表达基因中，肺转移病灶ASAP1表达相对于皮下瘤上调最为显著，进一步检测发现干扰ASAP1后, Wnt通路的β-catenin下调，上皮标志物E-cadherin上调。因此我们推测：ASAP1可能通过激活Wnt/β-catenin信号通路促进喉鳞癌发生上皮间质转化。我们拟：（1）验证喉鳞癌中ASAP1的功能；（2）通过表达谱芯片、免疫共沉淀等方法研究ASAP1调控的基因和蛋白，运用信号转导芯片等研究ASAP1对Wnt/β-catenin通路的调控，描绘ASAP1调控喉鳞癌EMT促进其侵袭转移的信号网络图；（3）评价ASAP1的表达与患者喉鳞癌转移及预后的关系。该课题将加深对喉癌侵袭转移机制的认识，为其防治提供新的靶分子和理论基础。

喉癌是上呼吸道常见的恶性肿瘤之一，占头颈部恶性肿瘤的25%，占全身恶性肿瘤发病率的1%-5%，严重危害人类的健康。病理分型上95%以上是鳞状细胞癌。尽管目前综合性治疗方法，包括手术治疗、放疗、化疗和免疫疗法等，广泛应用于喉癌治疗当中，但依然存在复发率高、易转移和预后差等缺陷，其远期疗效和预后并未带来突破性改变。喉癌的整体五年生存率近20年来无明显提高，而且晚期喉癌患者五年生存率有下降趋势[4]。其原因归咎于喉癌的侵袭和转移。ASAP1/DEF1(分化增强因子)是一种具有多个功能域的磷脂依赖性ARFGTP激活蛋白。其生物学活性广泛，其和肿瘤的增殖侵袭、迁移关系密切。目前在喉癌领域的研究尚属空白。本课题通过临床标本实验发现喉鳞癌中ASAP1表达相对癌旁组织显著上调，ASAP1与喉鳞癌的分化、临床进展、TNM分期、淋巴结转移相关，生存分析曲线分析ASAP1是喉鳞癌预后不良的重要标记物。下调ASAP1可以明显抑制喉鳞癌的增殖侵袭、迁移、周期凋亡等恶性表型。上皮间质转化（EMT）和肿瘤细胞的侵袭转移能力密切相关。干扰ASAP1可以明显抑制喉癌上皮间质转化。我们还发现ASAP1是通过抑制wnt通路的重要负性调控蛋白APC、MAP3K7，进而激活wnt通路的核心关键蛋白β-catenin，通过入核激活wnt通路下游进而促进喉鳞癌上皮间质转化，我们还发现ASAP1可以明显促进喉鳞癌的糖酵解。该项目的成功实施将有助于加深对喉癌侵袭转移机制的认识，为其防治提供新的靶分子和理论基础。