Sci. Signal., 10 January 2012
Wnt/β-Catenin Signaling and AXIN1 Regulate Apoptosis Triggered by Inhibition of the Mutant Kinase BRAFV600E in Human Melanoma
1 Department of Pharmacology, Howard Hughes Medical Institute, and the Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, WA 98109, USA.
Abstract: Because the Wnt/β-catenin signaling pathway is linked to melanoma pathogenesis and to patient survival, we conducted a kinome small interfering RNA (siRNA) screen in melanoma cells to expand our understanding of the kinases that regulate this pathway. We found that BRAF signaling, which is constitutively activated in many melanomas by the BRAFV600E mutation, inhibits Wnt/β-catenin signaling in human melanoma cells. Because inhibitors of BRAFV600E show promise in ongoing clinical trials, we investigated whether altering Wnt/β-catenin signaling might enhance the efficacy of the BRAFV600E inhibitor PLX4720. We found that endogenous β-catenin was required for PLX4720-induced apoptosis of melanoma cells and that activation of Wnt/β-catenin signaling synergized with PLX4720 to decrease tumor growth in vivo and to increase apoptosis in vitro. This synergistic enhancement of apoptosis correlated with reduced abundance of an endogenous negative regulator of β-catenin, AXIN1. In support of the hypothesis that AXIN1 is a mediator rather than a marker of apoptosis, siRNA directed against AXIN1 rendered resistant melanoma cell lines susceptible to apoptosis in response to treatment with a BRAFV600E inhibitor. Thus, Wnt/β-catenin signaling and AXIN1 may regulate the efficacy of inhibitors of BRAFV600E, suggesting that manipulation of the Wnt/β-catenin pathway could be combined with BRAF inhibitors to treat melanoma.
Introduction Back to Top
Most of both benign nevi and cutaneous melanomas harbor activating mutations in the BRAF oncogene, with BRAFV600E representing the most common of these mutations (1). The development of small-molecule compounds designed to specifically target BRAFV600E, including PLX4720 (2), PLX4032 (vemurafenib) (3, 4), and GSK2118436 (5), has led to clinical trials that demonstrated an unprecedented high response rate among patients with BRAFV600E tumors (5–7). However, despite the initial tumor response, only half of patients with BRAFV600E tumors meet established criteria for a confirmed objective clinical response. Furthermore, half of the patients exhibiting an initial response to BRAFV600E inhibitors develop resistant tumors and progressive disease within 6 months. These results highlight the need to identify regulatory interactions between BRAF signaling and other cellular pathways that may provide avenues for enhancing the long-term clinical effects of targeted BRAF inhibitors in melanoma treatment.
Activation of Wnt/β-catenin signaling promotes the nuclear functions of β-catenin (CTNNB1) (note that the HUGO nomenclature is provided parenthetically for proteins having substantially different abbreviations according to that convention), resulting in the regulation of cell proliferation, differentiation, and behavior (8). The exact role of Wnt/β-catenin signaling in melanoma progression remains controversial. Whereas transgenic mouse models expressing a melanocyte-specific, constitutively active mutant β-catenin did not display any spontaneous melanomas, coexpression of a constitutively active mutant Nras resulted in mice that exhibited enhanced immortalization of melanocytes and increased melanoma tumor promotion (9). By contrast, the decreased survival observed in patients exhibiting lower abundance of nuclear β-catenin in their tumors suggests that the loss of Wnt/β-catenin signaling plays an important role during melanoma evolution (10–14). Although benign nevi and a substantial number of melanoma tumors exhibit increased nuclear β-catenin (10, 11, 13, 14), activating mutations in the Wnt/β-catenin pathway are rare in melanoma (5–17). Thus, the mechanisms underlying the increase in β-catenin in melanoma are unresolved as is the functional significance of β-catenin in this context.
The mitogen-activated protein kinases (MAPKs), which are activated by multiple signals, represent another signaling pathway linked to melanoma (15). Signaling by the MAPK extracellular signal–regulated kinase (ERK) is mediated by RAS guanosine triphosphatases (GTPases) to activate kinases of the RAF family, which phosphorylate and activate the kinases mitogen-activated or extracellular signal–regulated protein kinase kinase 1 (MEK1) and MEK2 (collectively referred to as MEK1/2), which subsequently phosphorylate and activate the kinases extracellular signal–regulated kinase 1 (ERK1) and ERK2 (collectively referred to as ERK1/2). ERK1/2 phosphorylate and regulate numerous substrates leading to various cell type–specific and context-dependent responses (16). With regard to melanoma, constitutive activation of ERK1/2 by activating mutations in NRAS or BRAF is observed in most melanomas and plays an integral role in the regulation of tumor cell proliferation, invasiveness, and survival (17).
Several instances of crosstalk between Wnt/β-catenin and MAPK signaling have been reported, with most revealing that Wnt/β-catenin signaling positively regulates MAPK signaling (18). Conversely, others have reported that epidermal growth factor (EGF)–induced ERK activation in glioblastoma cell lines leads to phosphorylation of casein kinase II (CSNK2) and to disruption of the interaction between β-catenin and α-catenin (19). Disruption of this complex then enhances β-catenin target gene transactivation and subsequent tumor cell invasion.
