Research ArticleCancer

Wnt/β-Catenin Signaling and AXIN1 Regulate Apoptosis Triggered by Inhibition of the Mutant Kinase BRAFV600E in Human Melanoma

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Sci. Signal.  10 Jan 2012:
Vol. 5, Issue 206, pp. ra3
DOI: 10.1126/scisignal.2002274
  • Fig. 1

    BRAF signaling negatively regulates Wnt/β-catenin signaling in melanoma cells. (A) Scatter plot of a kinome-based siRNA screen in human A375 melanoma cells stably expressing the β-catenin–activated reporter (BAR) driving firefly luciferase, with each dot representing a known or predicted kinase. The blue and green dotted lines represent two mean absolute deviations above and below the mean, respectively. The full gene list is presented in databases S1 and S2. (B) An isobologram analysis shows a dose-dependent enhancement of Wnt/β-catenin signaling with the targeted BRAF inhibitor PLX4720 and WNT3A conditioned medium (CM) on BAR activity. (C) Immunoblot analysis of the dose-dependent inhibition of dual-phosphorylated ERK1/2 (ppERK1/2), Ser33/Ser37/Thr41-phosphorylated β-catenin (pCTNNB1), and Tyr216-phosphorylated GSK3 after PLX4720 treatment. (D) Two distinct MEK inhibitors, U0126 and AZD6244, both enhanced Wnt/β-catenin signaling in a dose-dependent manner. Symbols and error bars represent the mean and SD, respectively, of three biologic replicates. (E) Immunoblot analysis of the dose-dependent inhibition of ppERK1/2 by MEK1/2 inhibitors U0126 and AZD6244. (B to E) A375 cells were treated for 24 hours with the indicated conditions before harvesting. Data represent at least three independent experiments.

  • Fig. 2

    Wnt/β-catenin activation cooperates with targeted inhibition of mutant BRAF to inhibit tumor growth in vivo and in vitro. (A) WNT3A enhances the ability of the BRAF inhibitor PLX4720 to reduce tumor size in vivo. Human A375 melanoma cells expressing either GFP (A375-GFP) or WNT3A-IRES-GFP (A375-WNT3A) were grown as xenografts in NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice treated with either vehicle or PLX4720 (50 mg/kg) after tumors had reached an initial size of 100 mm3. For each treatment arm, the means and SEM are shown for five individual mice. (B) WNT3A enhances the ability of the BRAF inhibitor PLX4720 to reduce spheroid size in vitro. A375-GFP or A375-WNT3A were grown as spheroids in a three-dimensional collagen matrix and then treated with either DMSO or 2 μM PLX4720 for 72 hours before imaging. Representative spheroids of greater than 40 spheroids per treatment are shown in these light micrographs. (C) WNT3A synergizes with PLX4720 to inhibit the viability of A375 melanoma cells. A375 melanoma cells were treated with the indicated combinations of WNT3A CM and PLX4720 concentrations for 48 hours. Data represent at least three independent experiments with each data point assayed in triplicate.

  • Fig. 3

    Wnt/β-catenin activation synergistically enhances apoptosis with BRAF inhibition. (A) TUNEL staining was used to visualize apoptotic A375 cells after treatment with the indicated conditions, including WNT3A-CM (3A CM) or the control (L CM). (B) Spheroids generated from A375-GFP or A375-WNT3A cells were grown in a three-dimensional collagen matrix and treated with the indicated conditions. Simultaneously, GFP was used to image all cells and EtBr staining was used to identify dead cells. Representative spheroids of greater than 40 spheroids for each condition are shown in these panels. (C) A flow cytometry–based assay for cleaved caspase-3 (CASP3) was used to detect apoptotic cells after treatment with the indicated conditions in A375 cells. Red and blue peaks on the representative histograms indicate the distribution of cells that were negative and positive for caspase-3 staining, respectively. Numbers indicate the average percentage and SD of caspase-3–positive cells from three biological replicates. *P < 0.001 compared with all other conditions with one-way ANOVA and Tukey’s multiple comparison post-test. (D) Immunoblot analysis of the proapoptotic protein Bim in A375 cells treated with the indicated conditions. BimEL, BimL, and BimS represent the three major isoforms of Bim. (A to D) Cells were treated for 24 hours with the indicated conditions. Data represent at least three independent experiments. Where indicated, cells were treated with 2 μM PLX4720 with or without 100 μM Z-VAD-FMK.

