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• W2083944232 abstract "Tumor protein p53-induced nuclear protein 1 (TP53INP1) is involved in cell stress response. Its expression is lost at the pancreatic intraepithelial neoplasia 1b (PanIN1b)/PanIN2 stage of pancreatic carcinogenesis. Our objective was to determine whether TP53INP1 loss of expression contributes to pancreatic cancer formation in a conditional KrasG12D mouse model. We generated Kras-INP1KO mice using LSL-KrasG12D/+;Pdx1-Cre+/− mice (Kras mice) and TP53INP1−/− mice. Analysis of pancreases during ageing shows that in the presence of activated Kras, TP53INP1 loss of expression accelerated PanIN formation and increased pancreatic injury and the number of high-grade lesions as compared with what occurs in Kras mice. Moreover, cystic lesions resembling intraductal papillary mucinous neoplasm (IPMN) were observed as early as 2 months of age. Remarkably, TP53INP1 is down-regulated in human IPMN. Activation of the small GTPase Rac1 shows that more oxidative stress is generated in Kras-INP1KO than in Kras mice pancreas despite elevated levels of the Nrf2 antioxidant regulator. We firmly establish the link between Kras-INP1KO pancreatic phenotype and oxidative stress with rescue of the phenotype by the antioxidant action of N-acetylcysteine. Our data provide in vivo functional demonstration that TP53INP1 deficiency accelerates progression of pancreatic cancer, underlining its role in the occurrence of IPMN and highlighting the importance of TP53INP1 in the control of oxidative status during development of pancreatic cancer. Tumor protein p53-induced nuclear protein 1 (TP53INP1) is involved in cell stress response. Its expression is lost at the pancreatic intraepithelial neoplasia 1b (PanIN1b)/PanIN2 stage of pancreatic carcinogenesis. Our objective was to determine whether TP53INP1 loss of expression contributes to pancreatic cancer formation in a conditional KrasG12D mouse model. We generated Kras-INP1KO mice using LSL-KrasG12D/+;Pdx1-Cre+/− mice (Kras mice) and TP53INP1−/− mice. Analysis of pancreases during ageing shows that in the presence of activated Kras, TP53INP1 loss of expression accelerated PanIN formation and increased pancreatic injury and the number of high-grade lesions as compared with what occurs in Kras mice. Moreover, cystic lesions resembling intraductal papillary mucinous neoplasm (IPMN) were observed as early as 2 months of age. Remarkably, TP53INP1 is down-regulated in human IPMN. Activation of the small GTPase Rac1 shows that more oxidative stress is generated in Kras-INP1KO than in Kras mice pancreas despite elevated levels of the Nrf2 antioxidant regulator. We firmly establish the link between Kras-INP1KO pancreatic phenotype and oxidative stress with rescue of the phenotype by the antioxidant action of N-acetylcysteine. Our data provide in vivo functional demonstration that TP53INP1 deficiency accelerates progression of pancreatic cancer, underlining its role in the occurrence of IPMN and highlighting the importance of TP53INP1 in the control of oxidative status during development of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDA) displays an overall 5-year-survival rate of less than 5%. It is often not diagnosed before the cancer has metastasized and therefore becomes very challenging to treat. Identification of molecular pathways that cooperate in initiating PDA is essential for diagnosis and prevention. Recent research demonstrated that inflammation plays a contributory role in pancreatic carcinogenesis.1Greer J.B. Whitcomb D.C. Inflammation and pancreatic cancer: an evidence-based review.Curr Opin Pharmacol. 2009; 9: 411-418Crossref PubMed Scopus (130) Google Scholar, 2Raimondi S. Lowenfels A.B. Morselli-Labate A.M. Maisonneuve P. Pezzilli R. Pancreatic cancer in chronic pancreatitis; aetiology, incidence, and early detection.Best Pract Res Clin Gastroenterol. 