Our study reveals crosstalk between BRAF and Wnt/β-catenin signaling in regulating apoptosis and the abundance of the Wnt signaling inhibitory scaffolding protein AXIN1 in cultured melanoma cells. Specifically, we showed that activation of BRAF signaling by the BRAFV600E mutation inhibited Wnt/β-catenin signaling. Furthermore, endogenous β-catenin was required for the BRAFV600E inhibitor PLX4720 to induce apoptosis in cultured melanoma cells. Moreover, activation of Wnt/β-catenin signaling enhanced the ability of PLX4720 to reduce melanoma tumor growth in vivo and synergized with PLX4720 to reduce melanoma cell growth and to increase apoptosis in vitro. Mechanistically, inhibition of BRAFV600E enhanced the Wnt-mediated reduction in the abundance of AXIN1, which promoted Wnt/β-catenin signaling and increased β-catenin–mediated apoptosis of melanoma cells. Furthermore, knockdown of AXIN1 by small interfering RNA (siRNA) sensitized melanoma cell lines otherwise resistant to apoptosis after BRAFV600E inhibition. These results have implications for improving the efficacy of inhibitors of BRAFV600E in treating melanoma as well as revealing functional crosstalk between Wnt/β-catenin and BRAF signaling in melanoma.
Results Back to Top
BRAFV600E is a negative regulator of Wnt/β-catenin signaling in melanoma cells
To identify new regulators of Wnt/β-catenin signaling in melanoma, we used A375 human melanoma cells (which harbor the BRAFV600E mutation) stably expressing the β-catenin–activated reporter (BAR) (20). We used these cells in a high-throughput siRNA screen targeting 716 genes encoding known or predicted kinases. This screen revealed that BRAF siRNAs synergized with WNT3A to activate BAR (Fig. 1A, fig. S1, A and B, and databases S1 and S2). This result was validated with four independent siRNAs targeting BRAF, as well as with a published siRNA that specifically targets activated BRAFV600E (21) (fig. S2, A to C, and table S1). These data support the unexpected hypothesis that activated BRAFV600E negatively regulates Wnt/β-catenin signaling in melanoma cells.
We then asked whether the enhancement of Wnt/β-catenin signaling observed with BRAF siRNAs was phenocopied with PLX4720, a small molecule designed to selectively inhibit the constitutively active BRAFV600E mutant kinase (2). Indeed, PLX4720 enhanced Wnt/β-catenin signaling in a dose-dependent manner (Fig. 1B and fig. S3, A and B) at doses similar to its dose-dependent inhibition of dual-phosphorylated ERK1/2 (ppERK1/2) (Fig. 1C). Combination indices for WNT3A and PLX4720 were much less than 1 (fig. S3C), supporting a synergistic interaction between these two drugs with respect to Wnt/β-catenin activation. In further support of a synergistic interaction, the calculated WNT3A dose-reduction index showed that six times less WNT3A was needed when PLX4720 was present to achieve the median effective concentration (EC50) BAR response of WNT3A alone.
Consistent with its acting as an enhancer of Wnt/β-catenin signaling, PLX4720 treatment decreased phosphorylation of β-catenin at sites normally phosphorylated by glycogen synthase kinase-3 (GSK3) to target β-catenin for proteasomal degradation (Fig. 1C). In support of this observation, the activating autophosphorylation of GSK3 at Tyr216 was reduced upon treatment of cells with PLX4720 (Fig. 1C). Although these effects on phosphorylation of β-catenin and GSK3 did not result in increased abundance of cytosolic or nuclear β-catenin (fig. S4), recent findings have established that activation of β-catenin function in melanoma correlates with the same changes in phosphorylation shown here (Fig. 1C and fig. S4) rather than with changes in the abundance of β-catenin (22). Because BRAF signals through the downstream kinase MEK, we investigated the effects on β-catenin signaling of two independent small-molecule MEK inhibitors, U0126 and AZD6244 (23). We found that both drugs synergistically enhanced Wnt/β-catenin activation as measured by the BAR assay (Fig. 1D) and did so at doses similar to their dose-dependent inhibition of ppERK1/2 (Fig. 1E). Thus, BRAF signaling through MEK regulates Wnt/β-catenin signaling in melanoma cells.
WNT3A enhances the ability of an inhibitor of BRAFV600E to reduce tumor size
Because targeted inhibition of BRAFV600E or activation of Wnt/β-catenin signaling reduces melanoma tumor size (2, 11, 24), and because we showed that the inhibition of BRAFV600E enhanced Wnt/β-catenin signaling, we determined whether concurrent inhibition of BRAFV600E and activation of Wnt/β-catenin signaling would cooperate to reduce tumor size. Immunosuppressed mice harboring subcutaneous xenografts generated from human A375-GFP (green fluorescent protein) cells (controls) or A375-WNT3A cells (expressing WNT3A-IRES-GFP) were treated by oral gavage with either vehicle or PLX4720. Biochemical analysis of fine-needle aspirates sampled from tumors during treatment confirmed inhibition of ppERK1/2 in vivo after PLX4720 treatment (fig. S5, A and B). Results of the xenograft study revealed that treatment of A375-GFP tumors with PLX4720 decreased tumor growth compared to treatment with drug vehicle (Fig. 2A). The growth of A375-WNT3A tumors was slower compared to that of both A375-GFP tumors treated with vehicle and A375-GFP tumors treated with PLX4720. The effects of PLX4720 on A375-WNT3A tumor growth were even more pronounced than the effects on A375-GFP tumors, with near-complete suppression of A375-WNT3A tumor growth over 4 weeks. Growth curves were significantly different upon one-way analysis of variance (ANOVA) with a post-test for linear trend (P = 0.024). Direct comparisons of tumor volume between groups at day 23 (fig. S5C) revealed a highly significant difference upon one-way ANOVA with post-test for linear trend (P < 0.0001). These results paralleled the significant differences seen in mitotic index (P < 0.0001) upon histological analysis of the xenografts (fig. S5D). We conclude that WNT3A greatly enhanced the ability of PLX4720 to reduce melanoma tumor size in this xenograft assay.
