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The Loss of ATRX Increases Susceptibility to Pancreatic Injury and Oncogenic KRAS in Female But Not Male Mice

  • Claire C. Young
    Affiliations
    Department of Paediatrics, University of Western Ontario, London, Ontario, Canada

    Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

    Department of Oncology, University of Western Ontario, London, Ontario, Canada

    Children’s Health Research Institute, London, Ontario, Canada
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  • Ryan M. Baker
    Affiliations
    Department of Paediatrics, University of Western Ontario, London, Ontario, Canada

    Children’s Health Research Institute, London, Ontario, Canada
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  • Christopher J. Howlett
    Affiliations
    Department of Pathology and Laboratory Medicine, University of Western Ontario, London, Ontario, Canada
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  • Todd Hryciw
    Affiliations
    Department of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada

    Robarts Research Institute, London, Ontario, Canada
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  • Joshua E. Herman
    Affiliations
    Robarts Research Institute, London, Ontario, Canada
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  • Douglas Higgs
    Affiliations
    MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
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  • Richard Gibbons
    Affiliations
    MRC Molecular Haematology Unit, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
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  • Howard Crawford
    Affiliations
    Molecular & Integrative Physiology and Internal Medicine, University of Michigan, Ann Arbor, Michigan
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  • Arthur Brown
    Affiliations
    Department of Anatomy and Cell Biology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada

    Robarts Research Institute, London, Ontario, Canada

    Children’s Health Research Institute, London, Ontario, Canada
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  • Christopher L. Pin
    Correspondence
    Correspondence Address correspondence to: Christopher Pin, PhD, Department of Paediatrics, University of Western Ontario, Children’s Health Research Institute, 5th Floor, Victoria Research Laboratories, London, Ontario, Canada N6C 2V5. fax: (519) 685-8186.
    Affiliations
    Department of Paediatrics, University of Western Ontario, London, Ontario, Canada

    Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada

    Department of Oncology, University of Western Ontario, London, Ontario, Canada

    Children’s Health Research Institute, London, Ontario, Canada
    Search for articles by this author
Open AccessPublished:September 14, 2018DOI:https://doi.org/10.1016/j.jcmgh.2018.09.004

      Background

      Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in North America, accounting for >30,000 deaths annually. Although somatic activating mutations in KRAS appear in 97% of PDAC patients, additional factors are required to initiate PDAC. Because mutations in genes encoding chromatin remodelling proteins have been implicated in KRAS-mediated PDAC, we investigated whether loss of chromatin remodeler ɑ-thalassemia, mental-retardation, X-linked (ATRX) affects oncogenic KRAS’s ability to promote PDAC. ATRX affects DNA replication, repair, and gene expression and is implicated in other cancers including glioblastomas and pancreatic neuroendocrine tumors. The hypothesis was that deletion of Atrx in pancreatic acinar cells will increase susceptibility to injury and oncogenic KRAS.

      Methods

      Mice allowing conditional loss of Atrx within pancreatic acinar cells were examined after induction of recurrent cerulein-induced pancreatitis or oncogenic KRAS (KRASG12D). Histologic, biochemical, and molecular analysis examined pancreatic pathologies up to 2 months after induction of Atrx deletion.

      Results

      Mice lacking Atrx showed more progressive damage, inflammation, and acinar-to-duct cell metaplasia in response to injury relative to wild-type mice. In combination with KRASG12D, Atrx-deficient acinar cells showed increased fibrosis, inflammation, progression to acinar-to-duct cell metaplasia, and pre-cancerous lesions relative to mice expressing only KRASG12D. This sensitivity appears only in female mice, mimicking a significant prevalence of ATRX mutations in human female PDAC patients.

      Conclusions

      Our results indicate the absence of ATRX increases sensitivity to injury and oncogenic KRAS only in female mice. This is an instance of a sex-specific mutation that enhances oncogenic KRAS’s ability to promote pancreatic intraepithelial lesion formation.

      Graphical abstract

      Keywords

      Abbreviations used in this paper:

      ADM (acinar-to-duct cell metaplasia), ANOVA (analysis of variance), ATRX (ɑ-thalassemia, mental-retardation, X-linked), CIP (cerulein induced pancreatitis), CPA (carboxypeptidase), DAXX (death associated protein 6), ds (double stranded), EZH2 (Enhancer of Zeste Homologue 2, MKA, Mist1creERT/+KrasLSL-G12D/+AtrxflΔ18), PanIN (pancreatic intraepithelial lesion), PDAC (pancreatic ductal adenocarcinoma), WT (wild-type)
      See editorial on page 233.
      Female mice lacking ATRX in the pancreas have increased sensitivity to pancreatic cancer, whereas male mice without ATRX are protected. This study identifies such susceptibility in pancreatic cancer and highlights the need for sex-specific approaches in cancer treatment.
      Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in North America, with a 5-year survival rate of ∼9% (Pancreatic Cancer Facts, PANCAN). PDAC is characterized by increased genomic instability,
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      Results

