Ilse Rooman

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling. The genomes of 102 primary pancreatic neuroendocrine tumours have been sequenced, revealing mutations in genes with functions such as chromatin remodelling, DNA damage repair, mTOR activation and telomere maintenance, and a greater-than-expected contribution from germ line mutations. Pancreatic neuroendocrine tumours (PanNETs) are the second most common epithelial neoplasm of the pancreas. Aldo Scarpa, Sean Grimmond and colleagues report whole-genome sequencing of 102 primary PanNETs and present analysis of their mutational signatures as part of the International Cancer Genome Consortium. They find frequent mutations in genes with functions that include chromatin remodelling, DNA damage repair, activation of mTOR signalling, and telomere maintenance. They also identify mutational signatures, including one resulting from inactivation of the DNA repair gene MUTYH, and report a larger than expected germline contribution to PanNET development.

Beta-cell mass regulation represents a critical issue for understanding diabetes, a disease characterized by a near-absolute (type 1) or relative (type 2) deficiency in the number of pancreatic beta cells. The number of islet beta cells present at birth is mainly generated by the proliferation and differentiation of pancreatic progenitor cells, a process called neogenesis. Shortly after birth, beta-cell neogenesis stops and a small proportion of cycling beta cells can still expand the cell number to compensate for increased insulin demands, albeit at a slow rate. The low capacity for self-replication in the adult is too limited to result in a significant regeneration following extensive tissue injury. Likewise, chronically increased metabolic demands can lead to beta-cell failure to compensate. Neogenesis from progenitor cells inside or outside islets represents a more potent mechanism leading to robust expansion of the beta-cell mass, but it may require external stimuli. For therapeutic purposes, advantage could be taken from the surprising differentiation plasticity of adult pancreatic cells and possibly also from stem cells. Recent studies have demonstrated that it is feasible to regenerate and expand the beta-cell mass by the application of hormones and growth factors like glucagon-like peptide-1, gastrin, epidermal growth factor, and others. Treatment with these external stimuli can restore a functional beta-cell mass in diabetic animals, but further studies are required before it can be applied to humans.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome‐wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high‐density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non‐malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5′ region of genes (including the proximal promoter, 5′UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF‐β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT‐ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT‐PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT‐ROBO signaling and up‐regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer. Pancreatic ductal adenocarcinoma has a 5-year survival of <5%, with therapies offering only incremental benefit,1Vogelzang N.J. et al.J Clin Oncol. 2012; 30: 88-109Crossref PubMed Scopus (85) Google Scholar potentially due to the diversity of its genomic landscape.2Bailey P. et al.Nature. 2016; 531: 47-52Crossref PubMed Scopus (1973) Google Scholar, 3Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1379) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar Recent reports link high mutation burden with response to immune checkpoint inhibitors in several cancer types.5Le D.T. et al.N Engl J Med. 2015; 372: 2509-2520Crossref PubMed Scopus (6099) Google Scholar Defining tumors that are hypermutated with an increased mutation burden and understanding the underlying mechanisms in pancreatic cancer has the potential to advance therapeutic development, particularly for immunotherapeutic strategies. Whole genome sequencing (WGS, n = 180) and whole exome sequencing (n = 205) of 385 unselected predominantly sporadic pancreatic ductal adenocarcinoma (Supplementary Table 1) defined a mean mutation load of 1.8 and 1.1 mutation per megabase (Mb), respectively (Supplementary Table 2). Outlier analysis identified 20 tumors with the highest mutation burden (5.2%, 15 WGS and 5 exome) (Table 1 and Supplementary Figure 1A), 5 of which were considered extreme outliers and classified as hypermutated as they contained ≥12 somatic mutations/Mb, the defined threshold for hypermutation in colorectal cancer.6Cancer Genome Atlas NetworkNature. 2012; 487: 330-337Crossref PubMed Scopus (5894) Google Scholar Immunohistochemistry for mismatch repair (MMR) proteins (MSH2, MSH6, MLH1, and PMS2) identified 4 MMR-deficient tumors, all of which were hypermutated (n = 180, Figure 1).Table 1Clinical and Histologic Features and Proposed Etiology for Highly Mutated Pancreatic Ductal Adenocarcinoma Tumors (n = 20)Sample IDPersonal and family history of malignancyHistologyMutation load, mutations/MbIHC resultMSIsensor scoreKRAS mutationPredominant mutation signature (mutations/Mb)SV subtype (no. of events)Proposed etiologyHypermutation (extreme outliers) ICGC_0076aSample sequenced by WGS, other samples by exome sequencing.NoneMixed signet ring, mucinous and papillary adenocarcinoma38.55Absent MLH1 and PMS228.3p.G12VMMR (18.3)Scattered (131)MMR deficiency: >280 kb somatic homozygous deletion over MSH2. ICGC_0297aSample sequenced by WGS, other samples by exome sequencing.NoneUndifferentiated adenocarcinoma60.62Absent MSH2 and MSH627.33WTMMR (33.4)Scattered (75)MMR deficiency: Somatic MLH1 promoter hypermethylation. ICGC_0548aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, moderately differentiated30.13Absent MSH2 and MSH617.47WTMMR (16.6)Stable (49)MMR deficiency: >27 kb somatic inversion rearrangement disrupting MSH2. ICGC_0328aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma16.63Normal3.2p.G12DUnknown (11.9)Scattered (110)Cell line with signature: etiology unknown. ICGC_00901 FDR, father CRCDuctal adenocarcinoma, moderately differentiated12.9Absent MSH2 and MSH60.21p.G12CNANAMMR deficiency: somatic MSH2 splice site c.2006G>A.Highly mutated tumors ICGC_0054aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.52Normal0.01p.G12VHR deficiency (1.3)Unstable (310)HR deficiency: no germline or somatic cause found. ICGC_0290aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.54Not available0.07p.G12VHR deficiency (3.1)Unstable (558)HR deficiency: Germline BRCA2 mutation c.7180A>T, p.A2394*. Somatic CN-LOH. ICGC_0215aSample sequenced by WGS, other samples by exome sequencing.2 FDR lung cancer, 2 FDR prostate cancer. Previous CRC and melanomaDuctal adenocarcinoma, moderately differentiated6.27Normal0.01p.G12VHR deficiency (1.9)Scattered (111)HR deficiency: Germline ATM mutation c.7539_7540delAT, p.Y2514*. Somatic CN-LOH. ICGC_0324NoneDuctal adenocarcinoma, moderately differentiated6.24Normal0p.G12DNANAUndefined ICGC_0034aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated6.09Normal4.02p.G12DHR deficiency (3.4)Unstable (366)HR deficiency: Germline BRCA2 mutation c.5237_5238insT, p.N1747*. Somatic CN-LOH. ICGC_0131aSample sequenced by WGS, other samples by exome sequencing.Lung cancer after PCDuctal adenocarcinoma, moderately differentiated5.63Normal0p.G12DT>G at TT sites (3.0)Focal (147)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0006aSample sequenced by WGS, other samples by exome sequencing.1 FDR, father lung cancerAdenocarcinoma arising from IPMN, moderately differentiated5.29Normal0.01p.G12DHR deficiency (1.2)Unstable (211)HR deficiency: Somatic BRCA2 c.5351dupA, p.N1784KfsTer3. Somatic CN-LOH. ICGC_0321aSample sequenced by WGS, other samples by exome sequencing.2 FDR, mother and cousin breast cancerDuctal adenocarcinoma, poorly differentiated4.79Not available0p.G12DHR deficiency (2.1)Unstable (286)HR deficiency: Germline BRCA2 c.6699delT, p.F2234LfsTer7. Somatic CN loss- 1 copy. ICGC_0309aSample sequenced by WGS, other samples by exome sequencing.NoneAdenocarcinoma arising from IPMN, moderately differentiated4.74Normal0.03p.G12VT>G at TT sites (3.1)Unstable (232)T>G at TT sites signature: etiology potentially associated with DNA oxidation ICGC_0005aSample sequenced by WGS, other samples by exome sequencing.1 FDR, mother CRCDuctal adenocarcinoma, poorly differentiated4.72Not available1p.G12VHR deficiency (1.1)Focal (95)HR deficiency: No germline or somatic cause found. ICGC_0016aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.61Normal3.03p.G12VHR deficiency (1.7)Unstable (447)HR deficiency: potentially linked to Somatic RPA1 c.273G>T, p.R91S ICGC_00461 FDR, brother PCDuctal adenocarcinoma, poorly differentiated4.3Normal0p.Q61HNANAUndefined GARV_0668aSample sequenced by WGS, other samples by exome sequencing.NoneDuctal adenocarcinoma, poorly differentiated4.3Not available2.19p.G12VHR deficiency (1.6)Unstable (464)HR deficiency: Germline BRCA2 c.7068_7069delTC, p.L2357VfsTer2. Somatic CN loss - 1 copy. ICGC_0291NoneDuctal adenocarcinoma, well differentiated3.84Not available0.03p.G12RNANAHR deficiency: Somatic BRCA2 c.7283T>A, p.L2428*. ICGC_0256NoneDuctal adenocarcinoma, poorly differentiated3.72Not available0.06p.G12DNANAUndefinedCRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data.a Sample sequenced by WGS, other samples by exome sequencing. Open table in a new tab CRC, colorectal cancer; FDR, first-degree relative; IHC, immunohistochemistry; IPMN, intraductal papillary mucinous neoplasm; CN-LOH, copy neutral loss of heterozygosity; CN, copy number; PC, pancreatic cancer; NA, not applicable to exome data. KRAS mutation status and histopathologic characteristics have been associated with MMR-deficient pancreatic tumors.7Goggins M. et al.Am J Pathol. 1998; 152: 1501-1507PubMed Google Scholar Of the 4 MMR-deficient tumors in our cohort, 2 were KRAS wild-type; 3 had undifferentiated to moderately differentiated histology and one had a signet-ring component. These features were not predictive of MMR deficiency in our cohort, as 11 additional non−MMR-deficient tumors had a signet-ring cell component or colloid morphology, and 131 of 347 assessable tumors had poorly or undifferentiated histology. Mutational signature analysis can detect MMR deficiency indirectly based on the pattern of somatic mutations.8Alexandrov L.B. et al.Nature. 2013; 500: 415-421Crossref PubMed Scopus (6213) Google Scholar An MMR-deficient signature dominated the MMR-deficient tumors (with WGS), and was minimal in MMR intact tumors (Supplementary Figure 1). In addition, microsatellite instability (MSI), a hallmark of MMR deficiency in colorectal cancer, was detected in all three MMR deficient tumors with WGS using MSIsensor9Niu B. Ye K. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (294) Google Scholar (Supplementary Table 2). MSI was not identified for the fourth MMR deficient sample potentially due to the reduced number of microsatellite loci in exome data. The underlying causes of MMR deficiency in the 4 cases were private somatic events. For 2 cases, MSH2 was disrupted by different structural rearrangements, 1 case contained a missense MSH2 mutation and the last, methylation of the MLH1 promoter (Figure 1). The missense mutation caused an MSH2 splice acceptor site mutation that alters the same nucleotide results in a pathogenic skipping of exon 13 in germline studies.10Thompson B.A. et al.Nat Genet. 2014; 46: 107-115Crossref PubMed Scopus (346) Google Scholar Hypermethylation of the MLH1 promoter is the predominant mechanism of MSI in sporadic colon cancer.11Boland C.R. et al.Gastroenterology. 2010; 138: 2073-2087 e3Abstract Full Text Full Text PDF PubMed Scopus (1359) Google Scholar The remaining hypermutated tumor contained an intact MMR pathway, and was a cell line (ATCC, CRL-2551) with an unidentified mutational signature, therefore the high mutation burden in this sample may be the result of long-term cell culture. The 15 samples (11 WGS and 4 exome) identified in the outlier analysis with high mutation burden, but not hypermutated (∼4 to 12 mutations/Mb) contained no evidence of MMR deficiency. Mutational signature analysis of the WGS samples indicated homologous recombination (HR) repair deficiency as the most substantial (range, 1.0–3.4 mutations/Mb) contributor to the mutation burden for 8 WGS mutation load outlier tumors. In support of a HR defect4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1466) Google Scholar; 7 of these tumors contained high levels of genomic instability with >200 structural variants and mutations in genes involved in HR were present for 6 of 8 cases (Supplementary Table 2). In addition, 1 case that had undergone exome sequencing had a somatic BRCA2 nonsense mutation that likely contributed to HR deficiency in this case. A mutational signature associated with T>G mutations at TT sites previously described in other cancers, including esophageal cancer12Nones K. Waddell N. Wayte N. et al.Nat Commun. 2014; : 5Google Scholar was the major contributor (>3 mutations/Mb) in 2 samples. For these 2 and the remaining 4 cases, no potential causative event could be identified. Although germline defects in MMR genes are well reported in pancreatic cancer13Grant R.C. Selander I. et al.Gastroenterology. 2015; 148: 556-564Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar in our cohort, they did not contribute to MMR deficiency even in those with familial pancreatic cancer or a personal or family history of Lynch-related tumors. A germline truncating variant was detected in PMS2 in 1 case, but did not have loss of the second allele, had normal immunohistochemistry staining and did not display a MMR mutational signature (Supplementary Table 2). MMR deficiency is important in the evolution in a small, but meaningful proportion of pancreatic cancers with a prevalence of 1% (4 of 385) in our cohort. This is consistent with recent studies using the Bethesda polymerase chain reaction panel,14Laghi L. et al.PLoS One. 2012; 7: e46002Crossref PubMed Scopus (55) Google Scholar and with previous estimates of MSI prevalence of 2%−3%.15Nakata B. et al.Clin Cancer Res. 2002; 8: 2536-2540PubMed Google Scholar However, in tumors with low epithelial content that underwent exome sequencing, the sensitivity of somatic mutation detection is reduced, which will affect mutation burden and signature analysis. While cognizant of small numbers, immunohistochemistry was the most accurate in defining MMR due to multiple genomic mechanisms of MMR gene inactivation. Multiple methods to define MMR deficiency may be required for clinical trials that aim to recruit MMR-deficient participants to assess the potential efficacy of checkpoint inhibitors or other therapies in pancreatic cancer. Homologous recombination-deficient tumors, and those with a novel signature seen in esophageal cancer had an increased mutation burden, and need further evaluation as potential patient selection markers for clinical trials of checkpoint inhibitor and other therapies that target tumors with a high mutation burden. The authors would like to thank Cathy Axford, Deborah Gwynne, Mary-Anne Brancato, Clare Watson, Michelle Thomas, Gerard Hammond, and Doug Stetner for central coordination of the Australian Pancreatic Cancer Genome Initiative, data management, and quality control; Mona Martyn-Smith, Lisa Braatvedt, Henry Tang, Virginia Papangelis, and Maria Beilin for biospecimen acquisition; and Sonia Grimaldi and Giada Bonizzato of the ARC-Net Biobank for biospecimen acquisition. For a full list of contributors see Australian Pancreatic Cancer Genome Initiative: http://www.pancreaticcancer.net.au/apgi/collaborators. The cohort consisted of 385 patients with histologically verified pancreatic exocrine carcinoma, prospectively recruited between 2006 and 2013 through the Australian Pancreatic Cancer Genome Initiative (www.pancreaticcancer.net.au) as part of the International Cancer Genome Consortium.1Hudson T.J. et al.Nature. 2010; 464: 993-998Crossref PubMed Scopus (1689) Google Scholar Ethical approval was granted at all treating institutions and individual patients provided informed consent upon entry to the study. The clinicopathologic information for the cohort is described in (Supplementary Table 1), and the global mutation profile has previously been reported for some of these tumors (Supplementary Table 2). Tumor and normal DNA were extracted after histologic review from fresh frozen tissue samples collected at the time of surgical resection or biopsy, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar Tumor cellularity was determined from single-nucleotide polymorphism array data using qpure.3Song S. et al.PLoS One. 2012; 7: e45835Crossref PubMed Scopus (85) Google Scholar Tumors with epithelial content ≥40% underwent WGS lower cellularity tumors underwent whole exome sequencing. DNA from patient-derived pancreas cell lines and matched normal was also extracted. Exome and WGS were performed using paired 100-bp reads on the Illumina HiSeq 2000, as described previously.2Biankin A.V. et al.Nature. 2012; 491: 399-405Crossref PubMed Scopus (1513) Google Scholar, 4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar Regions of germline and somatic copy number change were detected using Illumina SNP BeadChips with GAP.5Popova T. et al.Genome Biol. 2009; 10 (R128−R128)Crossref PubMed Scopus (151) Google Scholar Somatic structural variants were identified from WGS reads using the qSV tool.4Waddell N. et al.Nature. 2015; 518: 495-501Crossref PubMed Scopus (1686) Google Scholar, 6Patch A.M. et al.Nature. 2015; 521: 489-494Crossref PubMed Scopus (930) Google Scholar Single nucleotide variants were called using 2 variant callers: qSNP7Kassahn K.S. et al.PLoS One. 2013; 8: e74380Crossref PubMed Scopus (52) Google Scholar and GATK.8McKenna A. et al.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14755) Google Scholar Mutations identified by both callers or, those that were unique to a caller but verified by an orthogonal sequencing approach, were considered high confidence and used in all subsequent analyses. Small indels (<200 bp) were identified using Pindel9Ye K. et al.Bioinformatics. 2009; 25: 2865-2871Crossref PubMed Scopus (1391) Google Scholar and each indel was visually inspected in the Integrative Genome Browser. The distribution of the total number of small somatic mutations (coding and noncoding single nucleotide and indel variants) identified per megabase for exome and WGS sequence data were analyzed separately. The group of samples with high mutation load, at the top of each distribution, were defined as the upper distribution outliers for mutations per megabase, that is, ≥75th centile + (1.5× interquartile range). The threshold for detecting outliers in the exome and WGS groups was 3.4 and 4.2 mutations/Mb, respectively. From within the highly mutated set of tumors, hypermutated samples were identified as those with a mutation rate exceeding the thresholds for extreme distribution outliers (≥75th centile + [5× interquartile range]) of 7.4 and 8.1 mutations/Mb for exome and WGS sequencing, respectively. MSIsensor was used to detect microsatellite instability by directly comparing microsatellite repeat lengths between paired normal and tumor sequencing data.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar A MSIsensor score of >3.5% of somatic microsatellites with repeat length shifts was the detection threshold used to indicate microsatellite instability as published for endometrial cancer.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar This correlated well with the 5 and 7 microsatellite panels recommended in the Bethesda guidelines.10Niu B. et al.Bioinformatics. 2014; 30: 1015-1016Crossref PubMed Scopus (378) Google Scholar, 11Umar A. et al.J Natl Cancer Inst. 2004; 96: 261-268Crossref PubMed Scopus (2461) Google Scholar Tissue microarrays were constructed using at least three 1-mm formalin-fixed, paraffin-embedded tumor cores. Immunohistochemistry for MSH6 and PMS2 proteins was performed on tissue microarray sections as a screen for MMR deficiency due to MMR proteins forming heterodimers with concordant mismatch repair loss (ie, loss of MLH1 and PMS2 or loss of MSH2 and MSH6).12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar Immunohistochemistry on full tumor sections for MSH2, MLH1, MSH6, and PMS2 was performed in those with abnormal staining in core sections. The immunohistochemistry was performed as described previously12Hall G. et al.Pathology. 2010; 42: 409-413Abstract Full Text PDF PubMed Scopus (98) Google Scholar and scored by a senior pathologist. Somatic mutational signatures were extracted from the whole genome sequenced samples using the framework described previously.13Alexandrov L.B. et al.Cell Rep. 2013; 3: 246-259Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar High confidence somatic substitutions were classified by the substitution change and sequence context, that is, the type of immediately neighboring bases to the variant. The framework processes the counts of somatic mutations at each context within each sample using non-negative factorization to produce the different signature profiles that are present in the data. The profiles identified were matched against reported signatures from the Cancer of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cosmic/signatures). The major contributory signatures, defined as the mutational signature with the highest number of contributing somatic substitution variants, is reported for highly mutated whole genome samples. Bisulfite-converted whole-genome amplified DNA was hybridized to Infinium Human Methylation 450K Beadchips according to the manufacturers protocol (Illumina). Methylation arrays were performed on DNA from 174 pancreatic ductal adenocarcinoma samples, which were compared to DNA from 29 adjacent nonmalignant pancreata. A subset of the methylation data has been published previously.14Nones K. et al.Int J Cancer. 2014; 135: 1110-1118Crossref PubMed Scopus (156) Google Scholar We examined the data for evidence of tumor-specific hypermethylation of the promoter region of MLH1 and MSH2 genes. The methylation array data have been deposited into the International Cancer Genome Consortium data portal (dcc.icgc.org, project PACA-AU). Download .xlsx (.08 MB) Help with xlsx files Supplementary Tables 1 and 2

