Activated Wnt/beta-catenin signaling in melanoma is associated with decreased proliferation in patient tumors and a murine melanoma model

Abstract

This study demonstrates that in malignant melanoma, elevated levels of nuclear beta-catenin in both primary tumors and metastases correlate with reduced expression of a marker of proliferation and with improved survival, in contrast to colorectal cancer. The reduction in proliferation observed in vivo is recapitulated in B16 murine melanoma cells and in human melanoma cell lines cultured in vitro with either WNT3A or small-molecule activators of beta-catenin signaling. Consistent with these results, B16 melanoma cells expressing WNT3A also exhibit decreased tumor size and decreased metastasis when implanted into mice. Genome-wide transcriptional profiling reveals that WNT3A up-regulates genes implicated in melanocyte differentiation, several of which are down-regulated with melanoma progression. These findings suggest that WNT3A can mediate transcriptional changes in melanoma cells in a manner reminiscent of the known role of Wnt/beta-catenin signaling in normal melanocyte development, thereby altering melanoma cell fate to one that may be less proliferative and potentially less aggressive. Our results may explain the observed loss of nuclear beta-catenin with melanoma progression in human tumors, which could reflect a dysregulation of cellular differentiation through a loss of homeostatic Wnt/beta-catenin signaling.

Fig. 1.

Nuclear ß-catenin levels predict improved survival in melanoma patients. (A) Representative tumors cores from the tissue microarray are shown to illustrate the localization of nuclear ß-catenin by AQUA. Tumor 1 (Upper panels) is representative of tumors with lower nuclear ß-catenin expression, whereas tumor 2 (Lower panels) is representative of tumors with higher nuclear ß-catenin expression. In the left panels, the orientation of the histospot used for analysis is oriented on the tumor core. The middle panels illustrate how S100 and DAPI are used to identify the cytoplasmic/membranous and nuclear compartments of the tumor, respectively. Staining with ß-catenin, shown in the right panels, is co-localized with either S100 or DAPI to generate measured values of ß-catenin staining in each subcellular compartment. (B) Primary tumors (n = 118) were stratified a priori into tertiles based on nuclear ß-catenin level (see also Fig. S1A). Patients with the highest nuclear ß-catenin levels (upper tertile) have a significantly higher survival probability by Kaplan-Meier analysis compared with those in the middle and lower tertiles (log-rank test). (C) Metastatic and recurrent tumors were separated into those with the highest nuclear ß-catenin levels (upper 20%; n = 46) and those with lower nuclear ß-catenin levels (remaining 80%; n = 179). The nuclear ß-catenin levels in the upper 20% of metastatic/recurrent tumors correspond with the levels seen in the upper tertile of primary tumors (Fig. S1B). Kaplan-Meier analysis demonstrated a significantly increased survival probability in patients with the highest nuclear ß-catenin levels (Gehan-Breslow-Wilcoxon test).

Fig. 2.

Activated Wnt/ß-catenin signaling is associated with decreased proliferation. (A) Nuclear ß-catenin level was measured in primary tumors staged by Breslow depth according to AJCC criteria. Tumor depth increases from T1 to T4. Bars representing the mean and SEM for tumors in each stage indicate decreasing nuclear ß-catenin level with increasing tumor depth. (B) In comparison, the proliferative index of tumors, as measured by %Ki-67, increases with tumor depth. Gray dots represent individual tumors, and the dotted red line indicates the average for the entire cohort. The changes in nuclear ß-catenin and Ki-67 were highly significant by ANOVA followed by a posttest for linear trend. (C) Histograms binned by 10% increments reveal the distribution of %Ki-67 within tumors stratified into tertiles by nuclear ß-catenin level. Note the increased number of tumors with higher %Ki-67 in the presence of lower nuclear ß-catenin levels (lower tertile), compared with the larger number of tumors with lower %Ki-67 seen in the presence of higher nuclear ß-catenin levels (upper tertile). The mean %Ki-67 for each tertile (shown above the histograms) increased significantly with lower expression of nuclear ß-catenin (*P < 0.0001 by ANOVA with posttest for linear trend). (D) Levels of nuclear ß-catenin and %Ki-67 in individual tumors were analyzed by Deming regression, revealing a slope of -1.089 ± 0.2374, suggesting that higher levels of nuclear ß-catenin are associated with decreased %Ki-67. In contrast, Deming regression comparing α-catenin and %Ki-67 revealed a slope not significantly different from zero (Fig. S2B).