      To determine whether ATRX deletion affected the phenotype of mature acinar cells, AtrxflΔ18 mice
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      Tamoxifen was administered to 2- to 4-month-old mice, and ATRX accumulation was assessed 7, 35, or 60 days after dosing (Figure 1B and C). Immunofluorescence analysis confirmed 98% of acinar cells were ATRX-negative at all time points, demonstrating efficient Atrx deletion and indicating mature acinar cells do not require ATRX for maintained viability (Figure 1C). Co-immunofluorescence for ATRX and insulin and identification of duct nuclei based on morphology confirmed Atrx deletion specifically in acinar cells (Figure 1D).
      Figure thumbnail gr1
      Figure 1Loss of ATRX in the pancreas is specific to acinar cells. (A) Schematic of Atrx deficient model. AtrxflΔ18 mice carrying loxP sites that flank exon 18. CreERT is expressed from the Mist1 promoter. On tamoxifen administration to Mist1creERT/+AtrxflΔ18 mice, cre recombinase becomes localized to the nucleus and produces deletion of exon 18 of the Atrx gene. This leads to degradation of full-length Atrx mRNA. (B) Immunofluorescence for ATRX in pancreatic tissue from Mist1creERT/+ (WT) mice 7, 35, or 60 days after tamoxifen gavage in Mist1creERT/+AtrxflΔ18 mice (AtrxflΔ18). White arrows indicate residual ATRX expression. Magnification bars = 25 μm. (C) Atrx deletion efficiency was quantified as the percentage of acinar cells lacking ATRX (n = 3 for all groups). (D) Localization of insulin (green) and ATRX (red) by co-immunofluorescence (IF) demonstrates acinar-specific knockout of ATRX expression in Mist1creERT/+AtrxflΔ18 mice 20 days after tamoxifen treatment. White arrows indicate ATRX expression in pancreatic duct cells. Sections were counterstained with DAPI to reveal nuclei. Magnification bar = 50 μm.
      Histologic analysis showed no obvious phenotypes regarding disorganization of acinar cells or injury/inflammation (Figure 2A), although intralobular adipocytes were observed at a higher frequency in Mist1creERT/+AtrxflΔ18 mice. Because loss of ATRX showed limited effects on overall pancreatic morphology, we focused specifically on the 60-day time point to determine whether any phenotypes occurred in response to loss of ATRX. Immunofluorescence analysis for proliferative markers Ki67 (Figure 2B) and pH3 (data not shown) or TUNEL analysis (Figure 2C) revealed increased numbers of proliferating and apoptotic cells after ATRX deletion. Because ATRX is involved in double stranded (ds) DNA repair,
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      Loss of ATRX leads to chromosome cohesion and congression defects.
      we examined γH2AX accumulation, a marker for unresolved dsDNA breaks. Mist1creERT/+AtrxflΔ18 pancreatic tissue showed an increase in the number of cells accumulating γH2AX (Figure 2D), suggesting loss of ATRX leads to an inability to resolve dsDNA breaks. These results suggest that short-term loss of ATRX in pancreatic acini has no overt consequences on pancreatic morphology but may increase susceptibility to events that require intact DNA repair pathways, such as pancreatic injury.
      Figure thumbnail gr2
      Figure 2Mature acinar cells do not require ATRX for maintenance, but ATRX loss induces low levels of pancreatic damage. (A) Representative H&E staining of Mist1creERT/+ (WT) and Mist1creERT/+AtrxflΔ18 (AtrxflΔ18) pancreatic tissue 60 days after last tamoxifen gavage. Intralobular adipocytes are indicated by *. (B–D) Representative images and quantification for (B) Ki67 immunofluorescence (Mist1creERT/+ [WT; n = 4]; Mist1creERT/+AtrxflΔ18 [ATRX–; n = 5]), (C) TUNEL (Mist1creERT/+, n = 9; Mist1creERT/+AtrxflΔ18, n = 10), or (D) γH2AX IF (Mist1creERT/+ [n = 7]; Mist1creERT/+AtrxflΔ18 [n = 11]) 60 days after tamoxifen treatment. White arrows indicate positive cells. Magnification bar = 50 μm. Data were assessed by using two-tailed t test with Tukey post hoc test. Error bars represent means ± standard error; *P < .005.
      To examine this possibility, Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 mice were subjected to recurrent pancreatic injury for 11 days and allowed to recover for 3 days. We chose a mild dosing regimen so damage in control mice would be limited. No significant differences were observed between Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 mice based on body weight, behavior, or gross tissue morphology 3 days after cessation of cerulein (data not shown). However, histologic analysis showed marked differences between the 2 genotypes in response to cerulein treatment. As expected, cerulein-treated Mist1creERT/+ mice show intra-acinar edema but no evidence of inflammation or fibrosis (Figure 3A), likely because of the mild nature of the cerulein treatment. Conversely, cerulein-treated Mist1creERT/+AtrxflΔ18 mice showed increased damage (Figure 3A) and fibrosis in female Mist1creERT/+AtrxflΔ18 mice relative to controls (Figure 3B and C), as indicated by H&E and trichrome histology, respectively. This enhanced cellular damage was further confirmed by the strong immunofluorescence signal for F4/80 antigen that was indicative of extensive macrophage infiltration in Mist1creERT/+AtrxflΔ18 mice relative to controls (Figure 3D). Quantification of the tissue damage confirmed increased sensitivity to recurrent cerulein induced pancreatitis (CIP) and indicated that female Mist1creERT/+AtrxflΔ18 mice are clearly more sensitive than male mice to these effects (Figure 3C, Table 2). Whereas analysis of acinar cell death by cleaved caspase-3 showed no difference between genotypes (Figure 4A), notably enhanced cell turnover based on cleaved caspase-3 (Figure 4B) or proliferative capacity by Ki67 staining (Figure 5E) was observed in Mist1creERT/+AtrxflΔ18 pancreata in areas showing classic features of ADM after CIP. No such areas of ADM were observed in Mist1creERT/+ mice (Table 2).
      Figure thumbnail gr3
      Figure 3Loss of ATRX sensitizes acinar tissue to recurrent pancreatic injury 3 days after cessation of pancreatic injury. (A) H&E staining of saline or cerulein (CIP) treated Mist1creERT/+ (WT) and Mist1creERT/+AtrxflΔ18 (AtrxflΔ18) mice. CIP-treated Mist1creERT/+ mice show vacuolation and intra-acinar edema relative to saline-treated mice. CIP-treated Mist1creERT/+AtrxflΔ18 mice demonstrated increased damage (black arrows) and putative ADM (white arrows) relative to CIP-treated Mist1creERT/+ mice. Magnification bar = 50 μm. (B) Trichrome stain of Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 tissue indicating fibrosis. Mist1creERT/+AtrxflΔ18 mice exhibit increased fibrosis (black arrow) and to a greater extent in female Mist1creERT/+AtrxflΔ18 mice. Magnification bar = 100 μm. (C) Quantification of fibrosis in pancreatic tissue from Mist1creERT/+ (WT) mice treated with saline (n = 4 female or 3 male) or cerulein (n = 4 female or 5 male) and Mist1creERT/+AtrxflΔ18 (AtrxflΔ18) mice treated with saline (n = 5 female or 3 male) or cerulein (n = 5 female and male). Male mice are denoted as black symbols and female mice as red symbols. Data were assessed using 2-way ANOVA with Tukey post hoc test. Error bars represent means ± standard error. When not considering sex (upper graph), no significant differences exist between groups. When sex of the mouse is considered (lower graph), female CIP-treated Mist1creERT/+AtrxflΔ18 mice are significantly different than all groups (*P < .05 vs all male mouse groups; **P < .01 vs female Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 saline-treated and female Mist1creERT/+ CIP-treated groups). Individual animals are denoted with black (male) or red (female) markers. (D) Representative immunofluorescent images of F4/80 accumulation in CIP-treated female Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 tissue (repeated 3 times). Arrows indicate macrophages. Sections were counterstained with DAPI. Magnification bar = 70 μm.
      Table 1Histology Grading Criteria
      ADM (based on worst pancreatic lobule)Fibrosis (based on trichrome stain)
      0None present0None present
      1>10% of lobule1<5% of tissue area
      210%–30% of lobule25%–10% of tissue area
      330%–50% of lobule310%–20% of tissue area
      4>50% of lobule4>20% of tissue area
      Inflammation
