The role of Wnt/β-catenin signaling pathway in melanoma epithelial-to-mesenchymal-like switching: evidences from patients-derived cell lines

Abstract

Deregulations or mutations of WNT/β-catenin signaling have been associated to both tumour formation and progression. However, contradictory results concerning the role of β-catenin in human melanoma address an open question on its oncogenic nature and prognostic value in this tumour. Changes in WNT signaling pathways have been linked to phenotype switching of melanoma cells between a highly proliferative/non-invasive and a slow proliferative/metastatic condition. We used a novel panel of cell lines isolated from melanoma specimens, at initial passages, to investigate phenotype differences related to the levels and activity of WNT/β-catenin signaling pathway. This in vitro cell system revealed a marked heterogeneity that comprises, in some cases, two distinct tumour-derived subpopulations of cells presenting a different activation level and cellular distribution of β-catenin. In cells derived from the same tumor, we demonstrated that the prevalence of LEF1 (high β-catenin expressing cells) or TCF4 (low β-catenin expressing cells) as β-catenin partner for DNA binding, is associated to the expression of two distinct profiles of WNT-responsive genes. Interestingly, melanoma cells expressing relative low level of β-catenin and an invasive markers signature were associated to the TNF-α-induced pro-inflammatory pathway and to the chemotherapy resistance, suggesting that the co-existence of melanoma subpopulations with distinct biological properties could influence the impact of chemo- and immunotherapy.

Keywords: WNT/β-catenin; melanoma heterogeneity; metastases; proliferation.

Figure 1. In vitro morphological differences of melanoma subpopulations isolated from the same patient

Phase contrast microscopic analysis of melanoma subpopulations isolated from the same biopsy evidenced marked morphological differences. In each case, one cell line showed a phenotype similar to that of normal melanocytes with elongated dendritic shape A, D, G. and abundant melanin C, F, I. and another one displayed a de-differentiated phenotype with low melanin (C, F, I) associated to an enlarged morphology similar to epithelial cells B-E. or to a rounded shape in the case of Mel8 floating cells H. Scale bar: 20 μm.

Figure 2. Analysis of Wnt/β-catenin signalling activation in freshly isolated low passage melanoma cell lines

Immunoblot analysis of β-catenin expression in a panel of melanoma cell lines A. and corresponding activation of its target genes AXIN2 and MITF B. The relative mRNA levels (± SD) of AXIN2 and MITF represent the amount of mRNA expression normalized to GAPDH mRNA expression. Total RNA was extracted from each melanoma cells at three different culture passages and was used as three independent experiments. Results were then combined for data analysis. Comparison of in vitro C. and ex-vivo D. level of β-catenin protein expression, evaluated by immunofluorescence and immunohistochemistry respectively. Phase contrast microscopic documentation of the morphological characteristics of the melanoma cell lines evaluated for β-catenin expression E. Scale bar: C: 50 μm; D: 100 μm and 50 μm for the higher magnification of the boxed areas; E: 20 μm.

Figure 3. Relative higher level of β-catenin protein is associated to increased proliferative rate of melanoma cells

Immunoblot A. and flow cytofluorimetric analysis B. of β-catenin level of expression in melanoma subpopulations isolated from the same biopsy. Quantitative evaluation of β-catenin level of expression in single cells, obtained by measuring FITC-β-catenin median fluorescence intensity (MFI), confirmed the homogeneous level of β-catenin protein in each cell lines. The diagram depicts a representative staining of cell line pairs. Cell growth evaluated by trypan blue exclusion assay at various time points C. Data are representative of three independent experiments. *p≤0.05 at the end time point. Immunofluorescence with anti-Ki67 antibody of Mel29 and Mel35 cell pairs D, E. Arrows point at cells detected by the nuclear 4′-6′-diamidino-2-phenyllindole (DAPI) staining, which are negative for Ki67. Scale bar: 20 μm. Percentage of Ki67-positive cells evaluated counting for each melanoma cell populations, a total of 500 cells observed in 10 fields F, G. Results are expressed as mean values ± SD. **p≤0.01.