  • Fig. 4

    Apoptosis mediated by Wnt/β-catenin signaling and BRAF inhibition requires β-catenin. (A) An immunoblot analysis of cleaved caspase-3 (CASP3) from A375 cells pretreated with control or β-catenin (CTNNB1) siRNA followed by treatment with the indicated conditions. Cells were transfected with siRNAs, incubated for 48 hours, and then treated with the indicated conditions for 48 hours. Where indicated, cells were treated with 2 μM PLX4720 in the presence or absence of WNT3A CM. (B) Immunoblot analysis of cleaved caspase-3 from A375 cells pretreated with control or β-catenin (CTNNB1) siRNA followed by treatment with the indicated conditions. Cells were transfected with siRNAs, incubated for 48 hours, and then treated with the indicated conditions for 36 hours. Where indicated, cells were treated with 2 μM PLX4720 in the presence or absence of 5 μM CHIR99021. Inhibition of GSK3 was confirmed by loss of the activating autophosphorylation at Tyr216. In (A) and (B), data represent at least three independent experiments.

  • Fig. 5

    Regulation of Wnt/β-catenin signaling by BRAF predicts apoptotic response to combined Wnt/β-catenin activation and BRAF inhibition. (A) In six melanoma lines harboring BRAFV600E mutations, synergistic enhancement of Wnt/β-catenin signaling by BRAFV600E inhibition was examined by quantitative PCR measurements of the endogenous target gene AXIN2. Data are expressed as copies of AXIN2 per 106 copies of GAPDH. (B) Flow cytometry detection of cleaved caspase-3 was used to measure apoptosis in several melanoma cell lines harboring BRAFV600E mutations. (A and B) Cells were treated with the indicated conditions for 24 hours and 2 μM PLX4720 was used where indicated. Columns and error bars represent the mean and SD, respectively, of three biologic replicates. *P < 0.001 by one-way ANOVA with Tukey’s post-test. Data represent at least three independent experiments.

  • Fig. 6

    AXIN1 abundance is positively regulated by BRAF signaling and reduction after BRAF inhibition predicts apoptotic response. (A) Immunoblot analysis of the same six melanoma cell lines analyzed in Fig. 5 after treatment with the indicated conditions. (B) The immunoblot of AXIN1 from (A) combined with immunoblots from two additional independent replicates were quantified by pixel intensity. P values were determined by two-tailed Student’s t test. *P < 0.005. (A and B) A375 cells were treated with the indicated conditions for 24 hours. (C) Reduced AXIN1 abundance after combined treatment with WNT3A CM and PLX4720 was rescued by the proteasome inhibitor MG132. A375 cells were treated with control conditions or WNT3A and PLX4720 in combination with DMSO, the proteasome inhibitor MG132, or the lysosome inhibitor chloroquine for 8 hours. In (A) to (C), 2 μM PLX4720, 10 μM MG132, and 10 μM chloroquine were used where indicated. Data represent at least three independent experiments.

  • Fig. 7

    AXIN1 depletion sensitizes melanoma cells to apoptosis mediated by BRAF inhibition. (A) Immunoblot analysis of a time course of A375 cells treated with WNT3A CM and PLX4720. (B) The decrease in AXIN1 abundance after WNT3A and PLX4720 treatment is not rescued by Z-VAD-FMK. A375 cells were treated for 24 hours with the indicated conditions. (C) PLX4720 enhancement of Wnt/β-catenin signaling is not dependent on caspase activation. A375 cells containing BAR were treated for 24 hours with the indicated conditions. *P < 0.001 compared to all other conditions by one-way ANOVA with Tukey’s post-test. (D) Flow cytometry detection of cleaved caspase-3 in SKMEL28 cells transfected with control or AXIN1 siRNA and treated with the indicated conditions for 24 hours. (E) Knockdown of AXIN1 by siRNA sensitizes SKMEL28 cells to PLX4720-induced apoptosis. Immunoblots show lysates from SKMEL28 cells transfected with either control or two nonoverlapping independent siRNAs targeting AXIN1 and treated with the indicated conditions for 24 hours. (F) Flow cytometry detection of cleaved caspase-3 in A375 cells transfected with control or AXIN1 siRNA and treated with the indicated conditions for 24 hours. (D and F) P values were determined by two-way ANOVA with Bonferroni post-test. *P < 0.05; **P < 0.001. n.s., not significant. In (A) to (F), 2 μM PLX4720, 10 μM MG132, and 100 μM Z-VAD-FMK were used where indicated, and data are representative of at least three independent experiments.