      0None present
      1Focal: small, contained areas
      2Mild: small, slightly diffuse areas
      3Moderate: diffuse areas
      4Severe: diffuse areas, significant presence throughout the tissue
      NOTE. Grading of pancreatic lesions: pancreatic lesions (ranging from ADM to PanIN grade 3) were quantified and classified into the following categories: ADM, PanIN grade 1, PanIN grade 2, PanIN grade 3, or PDAC based on morphologic characteristics including cell shape (cuboidal or columnar), presence of mucin accumulation, nuclear atypia, pseudostratification, and papillary or cribriform structure.
      Table 2Morphometric Analysis of Pancreatic Tissue After Recurrent Pancreatic Damage
      MaleFemale
      Mist1creERT/+ (5)Mist1creERT/+AtrxflΔ18(5)Mist1creERT/+ (4)Mist1creERT/+AtrxflΔ18 (5)
      Fibrosis0 ± 00 ± 00 ± 01.4 ± 0.51
      Inflammation0.2 ± 0.201.4 ± 0.250.75 ± 0.252.4 ± 0.40
      ADM0 ± 00.6 ± 0.250 ± 01.2 ± 0.37
      Total0.2 ± 0.22.0 ± 0.450.75 ± 0.255.0 ± 1.1a
      NOTE. (#) indicates n value; see methodology for scoring. Histopathologic assessment of pancreatic damage, as indicated by 3 factors: fibrosis, inflammation, and presence of ADM. Scores are represented on a grading scale from 0 to 4. Superscript letter “a” indicates groups that are statistically different (P < .01). Data were assessed using 2-way ANOVA and Tukey post hoc test.
      Figure thumbnail gr4
      Figure 4Mist1creERT/+AtrxflΔ18 pancreatic tissue shows increased apoptosis after recurrent CIP treatment. (A) Acinar cell–specific cleaved caspase-3 comparing Mist1creERT/+ (WT) and Mist1creERT/+AtrxflΔ18 (AtrxflΔ18) pancreatic tissue after saline (SAL) or cerulein (CIP) treatment. Quantification in pancreatic tissue from Mist1creERT/+ (WT) mice treated with saline (n = 4 female or 3 male) or cerulein (n = 4 female or 5 male) and Mist1creERT/+AtrxflΔ18 (AtrxflΔ18) mice treated with saline (n = 5 female or 4 male) or cerulein (n = 5 female and male). Data were assessed using 2-way ANOVA with Tukey post hoc test. Error bars represent means ± standard error. (B) Immunohistochemistry for cleaved caspase-3 in male and female Mist1creERT/+ (WT) or Mist1creERT/+AtrxflΔ18 pancreatic tissue 3 days after cessation of cerulein treatment. Arrows indicate apoptotic cells. Magnification bar = 50 μm.
      Figure thumbnail gr5
      Figure 5Mist1creERT/+AtrxflΔ18 pancreatic tissue shows reduced regeneration after recurrent CIP treatment. Tissue was examined 3 days after cessation of recurrent CIP treatment. (A) Western blot analysis for CPA, amylase, SOX9, and total ERK1/2 (tERK1/2; loading control). Increased accumulation of digestive enzymes was observed specifically in Mist1creERT/+ (WT) mice, whereas Mist1creERT/+AtrxflΔ18 (AtrxflΔ18) mice did not demonstrate similar accumulation. (B) Serum amylase levels in mice (n = 3–4 for each group). Data were assessed by using 2-way ANOVA with Tukey post hoc test. Bars represent mean ± standard error. No significant difference in body weight or amylase levels was observed between genotypes. (C) Immunohistochemistry for CPA in Mist1creERT/+ or Mist1creERT/+AtrxflΔ18 mice. CPA accumulation was decreased in female Mist1creERT/+AtrxflΔ18 mice (acinus is indicated by dotted line). (D) Representative immunofluorescence for SOX9 (green) expression was increased in male Mist1creERT/+AtrxflΔ18 and female Mist1creERT/+AtrxflΔ18 tissue (white arrows). Magnification bars = 50 μm. (E) Higher magnification images show SOX9 expression in duct, putative ADM (white arrows), and acinar cell (yellow arrowheads). Sections were co-stained for Ki-67 (red arrowheads) and counterstained with DAPI.
      Tissue histology also revealed a notable increase in ADM in Mist1creERT/+AtrxflΔ18 tissue based on the appearance of tubular complexes (Figure 3B). Western blot analysis (Figure 5A) for mature acinar cell markers amylase and pro-carboxypeptidase (CPA) confirmed a different response to recurrent CIP in Mist1creERT/+AtrxflΔ18 mice. Cerulein-treated Mist1creERT/+ mice exhibited increased accumulation of amylase and CPA compared with saline-treated controls, which was indicative of a regenerative response to injury. However, Mist1creERT/+AtrxflΔ18 mice did not show this recovery from injury (Figure 5A). This difference in enzyme accumulation was not due to increased release of enzymes in response to injury because circulating levels of amylase were not significantly different between genotypes (Figure 5B). Immunohistochemical analysis confirmed decreased CPA accumulation in both putative ADM as well as surrounding acinar cells (Figure 5C). Surprisingly, this decrease in CPA accumulation appeared to be more dramatic in cerulein-treated female Mist1creERT/+AtrxflΔ18 mice. Acini (delineated by a dotted line) show limited CPA accumulation in tissue sections from female mice (Figure 5C). We next examined expression of the progenitor/duct cell marker SOX9, a transcription factor that increases during regeneration and is required for ADM.
      • Kopp J.L.
      • von Figura G.
      • Mayes E.
      • Liu F.F.
      • Dubois C.L.
      • Morris J.P.
      • Pan F.C.
      • Akiyama H.
      • Wright C.V.
      • Jensen K.
      • Hebrok M.
      • Sander M.
      Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.
      In adult pancreatic tissue, SOX9 is expressed only in a subset of duct and centroacinar cells,
      • Kopp J.L.
      • von Figura G.
      • Mayes E.
      • Liu F.F.
      • Dubois C.L.
      • Morris J.P.
      • Pan F.C.
      • Akiyama H.
      • Wright C.V.
      • Jensen K.
      • Hebrok M.
      • Sander M.
      Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.
      • Prevot P.P.
      • Simion A.
      • Grimont A.
      • Colletti M.
      • Khalaileh A.
      • Van den Steen G.
      • Sempoux C.
      • Xu X.
      • Roelants V.
      • Hald J.
      • Bertrand L.
      • Heimberg H.
      • Konieczny S.F.
      • Dor Y.
      • Lemaigre F.P.
      • Jacquemin P.
      Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.
      which we confirmed by immunofluorescence on sections from saline-treated and cerulein-treated Mist1creERT/+ mice (Figure 5D and E). Conversely, increased SOX9 nuclear accumulation was observed in female and male Mist1creERT/+AtrxflΔ18 pancreatic tissue specifically after CIP treatment (Figure 5D), accumulating in ADM and some acinar cells (Figure 5D and E). Taken together, these data suggest loss of ATRX increased the sensitivity of acinar cells to recurrent cerulein exposure.
      These findings suggest that Mist1creERT/+AtrxflΔ18 mice will have increased susceptibility to oncogenic properties of mutated KRAS, because maintenance of the acinar phenotype constrains KRAS-induced transformation.
      • Shi G.
      • Direnzo D.
      • Qu C.
      • Barney D.
      • Miley D.
      • Konieczny S.F.
      Maintenance of acinar cell organization is critical to preventing Kras-induced acinar-ductal metaplasia.
      • von Figura G.
      • Fukuda A.
      • Roy N.
      • Liku M.E.
      • Morris J.P.
      • Kim G.E.
      • Russ H.A.
      • Firpo M.A.
      • Mulvihill S.J.
      • Dawson D.W.
      • Ferrer J.
      • Mueller W.F.
      • Busch A.
      • Hertel K.J.
      • Hebrok M.
      The chromatin regulator Brg1 suppresses formation of intraductal papillary mucinous neoplasm and pancreatic ductal adenocarcinoma.
      • Martinelli P.
      • Madriles F.
      • Canamero M.
      • Pau E.C.
      • Pozo N.D.
      • Guerra C.
      • Real F.X.
      The acinar regulator Gata6 suppresses KrasG12V-driven pancreatic tumorigenesis in mice.
      Therefore, we next introduced an inducible form of oncogenic KRAS (KrasLSL-G12D/+) into the Mist1creERT/+AtrxflΔ18 genotype (Mist1creERT/+KrasLSL-G12D/+AtrxflΔ18 hereafter referred to as MKA; Figure 6A). Cre-mediated induction of KRASG12D +/- deletion of Atrx was initiated in 2- to 4-month-old congenic Mist1creERT/+, Mist1creERT/+AtrxflΔ18, Mist1creERT/+KrasLSL-G12D/+, and MKA mice (Figure 6B), and acinar cell–specific loss of ATRX was confirmed by immunohistochemistry (Figure 6C). No significant differences were observed in body weight between groups during the course of the experiment (Figure 6D), and assessment of serum amylase levels at the time of death revealed no differences between genotypes (Figure 6E). Because of the presence of oral squamous papilloma tumors in KRASG12D-expressing mice, the experiment was terminated at 60 days after tamoxifen administration (data not shown). Gross morphologic examination revealed enlarged spleens in female Mist1creERT/+KrasLSL-G12D/+ and MKA mice (Figure 7).
