Research ArticleCancer

PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells

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Science Translational Medicine  18 Jul 2018:
Vol. 10, Issue 450, eaaq1093
DOI: 10.1126/scitranslmed.aaq1093
  • Fig. 1 PP2A inhibition drives kinase inhibitor and senescence resistance in KRAS-mutant lung cancer cells.

    (A) Drug sensitivities to 230 kinase inhibitors in A549 cells transfected with indicated siRNAs in triplicates (three siRNAs per gene) were measured by cell viability assay. The drug responses are ranked by ΔDSS, which quantifies the difference between cells with PP2A inhibition (PPP2R1A siRNA) and PP2A activation (CIP2A and PME-1 siRNAs). Statistically significant enrichment for differential sensitivity to certain inhibitor families is shown in the right. P values for enrichment were calculated similarly to Gene Set Enrichment Analysis (GSEA), as described in Materials and Methods. (B) Volcano plots showing the differential sensitivity of A549 cells to drugs for each condition based on data shown in (A). P values were calculated by two-tailed t test for each drug tested in three biological replicates, and the 20% false discovery rate (FDR) was determined by Benjamini-Hochberg correction. (C to E) Impact of PP2A modulation on ΔDSS in A549 cells treated with (C) MEKi (n = 11; blue dots) and ERK inhibitors (n = 2; green triangles), (D) EGFR inhibitors (n = 18), or (E) AURK inhibitors (n = 15). Data are extracted from (A) and shown as mean values for three replicate experiments with different siRNAs. (F) Responses of A549 cells and two other KRAS-mutant cell lines, NCI-H2122 and NCI-H460, with the indicated PPP2R1A genetic status to selected kinase inhibitors listed in table S2. Color coding of responses follows the same as in (A). (G) Average responses from (F). Data are shown as means ± SEM for 18 drugs. (H) Senescence-associated β-galactosidase (SA-β-gal) staining in two individual NCI-H460 clones after reverting one allele of the homozygous PPP2R1A E64D mutation back to wild-type (WT) sequence. (I) Fraction of β-galactosidase stain–positive and flattened cells ± 95% confidence intervals was calculated for pooled data from seven and five technical replicates, respectively, for clones 2 and 3 and their corresponding controls. ***P < 0.001 for χ2 test.

  • Fig. 2 PP2A inhibition confers MAPK pathway inhibitor resistance in KRAS-mutant lung cancer cells.

    PPP2R1A denotes RNA interference (RNAi)–mediated silencing of PPP2R1A in each panel. (A) Trametinib dose-response curve of A549 cells transfected with either control or PPP2R1A siRNA. Data are shown as means ± SEM for 11 biological replicate experiments with three technical replicates. (B) Colony formation assay for trametinib-treated A549 cells transfected with either scrambled or PPP2R1A siRNA. (C) Impact of PPP2R1A inhibition in trametinib response in colony formation assay. **P < 0.01, *P < 0.05, two-tailed t test. Data are shown as means ± SEM for six biological replicate experiments. (D) PPP2R1A protein content in control A549 cells (with four PPP2R1A alleles) or in cell clones in which CRISPR/Cas9 had selectively deleted one or two copies of the PPP2R1A gene. As a control, PPP2R1A expression was also inhibited by RNAi. Data are shown as means ± SEM for five biological replicate experiments. Scr., scramble; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Pearson’s correlation (P = 0.0076) between cell viability log[IC50 (half maximal inhibitory concentration)] for trametinib (eight biological replicate experiments with three technical replicates for the knockouts) and PPP2R1A protein expression based on (D). (F) Comparison of trametinib sensitivity of A549 (PPP2R1A WT) and NCI-H460 (PPP2R1A MUT) cells. Data are shown as means ± SEM for 11 biological replicate experiments with three technical replicates. (G) Dose-response curve of a clonogenic assay of H358 cells with shRNA knockdown of PPP2R5 (B56) family proteins or control vector (shCTRL). The graph represents the mean colony number ± SD relative to the vehicle control, three biological replicates with two technical replicates. For clarity, only shB56 knockdown lines, shPPP2R5B (gray) and shPPP2R5E (green), which have decreased sensitivity to trametinib, are shown in addition to the control. The cell colonies and data for other PPP2R5 shRNAs are shown in fig. S2 (E and F). (H) Cell viability assay of either scrambled or PPP2R1A siRNA–transfected A549 cells treated with increasing doses of trametinib and RAF inhibitor LY3009120. Data are shown as means ± SEM for three replicate experiments. (I) Colony formation assay of scrambled or PPP2R1A siRNA–transfected A549 cells treated with increasing concentrations of trametinib and RAF inhibitor LY3009120 either alone or in combination.

  • Fig. 3 AKT/mTOR signaling is a collateral MEKi resistance mechanism in PPP2R1A-depleted cells.

