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• W2769455020 abstract "•IKKα-deficiency-induced p62 accumulation drives PDAC progression•MDM2 links the autophagy substrate p62 to neoplastic progression in the pancreas•A p62-NRF2-MDM2 module acts via p53-dependent and -independent mechanisms•A p62-NRF2-MDM2 module converts acinar cells into progenitor-like cells Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals. Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals. PDAC is a highly aggressive malignancy that is refractory to most therapeutic interventions. Prevention and early treatment are likely to be more successful than treatment of established, metastatic tumors. Therefore, we studied how precursor PanIN lesions become fully malignant PDAC. We describe a p62-NRF2-MDM2 signaling pathway that controls malignancy in human and mouse PDAC. Inhibition of this pathway with MDM2-targeting drugs attenuates neoplastic progression and maintains acinar cellularity in stressed pancreata. Despite being a leading cause of cancer-related deaths, pancreatic ductal adenocarcinoma (PDAC) is relatively rare, with a worldwide incidence of 4.1 per 100,000 (Bray et al., 2013Bray F. Ren J.S. Masuyer E. Ferlay J. Global estimates of cancer prevalence for 27 sites in the adult population in 2008.Int. J. Cancer. 2013; 132: 1133-1145Crossref PubMed Scopus (1410) Google Scholar). However, pancreatic intraepithelial neoplasia 1 (PanIN1), a premalignant precursor lesion, is extremely common, found in 16% of healthy controls and 60% of chronic pancreatitis patients (Hruban et al., 2008Hruban R.H. Maitra A. Goggins M. Update on pancreatic intraepithelial neoplasia.Int. J. Clin. Exp. Pathol. 2008; 1: 306-316PubMed Google Scholar). Although most PanIN1 contain KRAS oncogenic mutations, only 1% of them ever progress to PDAC (Collins and Pasca di Magliano, 2013Collins M.A. Pasca di Magliano M. Kras as a key oncogene and therapeutic target in pancreatic cancer.Front. Physiol. 2013; 4: 407PubMed Google Scholar, Hruban et al., 2008Hruban R.H. Maitra A. Goggins M. Update on pancreatic intraepithelial neoplasia.Int. J. Clin. Exp. Pathol. 2008; 1: 306-316PubMed Google Scholar). Nonetheless, several risk factors greatly increase the likelihood that PanIN1 lesions will progress to PDAC, including having first-degree relatives with PDAC and chronic or cryptogenic pancreatitis (Becker et al., 2014Becker A.E. Hernandez Y.G. Frucht H. Lucas A.L. Pancreatic ductal adenocarcinoma: risk factors, screening, and early detection.World J. Gastroenterol. 2014; 20: 11182-11198Crossref PubMed Scopus (188) Google Scholar, Levy et al., 2014Levy P. Dominguez-Munoz E. Imrie C. Lohr M. Maisonneuve P. Epidemiology of chronic pancreatitis: burden of the disease and consequences.United European Gastroenterol. J. 2014; 2: 345-354Crossref PubMed Scopus (131) Google Scholar). Obesity, smoking, and alcohol consumption also increase PDAC risk. Thus, early PDAC screening may be economically justified in high-risk individuals and, together with effective chemoprevention, may reduce the enormous death toll associated with the disease. Such efforts, however, require improved understanding of the mechanisms that control PanIN1 to PDAC progression. Obesity, hypernutrition, alcohol consumption, tobacco smoking, and chronic pancreatitis have all been linked to impaired autophagic-lysosomal protein degradation in differentiated acinar cells, which specialize in production and secretion of digestive enzymes (Gukovsky et al., 2013Gukovsky I. Li N. Todoric J. Gukovskaya A. Karin M. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer.Gastroenterology. 