Transplantation of insulin-producing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulin-producing cells from adult rat exocrine cells cultured in the presence of growth factors.Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15.In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulin-immunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft.Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

It is still unclear which factors regulate pancreatic regeneration and beta-cell neogenesis and which precursor cells are involved. We evaluated the role of intravenously infused gastrin in regenerating pancreas of duct-ligated rats. The ligation of exocrine ducts draining the splenic half of the pancreas resulted in acinoductal transdifferentiation within the ligated part but not in the unligated part. We found that infusion of gastrin from day 7 to 10 postligation resulted in a doubling of the beta-cell mass in the ligated part as measured by morphometry. This increase in insulin-expressing cells was not associated with increased proliferation, hypertrophy, or reduced cell death of the beta-cells. Furthermore, we found an increased percentage of single, extra-insular beta-cells and small beta-cell clusters induced by gastrin infusion. These changes occurred only in the ligated part of the pancreas, where transdifferentiation of the exocrine acinar cells to ductlike cells (metaplasia) had occurred, and was not found in the normal unaffected pancreatic tissue. In conclusion, we demonstrate that administration of gastrin stimulates beta-cell neogenesis and expansion of the beta-cell mass from transdifferentiated exocrine pancreas.

In adult pancreatic regeneration models exocrine acini are found to transdifferentiate to duct-like complexes. This has also been associated with the formation of new endocrine islet cells. We aimed to establish an in vitro model in which this transdifferentiation process is characterised and can be modulated.Purified rat pancreatic acini were cultured in suspension. Differentiation was analysed by immunocytochemistry, electron microscopy, western blotting and RT-PCR.During culture acinar cells directly transdifferentiated without dividing, the cells lost their acinar phenotype and started to express cytokeratins 20 and 7 and fetal liver kinase-1 (Flk-1) receptors for vascular endothelial growth factor. Expression of the acinar pancreatic exocrine transcription factor (PTF-1) remained and the pancreatic duodenal homeobox-containing transcription factor (PDX-1) was induced. When transdifferentiation was completed, the cells started to express protein gene product 9.5, a panneuroendocrine marker. By combining these features, the transdifferentiated cells show similar characteristics to precursor cells during active beta-cell neogenesis. We were able to modulate the differentiation state by addition of nicotinamide or sodium butyrate, agents which are known to stimulate endocrine differentiation in other models.Here, we present an in vitro system in which the cellular differentiation of putative pancreatic endocrine precursor cells and their PDX-1 expression can be modulated, thereby providing a possible model for the study of beta-cell transdifferentiation.

Myc oncoproteins are commonly upregulated in human cancers of different organ origins, stabilized by Aurora A, degraded through ubiquitin-proteasome pathway-mediated proteolysis, and exert oncogenic effects by modulating gene and protein expression. Histone deacetylases are emerging as targets for cancer therapy. Here we demonstrated that the class III histone deacetylase SIRT2 was upregulated by N-Myc in neuroblastoma cells and by c-Myc in pancreatic cancer cells, and that SIRT2 enhanced N-Myc and c-Myc protein stability and promoted cancer cell proliferation. Affymetrix gene array studies revealed that the gene most significantly repressed by SIRT2 was the ubiquitin-protein ligase NEDD4. Consistent with this finding, SIRT2 repressed NEDD4 gene expression by directly binding to the NEDD4 gene core promoter and deacetylating histone H4 lysine 16. Importantly, NEDD4 directly bound to Myc oncoproteins and targeted Myc oncoproteins for ubiquitination and degradation, and small-molecule SIRT2 inhibitors reactivated NEDD4 gene expression, reduced N-Myc and c-Myc protein expression, and suppressed neuroblastoma and pancreatic cancer cell proliferation. Additionally, SIRT2 upregulated and small-molecule SIRT2 inhibitors decreased Aurora A expression. Our data reveal a novel pathway critical for Myc oncoprotein stability, and provide important evidences for potential application of SIRT2 inhibitors for the prevention and therapy of Myc-induced malignancies.

Purpose Individuals with adenocarcinoma of the ampulla of Vater demonstrate a broad range of outcomes, presumably because these cancers may arise from any one of the three epithelia that converge at that location. This variability poses challenges for clinical decision making and the development of novel therapeutic strategies. Patients and Methods We assessed the potential clinical utility of histomolecular phenotypes defined using a combination of histopathology and protein expression (CDX2 and MUC1) in 208 patients from three independent cohorts who underwent surgical resection for adenocarcinoma of the ampulla of Vater. Results Histologic subtype and CDX2 and MUC1 expression were significant prognostic variables. Patients with a histomolecular pancreaticobiliary phenotype (CDX2 negative, MUC1 positive) segregated into a poor prognostic group in the training (hazard ratio [HR], 3.34; 95% CI, 1.69 to 6.62; P &lt; .001) and both validation cohorts (HR, 5.65; 95% CI, 2.77 to 11.5; P &lt; .001 and HR, 2.78; 95% CI, 1.25 to 7.17; P = .0119) compared with histomolecular nonpancreaticobiliary carcinomas. Further stratification by lymph node (LN) status defined three clinically relevant subgroups: one, patients with histomolecular nonpancreaticobiliary (intestinal) carcinoma without LN metastases who had an excellent prognosis; two, those with histomolecular pancreaticobiliary carcinoma with LN metastases who had a poor outcome; and three, the remainder of patients (nonpancreaticobiliary, LN positive or pancreaticobiliary, LN negative) who had an intermediate outcome. Conclusion Histopathologic and molecular criteria combine to define clinically relevant histomolecular phenotypes of adenocarcinoma of the ampulla of Vater and potentially represent distinct diseases with significant implications for current therapeutic strategies, the ability to interpret past clinical trials, and future trial design.

Animal studies have indicated that pancreatic exocrine acinar cells have phenotypic plasticity. In rodents, acinar cells can differentiate into ductal precursors that can be converted to pancreatic ductal adenocarcinoma or insulin-producing endocrine cells. However, little is known about human acinar cell plasticity. We developed nongenetic and genetic lineage tracing methods to study the fate of human acinar cells in culture.Human exocrine tissue was obtained from organ donors, dissociated, and cultured. Cell proliferation and survival were measured, and cell phenotypes were analyzed by immunocytochemistry. Nongenetic tracing methods were developed based on selective binding and uptake by acinar cells of a labeled lectin (Ulex europaeus agglutinin 1). Genetic tracing methods were developed based on adenoviral introduction of a Cre-lox reporter system, controlled by the amylase promoter.Both tracing methods showed that human acinar cells can transdifferentiate into cells that express specific ductal markers, such as cytokeratin 19, hepatocyte nuclear factor 1β, SOX9, CD133, carbonic anhydrase II, and cystic fibrosis transmembrane conductance regulator. Within 1 week of culture, all surviving acinar cells had acquired a ductal phenotype. This transdifferentiation was decreased by inhibiting mitogen-activated protein kinase signaling.Human acinar cells have plasticity similar to that described in rodent cells. These results might be used to develop therapeutic strategies for patients with diabetes or pancreatic cancer.

Chronic pancreatitis predisposes to pancreatic cancer development and both diseases share a common etiology. A central role has been proposed for the digestive enzyme-secreting acinar cell that can undergo ductal metaplasia in the inflammatory environment of pancreatitis. This metaplastic change is now a recognised precursor of pancreatic cancer. Inflammatory molecules also foster tumour growth through autocrine and paracrine effects in the epithelium and the stroma. These insights have raised new opportunities such as the manipulation of inflammation as a preventive and/or therapeutic strategy for pancreatic cancer. Finally, we address the need for an in-depth study of the pancreatic acinar cells.

