Pancreatic adenocarcinoma up-regulated factor (PAUF), a novel up-regulated secretory protein in pancreatic ductal adenocarcinoma

Abstract

The identification of novel tumor-specific proteins or antigens is of great importance for diagnostic and therapeutic applications in pancreatic cancer. Using oligonucleotide microarrays, we identified a broad spectrum of differentially expressed pancreatic cancer-related genes. Of these, we selected an overexpressed expressed sequence taq and cloned a 721-bp full-length cDNA with an open reading frame of 196 amino acids. This novel gene was localized on the Homo sapiens 16p13.3 chromosomal locus, and its nucleotide sequence matched the Homo sapiens similar to common salivary protein 1 (LOC124220). We named the gene pancreatic adenocarcinoma up-regulated factor. The pancreatic adenocarcinoma up-regulated factor was secreted into the culture medium of pancreatic adenocarcinoma up-regulated factor-overexpressing Chinese hamster ovary cells, had an apparent molecular mass of approximately 25 kDa, and was N-glycosylated. The induction of pancreatic adenocarcinoma up-regulated factor in Chinese hamster ovary cells increased cell proliferation, migration, and invasion ability in vitro. Subcutaneous injection of mice with Chinese hamster ovary/pancreatic adenocarcinoma up-regulated factor cells resulted in 3.8-fold greater tumor sizes compared to Chinese hamster ovary/mock cells. Reverse transcription-polymerase chain reaction and western blotting with antirecombinant human pancreatic adenocarcinoma up-regulated factor antibodies confirmed that pancreatic adenocarcinoma up-regulated factor was highly expressed in six of eight pancreatic cancer cell lines. Immunohistochemical staining of human pancreatic cancer tissues also showed pancreatic adenocarcinoma up-regulated factor overexpression in the cytoplasm of cancer cells. Transfection with pancreatic adenocarcinoma up-regulated factor-specific small-interfering RNA reduced cancer cell migration and invasion in vitro. Treatment with antirecombinant human pancreatic adenocarcinoma up-regulated factor in vitro and in vivo reduced proliferation, migration, invasion, and tumorigenic ability. Collectively, our results suggest that pancreatic adenocarcinoma up-regulated factor is a novel secretory protein involved in pancreatic cancer progression and might be a potential target for the treatment of pancreatic cancer.

Figure 1

Expression of pancreatic adenocarcinoma up‐regulated factor (PAUF) in human tumor and non‐cancerous tissues. (a) Expression levels of PAUF in normal pancreas and pancreatic adenocarcinomas were compared with data acquired from Affymetrix HG‐U133 chip set analysis. PAUF was significantly up‐regulated in pancreatic adenocarcinomas compared to normal pancreatic tissue. (b) The differential expression of PAUF was validated by quantitative real‐time polymerase chain reaction in pancreatic cancer compared to adjacent normal pancreas. The relative expression value of PAUF was measured by threshold value relative to C‐terminal binding protein (CTBP). (c) The size of PAUF mRNA transcripts was measured using Human 12‐lane MTN blot containing various human normal tissues; only the placenta showed strong PAUF expression. (d) Northern blot analysis of PAUF from tumor tissues using MTN Human Tumor Blot. High expression of PAUF was found in colon, ovary, and stomach tumors.

Figure 2

Nucleotide and amino acid sequences of pancreatic adenocarcinoma up‐regulated factor (PAUF) cDNA. (a) PAUF contains 721 bp. An inframe termination codon is located at nucleotide positions 600–602 (), and the canonical 3′‐polyadenylation signal AATAAA is located at nucleotide positions 698–703 (_). PAUF encodes a 196‐amino acid protein, including a 40‐amino acid signal peptide (bold type; cleavage site, bold arrow) with putative post‐translational modification sites. Specifically, one N‐glycosylation site at amino acids 185–189 (); three phosphorylation sites at amino acids 7–9, 53–56, and 125–128 (); and three N‐myristorylation sites at amino acids 64–69, 90–95, and 137–143 () were predicted in silico. (b) The amino acid sequences of human PAUF, chimpanzee XP_523271 (similar to HRPE773), rhesus monkey XP_001086870 (similar to zymogen granule membrane protein 16 isoform 2), and mouse NP_064294 (demilone cell and parotid protein [Dcpp1]) were aligned by CLUSTAL W (1.83) multiple sequence alignment. Amino acid identities in all sequences are indicated by an asterisk while conserved and semiconserved substitutions are shown by a colon and a dot, respectively.

Figure 3

Subcellular localization and post‐translational modification of pancreatic adenocarcinoma up‐regulated factor (PAUF). The pcDNA3.1(+)‐PAUF‐Myc/His (CHO/PAUF) or pcDNA3.1(+)‐Myc/His vectors (CHO/Mock) were transfected stably into Chinese hamster ovary (CHO) cells. (a) Western blot analysis of the parental and transfected CHO cell lines: PAUF expression was detected with anti‐His antibody in cell lysates (CL) and culture medium (CM) from CHO/PAUF cells. (b) Secreted PAUF protein was purified from culture medium of CHO/PAUF and digested with Peptide‐N‐glycosidase F (PNGase F) for 1 h. The samples were electrophoresed and immunoblotted with anti‐His antibody. Treatment with PNGase F resulted in complete de‐glycosylation of PAUF as demonstrated by immunoreactivity of anti‐His antibodies with protein bands of lower molecular mass. (c) Secreted PAUF protein was purified from medium of 2‐µg/mL tunicamycin (TM) treated CFPAC‐1 cells. Treatment with tunicamycin resulted in complete de‐glycosylation of PAUF as demonstrated by immunoreactivity of antirhPAUF antibodies with protein bands of lower molecular mass.

