Differential involvement of LUBAC-mediated linear ubiquitination in intestinal epithelial cells and macrophages during intestinal inflammation.

Disruption of the intestinal epithelial barrier and dysregulation of macrophages are major factors contributing to the pathogenesis of inflammatory bowel diseases (IBDs). Activation of NF-κB and cell death are involved in maintaining intestinal homeostasis in a cell type-dependent manner. Although both are regulated by linear ubiquitin chain assembly complex (LUBAC)-mediated linear ubiquitination, the physiological relevance of linear ubiquitination to intestinal inflammation remains unexplored. Here, we used two experimental mouse models of IBD (intraperitoneal LPS and oral dextran sodium sulfate [DSS] administration) to examine the role of linear ubiquitination in intestinal epithelial cells (IECs) and macrophages during intestinal inflammation. We did this by deleting the linear ubiquitination activity of LUBAC specifically from IECs or macrophages. Upon LPS administration, loss of ligase activity in IECs induced mucosal inflammation and augmented IEC death. LPS-mediated death of LUBAC-defective IECs was triggered by TNF. IEC death was rescued by an anti-TNF antibody, and TNF (but not LPS) induced apoptosis of organoids derived from LUBAC-defective IECs. However, augmented TNF-mediated IEC death did not overtly affect the severity of colitis after DSS administration. By contrast, defective LUBAC ligase activity in macrophages ameliorated DSS-induced colitis by attenuating both infiltration of macrophages and expression of inflammatory cytokines. Decreased production of macrophage chemoattractant MCP-1/CCL2, as well as pro-inflammatory IL-6 and TNF, occurred through impaired activation of NF-κB and ERK via loss of ligase activity in macrophages. Taken together, these results indicate that both intraperitoneal LPS and oral DSS administrations are beneficial for evaluating epithelial integrity under inflammatory conditions, as well as macrophage functions in the event of an epithelial barrier breach. The data clarify the cell-specific roles of linear ubiquitination as a critical regulator of TNF-mediated epithelial integrity and macrophage pro-inflammatory responses during intestinal inflammation. © 2022 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.

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.

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.

Comparison between a rotatable sphincterotome and a conventional sphincterotome for selective bile duct cannulation.

BACKGROUND: Selective biliary cannulation (SBC) is the first challenge of endoscopic retrograde cholangiopancreatography (ERCP), especially for trainees, and a rotatable sphincterotome may be useful to guide the directional axis of the scope and SBC. METHODS: We performed a prospective randomized single-center trial, enrolling 200 patients with a native papilla who required therapeutic biliary ERCP. Patients were randomly assigned to the rotatable sphincterotome group (n = 100) or the conventional sphincterotome group (n = 100). The primary endpoint was successful SBC by the trainees within 10 minutes. RESULTS: The early and late cannulation success rates did not differ significantly between the groups (P = 0.46 and P > 0.99, respectively). For the patients in whom trainees failed to achieve SBC, the rotatable sphincterotome was used as a rescue cannulation technique in four patients from the conventional group; in no patients in the rotatable group was the conventional sphincterotome used for SBC. Post-ERCP pancreatitis (PEP) occurred in 11 patients (5.5 %; 6 mild, 5 moderate); the incidence did not differ significantly between the two groups (rotatable group 3 %, conventional group 8 %; P = 0.21). The two groups were thus combined for evaluation of the factors relating to cannulation difficulty for trainees, which revealed that orientation of the papilla was a significant factor (P < 0.001). CONCLUSIONS: The type of sphincterotome used did not affect the success of SBC by trainees. However, orientation of the papilla was revealed to be a significant factor relating to cannulation difficulty for trainees overall.

Pancreatic RECK inactivation promotes cancer formation, epithelial-mesenchymal transition, and metastasis.