2010; 24: 349-358Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar Data suggest that oxidative damage links inflammation and pancreatic carcinogenesis, but this link and the role of reactive oxygen species (ROS) are still only partially known.3Jackson L. Evers B.M. Chronic inflammation and pathogenesis of GI and pancreatic cancers.Cancer Treat Res. 2006; 130: 39-65Crossref PubMed Scopus (68) Google Scholar, 4Farrow B. Evers B.M. Inflammation and the development of pancreatic cancer.Surg Oncol. 2002; 10: 153-169Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 5DeNicola G.M. Karreth F.A. Humpton T.J. Gopinathan A. Wei C. Frese K. Mangal D. Yu K.H. Yeo C.J. Calhoun E.S. Scrimieri F. Winter J.M. Hruban R.H. Iacobuzio-Donahue C. Kern S.E. Blair I.A. Tuveson D.A. Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.Nature. 2011; 475: 106-109Crossref PubMed Scopus (1539) Google Scholar Tumor protein p53-induced nuclear protein 1 (TP53INP1) is a key stress-response protein highly expressed during pancreatitis.6Tomasini R. Samir A.A. Vaccaro M.I. Pebusque M.J. Dagorn J.C. Iovanna J.L. Dusetti N.J. Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death.J Biol Chem. 2001; 276: 44185-44192Crossref PubMed Scopus (76) Google Scholar Recent data7N’Guessan P. Pouyet L. Gosset G. Hamlaoui S. Seillier M. Cano C.E. Seux M. Stocker P. Culcasi M. Iovanna J.L. Dusetti N.J. Pietri S. Carrier A. Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.Antioxid Redox Signal. 2011; 15: 1639-1653Crossref PubMed Scopus (26) Google Scholar, 8Cano C.E. Gommeaux J. Pietri S. Culcasi M. Garcia S. Seux M. Barelier S. Vasseur S. Spoto R.P. Pebusque M.J. Dusetti N.J. Iovanna J.L. Carrier A. Tumor protein 53-induced nuclear protein 1 is a major mediator of p53 antioxidant function.Cancer Res. 2009; 69: 219-226Crossref PubMed Scopus (122) Google Scholar, 9Gommeaux J. Cano C. Garcia S. Gironella M. Pietri S. Culcasi M. Pebusque M.J. Malissen B. Dusetti N. Iovanna J. Carrier A. Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1.Mol Cell Biol. 2007; 27: 2215-2228Crossref PubMed Scopus (69) Google Scholar demonstrated the role of TP53INP1 in the control of oxidative status in vivo. Indeed, the level of ROS is increased in TP53INP1 knockout mice and their antioxidant defenses reduced, thus showing a chronic oxidative stress in correlation with an increased susceptibility to develop colitis and colitis-associated cancer.7N’Guessan P. Pouyet L. Gosset G. Hamlaoui S. Seillier M. Cano C.E. Seux M. Stocker P. Culcasi M. Iovanna J.L. Dusetti N.J. Pietri S. Carrier A. Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.Antioxid Redox Signal. 2011; 15: 1639-1653Crossref PubMed Scopus (26) Google Scholar, 8Cano C.E. Gommeaux J. Pietri S. Culcasi M. Garcia S. Seux M. Barelier S. Vasseur S. Spoto R.P. Pebusque M.J. Dusetti N.J. Iovanna J.L. Carrier A. Tumor protein 53-induced nuclear protein 1 is a major mediator of p53 antioxidant function.Cancer Res. 2009; 69: 219-226Crossref PubMed Scopus (122) Google Scholar, 9Gommeaux J. Cano C. Garcia S. Gironella M. Pietri S. Culcasi M. Pebusque M.J. Malissen B. Dusetti N. Iovanna J. Carrier A. Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1.Mol Cell Biol. 2007; 27: 2215-2228Crossref PubMed Scopus (69) Google Scholar TP53INP1 protein expression is lost during pancreatic cancer progression as early as pancreatic intraepithelial neoplasia 2 (PanIN2).10Gironella M. Seux M. Xie M.J. Cano C. Tomasini R. Gommeaux J. Garcia S. Nowak J. Yeung M.L. Jeang K.T. Chaix A. Fazli L. Motoo Y. Wang Q. Rocchi P. Russo A. Gleave M. Dagorn J.C. Iovanna J.L. Carrier A. Pebusque M.J. Dusetti N.J. Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development.Proc Natl Acad Sci U S A. 2007; 104: 16170-16175Crossref PubMed Scopus (481) Google Scholar Mice deficient for TP53INP1 did not reveal pancreatic abnormalities,9Gommeaux J. Cano C. Garcia S. Gironella M. Pietri S. Culcasi M. Pebusque M.J. Malissen B. Dusetti N. Iovanna J. Carrier A. Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1.Mol Cell Biol. 2007; 27: 2215-2228Crossref PubMed Scopus (69) Google Scholar therefore, we examined in this study the consequences of homozygous deletion of TP53INP1 and concomitant activation of the KrasG12D mutation on initiation of PDA. We show that TP53INP1 deficiency accelerates Kras-induced PanIN and cooperates with KrasG12D mutation to induce cystic lesions. We also show that an antioxidant treatment rescues this phenotype. The LSL-KrasG12D/+ mice were from the Mouse Models of Human Cancers Consortium Repository (National Cancer Institute, Frederick, MD) and the Pdx1-Cre+/− mice from the DA Melton Laboratory (Cambridge, MA).11Gu G. Dubauskaite J. Melton D.A. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.Development. 2002; 129: 2447-2457Crossref PubMed Google Scholar The TP53INP1−/− mice were previously described.7N’Guessan P. Pouyet L. Gosset G. Hamlaoui S. Seillier M. Cano C.E. Seux M. Stocker P. Culcasi M. Iovanna J.L. Dusetti N.J. Pietri S. Carrier A. Absence of tumor suppressor tumor protein 53-induced nuclear protein 1 (TP53INP1) sensitizes mouse thymocytes and embryonic fibroblasts to redox-driven apoptosis.Antioxid Redox Signal. 2011; 15: 1639-1653Crossref PubMed Scopus (26) Google Scholar, 9Gommeaux J. Cano C. Garcia S. Gironella M. Pietri S. Culcasi M. Pebusque M.J. Malissen B. Dusetti N. Iovanna J. Carrier A. Colitis and colitis-associated cancer are exacerbated in mice deficient for tumor protein 53-induced nuclear protein 1.Mol Cell Biol. 2007; 27: 2215-2228Crossref PubMed Scopus (69) Google Scholar LSL-KrasG12D/+ and Pdx1-Cre+/− mice were bred to generate LSL-KrasG12D/+;Pdx1-Cre+/− mice (Kras mice) and control wild-type mice.12Hingorani S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1800) Google Scholar LSL-KrasG12D/+;Pdx1-Cre+/−;TP53INP1−/− mice (Kras-INP1KO mice) were generated by interbreeding Kras mice with TP53INP1−/− mice. Control mice were Pdx1-Cre+/−;TP53INP1−/− or LSL-KrasG12D/+;TP53INP1−/− littermates (INP1KO mice). All procedures were approved by the animal care committee of the animal facility of INSERM-US006. Pancreases were fixed in 10% neutral buffered formalin and embedded in paraffin. For histopathological analysis, pancreases were serially sectioned (4 μm) and every 10 sections stained with H&E. Histopathological scoring of pancreatic lesions was performed by two pathologists (J.S. and T.A.S.) using serial H&E-stained sections (50 μm apart, six sections per pancreas). For quantification of acinoductal metaplasia (ADM)/PanIN/cystic lesions, one representative slide per mouse was imaged using a Hamamatsu NanoZoomer 2 slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan), and lesions were counted and measured on the entire section with the NanoZoomer Digital Pathology view software version 1.2.46.0 (Hamamatsu Photonics). Antibodies used for immunohistochemical studies were the following: anti-cytokeratin19 [rat monoclonal Troma III, developed by Rolf Kemler (Max-Planck Institute, Freiburg, Germany)] obtained from the Developmental Study Hybridoma Bank developed under the auspices of the National Institute of Child Health & Human Development and maintained by the University of Iowa, Department of Biology (Iowa City, IA), anti–α-smooth muscle actin (rabbit polyclonal, 1:100; Thermo Fischer Scientific, Kalamazoo, MI), anti–nuclear factor erythroid 2-related factor 2 (Nrf2) (C-20) (rabbit polyclonal, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), anti-active Rac1 (mouse monoclonal, 1:1000; NewEast Biosciences, King of Prussia, PA), anti–4-hydroxynonenal (HNE) (rabbit polyclonal, 1:250; Alpha Diagnostic International, San Antonio, TX). A25-E12 mouse monoclonal anti-TP53INP1 antibody was previously described.10Gironella M. Seux M. Xie M.J. Cano C. Tomasini R. Gommeaux J. Garcia S. Nowak J. Yeung M.L. Jeang K.T. Chaix A. Fazli L. Motoo Y. Wang Q. Rocchi P. Russo A. Gleave M. Dagorn J.C. Iovanna J.L. Carrier A. Pebusque M.J. Dusetti N.J. Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development.Proc Natl Acad Sci U S A. 2007; 104: 16170-16175Crossref PubMed Scopus (481) Google Scholar To ensure specific staining, control slides were stained without primary antibody. For Nrf2, we also preincubated the antibody with an excess of immunogenic peptide (sc-722 P; Santa Cruz Biotechnology). Human tissue samples containing intraductal papillary mucinous neoplastic (IPMN) lesions were provided by the Pathology Department of Beaujon Hospital (Clichy, France). A total of 46 paraffin-embedded IMPN were included in tissue microarray blocks. TP53INP1 expression was scored by combining an estimate of the percentage of immunoreactive cells (0 to 100) with an estimate of the staining intensity (0 = no staining; 1 = low; 2 = moderate; and 3 = high levels of staining) to obtain a score range between 0 and 300. N-acetylcysteine (NAC; Sigma-Aldrich, St. Louis, MO) (40 mmol/L) in drinking water was initiated during embryogenesis via treatment of parent mice and maintained throughout life.13Laurent G. Solari F. Mateescu B. Karaca M. Castel J. Bourachot B. Magnan C. Billaud M. Mechta-Grigoriou F. Oxidative stress contributes to aging by enhancing pancreatic angiogenesis and insulin signaling.Cell Metab. 2008; 7: 113-124Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Primary mouse embryonic fibroblasts from TP53INP1−/− embryos were prepared, cultured, and transformed by transduction as previously described.10Gironella M. Seux M. Xie M.J. Cano C. Tomasini R. Gommeaux J. Garcia S. Nowak J. Yeung M.L. Jeang K.T. Chaix A. Fazli L. Motoo Y. Wang Q. Rocchi P. Russo A. Gleave M. Dagorn J.C. Iovanna J.L. Carrier A. Pebusque M.J. Dusetti N.J. Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development.Proc Natl Acad Sci U S A. 2007; 104: 16170-16175Crossref PubMed Scopus (481) Google Scholar Mouse embryonic fibroblasts were resuspended in lysis buffer (50 mmol/L Hepes, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 10% glycerol, 1% triton X-100, 25 mmol/L NaF, 10 μmol/L ZnCl2). Fifty microgram of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Antibodies used for immunoblotting were anti-Nrf2 (sc-722; Santa Cruz Biotechnology), anti-TP53INP1 (rat monoclonal antibody generated in our laboratory, clone F8), and anti–β-tubulin (T4026-1; Sigma-Aldrich) as loading control. Proteins were detected using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA). Histopathological analysis of pancreas of mice euthanized at different time points showed that progression of more advanced mPanIN was significantly accelerated in the pancreas of Kras-INP1KO mice compared with KrasG12D/+;Pdx1-Cre+/− (Kras mice). We found mPanIN1a in 61%, mPanIN1b in 54%, and mPanIN2 in 7.7% of examined pancreases of young Kras-INP1KO mice. Interestingly, mPanIN2 were not observed in age-matched Kras animals (not shown and12Hingorani S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1800) Google Scholar). Remarkably, before 1 month of age, 50% of examined Kras-INP1KO displayed mPanIN1a (n = 4), which was only occasionally found in age-matched Kras animals (not shown and14Aguirre A.J. Bardeesy N. Sinha M. Lopez L. Tuveson D.A. Horner J. Redston M.S. DePinho R.A. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma.Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (803) Google Scholar). We detected mPanIN2 lesions in one animal age 17 days (Figure 1B). As the animals aged, there was an increase in the level of dysplasia and the frequency of mPanIN3 (Figure 1A). The total number of ductal lesions and their grades were scored in representative pancreas sections of cohorts of mice of 6 to 10 months of age (Figure 1C). The incidence of normal ducts relative to the total number of ducts (including ductal lesions) significantly decreased by twofold in Kras-INP1KO mice compared to Kras mice (P = 0.0011). Although approximately 