Metastasis is a complex process in which cancer cells spread from a primary tumor site to invade surrounding tissue, enter the bloodstream, and propagate into distant organs. One of the methods in which metastasis occurs is through Epithelial-to-Mesenchymal Transition, where the extracellular matrix is degraded and restructured; consequently, this enhances cellular migration, invasion, and ultimately, metastasis. A key component of this process is the formation of actin-rich protrusions called invadopodia that serve to break down the extracellular matrix, thereby facilitating cancer invasion. Meanwhile, the E3 ubiquitin ligase ITCH, a key regulator in ubiquitination, has an unexplored role in invadopodia formation. My project thus aims to elucidate ITCH's contribution to tumor invasion via invadopodia-mediated degradation across various cancer cell lines.

I initially focused on ITCH’s role in invadopodia-mediated degradation in the MDA-MB-435 (melanoma) and MDA-MB-231 (triple-negative breast cancer) cell lines, whose ability to form invadopodia was unknown. Degradation and immunofluorescence assays were conducted at various time points and cell confluences. Based on these results, the analysis was expanded to more aggressive cell lines: A673 (Ewing sarcoma), EWS-502 (Ewing sarcoma), TC-32 (neuroectodermal), and SK-N-MC (neuroblastoma). Degradation assays were repeated at different time points and concentrations. Promising results led to further investigation of A673 and EWS-502 by downregulating ITCH expression via lentivirus-mediated shRNA gene knockdown and repeating the degradation assays. Despite comprehensive analysis, the results were inconclusive. However, this project provided preliminary data and established a foundation for future studies to determine the precise role of ITCH in invadopodia formation and degradation, particularly in the A673 and EWS-502 cell lines.