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College, Institute, or Department
Eppley Institute for Research in Cancer
Prostate cancer (PC) is the second leading cause of death due to cancer among men in the United States. Metastatic disease is commonly seen in sites such as lymph nodes, bone and in visceral organs like lung and liver. The 5 year survival rate of patients with primary PC is almost 100%, however, this survival rate is drastically reduced to 30% in patients with metastatic disease. Therefore there is an unmet need to identify genes associated with metastatic progression and develop therapeutic strategies to increase survival rate in patients with aggressive disease. The loss of TAM kinase family receptor AXL is observed in metastatic sites, suggesting that AXL is a potential tumor suppressor in metastatic PC. Data from our laboratory further supports that loss of AXL gene is significantly co-deleted with tumor suppressors PTEN and TP53 in patients with metastatic PC. To test the functional role of AXL gene in metastasis, we generated lentiviral plasmid constructs to express a) doxycycline based inducible knockout of AXL (CRISPR-Cas9 gene editing, and b) overexpression of full length and domain mutants of AXL. Transformation efficiency indicated effective cloning, and long read sequencing with Oxford Nanopore technology confirmed that the cloned inserts were successful. A plasmid map was created for each construct using Snapgene software, and bioinformatic analysis of AXL gene was performed using NIH BLAST. Next, we transduced PC cell lines with lentivirus expressing the designed vectors. The results indicate that overexpression of full length Axl (mouse) in LNCaP-FGC cell line exhibited a rounded morphology with Axl expression (EGFP tag) confined to the plasma membrane and cytoplasm. Interestingly, cells expressing the kinase dead mutant of Axl protein (K561R in the ATP binding site) showed an elongated morphology with distinct plasma membrane localization. Future directions for research include a migration assay to probe the metastatic potential of cells with KO or overexpression of Axl. We will also compare full length vs kinase dead/domain mutants to see the rate of migration. Furthermore, we will also test the efficiency of doxycycline inducible expression of single guide RNAs to knockout AXL in PC cell lines. These systems can now be utilized to test our hypothesis that AXL loss induces metastasis in PC.
Metastatic prostate cancer, AXL, lentiviral transduction, CRISPR-Cas9 gene editing, molecular cloning
Bernal, Anastasia; Ryu, Borum; and Mathew, Grinu, "Defining the Role of AXL in Metastatic Prostate Cancer" (2022). Posters: 2022 Summer Undergraduate Research Program. 4.