50% of ducts were mPanIN1a in both strains, the incidence of mPanIN1b, mPanIN2, and mPanIN3 significantly increased in Kras-INP1KO mice compared to Kras mice (threefold, P = 0.004, 10-fold, P = 0.005, and sevenfold, P = 0.01, respectively) and represented 18.6%, 3%, and 2% of ducts, respectively. Taken together, these data suggest that TP53INP1 deficiency accelerates PanIN initiation and progression. Cystic lesions resembling IPMN occurred in the pancreas of 45.7% of analyzed Kras-INP1KO mice (n = 35). Cysts were multilocular (Figure 2A) and positive for cytokeratin19 (Figure 2B), and the presence of abundant quantities of apical mucins distinguished them from retention cysts (Figure 2C). They were observed as early as 2 months of age (23% of mice), suggesting that they probably develop during the early postnatal period; by contrast, age-matched Kras mice had no cystic lesions (Figure 2D). Examination of older mice (ages 3 to 6 months and 6 to 10 months) revealed a gradual increase in the frequency of these cystic lesions to 45% and 73% of examined pancreas, respectively, whereas the frequencies were 7% and 13% at these ages in Kras mice. The size of the cystic lesions was variable but unrelated to the strain or age. Very large cysts (>1 mm) were already observed at 2 months of age in Kras-INP1KO mice. However, the number of lesions was significantly increased in Kras-INP1KO mice, resulting in replacement of larger pancreatic areas than in Kras mice (Figure 2E). Positive expression of TP53INP1 was previously reported in IPMN with low dysplasia but was never explored in samples with higher dysplasia.10Gironella M. Seux M. Xie M.J. Cano C. Tomasini R. Gommeaux J. Garcia S. Nowak J. Yeung M.L. Jeang K.T. Chaix A. Fazli L. Motoo Y. Wang Q. Rocchi P. Russo A. Gleave M. Dagorn J.C. Iovanna J.L. Carrier A. Pebusque M.J. Dusetti N.J. Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development.Proc Natl Acad Sci U S A. 2007; 104: 16170-16175Crossref PubMed Scopus (481) Google Scholar We found TP53INP1 in the cytoplasm of acinar and ductal cells of human pancreas (Figure 2F). In IPMN, TP53INP1 staining was significantly lower in samples of moderate- and high-grade dysplasia than in samples of lower-grade dysplasia (Figure 2, G–I). Although the TP53INP1 staining score was high in 14% and 31% of IPMN of moderate and high grade, respectively, the proportion of individuals that did not express TP53INP1 increased with the degree of dysplasia (Figure 2J). Histological changes reflecting pancreatic injury were frequent in Kras-INP1KO mice with development of extensive fibrosis, infiltration of inflammatory cells, and acinar atrophy (Figure 3A and Figure 4G). Expansion of a reactive stroma strongly positive for α-smooth muscle actin was associated with several distinct metaplastic ductal lesions mirroring human chronic pancreatitis (Figure 3, B–D). ADM was detected at 2 weeks, and partial or complete ADM of several pancreatic lobules was observed in the majority of Kras-INP1KO mice (Figure 3E). Numerous ADM/mPanIN1a hybrid structures containing cells that coexpress amylase and CK19 were found (Figure 3F). Foci of cellular dysplasia suggestive of an in situ malignant behavior were also frequent in the pancreas of Kras-INP1KO mice (Figure 3, G and H). The area occupied by the abnormal pancreas represented 5% of the pancreas by the age of 4 to 5 months in Kras-INP1KO mice and reached 30% to 75% by 6 to 10 months, indicating that TP53INP1 deficiency is associated with an increased proliferation of both the preneoplastic epithelium and the stromal tissue.Figure 4Oxidative stress increases in the pancreas of Kras-INP1KO mice. A: Immunostaining of Kras mice pancreas with anti-active Rac1 antibody. Boxed area shows activity of Rac1 in the cytoplasm of acini (arrowhead) and of hybrid ADM/mPanIN1a (arrows), specificity of active Rac1 immunostaining was verified as indicated in Materials and Methods (not shown). B: