ELF3 suppresses gallbladder cancer development through downregulation of the EREG/EGFR/mTOR complex 1 signalling pathway.

The prognosis of gallbladder cancer (GBC) remains poor, and a better understanding of GBC molecular mechanisms is important. Genome sequencing of human GBC has demonstrated that loss-of-function mutations of E74-like ETS transcription factor 3 (ELF3) are frequently observed, with ELF3 considered to be a tumour suppressor in GBC. To clarify the underlying molecular mechanisms by which ELF3 suppresses GBC development, we performed in vivo analysis using a combination of autochthonous and allograft mouse models. We first evaluated the clinical significance of ELF3 expression in human GBC tissues and found that low ELF3 expression was associated with advanced clinical stage and deep tumour invasion. For in vivo analysis, we generated Pdx1-Cre; KrasG12D ; Trp53R172H ; Elf3f/f (KPCE) mice and Pdx1-Cre; KrasG12D ; Trp53R172H ; Elf3wt/wt (KPC) mice as a control and analysed their gallbladders histologically. KPCE mice developed larger papillary lesions in the gallbladder than those developed by KPC mice. Organoids established from the gallbladders of KPCE and KPC mice were analysed in vitro. RNA sequencing showed upregulated expression of epiregulin (Ereg) in KPCE organoids, and western blotting revealed that EGFR/mechanical targets of rapamycin complex 1 (mTORC1) were upregulated in KPCE organoids. In addition, ChIP assays on Elf3-overexpressing KPCE organoids showed that ELF3 directly regulated Ereg. Ereg deletion in KPCE organoids (using CRISPR/Cas9) induced EGFR/mTORC1 downregulation, indicating that ELF3 controlled EGFR/mTORC1 activity through regulation of Ereg expression. We also generated allograft mouse models using KPCE and KPC organoids and found that KPCE organoid allograft tumours exhibited poorly differentiated structures with mTORC1 upregulation and mesenchymal phenotype, which were suppressed by Ereg deletion. Furthermore, EGFR/mTORC1 inhibition suppressed cell proliferation and epithelial-mesenchymal transition in KPCE organoids. Our results suggest that ELF3 suppresses GBC development via downregulation of EREG/EGFR/mTORC1 signalling. EGFR/mTORC1 inhibition is a potential therapeutic option for GBC with ELF3 mutation. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Simultaneous activation of Kras-Akt and Notch pathways induces extrahepatic biliary cancer via the mTORC1 pathway.

Biliary tract cancer (BTC) has poor prognosis. The Notch receptor is aberrantly expressed in extrahepatic cholangiocarcinoma (eCCA). However, the role of Notch signaling in the initiation and progression of eCCA and gallbladder (GB) cancer remains unknown. Therefore, we investigated the functional role of Notch signaling during tumorigenesis of the extrahepatic bile duct (EHBD) and GB. Activation of Notch signaling and oncogenic Kras resulted in the development of biliary intraepithelial neoplasia (BilINs) in the EHBD and GB, which were premalignant lesions that progressed to adenocarcinoma in mice. The expression of genes involved in the mTORC1 pathway was increased in biliary spheroids from Hnf1b-CreERT2; KrasLSL-G12D ; Rosa26LSL-NotchIC mice and inhibition of the mTORC1 pathway suppressed spheroid growth. Additionally, simultaneous activation of the PI3K-AKT and Notch pathways in EHBD and GB induced biliary cancer development in mice. Consistent with this, we observed a significant correlation between activated NOTCH1 and phosphorylated Ribosomal Protein S6 (p-S6) expression in human eCCA. Furthermore, inhibition of the mTORC1 pathway suppressed the growth of Notch-activated human biliary cancer cells in vitro and in vivo. Mechanistically, the Kras/Notch-Myc axis activated mTORC1 through TSC2 phosphorylation in mutant biliary spheroids. These data indicate that inhibition of the mTORC1 pathway could be an effective treatment strategy for Notch-activated human eCCA. © 2023 The Pathological Society of Great Britain and Ireland.

