Molecular mechanism of INAFM2 in metastasis

PROJECT ABSTRACT Metastasis is the major cause of mortality in cancer patients and accounts for about 90% of cancer deaths, which has not changed in the past 50-years. This indicates a significant need for further study of metastasis pathways to find novel druggable targets. Our lab studies Ewing sarcoma (ES), which is an

aggressive childhood cancer that occurs in the bones or soft tissue. The five-year survival rate for Ewing sarcoma patients diagnosed with metastatic disease is still only 25% making ES a good model to use for metastasis study. To discover genes whose activation leads to increased metastasis, a genome-wide CRISPR/Cas9 transcriptional

activation screen was performed in a zebrafish xenograft model. The gene INAFM2 was chosen for further study and was overexpressed in six different cancer cell lines (four ES, one hepatocellular carcinoma, and one stomach adenocarcinoma). Overexpression of INAFM2 caused increased cell migration and chemotaxis in vitro, and

increased metastasis in zebrafish xenografts. INAFM2 knockdown in four cancer cell lines (two ES, one clear cell renal cell carcinoma, and one stomach adenocarcinoma) decreased chemotaxis and rescue of INAFM2 expression restored the cells’ ability to migrate. Analysis of patient tumor RNA sequencing data from many

different types of cancers revealed correlations between INAFM2 expression and both an increased metastatic phenotype and poor patient survival. This provides strong evidence for a substantial role of INAFM2 in cancer metastasis. However, there is no information currently available about the human INAFM2 protein. This grant

aims to address the gap in knowledge for this novel protein by pursuing the following two specific aims: The first major aim of this proposal is to discern the molecular pathways that mediate the role of INAFM2 in promoting metastasis using in vitro and in vivo models. To determine the mechanism underlying INAFM2’s

metastasis-promoting function, I performed RNA sequencing on ES cells following overexpression or knockdown of INAFM2, which revealed significant enrichment in the MAPK and Wnt pathways among others. I will study these pathways in vitro and in vivo by using the INAFM2 overexpression and knockdown cell lines that I already

generated. The second major aim of this proposal is to discover and characterize the functional significance of the direct protein binding partners and post-translational modifications (PTMs) for INAFM2. I will perform proximity-dependent biotinylation and mass spectrometry (MS) to identify binding partners and use tandem

MS/MS to identify glycosylation and phosphorylation sites on INAFM2. Overall, these two aims will fully confirm the novel protein INAFM2’s role in cancer metastasis and determine its mechanism of function, potentially revealing therapeutic vulnerabilities for design of future drug therapies.