To confirm and extend these results, we turned to a three-dimensional spheroid assay of tumor cell growth and invasion within a collagen matrix. Treatment of spheroids from either A375-GFP or A375-WNT3A cells with PLX4720 decreased spheroid size (Fig. 2B), paralleling the decreased tumor sizes observed in xenograft studies (Fig. 2A). Treatment of spheroids expressing WNT3A with PLX4720 led to a marked decrease in the number of invasive cells at 72 hours compared to either A375-WNT3A–derived spheroids treated with dimethyl sulfoxide (DMSO) or A375-GFP–derived spheroids treated with PLX4720 (Fig. 2B).
We also tested for synergistic inhibition of melanoma cell growth by WNT3A and PLX4720 in two-dimensional cell culture. Cell viability was measured in A375 melanoma cells treated with combinations of WNT3A and PLX4720 at various concentrations (Fig. 2C and fig. S6, A and B). The reduced number of viable cells after combined treatment with WNT3A and PLX4720 resulted in combination indices much less than 1 (fig. S6C). At 50% growth inhibition, the drug reduction indices were 8.1 for PLX4720 and 117.4 for WNT3A conditioned medium (CM), further supporting a synergistic effect of these two drugs. Together, these three different assays demonstrated that the simultaneous activation of Wnt/β-catenin signaling and the targeted inhibition of BRAFV600E by PLX4720 functionally cooperate to decrease melanoma cell growth both in vivo and in vitro.
WNT3A enhances the ability of an inhibitor of BRAFV600E to increase apoptosis
We next asked whether this reduction in tumor size that occurred in response to inhibition of BRAFV600E combined with activation of Wnt/β-catenin was due to apoptotic cell death. TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining of melanoma cells treated for 24 hours with WNT3A and PLX4720 indicated the presence of apoptotic cell death (Fig. 3A). Consistent with this finding, dead cells were only detected in A375 spheroids concurrently expressing WNT3A and treated with PLX4720 (Fig. 3B). Flow cytometry with an antibody that detects the cleaved (active) form of caspase-3 confirmed that cell death was the result of caspase-mediated apoptosis (Fig. 3C). Consistent with the TUNEL and the spheroid assays, we did not observe apoptosis in the presence of DMSO vehicle alone and detected only minimal increases in cleaved caspase-3 with either PLX4720 or WNT3A CM alone. However, in the presence of both WNT3A CM and PLX4720, the number of cells with cleaved caspase-3 increased ~5- to 20-fold (Fig. 3C). In support of a caspase-mediated apoptotic pathway, combined treatment with WNT3A and PLX4720 led to synergistic cleavage of the caspase-3 substrate, poly(ADP-ribose) polymerase 1 (PARP1) (Fig. 3D, lane 4 versus lanes 2 and 3). The pan-caspase inhibitor Z-VAD-FMK abolished PARP1 cleavage (Fig. 3D, lane 8 versus lane 4). To establish that the effects of PLX4720 on apoptosis were specifically due to inhibition of BRAFV600E, we showed that knockdown of BRAF by siRNA also enhanced the cleavage of caspase-3 in the presence of WNT3A CM (fig. S7). Together, these data demonstrated that simultaneous activation of Wnt/β-catenin signaling and inhibition of BRAFV600E functionally cooperated to induce caspase-mediated apoptosis of melanoma cells.
To understand how activation of Wnt/β-catenin signaling cooperated with inhibition of BRAFV600E to induce apoptosis in melanoma cells, we explored the Bcl-2 homology domain 3–only (BH3-only) protein Bim (BCL2L11). Bim is a regulator of apoptosis, including apoptosis of melanoma cells, that binds and inhibits prosurvival Bcl-2 family members (25–32). The three major isoforms of Bim, BimEL, BimL, and BimS, are generated by alternative splicing and vary in their proapoptotic activity, with BimS being the most potent followed by BimL (25). Melanoma cells expressing BRAFV600E treated with PLX4720 exhibited increased abundance of all Bim isoforms, and the increased abundance of BimS was the primary driver of apoptosis (26). We found that PLX4720 treatment of A375 melanoma cells resulted in an increase in the abundance of all Bim isoforms (Fig. 3D, lane 3 versus lane 1) and that co-treatment of cells with WNT3A led to an apparent further increase in BimL and BimS (Fig. 3D, lane 4 versus lane 3). These increases in BimL and BimS were not blocked by Z-VAD-FMK (Fig. 3D, lane 8 versus lane 4), consistent with its role as an upstream activator of caspase-3 during apoptosis. Thus, WNT3A increased the effectiveness of a BRAFV600E inhibitor to promote apoptosis in melanoma cells through a mechanism that may involve Bim.