Loss of Arid1a and Pten in Pancreatic Ductal Cells Induces Intraductal Tubulopapillary Neoplasm via the YAP/TAZ Pathway.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) arises from several types of premalignant lesions, including intraductal tubulopapillary neoplasm (ITPN); however, the molecular pathogenesis of ITPN remains unknown. METHODS: We performed studies with Hnf1b-CreERT2; Ptenf/f; Arid1af/f mice to investigate the consequence of genetic deletion of Arid1a in adult pancreatic ductal cells in the context of oncogenic PI3K/Akt pathway activation. RESULTS: Simultaneous deletion of Arid1a and Pten in pancreatic ductal cells resulted in the development of ITPN, which progressed to PDAC, in mice. Simultaneous loss of Arid1a and Pten induced dedifferentiation of pancreatic ductal cells and Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) pathway activation. Consistent with the mouse data, TAZ expression was found elevated in human ITPNs and ITPN-derived PDACs but not in human intraductal papillary mucinous neoplasms, indicating that activation of the TAZ pathway is a distinctive feature of ITPN. Furthermore, pharmacological inhibition of the YAP/TAZ pathway suppressed the dedifferentiation of pancreatic ductal cells and development of ITPN in Arid1a and Pten double-knockout mice. CONCLUSION: Concurrent loss of Arid1a and Pten in adult pancreatic ductal cells induced ITPN and ITPN-derived PDAC in mice through aberrant activation of the YAP/TAZ pathway, and inhibition of the YAP/TAZ pathway prevented the development of ITPN. These findings provide novel insights into the pathogenesis of ITPN-derived PDAC and highlight the YAP/TAZ pathway as a potential therapeutic target.

Innovative method for the diagnosis of bile duct lesions using a novel tapered-tip sheath system to facilitate biliary biopsies.

BACKGROUND AND AIMS: Pathologic evaluation of bile duct lesions is crucial for a definitive diagnosis and determination of an appropriate therapeutic strategy; however, current methods are limited by several challenges. This study evaluated the impact of a novel tapered-tip sheath system on biliary stricture diagnosis. METHODS: This observational study evaluated 47 consecutive patients who underwent transpapillary biliary stricture biopsy using the novel tapered-tip sheath system from July 2020 to March 2022 compared with 51 historical control subjects undergoing conventional biopsies. Technical success rate, total biopsy time, number of biopsy specimens, adequate tissue sampling rate, adverse events, and diagnostic performance for biliary strictures were assessed. RESULTS: The technical success rate was favorable in both groups, showing no significant difference (97.9% [46 of 47] vs 88.2% [45 of 51], P = .114). However, the total biopsy time was significantly shorter in the novel system group (3.7 vs 7.7 minutes, P < .001). The number of biopsy specimens did not differ between the groups; however, the novel system group had significantly more cases in which ≥3 tissue samples could be obtained (71.7% [33 of 46] vs 51.1% [23 of 45], P = .043), a higher adequate tissue sampling rate (88.2% vs 66.4%, P < .001), and fewer adverse events (6.4% vs 21.6%, P = .043). Although the diagnostic specificity of both groups was 100%, the novel system group had significantly higher diagnostic sensitivity and accuracy (82.1% vs 50% [P = .004] and 84.8% vs 55.5% [P = .005], respectively). CONCLUSIONS: The novel tapered-tip sheath system is a promising tool for biliary stricture diagnosis.

JNK pathway plays a critical role for expansion of human colorectal cancer in the context of BRG1 suppression.

Tumor stem cells (TSCs), capable of self-renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c-JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c-JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC.

Long-term Outcomes of Stent Placement Inside the Bile Duct for Biliary Strictures After Living Donor Liver Transplantation.

