PKCλ/ι inhibition activates an ULK2-mediated interferon response to repress tumorigenesis.

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.

Simultaneous Loss of Both Atypical Protein Kinase C Genes in the Intestinal Epithelium Drives Serrated Intestinal Cancer by Impairing Immunosurveillance.

Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.

Synergistic antitumor activity by dual blockade of CCR1 and CXCR2 expressed on myeloid cells within the tumor microenvironment.

BACKGROUND: Chemokine signaling within the tumor microenvironment can promote tumor progression. Although CCR1 and CXCR2 on myeloid cells could be involved in tumor progression, it remains elusive what effect would be observed if both of those are blocked. METHODS: We employed two syngeneic colorectal cancer mouse models: a transplanted tumor model and a liver metastasis model. We generated double-knockout mice for CCR1 and CXCR2, and performed bone marrow (BM) transfer experiments in which sub-lethally irradiated wild-type mice were reconstituted with BM from either wild-type, Ccr1-/-, Cxcr2-/- or Ccr1-/-Cxcr2-/- mice. RESULTS: Myeloid cells that express MMP2, MMP9 and VEGF were accumulated around both types of tumors through CCR1- and CXCR2-mediated pathways. Mice reconstituted with Ccr1-/-Cxcr2-/- BM exhibited the strongest suppression of tumor growth and liver metastasis compared with other three groups. Depletion of CCR1+CXCR2+ myeloid cells led to a higher frequency of CD8+ T cells, whereas the numbers of Ly6G+ neutrophils, FOXP3+ Treg cells and CD31+ endothelial cells were significantly decreased. Furthermore, treatment with a neutralizing anti-CCR1 mAb to mice reconstituted with Cxcr2-/- BM significantly suppressed tumor growth and liver metastasis. CONCLUSION: Dual blockade of CCR1 and CXCR2 pathways in myeloid cells could be an effective therapy against colorectal cancer.

Early detection of pancreatic cancer by comprehensive serum miRNA sequencing with automated machine learning.

BACKGROUND: Pancreatic cancer is often diagnosed at advanced stages, and early-stage diagnosis of pancreatic cancer is difficult because of nonspecific symptoms and lack of available biomarkers. METHODS: We performed comprehensive serum miRNA sequencing of 212 pancreatic cancer patient samples from 14 hospitals and 213 non-cancerous healthy control samples. We randomly classified the pancreatic cancer and control samples into two cohorts: a training cohort (N = 185) and a validation cohort (N = 240). We created ensemble models that combined automated machine learning with 100 highly expressed miRNAs and their combination with CA19-9 and validated the performance of the models in the independent validation cohort. RESULTS: The diagnostic model with the combination of the 100 highly expressed miRNAs and CA19-9 could discriminate pancreatic cancer from non-cancer healthy control with high accuracy (area under the curve (AUC), 0.99; sensitivity, 90%; specificity, 98%). We validated high diagnostic accuracy in an independent asymptomatic early-stage (stage 0-I) pancreatic cancer cohort (AUC:0.97; sensitivity, 67%; specificity, 98%). CONCLUSIONS: We demonstrate that the 100 highly expressed miRNAs and their combination with CA19-9 could be biomarkers for the specific and early detection of pancreatic cancer.

Understanding Flexdispersion: Structure-Function Relationship Studies of Organic Amphiphilic Ligands.

Since inorganic nanoparticles have unique properties that differ from those of bulk materials, their material applications have attracted attention in various fields. In order to utilize inorganic nanoparticles for functional materials, they must be dispersed without agglomeration. Therefore, the surfaces of inorganic nanoparticles are typically modified with organic ligands to improve their dispersibility. Nevertheless, the relationship between the tail group structure in organic ligands and the dispersibility of inorganic nanoparticles in organic solvents remains poorly understood. We previously developed amphiphilic ligands that consist of ethylene glycol chains and alkyl chains to disperse inorganic nanoparticles in a variety of organic solvents. However, the structural requirements for amphiphilic ligands to "flexibly" disperse nanoparticles in less polar to polar solvents are still unclear. Here, we designed and synthesized several phosphonic acid ligands for structure-function relationship studies of flexdispersion. Dynamic light scattering analysis and visible light transmittance measurements revealed that the ratio of alkyl/ethylene glycol chains in organic ligands alone does not determine the dispersibility of the nanoparticles in organic solvents, but the arrangement of the individual chains also has an effect. From a practical application standpoint, it is preferable to design ligands with ethylene glycol chains on the outside relative to the particle surface.

Reduction of butyrate-producing bacteria in the gut microbiome of Japanese patients with pancreatic cancer.

BACKGROUND: The incidence of pancreatic cancer is on the rise, and its prognosis remains poor. Recent reports have established a link between the gut and oral microbiome and pancreatic cancer. However, the intricacies of this association within the Japanese population remain unclear. In this study, we investigated the gut and oral microbiomes of Japanese patients with pancreatic cancer, comparing them with those of healthy individuals. METHODS: We recruited 30 patients with untreated pancreatic cancer and 18 healthy controls at Kyoto University Hospital (2018-2022). We performed a comprehensive 16S rRNA gene sequencing to analyze their gut and oral microbiomes. RESULTS: Analysis revealed that the diversity of the gut and oral microbiomes of patients with pancreatic cancer was reduced compared to that of the healthy controls. Specifically, we observed an increase in the genus Streptococcus in both the gut and oral microbiomes and a significant decrease in several butyrate-producing bacteria in fecal samples. Moreover, bacteria such as Streptococcus mitis and Holdemanella biformis were present in pancreatic cancer tissues, suggesting that they might influence the carcinogenesis and progression of pancreatic cancer. CONCLUSIONS: The gut and oral microbiome differed between patients with pancreatic cancer and healthy controls, with a notable decrease in butyrate-producing bacteria in the gut microbiome of the patients. This suggests that there may be a distinct microbial signature associated with pancreatic cancer in the Japanese population. Further studies are required to elucidate the microbiome's causal role in this cancer and help develop prognostic markers or targeted therapies.

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.

On the origin of gastric tumours: analysis of a case with intramucosal gastric carcinoma and oxyntic gland adenoma.

Most gastric cancers develop in inflamed gastric mucosa due to Helicobacter pylori infection, typically with metaplastic changes. However, the origins of gastric cancer remain unknown. Here, we present a case of intramucosal gastric carcinoma (IGC) and oxyntic gland adenoma (OGA) derived from spasmolytic polypeptide-expressing metaplasia (SPEM). Early gastric cancer adjacent to a polyp was found in the upper corpus of a 71-year-old woman without H. pylori infection and was endoscopically resected. Histological examination showed IGC and OGA, both of which had predominant MUC6 expression. Interestingly, gastric glands with enriched MUC6-positive mucous cells, referred to as SPEM, expanded between them. Whole-exome sequencing analysis revealed a truncating KRAS(G12D) mutation in IGC, OGA, and SPEM. In addition, TP53 and CDKN2A mutations and a loss of chromosome 17p were found in the IGC, whereas a GNAS mutation was observed in the OGA. These results indicated that IGC and OGA originated from the KRAS-mutated SPEM. © 2023 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.