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

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.


INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and is the fourth leading cause of cancer-related death in the western world [1,2]. The difficulty in early detection of PDAC and its resistance to conventional treatment options contribute to its dismal prognosis, as indicated by an overall 5-year survival rate of less than 10% [1,2]. Many researchers have attempted to elucidate the pathogenesis and carcinogenesis of PDAC.
However, the molecular basis for the aggressive nature of PDAC is incompletely understood, and the overall survival of patients has not improved significantly despite advances in treatment modalities and introduction of novel targeted therapies.
The clinical outcomes of PDAC may be poor because conventional therapies are directed at tumor cells that have limited tumorigenic potential instead of targeting the pancreatic cancer stem cells (CSCs) that have been identified in human PDAC [3]. Pancreatic CSCs are www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 44), pp: 76398-76407

Research Paper
identified by a variety of biomarkers, and transplantation assays using immune-deficient mice show that pancreatic CSCs self-renew and propagate the parental tumor. Li et al. has identified populations of CSCs in PDAC that express the cell surface markers CD44 + CD24 + ESA + or c-Met [3,4]. Apart from these markers, CD133 + and ALDH1 are regarded as other markers of CSCs in PDAC [5][6][7]. These CSCs are also characterized by their chemoresistance, as CD133 + pancreatic cancer cells have greater drug resistance to gemcitabine [6], and c-MET inhibitors have been shown to enhance antitumor effects in combination with gemcitabine [3].
Genome-wide analyses have uncovered pancreatic adenocarcinoma up-regulated factor (PAUF), a novel secretory protein associated with pancreatic cancer [8]. Although the mechanism through which PAUF contributes to cancer progression is not clearly established, previous reports suggest it plays critical roles in PDAC progression and metastasis processes, including cell proliferation and modulation of adhesion, migration, and invasion [9][10][11][12][13][14][15].
No studies have investigated the association between PAUF and CSCs and how it may contribute to drug sensitivity and resistance. Therefore, the purpose of the present study was to investigate the relationship between PAUF function and pancreatic CSCs. Our experiments provided evidence of PAUF expression in pancreatic CSCs and suggested that PAUF may contributes to multi-drug resistance in the pancreatic CSCs.

Pancreatic cancer spheres cultured from human pancreatic cancer cell lines
We used sphere culture to identify CSCs. This in vitro method involved culturing candidate pancreatic CSCs with serum-free media containing only EGF and bFGF under nonadherent conditions, and the resulting spheres indicated self-renewal consistent with a CSC phenotype. In this study, PDAC spheres cultured from the CFPAC-1 cells showed upregulated expression of the pluripotent stemness genes Oct4, Nanog, Stat3, and Sox2 relative to adherent CFPAC-1 cells ( Figure 1A).

PAUF overexpression in pancreatic CSCs
We previously reported PAUF as a novel secretory protein associated with pancreatic cancer [8]. To show the relationship between the PAUF and pancreatic CSCs, secretory PAUF expression were detected in spheres and adherent CFPAC-1 cells by western blot analysis. As Figure 1B, PAUF expressions were upregulated in spheres than adherent CFPAC-1 cells. Furthermore, in other pancreatic cancer cells such as CAPAN-1 and HPAC cells, PAUF expression were upregulated in spheres than adherent cells ( Figure 1B).

PAUF knockdown effects on pancreatic cancer stem cells in pancreatic cancer cell lines
To further access the specific role of PAUF in pancreatic CSCs function, we stably knocked down PAUF expression using shRNA targeting regions of PAUF.
The mRNA and protein expression of pluripotent stemness genes in shPAUF CFPAC-1 was measured using RT-PCR and western blot, respectively. The expression of stemness-related genes (including Oct4, Nanog, and Sox2), and CSC surface markers (such as CD44, CD133, and c-Met) were lower in the shPAUF CFPAC-1 than in the control CFPAC-1 cells ( Figure 3F). Protein expression www.impactjournals.com/oncotarget  of PAUF, Sox2, and ALDH were lower in shPAUF CFPAC-1 than in control cells ( Figure 3G).

Effects of PAUF on chemoresponse of pancreatic cancer cell lines
Past studies have suggested that pancreatic CSCs are more resistant to anti-cancer chemotherapy agents such as gemcitabine and 5-FU. To determine the role of PAUF in chemoresponse, we performed cell survival assays to validate cytotoxic effects of 5-FU or gemcitabine against PAUF-associated pancreatic cancer. Two transient siPAUF RNAs were used for this experiment.