In living donor liver transplantation (LDLT), anastomotic biliary stricture is a serious and refractory complication. In this study, we reviewed the transition of post-LDLT anastomotic biliary strictures and evaluated long-term outcomes of stent placement inside the bile duct, which is referred to as an "inside-stent." Of 805 consecutive adult LDLT recipients in our institution (2000-2018), we reviewed 639 patients with duct-to-duct biliary reconstruction and analyzed chronological changes of post-LDLT biliary strictures. Moreover, we focused on the year 2006 when various surgical modifications were introduced and compared the details of post-LDLT biliary strictures before and after 2006, especially focusing on the long-term outcome of inside-stent placement. The proportion of left lobe grafts had increased from 1.8% before 2005 to 39.3% after 2006 (P < 0.001) to maximize the living donor safety. Overall, post-LDLT anastomotic biliary strictures occurred in 21.3% of the patients with a median follow-up period of 106.1 months, which was decreased from 32.6% before 2005 to 12.8% after 2006 (P < 0.001). Anastomotic biliary strictures were less frequent in patients with left lobe grafts than with right lobe grafts (9.4% versus 25.4%; P < 0.001). The overall technical success rate of inside-stent placement was 82.4%, with an improvement from 75.3% before 2005 up to 95.7% after 2006 (P < 0.01). Furthermore, the stricture resolution rate remained high at approximately 90% throughout the observation period. Increased use of left lobe grafts with several surgical modifications significantly reduced post-LDLT anastomotic biliary strictures, leading to favorable long-term outcomes of inside-stent placements for this condition.

Brg1 is required to maintain colorectal cancer stem cells.

Tumor cells capable of self-renewal and continuous production of progeny cells are called tumor stem cells (TSCs) and are considered to be potential therapeutic targets. However, the mechanisms underlying the survival and function of TSCs are not fully understood. We previously reported that chromatin remodeling regulator Brg1 is essential for intestinal stem cells in mice and Dclk1 is an intestinal TSC marker. In this study, we investigated the role of Brg1 in Dclk1+ intestinal tumor cells for the maintenance of intestinal tumors in mice. Specific ablation of Brg1 in Dclk1+ intestinal tumor cells reduced intestinal tumors in ApcMin mice, and continuous ablation of Brg1 maintained the reduction of intestinal tumors. Lineage tracing in the context of Brg1 ablation in Dclk1+ intestinal tumor cells revealed that Brg1-null Dclk1+ intestinal tumor cells did not give rise to their descendent tumor cells, indicating that Brg1 is essential for the self-renewal of Dclk1+ intestinal tumor cells. Five days after Brg1 ablation, we observed increased apoptosis in Dclk1+ tumor cells. Furthermore, Brg1 was crucial for the stemness of intestinal tumor cells in a spheroid culture system. BRG1 knockdown also impaired cell proliferation and increased apoptosis in human colorectal cancer (CRC) cells. Microarray analysis revealed that apoptosis-related genes were upregulated and stem cell-related genes were downregulated in human CRC cells by BRG1 suppression. Consistently, high BRG1 expression correlated with poor disease-specific survival in human CRC patients. These data indicate that Brg1 plays a crucial role in intestinal TSCs in mice by inhibiting apoptosis and is critical for cell survival and stem cell features in human CRC cells. Thus, BRG1 represents a new therapeutic target for human CRC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical impact of a novel device delivery system in the diagnosis of bile duct lesions: A single-center experience.

BACKGROUND AND AIM: Pathological evaluation is essential for the diagnosis of biliary tract diseases. However, existing evaluation methods have various challenges in terms of operability and diagnostic performance. The present study aimed to evaluate the feasibility, utility, and safety of a novel device delivery system for bile duct biopsy. METHODS: This study was conducted as a retrospective, descriptive analysis at a single center. Overall, 25 examinations in 14 consecutive patients who underwent transpapillary biopsies for biliary lesions using the novel device delivery system from July to November 2020 were reviewed. Number and time of biopsy, technical success rate, adequate tissue sampling rate, adverse events, and diagnostic performance of bile duct biopsies using the novel device were evaluated. Moreover, negative surgical margins were assessed in patients who underwent surgical resection after mapping biopsy. RESULTS: The median number of biopsy samples was five (range: 2-13), with a median biopsy time of 11.6 min. The technical success rate was 100% (140/140), with an adequate sampling rate of 82.9% (116/140). These rates did not differ depending on the biopsy site or purpose. There were no serious adverse events related to the procedures. The diagnostic sensitivity, specificity, and accuracy of biliary stricture were 90%, 100%, and 92.3%, respectively. Negative surgical margins were confirmed in all patients undergoing surgical resection, including one patient with a surgical procedure changed based on the results of mapping biopsy. CONCLUSIONS: The novel device delivery system has potentials in diagnosing biliary tract diseases and determining appropriate treatment strategies.

Lineage tracing and targeting of IL17RB+ tuft cell-like human colorectal cancer stem cells.

Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1+ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1+ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB+ CSCs as a cancer therapeutic target.

Arid1a is essential for intestinal stem cells through Sox9 regulation.

Inactivating mutations of Arid1a, a subunit of the Switch/sucrose nonfermentable chromatin remodeling complex, have been reported in multiple human cancers. Intestinal deletion of Arid1a has been reported to induce colorectal cancer in mice; however, its functional role in intestinal homeostasis remains unclear. We investigated the functional role of Arid1a in intestinal homeostasis in mice. We found that intestinal deletion of Arid1a results in loss of intestinal stem cells (ISCs), decreased Paneth and goblet cells, disorganized crypt-villous structures, and increased apoptosis in adult mice. Spheroids did not develop from intestinal epithelial cells deficient for Arid1a Lineage-tracing experiments revealed that Arid1a deletion in Lgr5+ ISCs leads to impaired self-renewal of Lgr5+ ISCs but does not perturb intestinal homeostasis. The Wnt signaling pathway, including Wnt agonists, receptors, and target genes, was strikingly down-regulated in Arid1a-deficient intestines. We found that Arid1a directly binds to the Sox9 promoter to support its expression. Remarkably, overexpression of Sox9 in intestinal epithelial cells abrogated the above phenotypes, although Sox9 overexpression in intestinal epithelial cells did not restore the expression levels of Wnt agonist and receptor genes. Furthermore, Sox9 overexpression permitted development of spheroids from Arid1a-deficient intestinal epithelial cells. In addition, deletion of Arid1a concomitant with Sox9 overexpression in Lgr5+ ISCs restores self-renewal in Arid1a-deleted Lgr5+ ISCs. These results indicate that Arid1a is indispensable for the maintenance of ISCs and intestinal homeostasis in mice. Mechanistically, this is mainly mediated by Sox9. Our data provide insights into the molecular mechanisms underlying maintenance of ISCs and intestinal homeostasis.

SETDB1 Inhibits p53-Mediated Apoptosis and Is Required for Formation of Pancreatic Ductal Adenocarcinomas in Mice.

BACKGROUND & AIMS: SETDB1, a histone methyltransferase that trimethylates histone H3 on lysine 9, promotes development of several tumor types. We investigated whether SETDB1 contributes to development of pancreatic ductal adenocarcinoma (PDAC). METHODS: We performed studies with Ptf1aCre; KrasG12D; Setdb1f/f, Ptf1aCre; KrasG12D; Trp53f/+; Setdb1f/f, and Ptf1aCre; KrasG12D; Trp53f/f; Setdb1f/f mice to investigate the effects of disruption of Setdb1 in mice with activated KRAS-induced pancreatic tumorigenesis, with heterozygous or homozygous disruption of Trp53. We performed microarray analyses of whole-pancreas tissues from Ptf1aCre; KrasG12D; Setdb1f/f, and Ptf1aCre; KrasG12D mice and compared their gene expression patterns. Chromatin immunoprecipitation assays were performed using acinar cells isolated from pancreata with and without disruption of Setdb1. We used human PDAC cells for SETDB1 knockdown and inhibitor experiments. RESULTS: Loss of SETDB1 from pancreas accelerated formation of premalignant lesions in mice with pancreata that express activated KRAS. Microarray analysis revealed up-regulated expression of genes in the apoptotic pathway and genes regulated by p53 in SETDB1-deficient pancreata. Deletion of Setdb1 from pancreas prevented formation of PDACs, concomitant with increased apoptosis and up-regulated expression of Trp53 in mice heterozygous for disruption of Trp53. In contrast, pancreata of mice with homozygous disruption of Trp53 had no increased apoptosis, and PDACs developed. Chromatin immunoprecipitation revealed that SETDB1 bound to the Trp53 promoter to regulate its expression. Expression of an inactivated form of SETDB1 in human PDAC cells with wild-type TP53 resulted in TP53-induced apoptosis. CONCLUSIONS: We found that the histone methyltransferase SETDB1 is required for development of PDACs, induced by activated KRAS, in mice. SETDB1 inhibits apoptosis by regulating expression of p53. SETDB1 might be a therapeutic target for PDACs that retain p53 function.

A novel technique for mapping biopsy of bile duct cancer.