Research Article28 December 2016Open Access Source DataTransparent process ROCK signaling promotes collagen remodeling to facilitate invasive pancreatic ductal adenocarcinoma tumor cell growth Nicola Rath Nicola Rath Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Jennifer P Morton Jennifer P Morton Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Linda Julian Linda Julian Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Lena Helbig Lena Helbig Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Shereen Kadir Shereen Kadir Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Ewan J McGhee Ewan J McGhee Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Kurt I Anderson Kurt I Anderson Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Gabriela Kalna Gabriela Kalna Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Margaret Mullin Margaret Mullin Electron Microscopy Facility, School of Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Andreia V Pinho Andreia V Pinho Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Ilse Rooman Ilse Rooman Oncology Research Centre, Free University Brussels (VUB), Brussels, Belgium Search for more papers by this author Michael S Samuel Michael S Samuel Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, SA, Australia Search for more papers by this author Michael F Olson Corresponding Author Michael F Olson [email protected] orcid.org/0000-0003-3428-3507 Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Nicola Rath Nicola Rath Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Jennifer P Morton Jennifer P Morton Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Linda Julian Linda Julian Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Lena Helbig Lena Helbig Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Shereen Kadir Shereen Kadir Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Ewan J McGhee Ewan J McGhee Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Kurt I Anderson Kurt I Anderson Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Gabriela Kalna Gabriela Kalna Cancer Research UK Beatson Institute, Glasgow, UK Search for more papers by this author Margaret Mullin Margaret Mullin Electron Microscopy Facility, School of Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Andreia V Pinho Andreia V Pinho Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Ilse Rooman Ilse Rooman Oncology Research Centre, Free University Brussels (VUB), Brussels, Belgium Search for more papers by this author Michael S Samuel Michael S Samuel Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, SA, Australia Search for more papers by this author Michael F Olson Corresponding Author Michael F Olson [email protected] orcid.org/0000-0003-3428-3507 Cancer Research UK Beatson Institute, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Author Information Nicola Rath1, Jennifer P Morton1, Linda Julian1, Lena Helbig1, Shereen Kadir1, Ewan J McGhee1, Kurt I Anderson1, Gabriela Kalna1, Margaret Mullin2, Andreia V Pinho3, Ilse Rooman4, Michael S Samuel5 and Michael F Olson *,1,6 1Cancer Research UK Beatson Institute, Glasgow, UK 2Electron Microscopy Facility, School of Life Sciences, University of Glasgow, Glasgow, UK 3Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, NSW, Australia 4Oncology Research Centre, Free University Brussels (VUB), Brussels, Belgium 5Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, SA, Australia 6Institute of Cancer Sciences, University of Glasgow, Glasgow, UK *Corresponding author. Tel: +44 141 330 3654; Fax: +44 141 942 6521; E-mail: [email protected] EMBO Mol Med (2017)9:198-218https://doi.org/10.15252/emmm.201606743 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer death; identifying PDAC enablers may reveal potential therapeutic targets. Expression of the actomyosin regulatory ROCK1 and ROCK2 kinases increased with tumor progression in human and mouse pancreatic tumors, while elevated ROCK1/ROCK2 expression in human patients, or conditional ROCK2 activation in a KrasG12D/p53R172H mouse PDAC model, was associated with reduced survival. Conditional ROCK1 or ROCK2 activation promoted invasive growth of mouse PDAC cells into three-dimensional collagen matrices by increasing matrix remodeling activities. RNA sequencing revealed a coordinated program of ROCK-induced genes that facilitate extracellular matrix remodeling, with greatest fold-changes for matrix metalloproteinases (MMPs) Mmp10 and Mmp13. MMP inhibition not only decreased collagen degradation and invasion, but also reduced proliferation in three-dimensional contexts. Treatment of KrasG12D/p53R172H PDAC mice with a ROCK inhibitor prolonged survival, which was associated with increased tumor-associated collagen. These findings reveal an ancillary role for increased ROCK signaling in pancreatic cancer progression to promote extracellular matrix remodeling that facilitates proliferation and invasive tumor growth. Synopsis ROCK signaling contributes to the progression of pancreatic ductal adenocarcinoma (PDAC): Increased production and release of collagenases induce remodeling of the extracellular matrix to facilitate invasive tumor cell growth. Advanced pancreatic tumor stage and reduced patient survival correlate with elevated ROCK expression. Conditional ROCK activation decreased survival in a mouse PDAC model. ROCK signaling promoted invasion and proliferation of PDAC cells. ROCK activation induced matrix metalloproteinase expression and release via microvesicles to enable collagen remodeling. ROCK inhibitor treatment increased the survival time of PDAC mice. Introduction Patient survival from pancreatic cancer is the lowest of all common cancers, with 5-year survival rates below 5% in England for both men and women. Despite significant effort focused on developing pancreatic cancer targeted therapies, survival rates have not improved, emphasizing the need for additional therapeutic targets and treatment strategies. The predominant cancer form is pancreatic ductal adenocarcinoma (PDAC), characterized by dense desmoplasia with extensive myofibroblast proliferation and extracellular matrix (ECM) deposition, largely composed of bundled collagen fibers (Chu et al, 2007). The contribution of tumor-associated desmoplasia to PDAC growth and progression is unclear and controversial (Rath & Olson, 2016). The dense stroma serves as a barrier that impairs drug uptake (Feig et al, 2012); acute depletion of tumor-associated stroma using a Hedgehog pathway inhibitor (Olive et al, 2009); or enzymatic degradation of hyaluronic acid (Provenzano et al, 2012; Jacobetz et al, 2013) in mouse PDAC models increased drug uptake and promoted survival. Long-term inhibition or genetic deletion of Hedgehog signaling reduced stromal content and promoted tumor aggression in preclinical mouse models, while Hedgehog pathway activation increased desmoplasia and reduced epithelial cell proliferation (Lee et al, 2014; Rhim et al, 2014). Furthermore, depletion of α-smooth muscle actin (αSMA) expressing myofibroblasts resulted in invasive, undifferentiated tumors with reduced PDAC mouse survival, an observation paralleled by the association between fewer myofibroblasts and worse human patient survival (Ozdemir et al, 2014). Similarly, high PDAC stromal density has been linked with better patient survival (Bever et al, 2015). These observations are consistent with PDAC desmoplasia restraining tumor growth. However, it was reported recently that, although total and fibrillary collagen did not differ significantly in tumors at varying differentiation stages, collagen fiber diameters increased adjacent to poorly differentiated PDAC tumors, which was associated with short patient survival (Laklai et al, 2016). In addition, elevated levels of the collagen cross-linking enzyme lysyl oxidase (LOX) or fibrillar collagen were associated with reduced PDAC patient survival (Miller et al, 2015). These seemingly contradictory results illustrate the complex role of PDAC desmoplasia with the possibility of both negative and positive effects on tumor growth and progression, suggesting that the extent of desmosplasia alone is not directly responsible for tumor aggressiveness. By extension, properties that enabled PDAC cells to overcome potential inhibitory constraints on tumor growth imposed by the desmoplastic microenvironment would likely be positively selected, particularly in advanced tumors with well-established stromal components. Genomic analysis of pancreatic cancers identified core drivers including KRAS, TP53, SMAD4, and CDKN2A mutations as well as copy number variations including amplifications of MET and NOTCH1 (Waddell et al, 2015; Bailey et al, 2016). The ROCK1 locus on chromosome 18 was found to be amplified in 15% of pancreatic patient tumors (Biankin et al, 2012), which was accompanied by concordant copy number/gene expression changes (Bailey et al, 2016). The Rho GTPase-regulated ROCK1 and ROCK2 kinases control actomyosin contractility through phosphorylation of substrates including LIM kinases 1&2 (LIMK), myosin-binding subunit of the MLC phosphatase (MYPT1), and regulatory myosin light chain 2 (MLC2) (Rath & Olson, 2012; Julian & Olson, 2014). What role ROCK-mediated actomyosin contractility might play in PDAC has not been established, nor has it been determined whether ROCK2 expression is altered in pancreatic cancer. In this study, ROCK2 expression was found to increase with pancreatic cancer progression in human and KrasG12D-driven mouse tumors. Elevated ROCK1 and/or ROCK2 expression was associated with shorter survival in human pancreatic cancer patients, while conditional ROCK activation in KrasG12D-driven PDAC mice was sufficient to accelerate mortality. Conditional ROCK1 or ROCK2 activation in KrasG12D/p53+/− mouse PDAC tumor cells promoted collective invasion and proliferation in three-dimensional collagen matrices. Transcriptome analysis identified ROCK-induced differentially expressed gene networks associated with cell adhesion and cell–matrix interactions, with most highly induced transcripts being Mmp10 and Mmp13. ROCK-induced collagen degradation, collective invasion, and proliferation in three-dimensional collagen were blocked by MMP inhibition, indicating that ROCK signaling enables PDAC cells to overcome extracellular matrix imposed restraints on invasion and growth. Treatment of mice with KrasG12D-driven PDAC with the ROCK small molecule inhibitor Fasudil prolonged survival. These findings reveal an ancillary role for increased ROCK signaling in advanced pancreatic cancer to promote extracellular matrix remodeling that enables invasive tumor growth by overcoming microenvironmentally imposed proliferation restraints. An implication of these results is that ROCK inhibitor administration to pancreatic cancer patients might reverse the ability of pancreatic cancer cells to surmount the growth-restraining properties of tumor-associated desmoplasia. Results ROCK2 expression increases during pancreatic cancer progression To characterize ROCK2 expression in human pancreatic cancer progression, a tissue microarray containing 78 cases of pancreatic cancer and five normal pancreatic tissue samples was immunohistochemically stained with a ROCK2 antibody (Fig 1A). The specificity of the ROCK2 antibody had been validated in Pdx1-Cre; ROCK2fl/fl; Rosa26-LSL-RFP mouse pancreatic tissue, in which Cre-mediated recombination induced ROCK2 deletion and concomitant red fluorescent protein (RFP) expression (Appendix Fig S1A and B). ROCK2 levels rose with increasing tumor stage and grade, with the most progressed Stage III/IV having significantly higher levels than Normal or Stage I (Figs 1A and EV1A). In addition, ROCK2 was also present in tumor cells at resection margins (Fig EV1B). Analysis of The Cancer Genome Atlas (TCGA) Research Network (Cerami et al, 2012) pancreatic adenocarcinoma provisional dataset revealed that survival of patients with genomic amplification or significantly elevated mRNA for ROCK1 and/or ROCK2, or truncating ROCK1 mutations similar to previously described cancer-associated activating ROCK1 truncation mutations (Lochhead et al, 2010), was significantly shorter than in patients without ROCK1/ROCK2 alterations (median survival periods: 21 months for patients without alterations versus 17 months, P = 0.037) (Fig 1B). These observations paralleled the significantly elevated ROCK1 (Fig 1C) and ROCK2 (Fig 1D) mRNA levels observed in pancreatic tumors relative to normal tissue detected in publicly available datasets (Iacobuzio-Donahue et al, 2003; Segara et al, 2005; Badea et al, 2008) using Oncomine (Rhodes et al, 2004). Furthermore, TCGA Research Network data (Cerami et al, 2012) also revealed significantly coordinated ROCK1 and ROCK2 mRNA expression in pancreatic cancers (Fig 1E), consistent with an observed advantage associated with increased ROCK signaling in pancreatic cancer (Laklai et al, 2016). Figure 1. ROCK expression is elevated in pancreatic cancer and promotes disease progression A. ROCK2 immunohistochemistry-stained sections of normal and stages I, II, and III cases of human pancreas adenocarcinoma (left). Scale bar = 50 μm. Histoscores of ROCK2 staining (right) in normal pancreas (n = 5) and pancreas adenocarcinoma stage I (n = 22), stage II (n = 46), and stage III/IV (n = 4). One-way ANOVA with multiplicity adjusted exact P-value by post hoc Tukey multiple comparison test. B. Overall survival of 107 patients without alterations versus 37 patients with ROCK1 and/or ROCK2 gene amplification significantly increased mRNA or truncation mutation from TCGA research network. Survival P-value determined by log-rank test. C, D. Log2 median-centered ROCK1 or ROCK2 RNA expression in normal (n = 39) vs. PDAC (n = 39), normal (n = 5) vs. pancreatic adenocarcinoma (PAC) (n = 12), or normal (n = 6) vs. pancreatic carcinoma (PC) (n = 11) samples from indicated studies. Exact P-values determined by Mann–Whitney test. E. ROCK1 and ROCK2 mRNA expression in human PDAC (n = 146) from the TCGA research network. Significance (P-value) of slope deviation from 0 determined by Deming regression. F. Representative hematoxylin and eosin (H&E)- and ROCK2 immunohistochemistry-stained sections of normal mouse pancreas, acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) stages 1–3, and PDAC from mice with the indicated genotype. Scale bar = 50 μm. G. Quantification of ROCK2 staining in pancreatic cells of normal (wildtype), ADM/PanIN1 (KC), PanIN2 (KPC), PanIN3 (KPC), and PDAC (KPC) tissue (n = 5 mice per group). One-way ANOVA with multiplicity adjusted exact P-value by post hoc Tukey multiple comparison test. H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n = 21) or with conditional ROCK activation with tamoxifen citrate (n = 19). Survival P-value determined by log-rank test. Data information: Box (upper and lower quartiles divided by median value) and whisker (5th–95th percentile) plots show outliers as individual points. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. ROCK2 association with pancreas adenocarcinoma grade and expression in tumor cells in resection margin (related to Fig 1) Histoscores of ROCK2 staining (Fig 1A) in normal pancreas (n = 5), and pancreas adenocarcinoma grade 1 (n = 23), grade 2 (n = 27), and grade 3 (n = 13). Box (upper and lower quartiles divided by median value) and whisker (5th–95th percentile) plots show outliers as individual points. ROCK2-positive stained cells (indicated with arrows) found beyond the tumor in the resection margin of human PDAC. Download figure Download PowerPoint To further investigate ROCK2 expression in PDAC development and progression, genetically modified mouse models were used that closely recapitulate human PDAC (Gopinathan et al, 2015). Oncogenic KrasG12D, preceded by a Lox-Stop-Lox (LSL) transcriptional termination cassette that can be excised by Cre recombinase expressed from the pancreas-selective Pdx1 promoter (Pdx1-Cre), was combined with p53 deletion or mutation (Hingorani et al, 2003, 2005; Morton et al, 2010). ROCK2 immunostaining of normal pancreata as well as pancreatic tumors from LSL-KrasG12D; Pdx1-Cre (KC) or LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre (KPC) mice revealed weak ROCK2 expression in healthy normal tissue from wild-type mice, with progressive elevation in developing lesions of acinar-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) stages 1–3, and highest expression in PDAC tumors (Fig 1F and G). To determine whether increased ROCK signaling was sufficient to influence PDAC mouse survival, LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre (KPC) mice were crossed with genetically modified mice containing a Hprt-targeted LSL-ROCK2:ER transgene (Fig EV2A) (Samuel et al, 2016) to establish a RKPC mouse line (Fig EV2B). Fusion of the ROCK2 kinase domain with the estrogen receptor (ER) hormone-binding domain generates a ROCK2:ER chimeric protein that is inactive in the absence of ligand, but which can be conditionally activated by estrogen analogues such as 4-hydroxytamoxifen (4HT) or tamoxifen (Fig EV2C) both in vitro and in vivo (Croft et al, 2006; Samuel et al, 2009, 2011; Sanz-Moreno et al, 2011; Kumar et al, 2012). To conditionally activate ROCK2:ER in Pdx1-expressing KrasG12D/p53R172H pancreatic tumor cells (Appendix Figs S2 and S3), tamoxifen citrate or vehicle control was administered to RKPC cohorts for 9 weeks, starting at 10 weeks of age when KPC mice have typically progressed to the PanIN stage (Fig 1F) (Morton et al, 2010). Conditional ROCK activation significantly (P = 0.022) reduced RKPC mouse survival time relative to vehicle control-treated mice (Fig 1H), indicating that the additional ROCK activity in the PDAC mouse model paralleled the effect of increased ROCK1/ROCK2 expression on reduced human PDAC patient survival (Fig 1B). Click here to expand this figure. Figure EV2. Tissue-selective expression of conditionally activated ROCK2 (related to Fig 1) A CAG-LSL-Rock2:ER expression cassette was targeted to the mutant mouse Hprt gene locus together with the promoter and first exon of human HPRT and the second exon of murine Hprt to reconstitute an active chimeric Hprt locus. Not to scale. Generation of LSL-ROCK2:ER; KPC (RKPC) mice. Conditional activation of the ROCK2:ER fusion protein. Download figure Download PowerPoint ROCK kinases drive PDAC cell invasion and proliferation Pancreatic tumor masses are largely composed of stroma, of which ECM proteins including collagen are major constituents (Feig et al, 2012). Tumor cells must invade through the collagen-rich microenvironment to invade local tissue and to metastasize. A three-dimensional collagen matrix invasion assay, used previously to characterize PDAC cell invasive behavior (Timpson et al, 2011; Nobis et al, 2014), in which tumor cells move through a fibroblast-remodeled collagen meshwork toward serum-containing medium, was utilized to determine how ROCK signaling might influence invasion. Invasive KPC mouse PDAC cells (Appendix Fig S4) (Timpson et al, 2011) invaded significantly less into collagen matrix in the presence of ROCK-selective inhibitor H1152 (Fig 2A). Figure 2. ROCK activation induces PDAC cell invasion A. H&E-stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPC cells in the presence or absence of 10 μM H1152 ROCK inhibitor. Means ± SEM (n = 9 for untreated, n = 8 for H1152), P-value by unpaired t-test. B. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine-rich). Conditionally activated human ROCK1, human ROCK2, and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone-binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti-GFP antibody. C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 μM 4HT in the presence or absence of 1 μM or 10 μM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls. D. H&E-stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with 1 μM 4HT. Means ± SEM (n = 6), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Dunnett's multiple comparison test. E. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. F. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n = 30), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Dunnett's multiple comparison test. G. Ki67-positive cell percentages at the surface and within the collagen matrix. Means ± SEM (n = 30; n = 12 for GFP:ER/Matrix), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Dunnett's multiple comparison test. H, I. Viable cell relative to starting cell numbers were determined after 24 or 48 h treatment with vehicle (−) or 4HT on uncoated plastic surfaces (H) or collagen1-coated surfaces (I). Means ± SEM (n = 3). Source data are available online for this figure. Source Data for Figure 2 [emmm201606743-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint To understand how additional ROCK signaling input might promote PDAC progression and reduce survival in human patients and mouse PDAC models (Fig 1), we adopted a conditional gain-of-function approach. Conditionally activated ER-fusions with ROCK1 (ROCK1:ER) or ROCK2 (ROCK2:ER) kinase domains, or green fluorescent protein (GFP:ER), were stably retrovirally transduced and expressed in non-invasive PDAC tumor cells derived from Pdx1-Cre; LSL-KrasG12D; LSL-p53fl/+ (KPflC) mice (Fig 2B). While antibodies specific for epitopes in the carboxyl-terminal regions present in full-length endogenous ROCK1 or ROCK2 showed consistent levels, blotting with an antibody against a kinase-domain epitope shared by endogenous ROCK1, ROCK2, ROCK1:ER, or ROCK2:ER revealed comparable expression of the fusion proteins to endogenous ROCK kinases (Fig 2C). ROCK:ER fusions were induced and activated by 4HT, with consequent increased phosphorylation of MLC2 (pMLC2) that could be reversed by the ROCK-selective inhibitor H1152, in contrast to the lack of effect of 4HT treatment on GFP:ER-expressing cells (Fig 2C and Appendix Fig S5A). Consistent with previous observations (Croft et al, 2005), 4HT treatment of ROCK1:ER- or ROCK2:ER-expressing KPflC cells was sufficient to induce contraction and rounding compared to vehicle-treated controls, which were not observed for 4HT-treated GFP:ER-expressing cells (Appendix Fig S5B). Non-invasive KPflC mouse PDAC cells (Appendix Fig S4; Timpson et al, 2011) expressing GFP:ER and treated with 4HT did not invade the collagen matrix, while ROCK1:ER- or ROCK2:ER-expressing cells were significantly more invasive (Fig 2D). Comparable results were obtained if embedded fibroblasts were not removed prior to addition of ROCK1:ER- or ROCK2:ER-expressing KPflC cells to the collagen matrix surface (Fig EV3A). Further analysis revealed thicker ROCK1:ER and ROCK2:ER cell layers at collagen matrix surfaces (Fig 2D, inserts) with significantly more cells relative to GFP:ER-expressing cells (Fig 2E and F), associated with significantly increased cell proliferation as indicated by Ki67 immunoreactivity (Fig 2E and G). All three cell lines had high Ki67-positive percentages of cells that had successfully invaded collagen matrix, although both ROCK:ER-expressing cell lines tended to have higher percentages than GFP:ER controls (Fig 2G). However, when cells were grown on two-dimensional plastic (Fig 2H) or a thin collagen1-coated surface (Fig 2I), conditional ROCK activation did not affect viable proliferation. Furthermore, the invasion and proliferation of ROCK1:ER or ROCK2:ER-expressing cells were significantly reduced by addition of H1152 ROCK inhibitor to the collagen matrix invasion assay (Fig EV3B–E). Together, these data indicate that increased ROCK activity drives PDAC cell invasion, which enables proliferation by overcoming restraints imposed by three-dimensional collagen matrices that are not influential in two dimensions. Click here to expand this figure. Figure EV3. ROCK-induced collagen matrix invasion (related to Fig 2) H&E-stained sections of cell invasion into collagen matrix containing embedded fibroblasts after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with 1 μM 4HT. Means ± SEM (n = 6), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Dunnett's multiple comparison test. H&E-stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 μm. Invasion index of KPflC cells treated with EtOH vehicle, 1 μM 4HT, or 1 μM 4HT + 10 μM H1152. Means ± SEM (n = 6; n = 5 for ROCK2:ER/4HT), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Tukey multiple comparison test. Cell proliferation determined by Ki67 immunofluorescence. Scale bar = 20 μm. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n = 24), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Tukey multiple comparison test. Ki67-positive cell percentages at the surface. Means ± SEM (n = 24), one-way ANOVA with multiplicity adjusted exact P-value by post hoc Tukey multiple comparison test. Download figure Download PowerPoint ROCK activation induces collagen remodeling To determine whether ROCK-induced invasion (Fig 2) was associated with collagenolysis, collagen matrix sections were immunostained with Col1 3/4C antibody to detect a collagen1 neo-epitope at the C-terminal end of the ¾ fragment resulting from α1- and α2-chains cleavage at G775–I776 and G775–L776, respectively, and local triple helix denaturation. While collagen cleavage was minimal in GFP:ER-expressing cells, invasive ROCK1:ER and ROCK2:ER cells had considerable staining below collagen matrix surfaces and surrounding cell clusters (Fig 3A, left). To quantitatively assess fibrillar collagen organization in collagen matrices, second harmonic generation (SHG) microscopy, which takes advantage of optical properties of supramolecularly assembled collagen fibers (Chen et al, 2012), was performed at multiple z-planes below the collagen matrix surface (Fig 3A, second to fourth panels). When the intensity of fibrillary collagen detected by SHG was assessed, it was apparent that fibrillar collagen areas diminished more slowly with increasing depth of surface penetration in GFP:ER-expressing cells (Fig 3A, green line) compared to the greater decline observed in ROCK1:ER-expressing cells (Fig 3A, orange line). Gray-level correlation matrix (GLCM) texture analysis (Mostaço-Guidolin et al, 2013; Cameron et al, 2015; Miller et al, 2015) of SHG images confirmed that invading ROCK:ER cells had extensively remodeled fibrillar collagen (Fig 3B and C). Transmission electron microscopy (TEM) revealed that regions of collagen matrix surfaces not associated with invasive cell clusters lacked protrusions (Fig 3D), while surface cells near invasive clusters (Fig 3D, black border) or invading cell clusters (Fig 3D, red or green borders) had extensive protrusions resembling blebs and microvesicles. Tannic acid-glutaraldehyde fixation (Cotta-Pereira et al, 1976) allowed visualization of collagen fibers within the collagen matrix. Long fibers were often visible in regions away from cells or adjacent to non-invading cells at the collagen matrix surface (Fig 3E; left, yellow arrows), while there were decreased apparent long fibers and shorter collagen bundles observed adjacent to invading cell clusters, protrusions and microvesicles (Fig 3E; right, red arrows). These results reveal that ROCK:ER-expressing cells extensively re-modeled three-dimensional collagen matrices, likely resulting from proximal collagenolysis, physical force from actomyosin contraction, and pressure produced