2013; 144: 1199-1209.e4Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). In mice that conditionally express oncogenic Kras alleles in pancreatic epithelial cells (PECs), PanIN1 to PDAC progression, which is very inefficient, is strongly accelerated by cerulein, a pancreatic enzyme secretagogue that induces acinar cell damage and acute pancreatitis (Carriere et al., 2009Carriere C. Young A.L. Gunn J.R. Longnecker D.S. Korc M. Acute pancreatitis markedly accelerates pancreatic cancer progression in mice expressing oncogenic Kras.Biochem. Biophys. Res. Commun. 2009; 382: 561-565Crossref PubMed Scopus (161) Google Scholar, Guerra et al., 2011Guerra C. Collado M. Navas C. Schuhmacher A.J. Hernandez-Porras I. Canamero M. Rodriguez-Justo M. Serrano M. Barbacid M. Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence.Cancer Cell. 2011; 19: 728-739Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Cerulein also interferes with autophagy-dependent proteolysis (Mareninova et al., 2009Mareninova O.A. Hermann K. French S.W. O'Konski M.S. Pandol S.J. Webster P. Erickson A.H. Katunuma N. Gorelick F.S. Gukovsky I. et al.Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis.J. Clin. Invest. 2009; 119: 3340-3355PubMed Google Scholar), a process that is downregulated in human pancreatitis (Gukovsky et al., 2013Gukovsky I. Li N. Todoric J. Gukovskaya A. Karin M. Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer.Gastroenterology. 2013; 144: 1199-1209.e4Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). We postulated that insufficient autophagy, needed for protection of acinar cells from ER stress, to which they are highly susceptible (Antonucci et al., 2015Antonucci L. Fagman J.B. Kim J.Y. Todoric J. Gukovsky I. Mackey M. Ellisman M.H. Karin M. Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress.Proc. Natl. Acad. Sci. USA. 2015; 112: E6166-E6174Crossref PubMed Scopus (153) Google Scholar), could be responsible for enhancing PanIN1 to PDAC progression. Impaired autophagic degradation causes buildup of autophagy substrates, such as p62/SQSTM1, whose accumulation has been detected in mouse and human pancreatitis (Li et al., 2013Li N. Wu X. Holzer R.G. Lee J.H. Todoric J. Park E.J. Ogata H. Gukovskaya A.S. Gukovsky I. Pizzo D.P. et al.Loss of acinar cell IKKalpha triggers spontaneous pancreatitis in mice.J. Clin. Invest. 2013; 123: 2231-2243Crossref PubMed Scopus (91) Google Scholar). p62 aggregates are a common sign of chronic liver diseases that promote hepatocellular carcinoma (HCC) development (Denk et al., 2006Denk H. Stumptner C. Fuchsbichler A. Muller T. Farr G. Muller W. Terracciano L. Zatloukal K. Are the Mallory bodies and intracellular hyaline bodies in neoplastic and non-neoplastic hepatocytes related?.J. Pathol. 2006; 208: 653-661Crossref PubMed Scopus (54) Google Scholar). Recent studies have identified p62 as a key driver in HCC, whose high expression in non-tumor liver tissue predicts rapid recurrence after curative ablation (Umemura et al., 2016Umemura A. He F. Taniguchi K. Nakagawa H. Yamachika S. Font-Burgada J. Zhong Z. Subramaniam S. Raghunandan S. Duran A. et al.p62, upregulated during preneoplasia, induces hepatocellular carcinogenesis by maintaining survival of stressed HCC-initiating cells.Cancer Cell. 2016; 29: 935-948Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). In addition to being an autophagy receptor that recognizes poly-ubiquitinated proteins and organelles, p62 is a signaling adaptor that promotes activation of nuclear factor κB and NRF2 transcription factors (Komatsu and Ichimura, 2010Komatsu M. Ichimura Y. Physiological significance of selective degradation of p62 by autophagy.FEBS Lett. 2010; 584: 1374-1378Crossref PubMed Scopus (415) Google Scholar, Moscat and Diaz-Meco, 2009Moscat J. Diaz-Meco M.T. p62 at the crossroads of autophagy, apoptosis, and cancer.Cell. 2009; 137: 1001-1004Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar, Moscat et al., 2016Moscat J. Karin M. Diaz-Meco M.T. p62 in cancer: signaling adaptor beyond autophagy.Cell. 2016; 167: 606-609Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Given that NRF2 was shown to promote PanIN1 formation and proliferation in mice (DeNicola et al., 2011DeNicola 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. et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.Nature. 