BACKGROUND : Accurate preoperative assessment of the longitudinal extension of perihilar cholangiocarcinoma (PHCC) is essential for treatment planning. Mapping biopsies for PHCC remain challenging owing to technical difficulties and insufficient sample amounts. The aim of this study was to investigate the usefulness of a novel technique for mapping biopsies of PHCC. METHODS : Our novel method focused on a biliary stent delivery system for mapping biopsies. Fifty patients with PHCC undergoing endoscopic transpapillary mapping biopsy using the novel method were reviewed from August 2015 to June 2019. RESULTS : The median number of biopsy samples was six (range 1 - 17), and the rate of adequate sampling was 91.4 % (266 /291). Biopsy from the intrahepatic bile duct was possible in 82.0 % of patients (41 /50), and negative margins were confirmed in the resected specimens from 34 /39 patients who underwent surgery (87.2 %). None of the patients had post-endoscopic retrograde cholangiopancreatography pancreatitis. CONCLUSIONS : With our novel method, accurate assessment of the longitudinal extension of PHCC might be expected with minimal trauma to the duodenal papilla.

Visceral fat obesity is the key risk factor for the development of reflux erosive esophagitis in 40-69-years subjects.

BACKGROUND: Visceral fat obesity can be defined quantitatively by abdominal computed tomography, however, the usefulness of measuring visceral fat area to assess the etiology of gastrointestinal reflux disease has not been fully elucidated. METHODS: A total of 433 healthy subjects aged 40-69 years (234 men, 199 women) were included in the study. The relationship between obesity-related factors (total fat area, visceral fat area, subcutaneous fat area, waist circumference, and body mass index) and the incidence of reflux erosive esophagitis was investigated. Lifestyle factors and stomach conditions relevant to the onset of erosive esophagitis were also analyzed. RESULTS: The prevalence of reflux erosive esophagitis was 27.2% (118/433; 106 men, 12 women). Visceral fat area was higher in subjects with erosive esophagitis than in those without (116.6 cm2 vs. 64.9 cm2, respectively). The incidence of erosive esophagitis was higher in subjects with visceral fat obesity (visceral fat area ≥ 100 cm2) than in those without (61.2% vs. 12.8%, respectively). Visceral fat obesity had the highest odds ratio (OR) among obesity-related factors. Multivariate analysis showed that visceral fat area was associated with the incidence of erosive esophagitis (OR = 2.18), indicating that it is an independent risk factor for erosive esophagitis. In addition, daily alcohol intake (OR = 1.54), gastric atrophy open type (OR = 0.29), and never-smoking history (OR = 0.49) were also independently associated with the development of erosive esophagitis. CONCLUSIONS: Visceral fat obesity is the key risk factor for the development of reflux erosive esophagitis in subjects aged 40-69 years.

Utility of laser-cut covered self-expandable metal stents for unresectable malignant distal biliary obstruction: a single-center experience.

BACKGROUND: Few reports have evaluated the effectiveness of laser-cut, covered, self-expandable metal stents (LC-CSEMS) for unresectable malignant distal biliary obstruction (MDBO) and whether reintervention is feasible after placement. We describe our experience with LC-CSEMS placement for unresectable MDBO. METHODS: Patients undergoing LC-CSEMS placement for unresectable MDBO from November 2014 to December 2018 were reviewed. Recurrent biliary obstruction (RBO), median time to RBO (TRBO), and reintervention were analyzed. RESULTS: 52 patients who underwent LC-CSEMS placement for unresectable MDBO were included in the analysis. The RBO rate was 15 % and the median TRBO was 445 days. Reintervention was attempted in nine patients and stent removal was successful in eight patients. CONCLUSIONS: Our experience suggests the effectiveness of LC-CSEMS in patients with unresectable MDBO in terms of stent patency and feasibility of reintervention.

Nardilysin inhibits pancreatitis and suppresses pancreatic ductal adenocarcinoma initiation in mice.