Figure 4

Effects of pancreatic adenocarcinoma up‐regulated factor (PAUF) expression on cell proliferation, invasion, and tumorigenicity. (a) The transfected cells (2 × 10³ cells per well) were counted every 24 h using a hemocytometer. The Chinese hamster ovary (CHO)/PAUF () cells showed a higher proliferation rate compared to CHO/Mock cells (). (b) Cell migration ability was assessed based on the number of cells per microscopic field (×100) that migrated through the pores of a Transwell chamber to the under surface of the membrane after an incubation time of 24 h. In CHO/PAUF cells, cell migration increased compared to CHO/Mock cells. (c) Cell invasion ability was assessed by counting the number of cells per microscopic field (×100) that migrated through Matrigel invasion chambers after 48 h. Cell invasion ability increased in CHO/PAUF cells compared to CHO/Mock cells. (d) The CHO/PAUF cells were injected into the flank of 6‐week‐old female Balb/c nude mice. The CHO/Mock cells were used as a control. Mice were monitored for 32 days after injection (n = 5 for each). Data are shown as the mean ± SEM. Representative images of tumor xenografts after 32 days shows that grafting with CHO/PAUF cells produced larger tumors than CHO/Mock treatments.

Figure 5

Expression of pancreatic adenocarcinoma up‐regulated factor (PAUF) mRNA and protein in pancreatic cells and tissue. (A) Reverse transcription–polymerase chain reaction analysis of PAUF mRNA expression in various cell lines. Upper, the expression of PAUF mRNA in the normal cell lines: peripheral blood mononuclear cells (PBMC), PBMC_st, NIH/3T3, and bone marrow stromal (BMSC) cells. The CHO/PAUF cells served as a positive control for PAUF. Lower, the expression of PAUF mRNA pancreatic cancer cell lines and in parental, vector‐ (CHO/Mock), and CHO/PAUF cells. Beta‐actin (ACTB) served as an internal control. AsPC‐1; BxPC‐3; CFPAC‐1; HPAC, human pancreatic cancer cell line; MIAPaCa‐2; PANC‐1. (B) Western blot analysis of PAUF in pancreatic cancer cell lines; 20 µg of protein from culture media (CM) and cell lysates (CL) were probed with antirecombinant human PAUF (antirhPAUF) antibodies. For CHO/PAUF cells, which served as a positive control, 0.2 µg of protein from the culture medium was loaded. Molecular mass markers (kDa) are shown on the left. (C) Expression of PAUF in pancreatic tissue. Immunohistochemical staining with antirhPAUF antibodies revealed strong cytoplasmic overexpression of PAUF in cancer and islets cells. No significant staining was observed in normal ductal and acinar cells. (a) normal pancreas (×400); (b) pancreatic adenocarcinoma (×100); (c) pancreatic adenocarcinoma (×400); (d) metaplastic duct in chronic pancreatitis (×1000); (e) pancreatic adenocarcinoma (×1000); and (f) pancreatic adenocarcinoma (×1000).

Figure 6

Effects of inhibiting pancreatic adenocarcinoma up‐regulated factor (PAUF) in pancreatic cancer cells. Knockdown effect with PAUF small‐interfering RNA (siRNA)‐transfected CFPAC‐1 cells (a–c). PAUF siRNA were transiently transfected into CFPAC‐1 cells for 48 h. (a) At 48 h post‐transfection, cells were harvested and subjected to total RNA extraction, and reverse transcription–polymerase chain reaction was performed. Secreted protein was prepared from culture medium at 48 h post‐transfection. mRNA and protein levels of PAUF of siRNA‐transfected CFPAC‐1 cells decreased. ACTB, beta‐actin. (b) Cell migration ability was assessed based on the number of cells per microscopic field (×200) that migrated through the pores of a Transwell chamber to the under surface of the membrane after an incubation time of 24 h. In PAUF siRNA‐tranfected CFPAC‐1 cells, cell migration ability decreased compared to negative control cells. (c) Cell invasion ability was assessed by counting the number of cells per microscopic field (×200) that migrated through Matrigel invasion chambers after 48 h. Cell invasion ability was decreased in PAUF siRNA‐tranfected CFPAC‐1 cells compared to negative control cells. Effects of inhibiting PAUF with antirecombinant human PAUF (antirhPAUF) antibodies (d–f). The graphs (d,e) show the mean ± SD (% of antibody‐treated cells/non‐treated cells per microscopic field) from triplicate samples. (d) CFPAC‐1 cells were plated on a Boyden chamber and incubated for 24 h in the presence of the indicated concentration of antirhPAUF antibodies () or normal rabbit IgG (). AntirhPAUF antibodies effectively inhibited the migration of CFPAC‐1 in a dose‐dependent manner. (e) CFPAC‐1 cells were plated on Matrigel‐coated filters and incubated for 24 h in the presence of the indicated concentration of antirhPAUF antibodies () or normal rabbit IgG (). Invasion through Matrigel‐coated membranes was dose‐dependently inhibited by antirhPAUF antibodies. (f) Tumor‐bearing mice generated by the injection of CFPAC‐1 were treated with antirhPAUF antibodies () or normal rabbit IgG () via the tail vein twice a week (n = 6 for each) and tumor mass was monitored (left) for 32 days. Data are shown as the mean ± SEM. The starting day of antibody injection is represented by the arrow. The representative image shows the results of tumor xenografts at the end of the experiments.