Suppression of pancreatic adenocarcinoma upregulated factor (PAUF) increases the sensitivity of pancreatic cancer to gemcitabine and 5FU, and inhibits the formation of pancreatic cancer stem like cells

Abstract

Pancreatic cancer stem cells (CSCs) play a crucial role in tumorigenesis and chemoresistance of pancreatic ductal adenocarcinoma. Pancreatic adenocarcinoma up-regulated factor (PAUF), a novel secretory protein, has been shown to contribute to cancer progression and metastasis. Because the clinical relationship between PAUF and pancreatic CSCs is largely unknown, we investigated the associations between the functional role of PAUF and pancreatic CSCs. Pancreatic cancer sphere cultured from the CFPAC-1 cells showed elevated expression of PAUF and pluripotent stemness genes (Oct4, Nanog, Stat3, and Sox2), and the mRNA of PAUF were increased in CD44⁺CD24⁺ESA⁺ pancreatic CSCs. PAUF knockdown (shPAUF) CFPAC-1 diminished the number of spheres and decreased stemness genes and CSC surface markers (CD133, c-MET and ALDH1). In addition, siPAUF CFPAC-1 decreased the mRNA expression of multidrug resistant protein 5 (MRP5) and ribonucleotide reductase M2 (RRM2) and were more vulnerable to gemcitabine and 5-FU than negative control (p<0.05). In conclusion, PAUF was increased in pancreatic CSCs and the suppression of PAUF enhances chemotherapeutic response to gemcitabine and 5FU by decreasing MRP5 and RRM2 in pancreatic cancer cells.

Keywords: MRP5; PAUF; RRM2; cancer stem cells; pancreatic cancer.

Figure 1. PAUF overexpression in pancreatic cancer spheres

(A) RT-PCR showed that the overexpression of Oct4, Nanog, Stat3, and Sox2 genes in sphere formed from CFPAC-1 cells compare with adherent cells. (B) Up-regulation of secretory PAUF was determined by western blot in CFPAC-1, CAPAN-1 and HPAC spheres (Sp) than adherent (Ad) cells.

Figure 2. PAUF overexpression in CD24+/CD44+/ESA+ pancreatic CSCs

(A) CFPAC-1 cells were sorted using surface marker antibodies. CD24⁺/CD44⁺/ESA⁺ cells were 46% and CD24^-/CD44^-/ESA^- cells were 52%. (B) RT-PCR demonstrated that the mRNA expression of stemness-related genes and PAUF was higher in CD24⁺/CD44⁺/ESA⁺ pancreatic CSCs sorted from CFPAC-1 cells than in CD24^-/CD44^-/ESA^- CFPAC-1 cells. (C) Bar graph demonstrated that quantitative analysis of RT-PCR data. (D) HPAC-1 cells were sorted using surface marker antibodies. CD24⁺/CD44⁺/ESA⁺ cells were 19.1% and CD24^-/CD44^-/ESA^- cells were 19.2%. (E) RT-PCR demonstrated that the mRNA expression of stemness-related genes and PAUF was higher in CD24⁺/CD44⁺/ESA⁺ pancreatic CSCs sorted from HPAC-1 cells than in CD24^-/CD44^-/ESA^- HPAC-1 cells. (F) Bar graph demonstrated that quantitative analysis of RT-PCR data.

Figure 3. PAUF knockdown effects on pancreatic cancer stem cells in pancreatic cancer cell lines

(A) PAUF knockdown CFPAC-1 cells were established by shRNA transfection, and shPAUF CFPAC-1 cells were confirmed using RT-PCR and western blot. B-actin and GAPDH served as a loading control. (B) Migration assays showed that shPAUF CFPAC-1 cells had decreased migration ability compared with NC CFPAC-1 cells; 187.71 ± 41.33 cells/microscopic field in shPAUF vs. 278.67 ± 28.66 cells/microscopic field in NC (C) in vivo tumor cell xenograft assay showed shPAUF CFPAC-1 cells formed smaller size of tumor compared with NC CFPAC-1 cells; the average tumor size 114.5 ± 30.12 mm³ in shPAUF vs. 258.0 ± 103.0 mm³ in NC. (D) Sphere formation assays showed that shPAUF CFPAC-1 cells had a lower sphere-forming ability compared with NC CFPAC-1 cells. (E) Soft agar assays showed that shPAUF CFPAC-1 cells had lower number of colonies compared with NC CFPAC-1 cells; 106.25 ±0.24 colonies in shPAUF vs. 195.25 ± 0.10 colonies in NC (F) RT-PCR demonstrated that the expression of pluripotent stemness genes and cancer stem cell-related genes were reduced in shPAUF CFPAC-1 cells compared with NC CFPAC-1 cells. (G) Western blot showed a lower protein expression of PAUF and cancer stem cell-related proteins in shPAUF CFPAC-1 cells compared with NC CFPAC-1 cells.

Figure 4. Effects of PAUF on chemoresponse of pancreatic cancer cell lines

(A-B) PAUF knockdown effects on the cytotoxicity of 72 hours inoculation with gemcitabine and 5FU in CFPAC-1 cells. MTT assay showed significantly increased antitumor effect of gemcitabine (A) and 5FU (B) in PAUF-silenced CFPAC-1 cells compared with negative control CFPAC-1. Data are shown as the mean ± SD. (C) RT-PCR demonstrated mRNA expression of MRP5 and RRM2 was lower in the siPAUF CFPAC-1 cells than in the negative control CFPAC-1 cells. (D-E) PAUF knockdown effects on the cytotoxicity of 72 hours inoculation with gemcitabine and 5FU in AsPC-1 cells. MTT assay showed significantly increased antitumor effect of gemcitabine (D) and 5FU (E) in PAUF-silenced AsPC-1 cells compared with negative control CFPAC-1. (F) RT-PCR demonstrated mRNA expression of MRP5 and RRM2 was lower in the siPAUF AsPC-1 cells than in the negative control AsPC-1 cells