2011; 475: 106-109Crossref PubMed Scopus (1526) Google Scholar), we postulated that impaired acinar autophagy may stimulate neoplastic progression in the pancreas via a p62-NRF2 cascade. We therefore sought to determine how NRF2, which controls expression of enzymes that detoxify reactive oxygen species (ROS), overcomes the quiescent state of early PanINs. Of note, oncogene-induced senescence, which was suggested to be linked to ROS accumulation in K-Ras-transformed acinar cells (DeNicola et al., 2011DeNicola 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. et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.Nature. 2011; 475: 106-109Crossref PubMed Scopus (1526) Google Scholar), depends on activation of tumor suppressor p53 (Courtois-Cox et al., 2008Courtois-Cox S. Jones S.L. Cichowski K. Many roads lead to oncogene-induced senescence.Oncogene. 2008; 27: 2801-2809Crossref PubMed Scopus (289) Google Scholar), which controls transcription of cell-cycle inhibitors and apoptosis inducers. p53 also inhibits cellular reprogramming thereby preventing acquisition of stemness (Kawamura et al., 2009Kawamura T. Suzuki J. Wang Y.V. Menendez S. Morera L.B. Raya A. Wahl G.M. Izpisua Belmonte J.C. Linking the p53 tumour suppressor pathway to somatic cell reprogramming.Nature. 2009; 460: 1140-1144Crossref PubMed Scopus (892) Google Scholar, Marion et al., 2009Marion R.M. Strati K. Li H. Murga M. Blanco R. Ortega S. Fernandez-Capetillo O. Serrano M. Blasco M.A. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.Nature. 2009; 460: 1149-1153Crossref PubMed Scopus (842) Google Scholar), and is functionally inactivated in >80% of human PDAC (Waddell et al., 2015Waddell N. Pajic M. Patch A.M. Chang D.K. Kassahn K.S. Bailey P. Johns A.L. Miller D. Nones K. Quek K. et al.Whole genomes redefine the mutational landscape of pancreatic cancer.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1688) Google Scholar). Complete inhibition of autophagy accelerates PanIN1 progression to more proliferative PanIN2/3 lesions but blocks further malignant progression by inducing p53 accumulation (Rosenfeldt et al., 2013Rosenfeldt M.T. O'Prey J. Morton J.P. Nixon C. MacKay G. Mrowinska A. Au A. Rai T.S. Zheng L. Ridgway R. et al.p53 status determines the role of autophagy in pancreatic tumour development.Nature. 2013; 504: 296-300Crossref PubMed Scopus (521) Google Scholar). Here, we investigate how the p62-NRF2 cascade accelerates development of stress-induced PDAC and helps maintain the malignant phenotype. Immunohistochemistry revealed much more p62 in advanced PanIN2/3 lesions and PDAC epithelial cells than in normal or chronically inflamed pancreata (Figures 1A and S1A). p62 did not accumulate in peritumoral stroma. SQSTM1 gene transcription is stimulated by NRF2 (Komatsu and Ichimura, 2010Komatsu M. Ichimura Y. Physiological significance of selective degradation of p62 by autophagy.FEBS Lett. 2010; 584: 1374-1378Crossref PubMed Scopus (415) Google Scholar), a transcription factor proposed to protect K-Ras-transformed cells from ROS-induced senescence (DeNicola et al., 2011DeNicola 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. et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis.Nature. 2011; 475: 106-109Crossref PubMed Scopus (1526) Google Scholar). In turn, p62 sequesters Keap1, which recruits the CUL3 E3 ligase to NRF2 to promote its degradation, thereby increasing NRF2 abundance (Komatsu and Ichimura, 2010Komatsu M. Ichimura Y. Physiological significance of selective degradation of p62 by autophagy.FEBS Lett. 2010; 584: 1374-1378Crossref PubMed Scopus (415) Google Scholar). Congruently, expression of NRF2 and its target NQO1 paralleled p62 in PDAC specimens (Figures 1A and S1A). PanIN2/3 lesions and PDAC also exhibited elevated nuclear HES1, indicating active Notch signaling, a feature not obvious in chronic pancreatitis (Figure 1A). Expression of MDM2, which is mainly known as a negative regulator of p53 but has numerous other functions including Notch signaling activation (Fahraeus and Olivares-Illana, 2014Fahraeus R. Olivares-Illana V. MDM2's social network.Oncogene. 