Endogenous β-catenin is required for PLX4720 to induce apoptosis
Knockdown of β-catenin (CTNNB1) with siRNA completely prevented caspase-3 cleavage in A375 cells treated with PLX4720 (Fig. 4A, lane 3 versus lane 7), suggesting that the BRAFV600E inhibitor PLX4720 requires a functional Wnt/β-catenin pathway for its ability to induce apoptosis. Exogenous WNT3A failed to overcome this dependence on endogenous β-catenin for PLX4720-mediated caspase-3 cleavage (Fig. 4A, lane 4 versus lane 8). We then activated β-catenin signaling downstream of the Wnt-receptor complex by treating cells with the small-molecule GSK3 inhibitor CHIR99021. Like WNT3A, CHIR99021 enhanced caspase-3 cleavage when combined with PLX4720, and this was completely inhibited upon siRNA knockdown of β-catenin (Fig. 4B, lane 4 versus lane 8). These data support the unexpected conclusion that apoptosis mediated by targeted BRAF inhibition is dependent upon β-catenin, the primary downstream effector of Wnt/β-catenin signaling.
PLX4720-mediated enhancement of Wnt/β-catenin signaling predicts apoptosis among melanoma cell lines
Many patients with tumors harboring activating BRAF mutations do not exhibit an objective clinical response to targeted BRAF inhibitors (7), suggesting the involvement of as yet unidentified proteins or signaling pathways that determine cellular susceptibility to therapy. We therefore asked whether any new insights into the heterogeneity of the response to targeted BRAF inhibitors could be gleaned by examining the interaction between Wnt/β-catenin and BRAF signaling in multiple melanoma cell lines with the BRAFV600E mutation. The AXIN2 gene is a transcriptional target of Wnt/β-catenin signaling (11, 33) and thus can be used as a marker for activity of this pathway. In A375, MEL624, and COLO829 cells, treatment with WNT3A increased the abundance of mRNA for AXIN2, and co-treatment with PLX4720 further increased the amount of AXIN2 transcripts (Fig. 5A). In contrast, treatment with PLX4720 did not augment the WNT3A-mediated increases in AXIN2 transcripts in SKMEL5, SKMEL28, or A2058 cells (Fig. 5A), despite the fact that these cells also harbor the BRAFV600E mutation (table S2). Cell lines that displayed synergistic activation of Wnt/β-catenin signaling with WNT3A and PLX4720 also exhibited increased susceptibility to apoptosis as measured by cleaved caspase-3 (Fig. 5B). These data are consistent with a model in which Wnt/β-catenin signaling is a major determinant of the apoptotic response to targeted BRAF inhibition (Fig. 4, A and B). Notably, the discrepancy in response among these cell lines cannot be accounted for by the allelic status of the BRAFV600E mutation (table S2). These data might be relevant to the observed variations in clinical response to targeted inhibitors of BRAF among tumors carrying the BRAFV600E mutation (5–7).
Reduction of AXIN1 predicts apoptotic responsiveness with combined Wnt/β-catenin activation and BRAF inhibition
The correlation between Wnt/β-catenin signaling and apoptotic response (Fig. 5, A and B) led us to further investigate the underlying mechanisms. The three melanoma cell lines that displayed the greatest increase in caspase-3 cleavage in response to co-treatment with WNT3A plus PLX4720 (A375, MEL624, and COLO829), also displayed a significant reduction in the abundance of the β-catenin antagonist AXIN1 when compared to cells exposed to WNT3A alone (Fig. 6, A and B). (Like AXIN2, AXIN1 is an inhibitor of the Wnt/β-catenin pathway; unlike AXIN2, AXIN1 is not encoded by a Wnt target gene.) By contrast, in the melanoma cell lines that were resistant to apoptosis after treatment with WNT3A plus PLX4720 (SKMEL5, SKMEL28, and A2058), the relative abundance of AXIN1within each cell line did not significantly decrease when comparing treatment with WNT3A alone to WNT3A plus PLX4720 (Fig. 6, A and B). Thus, there is a correlation between susceptibility of melanoma cells to apoptosis and the reduction in AXIN1 abundance in the presence of WNT3A and PLX4720. Notably, it is not the baseline amounts of AXIN1 but the magnitude of reduction in AXIN1 abundance within each cell line that is predictive of apoptosis in cells exposed to both WNT3A and PLX4720. For example, MEL624 cells have relatively high baseline amounts of AXIN1 (Fig. 6A, lanes 9 and 10) and display a robust loss of AXIN1 and susceptibility to apoptosis (Fig. 5B) when treated with WNT3A and PLX4720. By contrast, SKMEL5 cells have very low baseline amounts of AXIN1, and neither the amount of AXIN1 (Fig. 6, A and B, lanes 5 and 6) nor susceptibility to apoptosis (Fig. 5B) changes markedly upon treatment with WNT3A and PLX4720.
