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Signal transducer and activator of transcription 3 (Stat3) has been shown to play a role in intestinal regeneration and colitis-associated colon carcinogenesis. However, the role of Stat3 in the Wnt-driven sporadic intestinal tumorigenesis remains poorly understood. We examined the roles of Stat3 in intestinal regeneration and tumorigenesis by organoid culture experiments using Stat3∆IEC mouse-derived intestinal epithelial cells in which Stat3 was disrupted. The regeneration of intestinal mucosa and organoid formation were significantly suppressed by Stat3 disruption, which was compensated by Wnt activation. Furthermore, once organoids were recovered, Stat3 was no longer required for organoid growth. These results indicate that Stat3 and Wnt signaling cooperatively protect epithelial cells at the early phase of intestinal regeneration. In contrast, intestinal tumorigenesis was not suppressed by Stat3 disruption in adenomatous polyposis coli (Apc)Δ716 and Apc∆716 Tgfbr2∆IEC mice, thus indicating that Stat3 is not required for Wnt activation-driven intestinal tumorigenesis. Mechanistically, Itga5 and Itga6 were down-regulated by Stat3 disruption, and focal adhesion kinase (FAK) activation was also suppressed. Notably, FAK inhibitor suppressed the organoid formation of wild-type epithelial cells. These results indicate that Stat3 is indispensable for the survival of epithelial cells through the activation of integrin signaling and the downstream FAK pathway; however, it is not required for the Wnt signaling-activated normal or tumor epithelial cells. [ABSTRACT FROM AUTHOR]

Genetic alterations in the TGFβ signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFβ signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined ApcΔ716 Tgfbr2ΔIEC compound mutant mice that carry mutations in Apc and Tgfbr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple Tgfbr2ΔIEC mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these Tgfbr2ΔIEC mice developed invasive colon cancer associated with chronic inflammation. We also found that TGFβ signaling is suppressed in human colitis-associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causing MMP2 activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFβ signaling-repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in Tgfbr2ΔIEC mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated Tgfbr2ΔIEC mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFβ signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans. [ABSTRACT FROM AUTHOR]

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11-7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide. Several genome sequencing studies have provided comprehensive CRC genomic datasets. Likewise, in our previous study, we performed genome-wide Sleeping Beautytransposon-based mutagenesis screening in mice and provided comprehensive datasets of candidate CRC driver genes. However, functional validation for most candidate CRC driver genes, which were commonly identified from both human and mice, has not been performed. Here, we describe a platform for functionally validating CRC driver genes that utilizes CRISPR-Cas9 in mouse intestinal tumor organoids and human CRC-derived organoids in xenograft mouse models. We used genetically defined benign tumor-derived organoids carrying 2 frequent gene mutations (Apc and Kras mutations), which act in the early stage of CRC development, so that we could clearly evaluate the tumorigenic ability of the mutation in a single gene. These studies showed that Acvr1b, Acvr2a, and Arid2could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-β (TGF-β) synergistically promote tumorigenesis, and shed light on the role of activin receptors in CRC. This experimental system can also be applied to mouse intestinal organoids carrying other sensitizing mutations as well as organoids derived from other organs, which could further contribute to identification of novel cancer driver genes and new drug targets. [ABSTRACT FROM AUTHOR]

Expression of the cytokine IL-11 is elevated in human Helicobacter pylori infection and progressively increases with worsening gastric pathology. Additionally, IL-11 is required for tumor development in STAT3-dependent murine models of gastric cancer (GC) and, when administered acutely, causes resolving atrophic gastritis. However, it is unclear whether locally elevated IL-11 ligand expression can, in isolation from oncogenic gp130-JAKSTAT pathway mutations, initiate GC pathogenesis. Here we developed a transgenic mouse model of stomach-specific (keratin 19 promoter) IL-11 ligand overexpression. Keratin 19 promoter-IL-11 transgenic (K19-IL11Tg) mice showed specific IL-11 overexpression in gastric corpus and antrum but not elsewhere in the gastrointestinal tract or in other tissues. K19-IL11Tg mice developed spontaneous premalignant disease of the gastric epithelium, progressing from atrophic gastritis to TFF2-positive metaplasia and severe epithelial hyperplasia, including adenoma-like lesions in a subset of older (1 yr old) animals. Although locally advanced, the hyperplastic lesions remained noninvasive. H. pylori infection in K19-IL11Tg mice accelerated some aspects of the premalignant phenotype. Finally, K19- IL11Tg mice had splenomegaly in association with elevated serum IL-11, with spleens showing an expanded myeloid compartment. Our results provide direct in vivo functional evidence that stomachspecific overexpression of IL-11, in isolation from germline gp130- JAK-STAT3 genetic drivers, is sufficient for premalignant progression. These findings have important functional implications for human GC, in which frequent IL-11 overexpression occurs in the reported absence of somatic mutations in gp130 signaling components. NEW & NOTEWORTHY We provide direct in vivo functional evidence that stomach-specific overexpression of the cytokine IL-11, in isolation from gp130-JAK-STAT3 pathway mutations, can trigger spontaneous atrophic gastritis progressing to locally advanced epithelial hyperplasia (but not dysplasia or carcinoma), which does not require, but may be accelerated by, concomitant Helicobacter pylori infection. [ABSTRACT FROM AUTHOR]