RECK is downregulated in various human cancers; however, how RECK inactivation affects carcinogenesis remains unclear. We addressed this issue in a pancreatic ductal adenocarcinoma (PDAC) mouse model and found that pancreatic Reck deletion dramatically augmented the spontaneous development of PDAC with a mesenchymal phenotype, which was accompanied by increased liver metastases and decreased survival. Lineage tracing revealed that pancreatic Reck deletion induced epithelial-mesenchymal transition (EMT) in PDAC cells, giving rise to inflammatory cancer-associated fibroblast-like cells in mice. Splenic transplantation of Reck-null PDAC cells resulted in numerous liver metastases with a mesenchymal phenotype, whereas reexpression of RECK markedly reduced metastases and changed the PDAC tumor phenotype into an epithelial one. Consistently, low RECK expression correlated with low E-cadherin expression, poor differentiation, metastasis, and poor prognosis in human PDAC. RECK reexpression in the PDAC cells was found to downregulate MMP2 and MMP3, with a concomitant increase in E-cadherin and decrease in EMT-promoting transcription factors. An MMP inhibitor recapitulated the effects of RECK on the expression of E-cadherin and EMT-promoting transcription factors and invasive activity. These results establish the authenticity of RECK as a pancreatic tumor suppressor, provide insights into its underlying mechanisms, and support the idea that RECK could be an important therapeutic effector against human PDAC.

Brg1 controls stemness and metastasis of pancreatic cancer through regulating hypoxia pathway.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. We previously reported that chromatin remodeler Brg1 is essential for acinar cell-derived PDAC formation in mice. However, the functional role of Brg1 in established PDAC and its metastasis remains unknown. Here, we investigated the importance of Brg1 for established PDAC by using a mouse model with a dual recombinase system. We discovered that Brg1 was a critical player for the cell survival and growth of spontaneously developed PDAC in mice. In addition, Brg1 was essential for metastasis of PDAC cells by inhibiting apoptosis in splenic injection and peritoneal dissemination models. Moreover, cancer stem-like property was compromised in PDAC cells by Brg1 ablation. Mechanistically, the hypoxia pathway was downregulated in Brg1-deleted mouse PDAC and BRG1-low human PDAC. Brg1 was essential for HIF-1α to bind to its target genes to augment the hypoxia pathway, which was important for PDAC cells to maintain their stem-like properties and to metastasize to the liver. Human PDAC cells with high BRG1 expression were more susceptible to BRG1 suppression. In conclusion, Brg1 plays a critical role for cell survival, stem-like property and metastasis of PDAC through the regulation of hypoxia pathway, and thus could be a novel therapeutic target for PDAC.

THBS1-producing tumor-infiltrating monocyte-like cells contribute to immunosuppression and metastasis in colorectal cancer.

Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

Concurrent Activation of Kras and Canonical Wnt Signaling Induces Premalignant Lesions That Progress to Extrahepatic Biliary Cancer in Mice.

Biliary cancer has long been known to carry a poor prognosis, yet the molecular pathogenesis of carcinoma of the extrahepatic biliary system and its precursor lesions remains elusive. Here we investigated the role of Kras and canonical Wnt pathways in the tumorigenesis of the extrahepatic bile duct (EHBD) and gall bladder (GB). In mice, concurrent activation of Kras and Wnt pathways induced biliary neoplasms that resembled human intracholecystic papillary-tubular neoplasm (ICPN) and biliary intraepithelial neoplasia (BilIN), putative precursors to invasive biliary cancer. At a low frequency, these lesions progressed to adenocarcinoma in a xenograft model, establishing them as precancerous lesions. Global gene expression analysis revealed increased expression of genes associated with c-Myc and TGFß pathways in mutant biliary spheroids. Silencing or pharmacologic inhibition of c-Myc suppressed proliferation of mutant biliary spheroids, whereas silencing of Smad4/Tgfbr2 or pharmacologic inhibition of TGFß signaling increased proliferation of mutant biliary spheroids and cancer formation in vivo. Human ICPNs displayed activated Kras and Wnt signals and c-Myc and TGFß pathways. Thus, these data provide direct evidence that concurrent activation of the Kras and canonical Wnt pathways results in formation of ICPN and BilIN, which could develop into biliary cancer. SIGNIFICANCE: This work shows how dysregulation of canonical cell growth pathways drives precursors to biliary cancers and identifies several molecular vulnerabilities as potential therapeutic targets in these precursors to prevent oncogenic progression.