A Pleiotropic RNA-Binding Protein Controls Distinct Cell Cycle Checkpoints to Drive Resistance of p53-Defective Tumors to Chemotherapy

Abstract

In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.

Figure 1. A focused screen implicates p27^Kip1 as an hnRNPA0-dependent G1/S regulator

(A) H1299 cells were depleted of hnRNPA0 or Gadd45α and treated with 2 μM doxorubicin for 4 hr followed by the addition of 250 ng/mL nocodazole for a further 24 hr. Cells were fixed and stained with propidium iodide and analyzed by flow cytometry. Yellow, blue and green arrows depict loss of the G1/S checkpoint in hnRNPA0 knockdown cells but not in Gadd45α knockdown cells. (B) Schematic representation of the known role of hnRNPA0 in the DNA damage response. (C) Cells were treated with doxorubicin for 16 hr, mRNA levels of the indicated targets were determined by qRT-PCR, and the data represented as fold-change vs. control siRNA vehicle-treated cells. (D) qRT-PCR analysis of H1299 cells, 16 hr post-doxorubicin treatment. Data are represented as fold change vs. control siRNA vehicle-treated cells. Error bars represent mean +/−SEM, n=3 experiments * p < 0.05, *** p < 0.001. (E) Western blots of H1299 cells 16 hr post-doxorubicin treatment. (F) H1299 cells were treated with doxorubicin for 16 hr followed by the addition of cycloheximide. Samples were taken at indicated time points and p27^Kip1 protein levels measured by immuno-blotting. (G) H1299 cells were treated with doxorubicin for 16 hr followed by the addition of actinomycin D. At indicated time points p27^Kip1 mRNA levels were determined by qRT-PCR. See also Figure S1.

Figure 2. p27^Kip1 controls the DNA damage-induced G1/S checkpoint in p53-deficient cells

(A) Western blots of H1299 cells transfected with p27^Kip1-specific siRNA or infected with a miR-30-based retroviral p27^Kip1-specific shRNA (shRNA#1). (B) Representative flow cytometry profiles of propidium iodide-stained H1299 cells transfected with a control siRNA or a p27^Kip1-specific siRNA 16 hr post-doxorubicin treatment. Red arrow indicates loss of the G1 peak in p27^Kip1 depleted cells. (C) Quantification of the percentage of G1 arrested cells from 3 independent experiments performed as in panel B. Error bars represent mean +/−SEM, *** p < 0.001. (D, E) Example of a typical EdU labeling experiment is shown in D. In panel E, loss of p27^Kip1 causes entry into S-phase despite the presence of DNA damage in a manner and magnitude comparable to that of hnRNPA0 knockdown. Quantified EdU incorporation assays were performed in H1299 cells transfected with a p27^Kip1-specific siRNA or transduced with a p27^Kip1 or hnRNPA0-specific shRNA retrovirus, at 15-16 hr following application of 1 μM doxorubicin. EdU incorporation values were normalized to those of control undamaged cells treated with vehicle alone (Fig S2B-C). Bars represent mean % of positive cells relative to vehicle treated cells (see Fig S2B) +/− SEM, n=3 experiments. * p < 0.05. See also Figure S2.

Figure 3. MK2-mediated phosphorylation of hnRNPA0 in response to DNA damage induces binding to its target RNAs and cytoplasmic localization

(A) Schematic representation of hnRNPA0 domain architecture and sequence surrounding Serine-84 in the linker region between the two RNA recognition motifs (RRMs). The MK2 phosphorylated residue, Serine-84, is highlighted in red. (B) Western blots of H1299 cells transduced with a control shRNA or an MK2-specific shRNA and treated with vehicle or doxorubicin. (C) qRT-PCR analysis of cells treated as in (B). (D) Stable H1299 cell lines harboring HA-tagged WT hnRNPA0 or a Ser-84-to-Ala mutant were treated with doxorubicin for 16 hr followed by HA-immuno-precipitation. Co-precipitated RNA was subjected to qRT-PCR for p27^Kip1 and Gadd45α mRNAs. Data shown as fold-change vs. the WT untreated control. Error bars represent mean +/− SEM, n=2 independent experiments. (E) Nuclear-cytoplasmic fractionation of H1299 cells following 16 hr doxorubicin treatment. (F) Immuno-flourescence microscopy of H1299 cells fixed 16 hr after 2 μM doxorubicin treatment. Scale bar represents 5 μm. (G) Quantification of hnRNPA0 localization immuno-flourescence from two independent experiments, error bars represent mean +/− SEM. See also Figure S3.

