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

项目说明/摘要 表皮生长因子受体(EGFR)在包括口腔鳞状细胞在内的大多数肿瘤中过度表达 癌(OSCC)。过度表达/激活的EGFR参与上皮-间充质转化 (EMT)通过促进肿瘤转移和化疗耐药而促进肿瘤进展。因此，EGFR拥有 成为口腔鳞癌的主要治疗靶点之一。内吞作用是内化的关键生物学途径。 配体激活的EGFR，随后通过内切分选将其送往溶酶体降解 招募和运输综合设施(ESCRT)机械。ESCRT是胞内囊泡的关键调节因子 贩运(EVT)。而囊泡运输缺陷导致激活的EGFR下调较差，持续 表面和细胞EGFR的表达和信号转导随后与癌症的发展有关。 令人惊讶的是，调节ESCRT途径的分子事件/机制对维持 EGFR的动态平衡以及预防EMT和肿瘤的发生在很大程度上是未知的。 在这里，基于可靠的初步数据，我们建议研究一种以前未知的调节机制 ESCRT依赖EVT、EGFR水平和信号、EMT和口腔鳞癌生长。我们发现1)耗尽了 CPAP导致口腔癌细胞EGFR表达延长，出现EMT样表型；2) CPAP缺失增强口腔鳞癌细胞系的致瘤性，CPAP在该细胞系中过表达 抑制其促肿瘤作用；3)CPAP过表达导致EGFR的从头产生 阳性的多囊泡小体，其生物发生需要ESCRT，是途径的必要中间体 EGFR对溶酶体的降解和信号的终止；4)CPAP的缺失导致 Vps4蛋白的细胞水平降低，这是一种与ESCRT相关的重要ATPase，有助于夹断 内吞小泡。这些集体观察表明，CPAP诱导了ESCRT的正向调节 途径和EVT维持EGFR的动态平衡，从而预防口腔鳞癌的EMT和肿瘤发生。 这一新的假设将在两个具体目标下得到系统检验。目标1的主要目标将是 明确CPAP积极调节口腔鳞癌EVT和EGFR动态平衡的分子机制。 这将通过使用口腔鳞癌和具有CPAP功能增减的正常口腔细胞以及通过 在EGFR动态平衡的背景下研究CPAP-ESCRT的相互作用。然后，我们将在目标2下确定 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- 口腔鳞癌细胞系的EMT特征、生长和致瘤特性的AND功能丧失，并通过研究 用条件性CPAP基因敲除小鼠研究口腔癌的易感性。总体而言，这项研究将描绘一部小说 ESCRT依赖的预防口腔鳞癌的机制。

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.