Activation of Wnt/β-catenin signaling increases apoptosis in melanoma cells treated with trail

Abstract

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.

Figure 1. WNT3A sensitizes melanoma cell lines to TRAIL in a β-catenin-dependent manner.

A) A375 melanoma cells were treated with indicated doses of rhTRAIL for 24 hours in the presence of WNT3A or control L-cell (L) CM (10%). Apoptotic cells were detected by Annexin V binding assay using FACS. Representative FACS histograms with Annexin V-positive gates are shown. Percent apoptotic cells are indicated. B) A375 cells were treated with indicated doses of rhTRAIL in the presence of WNT3A or L CM (10%). Data represents mean % apoptotic cells ± SEM as determined by Annexin V positivity at 24 hours. An (*) indicates that the difference between L and WNT3A CM treated cells at the indicated TRAIL dose is significant with a p-value of <0.01, calculated using Student’s t-test. C) A375 cells were treated with rhTRAIL and WNT3A in the absence and presence of the pan-caspase inhibitor zVAD-FMK (100 µM), and then analyzed for cleaved PARP at 24 hours. C) A375 cells were pre-treated with siRNA specific for β-catenin (CTNNB1) or non-targeting control siRNA for 48 hours. Cells were then treated with rhTRAIL (20 ng/mL) in the presence of WNT3A CM or L CM. Data represents mean percentages of Annexin V-positive cells (+/- SEM) at 24 hours post-treatment as detected by FACS. P-values were calculated using one way ANOVA and Tukey’s post-test analysis. A parallel immunoblot (lower panel) confirms knockdown of β-catenin. The experiments in A and B are representative of at least three independent experiments with similar results.

Figure 2. Melanoma cells exhibit diverse apoptotic response to WNT3A plus TRAIL.

Several human melanoma cell lines were assessed for apoptosis at 24 hours upon treatment with rhTRAIL (20 ng/ml) in the absence (10% L CM) and presence of 10% WNT3A CM. Statistically significant increases in Annexin V-positive cells were seen in three cell lines. Data represents the mean percentage of apoptotic cells for three replicates/group (* p<0.05; **p<0.01; ***p<0.001).

Figure 3. Wnt/β-catenin activation enhances the expression of the pro-apoptotic proteins BIM and PUMA and diminishes levels of anti-apoptotic MCL1.

A) A375 melanoma cells were treated with rhTRAIL (20 ng/mL) in the presence of WNT3A or L CM. To inhibit caspase activity, cells were also treated with 100µM zVAD-FMK. Immunoblotting analysis was performed with the indicated antibodies. B) A375 cells were pre-treated with β-catenin (CTNNB1) versus control siRNA. 48 hours later, the cells were treated with WNT3A CM or L CM. Immunoblot analysis was then performed with the indicated antibodies. C) Graphs represent the fold-change in protein expression in (B) from digitally-quantified immunoblots +/- SEM (n=5 blots from independent experiments). Each condition was normalized to control cells treated with L CM + control siRNA. D) A375 melanoma cells were treated with WNT3A CM versus L CM in the presence or absence of rhTRAIL (20ng/mL) in the presence of zVAD-FMK (100µM). After 24 hours, RNA was collected for cDNA synthesis and analysis by quantitative real-time PCR (qRT-PCR). Data represents mean relative expression of the indicated gene based on three replicates/group. Significant differences between groups are indicated.

Figure 4. Inhibition of GSK-3 sensitizes melanoma cells to TRAIL-mediated apoptosis.

A) A375 melanoma cells were treated with rhTRAIL at doses indicated on x-axis. Cells were concurrently treated with the GSK-3 inhibitor CHIR99021 (5µM) or DMSO vehicle control (v/v). Data represents the percentage of apoptotic cells measured by Annexin V-positive cells at 24 hours post-treatment by FACS. B) A375 cells were pre-treated with control siRNA or siRNA specific for β-catenin. 48 hours later, cells were treated with rhTRAIL (20ng/mL). 24 hours post-treatment, the percentage of apoptotic cells was determined by AnnexinV binding using FACS. Data represents mean percentage of apoptotic cells (+/- SEM). P-values were calculated by one-way ANOVA with a Tukey’s post-test analysis.

Figure 5. Depletion of AXIN1 sensitizes melanoma cells to TRAIL-mediated apoptosis.

Melanoma cell lines indicated were pre-treated with control versus AXIN1-specific siRNA for 48 hours. Cells were then treated either WNT3A CM or L CM in addition to rhTRAIL (20ng/mL). The percentage of apoptotic cells was determined by Annexin V binding using FACS. Data represents the mean percentage of apoptotic cells at 24 hours post-treatment +/- SEM. P-values were calculated by one-way ANOVA with a Tukey’s post-test analysis. * indicates p <0.05 compared to either L or WNT3A cells treated with control siRNA.