Objective Gastric cancer (GC) remains difficult to cure due to heterogeneity in a clinical challenge and the molecular mechanisms underlying this disease are complex and not completely understood. Accumulating evidence suggests that microRNAs (miRNAs) play an important role in GC, but the role of specific miRNAs involved in this disease remains elusive. We performed next generation sequencing (NGS)-based whole-transcriptome profiling to discover GC-specific miRNAs, followed by functional validation of results. Design NGS-based miRNA profiles were generated in matched pairs of GCs and adjacent normal mucosa (NM). Quantitative RT-PCR validation of miR-29c expression was performed in 274 gastric tissues, which included two cohorts of matched GC and NM specimens. Functional validation of miR-29c and its gene targets was undertaken in cell lines, as well as K19-C2mE and K19-Wnt1/C2mE transgenic mice. Results NGS analysis revealed four GC-specific miRNAs. Among these, miR-29c expression was significantly decreased in GC versus NM tissues (p<0.001). Ectopic expression of miR-29c mimics in GC cell lines resulted in reduced proliferation, adhesion, invasion and migration. High miR-29c expression suppressed xenograft tumour growth in nude mice. Direct interaction between miR-29c and its newly discovered target, ITGB1, was identified in cell lines and transgenic mice. MiR-29c expression demonstrated a stepwise decrease in wild type hyperplasia-dysplasia cascade in transgenic mice models of GC. Conclusions MiR-29c acts as a tumour suppressor in GC by directly targeting ITGB1. Loss of miR-29c expression is an early event in the initiation of gastric carcinogenesis and may serve as a diagnostic and therapeutic biomarker for patients with GC. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that platelet-type 12-lipoxygenase (p12-LOX) plays an important role in tumor development. However, how p12-LOX contributes to tumorigenesis is still not understood. The role of p12-LOX was therefore examined in tumor promotion using mouse epidermal JB6 P+ cells that are sensitive to 12-O-tetradecanoylphorbol-13-acetate-induced transformation. The expression of p12-LOX was significantly higher in JB6 P+ cells than in JB6 P− cells that were resistant to transformation, and its expression was further increased by tumor necrosis factor (TNF)-α. Importantly, the inhibition of p12-LOX in JB6 P+ cells by baicalein, a specific inhibitor or small interfering RNA significantly suppressed TPA-induced transformation. Moreover, treatment with 12(S)-hydroxyeicosatetraenoic acid (HETE), a metabolite of p12-LOX, enhanced TPA-induced neoplastic transformation either in the presence or absence of baicalein. These results indicate that p12-LOX is required for tumor promotion of epidermal cells and that 12(S)-HETE functions as a rate-limiting factor. Notably, treatment with baicalein significantly suppressed the proliferation of JB6 P+ cells when cells were seeded at a low density in a culture plate. Moreover, the cloning efficiency of JB6 P+ cells was dramatically decreased by inhibition of p12-LOX. In contrast, baicalein treatment did not affect the cloning efficiency of most malignant cancer cells. These results indicate that p12-LOX is induced by the inflammatory cytokine TNF-α in the early stage of tumorigenesis, and is required for tumor promotion through enhancing efficient proliferation of a small number of initiated cells. The present results suggest that the p12-LOX pathway may be an effective target of chemoprevention for skin carcinogenesis. [ABSTRACT FROM AUTHOR]