We next investigated how inhibition of BRAFV600E reduced the abundance of AXIN1. Analysis of A375 melanoma cells by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) revealed no change in the abundance of AXIN1 transcripts between cells treated with WNT3A or WNT3A and PLX4720 (fig. S8). Treatment of A375 cells with the proteasome inhibitor MG132, but not the lysosome inhibitor chloroquine, rescued the decrease in AXIN1 seen upon treatment with WNT3A and PLX4720 (Fig. 6C, lane 5 versus lane 4 and lane 6 versus lane 4). Together, these data reveal that BRAFV600E inhibition decreased the abundance of AXIN1 in the presence of WNT3A through a proteasome-dependent mechanism.
Loss of AXIN1 precedes apoptosis and can confer susceptibility to apoptosis with BRAF inhibition
To determine whether decreased AXIN1 abundance sensitized melanoma cells to PLX4720-mediated apoptosis, we investigated the temporal coordination of ppERK1/2 relative to changes in AXIN1 abundance and to the onset of apoptosis measured by caspase-3 cleavage. Time course analysis of A375 cells treated with WNT3A and PLX4720 showed that AXIN1 abundance decreased within 1 to 2 hours of treatment and that almost no detectable AXIN1 remained after 16 to 20 hours of treatment (Fig. 7A). This decrease in AXIN1 abundance followed the rapid inhibition of ppERK1/2, which occurred within 30 min of treatment. Apoptosis as measured by cleaved caspase-3 was first detected at 12 to 16 hours and increased for the duration of the experiment (Fig. 7A). These data suggest that loss of AXIN1 preceded caspase-mediated apoptosis. Furthermore, whereas the pan-caspase inhibitor Z-VAD-FMK inhibited apoptosis, measured as PARP1 cleavage, in these cells (Fig. 7B), it did not affect the loss of AXIN1, indicating that the decrease in AXIN1 was not a downstream consequence of caspase-3 activation. In agreement, inhibition of apoptosis by Z-VAD-FMK did not affect PLX4720-mediated enhancement of Wnt/β-catenin signaling as measured by BAR (Fig. 7C). These data suggested that the enhanced Wnt/β-catenin signaling observed in cell lines that exhibit enhanced apoptosis with co-treatment of WNT3A and PLX4720 (Fig. 5, A and B) is upstream of caspase-3 activation. We concluded that decreases in AXIN1 abundance precede, and are independent of, the onset of apoptosis.
Given that decreases in AXIN1 abundance (Figs. 6, A and B, and 7A) preceded apoptosis and seemed to predict both susceptibility to apoptosis in response to combined Wnt and PLX4720 treatment (Fig. 5B) and enhancement of Wnt/β-catenin signaling by BRAF inhibition (Fig. 5A), we hypothesized that reducing the abundance of AXIN1 in the three melanoma cell lines that were more resistant to apoptosis (SKMEL28, A2058, and SKMEL5) would render them newly susceptible to apoptosis in the presence of PLX4720. Indeed, siRNA-mediated knockdown of AXIN1 (fig. S9) increased apoptosis with PLX4720 as measured by cleaved caspase-3 in all three cell lines (Table 1 and Fig. 7D). This result is consistent with our hypothesis that the reduction of AXIN1 abundance seen with β-catenin activation facilitates PLX4720-mediated apoptosis. We confirmed the ability of AXIN1 knockdown to confer susceptibility to apoptosis in response to PLX4720 with two independent and validated siRNAs (Fig. 7E). In the A375 cell line that responded with robust apoptosis upon WNT3A and PLX4720 treatment, knockdown of AXIN1 by siRNA enhanced apoptosis in response to PLX4720 alone, whereas apoptosis triggered by either WNT3A or the combination of WNT3A and PLX4720 was not further enhanced by siRNA-mediated knockdown of AXIN1 (Fig. 7F). These results strongly argue that the decrease in AXIN1 abundance observed with the combination of BRAF inhibition and Wnt/β-catenin activation plays an important and previously unsuspected role in the regulation of apoptosis in melanoma cells.
Discussion Back to Top
The development and initial clinical success of targeted BRAF inhibitors, such as vemurafenib and GSK2118436, represent a milestone in cancer treatment that will likely pave the way for other mutation-specific cancer therapies. Although the response rates in early clinical trials with these two drugs are promising, there are still obstacles to achieving long-term disease control with this approach. For example, the variability of responses to targeted BRAF inhibitors among patients with BRAFV600E tumors remains unexplained (5–7). Our finding that AXIN1 abundance can predict the apoptotic response to inhibition of BRAFV600E begins to shed light on the mechanisms underlying these variable responses and points to potential approaches for enhancing the effectiveness of vemurafenib therapy.
Another ongoing clinical problem is the eventual development of resistant tumors and the progression of the disease even in patients who respond well to initial therapy (7). This raises the question of whether targeting multiple signaling pathways may lead to a durable clinical result. Although combination targeting of BRAF signaling has been suggested with other pathways implicated in melanoma, such as the phosphoinositide 3-kinase pathway (34, 35), our data support the evaluation of inhibition of BRAF signaling concurrent with activation of Wnt/β-catenin signaling.
The characterization of a large panel of melanoma cells treated with PLX4032 (36) supports the need for evaluating the efficacy of combination therapies for melanoma. Consistent with our observation that β-catenin was required for PLX4720 to promote apoptosis, the transcriptional profiling of melanoma lines revealed that cell lines that are more resistant to growth inhibition by PLX4032 exhibit the loss of genes related to active Wnt/β-catenin signaling (36). These observations may facilitate the identification of patients who will benefit from concurrent activation of Wnt/β-catenin signaling and inhibition of BRAFV600E.