Figure 4. Migration and invasion assays and expression of invasive markers

Cellular migration toward the scratched area was evaluated after creating a standardized cell-free area with a plastic pipette tip. Cells were allowed to grow for 0, 5, 24, 48 and 72 hours and then photographed under a microscope A, C. Images are representative of three separate experiments. Quantitative analysis of wound-induced migration B, D. Data are presented as means ± SD of three independent experiments. *p <0.05, **p≤0.01. Matrigel-invasion assay and count of the invaded cell number evaluated at 48 hours. Results are expressed as fold change of invasive cell subpopulation respect to the proliferative one, which was set as 1 for definition E. Data are presented as means ± SD of two independent experiments. **p≤0.01. Immunoblot analysis of N- and E-cadherin expression in Mel8, Mel29 and Mel35 cell line pairs F. Gene expression analysis of a panel of MMPs and VEGF normalized to GAPDH mRNA expression G. For each patient total RNA was extracted from cells cultures at three different passages. Values represent the means ± SD of fold-increase considering arbitrarily the proliferative subpopulation as control (value=1). Immunoblot analysis of VEGF expression H. Scale bar A, C, E: 50 μm.

Figure 5. Analysis of Wnt/β-catenin pathway-associated markers

Analysis of MITF A. LEF1 B. WNT5A C. and TCF4 D. at mRNA and protein level of expression in Mel8, 29 and 35 cell pairs. For mRNA quantification total RNA was extracted from cell cultures at three different passages. Values represent the means ± SD of fold-increase considering arbitrarily the proliferative subpopulation as control (value=1). LEF1 and TCF4 protein detection was performed by cytofluorimetric analysis (Mel8) or by immunofluorescence (Mel29 and Mel35). Immunoblots and cytofluorimetric analyses are representative of several independent experiments. Scale bars: 20 μm.

Figure 6. Immunohistochemical expression of β-catenin in primary and metastatic melanomas

An heterogeneous staining pattern for β-catenin both in intensity and localization is evident among the different tumour samples and within the same specimen. Examples of β-catenin immunoreactivity in primary melanomas (Mel35 in A. and enlarged view of the boxed areas in B. and C. Mel8 in D. and enlarged view of the boxed areas in E. and F. Mel29 in G. and enlarged view of the boxed areas in H. and I. and in lymphnode metastases (Mel 29 in K. and enlarged view of the boxed areas in L. and M.) are shown. Scale bar: A, D, G, K: 100 μm; B, C, E, F, H, I, L, M: 50 μm.

Figure 7. Immunohistochemical analysis of β-catenin, E-cadherin and N-cadherin expression

Serial sections of paraffin-embedded primary and lymph nodal metastases of patient 29 were analyzed for β-catenin A, D, G. E-cadherin B, E, H. and N-cadherin C, F, I. expression. Positive E-cadherin immunolabelling was mainly localized in tumour areas presenting high β-catenin reactivity whereas N-cadherin staining was weak and predominantly observed in tumour cells presenting feeble staining for β-catenin. Scale bar: 100 μm.

Figure 8. In vitro sensitivity of melanoma cells to cisplatin

Mel8, 29 and 35 cell pairs were cultured in the presence of different doses (20-40-60 μM) of cisplatin for 24 hours. Death and apoptosis were evaluated by FACS analysis using annexin V/iodide propidium staining. Dot plots show one representative experiment at the highest dose. For all the doses tested the number of surviving cells (Annexin V -/PI -) are reported in the Table. Data presented are the mean ± SD of three independent experiments. *p <0.05.

Figure 9. Analysis of the expression of TNF-α inducible genes in melanoma cell line subpopulations

Analysis of IL1-α IL-1β, IL6 and IL8 and TNF-αmRNA across melanoma cell lines pairs 8, 29 and 35. Total RNA was extracted from cell cultures at three different passages. Values represent the means ± SD of fold-increase considering arbitrarily the proliferative subpopulation as control (value=1).