2014; 33: 4365-4376Crossref PubMed Scopus (71) Google Scholar, Pettersson et al., 2013Pettersson S. Sczaniecka M. McLaren L. Russell F. Gladstone K. Hupp T. Wallace M. Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway.Biochem. J. 2013; 450: 523-536Crossref PubMed Scopus (38) Google Scholar), was also upregulated in human pancreatitis and PDAC (Figures 1A and S1A). MDM2 expression correlated with p62, NRF2, and NQO1 (R = 0.359, p < 0.001; R = 0.559, p < 0.001; and R = 0.403 and p < 0.001, respectively), suggesting a link between MDM2 and the p62-NRF2 axis. p62 expression in PDAC also overlapped that of Sox9 (Figure S1B), a transcriptional factor that controls acinar-to-ductal reprogramming and PDAC initiation (Kopp et al., 2012Kopp J.L. von Figura G. Mayes E. Liu F.F. Dubois C.L. Morris J.P.t. Pan F.C. Akiyama H. Wright C.V. Jensen K. et al.Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.Cancer Cell. 2012; 22: 737-750Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar). In mouse pancreatitis, p62 accumulation is promoted by PEC-specific IKKα ablation (Li et al., 2013Li N. Wu X. Holzer R.G. Lee J.H. Todoric J. Park E.J. Ogata H. Gukovskaya A.S. Gukovsky I. Pizzo D.P. et al.Loss of acinar cell IKKalpha triggers spontaneous pancreatitis in mice.J. Clin. Invest. 2013; 123: 2231-2243Crossref PubMed Scopus (91) Google Scholar). In concordance with these findings, decreased IKKα expression was observed in human PDAC (Figure 1A), and it statistically correlated with elevated p62 (R = −0.419, p < 0.01; Figure S1A). p62 was expressed in human MIA PaCa-2 PDAC cells and its small hairpin RNA (shRNA)-mediated silencing reduced NRF2, HES1, progenitor (SOX9), ductal (CK19), and proliferative (Ki67) markers and diminished tumorigenic growth in immunocompromised mice (Figures 1B and 1C). Accordingly, p62 ablation in MIA PaCa-2 and Capan-2 human PDAC cells (Figure 1D) decreased KRT19, SOX9, NES, PDX1, PROX1, EPCAM, CD24, CD44, PROM1, and ALDH1 mRNAs, the last five of which mark PDAC stem cells (Matsuda et al., 2012Matsuda Y. Kure S. Ishiwata T. Nestin and other putative cancer stem cell markers in pancreatic cancer.Med. Mol. Morphol. 2012; 45: 59-65Crossref PubMed Scopus (51) Google Scholar), along with NRF2-regulated (GSTM1 and NQO1) genes and Notch pathway components (NOTCH1, NOTCH2, NOTCH3, NOTCH4, and HES1; Figure S1C). p62 ablation also diminished MDM2 mRNA expression in both cell lines. In p53-mutated MIA PaCa-2 cells p62 ablation increased CDKN1B mRNA, which codes for the cell-cycle inhibitor p27. Despite the decrease in MDM2 mRNA, expression of p53 target genes, including CDKN1A, PMAIP1, and BBC3, was not increased (Figure S1C), congruent with the p53-deficient status of MIA PaCa-2 cells (Deer et al., 2010Deer E.L. Gonzalez-Hernandez J. Coursen J.D. Shea J.E. Ngatia J. Scaife C.L. Firpo M.A. Mulvihill S.J. Phenotype and genotype of pancreatic cancer cell lines.Pancreas. 