Drug-selected population in melanoma A2058 cells as melanoma stem-like cells retained angiogenic features - the potential roles of heparan-sulfate binding ANGPTL4 protein

Abstract

Malignant cancer may contain highly heterogeneous populations of cells, including stem-like cells which were resistant to chemotherapy agents, radiation, mechanical stress, and immune surveillance. The characterization of these specific subpopulations might be critical to develop novel strategy to remove malignant tumors. We selected and enriched small population of human melanoma A2058 cells by repetitive selection cycles (selection, restoration, and amplification). These subpopulation of melanoma cells persisted the characteristics of slower cell proliferation, enhanced drug-resistance, elevated percentage of side population as analyzed by Hoechst33342 exclusion, in vitro sphere formation, and in vivo xenograft tumor formation by small amount of tumor cells. The selected populations would be melanoma stem-like cells with high expression of stem cell markers and altered kinase activation. Microarray and bioinformatics analysis highlighted the high expression of angiopoietin-like 4 protein in drug-selected melanoma stem-like cells. Further validation by specific shRNA demonstrated the role of angiopoietin-like 4 protein in drug-selected subpopulation associated with enhanced drug-resistance, sphere formation, reduced kinase activation, in vitro tube-forming ability correlated with heparan-sulfate proteoglycans. Our finding would be applicable to explore the mechanism of melanoma stemness and use angiopoietin-like 4 as potential biomarkers to identify melanoma stem-like cells.

Keywords: angiopoietin-like 4 protein; drug resistance; melanoma stem-like cells.

Figure 1

Selected drug-resistant cells from melanoma A2058 cells showed reduced cell invasiveness and cell proliferation. (A) appearance of cell aggregates or cell spheres under detachment-impaired suspension cultures of different melanoma cells. (B) Difference in the phenotypes of elongated parental and pyramid-shaped drug-selected cells. (C) Comparison of transwell cell migration ability between parental and drug-selected cells show low cell invasiveness in drug-selected cells. (D) cell proliferation was slower in drug-selected cells than in parental cells.

Figure 2

Drug-selected melanoma cells persisted the characteristics of melanoma-stem like cells. (A) cell sphere forming assay under suspension culture for 2 weeks. Cell spheres were formed by drug-selected cells, while parental cells formed mostly cell aggregates. Spherocrit assay showed longer sphere length in capillary tube by cell spheres from drug-selected cells. (B) Quantification of sphere lengths from drug-selected cells through different selection cycles. (C) Side population analysis of parental and drug-selected cells showed more percentage of side population cells as determined by low dual fluorescence of hoechst33342-red and hoechst33342-blue. (D) Animal experiments of subcutaneous tumor formation showed larger tumors derived from drug-selected cells.

Figure 3

Expression of melanoma-stem markers in parental or drug-selected cells as analyzed by qPCR and western blot.

Figure 4

Drug-selected melanoma cells had lower levels of kinase activation than parental cells. (A) phosphor-kinase antibody array showed differential levels of different phosphor-proteins in parental or drug-selected cells. The quantification of blot intensities were shown (n=2; data were mean ±SD; *, p < 0.05.). (B) Western blot analysis of phosphor-tyrosine proteins in parental or drug-selected cells. Significantly lower levels of phosphor-proteins in drug-selected cells were indicated by arrows. Line-histograms were generated by Image J.

Figure 5

Higher expression of ANGPTL4 in drug-selected cells contributed to angiogenic activity through glycosaminoglycan modification on proteoglycans. (A) GSEA analysis showed that ANGPTL4 was the enriched in the gene sets related to oncogenic features. qPCR and western blot analysis demonstrated the higher expression level of ANGPTL4 in drug-selected cells. (B) Tube formation assay showed significantly connected and aligned cells by drug-selected cells, but not seen by parental cells. (C) Pretreatment with anti-ANGPTL4 antibody reduced tube-forming activity of drug-selected cells. (n=4; data were mean ±SD; **, p < 0.01; *, p < 0.05.) (D) qPCR analysis at expression of several heparan-sulfate proteoglycans (SDC1, SDC2, and SDC4) and sulfate transferases (HS2ST1 and NDST1) in parental and drug-selected cells. (E) Pretreatment with chemical inhibitors to reduce sulfate groups (by NaClO₃, sodium chlorate) or carbohydrate chain biosynthesis (by XylPyr, xylopyroside) inhibited tube-forming activity of drug-selected cells. (n=4; data were mean ±SD; **, p < 0.01; *, p < 0.05.).

Figure 6

Effects of suppression of ANGPTL4 expression on cellular activities in drug-selected cells. (A) ANGPTL4 expression level was reduced by transfection of ANGPTL4-specific shRNA as examined by qPCR and flow cytometry. (n=3; data were mean ±SD; **, p < 0.01) (B) in vitro tube forming ability of drug-selected cells was abolished by the suppression of ANGPTL4 expression. (C) cell proliferation assay showed no difference in cell number between parental and drug-selected cells. (n=3; data were mean ±SD; ns: not significant with p = 0.08). (D) upon suppression of ANGPTL4 expression in drug-selected cells, the sensitivity toward carmustine was increased as indicated by less survival. (n=6; data were mean ±SD; **, p < 0.01; *, p < 0.05.). (E) sphere forming ability was abolished by suppression of ANGPTL4 expression. (F) transwell assay showed no difference in migrated cells between parental and drug-selected cells. (n=6; data were mean ±SD).

Figure 7

Effects of suppression of ANGPTL4 expression on melanoma-stem marker expression and phosphor-protein expressions in drug-selected cells. (A) Effect of the ANGPTL4 suppression on expression of melanoma-stem markers in drug-selected cells as analyzed by qPCR. (B) Effect of the ANGPTL4 suppression on expression of phosphor-tyrosine proteins in drug-selected cells as analyzed by western blot analysis. Significantly higher levels of phosphor-proteins in drug-selected cells were indicated by arrows. Line-histograms were generated by Image J.