Although the notion of activating Wnt/β-catenin signaling in any cancer patient may seem initially contraindicated in light of its frequent role as an oncogenic pathway in colorectal carcinoma (37), it is likely that activating β-catenin has context-dependent effects that lead to distinct cellular responses. For example, context-dependent differences in the role of β-catenin in preventing versus promoting programmed cell death have been reported (38–41). Given that β-catenin signaling can elicit context-dependent effects, and given the lack of consensus on the effects of activating β-catenin signaling from in vitro cell models of melanoma (42–46), it is difficult to accurately predict the consequences of systemic activation of this pathway in melanoma patients, pointing to the need for additional research.
The observed effects of BRAF inhibition on AXIN1 abundance and GSK3 phosphorylation and activation intuitively predict that the abundance of β-catenin would likely change in response to BRAF inhibition. However, the ability of PLX4720 to enhance Wnt/β-catenin signaling, as monitored both by the luciferase reporter assay (BAR) and by monitoring the increased expression of the endogenous target gene AXIN2, does not require additional β-catenin accumulation (47–49). Although this seems perplexing on the basis of general models of Wnt signaling, such general models do not always fit with observed data. Indeed, the report that loss of phosphorylation of β-catenin at Thr41 is sufficient to enhance Wnt/β-catenin signaling in melanoma cells independent of detected increases in nuclear β-catenin is entirely consistent with our current observations (22).
These studies uncovered a previously unknown reciprocal relationship between Wnt/β-catenin and BRAF signaling in melanoma, highlighting the potential impact of both pathways on therapeutic efforts to target BRAF in metastatic melanoma with drugs such as vemurafenib. The discovery that regulation of AXIN1 provides the basis for mediating functional crosstalk between these two pathways provides not only a new model for studying Wnt/β-catenin and BRAF signaling in melanoma, but also a foundation for identifying tumor-specific determinants of the response to vemurafenib and other targeted BRAF inhibitors. The identification of these tumor-specific determinants may facilitate the development of new therapies and prognostic biomarkers that can further extend the promising clinical results seen with pioneering mutation-specific drugs such as vemurafenib, with the ultimate goal of developing therapies that provide sustained long-term clinical responses for patients with metastatic melanoma.
Materials and Methods Back to Top
Detailed information on BAR has been previously described (20). Briefly, pBAR is a lentiviral plasmid that contains 12 TCF/LEF binding sites (5'-AGATCAAAGG-3'), each separated by distinct 5–base pair linkers upstream of a minimal promoter and the firefly luciferase open reading frame. The reporter also contains a separate PGK (phosphoglycerate kinase) promoter that constitutively drives the expression of a puromycin resistance gene for mammalian cell selection. Transient transfection of siRNA was performed with RNAiMAX (Invitrogen), as directed by the manufacturer. siRNA sequences used are listed in table S1. Protease and phosphatase inhibitor tablets were purchased from Roche. Concanavalin A (Con A) Sepharose was purchased from GE Healthcare. U0126 was purchased from LC Laboratories. AZD6244 was purchased from Selleck Chemicals. PLX4720 was purchased from Symansis. CHIR99021 was purchased from Axon Medchem. Z-VAD-FMK was purchased from R&D Systems. Antibodies recognizing ERK (p42 and p44), phopho-ERK (p42/44), phospho–β-catenin S33/37/T41, BRAF, cleaved CASP3, cleaved PARP1, Bim, or cleaved CASP3 Alexa Fluor 488 conjugate were purchased from Cell Signaling. Antibodies recognizing β-tubulin or β-catenin were purchased from Sigma-Aldrich. The antibody recognizing phospho-GSK3 Y279/216 was purchased from Upstate Biotechnology. The antibody recognizing AXIN1 was purchased from R&D Systems. In Situ Cell Death Detection kit was purchased from Roche.
The human melanoma cell lines A375, A2058, and MEL624 were a gift from C. Yee (Fred Hutchinson Cancer Research Institute, Seattle, WA). The human melanoma cell lines COLO829, SKMEL28, and SKMEL5 were purchased from the American Type Culture Collection. Stable BAR cell lines were generated as previously described (20). BAR luciferase cell lines were also infected with a lentivirus carrying Renilla luciferase driven by a constitutive EF1α promoter.
The human melanoma lines A375 and A2058 were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 1% antibiotic. The human melanoma lines SKMEL5 and SKMEL28 were grown in Eagles minimum essential medium (EMEM) supplemented with 10% FBS and 1% antibiotic. The human melanoma lines COLO829 and MEL624 were grown in RPMI supplemented with 10% FBS and 1% antibiotic. Synthetic siRNAs were transfected into cultured cells at a final concentration of 20 nM with RNAiMAX (Invitrogen).
