Improvement of gemcitabine sensitivity of p53-mutated pancreatic cancer MiaPaCa-2 cells by RUNX2 depletion-mediated augmentation of TAp73-dependent cell death

Abstract

Pancreatic cancer exhibits the worst prognostic outcome among human cancers. Recently, we have described that depletion of RUNX2 enhances gemcitabine (GEM) sensitivity of p53-deficient pancreatic cancer AsPC-1 cells through the activation of TAp63-mediated cell death pathway. These findings raised a question whether RUNX2 silencing could also improve GEM efficacy on pancreatic cancer cells bearing p53 mutation. In the present study, we have extended our study to p53-mutated pancreatic cancer MiaPaCa-2 cells. Based on our current results, MiaPaCa-2 cells were much more resistant to GEM as compared with p53-proficient pancreatic cancer SW1990 cells, and there existed a clear inverse relationship between the expression levels of TAp73 and RUNX2 in response to GEM. Forced expression of TAp73α in MiaPaCa-2 cells significantly promoted cell cycle arrest and/or cell death, indicating that a large amount of TAp73 might induce cell death even in the presence of mutant p53. Consistent with this notion, overexpression of TAp73α stimulated luciferase activity driven by p53/TAp73-target gene promoters in MiaPaCa-2 cells. Similar to AsPC-1 cells, small interfering RNA-mediated knockdown of RUNX2 remarkably enhanced GEM sensitivity of MiPaCa-2 cells. Under our experimental conditions, TAp73 further accumulated in RUNX2-depleted MiaPaCa-2 cells exposed to GEM relative to GEM-treated non-silencing control cells. As expected, silencing of p73 reduced GEM sensitivity of MiPaCa-2 cells. Moreover, GEM-mediated Tyr phosphorylation level of TAp73 was much more elevated in RUNX2-depleted MiaPaCa-2 cells. Collectively, our present findings strongly suggest that knockdown of RUNX2 contributes to a prominent enhancement of GEM sensitivity of p53-mutated pancreatic cancer cells through the activation of TAp73-mediated cell death pathway, and also provides a promising strategy for the treatment of patients with pancreatic cancer bearing p53 mutation.

Figure 1

p53-mutated human pancreatic cancer MiaPaCa-2 cells are resistant to GEM. (a) Phase-contrast micrographs. MiaPaCa-2 cells were treated with the indicated concentrations of GEM. Forty-eight hours after treatment, representative pictures were taken. (b, c) MiaPaCa-2 cells undergo cell death following GEM exposure but to a lesser degree. MiaPaCa-2 cells were treated as in (a). Forty-eight hours after treatment, floating and attached cells were harvested and processed for FACS analysis (b) and trypan blue exclusion assay (c), respectively.

Figure 2

Inverse relationship between the expression levels of TAp73 and RUNX2 in response to GEM. MiaPaCa-2 cells were treated as in Figure 1a. Forty-eight hours after treatment, total RNA and cell lysates were prepared and analyzed by RT–PCR (a) and immunoblotting (b), respectively. GAPDH and actin were used as an internal control and a loading control, respectively.

Figure 3

Depletion of RUNX2 stimulates GEM-mediated reduction in number of viable cells. (a, b) siRNA-mediated silencing of RUNX2. MiaPaCa-2 cells were transfected with control siRNA or with siRNA targeting RUNX2. Twenty-four hours after transfection, cells were exposed to GEM (at a final concentration of 10 μm) or left untreated. Forty-eight hours after treatment, total RNA and cell lysates were extracted and subjected to RT–PCR (a) and immunoblotting (b), respectively. (c) Representative pictures.

Figure 4

Knockdown of RUNX2 enhances GEM sensitivity of MiaPaCa-2 cells. (a) FACS analysis. MiaPaCa-2 cells were treated as in Figure 3a. Forty-eight hours after treatment, floating and adherent cells were collected and analyzed by flow cytometry. (b) DNA fragmentation. MiaPaCa-2 cells were treated as in Figure 3a. Forty-eight hours after treatment, floating and adherent cells were collected and their genomic DNA was prepared according to the standard procedure. Genomic DNA was analyzed by 0.7% agarose gel electrophoresis and stained with ethidium bromide.

Figure 5

Depletion of RUNX2 enhances the sensitivity to GEM of MiaPaCa-2 cells. (a, b) Effects of RUNX2 knockdown on MiaPaCa-2 cells in the presence or absence of GEM. For trypan blue exclusion assay, MiaPaCa-2 cells were treated as in Figure 3a. Forty-eight hours after treatment, floating and attached cells were harvested and processed for trypan blue exclusion assay (a). For WST cell survival assay, MiaPaCa-2 cells were treated as in Figure 3a. Forty-eight hours after treatment, cells were subjected to WST cell survival assay (b). Results are presented as mean±s.d.

Figure 6

Silencing of RUNX2 further stimulates GEM-mediated induction of TAp73 and cleavage of PARP. (a, b) Expression of TAp73 and its target genes in RUNX2-depleted MiaPaCa-2 cells in response to GEM. MiaPaCa-2 cells were treated as in Figure 3a. Forty-eight hours after treatment, total RNA and cell lysates were prepared and analyzed by RT–PCR (a) and immunoblotting (b), respectively.

Figure 7

Depletion of p73 reduces GEM sensitivity of MiaPaCa-2 cells. (a) siRNA-mediated knockdown of p73. MiaPaCa-2 cells were transfected with control siRNA or with siRNA against p73. Forty-eight hours after transfection, total RNA and cell lysates were prepared and analyzed by RT–PCR and immunoblotting, respectively. (b) FACS analysis. p73-depleted and non-depleted MiaPaCa-2 cells were exposed to GEM or left untreated. Forty-eight hours after treatment, floating and attached cells were harvested and subjected to flow cytometric analysis. (c, d) Trypan blue exclusion assay. MiaPaCa-2 cells were treated as in (b). Forty-eight hours after GEM exposure, floating and attached cells were collected and subjected to trypan blue exclusion assay.

Figure 8

GEM-mediated Tyr phosphorylation of TAp73 is further stimulated in RUNX2-depleted MiaPaCa-2 cells. (a) GEM-mediated Tyr phosphorylation of TAp73. MiaPaCa-2 cells were treated with or without GEM. Forty-eight hours after treatment, cell lysates were prepared and immunoprecipitated with anti-p73 antibody or with normal rabbit serum (NRS). The immunoprecipitates were analyzed by immunoblotting with PY20. (b) GEM-mediated Tyr phosphorylation of TAp73 is augmented in RUNX2-depleted cells. MiaPaCa-2 cells were transfected with control siRNA or with siRNA against RUNX2. Twenty-four hours after transfection, cells were exposed to GEM for 48 h. After treatment, cell lysates were analyzed by co-immunoprecipitation experiments as in (a).