Pancreatic ductal adenocarcinoma (PDAC) has long been considered to arise from pancreatic ducts on the basis of its morphology, the occurrence of dysplasia in putative preneoplastic ductal lesions, and the absence of acinar dysplasia in the pancreas of patients with PDAC. However, evidence gathered through both in vitro studies and--more importantly--genetic mouse models of PDAC shows that ductal-type tumours can arise from acinar cells. These findings raise new important questions related to PDAC pathophysiology and call for in-depth studies of acinar cell differentiation in order to better understand PDAC biology. The authors review these issues and discuss how the novel findings should impact on future work aiming at early diagnosis and improved outcome of patients with PDAC.

Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.

Repurposing is a drug development strategy that seeks to use existing medications for new indications. In oncology, there is an increased level of activity looking at the use of non-cancer drugs as possible cancer treatments. The Repurposing Drugs in Oncology (ReDO) project has used a literature-based approach to identify licensed non-cancer drugs with published evidence of anticancer activity. Data from 268 drugs have been included in a database (ReDO_DB) developed by the ReDO project. Summary results are outlined and an assessment of clinical trial activity also described. The database has been made available as an online open-access resource (http://www.redo-project.org/db/).

Propranolol (PRO) is a well-known and widely used non-selective beta-adrenergic receptor antagonist (beta-blocker), with a range of actions which are of interest in an oncological context. PRO displays effects on cellular proliferation and invasion, on the immune system, on the angiogenic cascade, and on tumour cell sensitivity to existing treatments. Both pre-clinical and clinical evidence of these effects, in multiple cancer types, is assessed and summarised and relevant mechanisms of action outlined. In particular there is evidence that PRO is effective at multiple points in the metastatic cascade, particularly in the context of the post-surgical wound response. Based on this evidence the case is made for further clinical investigation of the anticancer effects of PRO, particularly in combination with other agents. A number of trials are on-going, in different treatment settings for various cancers.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylationlow tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylationlow/IFNsignhigh and Methylationhigh/IFNsignlow PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived KrasG12D/Trp53-/- mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylationlow/IFNsignhigh subtype potentially targetable by agents blocking intrinsic IFN signaling. SIGNIFICANCE: The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity.This article is highlighted in the In This Issue feature, p. 521.

Increasing beta-cell mass and/or function could restore glucose homeostasis in diabetes mellitus. Hitherto, trophic factors for beta-cell regeneration after toxic events have been difficult to identify. We evaluated the application of gastrin and epidermal growth factor after alloxan-induced pancreatic beta-cell damage. After alloxan treatment (70 mg/kg), mice were implanted with Alzet osmotic minipumps releasing gastrin and epidermal growth factor for one week. We monitored glycaemia, did histological analyses of the pancreata and quantified pancreatic beta-cell mass and insulin content. Alloxan treatment alone resulted in a persisting hyperglycaemic state. Combined gastrin and epidermal growth factor treatment restored normoglycaemia in 3 days, an effect which seemed permanent. Glucose tolerance tests showed normal glucose responsiveness. Gastrin on its own and epidermal growth factor on its own did not alleviate hyperglycaemia. Islet mass, islet density and pancreatic insulin content were higher in mice treated with gastrin and epidermal growth factor than in untreated mice with persisting hyperglycaemia. In normoglycaemic control mice treatment with gastrin and epidermal growth factor did not affect these parameters. We detected transitional cytokeratin-positive ductal to endocrine insulin-expressing cells and noted increased ductal but not beta-cell proliferation. Our results show that combined treatment with gastrin and epidermal growth factor can induce sufficient regeneration of a functional islet mass to restore glucose homeostasis.

<h3>Objective</h3> Acinar cells display plasticity in vitro and in vivo and can activate a variety of differentiation programmes that may contribute to pancreatic diseases. The aims were to determine: (1) the differentiation potential of acinar cells under conditions which favour stem cell survival, and (2) its relationship to the phenotypes acquired by pancreatic epithelial cells in chronic pancreatitis. <h3>Design</h3> Murine acinar cells were cultured in suspension and their molecular phenotype was characterised by qRT-PCR, chromatin immunoprecipitation, immunocytochemistry and global transcriptome analysis. These findings were compared to the changes occurring in experimental chronic pancreatitis induced by pancreatic duct ligation and chronic caerulein administration. <h3>Results</h3> Acinar cells in suspension culture acquired a dedifferentiated phenotype characteristic of pancreatic embryonic progenitors, consisting of the co-expression of Ptf1a and Pdx1, presence of an embryonic-type PTF1 transcriptional complex, activation of the Notch pathway, and expression of additional pancreatic progenitor cell markers such as CpA1, Sox9 and Hnf1b. A senescence programme, associated with activation of Ras and ERK signalling, limited the proliferative capacity of the cells. A similar progenitor-like phenotype with activation of a senescence programme was observed in experimental chronic pancreatitis induced by pancreatic duct ligation or repeated caerulein administration, with the concomitant and differential activation of proliferation and senescence in distinct cell populations. <h3>Conclusions</h3> Acinar cells dedifferentiate into an embryonic progenitor-like phenotype upon suspension culture. This is associated with the activation of a senescence programme. Both processes take place in experimental chronic pancreatitis where senescence may contribute to limit tumour progression.

Pancreatic acinar cells acquire in vitro a pancreatic progenitor phenotype associated with activation of p53, growth arrest and senescence. A similar program is also activated in chronic pancreatitis. To assess the mechanisms involved in this process, we cultured pancreatic acinar cells from wild-type, p53(-/-), p16(-/-) and p21(-/-) mice. Cultures from p53(-/-) mice, but not those from p16(-/-) or p21(-/-) mice, display an enhanced proliferation and can be expanded continuously for more than 20 passages. p53(-/-) cells also display features of stemness such as enhanced sphere formation, increased expression of pancreatic multipotent progenitor markers (Ptf1a, Pdx1, Cpa1, c-myc, Sox9 and Hnf1b), and of the stemness regulators Bmi1 and Klf4. Upon subculture, p53(-/-) cells undergo an epithelial-mesenchymal transition (EMT) and express high levels of vimentin and of the transcriptional regulators Snai1, Snai2, Twist, Zeb1 and Zeb2. Genetic lineage tracing unequivocally demonstrates the epithelial origin of the cells with mesenchymal phenotype. These cells express the endodermal markers Hhex, Pdx1, Sox9, Hnf1b, Foxa2, Gata6 and Sox17, and the stem cell markers c-myc, Bmi1 and Klf4. Cultures from p53(+/-) mice display intermediate levels of the transcription factors involved in EMT but do not surpass the growth arrest. Our findings support the notion that p53 controls both growth and epithelial cell differentiation in the pancreas. These observations have important implications regarding the mechanisms through which p53 inactivation in tumors may be associated with aggressive biological behavior.

<h3>Objective</h3> The transcription factor SOX9 was recently shown to stimulate ductal gene expression in pancreatic acinar-to-ductal metaplasia and to accelerate development of premalignant lesions preceding pancreatic ductal adenocarcinoma (PDAC). Here, we investigate how SOX9 operates in pancreatic tumourigenesis. <h3>Design</h3> We analysed genomic and transcriptomic data from surgically resected PDAC and extended the expression analysis to xenografts from PDAC samples and to PDAC cell lines. SOX9 expression was manipulated in human cell lines and mouse models developing PDAC. <h3>Results</h3> We found genetic aberrations in the <i>SOX9</i> gene in about 15% of patient tumours. Most PDAC samples strongly express SOX9 protein, and SOX9 levels are higher in classical PDAC. This tumour subtype is associated with better patient outcome, and cell lines of this subtype respond to therapy targeting epidermal growth factor receptor (EGFR/ERBB1) signalling, a pathway essential for pancreatic tumourigenesis. In human PDAC, high expression of SOX9 correlates with expression of genes belonging to the ERBB pathway. In particular, ERBB2 expression in PDAC cell lines is stimulated by SOX9. Inactivating Sox9 expression in mice confirmed its role in PDAC initiation; it demonstrated that Sox9 stimulates expression of several members of the ERBB pathway and is required for ERBB signalling activity. <h3>Conclusions</h3> By integrating data from patient samples and mouse models, we found that SOX9 regulates the ERBB pathway throughout pancreatic tumourigenesis. Our work opens perspectives for therapy targeting tumourigenic mechanisms.