      Figure thumbnail gr6
      Figure 6Induction of oncogenic KRAS with Atrx deletion. (A) Schematic of Atrx-deficient mouse model with oncogenic KRAS activation. (B) Experimental timeline for tamoxifen gavage in congenic Mist1creERT/+, Mist1creERT/+AtrxflΔ18, Mist1creERT/+KrasLSL-G12D/+, and MKA mice. Names used for each line within the figures are depicted in bold. (C) Immunohistochemistry for ATRX in acinar (black arrow) and duct (red arrow) cells of Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 mice, ATRX expression is limited to islet (open arrow; delineated by dotted line) and duct cells (red arrow). (D) Change in body weight (%) of mice after Atrx deletion ± oncogenic KRAS activation. Mouse genotypes include Mist1creERT/+ (WT; n = 13), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18; n = 12), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+; n = 17), and MKA (n = 16). Points are represented as mean weight ± standard error. (E) Serum amylase levels in WT (n = 7 male or 3 female), AtrxflΔ18 (n = 7 male or 5 female), KrasLSL-G12D/+ (n = 6 male or 5 female), and MKA (n = 8 male or 6 female) mice 60 days after Atrx deletion ± oncogenic KRAS activation. Data were assessed using 2-way ANOVA with Tukey post hoc test. Bars represent mean ± standard error.
      Figure thumbnail gr7
      Figure 7Gross morphology does not reveal significant differences on dissection. Mist1creERT/+ (WT), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+), or MKA mice. Although female MKA mice may show some edema, the only easily identified difference is splenomegaly (*), which consistently appeared in Mist1creERT/+KrasLSL-G12D/+ and MKA mice.
      Histologic examination revealed normal pancreatic morphology in Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 mice (Figure 8A). Mist1creERT/+KrasLSL-G12D/+ mice also showed typical pancreatic morphology for the most part, with a few instances of ADM or PanINs (Figure 8A). This is consistent with previous reports that activation of oncogenic KRASG12D in mature acinar cells was insufficient on its own to cause widespread pancreatic damage.
      • Morris JPt
      • Cano D.A.
      • Sekine S.
      • Wang S.C.
      • Hebrok M.
      Beta-catenin blocks Kras-dependent reprogramming of acini into pancreatic cancer precursor lesions in mice.
      Conversely, MKA mice demonstrated a variable phenotype based on sex (Figure 8A). Male MKA mice (n = 10) appeared phenotypically normal with negligible PanIN formation and few pockets of fibrosis relative to Mist1creERT/+KrasLSL-G12D/+ mice. Conversely, all female MKA mice (n = 6) developed PanINs and fibrosis, with some mice displaying extensive inflammation and fibrosis, along with disruptions in acinar cell organization consistent with a chronic pancreatitis phenotype (Figure 8A). Trichrome staining confirmed increased fibrosis only in female MKA pancreatic tissue relative to Mist1creERT/+KrasLSL-G12D/+ mice (Figure 9A), and Alcian blue histology confirmed increases in neoplastic PanIN lesions (Figure 9B). The tissue showed significant variability in fibrosis between mice in both MKA and Mist1creERT/+KrasLSL-G12D/+ cohorts (Figure 9C), although female MKA mice in general had increased damage (MKA = 15.2% ± 9.2% damaged area vs Mist1creERT/+KrasLSL-G12D/+ = 5.0% ± 1.6%), whereas male MKA mice had decreased damage (MKA = 0.2% ± 0.2% vs Mist1creERT/+KrasLSL-G12D/+ = 5.8% ± 3.8%). On the basis of a 2-way ANOVA, no significant differences were observed between any group regarding damaged (ie, lesion) area. However, quantification of lesion type (Figure 9D) indicated significantly more pre-cancerous lesions develop in female MKA mice relative to all groups except Mist1creERT/+KrasLSL-G12D/+ male mice. Quantification of overall pancreatic fibrosis, inflammation, and ADM, as described earlier, confirmed increased injury in MKA females relative to Mist1creERT/+KrasLSL-G12D/+ counterparts (Table 3).
      Figure thumbnail gr8
      Figure 8Loss of ATRX enhances KRASG12D’s ability to promote pre-cancerous lesions in female mice. (A) H&E images of pancreatic tissue from male and female Mist1creERT/+ (WT), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+), and MKA mice 60 days after tamoxifen treatment. Arrows indicate focal ADM or PanIN lesions. Dotted white line delineates significant lesion area from acinar tissue. Islets are indicated by I. Magnification bar = 200 μm. (B) Average percentage of lobules containing at least one instance of ADM, PanIN1, or PanIN2 in KrasLSL-G12D/+ (n = 7 male or 10 female) and MKA (n = 10 male or 6 female) mice. Data were assessed by using 2-way ANOVA with Tukey post hoc test. Representative examples of ADM, PanIN1, and PanIN2 lesions from female MKA mice. Magnification bars = 50 μm.
      Figure thumbnail gr9
      Figure 9Histology of pancreatic tissue after Atrx deletion. Representative (A) trichrome or (B) Alcian blue images of pancreatic tissue from male and female Mist1creERT/+ (WT), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+), and MKA mice 60 days after tamoxifen treatment. Increased tissue fibrosis was routinely observed in MKA females (arrows). Magnification bar = 200 μm. Increased fibrosis (open arrow) and duct metaplasia are observed in female MKA tissue. Quantification of (C) damaged area as percentage of total pancreatic area based on appearance of PanINs or fibrosis, or (D) number of ADM and PanINs (lesions) observed within Mist1creERT/+ (WT; n = 8 male or 5 female), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18; n = 8 male or 5 female), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+; n = 7 male or 10 female), and MKA (n = 10 male or 6 female) mice 60 days after tamoxifen treatment. Number of ADM and PanINs (lesions) was significantly increased in MKA females compared with all other groups except male Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+) mice. Data were assessed by using 2-way ANOVA with Tukey post hoc test. Bars represent mean ± standard error; *P < .05.
      Table 3Morphometric Analysis of Pancreatic Tissue 60 Days After Activation of KRASG12D and Loss of Atrx
      Mist1creERT/+Mist1creERT/+AtrxflΔ18Mist1creERT/+KrasLSL-G12D/+MKA
      Male (8)Female (5)Male (8)Female (5)Male (7)Female (10)Male (10)Female (6)
      Fibrosis0 ± 00 ± 00 ± 00 ± 01 ± 0.660.7 ± 0.470 ± 01.83 ± 0.48
      Inflammation0 ± 00 ± 00.14 ± 0.140.33 ± 0.211 ± 0.541 ± 0.520.2 ± 0.132.17 ± 0.70
      ADM0 ± 00.2 ± 0.20 ± 00 ± 01.57 ± 0.651.5 ± 0.450.4 ± 0.163 ± 0.45
      Total0 ± 0a0.2 ± 0.2a0.14 ± 0.14a0.33 ± 0.21a3.57 ± 1.8a,b3.2 ± 1.4a,b0.6 ± 0.27a7 ± 1.57b
      NOTE. (#) indicates n value; see methodology for scoring. Histopathologic assessment of pancreatic damage, as indicated by 3 factors: fibrosis, inflammation, and presence of ADM. Scores are represented on a grading scale from 0 to 4. Superscript letters “a” and “b” indicate groups that are statistically different (P < .01). Data were assessed using 2-way ANOVA and Tukey post hoc test.
      To assess PanIN formation within Mist1creERT/+KrasLSL-G12D/+ and MKA pancreatic tissue, the percentage of pancreatic lobules containing at least one instance of each lesion type (ranging from ADM to PanIN3) was quantified on H&E stained sections (Figure 8B) and statistically compared by 2-way ANOVAs (Table 4). MKA female mice had significantly fewer lobules consisting only of normal acini relative to all other groups (Table 4, P < .05). Whereas both Mist1creERT/+KrasLSL-G12D/+ and MKA mice exhibited ADM, the incidence of PanIN1 in female MKA mice was 2.5-fold higher (16.21% ± 8.3% of lobules; n = 10) relative to female Mist1creERT/+KrasLSL-G12D/+ mice (6.52% ± 3.5%; n = 6), and female MKA mice contained significantly more lobules with PanIN2 lesions than all other genotypes and sexes (Figure 8B, Table 4; P < .01). Interestingly, histologic analysis of male MKA mice revealed no PanIN1 or PanIN2 lesions (Figure 8B, Table 4).