DISCUSSION
Recent studies have demonstrated the presence of pancreatic CSCs, which are capable of self-renewal and production of differentiated progeny, in PDAC. Although the isolation and culturing of pancreatic CSCs remains difficult, the role of CSCs in development, maintenance, and metastasis of PDAC is increasingly recognized. In this study, we cultured the pancreatic cancer cells in serum-free media and obtained pancreatic cancer sphere cells, which had self-renewal capabilities. Relative to the adherent cells, PDAC spheres showed increased mRNA and protein expression of PAUF and the pluripotent stemness genes Oct4, Nanog, Sox2 and STAT3. Additionally, CD44 + CD24 + ESA + pancreatic CSCs had increased mRNA expression of PAUF.
We recently identified PAUF, (also known as a paralog of ZG16B), a novel 27-kDa secretory protein highly expressed in human PDAC [8]. As an autocrine factor, PAUF is involved in altered migration, invasion, proliferation, and metastasis [9,11,16]. PAUF also acts as a paracrine factor by causing stromal changes in the tumor microenvironment that promotes tumor evasion of the immune surveillance system [12]. PAUF also modulates permeability of the endothelial cells and vasculature to promote tumor angiogenesis [17]. Although only a few preclinical studies have investigated anti-PAUF monoclonal antibodies, PAUF-specific RNA aptamers (P12FR2), and adenoviral PAUF-targeting trans-splicing ribozymes (TSR) for PDAC, PAUF-targeted therapies may be an alternative approach for cancer treatment [10,14,15]. The above-mentioned studies suggest functional roles of PAUF in pancreatic cancer; however, little is known about the clinical relationship between PAUF and pancreatic CSCs. Therefore, we show the association between PAUF expression and characteristics of CSCs to determine the contribution of PAUF activity to chemoresponse in pancreatic CSCs, Of clinical importance, recent research suggests CSCs are resistant to conventional chemotherapy and radiation [4]. Enhanced expression of ATP-binding cassette (ABC) family of transporters has been shown to be characteristic of side population cells with CSC features. These side population cells are more resistant to chemotherapeutic agents and may play a key role in tumor progression, recurrence, and metastasis [18,19]. To identify the relationship between chemoresponse and the functional role of PAUF, we conducted cytotoxic studies with gemcitabine and 5-FU in siPAUF CFPAC-1/siPAUF AsPC-1 cells and negative controls. Our study showed that siPAUF CFPAC-1/siPAUF AsPC-1 cells were significantly more vulnerable to both cytotoxic drugs. Similar to our results, recent study reported that silencing of PAUF leads to increase the sensitivity to gemcitabine in BxPC-3 cells and this mechanism may involve drug transporter such as MRP2, MRP3, and MDR1 genes [20]. However, because both chemosensitivity and chemoresistance can occur through intracellular enzymes regardless of drug transporters, we measured the mRNA expression of genes involved in drug metabolism (such as dCK, CDA, RRM1, and RRM2) in addition to transporters (including MRP3, MRP4, MRP5, and hENT1). In the present study, the both siPAUF CFPAC-1 and siPAUF AsPC-1 cells decreased mRNA expression of MRP5 and RRM2. MRP5 (also known as ABCC5) is a member of the multidrug resistance-associated protein subfamily of ABC transporters and confers resistance against chemotherapeutic drugs such as etoposide, 5-FU, and  [21][22][23]. RRM2 is the catalytic subunit of ribonucleotide reductase, a dimeric enzyme that supplies deoxynucleotides essential for DNA replication and cell growth. The overexpression of RRM2 has been associated with gemcitabine resistance and increased cellular invasiveness in vitro [24], [25] and high expression of RRM2 in pancreatic tumors is associated with reduced overall survival after resection [26].
Taken together, our study indicated that PAUF signaling was required for the formation of pancreatic CSCs. In addition, the suppression of PAUF may contribute to increase drug sensitivity through mechanisms involving MRP5 and RRM2, thus making it a novel target for treatment of PDAC. However, our study had some limitations. First, we could not demonstrate a PAUF mediated signal transduction mechanism by which PAUF induces CSCs phenotypes. Second, we used pancreatic cancer cell lines rather than patient-derived pancreatic cancer cells and only investigated the effect of PAUF knockdown in PDAC cancer cell lines rather than in pancreatic CSCs derived from human tissues. Third, we did not perform the experimental study using in vivo models of human PDAC, which are required to prove a causal relationship between PAUF and target proteins such as MRP5 and RRM2. Thus, the possible connection between PAUF, drug transporters, and cell signaling pathways should be investigated in future studies.
In conclusion, we showed that PAUF is a novel protein overexpressed in human pancreatic CSCs that may contribute to chemosensitivity by decreasing MRP5 and RRM2. Thus, PAUF may constitute a therapeutic target to enhance the drug sensitivity of pancreatic CSCs.