Different histological and molecular subtypes of pancreatic ductal adenocarcinoma (PDAC), with different molecular composition and survival statistics, have recently been recognised.This review describes the currently available studies regarding molecular and histological subtypes in PDAC. Studies from major cohorts such as International Cancer Genome Consortium as well as smaller cohorts are reviewed. We discuss where the described subtypes overlap, where the discrepancies are and which paths forward could be taken regarding diagnosis, ontogeny and therapy.Four molecular subtypes with strong overlap among the different studies can be found, next to a list of mixed findings. Two of the four subtypes (epithelial classical and mesenchymal basal-like) were represented in every study and were often discriminated in other solid tumours as well. These two subtypes differ substantially in prognosis. One biomarker has been discovered, only discriminating these two subtypes, and insights into subtype-specific therapeutic vulnerabilities are scarce.Subtypes can be reproducibly detected in cohorts of PDAC patients and two of them directly relate with prognosis. A consensus on the subtypes is warranted. Further discovery and validation studies are needed to identify strong biomarkers, to comprehend subtype ontogeny and to define strategies for precision medicine.

Repurposing of medicines has gained a lot of interest from the research community in recent years as it could offer safe, timely, and affordable new treatment options for cancer patients with high unmet needs. Increasingly, questions arise on how new uses will be translated into clinical practice, especially in case of marketed medicinal products that are out of basic patent or regulatory protection. The aim of this study was to portray the regulatory framework relevant for making repurposed medicines available to cancer patients in Europe and propose specific policy recommendations to address the current regulatory and financial barriers. We outlined two routes relevant to the clinical adoption of a repurposed medicine. First, a new indication can be approved, and thus brought on-label, via the marketing authorization procedures established in European and national legislation. Such procedures initiate a detailed and independent assessment of the quality and the benefit-risk balance of a medicinal product in a specific indication, benefiting both prescribers and patients as it reassures them that the scientific evidence is robust. However, the process of marketing authorization for new therapeutic indications entails a high administrative burden and significant costs while the return-on-investment for the pharmaceutical industry is expected to be low or absent for medicines that are out of basic patent and regulatory protection. Moreover, most of the repurposing research is conducted by independent or academic researchers who do not have the expertise or resources to get involved in regulatory procedures. A second option is to prescribe a medicine off-label for the new indication, which is managed at the national level in Europe. While off-label use could provide timely access to treatments for patients with urgent medical needs, it also entails important safety, liability and financial risks for patients, physicians, and society at large. In view of that, we recommend finding solutions to facilitate bringing new uses on-label, for example by developing a collaborative framework between not-for-profit and academic organizations, pharmaceutical industry, health technology assessment bodies, payers, and regulators.

When pancreatic tissue is injured after duct obstruction, acinoductal metaplasia is observed. Similar metaplastic changes occur when exocrine pancreatic cells are isolated and cultured. We demonstrate that under these experimental conditions the exocrine acinar cells lose their differentiated characteristics: expression of the acinar transcription factors p48/Ptf1alpha and Mist1 is decreased or lost, whereas expression of the embryonic transcription factor Pdx1 is increased. The receptors Notch1 and Notch2, members of the DSL family of Notch ligands, and the target genes in the Notch-signaling pathway Hes1, Hey1, and Hey2 become strongly up-regulated. We noted also reduced expression of Sel1L, a Notch repressor that is normally highly expressed in exocrine pancreas. Stimulation of Notch by its ligand Jagged1 diminished the proliferation of cultured metaplastic exocrine cells. Chemical inhibition of Notch signaling resulted in increased proliferation and induction of the cell-cycle regulator p21Cip1. This effect seems to be Hes1-independent and mainly coincides with decreased Hey1 and Hey2 mRNA expression. In conclusion, we demonstrate that during acinoductal metaplasia the Notch-signaling pathway is activated concomitantly with changes in transcription factor expression of pancreatic acinar cells. In addition, we show that Notch signaling is implicated in the suppression of proliferation of these metaplastic exocrine cells. The latter may be important in protection from neoplastic transformation.

Background & AimsExocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine β-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown.MethodsUsing rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-β-cell conversion.ResultsWe report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing β-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to β-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves β-cell neoformation with approximately 30% of acinar cells that convert to β-cells. The newly formed β-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients.ConclusionsWe report for the first time an efficient way to reprogram one third of the acinar cells to β-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models. Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine β-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown. Using rat acinar cell cultures, we studied the role of Notch signaling in a model of acinar-to-β-cell conversion. We report a novel lectin-based cell labeling method to demonstrate the acinar origin of newly formed insulin-expressing β-cells. This method allows for specific tracing of the acinar cells. We demonstrate that growth factor-induced conversion of adult acinar cells to β-cells is negatively regulated by Notch1 signaling. Activated Notch1 signaling prevents the reexpression of the proendocrine transcription factor Neurogenin-3, the key regulator of endocrine development in the embryonic pancreas. Interfering with Notch1 signaling allows modulating the acinar cell susceptibility to the differentiation-inducing factors. Its inhibition significantly improves β-cell neoformation with approximately 30% of acinar cells that convert to β-cells. The newly formed β-cells mature when transplanted ectopically and are capable of restoring normal blood glycemia in diabetic recipients. We report for the first time an efficient way to reprogram one third of the acinar cells to β-cells by adult cell type conversion. This could find application in cell replacement therapy of type 1 diabetes, provided that it can be translated from rodent to human models.

The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD(+)-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis.

Abstract Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1 Cre ;Robo2 F/F ) show increased activation of Robo1 + myofibroblasts and induction of TGF-β and Wnt pathways. During pancreatitis, Pdx1 Cre ;Robo2 F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-β inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2 low ;ROBO1 high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-β inhibitors or other stroma /immune modulating agents.

Maintenance of the pancreatic acinar cell phenotype suppresses tumor formation. Hence, repetitive acute or chronic pancreatitis, stress conditions in which the acinar cells dedifferentiate, predispose for cancer formation in the pancreas. Dedifferentiated acinar cells acquire a large panel of duct cell-specific markers. However, it remains unclear to what extent dedifferentiated acini differ from native duct cells and which genes are uniquely regulating acinar cell dedifferentiation. Moreover, most studies have been performed on mice since the availability of human cells is scarce. Here, we applied a non-genetic lineage tracing method of human pancreatic exocrine acinar and duct cells that allowed cell-type-specific gene expression profiling by RNA sequencing. Subsequent to this discovery analysis, one transcription factor that was unique for dedifferentiated acinar cells was functionally characterized. RNA sequencing analysis showed that human dedifferentiated acinar cells expressed genes in "Pathways of cancer" with a prominence of MECOM (EVI-1), a transcription factor that was not expressed by duct cells. During mouse embryonic development, pre-acinar cells also transiently expressed MECOM and in the adult mouse pancreas, MECOM was re-expressed when mice were subjected to acute and chronic pancreatitis, conditions in which acinar cells dedifferentiate. In human cells and in mice, MECOM expression correlated with and was directly regulated by SOX9. Mouse acinar cells that, by genetic manipulation, lose the ability to upregulate MECOM showed impaired cell adhesion, more prominent acinar cell death, and suppressed acinar cell dedifferentiation by limited ERK signaling. In conclusion, we transcriptionally profiled the two major human pancreatic exocrine cell types, acinar and duct cells, during experimental stress conditions. We provide insights that in dedifferentiated acinar cells, cancer pathways are upregulated in which MECOM is a critical regulator that suppresses acinar cell death by permitting cellular dedifferentiation.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating type of cancer. While many studies have shed light into the pathobiology of PDAC, the nature of PDAC's cell of origin remains under debate. Studies in adult pancreatic tissue have unveiled a remarkable exocrine cell plasticity including transitional states, mostly exemplified by acinar to ductal cell metaplasia, but also with recent evidence hinting at duct to basal cell transitions. Single-cell RNA sequencing has further revealed intrapopulation heterogeneity among acinar and duct cells. Transcriptomic and epigenomic relationships between these exocrine cell differentiation states and PDAC molecular subtypes have started to emerge, suggesting different ontogenies for different tumor subtypes. This review sheds light on these diverse aspects with particular focus on studies with human cells. Understanding the "masked ball" of exocrine cells at origin of PDAC and leaving behind the binary acinar vs duct cell classification may significantly advance our insights in PDAC biology.

Ductular metaplastic cells are observed during pancreas injury. Growth control by gastrin and expression of gastrin/cholecystokinin (CCK) B receptors were evaluated in these cells.Acinoductal transdifferentiation was induced in vitro by culturing of acinar cells, and ductular metaplasia was obtained in vivo by ligation of the pancreatic ducts. Mitogenic effects of gastrin I on ductal complexes in vivo and of tetragastrin, pentagastrin, and gastrin I and II, with or without the CCK-B receptor antagonist L-365,260, on duct-like cells in vitro were analyzed by 5-bromo-2'-deoxyuridine labeling. Immunocytochemistry, Western blotting, and reverse-transcription polymerase chain reaction were applied for detection of the CCK-B receptor.Gastrin analogues induced a mitogenic stimulus in the duct-like cells in vitro and in ductal complexes in duct-ligated rat pancreas. Immunocytochemistry showed expression of CCK-B receptors in these models and in fetal but not normal adult exocrine pancreas. Additionally, up-regulation of CCK-B receptors during ductular metaplasia was shown by Western blotting and reverse-transcription polymerase chain reaction.Duct-like pancreatic epithelial cells in vitro and ductal complexes in vivo express gastrin/CCK-B receptors and proliferate in response to gastrin.

Under certain experimental conditions, hepatocytes can arise in the pancreas. It has been suggested that the pancreas retains a source of hepatocyte progenitor cells. However, such cells have not been yet identified in the adult pancreas. We describe here the transdifferentiation of primary rat pancreatic exocrine cells into hepatocyte-like cells during 5 days of tissue culture in the presence of dexamethasone (DX). Using reverse-transcription polymerase chain reaction and immunocytochemistry, it was observed that DX treatment induced albumin RNA and protein expression in the cells. Coexpression of albumin and amylase, and the absence of cell proliferation, demonstrated a direct transdifferentiation of acinar cells to hepatocytic cells. CCAAT enhancer-binding protein-ss protein, a liver-enriched transcription factor that is considered to be the master switch in pancreatohepatic transdifferentiation, and alpha-fetoprotein were markedly upregulated in the cells after treatment with DX. We compared transcriptional profiles of freshly isolated exocrine cells and DX-treated cells using oligonucleotide microarrays and found that multiple liver-specific genes are induced along with albumin, and that certain pancreatic genes are downregulated in the DX-treated cells. In conclusion, these observations support the notion of plasticity in the adult pancreas and that exocrine cells can be reprogrammed to transdifferentiate into other cell types such as hepatocytes.

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up-regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K-Ras(G12V) conditional knock-in and caerulein-treated K-Ras(G12V) mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells-but not acinar cells-in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar-ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up-regulated in high-grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development.

Abstract Human pancreatic exocrine cells were cultured in 3D suspension and formed pancreatospheres composed of acinar-derived and duct-like cells. We investigated, up to 6 days, the fate of human pancreatic acinar cells using fluorescein-conjugated Ulex Europaeus Agglutinin 1 lectin, a previously published acinar-specific non-genetic lineage tracing strategy. At day 4, fluorescence-activated cell sort for the intracellularly incorporated FITC-conjugated UEA1 lectin and the duct-specific CA1 9.9 surface marker, distinguished acinar-derived cells ( UEA1 + CA1 9.9 − ) from duct-like cells ( UEA1 − CA1 9.9 + ) and acinar-to-duct-like transdifferentiated cells ( UEA1 + CA1 9 . 9 + ). mRNA expression analysis of the acinar-derived ( UEA1 + CA19 . 9 − ) and duct-like ( UEA1 - CA19 . 9 + ) cell fractions with concomitant immunocytochemical analysis of the pancreatospheres revealed acquisition of an embryonic signature in the UEA1 + CA19 . 9 − acinar-derived cells characterized by de novo expression of SOX9 and CD142 , robust expression of PDX1 and surface expression of GP2 . The colocalisation of CD142, a multipotent pancreatic progenitor surface marker, PDX1, SOX9 and GP2 is reminiscent of a cellular state present during human embryonic development. Addition of TGF-beta signalling inhibitor Alk5iII, induced a 28-fold increased KI67 -labeling in pancreatospheres, more pronounced in the CD142 + GP2 + acinar-derived cells. These findings with human cells underscore the remarkable plasticity of pancreatic exocrine acinar cells, previously described in rodents, and could find applications in the field of regenerative medicine.

We investigated the expression and function of netrin-1, a diffusible laminin-like protein known to regulate neuronal-cell migration in the pancreas. We questioned whether this factor regulates migration of pancreatic epithelial cells and whether this could be involved in islet neogenesis.We studied fetal and adult rat pancreas wherein duct ligation induced islet neogenesis. Netrin-1 expression was analysed by RT-PCR, western blot and immunohistochemistry. In vitro cell migration was measured with a human pancreatic duct cell line (CAPAN-2) and with fetal porcine islet cells. We also studied the expression of two netrin-receptors, neogenin and deleted in colorectal cancer.We found a transient expression of netrin-1 mRNA and protein in fetal pancreas from E15 to E18, and in adult pancreas after duct ligation. In normal adult pancreas there was very little netrin-1 expression. Netrin-1 expression was observed both in endocrine and exocrine cells. At the immunohistochemical level, it was expressed by islet cells during tissue regeneration. We could show that netrin-1 increases the migration of fetal islet cells and of a ductal cell line, mainly via a chemokinetic effect. From the two well-established netrin receptors, DCC and neogenin, we only found neogenin to be expressed in the pancreas. Neogenin expression coincided with the period of netrin-1 up-regulation.Netrin-1 is involved in pancreatic morphogenesis and tissue remodelling and plays a role in the regulation of duct-cell and fetal-islet cell migration. This can be of importance in islet regeneration, where migration of islet precursors takes place.

Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here, we determine the contribution of epigenetically silenced genes to the development of PDAC. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway.

Metabolic reprogramming is a feature of neoplasia and tumor growth.Sirtuin 1 (SIRT1) is a lysine deacetylase of multiple targets including metabolic regulators such as p53.SIRT1 regulates metaplasia in the pancreas.Nevertheless, it is unclear if SIRT1 affects the development of neoplastic lesions and whether metabolic gene expression is altered.To assess neoplastic lesion development, mice with a pancreas-specific loss of Sirt1 (Pdx1-Cre;Sirt1-lox) were bred into a Kras G12D mutant background (KC) that predisposes to the development of pancreatic intra-epithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC).Similar grade PanIN lesions developed in KC and KC;Sirt1-lox mice but specifically early mucinous PanINs occupied 40% less area in the KC;Sirt1-lox line, attributed to reduced proliferation.This was accompanied by reduced expression of proteins in the glycolysis pathway, such as GLUT1 and GAPDH.The stimulatory effect of SIRT1 on proliferation and glycolysis gene expression was confirmed in a human PDAC cell line.In resected PDAC samples, higher proliferation and expression of glycolysis genes correlated with poor patient survival.SIRT1 expression per se was not prognostic but low expression of Cell Cycle and Apoptosis Regulator 2 (CCAR2), a reported SIRT1 inhibitor, corresponded to poor patient survival.These findings open perspectives for novel targeted therapies in pancreatic cancer.