OBJECTIVE: Nardilysin (NRDC), a zinc peptidase, exhibits multiple localisation-dependent functions including as an enhancer of ectodomain shedding in the extracellular space and a transcriptional coregulator in the nucleus. In this study, we investigated its functional role in exocrine pancreatic development, homeostasis and the formation of pancreatic ductal adenocarcinoma (PDA). DESIGN: We analysed Ptf1a-Cre; Nrdcflox/flox mice to investigate the impact of Nrdc deletion. Pancreatic acinar cells were isolated from Nrdcflox/flox mice and infected with adenovirus expressing Cre recombinase to examine the impact of Nrdc inactivation. Global gene expression in Nrdc-cKO pancreas was analysed compared with wild-type pancreas by microarray analysis. We also analysed Ptf1a-Cre; KrasG12D; Nrdcflox/flox mice to investigate the impact of Nrdc deletion in the context of oncogenic Kras. A total of 51 human samples of pancreatic intraepithelial lesions (PanIN) and PDA were examined by immunohistochemistry for NRDC. RESULTS: We found that pancreatic deletion of Nrdc leads to spontaneous chronic pancreatitis concomitant with acinar-to-ductal conversion, increased apoptosis and atrophic pancreas in mice. Acinar-to-ductal conversion was observed mainly through a non-cell autonomous mechanism, and the expression of several chemokines was significantly increased in Nrdc-null pancreatic acinar cells. Furthermore, pancreatic deletion of Nrdc dramatically accelerated KrasG12D -driven PanIN and subsequent PDA formation in mice. These data demonstrate a previously unappreciated anti-inflammatory and tumour suppressive functions of Nrdc in the pancreas in mice. Finally, absence of NRDC expression was observed in a subset of human PanIN and PDA. CONCLUSION: Nrdc inhibits pancreatitis and suppresses PDA initiation in mice.

ARID1A Maintains Differentiation of Pancreatic Ductal Cells and Inhibits Development of Pancreatic Ductal Adenocarcinoma in Mice.

BACKGROUND & AIMS: The ARID1A gene encodes a protein that is part of the large adenosine triphosphate (ATP)-dependent chromatin remodeling complex SWI/SNF and is frequently mutated in human pancreatic ductal adenocarcinomas (PDACs). We investigated the functions of ARID1A during formation of PDACs in mice. METHODS: We performed studies with Ptf1a-Cre;KrasG12D mice, which express activated Kras in the pancreas and develop pancreatic intraepithelial neoplasias (PanINs), as well as those with disruption of Aird1a (Ptf1a-Cre;KrasG12D;Arid1af/f mice) or disruption of Brg1 (encodes a catalytic ATPase of the SWI/SNF complex) (Ptf1a-Cre;KrasG12D; Brg1f/fmice). Pancreatic ductal cells (PDCs) were isolated from Arid1af/f mice and from Arid1af/f;SOX9OE mice, which overexpress human SOX9 upon infection with an adenovirus-expressing Cre recombinase. Pancreatic tissues were collected from all mice and analyzed by histology and immunohistochemistry; cells were isolated and grown in 2-dimensional and 3-dimensional cultures. We performed microarray analyses to compare gene expression patterns in intraductal papillary mucinous neoplasms (IPMNs) from the different strains of mice. We obtained 58 samples of IPMNs and 44 samples of PDACs from patients who underwent pancreatectomy in Japan and analyzed them by immunohistochemistry. RESULTS: Ptf1a-Cre;KrasG12D mice developed PanINs, whereas Ptf1a-Cre;KrasG12D;Arid1af/f mice developed IPMNs and PDACs; IPMNs originated from PDCs. ARID1A-deficient IPMNs did not express SOX9. ARID1A-deficient PDCs had reduced expression of SOX9 and dedifferentiated in culture. Overexpression of SOX9 in these cells allowed them to differentiate and prevented dilation of ducts. Among mice with pancreatic expression of activated Kras, those with disruption of Arid1a developed fewer PDACs from IPMNs than mice with disruption of Brg1. ARID1A-deficient IPMNs had reduced activity of the mTOR pathway. Human IPMN and PDAC specimens had reduced levels of ARID1A, SOX9, and phosphorylated S6 (a marker of mTOR pathway activation). Levels of ARID1A correlated with levels of SOX9 and phosphorylated S6. CONCLUSIONS: ARID1A regulates expression of SOX9, activation of the mTOR pathway, and differentiation of PDCs. ARID1A inhibits formation of PDACs from IPMNs in mice with pancreatic expression of activated KRAS and is down-regulated in IPMN and PDAC tissues from patients.