Figure 4. Synthetic lethality between hnRNPA0-loss and a defective p53 pathway in response to chemotherapy

(A,B) H1299 (p53 null) cells were transfected with a control or hnRNPA0-specific siRNA and treated with doxorubicin or cisplatin as indicated, followed by western blotting for cleaved caspase 3. Data are represented as fold-change vs control siRNA vehicle-treated cells. Error bars represent mean +/− SEM, n=3 experiments. * p < 0.05 ** p < 0.01 *** p < 0.001 (C) K-Ras^G12D;p53^−/− KP7B murine lung adenocarcinoma cells were transduced with a miR-30 retrovirus expressing an hnRNPA0-specifc shRNA and treated as indicated. (D) A panel of p53 mutant and WT cell lines were transduced or transfected with hnRNPA0-targetting shRNA or siRNA and cell death in response to cisplatin assayed by measuring cleaved caspase 3. Shown is fold change in cell death in cisplatin treated hnRNPA0-knockdown cells relative to cisplatin treated control siRNA/shRNA cells. Bars represent mean +/− SEM n=3-5. * p < 0.05. (E) Heatmap representation of Log₂ fold change in the mRNA levels of the indicated p53-target genes after 16 hr of 10 μM cisplatin treatment. Lower panel is a p53 western blot of the indicated cell lines treated or not with 10 μM cisplatin for 16 hr. (F) p53 levels from untreated cells from (E) were normalized to beta-actin, Log₂ transformed, and plotted against the Log₂ transformed data from (D). Shown is the linear regression and the pearson R². (G) p53 WT H1944 cells were transfected with the indicated siRNAs for 48 hr followed by treatment with 10 μM cisplatin for 24 hr and measurement of cleaved caspase 3. Bars represent mean +/− SEM n=3. * p = < 0.05. See also Figure S4.

Figure 5. Decreased hnRNPA0 activity promotes cisplatin efficacy against p53-defective non-small cell lung cancer (NSCLC) in vivo

(A) Schematic representation of the transplantable NSCLC model. (B) Representative bio-luminescence images before and after cisplatin treatment on Day 0 and 7. Red arrows indicate timing of cisplatin dosing. (C) Quantification of lung bio-luminecence pre-treatment and 5 days post-cisplatin treatment. Error bars represent mean +/− SEM, 3-4 animals per condition * p < 0.05. (D) Post-treatment Kaplan-Meier survival analysis of control tumor-bearing mice with or without cisplatin treatment, as indicated. (Vector n=7, vector + Cis n=7). (E) Post-treatment Kaplan-Meier survival analysis of shA0 tumor-bearing mice with or without cisplatin treatment, as indicated. (shA0(3) n=7, shA0(3) + Cis n=9). p values in D and E were calculated using the log-rank test. See also Figure S5.

Figure 6. hnRNPA0 promotes resistance to chemotherapy through p27^Kip1 and Gadd45α

(A) Model depicting the role of MK2, hnRNPA0, p27^Kip1 and Gadd45α in the DNA damage response, limiting the efficacy of anti-cancer chemotherapy. (B) H1299 (p53 null) cells were transfected with the indicated siRNAs and analyzed as in (Figure 4). Error bars represent mean +/− SEM, n=3 experiments. ** p < 0.01, *** p < 0.001, n.s.=not significant. Control siRNA data from Figure 4B are shown again here for comparison. (C, D) Stage II patients from the JBR.10 lung cancer adjuvant chemotherapy trial were clustered into two groups based on expression of both p27^Kip1 and Gadd45α. Patients in this trial were either observed (Obs, orange line) or treated with cisplatin/vinorelbine-based chemotherapy (Chemo, green line). Kaplan-Meier analysis of lung cancer patients based on expression of MK2/hnRNPA0-target mRNAs demonstrates that only those patients with low levels of these mRNAs benefit significantly from adjuvant chemotherapy. HR=hazard ratio, 95% CI=95% confidence interval. p values were calculated using the log-rank test. See also Figure S6.

Figure 7. The primary p53/p21 axis actively suppresses the sucessor hnRNPA0

(A) p53 WT A549 cells were transfected with a control siRNA or a p53-targeting siRNA for 48 hr, followed by treatment with 2 μM doxorubicin for 24 hr. hnRNPA0 protein expression was measured by western blotting. Data are plotted as fold-change in hnRNPA0 protein (normalized to actin) relative to vehicle-treated control siRNA transfected cells, bars represent mean +/− SEM, n=3 experiments. ** p < 0.01. (B) p53 WT A549 cells were transfected with siRNAs as in (A), then treated with 10 μM MDM-2 inhibitor/p53 activator Nutlin 3a for 24 hr. Left panel: data are plotted as fold-change in hnRNPA0 protein (normalized to actin) relative to vehicle-treated control siRNA transfected cells, bars represent mean +/− SEM, n=4 experiments. * p < 0.05. Right panel, data are plotted as fold-change in hnRNPA0 mRNA relative to vehicle-treated control siRNA transfected cells, bars represent mean +/− SEM, n=3 experiments. ** p < 0.01. (C) A549 cells were treated as in (B) except that at 24 hr Actinomycin D was added to the culture medium and time points taken for RNA analysis by qRT-PCR. Data represent percent remaining hnRNPA0 mRNA for each condition relative to the 24 hr time point where Actinomycin was added. (D) Slopes of the lines from 3 independent experiments performed as in (C) were calculated and used to determine the fold change in mRNA half-life upon p53 activation. Bars represent mean +/− SEM. (E) A549 cells were transfected with a control siRNA or a p21-targeting siRNA for 48 hr, followed by treatment with 10 μM Nutlin 3a for 24 hr. p21 and hnRNPA0 mRNA expression were measured by qRT-CPR. Data are plotted as fold-change in mRNA levels relative to vehicle-treated control siRNA transfected cells, bars represent mean +/− SEM, n=3 experiments. * p < 0.05, ** p < 0.01. (F) Model representation of the interplay between the p53 and MK2/hnRNPA0 pathways in response to DNA damaging chemotherapy. See also figure S7.