Objective The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63 + basal cells reportedly do not exist in the normal pancreas. Design We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. Results In normal human pancreas, rare ΔNp63 + cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63 + cells are atypical KRT19 + duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9 low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. Conclusion In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers.We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays.After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells.We propose human FXYD2gammaa as a novel beta cell-specific biomarker.

Pancreatic acinar cells accumulate amino acids against a marked concentration gradient to synthesize digestive enzymes. Thus, the function of acinar cells depends on amino acid uptake mediated by active transport. Despite the importance of this process, pancreatic amino acid transporter expression and cellular localization is still unclear. We screened mouse pancreas for the expression of genes encoding amino acid transporters. We showed that the most highly expressed transporters, namely sodium dependent SNAT3 (Slc38a3) and SNAT5 (Slc38a5) and sodium independent neutral amino acids transporters LAT1 (Slc7a5) and LAT2 (Slc7a8), are expressed in the basolateral membrane of acinar cells. SNAT3 and SNAT5, LAT1 and LAT2 are expressed in acinar cells. Additional evidence that these transporters are expressed in mature acinar cells was gained using acinar cell culture and acute pancreatitis models. In the acute phase of pancreatic injury, when acinar cell loss occurs, and in an acinar cell culture model, which mimics changes occurring during pancreatitis, SNAT3 and SNAT5 are strongly down-regulated. LAT1 and LAT2 were down-regulated only in the in vitro model. At protein level, SNAT3 and SNAT5 expression was also reduced during pancreatitis. Expression of other amino acid transporters was also modified in both models of pancreatitis. The subset of transporters with differential expression patterns during acute pancreatitis might be involved in the injury/regeneration phases. Further expression, localization and functional studies will follow to better understand changes occurring during acute pancreatitis. These findings provide insight into pancreatic amino acid transport in healthy pancreas and during acute pancreatitis injury.

ABSTRACT Chromatin structure has a major influence on the cell-specific density of somatic mutations along the cancer genome. Here, we present a pan-cancer study in which we searched for the putative cancer cell-of-origin of 2,550 whole genomes, representing 32 cancer types by matching their mutational landscape to the regional patterns of chromatin modifications ascertained in 104 normal tissue types. We found that, in almost all cancer types, the cell-of-origin can be predicted solely from their DNA sequences. Our analysis validated the hypothesis that high-grade serous ovarian cancer originates in the fallopian tube and identified distinct origins of breast cancer subtypes. We also demonstrated that the technique is equally capable of identifying the cell-of-origin for a series of 2,044 metastatic samples from 22 of the tumor types available as primaries. Moreover, cancer drivers, whether inherited or acquired, reside in active chromatin regions in the respective cell-of-origin. Taken together, our findings highlight that many somatic mutations accumulate while the chromatin structure of the cell-of-origin is maintained and that this historical record, captured in the DNA, can be used to identify the often elusive cancer cell-of-origin.

Finding new therapeutic uses for existing medicines could lead to safe, affordable and timely new treatment options for patients with high medical needs. However, due to a lack of economic incentives, pharmaceutical developers are rarely interested to invest in research with approved medicines, especially when they are out of basic patent or regulatory protection. Consequently, potential new uses for these medicines are mainly studied in independent clinical trials initiated and led by researchers from academia, research institutes, or collaborative groups. Yet, additional financial support is needed to conduct expensive phase III clinical trials to confirm the results from exploratory research.In this study, scientific and grey literature was searched to identify and evaluate new mechanisms for funding clinical trials with repurposed medicines. Semi-structured interviews were conducted with 16 European stakeholders with expertise in clinical research, funding mechanisms and/or drug repurposing between November 2018 and February 2019 to consider the future perspectives of applying new funding mechanisms.Traditional grant funding awarded by government and philanthropic organisations or companies is well known and widely implemented in all research fields. In contrast, only little research has focused on the application potential of newer mechanisms to fund independent clinical research, such as social impact bonds, crowdfunding or public-private partnerships. Interviewees stated that there is a substantial need for additional financial support in health research, especially in areas where there is limited commercial interest. However, the implementation of new funding mechanisms is facing several practical and financial challenges, such as a lack of expertise and guidelines, high transaction costs and difficulties to measure health outcomes. Furthermore, interviewees highlighted the need for increased collaboration and centralisation at a European and international level to make clinical research more efficient and reduce the need for additional funding.New funding mechanisms to support clinical research may become more important in the future but the unresolved issues identified in the current study warrant further exploration.

Sirtuin 1 (Sirt1) has been reported to be a critical positive regulator of glucose-stimulated insulin secretion in pancreatic beta-cells. The effects on islet cells and blood glucose levels when Sirt1 is deleted specifically in the pancreas are still unclear.This study examined islet glucose responsiveness, blood glucose levels, pancreatic islet histology and gene expression in Pdx1Cre; Sirt1ex4F/F mice that have loss of function and loss of expression of Sirt1 specifically in the pancreas.We found that in the Pdx1Cre; Sirt1ex4F/F mice, the relative insulin positive area and the islet size distribution were unchanged. However, beta-cells were functionally impaired, presenting with lower glucose-stimulated insulin secretion. This defect was not due to a reduced expression of insulin but was associated with a decreased expression of the glucose transporter Slc2a2/Glut2 and of the Glucagon like peptide-1 receptor (Glp1r) as well as a marked down regulation of endoplasmic reticulum (ER) chaperones that participate in the Unfolded Protein Response (UPR) pathway. Counter intuitively, the Sirt1-deficient mice did not develop hyperglycemia. Pancreatic polypeptide (PP) cells were the only other islet cells affected, with reduced numbers in the Sirt1-deficient pancreas.This study provides new mechanistic insights showing that beta-cell function in Sirt1-deficient pancreas is affected due to altered glucose sensing and deregulation of the UPR pathway. Interestingly, we uncovered a context in which impaired beta-cell function is not accompanied by increased glycemia. This points to a unique compensatory mechanism. Given the reduction in PP, investigation of its role in the control of blood glucose is warranted.

Background and aims: Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. Methods: We used scRNA-seq to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. Results: We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro, including Wnt-responsive-population, ciliated-population and FLRT3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples, highlighting a putative role of WNT-responsive, IFN-responsive and EMT-populations in pancreatic exocrine pathogenesis as their expression increases in chronic pancreatitis and PanIN lesions. Conclusions: In light of our discovery of previously unidentified ductal populations, we unmask the potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis.

xCT (Slc7a11), the specific subunit of the cystine/glutamate antiporter system xc-, is present in the brain and on immune cells, where it is known to modulate behavior and inflammatory responses. In a variety of cancers -including pancreatic ductal adenocarcinoma (PDAC)-, xCT is upregulated by tumor cells to support their growth and spread. Therefore, we studied the impact of xCT deletion in pancreatic tumor cells (Panc02) and/or the host (xCT-/- mice) on tumor burden, inflammation, cachexia and mood disturbances. Deletion of xCT in the tumor strongly reduced tumor growth. Targeting xCT in the host and not the tumor resulted only in a partial reduction of tumor burden, while it did attenuate tumor-related systemic inflammation and prevented an increase in immunosuppressive regulatory T cells. The latter effect could be replicated by specific xCT deletion in immune cells. xCT deletion in the host or the tumor differentially modulated neuroinflammation. When mice were grafted with xCT-deleted tumor cells, hypothalamic inflammation was reduced and, accordingly, food intake improved. Tumor bearing xCT-/- mice showed a trend of reduced hippocampal neuroinflammation with less anxiety- and depressive-like behavior. Taken together, targeting xCT may have beneficial effects on pancreatic cancer-related comorbidities, beyond reducing tumor burden. The search for novel and specific xCT inhibitors is warranted as they may represent a holistic therapy in pancreatic cancer.

Abstract Acinar to ductal metaplasia (ADM) or acinar cell dedifferentiation is one of the most notable features of chronic pancreatitis. It is also considered the initial step of pancreatic cancer development when oncogenic mutations accumulate. However, its precise mechanism and regulatory pathways remain unclear. This study profiled the transcriptome of dedifferentiated acinar cells from both humans and mice. Differential expression analysis integrated with a systematic literature search revealed several potential regulators (e.g., GDF15, GFRA1, MXRA5, CXCL17), of which the expression pattern over time was validated by qRT-PCR. We also focused on multiple conserved pathways of cell survival, among which SLC7A11 (xCT) is transiently upregulated in both species. The xCT subunit, integral to the cystine/glutamate antiporter system xc-, plays a critical role in cell survival across various cell types. To elucidate its role in acinar dedifferentiation, we employed gene silencing, pharmacological inhibition, and a knock-out mouse model. Acinar cells with diminished xCT function exhibit increased ferroptosis linked to lipid peroxidation. Antioxidants such as N-acetylcysteine and ferroptosis inhibitors like Ferrostatin-1 can mitigate lower glutathione levels and lipid ROS accumulation. Similarly, in mouse acute pancreatitis, xCT prevents lipid peroxidation. Our findings indicate that xCT fuels the glutathione pool and maintains ROS balance during ADM, thereby preventing pancreatic acinar cells from ferroptosis, a form of cell death rarely reported in relation to ADM. Next to xCT, a future and similar comprehensive study of the aforementioned genes could provide valuable insights into the dynamic changes that occur during acinar cell dedifferentiation and pave the way to the discovery of new intervention targets to suppress tumorigenesis in chronic pancreatitis. Citation Format: Zhaolong Pan, Jan-Lars Van den Bossche, Eva Rodriguez-Aznar, Pauline Janssen, Olaya Lara, Gamze Ates, Ann Massie, Diedert Luc De Paep, Isabelle Houbracken, Marco Mambretti, Ilse Rooman. Acinar cell susceptibility for tumor development in the pancreas: Targeting the critical regulators for therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3378.

The loss of β-catenin inhibitory components is a well-established mechanism of carcinogenesis but β-catenin hyperactivity can also be enhanced through its coactivators. Here we first interrogated a highly validated genomic screen and the largest repository of cancer genomics data and identified JRK as a potential new oncogene and therapeutic target of the β-catenin pathway. We proceeded to validate the oncogenic role of JRK in colon cancer cells and primary tumors. Consistent with a β-catenin activator function, depletion of JRK in several cancer cell lines repressed β-catenin transcriptional activity and reduced cell proliferation. Importantly, JRK expression was aberrantly elevated in 21% of colorectal cancers, 15% of breast and ovarian cancers and was associated with increased expression of β-catenin target genes and increased cell proliferation. This study shows that JRK is required for β-catenin hyperactivity regardless of the adenomatous polyposis coli/β-catenin mutation status and targeting JRK presents new opportunities for therapeutic intervention in cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest tumors with slow progress in systemic therapies due to its peculiar and resistant tumor microenvironment. Inclusion of isotoxic high-dose stereotactic body radiation therapy (iHD-SBRT) into a total neoadjuvant strategy (TNT) is promising for the treatment of localized PDAC. However, the histo-molecular effects of iHD-SBRT are still poorly explored. In this study, we have shown that TNT, associating FOLFIRINOX [FFX] followed by iHD-SBRT, leads to significant and long-lasting remodeling of PDAC, affecting its stromal, metabolic, and molecular features. Contrary to FFX alone, TNT is able to enrich tumors with Classical and Inactive stromal signatures associated with better prognosis. Furthermore, iHD-SBRT seems capable to counteract several of the detrimental modulatory effects induced by FFX such as Epithelial-to-Mesenchymal Transition or angiogenesis. Additionally, we identified inflammatory cancer-associated fibroblasts signatures as an important prognostic factor. This work provides new rationale to sequentially combine FFX with iHD-SBRT and suggests new pathways that can be targeted in combination with a TNT.

Activation of embryonic signaling pathways quiescent in the adult pancreas is a feature of pancreatic cancer (PC). These discoveries have led to the development of novel inhibitors of pathways such as Notch and Hedgehog signaling that are currently in early phase clinical trials in the treatment of several cancer types. Retinoid signaling is also essential for pancreatic development, and retinoid therapy is used successfully in other malignancies such as leukemia, but little is known concerning retinoid signaling in PC.We investigated the role of retinoid signaling in vitro and in vivo in normal pancreas, pancreatic injury, regeneration and cancer. Retinoid signaling is active in occasional cells in the adult pancreas but is markedly augmented throughout the parenchyma during injury and regeneration. Both chemically induced and genetically engineered mouse models of PC exhibit a lack of retinoid signaling activity compared to normal pancreas. As a consequence, we investigated Cellular Retinoid Binding Protein 1 (CRBP1), a key regulator of retinoid signaling known to play a role in breast cancer development, as a potential therapeutic target. Loss, or significant downregulation of CRBP1 was present in 70% of human PC, and was evident in the very earliest precursor lesions (PanIN-1A). However, in vitro gain and loss of function studies and CRBP1 knockout mice suggested that loss of CRBP1 expression alone was not sufficient to induce carcinogenesis or to alter PC sensitivity to retinoid based therapies.In conclusion, retinoid signalling appears to play a role in pancreatic regeneration and carcinogenesis, but unlike breast cancer, it is not mediated directly by CRBP1.

xCT is the specific subunit of System xc-, an antiporter importing cystine while releasing glutamate. Although xCT expression has been found in the spinal cord, its expression and role after spinal cord injury (SCI) remain unknown. The aim of this study was to characterize the role of xCT on functional and histological outcomes following SCI induced in wild-type (xCT+/+) and in xCT-deficient mice (xCT-/-). In the normal mouse spinal cord, slc7a11/xCT mRNA was detected in meningeal fibroblasts, vascular mural cells, astrocytes, motor neurons and to a lesser extent in microglia. slc7a11/xCT gene and protein were upregulated within two weeks post-SCI. xCT-/- mice recovered muscular grip strength as well as pre-SCI weight faster than xCT+/+ mice. Histology of xCT-/- spinal cords revealed significantly more spared motor neurons and a higher number of quiescent microglia. In xCT-/- mice, inflammatory polarization shifted towards higher mRNA expression of ym1 and igf1 (anti-inflammatory) while lower levels of nox2 and tnf-a (pro-inflammatory). Although astrocyte polarization did not differ, we quantified an increased expression of lcn2 mRNA. Our results show that slc7a11/xCT is overexpressed early following SCI and is detrimental to motor neuron survival. xCT deletion modulates intraspinal glial activation by shifting towards an anti-inflammatory profile.

p48, also called Ptf1a (pancreas-specific transcription factor 1a), is a tissue-restricted bHLH (basic helix loop helix) transcription factor which is critical for pancreatic commitment during development and for the activation and maintenance of the acinar differentiation programme in the exocrine pancreas. High-level expression of exocrine digestive enzymes, a hallmark of mature acinar cells, depends largely on the trimeric complex PTF1, formed by p48, RBP-L (recombination signal-binding protein 1-like) and a class A bHLH protein. In addition, p48 induces cell-cycle exit by controlling G(1)/S-phase progression. However, the mechanisms that mediate PTF1-dependent gene activation are poorly understood. In the present study, we report that p48 increases transcription through two activation domains located in its N-terminal region by recruiting transcriptional co-activators. The histone acetyltransferase cofactor p/CAF {p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein]-associated factor} interacts with p48 in acinar cells in vivo and is associated with the promoter region of acinar genes targeted by the PTF1 complex. p/CAF potentiates PTF1 transcriptional activity by enhancing selectively the p48 transactivation activity. p/CAF promotes the nuclear accumulation of p48 and its in vivo acetylation in Lys(200). The K200R mutation abolishes the transcriptional activity of p48, as well as its capacity to functionally co-operate with RBP-L to ensure effective PTF1-driven transcription, indicating that p/CAF-mediated acetylation of p48 is required for the full transcriptional activity of PTF1. In contrast, p/CAF did not co-operate with p48 in its growth regulatory effects. These results support a critical and selective role of p/CAF in PTF1-dependent gene activation during acinar differentiation.