2010; 39: 425-435Crossref PubMed Scopus (625) Google Scholar). Thus, p62 upregulation is important for maintaining malignant behavior even in p53-deficient PDAC. In Capan-2 cells, which harbor wild-type (WT) p53, the p62 deficiency increased expression of the cell-cycle inhibitors and p53 targets CDKN1A, PMAIP1, and BBC3. p62 ablation in both cell lines reduced sphere formation, a surrogate readout for stemness (Figures 1D and S1D). By contrast, IKKα silencing enhanced sphere formation and modestly elevated p62 expression (Figure 1E). Of note, IKKα silencing in MIA PaCa-2 cells increased tumorigenic growth in immunocompromised mice, and its effect on spheroid formation was blocked by p62 depletion, supporting the epistatic relationship between the two proteins (Figures 1E and 1F). Ablation of NRF2, encoded by the Nfe2l2 gene, was also accompanied by decreased sphere formation and tumorigenic growth in immunocompromised mice (Figures 1G, 1H, and S1E). Since cellular senescence is an established PDAC-suppressive mechanism (Guerra et al., 2011Guerra C. Collado M. Navas C. Schuhmacher A.J. Hernandez-Porras I. Canamero M. Rodriguez-Justo M. Serrano M. Barbacid M. Pancreatitis-induced inflammation contributes to pancreatic cancer by inhibiting oncogene-induced senescence.Cancer Cell. 2011; 19: 728-739Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar), we used senescence-associated β-galactosidase (SA-β-gal) and histone γ-H2AX to enumerate senescent cells. The results suggested that p62 and NRF2 deletions enhanced cellular senescence (Figures S1F and S1G). NRF2 activation, induced by KEAP1 ablation, restored sphere formation and decreased senescence in p62-deficient human MIA PaCa-2 and Capan-2 PDAC cells (Figures S1H and S1I), suggesting that p62 promotes a cancer stem/progenitor cell character via NRF2. We used mice to investigate whether conditions that promote p62 accumulation contribute to acinar-to-ductal metaplasia (ADM) and neoplastic progression. Since by interfering with autophagic proteolysis, PEC-specific IKKα ablation causes chronic pancreatitis accompanied by p62 accumulation (Li et al., 2013Li N. Wu X. Holzer R.G. Lee J.H. Todoric J. Park E.J. Ogata H. Gukovskaya A.S. Gukovsky I. Pizzo D.P. et al.Loss of acinar cell IKKalpha triggers spontaneous pancreatitis in mice.J. Clin. Invest. 2013; 123: 2231-2243Crossref PubMed Scopus (91) Google Scholar), we crossed ChukΔpan (hereafter IkkαΔpan) mice with Pdx1-Cre;LSL-KrasG12D/+ (hereafter KrasG12D) mice, which develop PanIN1 lesions by 2 weeks of age but rarely progress to PDAC within their first year (Hingorani et al., 2003Hingorani 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. et al.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 (1793) Google Scholar). KrasG12D;IkkαΔpan mice were obtained at the expected frequency but manifested weight loss and abdominal distension at 4–5 weeks, and at 8 weeks were obviously distressed. Necropsy revealed a large, irregularly shaped white mass occupying the entire pancreas and compressing the duodenum, with gallbladder dilation and splenomegaly (Figure 2A). Age-matched KrasG12D mice displayed only one to two small, white nodules on a normal pancreas. Eight-week-old KrasG12D;IkkαΔpan mice lacked normal acinar tissue, and already at 5 weeks exhibited multiple tumor nodules. Histology revealed abundant ADM, which is used to repair pancreatic injuries (Murtaugh and Keefe, 2015Murtaugh L.C. Keefe M.D. Regeneration and repair of the exocrine pancreas.Annu. Rev. Physiol. 2015; 77: 229-249Crossref PubMed Scopus (118) Google Scholar), and PanIN lesions of different stages in KrasG12D;IkkαΔpan pancreata, whereas KrasG12D pancreata contained only low-grade PanIN1 at 5 weeks of age (Figure 2B). These pronounced pathological changes were accompanied by rapid neoplastic progression in KrasG12D;IkkαΔpan animals, which showed multiple PanIN2/3 lesions by 8 weeks and hardly any normal acinar cells, dramatically shortened tumor latency, and aggressive PDAC by 2 months, with a median 5-month survival (Figures 2B–2D). KrasG12D;IkkαΔpan tumors displayed elevated p62 and LC3II in transformed acinar cells (Figure 2E), but little further increase in Sqstm1/p62 mRNA, which was already elevated in KrasG12D mice (Figure 2F). Using ductal-specific Dolichos biflorus agglutinin and acinar-specific Ulex europaeus agglutinin I lectin labeling we isolated ductal and acinar cells from mouse pancreata (Reichert et al., 2013Reichert M. Takano S. Heeg S. Bakir B. Botta G.P. Rustgi A.K. