A novel ESCRT-associated regulatory mechanism of EMT in oral cancer

项目描述/摘要 口腔鳞状细胞癌占头颈部鳞状细胞癌的90 （HNSCC）。表皮生长因子受体（EGFR）在包括OSCC在内的大多数肿瘤中过表达。的 过度表达/过度活化的EGFR促进癌症进展和上皮-间质转化 （EMT）。EGFR还有助于肿瘤转移和对化疗的抵抗，并且已经成为肿瘤转移的重要因素之一。 OSCC的主要治疗靶点。内吞作用是配体活化的内源性内吞的关键生物学途径。 EGFR，随后通过内体分选复合物进行溶酶体降解以进行募集 和运输（ESCRT）机械。ESCRT是囊泡运输的关键介质。而囊泡运输缺陷 导致活化的EGFR、持续的表面和细胞EGFR表达和信号传导的不良下调 与癌症的发展有关。令人惊讶的是，与ESCRT相关的分子事件 途径和EMT在很大程度上未知。 在这里，我们建议研究一种以前未知的调节EGFR水平和信号传导的机制， EMT和OSCC生长。我们发现：1）CPAP耗竭导致EGFR和EMT表达延长， 2）CPAP的过度表达导致EGFR阳性细胞的重新产生; 多泡体（MVB），其生物发生需要ESCRT，并且对于将EGFR路由至 溶酶体降解和终止其信号传导;和3）CPAP的缺乏导致减少 细胞水平的VPS 4蛋白，一种必需的ESCRT相关ATP酶，对囊泡分选至关重要。这些 集体观察表明，CPAP调节ESCRT通路和EGFR表达，以避免EMT， 口腔鳞癌细胞。具体目标1的主要目标是确定功能相互作用的动力学 通过a）确定CPAP、VPS 4和EGFR在口腔癌中的表达水平和功能相互作用 CPAP和VPS 4与囊泡运输和OSCC细胞中EGFR降解的概况，和B）研究CPAP和VPS 4与囊泡运输和OSCC细胞中EGFR降解的关系。 CPAP，VPS 4和EGFR丰度和激活在正常和OSCC细胞中的动态， 急救员具体目标2：确定CPAP是否在预防EGFR依赖性EMT和肿瘤发生中发挥作用。 OSCC细胞将集中于a）检查CPAP功能的获得和丧失对VPS 4和EGFR的影响， 丰度和活性，以及生长特性和EMT特征，和B）确定CPAP的获得和丧失 在体内对非侵袭性和侵袭性OSCC细胞的致瘤性有影响。

PROJECT DESCRIPTION/ABSTRACT Oral squamous cell carcinoma (OSCC) accounts for 90% of the head and neck squamous cell carcinoma (HNSCC). Epidermal growth factor receptor (EGFR) is overexpressed in majority of tumors including OSCC. The over-expressed/over-activated EGFR contributes to cancer progression and epithelial-mesenchymal transition (EMT). EGFR also contributes to tumor metastasis and resistance to chemotherapy and 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 vesicle trafficking. While vesicle trafficking defects result in 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 associated with ESCRT pathway and EMT are largely unknown. Here, we propose to investigate a previously unknown mechanism that regulates EGFR levels and signaling, EMT, and OSCC growth. We found that 1) depletion of CPAP caused prolonged expression of EGFR and EMT- like phenotype in oral cancer cells; 2) overexpression of CPAP caused the de novo generation of EGFR-positive multivesicular bodies (MVBs), whose biogenesis requires ESCRT and are essential for routing EGFR to lysosomes for degradation and termination of its signaling; and 3) the absence of CPAP resulted in diminished cellular levels of VPS4 protein, an essential ESCRT associated ATPase that is critical for vesicle sorting. These collective observations suggest that CPAP regulates ESCRT pathway and EGFR expression to avoid EMT in OSCC cells. The primary goals of specific aim 1 will be to determine the dynamics of functional interactions between CPAP, VPS4 and EGFR in oral cancer by a) determining the expression levels and functional interaction profiles of CPAP and VPS4 with vesicle trafficking and degradation of EGFR in OSCC cells, and b) studying the dynamics of CPAP, VPS4 and EGFR abundance and activation in normal and OSCC cells that are undergoing EMT. Specific aim 2 to determine if CPAP has a role in preventing EGFR dependent EMT and tumorigenesis in OSCC cells will focus on a) examining the effects of gain- and loss-of CPAP function on VPS4 and EGFR abundance and activity, and growth properties and EMT features and b) determining if gain- and loss-of CPAP has an impact on tumorigenic properties of non-aggressive and aggressive OSCC cells in vivo.