Impact of RUNX2 gene silencing on the gemcitabine sensitivity of p53­mutated pancreatic cancer MiaPaCa­2 spheres.

ABSTRACT

Recently, it has been well­recognized that the response toward anticancer drugs differs between two­ and three­dimensional (2D and 3D) in vitro cancer cell growth models. In the present study, we have demonstrated that, similar to the conventional 2D monolayer culture systems which often lack in vivo physiological insights, RUNX2 gene silencing increases the gemcitabine (GEM) sensitivity of the 3D spheres generated from p53­mutated pancreatic cancer MiaPaCa­2 cells. According to our results, MiaPaCa­2 cells, but not p53­wild­type pancreatic cancer SW1990 cells efficiently formed sphere structures in serum­free sphere­forming medium. Although GEM treatment caused a marked induction of TAp73/TAp63 in MiaPaCa­2 spheres accompanied by the transcriptional activation of p53 family­target genes such as p21WAF1 and NOXA, only 20% of cells underwent cell death. Under these experimental conditions, mutant p53 expression level was increased in response to GEM and RUNX2 remained unchanged at the protein level regardless of GEM exposure, which may suppress the pro­apoptotic activity of TAp73/TAp63. Notably, RUNX2 gene silencing markedly augmented GEM­mediated cell death of MiaPaCa­2 spheres compared to that of non­depleted ones. Expression analyses revealed that forced depletion of RUNX2 further stimulated GEM­induced upregulation of TAp63 as well as its downstream target genes such as p21WAF1 and NOXA. In summary, our observations strongly indicated that, similarly to 2D monolayer culture, RUNX2 gene silencing increased GEM sensitivity of MiaPaCa­2 spheres and highlighted the therapeutic potential of RUNX2 in pancreatic cancer with p53 mutation.