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells.Nat. Protoc. 2013; 8: 1354-1365Crossref PubMed Scopus (65) Google Scholar, Xiao et al., 2016Xiao X. Fischbach S. Fusco J. Zimmerman R. Song Z. Nebres P. Ricks D.M. Prasadan K. Shiota C. Husain S.Z. et al.PNA lectin for purifying mouse acinar cells from the inflamed pancreas.Sci. Rep. 2016; 6: 21127Crossref PubMed Scopus (6) Google Scholar). NRF2 target genes were upregulated in KrasG12D relative to WT mice, but IkkαΔpan ablation resulted in significant further increase in gene expression in both cell fractions (Figure 2G). Although ADM can give rise to PanINs that may further progress to PDAC, it usually forms in response to pancreatic damage (Murtaugh and Keefe, 2015Murtaugh L.C. Keefe M.D. Regeneration and repair of the exocrine pancreas.Annu. Rev. Physiol. 2015; 77: 229-249Crossref PubMed Scopus (118) Google Scholar). ADM and PanIN multiplicity, grade and size were markedly elevated in KrasG12D;IkkαΔpan mice relative to KrasG12D mice already at 5 weeks of age (Figures 2H and 2I). IKKα-deficient specimens contained many more proliferative cells (Ki67, PCNA, cyclin D1, and pERK positive) and showed an increase in Ck19-, SOX9-, or Alcian blue-positive ductal precursor lesions compared with IKKα-expressing KrasG12D counterparts (Figures 2J–2L and S2A). Furthermore, the NRF2 target NQO1, MDM2, and HES1 were more strongly expressed in IKKα-deficient ductal precursor lesions (Figure 2M). Unlike Atg7 deletion, which completely blocks autophagy and induces massive p53 accumulation (Rosenfeldt et al., 2013Rosenfeldt M.T. O'Prey J. Morton J.P. Nixon C. MacKay G. Mrowinska A. Au A. Rai T.S. Zheng L. Ridgway R. et al.p53 status determines the role of autophagy in pancreatic tumour development.Nature. 2013; 504: 296-300Crossref PubMed Scopus (521) Google Scholar), IKKα deficiency slightly reduced p53 protein expression (Figure 2N). Serum amylase and lipase were dramatically elevated in KrasG12D;IkkαΔpan mice (Figure S2B), indicating extensive acinar injury. KrasG12D;IkkαΔpan acinar and ductal cells, exhibited markedly elevated expression of ductal (Krt19)-, progenitor (Sox9, Nes, Pdx1, and Prox1)-, and stemness (Epcam, Cd24, Cd44, Prom1, and Aldh1)-related transcripts along with Notch (Notch1, Notch2, Notch3, Notch4, and Hes1) targets and components (Figure S2C). Of note, the fold-increase in ductal (Krt19) and certain progenitor markers (Nes, Pdx1, and Prox1) was much greater in the IKKα-deficient acinar fraction due to lower basal expression relative to ductal cells. Expression of pro-apoptotic p53 targets (Pmaip1 and Bbc3) was decreased along with Cdkn1b mRNA in both fractions, while Trp53 mRNA remained unaltered and Mdm2 mRNA was upregulated on IKKα ablation, especially in the ductal fraction (Figure S2C). Elevated Sirius Red and α smooth muscle actin staining, indicative of stellate cell activation and fibrosis, massive immune cell infiltration, primarily F4/80+ macrophages, and accumulation of fibrosis- and inflammation-related transcripts (Acta2, Col1a1, Col3a1, Tgfb1, Timp1, Ccl2, Ccl5, Tnf, Il6, and Il1b) also accompanied IKKα loss (Figures S2D and S2E). p62 ablation in KrasG12D;IkkαΔpan mice reduced ADM, inhibited appearance of advanced PanIN lesions, and reversed acinar cell loss (Figures 3A and 3B ). Notably, p62 ablation also prolonged KrasG12D;IkkαΔpan mouse survival by approximately 50% (Figure 3C), and reduced the number of proliferative (Ki67+) cells (Figures 3A and S3A). Ductal progenitor and stemness markers were downregulated along with MDM2 and NRF2 and Notch targets upon p62 ablation, in both acinar and ductal cells (Figures 3B, 3D, and 3E). The effect of p62 ablation on progenitor and ductal markers was most striking in the acinar cell fraction. Furthermore, Cdkn1a (p21) and Cdkn1b (p27) mRNAs were elevated, especially