Activity of Rac1 in the pancreas of Kras-INP1KO mice. Boxed area shows intense labeling of acini (arrowhead), tubular complexes (asterisks), and heterogeneous activity in mPanIN (arrows). C: Nrf2 levels in Kras mice pancreas. Boxed area shows basolateral expression of Nrf2 in acinar cells and nuclear immunostaining of mPanIN (arrows). Islets (i) are negative, and specificity of Nrf2 immunostaining was verified as indicated in Materials and Methods (not shown). D: Nrf2 levels in Kras-INP1KO mice pancreas. Boxed area shows high levels of Nrf2 in the nuclei of most of the cells. E: TP53INP1−/−–transformed mouse embryonic fibroblasts were transduced with empty murine stem cell virus (MSCV-neo), MSCV-TP53INP1α, or MSCV-TP53INP1β retroviral vectors. Nrf2 protein level was analyzed by Western blot in total cell lysates from TP53INP1−/−–transformed mouse embryonic fibroblasts, re-expressing one or the other isoform of TP53INP1 (upper panel). Re-expression of TP53INP1 is shown by immunoblotting using anti-TP53INP1 antibodies (lower panel). F: Representative histology of Kras-INP1KO pancreas without NAC treatment (10 months of age). G: NAC-treated Kras-INP1KO mice pancreas at 10 months of age. H: Proportion of untreated (NAC−) (n = 6) and treated (NAC+) (n = 6) 10-month-old Kras-INP1KO mice with pancreatic mPanIN, cystic lesions (CL), and ADM. I: Quantitative analysis of mPanIN lesions. Percentage of pancreatic ducts with no pathology (normal), mPanIN1a, mPanIN1b, mPanIN2, and mPanIN3 in NAC− (n = 6) and NAC+ Kras-INP1KO mice (n = 6) at 10 months of age. ∗P > 0.01 and < 0.05, ∗∗P > 0.001 and < 0.01. J: Nrf2 levels in NAC-treated Kras-INP1KO mice pancreas. Boxed area shows basolateral expression of Nrf2 in acinar cells and nuclear immunostaining of mPanIN (arrows).View Large Image Figure ViewerDownload Hi-res image Download (PPT) On the basis of the known antioxidant function of TP53INP1, we reasoned that oxidative stress could play a major role in the pancreatic phenotype of Kras-INP1KO mice and investigated the activation state of Rac1, a known mediator of ROS production via NADPH oxidase, by performing immunostaining with an antibody against active Rac1. We observed a more intense staining in Kras-INP1KO pancreas than in Kras mice (Figure 4, A and B). In both strains, staining of acinar cells was cytoplasmic and apical, ducts were negative. Activity of Rac1 was heterogeneous in mPanIN and IPMN cells. ADM and hybrid ADM/mPanIN1a were positive. Similar staining patterns were observed with an antibody against HNE, an aldehydic lipid peroxidation product that is a good marker for oxidative stress. HNE was not detected in the exocrine pancreas of LSL-KrasG12D/+ and INP1KO control littermates (Supplemental Figure S1, A and B). Staining was heterogeneous in mPanIN in both Kras and Kras-INP1KO pancreas (Supplemental Figure S1, C and D). Interestingly HNE was detected in acinar cells, ADM, and hybrid ADM/mPanIN1a of Kras-INP1KO mice (Supplemental Figure S1, D–F). Because the Nrf2 antioxidant program was shown to promote KrasG12D pancreatic carcinogenesis, we examined whether expression of Nrf2 was modulated by TP53INP1 deficiency. Normal ducts (not shown) and islets did not express detectable levels of Nrf2 in Kras mice pancreas, whereas Nrf2 expression was cytoplasmic, basolaterally found in acini, and nuclear in mPanIN cells; most stromal cells were negative (Figure 4C). Intense Nrf2 signal was found in Kras-INP1KO mice pancreas in the nucleus of most cells, including acini (Figure 4D). Western blot analysis further demonstrated that Nrf2 levels are increased in TP53INP1-deficient cells (Figure 4E). Finally, we examined the impact of prolonged treatment with NAC, a well-known antioxidant. Because loss of TP53INP1 accelerates Kras-induced development of mPanIN, NAC treatment was initiated early during Kras-INP1KO mouse embryogenesis. After 10 months and consistent with the data reported above, histological