  • Table 1

    AXIN1 siRNAs confer sensitivity to PLX4720-mediated apoptosis in previously resistant cell lines. A2058, SKMEL5, and SKMEL28 cells were transfected with control siRNA or siRNA targeting AXIN1. Forty-eight hours after transfection, cells were treated with the indicated conditions for 24 hours and analyzed for apoptosis by flow cytometry for cleaved caspase-3. L CM and W3A CM indicate control and WNT3A-conditioned media, respectively. PLX4720 (2 μM) was used where indicated. Values represent the average number of cleaved caspase-3–positive cells ± SD in three or more biologic replicates. Statistically significant differences between control and AXIN1 siRNA for different conditions within each cell line are indicated by an asterisk (*) denoting a P < 0.001 by two-way ANOVA with Bonferonni’s post-test comparison.

    Melanoma cell
    line
    Control siRNAAXIN1 siRNA
    L CM
    DMSO
    W3A CM
    DMSO
    L CM
    PLX4720
    W3A CM
    PLX4720
    L CM
    DMSO
    W3A CM
    DMSO
    L CM
    PLX4720
    W3A CM
    PLX4720
    A20581.18 ± 0.301.37 ± 0.082.20 ± 0.433.16 ± 0.403.78 ± 0.515.23 ± 0.9421.27 ± 2.11*21.77 ± 4.36*
    SKMEL281.13 ± 0.251.00 ± 0.401.81 ± 0.355.34 ± 1.551.03 ± 0.240.71 ± 0.3222.20 ± 5.43*30.97 ± 3.65*
    SKMEL51.01 ± 0.140.96 ± 0.152.20 ± 0.172.72 ± 0.182.25 ± 0.522.45 ± 0.1210.80 ± 1.06*12.10 ± 1.15*

Additional Files

  • Supplementary Materials for:

    Wnt/β-Catenin Signaling and AXIN1 Regulate Apoptosis Triggered by Inhibition of the Mutant Kinase BRAFV600E in Human Melanoma

    Travis L. Biechele, Rima M. Kulikauskas, Rachel A. Toroni, Olivia M. Lucero, Reyna D. Swift, Richard G. James, Nick C. Robin, David W. Dawson, Randall T. Moon,* Andy J. Chien*

    *To whom correspondence should be addressed. E-mail: andchien{at}uw.edu (A.J.C.); rtmoon{at}uw.edu (R.T.M.)

    This PDF file includes:

    • Fig. S1. BRAF and other members of the MAPK family are identified as regulators of Wnt/β-catenin signaling in melanoma cells.
    • Fig. S2. BRAF is a negative regulator of Wnt/β-catenin signaling and inhibition of BRAF decreases the phosphorylation of β-catenin at Ser33/Ser37/Thr41 in melanoma cells.
    • Fig. S3. Isobologram analysis of WNT3A and PLX4720 shows a synergistic activation of Wnt/β-catenin signaling in melanoma cells.
    • Fig. S4. BRAF inhibition reduces the abundance of phosphorylated Ser33/Ser37/Thr41 β-catenin with no effect on total β-catenin abundance.
    • Fig. S5. Activation of Wnt/β-catenin signaling cooperates with BRAF inhibition to inhibit proliferation of melanoma cells in vivo.
    • Fig. S6. Isobologram analysis of WNT3A and PLX4720 shows a synergistic inhibition of melanoma cell viability.
    • Fig. S7. Activation of Wnt/β-catenin signaling combined with siRNA-mediated knockdown of BRAF promotes apoptosis of melanoma cells.
    • Fig. S8. BRAF inhibition does not regulate AXIN1 mRNA abundance.
    • Fig. S9. siRNA targeting AXIN1 effectively reduces AXIN1 protein abundance.
    • Table S1. siRNA sequences used for the described studies.
    • Table S2. Allelic status of BRAFV600E in different melanoma cell lines.
    • Database S1. A375 melanoma cells expressing the BAR reporter were screened using siRNAs for kinases that regulate Wnt/β-catenin signaling.
    • Database S2. Screen data from database S1 without resazurine normalization.

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    Other Supplementary Material for this manuscript includes the following:

    • Database S1 (Microsoft Excel format). A375 melanoma cells expressing the BAR reporter were screened using siRNAs for kinases that regulate Wnt/β-catenin signaling.
    • Database S2 (Microsoft Excel format). Screen data from database S1 without resazurine normalization.
    [Download Databases S1 to S2 (Compressed)]

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    Citation: T. L. Biechele, R. M. Kulikauskas, R. A. Toroni, O. M. Lucero, R. D. Swift, R. G. James, N. C. Robin, D. W. Dawson, R. T. Moon, A. J. Chien, Wnt/β-Catenin Signaling and AXIN1 Regulate Apoptosis Triggered by Inhibition of the Mutant Kinase BRAFV600E in Human Melanoma. Sci. Signal. 5, ra3 (2012).

    © 2012 American Association for the Advancement of Science

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