M. Sharma2, P. Jackson3, J. Huang2, D. Grabski1, D. Rekosh2,4, M. Hammarsjkold2,4, S. K. Rasmussen1 1University of Virginia,Surgery,Charlottesville, VA, USA 2University of Virginia,Microbiology, Immunology, And Cancer Biology,Charlottesville, VA, USA 3University of Virginia,Internal Medicine,Charlottesville, VA, USA 4University of Virginia,Thaler Center For HIV And Retrovirus Research,Charlottesville, VA, USA
Introduction: Melanoma is a skin cancer that is responsible for >9,000 deaths In the U.S. yearly; 5-year survival from advanced disease is <40%. Despite advances in the understanding and treatment of this cancer, there are limitations in treatments, and new targets for therapy are needed. More insight into neoantigen expression in melanoma will be beneficial to bringing new innovations to fruition.
Expression from the youngest class of human endogenous retroviruses (HERV-K) is known to be upregulated in melanoma. HERVs are derived from ancient retroviral infections and they comprise 8% of the genome. The effect of HERV-K upregulation in melanomas is unknown. However, HERV-Ks encode several different proteins that can be expressed in human cells. One of these proteins is the regulatory protein Rec. We probed the function of this protein in melanoma cell lines using a novel Lentiviral GFP reporter.
HERV-K Rec is a nuclear-export factor that exports mRNA with retained introns. Some studies postulate that Rec protein may have oncogenic properties, which makes it a provocative target for investigation. Many of the rec sequences in our genome have been subject to deletions and other inactivating mutations during genomic evolution; it remains to be shown if any of the Rec proteins expressed in melanoma cells are functional.
Methods: To analyze the expression of Rec in the SK-28 Mel cell line, we isolated, amplified, and cloned rec mRNAs expressed in these cells. Multiple cDNA clones were then sequenced. The most commonly detected variants were cloned and tested for functionality. Next, we utilized a lentiviral vector that had been engineered to express green fluorescent protein (GFP) only in the presence of functional Rec protein, to determine if Rec protein produced by melanoma cell lines was functional. We utilized a protein that inactivates Rec (Td-Rev) to eliminate native cellular Rec activity, in order to confirm that the induced GFP expression from our vector was Rec-dependent.
Results:65% of Rec cDNA clones obtained from the SK-28 cells had an open reading frame identical to a previously identified functional Rec, whereas 35% of the clones contained mutations, implying that multiple rec loci were expressed in this cell line. Three rec mutants were tested in transfection assays. One was shown to have decreased activity, whereas the other two were non-functional. We found that the Lentiviral vector that expresses Rec-dependent GFP showed significant activity in SK-28 cells, indicating expression of functional Rec protein in these cells. When Td-Rev, a protein that inhibits Rec activity, was co-transfected, GFP expression was reduced to background levels.
Conclusion:SK-Mel28 cells express functional Rec protein as well as non-functional Rec proteins from mutated loci. Furthermore, expression of a Rec dependent GFP reporter can be inhibited by targeting Rec. Further studies on the role of Rec in melanoma are warranted, as Rec may be a viable therapeutic target.