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

M Nakamura,H Sugimoto,O Shimozato,Y Zhu,M Sang,T Ogata,T Ozaki,K Hiraoka,M Yu,H Yoda

doi:10.1038/oncsis.2016.40

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.

Highlights

Human pancreatic cancer is a serious disease with 5-year survival rate of o 5% and its incidence is increasing annually.[1,2] In this connection, pancreatic cancer is expected to be the second leading cause of cancer-related death by 2030.3 surgical resection is the preferred treatment for pancreatic cancer patients and it has been significantly improved, most cases are found at a late advanced unresectable stage
We have examined whether silencing of runt-related transcription factor 2 (RUNX2) in p53-mutated pancreatic cancer MiaPaCa-2 cells could enhance their GEM sensitivity
Our recent findings strongly indicate that depletion of RUNX2 improves the anti-cancer drug sensitivity of p53-proficient osteosarcoma U2OS cells as well as p53-deficient pancreatic cancer AsPC-1 cells in a p53 family-dependent manner.[37,38,39]

Summary

INTRODUCTION

Human pancreatic cancer is a serious disease with 5-year survival rate of o 5% and its incidence is increasing annually.[1,2] In this connection, pancreatic cancer is expected to be the second leading cause of cancer-related death by 2030.3 surgical resection is the preferred treatment for pancreatic cancer patients and it has been significantly improved, most cases are found at a late advanced unresectable stage. As mutant p53 acquires pro-oncogenic activity and contributes at least in part to drug resistance of aggressive cancers, expression of angiogenic VEGF-A under tumor-relevant hypoxic we have sought to examine GEM sensitivity of p53-mutated condition These observations indicate that ΔN isoforms might human pancreatic cancer MiaPaCa-2 cells and p53-proficient have their own target genes involved in carcinogenesis. Immunoprecipitation/immunoblotting experiments demonstrated that GEM-mediated Tyr phosphorylation level of TAp73 remarkably elevates in RUNX2-depleted MiaPaCa-2 cells relative to nondepleted MiaPaCa-2 cells (Figure 8b). These results suggest that RUNX2 attenuates GEM-dependent phosphorylation of TAp73 at Tyr residues, and thereby suppressing its pro-apoptotic activity

DISCUSSION

Findings

MATERIALS AND METHODS