    (A) Log2 fold change in average of predicted ERK target site phosphorylation in A549 liquid chromatography (LC)–MS/MS data: All targets are indicated with black, PPP2R1A-responsive subset with red, and trametinib-responsive subset with blue. Data are shown as means ± SEM for mean values for peptides indicated in the legend. Each condition was analyzed as three biological replicates with different siRNA sequences (B) LC-MS/MS data of phosphorylation of AKT targets in cells treated as indicated. The shaded area highlights the shift in AKT target phosphorylation in PPP2R1A siRNA–transfected cells treated with trametinib. Distribution of mean log fold changes for three biological replicate experiments is shown. (C) Western blot analysis of selected AKT/mTOR pathway components in trametinib-treated and control cells with or without PPP2R1A depletion. (D) Cotreatment with AKT inhibitor perifosine in PPP2R1A-depleted A549 cells. Data are shown as means ± SEM for three biological replicate experiments with three technical replicates. (E) The impact of PPP2R1A inhibition on A549 cell response to combination of trametinib and mTOR inhibitor temsirolimus. Data are shown as means ± SEM for three biological replicate experiments with three technical replicates. (F) Colony growth assay validation of the results shown in (E). Red circle denotes reversal of PPP2R1A inhibition–mediated resistance by temsirolimus combination with the highest trametinib concentration. (G) Western blot analysis of selected MAPK and AKT/mTOR pathway components in trametinib- and temsirolimus-treated cells. For selected proteins, different exposures for parts of the same blots are shown in (C). (H) MYC expression in PPP2R1A-depleted A549 cells treated with the combination of trametinib (tra) and temsirolimus (tem). Quantification from data is shown as mean fold changes ± SEM for three biological replicate experiments from (G). *P < 0.05, two-tailed t test. (I) Representative image of a clonogenic assay of H358 cells overexpressing mutant MYC-S62D or enhanced green fluorescent protein (EGFP) control. The graph represents the mean colony number ± SD relative to the vehicle control, three biological replicates with two technical replicates; two-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test, ****P < 0.0001. DMSO, dimethyl sulfoxide.

  • Fig. 4 PP2A inhibition is a druggable MEKi resistance mechanism in vivo.

    (A and B) Clonogenic assay of (A) H441 and (B) H358 cells treated with increasing doses of DT-061 (5 μM and 10 μM), AZD6244 (200 nM and 1 μM), or the combination of DT-061 and AZD6244 for 3 weeks. (C) Relative caspase-3/7 activity in H358 and H441 cells 24 hours after DT-061, AZD6244, or DT-061 + AZD6244 treatment. Error bars represent SEM of two to three independent experiments. *P < 0.05 for both pairs of groups, t test. (D) H441 cells (5 × 106) were subcutaneously injected into nude mice and allowed to grow to an average of 100 mm3. Mice were treated by oral gavage with vehicle control (n = 7), DT-061 (5 mg/kg; n = 7), AZD6244 (25 mg/kg; n = 7), or the combination of DT-061 (5 mg/kg) and AZD6244 (25 mg/kg; n=7) for 4 weeks. Graph shows the waterfall plot for H441 xenograft responses. Two-tailed t test. (E) H358 cells (1 × 107) were subcutaneously injected into nude mice and allowed to grow to an average of 100 mm3. Mice were treated by oral gavage with vehicle control (n = 9), DT-061 (5 mg/kg; n = 9), AZD6244 (25 mg/kg; n = 9), or the combination of DT-061 and AZD6244 (n = 9) for 4 weeks. Graph shows the waterfall plot for H358 xenograft responses. Two-tailed t test. PD, progressive disease; SD, stable disease; PR, partial response; CR, complete response. (F) Analysis of xenograft data based on RECIST criteria. (G to I) H441 xenograft tumors were stained for PCNA (proliferation), TUNEL (apoptosis), and MYC. Nuclear staining was quantified as means ± SD (one-way ANOVA). (J) Tumor volume growth curves for H358-GFP xenograft. Mice were treated by oral gavage with vehicle control (n = 8), DT-061 (5 mg/kg; n = 8), or the combination of AZD6244 (24 mg/kg) and MK2206 (6 mg/kg; n = 8). Data are shown as means ± SEM. (K) Tumor volume growth curves for H358-WT MYC xenograft. Mice were treated by oral gavage with vehicle control (n = 8), DT-061 (5 mg/kg; n = 8), or the combination of AZD6244 (24 mg/kg) and MK2206 (6 mg/kg; n = 8). Data are shown as means ± SEM. (L) Tumor volume growth curves for H358-S62D xenograft. Mice were treated by oral gavage with vehicle control (n = 8), DT-061 (5 mg/kg; n = 8), or the combination of AZD6244 (24 mg/kg) and MK2206 (6 mg/kg; n = 8). Data are shown as means ± SEM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/450/eaaq1093/DC1

    Materials and Methods

    Fig. S1. PP2A activity restricts KRAS-mutant lung cancer cell line proliferative potential.

    Fig. S2. MEKi resistance in KRAS-mutant lung cancer cells by PP2A inhibition.

    Fig. S3. PPP2R1A inhibition drives MEKi resistance by increased AKT/mTOR pathway activities.

    Fig. S4. Validation of PP2A inhibition as a druggable MEKi resistance mechanism in vivo.

    Fig. S5. Analysis of xenograft tissues with respect to proliferation, apoptosis, and MYC protein expression.

    Fig. S6. Pharmacological PP2A activation does not induce weight loss in animals.

    Table S1. A549 cell DSRT.

    Table S2. Validation drug screen in NCI-H460 and NCI-H2122 cells.

    Table S3. Oligonucleotide sequences and antibody information.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. PP2A activity restricts KRAS-mutant lung cancer cell line proliferative potential.
    • Fig. S2. MEKi resistance in KRAS-mutant lung cancer cells by PP2A inhibition.
    • Fig. S3. PPP2R1A inhibition drives MEKi resistance by increased AKT/mTOR pathway activities.
    • Fig. S4. Validation of PP2A inhibition as a druggable MEKi resistance mechanism in vivo.
    • Fig. S5. Analysis of xenograft tissues with respect to proliferation, apoptosis, and MYC protein expression.
    • Fig. S6. Pharmacological PP2A activation does not induce weight loss in animals.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). A549 cell DSRT.
    • Table S2 (Microsoft Excel format). Validation drug screen in NCI-H460 and NCI-H2122 cells.
    • Table S3 (Microsoft Excel format). Oligonucleotide sequences and antibody information.

    [Download Tables S1 to S3]

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