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.
Although signaling and endocytic trafficking of the epidermal growth factor receptor (EGFR) have been studied in the context of mitogenic activation of cells and tumorigenesis, the molecular events that regulate EGFR levels and signaling, and their contribution to oral tumorigenesis are largely unknown. Studies proposed in this application will determine for the first time how an essential cellular protein, CPAP regulates EGFR signaling and EMT of oral cancer cells, independent of its function on the centrioles.