Background Treatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B 3 ), in mice with pancreatic cancer. Methods Various mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (Kras G12D , p53 R172H , Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope. Results A significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading. Conclusion This study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

This corrects the article DOI: 10.1038/nature21063.

Introduction Pancreatic cancer is the fourth-leading cause of cancer-related death in developed countries. Despite advances in systemic chemotherapy, the mainstay of curative therapy for non-metastatic disease is surgical resection. However, the perioperative period is characterised by stress and inflammatory reactions that can contribute to metastatic spread and disease recurrence. Catecholamines and prostaglandins play a crucial role in these reactions. Therefore, a drug repurposing of betablockers and cyclooxygenase inhibitors seems reasonable to attenuate tumour-associated inflammation by inhibiting psychological, surgical and inflammatory stress responses. This may cause a relevant antitumourigenic and antimetastatic effect during the perioperative period, a window for cancer-directed therapy that is currently largely unexploited. Methods and analysis This is a prospective, single-centre, two-arm randomised, patient and observer blinded, placebo-controlled, phase-II trial evaluating safety and feasibility of combined perioperative treatment with propranolol and etodolac in adult patients with non-metastatic cancer of the pancreatic head undergoing elective pancreatoduodenectomy. 100 patients fulfilling the eligibility criteria will be randomised to perioperative treatment for 25 days perioperatively with a combination of propranolol and etodolac or placebo. Primary outcome of interest will be safety in terms of serious adverse events and reactions within 3 months. Furthermore, adherence to trial medication will be assessed as feasibility outcomes. Preliminary efficacy data will be evaluated for the purpose of power calculation for a potential subsequent phase-III trial. The clinical trial is accompanied by a translational study investigating the mechanisms of action of the combined therapy on a molecular basis. Ethics and dissemination The PROSPER-trial has been approved by the German Federal Institute for Drugs and Medical Devices (reference number 4042875) and the Ethics Committee of the Medical Faculty of the University of Heidelberg (reference number AFmo-385/2018). The final trial results will be published in a peer-reviewed journal and will be presented at appropriate national and international conferences. Trial registration numbers DRKS00014054; EudraCT number: 2018-000415-25.

Earlier data on liver development demonstrated that morphogenesis of the bile duct, portal mesenchyme and hepatic artery is interdependent, yet how this interdependency is orchestrated remains unknown. Here, using 2D and 3D imaging, we first describe how portal mesenchymal cells become organised to form hepatic arteries. Next, we examined intercellular signalling active during portal area development and found that axon guidance genes are dynamically expressed in developing bile ducts and portal mesenchyme. Using tissue-specific gene inactivation in mice, we show that the repulsive guidance molecule BMP co-receptor A (RGMA)/neogenin (NEO1) receptor/ligand pair is dispensable for portal area development, but that deficient roundabout 2 (ROBO2)/SLIT2 signalling in the portal mesenchyme causes reduced maturation of the vascular smooth muscle cells that form the tunica media of the hepatic artery. This arterial anomaly does not impact liver function in homeostatic conditions, but is associated with significant tissular damage following partial hepatectomy. In conclusion, our work identifies new players in development of the liver vasculature in health and liver regeneration.

Acinar cell dedifferentiation is one of the most notable features of acute and chronic pancreatitis. It can also be the initial step that facilitates pancreatic cancer development. In the present study, we further decipher the precise mechanisms and regulation using primary human cells and murine experimental models. Our RNAseq analysis indicates that, in both species, early acinar cell dedifferentiation is accompanied by multiple pathways related to cell survival that are highly enriched, and where SLC7A11 (xCT) is transiently upregulated. xCT is the specific subunit of the cystine/glutamate antiporter system xC-. To decipher its role, gene silencing, pharmacological inhibition and a knock-out mouse model were used. Acinar cells with depleted or reduced xCT function show an increase in ferroptosis relating to lipid peroxidation. Lower glutathione levels and more lipid ROS accumulation could be rescued by the antioxidant N-acetylcysteine or the ferroptosis inhibitor ferrostatin-1. In caerulein-induced acute pancreatitis in mice, xCT also prevents lipid peroxidation in acinar cells. In conclusion, during stress, acinar cell fate seems to be poised for avoiding several forms of cell death. xCT specifically prevents acinar cell ferroptosis by fueling the glutathione pool and maintaining ROS balance. The data suggest that xCT offers a druggable tipping point to steer the acinar cell fate in stress conditions.

Heterotopia of the salivary gland occurs mainly in the head and neck region of the human body, rarely in regions such as the rectum, but has never been demonstrated in the pancreas. Within a screening effort of pancreatic samples for detecting ΔNp63 expression, we discovered two pancreatic samples from a 35-year-old male showing salivary gland heterotopia. Immunohistochemical stainings were done for markers of healthy and neoplastic salivary glands and showed expression of calponin, CD142 and KRT14 but not of S100p, GFAP or CD117. A PAS-staining and Alcian Blue staining showed the presence of acid mucins. These staining patterns were consistent with non-neoplastic submandibular gland tissue comprised of abundant seromucous glands, basal cells and myoepithelial cells, all features typically absent in the pancreas. Also, no pancreatic islets of Langerhans were detected. We show for the first time that salivary gland heterotopia can occur at the location of the pancreas.

Purpose: Total body irradiation (TBI) followed by bone marrow transplantation (BMT) is used in pre-clinical research to generate mouse chimeras that allow to study the function of a protein specifically on immune cells.Adverse consequences of irradiation on the juvenile body and brain are well described and include general fatigue, neuroinflammation, neurodegeneration and cognitive impairment.Yet, the long-term consequences of TBI/BMT performed on healthy adult mice have been poorly investigated. Material and Methods:We developed a robust protocol to achieve near complete bone marrow replacement in mice using 2x550cGy TBI and evaluated the impact of the procedure on their general health, mood disturbances, memory, brain atrophy, neurogenesis, neuroinflammation and blood-brain barrier (BBB) permeability 2 and/or 16 months post-BMT.Results: We found a persistent decrease in weight along with long-term impact on locomotion after TBI and BMT.Although the TBI/BMT procedure did not lead to anxiety-or depressive-like behavior 2-or 16-months post-BMT, long-term spatial memory of the irradiated mice was impaired.We also observed radiation-induced impaired neurogenesis and cortical microglia activation 2 months post-BMT.Moreover, higher levels of hippocampal IgG in aged BMT mice suggest an enhanced age-related increase in BBB permeability that could potentially contribute to the observed memory deficit.Conclusions: Overall health of the mice did not seem to be majorly impacted by TBI followed by BMT during adulthood.Yet, TBI-induced alterations in the brain and behavior could lead to erroneous conclusions on the function of a protein on immune cells when comparing mouse chimeras with different genetic backgrounds that might display altered susceptibility to radiation-induced damage.Ultimately, the BMT model we here present could also be used to study the related long-term consequences of TBI and BMT seen in patients.

Sirtuin 1 is a protein deacetylase that regulates a large number of proteins often functionally implicated in tumor development and progression. Its pleiotropic function has turned SIRT1 into an attractive chemotherapeutic target, underscored by very promising preclinical results with SIRT1 inhibitors in the treatment of chronic myeloid leukemia. Here, we revisit the studies on SIRT1 as an emerging target for therapy in pancreatic cancer, a tumor with dismal outcomes for which currently few therapeutic options are available. We highlight those potential SIRT1 target genes that are commonly affected in pancreatic cancer according to recent genomic analyses.

Cancers affecting the gastrointestinal system are highly prevalent and their incidence is still increasing. Among them, gastric and pancreatic cancers have a dismal prognosis (survival of 5-20%) and are defined as difficult-to-treat cancers. This reflects the urge for novel therapeutic targets and aims for personalised therapies. As a prerequisite for identifying targets and test therapeutic interventions, the development of well-established, translational and reliable preclinical research models is instrumental. This review discusses the development, advantages and limitations of both patient-derived organoids (PDO) and patient-derived xenografts (PDX) for gastric and pancreatic ductal adenocarcinoma (PDAC). First and next generation multicellular PDO/PDX models are believed to faithfully generate a patient-specific avatar in a preclinical setting, opening novel therapeutic directions for these difficult-to-treat cancers. Excitingly, future opportunities such as PDO co-cultures with immune or stromal cells, organoid-on-a-chip models and humanised PDXs are the basis of a completely new area, offering close-to-human models. These tools can be exploited to understand cancer heterogeneity, which is indispensable to pave the way towards more tumour-specific therapies and, with that, better survival for patients.

&lt;p&gt;PDF file - 790K, S Table 1. Primary and secondary antibodies used in immunostaining and Western Blotting experiments S Table 2. Antibodies used for immunoprecipitation experiments S Table 3. Primers used in real time RT-PCR and PCR of the ChIP experiments S Figure 1: The expression pattern of Sirt1 and beta-catenin changes in Caerulein induced pancreatitis. S Figure 2: The expression pattern of Sirt1 and beta-catenin, but not Dbc1, changes in pancreatic duct ligation. S Figure 3: In an in vitro ADM model, Sirt1 undergoes transient changes in subcellular localisation without changes in expression levels. S Figure 4: Deletion of functional Sirt1 in Pdx1-Cre;Sirt1ex4lox/lox pancreas impacts on acinar cell differentiation during ADM. S Figure 5: Acinar cell differentiation is not altered during ADM in Sirt1 transgenic (SirtTg) pancreas. S Figure 6: Nicotinamide represses ADM of human cells in culture. S Figure 7: Sirt1 co-localizes and interacts with beta-Catenin in acinar cells. S Figure 8: Nicotinamide increases beta-catenin/Wnt signaling in ADM cultures. S Figure 9: Dbc1 expression is decreased in PDAC but not in chronic pancreatitis.&lt;/p&gt;

&lt;p&gt;PDF file - 790K, S Table 1. Primary and secondary antibodies used in immunostaining and Western Blotting experiments S Table 2. Antibodies used for immunoprecipitation experiments S Table 3. Primers used in real time RT-PCR and PCR of the ChIP experiments S Figure 1: The expression pattern of Sirt1 and beta-catenin changes in Caerulein induced pancreatitis. S Figure 2: The expression pattern of Sirt1 and beta-catenin, but not Dbc1, changes in pancreatic duct ligation. S Figure 3: In an in vitro ADM model, Sirt1 undergoes transient changes in subcellular localisation without changes in expression levels. S Figure 4: Deletion of functional Sirt1 in Pdx1-Cre;Sirt1ex4lox/lox pancreas impacts on acinar cell differentiation during ADM. S Figure 5: Acinar cell differentiation is not altered during ADM in Sirt1 transgenic (SirtTg) pancreas. S Figure 6: Nicotinamide represses ADM of human cells in culture. S Figure 7: Sirt1 co-localizes and interacts with beta-Catenin in acinar cells. S Figure 8: Nicotinamide increases beta-catenin/Wnt signaling in ADM cultures. S Figure 9: Dbc1 expression is decreased in PDAC but not in chronic pancreatitis.&lt;/p&gt;

&lt;p&gt;PDACvsNP enrichment results&lt;/p&gt;

&lt;p&gt;Patient characteristics and sequencing QC results&lt;/p&gt;

&lt;p&gt;MC1vsMC2 DE genes&lt;/p&gt;

&lt;p&gt;MC1vsMC2 DE genes&lt;/p&gt;

&lt;p&gt;PDACvsNP DMRs&lt;/p&gt;

&lt;p&gt;Supplementary Figures S1 to S15&lt;/p&gt;

&lt;p&gt;Supplementary Methods Figure 1&lt;/p&gt;

&lt;p&gt;Supplementary Methods Figure 1&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylation&lt;sup&gt;low&lt;/sup&gt; tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylation&lt;sup&gt;low&lt;/sup&gt;/IFNsign&lt;sup&gt;high&lt;/sup&gt; and Methylation&lt;sup&gt;high&lt;/sup&gt;/IFNsign&lt;sup&gt;low&lt;/sup&gt; PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived &lt;i&gt;Kras&lt;/i&gt;&lt;sup&gt;G12D&lt;/sup&gt;/&lt;i&gt;Trp53&lt;/i&gt;&lt;sup&gt;−/−&lt;/sup&gt; mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylation&lt;sup&gt;low&lt;/sup&gt;/IFNsign&lt;sup&gt;high&lt;/sup&gt; subtype potentially targetable by agents blocking intrinsic IFN signaling.&lt;/p&gt;Significance:&lt;p&gt;The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity.&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 521&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Patient characteristics and sequencing QC results&lt;/p&gt;

&lt;p&gt;PDACvsNP enrichment results&lt;/p&gt;

&lt;p&gt;Genesets used in the study&lt;/p&gt;

&lt;p&gt;MC2vsMC1 DMRs&lt;/p&gt;

&lt;p&gt;MC2vsMC1 DMRs&lt;/p&gt;

&lt;p&gt;Genesets used in the study&lt;/p&gt;

&lt;p&gt;PDACvsNP DMRs&lt;/p&gt;

&lt;p&gt;Extended Data Figures 1-3&lt;/p&gt;

&lt;p&gt;Supplementary Figures S1 to S15&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive desmoplasia, which challenges the molecular analyses of bulk tumor samples. Here we FACS-purified epithelial cells from human PDAC and normal pancreas and derived their genome-wide transcriptome and DNA methylome landscapes. Clustering based on DNA methylation revealed two distinct PDAC groups displaying different methylation patterns at regions encoding repeat elements. Methylation&lt;sup&gt;low&lt;/sup&gt; tumors are characterized by higher expression of endogenous retroviral transcripts and double-stranded RNA sensors, which lead to a cell-intrinsic activation of an interferon signature (IFNsign). This results in a protumorigenic microenvironment and poor patient outcome. Methylation&lt;sup&gt;low&lt;/sup&gt;/IFNsign&lt;sup&gt;high&lt;/sup&gt; and Methylation&lt;sup&gt;high&lt;/sup&gt;/IFNsign&lt;sup&gt;low&lt;/sup&gt; PDAC cells preserve lineage traits, respective of normal ductal or acinar pancreatic cells. Moreover, ductal-derived &lt;i&gt;Kras&lt;/i&gt;&lt;sup&gt;G12D&lt;/sup&gt;/&lt;i&gt;Trp53&lt;/i&gt;&lt;sup&gt;−/−&lt;/sup&gt; mouse PDACs show higher expression of IFNsign compared with acinar-derived counterparts. Collectively, our data point to two different origins and etiologies of human PDACs, with the aggressive Methylation&lt;sup&gt;low&lt;/sup&gt;/IFNsign&lt;sup&gt;high&lt;/sup&gt; subtype potentially targetable by agents blocking intrinsic IFN signaling.&lt;/p&gt;Significance:&lt;p&gt;The mutational landscapes of PDAC alone cannot explain the observed interpatient heterogeneity. We identified two PDAC subtypes characterized by differential DNA methylation, preserving traits from normal ductal/acinar cells associated with IFN signaling. Our work suggests that epigenetic traits and the cell of origin contribute to PDAC heterogeneity.&lt;/p&gt;&lt;p&gt;&lt;i&gt;This article is highlighted in the In This Issue feature, p. 521&lt;/i&gt;&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;Extended Data Figures 1-3&lt;/p&gt;