Screening was performed at the Quellos High-Throughput Screening Facility at the University of Washingtons Institute for Stem Cells and Regenerative Medicine (Seattle, WA). A library of siRNAs targeting primarily the human kinome was screened in A375 melanoma cells stably expressing BAR. The kinome siRNA library was purchased from Sigma-Aldrich and resuspended in ribonuclease-free water. The library consists of a pool of three independent nonoverlapping siRNAs for each mRNA target. siRNA pools were screened in quadruplicate at 9.5, 1.9, 0.38, and 0.08 nM final concentration. Cell viability was assessed by adding resazurine (Sigma-Aldrich) at a final concentration of 1.25 μg/ml [phosphate-buffered saline (PBS) vehicle] and measuring fluorescence intensity (Ex = 530 nM, Em = 580 nM) on an Envision multilabel plate reader (PerkinElmer). Luciferase activity was assessed by adding Steady-Glo (5 μl per well; Promega) and measuring total luminescence on an Envision multilabel plate reader (PerkinElmer). The screen workflow was as follows.
On day 1, 1.5 μl of the appropriate concentration of siRNA was added to 28.5 μl of Opti-MEM (Invitrogen) containing RNAiMAX (3.125 μl/ml; Invitrogen). Five microliters of this mix was transferred to a 384-well plate containing 15 μl of growth medium (DMEM/5% FBS/1% penicillin-streptomycin). Twenty microliters of cells at 75 cells/μl was added to each well for a final cell number of 1500 cells per well. On day 3, 10 μl of WNT3A CM diluted 1:12.8 with growth medium was added for a final dilution of 1:64. On day 4, 10 μl of 6x resazurine was added to each well, incubated at 37°C for 3 hours, and fluorescence intensity was measured. Immediately after, 5 μl of Steady-Glo was added, incubated at room temperature for 10 min, and total luminescence was measured. Data are represented as a ratio of BAR activity (luminescence) to cell viability (resazurine fluorescence intensity).
Low-throughput BAR reporter assays
Cells were plated in 96-well plates. Twenty-four hours after plating, cells were treated with the indicated conditions, and luciferase activity was measured 24 hours later with a Dual-Luciferase Reporter Assay kit (Promega) and an Envision multilabel plate reader (PerkinElmer) per manufacturers suggestions. For BAR assays involving siRNAs, siRNAs were transfected 48 hours before treatment.
Low-throughput siRNA transfections
Cells were reverse-transfected with 20 nM siRNA (final concentration) in six-well plates with RNAiMAX reagent per manufacturers suggestions (Invitrogen). Cells were incubated for 48 hours after transfection and then treated with the indicated conditions for the indicated amount of time.
Cytosolic and nuclear β-catenin fractionation
Cells were plated in 100-mm dishes. Twenty-four hours after plating, cells were treated with the indicated conditions for 24 hours. Cells were gently rinsed with PBS and harvested by scraping in 500 μl of hypotonic lysis buffer [50 mM Hepes (pH 8.0), 1 mM EDTA, and 1 mM dithiothreitol] containing protease and phosphatase inhibitors. Cells were swelled on ice for 30 min and then passed through a 27-gauge needle 10 times and checked for complete lysis with a microscope. Lysates were centrifuged at 10,000g for 20 min and supernatant was collected as the cytosolic fraction. Pelleted membranes were washed five times with hypotonic lysis buffer and then solubilized with solubilization buffer [50 mM tris (pH 8.0), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100] containing protease and phosphatase inhibitors. After a 30-min incubation on ice, lysates were centrifuged at 16,000g for 20 min. The protein concentration of the cleared supernatant was determined by bicinchoninic acid (BCA) analysis, and an equal amount of protein and volume was then incubated with prewashed Con A Sepharose beads overnight at 4°C. Supernatant was collected as the nuclear fraction.
RNA purification and qRT-PCR analysis
RNA was purified with the RNeasy kit according to the manufacturers protocol (Qiagen). Complementary DNA (cDNA) was synthesized with RevertAid M-MuLV Reverse Transcriptase (Fermentas). LightCycler FastStart DNA Master SYBR Green 1 (Roche) was used for real-time PCR as previously described (50). Quantitative PCR results presented in the manuscript are averages of a minimum of three biologic replicates.
Isobologram analysis of cell viability
A375 melanoma cells were seeded in 96-well plates at a concentration of 8000 cells per well in 100 μl of growth medium. Twenty-four hours after plating, cells were treated with all combinations of twofold dilutions of WNT3A CM ranging from 20 to 0% and twofold dilutions of PLX4720 ranging from 5 to 0 μM for 48 hours. CellTiter-Glo (10 μl; Promega) was added to each well and total luminescence was measured on an Envision multilabel plate reader (PerkinElmer). Each condition within an experiment was assayed in triplicate wells and three independent experiments were performed.
Flow cytometry for active caspase-3
Cells were seeded in a six-well dish at a density to achieve 90 to 100% confluence at harvest. Twenty-four hours after seeding, cells were treated with the indicated conditions for the indicated amount of time. At the time of collection, supernatants were collected and pooled with trypsinized cells. Cells were fixed with 4% paraformaldehyde and permeabilized according to the vendors protocol for cleaved caspase-3 (Asp175) antibody (Alexa Fluor 488 conjugate) (Cell Signaling). The antibody was used at a final dilution of 1:100. Flow was performed on a BD FACSCanto II, and data were analyzed with FlowJo 8.8.6 (Tree Star) software. Experiments were performed in biological triplicate, and data are representative of at least three independent experiments.