      Table 4Classification of ADM and PanIN Lesions 60 Days After Activation of KRASG12D and Loss of Atrx
      Percent of lobules determined by highest lesion grade
      GenotypeSexNormalADMPanIN1PanIN2
      KRASLSL-G12DMale (7)83.1 ± 7.810.8 ± 4.24.9 ± 3.61.3 ± 1.3
      Female (10)60.1 ± 8.233.3 ± 6.36.5 ± 3.50 ± 0
      MKAMale (10)81.2 ± 7.818.8 ± 7.80 ± 00 ± 0
      Female (6)46.9 ± 11.9
      Difference from all other groups (P < .05).
      25.2 ± 5.716.2 ± 8.3
      Difference from male MKA mice (P < .05).
      11.7 ± 6.3
      Difference from all other groups (P < .05).
      NOTE. (#) indicates n values. Data were assessed using 2-way ANOVA and Tukey post hoc test. These data are presented in Figure 8B.
      a Difference from all other groups (P < .05).
      b Difference from male MKA mice (P < .05).
      To determine whether presumptive ADM and PanINs were arising from ATRX null acinar cells, we examined the expression of transcription factors involved in ADM. SOX9 (Figure 10A and B) and PDX1 (data not shown) accumulation was assessed by immunofluorescence and immunohistochemistry, respectively. As observed earlier, no SOX9+ acinar cells were observed in Mist1creERT/+ and Mist1creERT/+AtrxflΔ18 mice. Similarly, Mist1creERT/+KrasLSL-G12D/+ tissue was devoid of SOX9+ acinar cells, although pockets of putative SOX9+ ADM were observed (Figure 10A). Whereas male MKA mice showed few SOX9-expressing cells, SOX9+ cells and ADM were readily apparent in female MKA mice (Figure 10A and B). This widespread SOX9 accumulation in both ADM and acinar cells adjacent to areas of damage suggested SOX9 expression precedes ADM, which is in support of previous studies indicating SOX9 expression is required for ADM. Similar increases for PDX1 were observed in female MKA tissue, with PDX1+ cells readily observed in ADM and PanINs (data not shown) compared with all other genotypes. In many cases, cells within ADMs and PanIN lesions also stained for Ki67 (Figure 10B), indicating an increase in proliferation in these areas. Quantification of Ki67+ acinar cells showed no significant difference between genotypes, suggesting that proliferation likely occurs after ADM (Figure 10C).
      Figure thumbnail gr10
      Figure 10PanIN lesions in Mist1creERT/+AtrxflΔ18 mice are derived from ATRX– acinar cells undergoing ADM. (A) Immunofluorescent images for SOX9 in male and female Mist1creERT/+ (WT), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+), and MKA mice 60 days after tamoxifen treatment. Insets show nuclear localization of SOX9 in putative ADM in MKA mice. (B) Co-immunofluorescence of putative ADM in female MKA pancreatic tissue shows SOX9 nuclear accumulation in proliferating cells based on Ki67 accumulation (red). White arrows indicate cells expressing Ki67 and SOX9. Sections were counterstained for DAPI. Magnification bars = 50 μm. (C) Quantification of Ki67+ acinar cells in Mist1creERT/+, Mist1creERT/+AtrxflΔ18, Mist1creERT/+KrasLSL-G12D/+, and MKA mice. Bars represent mean ± standard error. N = 3 for each group. (D) Immunohistochemistry for ATRX in putative ADM and PanINs in male and female Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+) and MKA mice. ATRX+ lesions are identified in Mist1creERT/+KrasLSL-G12D/+ mice (black arrow), whereas female MKA revealed both ATRX+ (*) and ATRX– (**) cells within the PanINs. ATRX+ cells are found in ducts (D) and islets (I) of MKA males and in interstitial fibroblasts in MKA females. (E) Quantification of lesions with at least 1 ATRX+ cell (black) or no ATRX+ cells (gray) in Mist1creERT/+ (WT; n = 3 for male and female), Mist1creERT/+AtrxflΔ18 (AtrxflΔ18; n = 3 for male and female), Mist1creERT/+KrasLSL-G12D/+ (KrasLSL-G12D/+; n = 3 for male or n = 6 for female), and MKA (n = 4 for male or n = 3 for female) mice. Bars represent mean % ± standard error.
      This expression pattern of SOX9 suggests normal progression through ADM in MKA mice but does not indicate whether ADM arises from ATRX-positive cells. Therefore, ATRX accumulation was examined to confirm an acinar cell origin for ADM and PanINs. All presumptive ADM observed in male Mist1creERT/+KrasLSL-G12D/+ mice tissue was ATRX positive. Similarly, all ADM and PanINs in female Mist1creERT/+KrasLSL-G12D/+ tissue contained exclusively ATRX-positive cells. In Mist1creERT/+KrasLSL-G12D/+AtrxflΔ18/x female mice, which harbor one transcriptionally active copy of the Atrx gene, ADM and PanINs contained mixed populations of ATRX+ and ATRX– cells. Fifty-nine percent ± 17% of lesions contained at least 1 ATRX+ cell, with the other 41% ± 17% lesions containing only ATRX-negative cells (Figure 10D and E). The ATRX– lesions likely arise from cells in which the non-targeted Atrx gene has been silenced as part of X chromosome inactivation. Although some ATRX+ cells were observed in PanINs and ADMs of MKA mice, the majority of lesions were completely devoid of ATRX expression in male (82% ± 7%) and female (73% ± 10%) mice. Because ATRX is ubiquitously expressed, the absence of ATRX in PanINs and ADM suggests the origin of these lesions in MKA mice as acinar cells.
      Finally, to determine whether sex-specific susceptibility conferred by the absence of ATRX on KRAS mice translates to humans, we queried the International Cancer Genome Consortium (dcc.icgc.org) database for ATRX mutations (Figure 11). The International Cancer Genome Consortium database includes whole genome sequence analysis for 729 patients from Australian and Canadian tumor sequencing studies and consists of 324 female patients (42%) and 405 male patients (53%). Gender was not identified in 5% of the patients. Therefore, the proportion of PDAC patients reported as female was 0.44. KRAS mutations were identified in 591 patients, with a similar ratio of male (55%) to female (45%) patients. Two hundred sixty-eight mutations were observed within the ATRX gene, encompassing 145 (∼19%) PDAC patients, with 68% of ATRX mutations in female patients. Therefore, the proportion of PDAC patients carrying ATRX mutations that were female was 0.68. The difference in proportions is significant, χ2(1, N = 729) = 41.633; P < .00001 (Figure 11B), suggesting ATRX mutations are related to the sex of the patient.
      Figure thumbnail gr11
      Figure 11Mutation analysis on PDAC patients based on data from the International Cancer Genome Consortium. (A) Comparison of gender breakdown for all PDAC cases, or for mutations in ATRX, known driver mutations for PDAC (KRAS, CDNK2/P16), or for DAXX. Impact ATRX mutations refer to those mutations that occur with the protein coding region and are predicted to have a negative impact on protein function. (B and C) Chi-squared analyses determining if ATRX mutations and sex are independent variables in PDAC (B) or pancreatic neuroendocrine tumors (PNETs; C).
      Most ATRX mutations in both sexes are found in non-coding regions, and the impact on expression is unknown. However, 8 patients harbor ATRX mutations with a predicted impact on protein function. All but one of these mutations occur in female patients, suggesting a sex-specific susceptibility in the human patient population. Pancreatic neuroendocrine and glioblastoma patient populations show 46% and 42% of the identified ATRX mutations are in female populations. Conversely, 60% of the ATRX mutations found in the pediatric brain tumor population occur in female patients. However, χ2 analysis indicates the proportions of ATRX mutations are independent of sex (Figure 11C and data not shown). Interestingly, mutations in DAXX, the partner for ATRX in depositing H3.3 variant histones into chromatin,
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      • Elsaesser S.J.
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      Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres.
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      • Banaszynski L.A.
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      • Dewell S.
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      • Wen D.
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      Distinct factors control histone variant H3.3 localization at specific genomic regions.
      are rare in PDAC and show no sex bias. Other common mutations linked to PDAC, including P16/CDNK2 (Figure 11A) and SMAD4 (data not shown), showed no sex bias.

