A TRAIL receptor-dependent synthetic lethal relationship between MYC activation and GSK3beta/FBW7 loss of function

Abstract

The MYC protooncogene is frequently deregulated in human cancers. Here, by screening a kinase-directed library of small inhibitory RNAs, we identify glycogen synthase kinase 3beta (GSK3beta) as a gene whose inactivation potentiates TNF-related apoptosis-inducing ligand death receptor-mediated apoptosis specifically in MYC-overexpressing cells. Small inhibitory RNA-induced silencing of GSK3beta prevents phosphorylation of MYC on T58, thereby inhibiting recognition of MYC by the E3 ubiquitin ligase component FBW7. Attenuating the GSK3beta-FBW7 axis results in stabilization of MYC, up-regulation of surface levels of the TNF-related apoptosis-inducing ligand death receptor 5, and potentiation of death receptor 5-induced apoptosis in vitro and in vivo. These results identify GSK3beta and FBW7 as potential cancer therapeutic targets and MYC as a critical substrate in the GSK3beta survival-signaling pathway. The results also demonstrate paradoxically that MYC-expressing tumors might be treatable by drug combinations that increase rather than decrease MYC oncoprotein function.

Fig. 1.

siRNA screen for genes synthetically lethal with MYC activation in the presence of suboptimal doses of DR5 agonists. (a) Screen schematic. Arrayed siRNAs were reverse-transfected into HA1E or HA1E-MYC cells and assayed for sensitivity to DR5-A (see Methods). (b) Genotype-dependent effects of siRNAs on cell viability. siRNAs were ranked in increasing order on the x axis by their ability to sensitize HA1E-MYC cells to DR5-A-induced apoptosis relative to the sensitization of HA1E cells (see Supporting Methods). The siRNA for GSK3β (siGSK3β) is indicated.

Fig. 2.

Depletion of GSK3β but not GSK3α potentiates DR5-mediated apoptosis specifically in MYC-overexpressing cells. (a and b) Multiple siRNAs targeting nonoverlapping sequences of GSK3β (siGSKβ) or GSK3α (siGSKα) were reverse-transfected into HA1E or HA1E-MYC cells and assayed for sensitivity to DR5-A. Numbers designate distinct siRNAs, and sp denotes a “smartpool” of four distinct siRNAs. Error bars indicate standard deviations of triplicate measurements. siGL3 were used as controls. (c) siRNA efficacy was determined by Western blot analysis using antibodies specific for each siRNA target.

Fig. 3.

GSK3β regulates sensitivity to DR5-mediated apoptosis via phosphorylation of MYC T58. (a and b) Mutation of the GSK3β MYC T58 phosphorylation site mimics GSK3β or FBW7 loss of function. Derivatives of HA1E cells expressing retrovirally transduced WT MYC (b) or the T58 phosphorylation site mutant MYCT58A (a) were transfected with the indicated siRNAs and treated with DR5-A and assayed for viability (see Fig. 10 a and b for comparison with higher DR5-A concentrations). (c) In HA1E-MYC cells transfected with siRNAs directed against GSK3α (siGSK3α), GSK3β (siGSK3β), or FBW7 (siFBW7), MYC protein levels and T58 phosphorylation status were determined by Western blot analysis using MYC or MYCT58-phospho-specific antibodies. By densitometry, siRNAs directed against GSK3α, GSK3β, and FBW7 reduced MYC T58 phosphorylation by 1.7-fold, >100-fold, and 1.5-fold, respectively, relative to siGl3.

Fig. 4.

The GSK3β–FBW7 MYC degradation pathway determines sensitivity to DR5-A-induced apoptosis. (a and c) HA1E-MYC (a) and HA1E (c) cells were transfected with one of two nonoverlapping siRNAs targeting the MYC ubiquitination component FBW7 (siFBW7-1 or siFBW7-2) and assayed for sensitivity to DR5-A. (b and d) siRNA efficacy in HA1E-MYC (b) and HA1E (d) cells was determined by Western blot analysis. Note that depletion of FBW7 or GSK3β, but not GSK3α, results in increased MYC protein levels in only HA1E-MYC cells.

Fig. 5.

Depletion or mutation of the FBW7 tumor suppressor enhances DR5-A sensitivity in tumor-derived cell lines in a MYC-dependent manner. (a) The colon carcinoma-derived cell line, HCT116, was transfected with the indicated siRNAs as previously described, and sensitivity to DR5-A was compared with genetically engineered HCT116 cells (see Methods) heterozygous (+/-) or homozygous (-/-) for FBW7. Confirmation of the efficacy of siRNA-mediated knockdown and of FBW7 genotypic status is shown in Fig. 9. (b) HCT116 FBW7^-/- cells were transfected with siRNAs and analyzed as in a with lower DR5-A concentrations (see Fig. 12 for comparison with higher DR5-A concentrations). (c) HCT116 FBW7^+/+, FBW7^+/-, and FBW7^-/- cells were transfected with control siRNAs (siGL3), two distinct nonoverlapping siRNAs targeting MYC (siMYC1 and siMYC2), and an siRNA specific for cyclin E (siCyclinE). After 48 h, transfected cells were treated with DR5-A for 20 h, and cell viability was determined.

Fig. 6.

Inactivation of the GSK3β–FBW7 axis increases DR5 receptor levels and stimulates DR5-dependent caspase-8 processing in MYC-expressing cells. (a) DR5 cell surface expression was measured by flow cytometry in HA1E-MYC cells 48 h after siRNA transfection with the denoted siRNAs. (b) Caspase-8 activation was monitored by proteolytic processing of the caspase-8 zymogen [pro-form, arrow labeled Pro-casp-8 (p55/53); activated form, arrow labeled p18; transient p43/41 form, arrow labeled p43/41]. *, nonspecific band. (c–e) HCT116 FBW7^+/+ (c), FBW7^+/- (d), and FBW7^-/- (e) cells were examined for cell surface DR5 levels by FACS analysis. For comparison, each cell line was transfected with siRNAs specific for DR5, GL3 (control shaded in blue), GSK3α, GSK3β, and FBW7.(Right) Correlation to MYC protein levels. (Left) The yellow line in each FACS panel denotes the control levels of DR5 receptor, which increases with decreasing FBW7 gene dosage. (f and g) FBW7 or GSK3β depletion specifically enhances apoptosis induced through DR5 (f) but not DR4 (g). Apoptosis was induced in siRNA-transfected or compound-treated HA1E-MYC cells of an agonistic antibody against DR4 (f) or DR5 (g). Note that treatments disrupting the GSK3β–FBW7 axis sensitize specifically to DR5-A, whereas depletion of FLIP or treatment with the translation inhibitor cycloheximide (CHX) sensitize to DR4- and DR5-mediated apoptosis. Data were normalized to antibody-treated cells transfected with a control siRNA (siGL3). Treatment with the DR4 and DR5 antibodies alone resulted in ≈5% and ≈30% reduction in viability under the conditions used for these assays.

Fig. 7.

Response of FBW7^+/+ and FBW7^+/- tumor xenografts to DR5-A therapy in vivo. HCT116 FBW7^+/+ or FBW7^+/- cells (n = 3 × 10⁶ each) were implanted in the flank of BALB/c nude mice (see Methods). At a tumor volume of 80 mm³, four animals per group were i.p. injected with 100 μg of DR5-A or buffer every other day for a total of seven treatments. Tumor sizes were measured with calipers in three dimensions. Arrows indicate the days of treatment. Error bars represent standard deviations.