PAP1 signaling involves MAPK signal transduction

El pdf del artículo es la versión post-print.-- et al.Pancreatitis-associated protein 1 (PAP1) belongs to the Reg family of secretory proteins. Several important biological roles have been attributed to PAP1 but the signaling pathways activated by this protein remain only partially understood. Here, we describe the intracellular pathways triggered by PAP1 in a pancreatic acinar cell line. Taking advantage of the fact that PAP1 induces its own transcription, we performed ChIP assays to analyze the recruitment of transcriptional factors on its promoter. Our results show that PAP1 increased the transactivation activity of pap1 and the binding on its promoter of the nuclear factors C/EBPβ, P-CREB, P-ELK1, EGR1, STAT3, and ETS2, which are downstream targets of MAPK signaling. p44/42, p38, and JNK MAPKs activity increased after PAP1 treatment. In addition, pharmacological inhibition of these kinases markedly inhibited the induction of pap1 mRNA. Taken together, these results indicated that the mechanism of PAP1 action involves the activation of the MAPK superfamily.This work was supported by the FIS PI050599 and FIS PI081608 projects to E. Folch-Puy, Acción Integrada HF2006-0092 and CIBERehd to D. Closa and Spanish Ministry of Science and Technology BFU2007-63120 and CSD2006-49 to G. López-Rodas. CIBERehd is funded by the Instituto de Salud Carlos III. E. Folch-Puy is the recipient of a Ramón y Cajal contract from the Spanish Ministry of Education and Science. M. Ferrés-Masó is the recipient of a FIS-Instituto de Salud Carlos III contract PI050599.Peer reviewe

The reg4gene, amplified in the early stages of pancreatic cancer development, is a promising therapeutic target

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: The aim of our work was to identify the genes specifically altered in pancreatic adenocarcinoma and especially those that are altered early in cancer development.[Methodology/Principal Findings]: Gene copy number was systematically assessed with an ultra-high resolution CGH oligonucleotide microarray in DNA from samples of pancreatic cancer. Several new cancer-associated variations were observed. In this work we focused on one of them, involving the reg4 gene. Gene copy number gain of the reg4 gene was confirmed by qPCR in 14 cancer samples. It was also found with increased copy number in most PanIN3 samples. The relationship betweena gain in reg4 gene copy number and cancer development was investigated on the human pancreatic cancer cell line Mia-PaCa2 xenografted under the skin of nude mice. When cells were transfected with a vector allowing reg4 expression, they generated tumors almost twice larger in size. In addition, these tumors were more resistant to gemcitabine treatment than control tumors. Interestingly, weekly intraperitoneal administration of a monoclonal antibody to reg4 halved the size of tumors generated by Mia-PaCa2 cells, suggesting that the antibody interfered with a paracrine/autocrine mechanism involving reg4 and stimulating cancer progression. The addition of gemcitabine resulted in further reduction, tumors becoming 5 times smaller than control. Exposure to reg4 antibody resulted in a significant decrease in intra-tumor levels of pAkt, Bcl-xL, Bcl-2, survivin and cyclin D1.[Conclusions/Significance]: It was concluded that adjuvant therapies targeting reg4 could improve the standard treatment of pancreatic cancer with gemcitabine.This work was supported by grants from INSERM, Ligue Contre le Cancer and INCA to JLI and by the FIS PI081608 project, Acción Integrada HF2006-0092 and CIBERehd. CIBERehd is funded by the Instituto de Salud Carlos III to EF-P and DC. EF-P is the recipient of a Ramón y Cajal contract from the Spanish Ministry of Education and Science and MF-M is the recipient of a FIS-Instituto de Salud Carlos III contract PI050599.Peer reviewe

The reg4 Gene, Amplified in the Early Stages of Pancreatic Cancer Development, Is a Promising Therapeutic Target

BACKGROUND: The aim of our work was to identify the genes specifically altered in pancreatic adenocarcinoma and especially those that are altered early in cancer development. METHODOLOGY/PRINCIPAL FINDINGS: Gene copy number was systematically assessed with an ultra-high resolution CGH oligonucleotide microarray in DNA from samples of pancreatic cancer. Several new cancer-associated variations were observed. In this work we focused on one of them, involving the reg4 gene. Gene copy number gain of the reg4 gene was confirmed by qPCR in 14 cancer samples. It was also found with increased copy number in most PanIN3 samples. The relationship betweena gain in reg4 gene copy number and cancer development was investigated on the human pancreatic cancer cell line Mia-PaCa2 xenografted under the skin of nude mice. When cells were transfected with a vector allowing reg4 expression, they generated tumors almost twice larger in size. In addition, these tumors were more resistant to gemcitabine treatment than control tumors. Interestingly, weekly intraperitoneal administration of a monoclonal antibody to reg4 halved the size of tumors generated by Mia-PaCa2 cells, suggesting that the antibody interfered with a paracrine/autocrine mechanism involving reg4 and stimulating cancer progression. The addition of gemcitabine resulted in further reduction, tumors becoming 5 times smaller than control. Exposure to reg4 antibody resulted in a significant decrease in intra-tumor levels of pAkt, Bcl-xL, Bcl-2, survivin and cyclin D1. CONCLUSIONS/SIGNIFICANCE: It was concluded that adjuvant therapies targeting reg4 could improve the standard treatment of pancreatic cancer with gemcitabine

Overexpression of the REG4 protein in Mia-PaCa2 cells.

