Abstract
Runt-related transcription factor 2 (RUNX2) has been considered to be one of master regulators for osteoblast differentiation and bone formation. Recently, we have described that RUNX2 attenuates p53/TAp73-dependent cell death of human osteosarcoma U2OS cells bearing wild-type p53 in response to adriamycin. In this study, we have asked whether RUNX2 silencing could enhance gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells. Under our experimental conditions, GEM treatment increased the expression level of p53 family TAp63, whereas RUNX2 was reduced following GEM exposure, indicating that there exists an inverse relationship between the expression level of TAp63 and RUNX2 following GEM exposure. To assess whether TAp63 could be involved in the regulation of GEM sensitivity of AsPC-1 cells, small interfering RNA-mediated knockdown of TAp63 was performed. As expected, silencing of TAp63 significantly prohibited GEM-dependent cell death as compared with GEM-treated non-silencing cells. As TAp63 was negatively regulated by RUNX2, we sought to examine whether RUNX2 knockdown could enhance the sensitivity to GEM. Expression analysis demonstrated that depletion of RUNX2 apparently stimulates the expression of TAp63, as well as proteolytic cleavage of poly ADP ribose polymerase (PARP) after GEM exposure, and further augmented GEM-mediated induction of p53/TAp63-target genes, such as p21WAF1, PUMA and NOXA, relative to GEM-treated control-transfected cells, implying that RUNX2 has a critical role in the regulation of GEM resistance through the downregulation of TAp63. Notably, ablation of TAp63 gave a decrease in number of γH2AX-positive cells in response to GEM relative to control-transfected cells following GEM exposure. Consistently, GEM-dependent phosphorylation of ataxia telangiectasia-mutated protein was remarkably impaired in TAp63 knockdown cells. Collectively, our present findings strongly suggest that RUNX2-mediated repression of TAp63 contributes at least in part to GEM resistance of AsPC-1 cells, and thus silencing of RUNX2 may be a novel strategy to enhance the efficacy of GEM in p53-deficient pancreatic cancer cells.
Highlights
Human pancreatic cancer is a highly aggressive, as well as metastatic tumor, and represents the fourth and fifth leading causes of cancer-related death in the United States and Japan, respectively, whose incidence is increasing.[1,2] the best chance of long-term survival is the complete surgical resection, most patients are not amenable to surgery at the time of diagnosis because of its difficulty in early detection.[3]
We have focused on p53-deficient pancreatic cancer AsPC-1 cells and found that depletion of runt-related transcription factor 2 (RUNX2) enhances the sensitivity to GEM of AsPC-1 cells in association with a significant stimulation of TAp63
Similar results were obtained in p53-mutated human pancreatic cancer MiaPaCa-2 cells (Nakamura et al, manuscript in preparation), indicating that RUNX2 depletion-mediated enhancement of GEM sensitivity may not be restricted to p53deficient AsPC-1 cells. These results suggest that silencing of RUNX2 augments GEM-mediated cell death in AsPC-1 cells
Summary
Human pancreatic cancer is a highly aggressive, as well as metastatic tumor, and represents the fourth and fifth leading causes of cancer-related death in the United States and Japan, respectively, whose incidence is increasing.[1,2] the best chance of long-term survival is the complete surgical resection, most patients (over 80%) are not amenable to surgery at the time of diagnosis because of its difficulty in early detection.[3]. To shed light on the molecular mechanism(s) behind GEMresistant phenotype of AsPC-1 cells, we have examined the expression pattern of p53 family-related gene products following GEM exposure. In these experiments, cleavage of PARP and accumulation of γH2AX were checked as cell death and DNA damage markers, respectively. We have demonstrated for the first time that RUNX2 attenuates p53 and/or TAp73-dependent cell death in p53proficient osteosarcoma U2OS cells following DNA damage inducer adriamycin (ADR) exposure.[47,48] In this study, we have focused on p53-deficient pancreatic cancer AsPC-1 cells and found that depletion of RUNX2 enhances the sensitivity to GEM of AsPC-1 cells in association with a significant stimulation of TAp63-.
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