A 'classical' and a 'basal-like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin-embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter-patient and intra-patient inter-ductal heterogeneity, intraductal spatial phenotypes exist with anti-correlating expression levels of GATA6 and KRASG12D . The basal-like mRNA panel better captured the basal-like cell states than widely used protein markers. The panels corroborated the co-existence of the classical and basal-like cell states in a single tumour duct with functional diversification, i.e. proliferation and epithelial-to-mesenchymal transition respectively. Mutant KRASG12D detection ascertained an epithelial origin of vimentin-positive cells in the tumour. Uneven spatial distribution of cancer-associated fibroblasts could recreate similar intra-organoid diversification. This extensive heterogeneity with functional cooperation of plastic tumour cells poses extra challenges to therapeutic approaches. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

&lt;div&gt;Abstract&lt;p&gt;The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD&lt;sup&gt;+&lt;/sup&gt;-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. &lt;i&gt;Cancer Res; 73(7); 2357–67. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;div&gt;Abstract&lt;p&gt;The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD&lt;sup&gt;+&lt;/sup&gt;-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. &lt;i&gt;Cancer Res; 73(7); 2357–67. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

Supplementary Material 1

Sirtuin 1 (Sirt1) is a gene with diverse and complex roles in metabolic regulation, tumorigenesis and longevity, the latter being highly debated. Sirt1 is one of seven sirtuins that are NAD+ dependent and that, depending on the family member, modify histone and non-histone proteins by deacetylation and/or mono-ADP-ribosylation. Excellent reviews detail the long list of Sirt1 targets for deacetylation and capture its functions in homeostasis and disease [1, 2]. Recently, we reported a novel role for Sirt1 in pancreatic disease [3]. Until then, Sirt1 studies in the pancreas were confined to insulin producing endocrine cells. We investigated Sirt1 in the acinar cells of the pancreas that secrete digestive enzymes, and are prominent players in exocrine pancreatic disease. Acinar cells undergo ductal metaplasia when under stress, exemplified by the events in chronic pancreatitis where this can be a precursor to pancreatic ductal adenocarcinoma (PDAC) [4]. PDAC is the most prevalent form of pancreatic cancer and a lethal tumour that limits the median patient survival to less than 6 months after diagnosis. An increased understanding of pancreatic biology, both in pancreatitis and pancreatic cancer is warranted. Our work described Sirt1's expression related to that of Ccar2 (Cell cycle and apoptosis regulator 2, also named Deleted in breast cancer 1 or KIAA1967), a protein that directly inhibits the deacetylation activity of Sirt1 (Figure ​(Figure1).1). While in the unstressed pancreas both proteins colocalize in the nucleus, in acinar to ductal metaplasia Sirt1 temporary relocates to the cytoplasm while Ccar2 remains nuclear. This allows altered interaction of Sirt1 with its target genes, here, Pancreatic transcription factor 1a and beta-Catenin that critically regulate acinar cell differentiation and ductal metaplasia [4], and possibly other proteins. While SIRT1 is variably expressed in most of the human PDAC, CCAR2 is downregulated in a subset. These observations suggest that altered Sirt1 activity through changed intracellular localization and interaction with Ccar2 is important in different stages of pancreatic cancer development, revealing potential therapeutic opportunities. Indeed, Sirt1 inhibitors have generated excitement as therapeutic agents [1]. One of them, nicotinamide, attenuates the incidence of hepatocellular carcinoma without any evidence of toxic effects for the mice and is being tested in a clinical trial as a preventative agent for skin cancer. Our study showed that addition of nicotinamide partially repressed pancreatic acinar to ductal metaplasia. Other Sirt1 inhibitors, like Tenovin-6, showed anti-cancer effects in cell lines and mouse models, through activation of p53 and other mechanisms [1]. Another Sirt1 inhibitor (EX-527) is in clinical trial, albeit for Huntington's Disease and not (yet) for cancer [1]. In our study, several PDAC cell lines were sensitive to Sirt1 inhibition, an observation that is extended by others who showed that Sirt1 inhibitors increase chemosensitivity of PDAC cells to the clinically applied drug gemcitabine [5, 6]. Figure 1 Graphic illustration of the expression of Sirt1 and its endogenous inhibitor Cacr2 in pancreatic acinar to ductal metaplasia and in progressed pancreatic ductal adenocarcinoma The latter data strongly suggest an oncogenic role of Sirt1 in late stage PDAC. Several Sirt1 mouse models are available to shed light on its potential contribution to PDAC development. The mouse models are based on impairment of Sirt1's catalytic subunit through deletion of a critical exon or point mutation, and have been used in a whole body as well as in a tissue-restricted context [1, 2]. Complementary, a Sirt1 transgenic mouse has been widely studied [1, 2]. These mouse models, however, have illustrated the complexity of Sirt1 and have not uncommonly generated data that are hard to reconcile. One such example is the role in the intestine where both Sirt1 inactivation [7] and Sirt1 overexpression [1, 2] in the APC Min model of colorectal cancer led to reduced tumor development. For PDAC studies, several important aspects may be missing in the genetically engineered mice, such as the contextual effects of Sirt1 being shuttled in and out of the nucleus impacting on Sirt1's accessibility to different targets for deacetylation. Another factor is the interaction with endogenous regulators such as Ccar2, as well as possible compensatory effects by the closely related Sirt2, mostly located in the cytoplasm and shown to have a tumor suppressive role [1]. Nevertheless, an integrative study using complementary models will provide new insights. The in vivo part of our study to date was limited to experimental acute pancreatitis by injections of the cholecystokinine analogue caerulein. Our paper demonstrates that the caerulein treated pancreas with Sirt1 inactivation has more pronounced acinar to ductal metaplasia and after a week shows a reduced size compared to controls. The latter could be easily explained by Sirt1's well known inactivating role on stress-induced p53 mediated apoptosis and proliferation block [8]. We attribute the persistence of acinar to ductal metaplasia to the depletion of nuclear Sirt1, with our results suggesting that lack of nuclear Sirt1 and increase in cytoplasmic Sirt1 both independently contribute to acinar to ductal metaplasia. In no doubt, this study warrants further investigation of the role of Sirt1 in pancreatic cancer development as well as of the chemotherapeutic efficacy of Sirt1 inhibitors in preclinical models of human PDAC, guided by the expression of Sirt1 and Ccar2. Not only may a unique opportunity arise from the use of Sirt1 inhibitors, our studies also caution the proposed use of Sirt1 activators as anti-ageing therapy as long as uncertainties persist on the role of a pleiotrophic protein such as Sirt1.

It is becoming increasingly clear that differentiated adult somatic cells retain the capacity to be reprogrammed into other cell types. In the case of the pancreas, a switch from an acinar to a β-cell phenotype in vitro can be induced by soluble agents, such as growth factors and cytokines. We found that the combination of epidermal growth factor and leukemia inhibitory factor stimulated the transdifferentiation of rat acinar cells into β cells in culture. The transdifferentiation, or cellular reprogramming, appears to recapitulate embryonic events, such as expression of the transcription factor NGN3, which is characteristic of pancreas proendocrine progenitor cells. The NOTCH-signaling pathway, whose activity is normally restricted to embryonic pancreas development, is also reactivated during transdifferentiation. Inhibition of this pathway in the same experimental model leads to further stimulation of β-cell neogenesis from adult acinar cells. Engraftment of the acinar-derived β cells results in correction of glycemia in alloxan-diabetic mice. The phenotype of the transdifferentiated cells is stable in vivo, resulting in normal and safe function following transplantation. This approach opens ways for β-cell replacement therapy by transplantation or regeneration.

Abstract The transcription factor SOX9 has recently been shown to have a role in the ontogenesis of pancreatic ductal adenocarcinoma (PDAC) through metaplastic changes in pancreatic acinar cells. Nevertheless, the mechanisms through which SOX9 operates remain to be explored. We analyzed genomic and transcriptomic data from a cohort of surgically resected cases of PDAC (n=90) from the Australian Pancreatic cancer Genome Initiative (APGI). Genetic aberrations (mutation and copy number changes) in the SOX9 gene were found in 15% of patient tumors. Protein expression analysis revealed that most PDAC samples strongly express SOX9 protein and differential SOX9 expression correlates with patient survival. SOX9 gene expression was higher in the classical PDAC subtype, which relates with better patient outcome and increased response to epidermal growth factor receptor (EGFR)-directed therapy. In patient tumors, we found that SOX9 expression correlates with expression of EGFR signaling pathway genes, including ErbB2. Analysis of patient-derived xenografts confirmed strong correlation of SOX9 and ErbB2 expression and manipulation of human tumor cell lines demonstrated that Sox9 regulates the expression of ErbB2. We extended these findings by using a mouse model lacking pancreatic Sox9 expression where we found reduced expression of Egfr/ErbB signaling components. Sox9-deficient pancreatic acinar cells failed to generate PDAC precursor lesions and did not respond to EGF, indicating that Sox9 is required for Egfr/ErbB signaling pathway activity. In conclusion, by using an integrative approach that combines human and mouse models data, we discovered a new function of SOX9 in the regulation of EGFR/ERBB signaling throughout pancreatic tumorigenesis. This provides a novel mechanistic insight of potential therapeutic significance for pancreatic cancer patients. Citation Format: Andreia V. Pinho, Adrien Grimont, Mark J. Cowley, Cecile Augereau, Amanda Mawson, Marc Giry-Laterriere, Geraldine Van den Steen, Nicola Waddell, Marina Pajic, Christine Sempoux, Australian Pancreatic Cancer Genome Initiative, Jianmin Wu, Sean M. Grimmond, Andrew V. Biankin, Frederic P. Lemaigre, Patrick Jacquemin, Ilse Rooman. SOX9 regulates EGFR/ERBB signaling in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-73. doi:10.1158/1538-7445.AM2014-LB-73

Abstract Pancreatic cancer is one of the leading causes of cancer death and despite advances in chemotherapeutic regimens the overall 5-year survival rate remains less than 5%. The actomyosin-regulating ROCK1 and ROCK2 kinases are downstream-targets of the Rho GTPase pathway. They contribute to various processes, such as cell adhesion, motility, proliferation, differentiation and survival, which then influence numerous stages of cancer growth and progression. Interestingly, exome sequencing of pancreatic cancer genomes revealed that 15% of pancreatic cancer patients carry an amplification of the ROCK1 gene (1). Moreover, we found significant increases in ROCK1 and ROCK2 RNA expression in pancreatic cancer datasets obtained using Oncomine. When we further determined the protein expression of ROCK2 in pancreatic ductal adenocarcinoma (PDAC) we found an up-regulation of ROCK2 during disease progression. Higher ROCK2 levels also correlated with less differentiated tumors. A characteristic of advanced stages of pancreatic cancer is a collagen and fibroblast enriched stroma. To analyze the effect of ROCK kinases on cancer cell invasion, we performed 3-dimensional organotypic assays. Our results demonstrate that increased ROCK signaling was sufficient to convert non-invasive PDAC cells into ones capable of invasion into organotypic collagen matrix. Using an RNA sequencing approach, we investigated the effect of ROCK activation on pancreatic cancer cell gene transcription. Differential expression analysis revealed an enrichment of gene sets that are involved in cell-matrix interaction, and we found a higher release of matrix metalloproteinases MMP10 and MMP13 upon ROCK activation. In addition, our organotypic studies revealed extensive tissue remodeling and an accumulation of cleaved collagen bundles at the sites of PDAC cell invasion. Furthermore, collagen degradation and cell invasion into organotypic matrices were significantly reduced by application of a broad-spectrum MMP inhibitor, confirming that ROCK-induced invasion is dependent on MMP activity. Interestingly, our studies also indicated that ROCK-driven invasion of PDAC cells into collagen matrix enabled cell growth. To study the function of ROCK kinase signaling in pancreatic cancer in vivo, we expressed conditionally active ROCK2 in a mouse model of PDAC. We found that an increase of ROCK activity in pancreatic cancer cells accelerated PDAC progression, which resulted in reduced survival. Contrary, the administration of a ROCK inhibitor during tumor progression had a beneficial effect on survival. In summary, our results suggest that targeting ROCK kinases should be considered for chemotherapy of invasive pancreatic cancer. (1) Biankin et al. Nature. 2012 Nov 15;491(7424):399-405 Citation Format: Nicola Rath, Shereen Kadir, Jennifer P. Morton, Andreia V. Pinho, Lena Helbig, Linda Julian, Ewan J. McGhee, Gabriela Kalna, Alexei Vazquez, Kurt I. Anderson, Ilse Rooman, Michael S. Samuel, Michael F. Olson. ROCK kinases drive invasive pancreatic tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C22.

305 Background: The most important modifiable risk factor for the development of pancreas cancer is smoking, which accounts for up to 25% of pancreatic ductal adenocarcinomas (PDAC; Maisonneuve P, Lowenfels AB: Epidemiology of pancreatic cancer: an update. Digestive Diseases 28:645-656, 2010), and the incidence of PDAC correlates with smoking prevalence (Weiss W, Benarde MA: The temporal relation between cigarette smoking and pancreatic cancer. American journal of public health 73:1403-1404, 1983). A recently published Ingenuity Pathway Analysis of GWAS genotype and risk factor data from the Pancreatic Cancer Case Control Consortium demonstrated that axon guidance signalling genes were significantly overrepresented in smokers (Tang H, Wei P, Duell EJ, et al: Axonal guidance signaling pathway interacting with smoking in modifying the risk of pancreatic cancer: A gene and pathway-based interaction analysis of GWAS data. Carcinogenesis:bgu010, 2014) and we aimed to investigate this link in our cohort. Methods: Tissue from resected PDAC were obtained from 200 patients via the Australian Pancreatic Cancer Genome Initiative with patient consent and ethical approval. Immunohistochemistry was performed on TMAs with the anti-Robo1 antibody (ab7279). The sections were scored on a 4-point scale of 0, 1, 2 and 3 intensity. All sections were scored blindly by two independent reviewers. Results: Robo1 protein expression is found in the normal pancreas, predominantly in acinar cells. In PDAC, Robo1 expression is predominantly in epithelial ductal cells. Most PDACs have Robo1 expression with an overall mean score of 1.65±0.05. 20 out of 200 patients were current smokers at the time of their pancreatectomy, 97 were never smokers, the remainder were ex-smokers. The mean score for smokers was 2.1± 0.1 and for never smokers 1.6±0.1 (p = 0.0003). Genomic analysis did not demonstrate any mutations in the Robo1 gene. There were 7 cases of loss of heterozygosity for Robo1; none of these were current smokers and they had an average score of 1.25±0.17. Conclusions: In addition to our genomic (Biankin AV, Waddell N, Kassahn KS, et al: Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes. Nature 491:399-405, 2012) and methylation data (Nones K, Waddell N, Song S, et al: Genome‐wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT‐ROBO, ITGA2 and MET signaling. International Journal of Cancer, 2014), we provide the results of protein expression of Robo1 in a clinically annotated cohort of 200 cases of PDAC. We demonstrate that patients who are currently smoking have enhanced Robo1 expression. Preliminary results indicate that this may confer a poorer prognosis when coupled with high SLIT2 expression.