Fig. 3.

Wnt/ß-catenin activation is associated with decreased proliferation in melanoma. (A) Immunofluorescent staining demonstrates increased nuclear ß-catenin in B16 cells expressing WNT3A, but not in cells expressing either GFP or WNT5A. (B) CM from B16:WNT3A cells activated a Wnt/ß-catenin reporter in UACC1273 human melanoma cells, indicating that these cells secrete active WNT3A. No activation was seen with CM isolated from B16:GFP or B16:WNT5A cells. (C) Proliferation of B16:GFP, B16:WNT3A, or B16:WNT5A cells was measured by hematocytometry after 6 days of culture (black bars, left y-axis) or by MTT assay after 3 days of culture (white bars, right y-axis). Bars represent the average and standard deviation of 3–6 biological replicates. The inhibition of proliferation seen with WNT3A cells was highly significant by ANOVA with both proliferation assays (*P < 0.001). (D) In cell cycle analysis, B16:WNT3A cells were decreased in S phase and increased in G1 compared with B16:GFP or B16:WNT5A cells. Bars indicate the average and standard deviation of 3 biological replicates, and the data shown are representative of 5 individual experiments, each with at least 3 biological replicates per condition. The changes observed in B16:WNT3A cells are extremely significant by ANOVA (*P < 0.001). (E) Tumor grafts demonstrate that B16 cells expressing WNT3A form smaller tumors than cells expressing GFP or WNT5A. Data are expressed as mean and standard deviation from 4 mice for each cell line tested. The experiment shown is representative of 4 independent experiments with the same result, with each experiment involving at least 4 mice for each cell line tested. The decrease in tumor size with WNT3A was found to be highly significant by ANOVA at 14 days postimplantation (*P = 0.004). (F) Metastases to the popliteal sentinel lymph node bed were evaluated by firefly luciferase assay, demonstrating significantly decreased metastases in tumors expressing WNT3A. Bars represent mean and standard deviation, and the data shown are representative of 4 independent experiments.

Fig. 4.

Activation of Wnt/ß-catenin signaling alters cell fate in melanoma cells. (A) B16:GFP, B16:WNT3A, or B16:WNT5A cells were isolated at equivalent confluency, spun down, and photographed in a 96-well plate, demonstrating the marked difference in pigmentation seen in melanoma cells expressing WNT3A. Increased pigmentation also was seen in B16 cells treated for 4 days with WNT3A-conditioned L-cell media (L-WNT3A) compared with control L-cell media (L-control). (B) Whole genome expression profiles of B16:WNT3A or B16:WNT5A cells were initially compared with gene expression in B16:GFP cells, with 3 biological replicates analyzed for each cell line. The heatmap illustrates the differences between the most significant regulated genes in B16:WNT3A cells compared with B16:WNT5A cells by subsequent unpaired t-test. Genes that were among the most significantly regulated in B16:WNT3A cells are listed with their normalized fold-change [log (2)] compared with B16:GFP cells shown in parentheses; these include known Wnt/ß-catenin targets, genes involved in melanocyte and neural crest differentiation, and genes implicated in melanoma prognosis or therapy. (C) Various genes were selected for validation using quantitative RT-PCR (qRT-PCR), including genes implicated in melanocyte differentiation (Met, Kit, Sox9, Mitf, Si/Gp100) and melanoma biology (Trpm1, Kit, Mme, Mlze), as well as known Wnt target genes (Axin2, Met, Sox9). All genes that were up-regulated in B16:WNT3A cells by transcriptional profiling were up-regulated by qRT-PCR, whereas genes that were down-regulated in B16:WNT3A cells on the array (Mlze, Mme) also were down-regulated by qRT-PCR. Genes up-regulated in B16:WNT3A cells were universally down-regulated in the B16:WNT5A cells, providing evidence that WNT5A can antagonize transcription of Wnt/ß-catenin gene targets in melanoma cells, even in the absence of WNT3A. Data are expressed as log (2)–transformed fold-change (with standard error) compared with B16:GFP cells and are representative of 3 or more experiments with similar results. (D) Gene changes induced in B16:WNT3A cells were antagonized on treatment with ß-catenin siRNA (20 nM) compared with control siRNA (20 nM). Data are expressed as log (2)–transformed fold-change (with standard error) in cells treated with ß-catenin siRNA compared with control siRNA.