<p>The entire coding sequence of the reg4 cDNA was subcloned into the pLPCX retroviral vector. Phoenix Amphotropic packaging cells were transfected with the retroviral plasmid to obtain the supernatant containing retroviral particles. Target Mia-PaCa2 cells were plated in the presence of the supernatant containing retroviral particles as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007495#s4" target="_blank">Material and Methods</a> section. The populations of reg4-expressing Mia-PaCa2 (Mia-PaCa2/reg4) and control cells (Mia-PaCa2/empty), infected with the empty vector, were isolated by puromycin selection. Expression of REG4 was measured by western blot on cell extracts, using the anti-reg4 monoclonal antibody. After development, the membrane was stripped and re-probed for β-actin.</p

Growth and response to gemcitabine of pancreatic tumors treated with a specific anti-REG4 antibody.

<p>Approximately 2×10⁶ Mia-PaCa-2 cells were inoculated subcutaneously with 0.1 ml of Matrigel to nude mice. One day before tumoral cell inoculation, the control group received an intraperitoneal injection of 150 µl of PBS, whereas the assay group received 0.25 mg of reg4 monoclonal antibody in 150 µl of PBS. Vehicle buffer or anti-reg4 antibody was injected weekly in animals. Tumor dimensions were measured once weekly and tumor volumes calculated with the formula length × width × depth ×0.5236. Gemcitabine (100 mg/kg) was injected intraperitoneally twice a week. Values are expressed as mean +/− SE (n = 6).</p

<i>reg4</i> gene amplification in pancreatic cancer and precancerous lesions measured by quantitative PCR.

<p>Homogeneous populations of cells were carefully microdissected using a Laser Capture Microdissection System. PanINs were graded 1 to 3 as a function of the importance of architectural and cytological abnormalities. DNA from pancreatic cancer samples obtained by endoscopic ultrasound (EUS)-guided fine needle aspiration was used. DNA from blood leucocytes was used as control. <i>reg4</i> gene copy number was determined by qPCR performed in triplicate and results were analyzed using the RealQuant software.</p

Expression of phosphorylated AKT, Bcl-xL, Bcl-2, surviving and cyclin D1 in pancreatic tumors treated with a specific anti-REG4 antibody.

<p>Tumor tissue samples were lysed in a homogenization buffer containing a cocktail of antiproteases. Cell debris and insoluble materials were eliminated by centrifugation and soluble fractions were loaded in Laemmli buffer onto a 12.5% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose membrane and membrane incubated with the anti phosphorylated AKT (phospho-Ser473), anti-pan AKT, anti-Bcl-2, anti-Bcl-xL, anti-survivin or anti-cyclin D1 antibodies. After development, the membrane was stripped and reprobed for β-actin.</p

The <i>reg4</i> gene is found with increased copy number in pancreatic cancer.

<p>Allelic ratios were calculated with the Partek Genomics Suite, version 6.4. HapMap (270 samples) was used to create baseline copy number. Genomic segmentation was utilized to detect copy number gain or loss. Regions were detected using the following segmentation parameters: minimum of 10 genomic markers; segmentation p-value threshold lower than 0.001; a signal to noise equal to 0.3. A. Schematic representation of the <i>reg4</i> locus. Positions of the 7 genes present in this locus are indicated: from left to righ: <i>ZNF697</i>, <i>PHGDH</i>, <i>HMGCS2</i>, <i>REG4</i>, <i>NBPF7</i>, <i>ADAM30</i> and <i>NOTCH2</i>. B. Position of the copy number gain segment found in all 14 DNA samples from pancreatic cancer, which includes the <i>reg4</i> gene. C. Genomic changes in the amplified segment of the <i>reg4</i> locus, determined by the Affymetrix Genome-Wide Human SNP Array 6.0 analysis in the 14 pancreatic cancer samples. Genetic gains are shown as red bars and losses as blue bars, grey bars corresponding to 2 copies as indicated in the scale shown underneath. Note the predominance of gains (red) in the <i>reg4</i> region. D. Detail of gains/losses in the <i>reg4</i> gene and its flanking regions for all 14 analyzed samples.</p

Growth and response to gemcitabine of xenografted Mia-PaCa2 cells overexpressing REG4.

<p>Approximately 2×10⁶ Mia-PaCa-2 cells were inoculated subcutaneously with 0.1 ml of Matrigel to nude mice. Gemcitabine (100 mg/kg) was injected intraperitoneally twice a week. Tumor dimensions were measured once weekly and tumor volumes calculated with the formula length × width × depth ×0.5236. Values are expressed as the mean +/− SE of six measurements.</p

Influence of a reg4 siRNA or a REG4 antibody on growth and response to gemcitabine of Mia-PaCa2 cells overexpressing REG4.

<p>Mia-PaCa2/reg4 and Mia-PaCa2/empty cells were transfected with a specific siRNA against reg4 (A) or incubated in the presence of an anti-REG4 monoclonal antibody (B) and their growth and their resistance to gemcitabine treatment was measured by the MTS assay. Results were expressed as percent of untreated Mia-PaCa2/empty cells.</p