Depletion of runt-related transcription factor 2 (RUNX2) enhances SAHA sensitivity of p53-mutated pancreatic cancer cells through the regulation of mutant p53 and TAp63

Abstract

Suberoylanilide hydroxamic acid (SAHA) represents one of the new class of anti-cancer drugs. However, multiple lines of clinical evidence indicate that SAHA might be sometimes ineffective on certain solid tumors including pancreatic cancer. In this study, we have found for the first time that RUNX2/mutant p53/TAp63-regulatory axis has a pivotal role in the determination of SAHA sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our present results, MiaPaCa-2 cells responded poorly to SAHA. Forced depletion of mutant p53 stimulated SAHA-mediated cell death of MiaPaCa-2 cells, which was accomapanied by a further accumulation of γH2AX and cleaved PARP. Under these experimental conditions, pro-oncogenic RUNX2 was strongly down-regulated in mutant p53-depleted MiaPaCa-2 cells. Surprisingly, RUNX2 silencing augmented SAHA-dependent cell death of MiaPaCa-2 cells and caused a significant reduction of mutant p53. Consistent with these observations, overexpression of RUNX2 in MiaPaCa-2 cells restored SAHA-mediated decrease in cell viability and increased the amount of mutant p53. Thus, it is suggestive that there exists a positive auto-regulatory loop between RUNX2 and mutant p53, which might amplify their pro-oncogenic signals. Intriguingly, knockdown of mutant p53 or RUNX2 potentiated SAHA-induced up-regulation of TAp63. Indeed, SAHA-stimulated cell death of MiaPaCa-2 cells was partially attenuated by p63 depletion. Collectively, our present observations strongly suggest that RUNX2/mutant p53/TAp63-regulatory axis is one of the key determinants of SAHA sensitivity of p53-mutated pancreatic cancer cells.

Fig 1. p53-mutated human pancreatic cancer MiaPaCa-2 and Panc-1 cells respond poorly to SAHA.

MiaPaCa-2 (solid boxes), Panc-1 (grey boxes) and p53-proficient human breast cancer MCF-7 (open boxes) cells were exposed to DMSO, the indicated concentrations of SAHA or left untreated (W/O). Twenty-four (A) and 48 h (B) after treatment, cell viability was examined by standard WST cell survival assay.

Fig 2. Inverse relationship between the expression levels of mutant p53/RUNX2 and TAp63 in MiaPaCa-2 cells following SAHA exposure.

MiaPaCa-2 cells were treated with DMSO, the increasing amounts of SAHA or left untreated. Forty-eight hours after treatment, cell lysates and total RNA were prepared and subjected to immunoblotting (A) and RT-PCR (B), respectively. Actin and GAPDH were used as a loading and an internal control, respectively.

Fig 3. SAHA-dependent induction of γH2AX as well as p63 and reduction of mutant p53.

MiaPaCa-2 cells were treated with DMSO, the indicated concentrations of SAHA or left untreated. Forty-eight hours after treatment, cells were fixed and incubated with anti-γH2AX (A), anti-p63 (B) or with anti-p53 (C) antibody (green). Cell nuclei were stained with DAPI (blue) (left panels). Based on the results obtained from the immunostaing experiments, number of γH2AX-, p63- or mutant p53-positive cells was scored (right bar graphs).

Fig 4. Silencing of mutant p53 in MiaPaCa-2 cells stimulates SAHA-dependent decrease and increase in cell viability and cell death, respectively.

(A) Phase-contrast micrographs. MiaPaCa-2 cells were transfected with control siRNA or with siRNA against p53, and then treated with DMSO, 1 μM of SAHA or left untreated. Forty-eight hours after treatment, the representative pictures were taken. (B) WST assay. MiaPaCa-2 cells were transfected with control siRNA or with siRNA against p53, and treated with DMSO or with the indicated concentrations of SAHA. Forty-eight hours after SAHA exposure, cells were analyzed by the standard WST cell survival assay. Solid and grey boxes indicate control siRNA- and p53 siRNA-transfected cells, respectively. (C) FACS analysis. MiaPaCa-2 cells were transfected with control siRNA or with siRNA against p53, and treated with DMSO or with the indicated concentrations of SAHA. Forty-eight hours after treatment, floating and adherent cells were harvested and subjected to flow cytometric analysis. Solid and grey boxes indicate control siRNA- and p53 siRNA-transfected cells, respectively.

Fig 5. Forced depletion of mutant p53 augments SAHA-mediated accumulation of TAp63 and reduction of RUNX2.

MiaPaCa-2 cells were transfected and treated as in Fig 4A. Forty-eight hours post-treatment, cell lysates and total RNA were prepared and analyzed by immunoblotting (A) and RT-PCR (B), respectively. Actin and GAPDH were used as a loading and an internal control, respectively.

Fig 6. Knockdown of RUNX2 potentiates SAHA-dependent cell death of MiaPaCa-2 cells.

(A) Representative photos. MiaPaCa-2 cells were transfected with control siRNA or with siRNA targeting RUNX2. Twenty-four hours after transfection, cells were treated with DMSO, 1 μM of SAHA or left untreated. Forty-eight hours after treatment, the representative pictures were taken. (B) Cell survival assay. MiaPaCa-2 cells were transfected with control siRNA or with siRNA targeting RUNX2, and treated with DMSO or with the indicated concentrations of SAHA. Forty-eight hours after treatment, cells were analyzed by WST cell survival assay. Solid and grey boxes indicate control siRNA- and RUNX2 siRNA-transfected cells, respectively. (C) FACS analysis. MiaPaCa-2 cells were transfected with control siRNA or with siRNA targeting RUNX2, and treated with DMSO or with the indicated concentrations of SAHA. Forty-eight hours after treatment, floating and adherent cells were harvested and subjected to flow cytometric analysis. Solid and grey boxes indicate control siRNA- and RUNX2 siRNA-transfected cells, respectively.

Fig 7. Silencing of RUNX2 stimulates SAHA-induced accumulation of TAp63 and reduction of mutant p53.

MiaPaCa-2 cells were transfected and treated as in Fig 6A. Forty-eight hours after treatment, cell lysates and total RNA were extracted and processed for immunoblotting (A) and RT-PCR (B), respectively. Actin and GAPDH were used as a loading and an internal control, respectively.

Fig 8. Forced expression of RUNX2 restores SAHA-mediated decrease in cell viability and further increases the amount of mutant p53.

(A) Overexpression of RUNX2. MiaPaCa-2 cells were transfected with pcDNA3 or with the expression plasmid for RUNX2. Forty-eight hours after transfection, cell lysates and total RNA were prepared and subjected to immunoblotting (upper panels) and RT-PCR (lower panels), respectively. Actin and GAPDH were used as a loading and an internal control, respectively. (B) WST assay. MiaPaCa-2 cells were transfected as in (A). Twenty-four hours after transfection, cells were treated with DMSO, the indicated concentrations of SAHA or left untreated. Forty-eight hours after treatment, cell viability was examined by WST cell survival assay. (C) Immunoblotting. MiaPaCa-2 cells were transfected as in (A). Twenty-four hours after transfection, cells were treated with DMSO, 1 μM of SAHA or left untreated. Forty-eight hours ater treatment, cell lysates were extracted and analyzed by immunoblotting with the indicated antibodies. Actin was used as a loading control.