K. M. Leick1,2, K. Du1, M. Parlak1, T. Abbas1, V. H. Engelhard1, C. L. Slingluff1 1University of Virginia,Charlottesville, VA, USA 2University of Iowa,Iowa City, IA, USA
Introduction:
We have previously reported that survival of patients with melanoma is reduced when melanomas express a set of genes including filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), which encode proteins that mediate mechanical barrier function in normal skin. To determine whether these genes have a causative role in melanoma progression, rather than just being markers associated with poor outcome, we evaluated their effects on melanoma growth in culture and in mice following their deletion using CRISPR/Cas9 or ectopic expression by lentivirus transduction. We hypothesized that overexpression of FLG, DST, PKP3, and JUP may directly enhance melanoma cell growth in vitro and decrease host survival in a murine model, while their deletion will decrease tumor burden and improve survival.
Methods:
Co-knockout of both FLG and DST using CRISPR/Cas9 and sgRNA and control Empty Vector (EV) was performed in human DM93 melanoma cells and in murine B16-F1 melanoma cells. Proliferation of cultured control DM93 EV and sgFLGDST was measured by MTT assay (n=1), and B16-F1 EV and sgFLGDST tumors were inoculated subcutaneously into mice (n=3). Overexpression models of PKP3 and of JUP were also generated by lentiviral transduction of murine B16-AAD melanoma cells, which were implanted intraperitoneally into mice. Tumors were harvested at day 14 and weighed (n=3) to assess tumor burden.
Results:
In vitro studies with human DM93 melanoma demonstrated decreased proliferation after FLG/DST knockout (Figure 1a), suggesting that these genes confer a tumor growth advantage for human melanoma. Similarly, FLG/DST knockout in murine B16-F1 led to significantly decreased tumor burden compared to control in vivo (p=0.02, Figure 1b). Also, overexpression of JUP in murine B16-AAD melanoma resulted in significantly greater tumor burden compared to control (p=0.0001) and compared to PKP3 overexpression (p < 0.0001); however, PKP3 overexpression did not significantly impact tumor growth (Figure 1c).
Conclusion:
FLG, DST, and JUP appear to support melanoma cell growth and survival both in vitro and in vivo. These findings raise the possibility that overexpression of these selected genes may activate pro-survival or proliferation pathways, create stronger intercellular junctions, or may support immune cell exclusion from melanomas. Therefore, FLG, DST, and JUP warrant further study to identify regulatory targets of these genes in order to intervene on their expression to confer clinical benefit.
