Abstract C03: Role of human endogenous retrovirus-K in phenotype-switching of metastatic melanoma cells during microenvironment alterations
Authors: Argaw-Denboba Ayele , Balestrieri Emanuela , Serafino Annalucia , Bucci Ilaria , Cipriani Chiara , Sorrentino Roberta , Spadafora Corrado , Sinibaldi-Vallebona Paola , Matteucci Claudia 
Published: October 2016

Abstract
Background: Malignant melanoma is one of the most aggressive types of skin cancers and its etiology is not yet clear. Phenotypic-switching has been associated with melanoma tumor aggressiveness and metastasis. Microenvironmental factors such as estrogen, cytokines and other stress conditions determine epigenetic events, as hypo-methylation, which are involved in cell transformation. Interestingly, endogenous retroviruses contribute to melanoma progression and are transcriptionally regulated by epigenetic events. Previously, we demonstrated that the aggressiveness and immune evasion of metastatic melanoma is partly depends on human endogenous retrovirus-K (HERV-K) activation. Thus, studying the role of HERV-K activation in melanoma phenotypic-switching is important to understand melanomagenesis and find possible therapeutic targets.
Objective: To investigate the potential role of HERV-K activation in melanoma cells phenotypic-switching in response to change in microenvironment conditions.
Methods: the TVM-A12 cell line, isolated in our laboratory from a metastatic melanoma lesion, and other commercial melanoma cell lines (WM-266-4, WM-115 and A375) were cultured in standard, differentiation and serum-free cell culture media. RNA interference, flow cytometry, qRT-PCR, self-renewing assay, sphere-forming assay and migration/invasion assays were performed. Data were analyzed using SPSS software version 17.
Results: TVM-A12 showed high cellular plasticity as capable to acquire different phenotypes depending on the modification of the microenvironment. Indeed, in differentiation media the TVM-A12 modified the morphology towards a more differentiated phenotype, but, surprisingly, the cells were not committed to a final stage of differentiation and were able to revert to the original phenotype. The exposure of TVM-A12 to a serum-free medium, induced changes in cell growth and morphology, from adherent towards sphere-like cellular aggregates, characterized by an increased activation of HERV-K expression and generation of a CD133+ subpopulation of melanoma cells. Under this stress-condition, the silencing of HERV-K expression in TVM-A12 cells by RNA interference, significantly abolished the generation of the CD133+ subpopulation, dysregulated the cellular aggregates phenotype and suppressed their proliferation. More appreciably, the induction of HERV-K expression and the stress-mediated generation of CD133+ subpopulation of melanoma cells were also seen in other primary and metastatic melanoma cell lines like WM-115, WM-266-4 and A375 with a considerable variability. Furthermore, theTVM-A12-CD133+ cells, sorted from TVM-A12, showed dynamic cell plasticity upon the modification of the microenvironments and displayed a significantly higher self-renewing, migration and invasion capacity than the parental TVM-A12 cells. Moreover, treatment of TVM-A12 and TVM-A12-CD133+ with the non-nucleoside reverse transcriptase inhibitors (NNRTIs), nevirapine and efavirenz, inhibited the expression of HERV-K and significantly induced high levels of apoptosis in TVM-A12-CD133+ cells.
Conclusions: these results demonstrated for the first time that HERV-K has a decisive role on phenotype-switching in metastatic melanoma cells, and the generation of the more aggressive CD133+ subpopulation in stressful microenvironments. Moreover, NNRTIs treatments were able to inhibit the expression of HERV-K and affect the stress-mediated generation of the CD133+ subpopulation. Thus, the further understanding of the dynamics of HERV-K in melanoma will help to understand melanomagenesis and find possible new therapeutic targets.
Citation Format: Ayele Argaw-Denboba, Emanuela Balestrieri, Annalucia Serafino, Ilaria Bucci, Chiara Cipriani, Roberta Sorrentino, Corrado Spadafora, Paola Sinibaldi-Vallebona, Claudia Matteucci. Role of human endogenous retrovirus-K in phenotype-switching of metastatic melanoma cells during microenvironment alterations. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C03.