      Discussion

      Pancreatic ductal adenocarcinoma is currently the third leading cause of cancer-related deaths in North America (www.pancan.org). Five-year survival rates have increased only marginally in the last 30 years because of late stage of diagnosis and insensitivity to conventional chemotherapeutics. Therefore, detecting factors that increase sensitivity of pancreatic tissue to the oncogenic properties of mutated KRAS is important in identifying alternative therapeutic and diagnostic options. In this study, we have shown that acinar-specific loss of ATRX, a chromatin remodelling protein, affects the tissue’s response to injury and constitutive mutant KRAS activity. Using a novel mouse line that allows for acinar-specific ablation of Atrx, we show loss of ATRX increased the sensitivity for pancreatic injury. In addition, we showed loss of Atrx dramatically enhanced the ability of oncogenic KRAS to promote precancerous lesions in the pancreas. Importantly, these effects were observed in a sex-specific fashion, with only female mice displaying sensitivity to loss of ATRX. Our results also suggest that loss of ATRX may reduce the sensitivity to oncogenic KRAS in male mice. This is evidence of a sex-specific susceptibility factor and suggests stratification of PDAC based on their molecular profile may identify new targets for therapy and diagnosis.
      Although we have not defined a role for ATRX in normal acinar cell physiology, it appears ATRX is dispensable for maintaining the acinar cell phenotype in the adult. This is consistent with previous studies that identified roles for ATRX only in mitotically active tissue, where loss of ATRX maintains genomic stability and regulates cell cycle processes including proper chromosome segregation during mitosis.
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      ATRX dysfunction induces replication defects in primary mouse cells.
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      Loss of ATRX leads to chromosome cohesion and congression defects.
      We did observe small, yet significant increases in acinar cell apoptosis, dsDNA damage, and proliferation in Mist1creERT/+AtrxflΔ18 mice, suggesting during a longer period of time (>2 months), the absence of ATRX may lead to more overt damage. Acinar cell division in the adult rodent pancreas is <2%,
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      Induction of pancreatic acinar cell proliferation by thyroid hormone.
      which is similar to the observed rates of apoptosis and dsDNA damage. However, targeted ablation of other factors, including Xbp1
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      • Lee A.H.
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      Extensive pancreas regeneration following acinar-specific disruption of Xbp1 in mice.
      and Pdx1,
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      PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance.
      results in rapid loss of acinar tissue, so acinar cell division is clearly not a prerequisite for development of overt pancreatic pathologies. Mild damage in acinar tissue with ATRX loss suggested acinar cells may be sensitive to other factors known to promote pancreatic pathologies.
      When exposed to recurrent cerulein treatment, only Mist1creERT/+AtrxflΔ18 mice showed fibrosis, inflammatory cell infiltration, and regions of ADM, with the effects more dramatic in female mice. It is unclear whether loss of ATRX leads to increased damage or if regeneration is impaired in Mist1creERT/+AtrxflΔ18 mice. Unpublished work from our laboratory using an acute pancreatitis regimen indicated similar amounts of damage in control and Mist1creERT/+AtrxflΔ18 mice immediately after injury, suggesting loss of ATRX impairs the regenerative process after injury, and is consistent with studies in which other chromatin remodelling proteins (EZH2
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      EZH2 couples pancreatic regeneration to neoplastic progression.
      and BMI1
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      Bmi1 is required for the initiation of pancreatic cancer through an Ink4a-independent mechanism.
      ) are required for proper pancreatic regeneration. It is possible that defects from Atrx loss become more widespread once injury is induced, and increased DNA damage and/or replicative defects provide a barrier to acinar tissue regeneration. However, we found no differences in apoptosis and proliferation in cerulein-treated mice, suggesting these are not contributing factors through which ATRX loss promotes damage.
      The combination of Atrx deletion with oncogenic KRAS activation produced extensive fibrosis and inflammation, pancreatic damage indicative of chronic pancreatitis, as well as PanIN lesions up to grade 2. In this instance, damage was exclusive to female mice. Consistent with previous studies,
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      we observed minimal fibrosis and PanIN lesions in KRAS mice that indicate oncogenic KRAS activation in adult mice required another stimulus, such as chronic pancreatitis, to produce invasive PDAC. The pancreatic injury observed in the female MKA mice suggests that chronic pancreatitis occurs in these mice, and the inflammation observed may contribute to increased susceptibility to active KRAS. Loss of function mutations in ATRX have been identified in other cancer types, most notably those involving up-regulation of the alternative lengthening of telomeres pathway.
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      Loss of ATRX, genome instability, and an altered DNA damage response are hallmarks of the alternative lengthening of telomeres pathway.
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      Loss of ATRX or DAXX expression and concomitant acquisition of the alternative lengthening of telomeres phenotype are late events in a small subset of MEN-1 syndrome pancreatic neuroendocrine tumors.
      Mutations in ATRX or binding partner DAXX are often observed in pancreatic neuroendocrine tumors and glioblastomas, but neither cancer shows a gender preference with or without ATRX mutation. Studies examining PDAC tumors confirmed an absence of alternative lengthening of telomeres in every case,
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      • Papadopoulos N.
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      Altered telomeres in tumors with ATRX and DAXX mutations.
      and PDAC is typically characterized by telomere attrition.
      • van Heek T.
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      • Offerhaus G.J.
      • McCarthy D.M.
      • Goggins M.
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      K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: a molecular panel correlates with and supplements cytologic diagnosis.
      Therefore, we suggest that ATRX is affecting an alternative pathway in PDAC, possibly in a DAXX-independent manner. ATRX interacts with enhancer of zeste homologue 2 (EZH2), a member of polycomb repressor complex 2, leading to altered gene expression,
      • Cardoso C.
      • Timsit S.
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      Specific interaction between the XNP/ATR-X gene product and the SET domain of the human EZH2 protein.
      and loss of EZH2 also increases sensitivity to oncogenic KRAS.
      • Mallen-St Clair J.
      • Soydaner-Azeloglu R.
      • Lee K.E.
      • Taylor L.
      • Livanos A.
      • Pylayeva-Gupta Y.
      • Miller G.
      • Margueron R.
      • Reinberg D.
      • Bar-Sagi D.
      EZH2 couples pancreatic regeneration to neoplastic progression.
      The incidence of human PDAC between sexes is relatively equal, with approximately the same number of cases occurring in men and women (Canadian Cancer Statistics, 2016). Assessment of International Cancer Genomic Consortium database revealed ATRX single nucleotide polymorphisms in almost 20% of PDAC cases (145/729), although most were in non-coding regions of the gene. However, whether in the coding or non-coding regions, ATRX mutations had a higher than expected frequency in female patients, even when taking into consideration that it is an X-linked gene. Therefore, it is possible that ATRX loss defines a unique subtype of PDAC, in which female patients are more susceptible, or that loss of ATRX function in male patients does not allow progression through to a PDAC phenotype. Although we have observed decreased acinar cell sensitivity to oncogenic KRAS in male mice, female MKA mice show significantly increased progression to PanIN1 and PanIN2 lesions, and the mechanisms underlying the extensive pancreatic damage specifically in female MKA mice are unclear. It is possible that loss of ATRX enhances KRAS activity and leads to altered hormonal signalling. Sex hormone receptors, including estrogen receptors, play a role in the progression of other cancers such as colorectal cancer.
      • Nussler N.C.
      • Reinbacher K.
      • Shanny N.
      • Schirmeier A.
      • Glanemann M.
      • Neuhaus P.
      • Nussler A.K.
      • Kirschner M.
      Sex-specific differences in the expression levels of estrogen receptor subtypes in colorectal cancer.
      However, the Atrx gene is located on the X chromosome and is a target of X inactivation. Therefore, it would be expected that female mice heterozygous (AtrxflΔ18/x) for the mutant Atrx allele would also show similar effects because approximately half of the acinar cells lose ATRX expression. Immunohistochemistry for ATRX confirmed that at least a portion of acinar cells in heterozygous AtrxflΔ18/x mice did lose ATRX expression (data not shown), but these mice did not demonstrate increased damage or susceptibility to oncogenic KRAS. These results suggest complete loss of ATRX is required for enhanced KRAS activity and pancreatic damage to occur in female mice. However, such a model also does not account for decreased sensitivity in male MKA mice.
      Sex-specific mechanisms could also be explained by a difference in inflammatory response. It is possible that female AtrxflΔ18/ flΔ18 mice are more susceptible to factors promoting inflammation. During recurrent pancreatic injury, AtrxflΔ18/ flΔ18 mice showed increased inflammation in comparison with male counterparts, resulting in higher levels of damage. In combination with oncogenic KRAS, increased inflammation in AtrxflΔ18/ flΔ18 mice may amplify KRAS activity and activation of downstream pathways, including MAPK and PI3K-PDK1-Akt signaling, leading to increased cell survival and proliferation. Accordingly, increased KRASG12D activity by inflammatory cytokines (nuclear factor kappa B, interleukin 6) has been demonstrated previously.
      • Daniluk J.
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      • Chu J.
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      • Wang H.
      • Ji B.
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      An NF-kappaB pathway-mediated positive feedback loop amplifies Ras activity to pathological levels in mice.
      • Baumgart S.
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      Inflammation-induced NFATc1-STAT3 transcription complex promotes pancreatic cancer initiation by KrasG12D.
      The presence of an inflammatory response in female AtrxflΔ18/ flΔ18 mice, which is not observed in male AtrxflΔ18/y mice, could lead to increased damage.
      It is also possible that sex-specific differences exist regarding the function of SOX9. Previous work suggests SOX9 is required for initiation of ADM,
      • Kopp J.L.
      • von Figura G.
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      • Sander M.
      Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma.
      and we show increased SOX9 accumulation in female MKA acinar cells surrounding damage. Increased susceptibility in female MKA mice could include sex-specific hormonal or inflammatory pathways that provoke increased SOX9 expression. As mentioned, female AtrxflΔ18/ flΔ18 mice have an amplified inflammatory response, and inflammatory signaling pathways can influence SOX9 expression during development.
      • Akiyama H.
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      Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice.
      Alternatively, hormonal factors could play a role in sex-specific Sox9 regulation. Recently, up-regulation of estrogen receptor α-receptor activity in breast cancer cells has been associated with increased SOX9 expression, although this study occurs in the context of estrogen deprivation.
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      It would be interesting to observe the long-term effects of ATRX deletion on oncogenic KRAS-mediated PDAC formation. Because of the prevalence of tumors developing in the oral mucosa, we were forced to kill MKA mice before overt PDAC development. These tumors likely arise because of the expression of Mist1creERT in other tissues, and we are currently generating AtrxflΔ18/ flΔ18 mice with a pancreas-specific inducible cre-recombinase, which will allow for longer-term analysis. It is possible that having only a single copy of Mist1 contributes to the Mist1creERT/+AtrxflΔ18 and MKA phenotypes. Although previous studies
      • Karki A.
      • Humphrey S.E.
      • Steele R.E.
      • Karki A.
      • Humphrey S.E.
      • Steele R.E.
      • Hess D.A.
      • Taparowsky E.J.
      • Konieczny S.F.
      Silencing Mist1 gene expression is essential for recovery from acute pancreatitis.
      and unpublished work from our laboratory demonstrated no difference in the phenotype between mice heterozygous or wild-type (WT) for MIST1 expression, using a different cre-recombinase (such as Ptf1acreERT) would rule out any contribution of Mist1 haploinsufficiency to the results observed in this study.
      In summary, we identified that loss of ATRX enhanced pancreatic injury and susceptibility to KRAS-mediated pancreatic damage. Potential gender-specific factors within AtrxflΔ18/ flΔ18 mice (including hormonal factors or increased inflammation) provide an additional driving factor for KRAS activity and pancreatic damage, leading to a female-specific phenotype.