analysis of untreated Kras-INP1KO (n = 6) shows large areas of pancreas replaced by ADM, mPanIN, and cystic lesions with abundant surrounding stroma (Figure 4F), whereas antioxidant treatment strongly reduced the incidence of mPanIN and cystic lesions (Figure 4, G and H). mPanIN1a represented 22.4 ± 10.1% of ducts (2.6-fold reduction compared to untreated, P = 0.007), mPanIN1b represented 1.7 ± 1.6% of ducts (11.2-fold reduction, P = 0.004); no mPanIN2 and mPanIN3 were found (Figure 4I). One cystic lesion <1 mm in size was found in one treated mouse (Figure 4H). Focal ADM represented 0.3 ± 0.2% of total area in 50% of treated mice, whereas it was 7.5 ± 2.7% of the pancreas in all untreated mice (P = 0.013). The area occupied by the abnormal pancreas represented <1% of the pancreas in treated mice (n = 6). Interestingly, Nrf2 immunostaining performed in treated Kras-INP1KO mice (Figure 4J) showed decreased Nrf2 levels compared to untreated Kras-INP1KO mice (Figure 4D). The immunostaining pattern was similar to the one in untreated Kras mice, with Nrf2 expressed in the cytoplasm of acini and in the nuclei of mPanIN cells (Figure 4C). Taken together, these results demonstrate that oxidative stress mediates pancreatic damage in Kras-INP1KO mice. Results of our in vivo study provide clear genetic evidence for a role of TP53INP1 in initiation steps and progression of pancreatic cancer. We show that the deficiency of TP53INP1 in the context of an activated KrasG12D promotes the formation of PanIN in young mice, indicating that TP53INP1 may impede transformation of embryonic pancreatic cells by oncogenic Kras. Moreover, both accelerated development of PanIN and increased incidence of high-grade lesions demonstrate the functional impact of TP53INP1 deletion on pancreatic cancer evolution, with a higher susceptibility to develop preneoplastic lesions. Our results also indicate that TP53INP1 can restrain the initiation of cystic lesions in the context of KrasG12D heterozygosity in mice, thus establishing a novel genotype–phenotype relationship underlying the formation of these neoplasms.15Delpu Y. Hanoun N. Lulka H. Sicard F. Selves J. Buscail L. Torrisani J. Cordelier P. Genetic and epigenetic alterations in pancreatic carcinogenesis.Curr Genomics. 2011; 12: 15-24Crossref PubMed Scopus (85) Google Scholar IPMN do not develop in young Kras mice with or without loss of a tumor suppressor such as tumor protein 53 (Tp53), p16Ink4a, or p19Arf,12Hingorani S.R. Petricoin E.F. Maitra A. Rajapakse V. King C. Jacobetz M.A. Ross S. Conrads T.P. Veenstra T.D. Hitt B.A. Kawaguchi Y. Johann D. Liotta L.A. Crawford H.C. Putt M.E. Jacks T. Wright C.V. Hruban R.H. Lowy A.M. Tuveson D.A. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1800) Google Scholar, 14Aguirre A.J. Bardeesy N. Sinha M. Lopez L. Tuveson D.A. Horner J. Redston M.S. DePinho R.A. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma.Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (803) Google Scholar, 16Bardeesy N. Aguirre A.J. Chu G.C. Cheng K.H. Lopez L.V. Hezel A.F. Feng B. Brennan C. Weissleder R. Mahmood U. Hanahan D. Redston M.S. Chin L. Depinho R.A. Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse.Proc Natl Acad Sci U S A. 2006; 103: 5947-5952Crossref PubMed Scopus (456) Google Scholar, 17Hingorani S.R. Wang L. Multani A.S. Combs C. Deramaudt T.B. Hruban R.H. Rustgi A.K. Chang S. Tuveson D.A. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar but are induced by loss of SMAD4 or transcriptional intermediary factor 1 gamma (TIF1γ) or overexpression of transforming growth factor alpha (TGFα).18Bardeesy N. Cheng K.H. Berger J.H. Chu G.C. Pahler J. Olson P. Hezel A.F. Horner J. Lauwers G.Y. Hanahan D. DePinho R.A. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer.Genes Dev. 2006; 20: 3130-3146Crossref PubMed Scopus (512) Google Scholar, 19Kojima K. Vickers S.M." @default.
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