ONCOLOGY REPORTS 39: 2749-2758, 2018 Impact of RUNX2 gene silencing on the gemcitabine sensitivity of p53‑mutated pancreatic cancer MiaPaCa‑2 spheres MEIJIE SANG1,2, MIZUYO NAKAMURA1, TAKEHIRO OGATA1, DAN SUN3, OSAMU SHIMOZATO1, TOSHIO NIKAIDO2 and TOSHINORI OZAKI1 1Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba 260-8717; 2Department of Regenerative Medicine, Graduate School of Medicine, University of Toyama, Toyama 930-0194, Japan; 3Department of Urology, First Hospital of China Medical University, Shenyang, Liaoning110001, P.R. China Received September 26, 2017; Accepted March 20, 2018 DOI: 10.3892/or.2018.6344 Abstract. Recently, it has been well-recognized that the stimulated GEM-induced upregulation of TAp63 as well as response toward anticancer drugs differs between two- and its downstream target genes such as p21WAF1 and NOXA. In three-dimensional (2D and 3D) in vitro cancer cell growth summary, our observations strongly indicated that, similarly models. In the present study, we have demonstrated that, to 2D monolayer culture, RUNX2 gene silencing increased similar to the conventional 2D monolayer culture systems GEM sensitivity of MiaPaCa-2 spheres and highlighted the which often lack in vivo physiological insights, RUNX2 gene therapeutic potential of RUNX2 in pancreatic cancer with p53 silencing increases the gemcitabine (GEM) sensitivity of the mutation. 3D spheres generated from p53-mutated pancreatic cancer MiaPaCa-2 cells. According to our results, MiaPaCa-2 cells, Introduction but not p53‑wild‑type pancreatic cancer SW1990 cells effi- ciently formed sphere structures in serum-free sphere-forming Pancreatic cancer is the fourth leading cause of cancer-related medium. Although GEM treatment caused a marked induc- deaths in Japan and its overall 5-year survival rate remains very tion of TAp73/TAp63 in MiaPaCa-2 spheres accompanied low (less than 10%) (1). Although surgical resection provides by the transcriptional activation of p53 family-target genes complete cure for patients with pancreatic cancer, over 80% such as p21WAF1 and NOXA, only 20% of cells underwent of cases are judged to be inoperable at the time of initial diag- cell death. Under these experimental conditions, mutant nosis, due to advanced disease or distant metastasis, and thus p53 expression level was increased in response to GEM and subjected to chemotherapy and/or radiotherapy (2). The DNA RUNX2 remained unchanged at the protein level regardless damaging anticancer drug, gemcitabine (GEM) is a first‑line of GEM exposure, which may suppress the pro-apoptotic chemotherapy agent for patients. However, most of them do activity of TAp73/TAp63. Notably, RUNX2 gene silencing not respond well to GEM treatment, followed by subsequent markedly augmented GEM-mediated cell death of MiaPaCa-2 disease progression (3,4). To overcome this serious issue, a spheres compared to that of non-depleted ones. Expression variety of combinations of GEM with other anticancer drugs analyses revealed that forced depletion of RUNX2 further such as Nab‑paclitaxel (Nab‑P) and 5‑fluorouracil (5‑FU) have been assessed. Unfortunately, these combinations have resulted in limited clinical benefits for patients compared to patients treated with GEM alone (5). Since the vast majority of cases Correspondence to: Dr Toshinori Ozaki, Laboratory of DNA carry loss of function mutations in tumor suppressor gene p53, Damage Signaling, Chiba Cancer Center Research Institute, 666-2 as well as CDKN2A and gain of function (GOF) mutations in Nitona, Chuoh-ku, Chiba 260-8717, Japan proto-oncogene K-Ras (6), it is probable that these mutations E-mail: [email protected] may contribute to the acquisition of resistance of pancreatic cancer tissues to GEM. Considering this, the understanding of Abbreviations: DTT, dithiothreitol; EGF, epidermal growth the precise molecular basis behind the GEM-resistant property factor; EMT, epithelial-mesenchymal transition; bFGF, fibroblast of pancreatic cancer is urgently required. growth factor-basic; GEM, gemcitabine; MRP5, multidrug resistant RUNX2 (runt-related transcription factor 2) has been widely protein 5; PARP, poly (ADP-ribose) polymerase; PI, propidium iodide; RPM2, ribonucleotide reductase M2; RUNX2, runt-related accepted to be one of the master regulators of osteoblast differ- transcription factor 2; siRNA, small interfering RNA; TBS, entiation and bone formation. Previous studies have revealed Tris-buffered saline that RUNX2‑deficient mice lack a mineralized skeleton and its forced expression potentiates the osteoblast transcription Key words: gemcitabine, pancreatic cancer, p53 family, RUNX2, program (7,8). As expected, RUNX2 transactivates its multiple sphere downstream target gene promoters implicated