      Materials and Methods

      Animal Generation and Cre Induction

      Mouse experiments were approved by the Animal Care and Use Committee at Western University (Protocol #2017-001). All mice used in this study were maintained in a C57Bl6 background. Mice expressing creERT from the Mist1 locus (Mist1creERT/+)
      • Shi G.
      • Direnzo D.
      • Qu C.
      • Barney D.
      • Miley D.
      • Konieczny S.F.
      Maintenance of acinar cell organization is critical to preventing Kras-induced acinar-ductal metaplasia.
      were crossed with mice harboring an Atrx allele with exon 18 flanked by loxP sites,
      • Berube N.G.
      • Mangelsdorf M.
      • Jagla M.
      • Vanderluit J.
      • Garrick D.
      • Gibbons R.J.
      • Higgs D.R.
      • Slack R.S.
      • Picketts D.J.
      The chromatin-remodeling protein ATRX is critical for neuronal survival during corticogenesis.
      producing male (Mist1creERT/+AtrxflΔ18/y) and female (Mist1creERT/+AtrxflΔ18/fl Δ18) mice, collectively referred to as Mist1creERT/+AtrxflΔ18. Mist1creERT/+AtrxflΔ18 mice were crossed to mice containing an inducible oncogenic KRAS (loxP-STOP-loxP (LSL)-KRASG12D)
      • Jackson E.L.
      • Willis N.
      • Mercer K.
      • Bronson R.T.
      • Crowley D.
      • Montoya R.
      • Jacks T.
      • Tuveson D.A.
      Analysis of lung tumor initiation and progression using conditional expression of K-ras.
      to produce Mist1creERT/+KrasLSL-G12D/+AtrxflΔ18 mice (referred to as MKA). Furthermore, female mice containing one (Mist1creERT/+AtrxflΔ18/x) or two (Mist1creERT/+Atrxx/x) copies of the Atrx allele showed no obvious phenotypic differences and were combined as a single Mist1creERT/+ control group. Female mice expressing KRASG12D that were heterozygous (Mist1creERT/+KrasLSL-G12D/+AtrxflΔ18/x) or homozygous for Atrx (Mist1creERT/+KrasLSL-G12D/+Atrxx/x) also showed no obvious morphologic differences and were collectively referred to as Mist1creERT/+KrasLSL-G12D/+ mice.
      Tamoxifen (Sigma-Aldrich, St Louis, MO; cat. #T5648) was administered through oral gavage (2 mg/mouse) 3 times over 5 days. This resulted in >95% recombination in acinar cells and no recombination in duct cells.
      • Johnson C.L.
      • Peat J.M.
      • Volante S.N.
      • Wang R.
      • McLean C.A.
      • Pin C.L.
      Activation of protein kinase C delta leads to increased pancreatic acinar cell de-differentiation in the absence of MIST1.
      Mice were monitored for 60 days from first tamoxifen gavage, and body weight was measured weekly.

      Cerulein Induced Pancreatitis

      To induce recurrent pancreatic injury, control (Mist1creERT/+) and Mist1creERT/+AtrxflΔ18 mice were given intraperitoneal injections of saline or cerulein (75 μg/kg body weight; Sigma-Aldrich; cat. #C9026) twice daily for 11 days, followed by a 3-day recovery period. Mice were weighed daily throughout the injury protocol. Mice were killed and processed for histologic, molecular, biochemical, and blood serum analysis.
      • Fazio E.N.
      • Young C.C.
      • Toma J.
      • Levy M.
      • Berger K.R.
      • Johnson C.L.
      • Mehmood R.
      • Swan P.
      • Chu A.
      • Cregan S.P.
      • Dilworth F.J.
      • Howlett C.J.
      • Pin C.L.
      Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.
      Serum amylase was quantified by using Phadebas tablets (Magle Life Sciences, Lund, Sweden; cat. #1302) following manufacturer’s instructions.

