HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features

Abstract

Background: Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications.

Methods: TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression.

Results: Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells.

Conclusions: HERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.

Keywords: CD133; Cancer stem cell; HERV-K; Melanoma; Microenvironment; Retroelements.

Fig. 1

TVM-A12 cells exhibit adaptive plasticity, phenotype switching and concomitant up-regulation of HERV-K expression upon microenvironmental modifications. a Phase contrast microscopy of TVM-A12 grown in RPMI-1640 complete medium, in stem cell medium (X-VIVO) or upon the addition of increasing percentage of serum (up to 10% of FBS), where cells reverse their original phenotype within 72 h; original magnification: left panels, 10x; right panels, 20x. b Flow cytometry analysis after extracellular staining of TVM-A12 melanoma cells undergoing from adherent growth, in RPMI-1640 complete medium, to grape-like cellular aggregates in X-VIVO medium. c Flow cytometry analysis after intracellular staining of TVM-A12 melanoma cells undergoing from adherent growth, in RPMI-1640 complete medium, to grape-like cellular aggregates in X-VIVO medium. d Fluorescence microscopic analysis of Hoechst 33342 staining on living TVM-A12 cells growing in low serum X-VIVO medium (2% FBS); arrows point to putative cancer stem cells exhibiting low or negative Hoechst positivity in the nuclei. Original magnification: 40x. e Shows identification and characterization of the SP cells from TVM-A12 melanoma cells cultured for 72 h in RPMI-1640 complete medium or X-VIVO using Hoechst 33342 nucleic acid stain by FACS analysis. The FACS dot-plots displays the SP cells co-stained with CD133 marker preincubated with verapamil (left panel) and in the absence of verapamil (middle panel). Likewise, the FACS histograms plots (right panel) displays the percentage of CD133+ cells and the median of the relative fluorescence within the total SP cells in the absence of verapamil. f Relative mRNA expression of HERV-K env gene in TVM-A12 melanoma cells upon modification of culture conditions (** p < 0.001). Data represent the results of three independent experiments

Fig. 2

Cloned TVM-A12-CD133+ cells show adaptive plasticity similar to the parental TVM-A12 melanoma cells. a Sorting of CD133+ subpopulation from TVM-A12 cells by flow cytometry-based side population technique. b Phase contrast microscopy image of adherent cells (left panel) and grape-like cellular aggregates (right panel) of TVM-A12-CD133+ in RPMI-1640 and X-VIVO medium, respectively: magnification 10x. c Flow cytometry analysis of CD133 expression of adherent (left panel) and non-adherent (right panel) TVM-A12-CD133+ cells. Data represent the results of three independent experiments

Fig. 3

TVM-A12-CD133+ cells are endowed with CSCs features more than the parental TVM-A12 cells. a TVM-A12-CD133+ melanoma cells show higher self-renewing potential than TVM-A12 cells. Top panels represent the microscopy pictures of melanospheres from TVM-A12 cells (left) and TVM-A12-CD133+ cells (right); magnification 10x. Bar graph displays the self-renewing efficiency difference between TVM-A12 and TVM-A12-CD133+ cells in 2nd passage (p = 0.004) and 3rd passage (p = 0.012) and between 1st and 3rd passage of TVM-A12-CD133+ cells (** p < 0.001). b TVM-A12-CD133+ cells display higher migratory capacity than TVM-A12 cells. Top panels represent the microscopy pictures of TVM-A12 and TVM-A12-CD133+ migrated through the transwell insert: magnification 20x. Bar graph shows the migration capacity of TVM-A12 and TVM-A12-CD133+ cell lines in the presence or not of 40 ng/ml HGF (** p < 0.001). c TVM-A12-CD133+ cells display higher invasive capacity than TVM-A12 cells. Top panels represent the microscopy pictures of TVM-A12 and TVM-A12-CD133+ invasive cells through Matrigel coated transwell inserts: magnification 20x. Bar graph shows the invasive capacity difference between TVM-A12 and TVM-A12-CD133+ cell lines in the presence or not of 40 ng/ml HGF (** p < 0.001). ImageJ software was used to count total migrated and invasive cells. p-values (* p ≤ 0.050; ** p < 0.001). d Expression of the core stem cells transcriptional factor Oct4 (left panel) and Nanog (right panel). Data represent the results of three independent experiments

Fig. 4

Different melanoma cell lines increase CD133 marker upon activation of HERV-K. a Morphological transition (left panel) and CD133 expression by flow cytometry (right panel) of human melanoma cell lines (G-361, WM-155, WM-266-4 and A375) upon exposure to X-VIVO medium: magnification 10x. Relative mRNA expression of CD133 (b) and HERV-K (c) in different melanoma cell lines upon modification of culture conditions. p-values (* p ≤ 0.050; ** p < 0.001). Data represent the results of three independent experiments

Fig. 5

Downregulating of HERV-K expression by RNAi abolishes the expansion of CD133+ subpopulation. a Phase contrast microscopy image of adherent cells in RPMI-1640 medium (upper panels) and grape-like cellular aggregates in X-VIVO medium (lower panels) from TVM-A12pS-puro (non-interfered) and TVM-A12pS-H-Ki (interfered) cells: magnification 20x. b Cell counts. Relative mRNA expression of HERV-K env gene (c) and CD133 (d) in TVM-A12pS-puro and TVM-A12pS-H-Ki cells upon modification of culture conditions. e Flow cytometry analysis of CD133 expression shows HERV-K RNAi abolished the expansion and maintenance of CD133+ subpopulation in X-VIVO medium. Data represent the results of three independent experiments

Fig. 6

NNRTIs are effective to restrain HERV-K activation and induce apoptosis in TVM-A12 CD133+ cells. Effects of NNRTIs on the expansion and maintenance of CD133+ cells in TVM-A12 (a) and TVM-A12-CD133+ (b) cell lines analyzed by flow cytometry analysis. Effects of NNRTIs on apoptosis levels in TVM-A12 (c) and TVM-A12-CD133+ (d) analyzed by flow cytometry analysis after nuclei staining with propidium iodide. Relative mRNA expression of HERV-K env gene (e) and CD133 (f) analyzed by Real-time PCR. Data represent the results of three independent experiments