in acinar cells. In line with this, p53 protein was higher in KrasG12D;Ikkα/Sqstm1Δpan (hereafter KrasG12D;Ikkα/p62Δpan) than in KrasG12D;IkkαΔpan mice (Figure 3F). By contrast, the stem cell marker ALDH, whose expression was elevated in KrasG12D;IkkαΔpan cells, was lower in KrasG12D;Ikkα/p62Δpan double knockouts (Figures 3G and S3B). Sorted EpCAM+;ALDH+ KrasG12D;IkkαΔpan PECs exhibited much higher sphere-forming ability than KrasG12D;Ikkα/p62Δpan EpCAM+;ALDH+ cells (Figures 3H and S3C), underscoring p62 importance in augmenting stemness or proliferative capacity. Coactivation of Kras and Notch promotes rapid reprogramming of acinar cells to a duct-like phenotype and PanIN formation (De La et al., 2008De La O.J. Emerson L.L. Goodman J.L. Froebe S.C. Illum B.E. Curtis A.B. Murtaugh L.C. Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia.Proc. Natl. Acad. Sci. USA. 2008; 105: 18907-18912Crossref PubMed Scopus (312) Google Scholar, Thomas et al., 2014Thomas M.M. Zhang Y. Mathew E. Kane K.T. Maillard I. Pasca di Magliano M. Epithelial Notch signaling is a limiting step for pancreatic carcinogenesis.BMC Cancer. 2014; 14: 862Crossref PubMed Scopus (24) Google Scholar). Conversely, Notch inhibition suppresses PDAC cell proliferation and tumor growth (Wang et al., 2006Wang Z. Zhang Y. Li Y. Banerjee S. Liao J. Sarkar F.H. Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells.Mol. Cancer Ther. 2006; 5: 483-493Crossref PubMed Scopus (270) Google Scholar, Yabuuchi et al., 2013Yabuuchi S. Pai S.G. Campbell N.R. de Wilde R.F. De Oliveira E. Korangath P. Streppel M.M. Rasheed Z.A. Hidalgo M. Maitra A. et al.Notch signaling pathway targeted therapy suppresses tumor progression and metastatic spread in pancreatic cancer.Cancer Lett. 2013; 335: 41-51Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Having shown in human PDAC cell lines and mice that p62 promotes progenitor and stem cell features and PanIN formation, we postulated that p62 affects Notch signaling during PDAC development. Indeed, overexpression of human Notch1 intracellular domain (NICD1) abrogated the decrease in progenitor and stem cell markers in p62-deficient PDAC cells and suppressed senescence, regardless of p53 status (Figures 3I, 3J, and S3D). Cerulein-induced pancreatitis accelerates PDAC development in KrasG12D mice (Carriere et al., 2009Carriere C. Young A.L. Gunn J.R. Longnecker D.S. Korc M. Acute pancreatitis markedly accelerates pancreatic cancer progression in mice expressing oncogenic Kras.Biochem. Biophys. Res. Commun. 2009; 382: 561-565Crossref PubMed Scopus (161) Google Scholar). To determine the role of p62 in this model, KrasG12D and KrasG12D;p62Δpan mice were treated with PBS or cerulein and were analyzed 2 or 12 days later. At day 2, both strains exhibited damage-induced ADM development, but on day 12, when KrasG12D pancreata displayed big fibrotic nodules containing ADM, advanced PanIN, and few remaining acinar cells, KrasG12D;p62Δpan pancreata exhibited fewer lesions and little amylase+ acinar cell loss (Figures 4A–4D ). As described (Guerra et al., 2007Guerra C. Schuhmacher A.J. Canamero M. Grippo P.J. Verdaguer L. Perez-Gallego L. Dubus P. Sandgren E.P. Barbacid M. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice.Cancer Cell. 2007; 11: 291-302Abstract Full Text Full Text PDF PubMed Scopus (872) Google Scholar), cerulein treatment decreased senescent (SA-β-gal+) and increased proliferative (Ki67+) cells in KrasG12D PanINs, but this was not seen in KrasG12D;p62Δpan PanINs (Figures 4E–4G). Furthermore, p62 ablation in KC cells, originally derived from developed K" @default.
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• W2769455020 date "2017-12-01" @default.
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• W2769455020 title "Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas" @default.
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