Establishment of stable PAUF knockdown cell line using shRNA
The shRNA-expressing plasmid targeting human PAUF and negative control plasmid were purchased from SABiosciences. The human PAUF shRNA sequence was 5'-ACACCAGCAAGGACCGCTATT -3' and the control shRNA sequence 5'-GGAATCTCATTCGATGCATAC -3'. For shRNA transfection, CFPAC-1 cells were plated into 6-well plates at a density of 5×10 4 cells/well the day before transfection. Transfection was performed using Lipofectamine2000 reagent according to manufacturer's instructions, and stable knockdown clones were selected using neomycin.

Transient transfection of small interfering RNA (siRNA) targeted for PAUF
The two sets of 25-nucleotide stealth RNAi-targeting PAUF were synthesized customarily by Invitrogen. Stealth RNAi duplexes, which have a similar GC content to that of the duplex siRNA obtained from Invitrogen, were used as negative controls. Stealth RNAi-targeting PAUF were transfected into CFPAC-1 cells using Lipectamine™ RNAi Max transfection agent (Invitrogen) according to the manufacturer's protocol. Cells were harvested 72 h post-transfection and subjected to total RNA extraction and a migration and invasion assay. Secreted protein was prepared from culture medium 48 h post-transfection.

MTT assay
After incubation at 37˚C overnight, cells were treated with various concentrations of gemcitabine or fluorouracil (5-FU) in complete growth media and incubated for 72 h at 37˚C. A 3-(4,5-dimenthelthiazol-2-ly)-2,5-diphenyltetrasolium bromide-based assay (absorbance 570 nm) was used to measure the number of metabolically active cells.

Soft agar assay
A suspension of 500 single cells containing 0.3% agar medium was overlaid on 0.6% agar medium in 24well plates. Each well was covered with complete medium, and the plates were incubated for 4 weeks. Colonies were stained with crystal violet and counted. Experiments were done in triplicate.

Cell proliferation, migration and invasion assay
For cell proliferation, 2 x 10 3 cells were seeded per sell into 24 well plates. Every 24 h, the number of cells was counted. The experimental was done in triplicate to determine the number of cells at each time point. The migration assays were performed in 6-well Transwell plates (Costar, Cambridge, MA). Cells (1 × 10 5 ) were seeded in triplicate in the upper compartment. The media incubated the NIH/3T3 with DMEM for 24 hours was added to the lower wells. Following 24-hour incubation at 37C in a 5% CO 2 humidified incubator the cells in the upper chamber and on the surface of the filter were completely removed by wiping with a moist cotton swab. Cells that had migrated through the filter and adhered to the outer surface were fixed with methanol and stained with Toluidine Blue. Invading cells were examined, counted and photographed by microscopy at 100× magnification. The invasive abilities were tested by Matrigel invasion assay. Assays were performed in 6-well Transwell plates (Costar, Cambridge, MA) with an 8 μm pore size were coated with Matrigel (Becton Dickinson). Cells (1 × 10 5 ) were then seeded in triplicate in the upper compartment chamber coated with Matrigel. The media incubated the NIH/3T3 with DMEM for 24 hours was added to the lower wells. Following 48-hour incubation at 37C in a 5% CO 2 humidified incubator the cells in the upper chamber and on the surface of the filter were completely removed by wiping with a moist cotton swab. Cells that had invaded the Matrigel, migrated through the filter and adhered to the outer surface were fixed with methanol and stained with Toluidine Blue. Invading cells were examined, counted and photographed by microscopy at 100× magnification.

In vivo tumorigenic assay
Cells were resuspended at a cell density of 3x 10 6 cells in 150 μl of serum-free culture medium and injected subcutaneously into the BALB/c nude female mice (6 weeks of age). Tumor formation was monitored twice a week measuring the width and length. Tumor volumes were calculated by the formula V (mm 3 ) = A x B 2 , where A is the largest dimension and B is the perpendicular diameter. Tumor xenografts were recovered from mice, fixed in 4% paraformaldehyde, and embedded in paraffin.

Statistical analysis
Mann-Whitney U test and independent t-test were used to compare cell survival between siPAUF and control groups. Statistical calculations were performed using SPSS (version 12.0 for Windows; SPSS, Inc., Chicago, IL, USA). Values of p < 0.05 were considered statistically significant. For quantitative analysis western blot and RT-PCR data, we used ImageJ softwere (U.S. National Institutes of Health, Bethesda, MD, https://imagej.nih. gov/ij). www.impactjournals.com/oncotarget enhances pancreatic adenocarcinoma chemosensitivity to gemcitabine.