Pharmacologic Inhibition of Ezrin-Radixin-Moesin Phosphorylation is a Novel Therapeutic Strategy in Rhabdomyosarcoma

Abstract

Intermediate and high-risk rhabdomyosarcoma (RMS) patients have poor prognosis with available treatment options, highlighting a clear unmet need for identification of novel therapeutic strategies. Ezrin-radixin-moesin (ERM) family members are membrane-cytoskeleton linker proteins with well-defined roles in tumor metastasis, growth, and survival. ERM protein activity is regulated by dynamic changes in the phosphorylation at a conserved threonine residue in their C-terminal actin-binding domain. Interestingly, ERM family member, ezrin, has elevated expression in the RMS tissue. Despite this, the translational scope of targeting ERM family proteins in these tumors through pharmacological inhibition has never been considered. This study investigates the inhibition of ERM phosphorylation using a small molecule pharmacophore NSC668394 as a potential strategy against RMS. Upon in vitro treatment with NSC668394, RMS cells exhibit a dose-dependent decrease in cell viability and proliferation, with induction of caspase-3 cleavage and apoptosis. siRNA-mediated knockdown of individual ERM protein expression revealed that each regulates RMS survival to a different degree. In vivo administration of NSC668394 in RMS xenografts causes significant decrease in tumor growth, with no adverse effect on body weight. Collectively, this study highlights the importance of the active conformation of ERM proteins in RMS progression and survival and supports pharmacologic inhibition of these proteins as a novel therapeutic approach.

Figure 1

NSC668394 dephosphorylates ezrin and reduces cell viability and metabolism in RMS cells. (a) Western blot showing levels of phosphorylated ezrin-radixin-moesin (pERM) and total ezrin in untreated RMS cell lines. (b) Levels of pERM and total ezrin in RD and Rh41 whole cell lysates following treatment with indicated concentrations of NSC668394 or DMSO (D) for 1 h. (c) RMS cell viability following treatment with increasing concentrations of NSC668394 for 0–96 h as determined by trypan blue (d) RMS cell metabolism following treatment with increasing concentrations of NSC668394 as determined by MTT assay. (e) Variable slope nonlinear regression of 96 h MTT data was used to extrapolate IC50 values for inhibition of cell metabolism. All individual data points are represented as mean ± SEM and are representative of at least three independent experiments. Asterisks represent a significant difference between vehicle control DMSO and NSC668394 treated groups (^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and p < 0.0001=^∗∗∗∗) for the given time point as determined by two-way ANOVA with Dunnett's multiple comparisons test.

Figure 2

NSC668394 treatment triggers caspase-induced apoptosis in RMS cells. (a) Representative flow cytometry dot plots of RMS cell lines stained with annexin V and 7-AAD following treatment with 10 μM NSC668394 or DMSO for 96 h. (b) Percentage of cells determined to be annexin V+ or annexin V+/7AAD+ were quantified after 0, 48, and 96 h after treatment with DMSO or NSC668394. Individual bars are represented as mean ± SEM and representative of three independent experiments. Asterisks represent a significant difference between vehicle and treated groups (^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and p < 0.0001=^∗∗∗∗) for the given time point, as determined by two-way ANOVA with Dunnett's multiple comparisons test. (c) Representative Western blot of cleaved caspase-3 levels in RD or Rh41 cells treated with 10 μM NSC668394 for indicated times. Relative band intensity was determined using ImageJ software and depicted in the bar graph. Blots are representative of three independent experiments.

Figure 3

siRNA-mediated interference of ERM proteins reduces RMS cell viability. (a) Representative Western blots showing relative expression of ezrin, radixin, or moesin in RD cells treated with pooled siRNAs against ezrin, radixin, moesin, or all three together. Nontargeting scrambled (control) siRNA was used as a negative control for target protein expression, and β-actin was used as a loading control. (b) Quantification of ezrin, radixin, and moesin expression in cells treated with siRNA against ERM proteins relative to the corresponding target expression in control siRNA-treated cells. Expression is plotted as a function of relative band intensities determined using ImageJ software. (c) Viable cell counts as determined by trypan blue staining 96 h after treatment of RD cells with siRNA against ERM proteins or nontargeting control siRNA. Bars represent mean ± SEM, and asterisks represent a significant difference between control siRNA treatment group and ERM siRNA treatment groups as determined by one-way ANOVA with Dunnett's multiple comparisons tests (^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and p < 0.0001=^∗∗∗∗). All data are representative of two independent experiments.

Figure 4

NSC668394 inhibits the growth of RD xenograft in vivo. (a) Subcutaneous (SQ) RD tumor volumes in NSG mice subjected to daily intraperitoneal administration of either 20 mg/kg NSC668394 (n = 6) or DMSO (n = 6). (b) Orthotopic intramuscular (IM) RD tumor volumes in NSG mice subjected to daily intraperitoneal administration of 40 mg/kg NSC668394 (n = 10) or DMSO (n = 10). Total body weight of NSG mice bearing subcutaneous (c) and orthotopic (d) RD tumors on the indicated days. Negative values shown on the x-axis of total body weight plots represent days prior to starting NSC668384 treatment. Individual data points are represented as mean ± SEM. Asterisks represent a significant difference between treatment groups (^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0005, and p < 0.0001=^∗∗∗∗) for the given time point as determined by RM two-way ANOVA with Holm-Šídák multiple comparisons tests. Representative images of subcutaneous xenograft sections stained for Ki67 (e) or cleaved caspase-3 (f). Each stain was quantified as optical density using Fiji extension of ImageJ and plotted as bar graphs. Bars represent mean ± SEM, and asterisks represent a significant difference between treatment groups as determined by the unpaired t-test (^∗p < 0.05 and ^∗∗p < 0.005).