For experiments involving siRNAs, cells were reverse-transfected with 20 nM siRNA in six-well dishes in triplicate with RNAiMAX according to the manufacturers protocol. Forty-eight hours after transfection, cells were treated with the indicated conditions for 24 hours and then harvested for analysis. Cells were harvested, stained, and analyzed as described above.
Glass coverslips were coated with poly-L-lysine in a 24-well dish, rinsed with PBS, and dried. Cells were seeded at a density to achieve 90 to 100% at harvest. Twenty-four hours after seeding, cells were treated with the indicated conditions and incubated for 24 hours. TUNEL staining was performed according to the vendors protocol (Roche). Briefly, the medium was gently aspirated and cells were fixed in 4% paraformaldehyde for 1 hour at room temperature. Cells were gently rinsed twice with PBS and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Cells were rinsed twice with PBS and 40 μl of TUNEL reaction mixture was added directly on top of the slide; cells were incubated for 1 hour at 37°C in a humidified incubator. Slips were rinsed three times and mounted on Superfrost Plus glass slides with ProLong Gold anti-fade mounting medium containing 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen). Images were obtained on a Nikon TiE inverted wide-field high-resolution microscope.
A375 cells were used for the spheroid assays. Spheroids were formed and implanted in collagen as previously described (51). Spheroids were treated with indicated conditions 30 min after collagen polymerization. Images were obtained on a Nikon TiE inverted wide-field high-resolution microscope. For comparison of growth effects (such as shown in Fig. 2), spheroids were imaged at 72 hours after spheroid implantation. For live-dead imaging assays (such as shown in Fig. 3), imaging was performed at 24 hours after spheroid implantation.
NSG [NOD/SCID/IL2r- (null)] mice were injected with 5 x 105 A375 cells stably expressing GFP or 5 x 105 A375 cells stably expressing WNT3A-IRES-GFP. Tumors were allowed to establish to about 100 mm3, after which mice were tumor size–matched and allocated to five per treatment group (vehicle or PLX4720). WNT3A-IRES-GFP tumors grew slower, and, therefore, the first day of treatment was day 14, whereas GFP-expressing tumors were first treated on day 9. Treatment was by oral gavage once daily with 5% DMSO in 1% carboxymethyl cellulose or PLX4720 (50 mg/kg) in 1% carboxymethyl cellulose (604 mM PLX4720 in DMSO was diluted 1:20 in 1% carboxymethyl cellulose). Tumor size was determined by caliper measurements of tumor length and width every 3 to 4 days. Tumor volume was then calculated with the following formula: volume = (width)2 x length/2. Tumors were harvested 2 hours after the last dose and fixed in neutral-buffered formalin overnight at room temperature.
Hematoxylin and eosin–stained tumor sections were scored for mitotic activity by a board-certified pathologist who was blinded to the treatment conditions. For each treatment condition, five tumors were evaluated and a range of 26 to 60 high-powered fields (hpfs) per individual tumor were scored (average of 44 hpfs per tumor). Areas with fixation artifact were excluded a priori from the final analysis, accounting for differences in the number of hpfs per individual tumor. Analysis was performed with a one-way ANOVA followed by a post-test for linear trend.
Standard statistical analysis was performed with GraphPad Prism version 5.01 (GraphPad Software Inc.). Dose-effect analyses, including combination indices, dose reduction indices, and median-effect analysis, were performed using the method of Chou and Talalay (52) with the CalcuSyn software suite version 2.1 (Biosoft).
Supplementary Materials Back to Top
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.
References and Notes Back to Top
Acknowledgments: We thank C. Yee (Fred Hutchinson Research Institute, Seattle, WA) for cell lines and J. D. Lebowski for administrative assistance with the preparation of the manuscript. Funding: T.L.B. is funded in part through a training grant from NIH/National Institute of Arthritis and Musculoskeletal and Skin (NIAMS) (T32AR056969). A.J.C. is funded by the NIH/National Cancer Institute (NCI) (K08CA128565). R.M.K. is supported by an administrative supplemental grant through the American Recovery and Relief Act and the NIH/NCI. R.G.J. is funded in part by NIH/National Heart, Lung, and Blood Institute K99/R00 grant 1K99HL103768-01. R.T.M. is an Investigator of the Howard Hughes Medical Institute. We are indebted to these funding agencies for their continued support of our work. The contents of this manuscript are the sole responsibility of the authors and do not necessarily represent the official views of the NIAMS, NCI, NIH, or the Howard Hughes Medical Institute. Author contributions: T.L.B., A.J.C., and R.T.M. conceived the project. T.L.B., A.J.C., and R.T.M. designed the experiments and analyzed the data. T.L.B. and R.M.K. performed most of the experiments. R.A.T. performed the in vivo studies. O.M.L. and R.D.S. performed in vitro spheroid assays. R.G.J. and N.C.R. helped with the siRNA screening. D.W.D. analyzed the xenograft histology. T.L.B., A.J.C., and R.T.M. wrote the manuscript. Competing interests: R.T.M. is a cofounder of Fate Therapeutics (San Diego, CA) and a consultant for Theriac Inc. (Seattle, WA). The following provisional patents were filed based on this work: (i) combination therapies involving Wnt/β-catenin signaling and stratification of patients based on Axin levels and (ii) use of WNT activators as synergistic enhancers of targeted BRAF inhibitors in melanoma.
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).
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