ESCRT-dependent novel regulatory mechanism of EMT and tumorigenesis in oral cancer

项目描述/摘要 表皮生长因子受体（EGFR）在包括口腔鳞状细胞癌在内的大多数肿瘤中过表达， 癌（OSCC）。EGFR的过度表达/激活促进上皮-间质转化 (EMT)以及通过促进肿瘤转移和化疗抗性而导致肿瘤进展。因此，EGFR具有 成为口腔鳞癌的主要治疗靶点之一。内吞作用是细胞内化的重要生物学途径 配体激活的EGFR，随后通过内体分选将其路由至溶酶体降解 招募和运输综合体（ESCRT）。ESCRT是内吞囊泡的关键介质 贩运人口（EVT）。虽然囊泡运输缺陷导致激活的EGFR的不良下调，但持续性的EGFR下调可能是一个潜在的原因。 表面和细胞EGFR表达和信号传导随后与癌症的发展相关联。 令人惊讶的是，调控ESCRT途径的分子事件/机制对于维持ESCRT的稳定至关重要。 EGFR稳态和预防EMT和肿瘤发生在很大程度上尚不清楚。 在这里，基于坚实的初步数据，我们建议调查一个以前未知的机制，调节 ESCRT依赖性EVT、EGFR水平和信号传导、EMT和OSCC生长。我们发现：（1） CPAP导致口腔癌细胞EGFR表达延长和EMT样表型; 2） CPAP的缺失增强了OSCC细胞系的致瘤性，CPAP在该细胞系中的过表达 抑制其肿瘤诱导潜力; 3）CPAP的过度表达导致EGFR的重新产生- 阳性多泡体，其生物发生需要ESCRT，并且是途径 EGFR对溶酶体的降解和终止其信号传导;和4）CPAP的缺乏导致 VPS 4蛋白的细胞水平降低，VPS 4蛋白是一种重要的ESCRT相关ATP酶， 内吞囊泡的结构这些共同的观察结果表明，CPAP诱导的ESCRT的积极调节， EMT通路和EVT维持EGFR稳态，从而预防OSCC中的EMT和肿瘤发生。 这一新的假设将在两个特定的目标下进行系统的测试。目标1的主要目标是 明确CPAP积极调节OSCC中EVT和EGFR稳态的分子机制。 这将通过使用OSCC和具有CPAP功能获得和丧失的正常口腔细胞以及通过 研究EGFR稳态背景下CPAP-ESCRT相互作用。然后，我们将根据目标2，确定 CPAP在预防EMT和口腔肿瘤发生中的作用。这将通过表征CPAP增益来实现- 和功能丧失的OSCC细胞系的EMT特征，生长和致瘤特性，并通过研究 使用条件性CPAP敲除小鼠的口腔癌易感性。总的来说，这项研究将描绘一部小说 ESCRT依赖的机制，防止口腔鳞状细胞癌。

PROJECT DESCRIPTION/ABSTRACT Epidermal growth factor receptor (EGFR) is overexpressed in majority of tumors including oral squamous cell carcinoma (OSCC). The over-expressed/-activated EGFR contributes to epithelial-mesenchymal transition (EMT) and tumor progression by contributing to tumor metastasis and chemo-resistance. Hence, EGFR has become one of the major therapeutic targets for OSCC. Endocytosis is a key biological pathway for internalization of ligand activated EGFR, following which it gets routed for lysosomal degradation by the endosomal sorting complex for recruitment and transport (ESCRT) machinery. ESCRT is a key mediator of endocytic vesicle trafficking (EVT). While vesicle trafficking defects result in the poor downregulation of activated EGFR, persistent surface and cellular EGFR expression and signaling is subsequently linked to the development of cancer. Surprisingly, the molecular events/mechanisms that regulate ESCRT pathway which is critical for maintaining EGFR homeostasis and preventing EMT and tumorigenesis are largely unknown. Here, based on solid preliminary data, we propose to investigate a previously unknown mechanism that regulates ESCRT dependent EVT, EGFR levels and signaling, EMT, and OSCC growth. We found that 1) depletion of CPAP caused the prolonged expression of EGFR and an EMT-like phenotype in oral cancer cells; 2) while depletion of CPAP enhanced the tumorigenicity of an OSCC cell line, overexpression of CPAP in this cell line suppressed its tumor inducing potential; 3) overexpression of CPAP caused the de novo generation of EGFR- positive multivesicular bodies, whose biogenesis requires ESCRT and are essential intermediates that route EGFR to lysosomes for degradation and termination of its signaling; and 4) the absence of CPAP resulted in diminished cellular levels of VPS4 protein, an essential ESCRT associated ATPase that facilitates pinching off of endocytic vesicles. These collective observations suggest that CPAP induced positive regulation of ESCRT pathway and EVT maintains EGFR homeostasis, resulting in prevention of EMT and tumorigenesis in OSCC. This novel hypothesis will be tested systematically under two specific aims. The primary goals of aim 1 will be to define the molecular mechanisms by which CPAP positively regulates EVT and EGFR homeostasis in OSCC. This will be done by using OSCC and normal oral cells with gain-and-loss-of-function of CPAP as well as by studying CPAP-ESCRT interaction in the context of EGFR homeostasis. We will then, under aim 2, determine the role of CPAP in preventing EMT and oral tumorigenesis. This will be achieved by characterizing CPAP gain- and-loss-of-function in OSCC cell lines for EMT features, and growth and tumorigenic properties, and by studying the oral cancer susceptibility using a conditional CPAP-knockout mice. Overall, this study will delineate a novel ESCRT dependent mechanism that prevents OSCC.