in osteogenesis such as osteocalcin (OCN), type I collagen, osteopontin (OPN) and collagenase 3 (9). In addition to osteogenesis, a growing 2750 SANG et al: DEPLETION OF RUNX2 INCREASES GEMCITABINE SENSITIVITY OF PANCREATIC CANCER SPHERES body of evidence indicates that RUNX2 has a pro-oncogenic Lafayette, CO, USA) using Lipofectamine 2000 transfec- function. For example, RUNX2 has been demonstrated to tion reagent according to the manufacturer's instructions be aberrantly overexpressed in numerous cancer tissues (Invitrogen; Thermo Fisher Scientific, Inc.). The final concen- compared to their corresponding normal ones (10). Consistent tration of each siRNA was 10 nM. RUNX2 gene silencing was with these observations, forced expression and depletion of evaluated by immunoblotting and RT-PCR. RUNX2 promoted and suppressed malignant phenotypes of cancer cells of various origins, respectively (10,11). Immunoblotting. Cells were lysed in 1X Laemmli buffer supple- Recently, we have demonstrated that RUNX2 gene mented with 0.1 M DTT and the commercial protease inhibitor silencing significantly increased chemosensitivity of human mixture (product no. P8340; Sigma-Aldrich; Merck KGaA, osteosarcoma and pancreatic cancer cells through the augmen- Darmstadt, Germany). Equal amounts of cell lysates (30 µg tation of the tumor suppressor p53 family-dependent cell death of protein) were analyzed by 10% SDS-polyacrylamide gel pathway (12-15). Tumor suppressor p53 family is composed electrophoresis, transferred onto membrane filter (Immobilon; of three members including p53, TAp73 and TAp63 (16,17). Merck Millipore, Amsterdam, The Netherlands), and incu- According to our observations, RUNX2 knockdown mark- bated with Tris-buffered saline containing 0.1% Tween-20 edly increased GEM sensitivity of p53-mutated pancreatic (TBS‑T) plus 5% of non‑fat dry milk at 4˚C overnight. The cancer cells (14,15). Since it has been well known that mutant membranes were probed with mouse monoclonal anti-p53 p53 with an extended half-life participates in the acquisi- (DO-1; 1:4,000; cat. no. sc126; Santa Cruz Biotechnology) tion/maintenance of chemoresistant properties of aggressive which recognizes both wild-type and mutant p53, rabbit cancers (16,18), it is indicative that depletion of RUNX2 may polyclonal anti-TAp73 (1:1,000; cat. no. GTX-109045; override at least in part, the negative effect of mutant p53 on GeneTex, Inc., Irvine, CA, USA), rabbit polyclonal anti-TAp63 their chemosensitivity. To further validate our prior results, we (1:1,000; cat. no. GTX-102425; GeneTex), rabbit monoclonal sought to address these issues under the anchorage-indepen- anti-RUNX2 (1:1,000; cat. no. 8486; Cell Signaling Technology, dent growth of pancreatic cancer cells (spheres) instead of the Beverley, CA, USA), rabbit polyclonal anti-E2F-1 (1:1,000; conventional two-dimensional (2D) monolayer culture condi- cat. no. 3742; Cell Signaling Technology), rabbit polyclonal tions (19). It has been considered that multicellular spheres anti-PARP (1:1,000; cat. no. 9542; Cell Signaling Technology), mimic the in vivo physiological microenvironments of cancer mouse monoclonal anti-Itch (1:2,000; cat. no. 611199; BD tissues (20). Transduction Laboratories, Lexington, KY, USA), mouse In the present study, we took advantage of the clinically‑­ monoclonal anti-Lamin B (1:2,000; cat. no. NA12; 101-B7; relevant in vitro sphere culture system and demonstrated Calbiochem; Merck KGaA, St. Louis, MO, USA), mouse that RUNX2 gene silencing increased the GEM sensitivity of monoclonal anti-γH2AX (2F3; 1:2,000; cat. no. 613401; p53-mutated pancreatic cancer MiaPaCa-2 spheres through BioLegend, San Diego, CA, USA) or with mouse monoclonal the stimulation of TAp63-dependent cell death pathway. anti-actin antibody (C-2; 1:2,000; cat. no. sc-8432; Santa Cruz Biotechnology) at room temperature for 1 h. Actin was used Materials and methods as a loading control. After washing in TBS-T, the membranes were incubated with horseradish peroxidase-conjugated goat Cells and cell culture. Human pancreatic cancer-derived anti-mouse (cat. no. 31430) or anti-rabbit IgG (cat. no. 31466) SW1990 and MiaPaCa-2 cells obtained from The American (1:4,000; Invitrogen; Thermo Fisher Scientific, Inc.) at room Type Culture Collection (Manassas, VA, USA) were main- temperature for 1 h. Immunoreactive signals were detected tained in Dulbecco's modified Eagle's medium (DMEM; with an enhanced chemiluminescence detection system (ECL; Wako Pure Chemical Industries, Ltd., Osaka, Japan) supple- Ge Healthcare Life Science, Piscataway, NJ, USA). mented with heat-inactivated 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., Carlsbad, CA, RNA preparation and RT‑PCR. Total RNA was extracted USA) and