      Histology, Immunohistochemistry, and Immunofluorescent Analysis

      The head of the pancreas was used for paraffin sections, and cryostat sections were obtained from the middle portion of pancreas. Tissue was washed 2 times in phosphate-buffered saline and then dehydrated through a series of alcohol washes for embedding into paraffin. Paraffin sections (5 μm) were stained by using standard H&E, Alcian blue (Mucin Stain; Abcam Inc, Cambridge, MA; cat. #ab150662) or Masson’s trichrome stain (Abcam Inc; cat. #ab150686) protocols. Sections were imaged by using the Aperio CS2 Digital Scanner and Aperio ImageScope software (Leica Biosystems Imaging Inc, San Diego, CA). Total tissue area was quantified by using the Fiji software,
      • Schindelin J.
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      • Frise E.
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      • Longair M.
      • Pietzsch T.
      • Preibisch S.
      • Rueden C.
      • Saalfeld S.
      • Schmid B.
      • Tinevez J.Y.
      • White D.J.
      • Hartenstein V.
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      Fiji: an open-source platform for biological-image analysis.
      and area of damage was quantified as a percentage of total area. Levels of pancreatic damage were assessed by using a grading scale based on 3 factors: fibrosis, inflammation, and presence of acinar to ductal metaplasia. Tissue sections were scored by multiple individuals blinded to mouse genotypes on a scale from 0 to 4. Descriptions of each score can be found in Table 1 along with a description of scoring PanIN lesions.
      For immunohistochemical analysis, paraffin tissue sections were stained by using the ABC staining system (Santa Cruz Biotechnology Inc, Dallas, TX) or the VectaStain ABC HRP kit with ImmPACT DAB Peroxidase (HRP) Substrate (Vector Laboratories, Brockville, ON, Canada; cat. #SK-4105) according to kit instructions. Cleaved caspase-3 (rabbit 1:100; Cell Signaling Technology, Danvers, MA; cat. #966455) staining was completed by using the Ventana Discovery Ultra XT autostainer (Ventana Medical Systems Inc, Tucson, AZ). Primary antibodies used are specific to ATRX (rabbit; diluted 1:100 in 1.5% mouse blocking serum in phosphate-buffered saline; Santa Cruz Biotechnology Inc; cat. #sc15408), PDX1 (rabbit; 1:1000; Abcam Inc; cat. #ab47267), Ki67 (rabbit 1:500; Abcam Inc; cat. #ab15580). For immunofluorescence, cryostat sections were processed as previously described.
      • Fazio E.N.
      • Young C.C.
      • Toma J.
      • Levy M.
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      • Johnson C.L.
      • Mehmood R.
      • Swan P.
      • Chu A.
      • Cregan S.P.
      • Dilworth F.J.
      • Howlett C.J.
      • Pin C.L.
      Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.
      Primary antibodies used were specific to ATRX (rabbit; diluted 1:100 in blocking solution; Santa Cruz Biotechnology Inc), MIST1 (rabbit; 1:500),
      • Pin C.L.
      • Bonvissuto A.C.
      • Konieczny S.F.
      Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis.
      β-catenin (mouse; 1:500; BD Biosciences, Mississauga, ON, Canada; cat. #610153, lot#5121508), SOX9 (rabbit; 1:500; MilliporeSigma, Etobicoke, ON, Canada; cat. #AB5535, lot#3018860), insulin (mouse 1:500; Sigma; cat. #I2018; lot#092K4841), or γH2AX (rabbit; 1:200; Santa Cruz Biotechnology Inc; cat. #sc-101696, lot#12613). Secondary antibodies used include anti-rabbit FITC (cat. #111-545-003, lot#125266) and anti-mouse FITC (cat. #115-025-003, lot#125278; 1:250; Jackson ImmunoResearch, West Grove, PA). Sections were counterstained with 4',6-diamidino-2-phenylindole and imaged using a Leica DM5500B microscope with LAS V4.4 software (Leica Microsystems Ltd, Wetzlar, Germany).

      Protein Isolation and Western Blot Analyses

      Protein was isolated from the middle portion of the pancreata and homogenized on ice using a Potter Elvehiem Homogenizer in extraction buffer (50 mmol/L Tris [pH 7.2], 5 mmol/L MgCl2, 1 mmol/L CaCl2, 1% NP-40, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, 10 mmol/L NaF, 2 mmol/L NaVO4, 150 nmol/L aprotinin, 10 μmol/L, pepstatin, 50 μmol/L leupeptin).
      • Fazio E.N.
      • Young C.C.
      • Toma J.
      • Levy M.
      • Berger K.R.
      • Johnson C.L.
      • Mehmood R.
      • Swan P.
      • Chu A.
      • Cregan S.P.
      • Dilworth F.J.
      • Howlett C.J.
      • Pin C.L.
      Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.
      Homogenates were sonicated for 20 seconds on ice (level 4 Fisher Sonic Dismemberator) and centrifuged 10 minutes at 4°C at 14,000g. Supernatants were taken and frozen at –80°C until used. Isolated protein was resolved by sodium dodecylsulfate–gel electrophoresis in 10% acrylamide gels and transferred to a polyvinylidene difluoride membrane (Bio-Rad; cat. #162-0177) for Western blot analyses.
      • Pin C.L.
      • Bonvissuto A.C.
      • Konieczny S.F.
      Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis.
      Primary antibodies were specific for total MAPK (tERK1/2) (rabbit 1:2500 in 5% BSA-0.1% Tween20; Cell Signaling Technology; cat. #9102, lot #26), carboxypeptidase (CPA) (rabbit 1:1000 in 5% NFDM; R&D Systems, Minneapolis, MN; cat. #AF2765, lot #wo00117071), and amylase (rabbit 1:1000 in 5% NFDM; Abcam; cat. #ab21156). After overnight incubation, blots were incubated in secondary antibody (anti-rabbit HRP, 1:10,000; Jackson Labs, Bar Harbor, ME; cat. #111-035-144) diluted in 5% NFDM for 1 hour at room temperature. Blots were developed by using Clarity Western blot ECL kit (Biorad; cat. #1705061) and visualized by using the VersaDoc system with Quantity One 1-D analysis software (Bio-Rad).

      TUNEL Assay

      To assess apoptosis, cryostat sections were processed using the In Situ Cell Death detection kit (Roche, Laval, QC, Canada; cat. #11684795910) per manufacturer’s directions. Sections were counterstained with DAPI. The number of TUNEL-positive cells was quantified using 7 random fields of view from each mouse and calculated as percentage of TUNEL-positive cells compared with DAPI counts.

      Statistical Analysis

      In all cases, data were analyzed for significance by using an unpaired, two-tailed t test or 2-way analysis of variance (ANOVA) with Tukey post hoc test. Values are depicted as means ± standard error of the mean. Significance is considered P < .05.

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      Linked Article

      • ATRX Links Chromatin Remodeling to Inflammation and Tumorigenesis in the Pancreas
        Cellular and Molecular Gastroenterology and HepatologyVol. 7Issue 1
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          A link between inflammation and cancer is clear, yet the details of how each influences the other remain incomplete. Young et al1 have added new insight implicating a link between inflammatory responses and chromatin remodeling in susceptibility to neoplastic changes in the pancreas. Studying mouse models of pancreatic inflammation and of pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, Young et al1 found that the ATRX chromatin remodeling protein is critical both to suppress pancreatitis in response to damage and to suppress progression to precancerous neoplastic lesions in the presence of the KrasG12D oncogene.
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