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

项目说明/摘要 口腔鳞癌(OSCC)占头颈部鳞状细胞癌的90% (HNSCC)。表皮生长因子受体(EGFR)在包括口腔鳞癌在内的大多数肿瘤中都有过表达。这个 过度表达/过度激活的EGFR在肿瘤进展和上皮间质转化中的作用 (EMT)。EGFR还导致肿瘤转移和对化疗的耐药性，已成为 口腔鳞癌的主要治疗靶点。内吞作用是激活配体内化的关键生物学途径 EGFR，之后它被内体分选复合体运送到溶酶体降解以进行招募 和运输(ESCRT)机械。ESCRT是囊泡运输的关键调节因子。而水泡运输缺陷 导致活化的EGFR、持续性表面和细胞的EGFR表达和信号下调较差 随后与癌症的发展有关。令人惊讶的是，与ESCRT相关的分子事件 通路和EMT在很大程度上是未知的。 在这里，我们建议研究一种以前未知的调节EGFR水平和信号转导的机制， EMT，和口腔鳞癌的增长。我们发现：1)CPAP耗竭导致EGFR和EMT-1表达延长。 口腔癌细胞的表型相似；2)CPAP的过表达导致EGFR阳性细胞的新生产生 多泡小体(MVB)，其生物发生需要ESCRT，并且是将EGFR路由到 溶酶体对其信号的降解和终止；3)CPAP的缺失导致其功能减弱 Vps4蛋白的细胞水平，这是一种与ESCRT相关的重要ATPase，对囊泡分类至关重要。这些 集体观察提示CPAP调节ESCRT途径和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态 口腔癌组织中CPAP、Vps4和EGFR的表达水平及功能相互作用 CPAP和Vps4在口腔鳞癌细胞中的囊泡转运和EGFR降解的分布，以及b)研究 正常和口腔鳞癌细胞中CPAP、Vps4和EGFR丰度和活化的动态变化 急诊室。特异性目标2确定CPAP是否在预防EGFR依赖的EMT和肿瘤发生中发挥作用 口腔鳞状细胞癌细胞将专注于a)检测CPAP功能的增减对Vps4和EGFR的影响 丰度和活性、生长特性和EMT特征以及b)决定CPAP的得失 对体内非侵袭性和侵袭性口腔鳞癌细胞的致瘤特性有影响。

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.