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Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide. Chemotherapies against gastric cancer often fail, with cancer recurrence due potentially to the persistence of cancer stem cells. This unique subpopulation of cells in tumors possesses the ability to self-renew and dedifferentiate. These cancer stem cells are critical for initiation, maintenance, metastasis, and relapse of cancers; however, the molecular mechanisms supporting cancer stemness remain largely unknown. Increased kinase and decreased phosphatase activity are hallmarks of oncogenic signaling. Protein phosphatase 2A (PP2A) functions as a tumor-suppressor enzyme, and elevated levels of SET/I2PP2A, an endogenous PP2A protein inhibitor, are correlated with poor prognosis of several human cancers. Here, it was determined that SET expression was elevated in tumor tissue in a gastric cancer mouse model system, and SET expression was positively correlated with poor survival of human gastric cancer patients. Mechanistically, SET knockdown decreased E2F1 levels and suppressed the stemness of cancer cell lines. Immunoprecipitations show SET associated with the PP2A–B56 complex, and the B56 subunit interacted with the E2F1 transcription factor. Treatment of gastric cancer cells with the SET-targeting drug OP449 increased PP2A activity, decreased E2F1 protein levels, and suppressed stemness of cancer cells. These data indicate that a SET/PP2A/E2F1 axis regulates cancer cell stemness and is a potential target for gastric cancer therapy.Implications: This study highlights the oncogenic role of SET/I2PP2A in gastric cancer and suggests that SET maintains cancer cell stemness by suppressing PP2A activity and stabilizing E2F1. Mol Cancer Res; 16(3); 554–63. ©2018 AACR.

Suppression of NF-κB and Wnt signaling by Myd88 disruption in BMDCs.

Expression levels of MYD88 and IRF5 in human and mouse gastric cancer.

Expression levels of MyD88-dependent unregulated genes in the gp130F/F mice.

Supplemental Figure S1. Parietal cells loss induced by tamoxifen injection in WT mouse.

Supplementary Materials and Methods.

Induction of Tlr2 and Cd14 in regenerating gastric mucosa.

It has been established that COX-2 and downstream signaling by prostaglandin E2 (PGE2) play a key role in tumorigenesis through generation of inflammatory microenvironment. Toll-like receptor (TLR) signaling through myeloid differentiation factor 88 (MyD88) also regulates inflammatory responses in tumors. However, the relationship between these distinct pathways in tumorigenesis is not yet fully understood. We herein investigated the role of MyD88 in gastric tumorigenesis using Gan mice, which develop inflammation-associated gastric tumors due to the simultaneous activation of the COX-2/PGE2 pathway and Wnt signaling. Notably, the disruption of Myd88 in Gan mice resulted in the significant suppression of gastric tumorigenesis with the inhibition of inflammatory responses, even though COX-2/PGE2 pathway is constitutively activated. Moreover, Myd88 disruption in bone marrow–derived cells (BMDCs) in Gan mice also suppressed inflammation and tumorigenesis, indicating that MyD88 signaling in BMDCs regulates the inflammatory microenvironment. We also found that expression of Tlr2 and its coreceptor Cd14 was induced in tumor epithelial cells in Gan mice, which was suppressed by the disruption of Myd88. It has already been shown that TLR2/CD14 signaling is important for stemness of intestinal epithelial cells. These results indicate that MyD88 in BMDCs, together with COX-2/PGE2 pathway, plays an essential role in the generation of the inflammatory microenvironment, which may promote tumorigenesis through induction of TLR2/CD14 pathway in tumor epithelial cells. These results suggest that inhibition of TLR/MyD88 signaling together with COX-2/PGE2 pathway will be an effective preventive strategy for gastric cancer. Cancer Prev Res; 9(3); 253–63. ©2016 AACR.

Supplementary Figure Legends.

Supplementary Methods and Figure legend.

Expression levels of TLR2 and CD14 in human and mouse gastric tumors.

IPA analysis of human gastric cancer subtypes.

This file contains detailed material and methods, and supplemental data related with Fig. 1g, Fig. 2a, b, Fig. 3c, d, Fig. 4a, b, c, Fig. 5c

Loss of parietal cells initiates the development of spasmolytic polypeptide–expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38MAPK signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38MAPK signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis. Cancer Prev Res; 8(6); 492–501. ©2015 AACR.

Histological diagnosis of the primary tumors

Increased number of small adenomas by Kras mutation

The content of this file containing Supplementary Figures (and a Supplementary Table) provides additional (and essential) data supporting our conclusions that the ASC inflammasome/caspase-1/IL-18 axis promotes gastric tumorigenesis.

Colorectal cancer is driven by the accumulation of driver mutations, but the contributions of specific mutations to different steps in malignant progression are not fully understood. In this study, we generated mouse models harboring different combinations of key colorectal cancer driver mutations (Apc, Kras, Tgfbr2, Trp53, Fbxw7) in intestinal epithelial cells to comprehensively investigate their roles in the development of primary tumors and metastases. ApcΔ716 mutation caused intestinal adenomas and combination with Trp53R270H mutation or Tgfbr2 deletion induced submucosal invasion. The addition of KrasG12D mutation yielded epithelial–mesenchymal transition (EMT)-like morphology and lymph vessel intravasation of the invasive tumors. In contrast, combinations of ApcΔ716 with KrasG12D and Fbxw7 mutation were insufficient for submucosal invasion, but still induced EMT-like histology. Studies using tumor-derived organoids showed that KrasG12D was critical for liver metastasis following splenic transplantation, when this mutation was combined with either ApcΔ716 plus Trp53R270H or Tgfbr2 deletion, with the highest incidence of metastasis displayed by tumors with a ApcΔ716 KrasG12D Tgfbr2−/− genotype. RNA sequencing analysis of tumor organoids defined distinct gene expression profiles characteristic for the respective combinations of driver mutations, with upregulated genes in ApcΔ716 KrasG12D Tgfbr2−/− tumors found to be similarly upregulated in specimens of human metastatic colorectal cancer. Our results show how activation of Wnt and Kras with suppression of TGFβ signaling in intestinal epithelial cells is sufficient for colorectal cancer metastasis, with possible implications for the development of metastasis prevention strategies.Significance: These findings illuminate how key driver mutations in colon cancer cooperate to drive the development of metastatic disease, with potential implications for the development of suitable prevention strategies. Cancer Res; 78(5); 1334–46. ©2017 AACR.

Inflammasomes are key regulators of innate immunity in chronic inflammatory disorders and autoimmune diseases, but their role in inflammation-associated tumorigenesis remains ill-defined. Here we reveal a protumorigenic role in gastric cancer for the key inflammasome adaptor apoptosis-related speck-like protein containing a CARD (ASC) and its effector cytokine IL18. Genetic ablation of ASC in the gp130F/F spontaneous mouse model of intestinal-type gastric cancer suppressed tumorigenesis by augmenting caspase-8-like apoptosis in the gastric epithelium, independently from effects on myeloid cells and mucosal inflammation. This phenotype was characterized by reduced activation of caspase-1 and NF-κB activation and reduced expression of mature IL18, but not IL1β, in gastric tumors. Genetic ablation of IL18 in the same model also suppressed gastric tumorigenesis, whereas blockade of IL1β and IL1α activity upon genetic ablation of the IL1 receptor had no effect. The specific protumorigenic role for IL18 was associated with high IL18 gene expression in the gastric tumor epithelium compared with IL1β, which was preferentially expressed in immune cells. Supporting an epithelial-specific role for IL18, we found it to be highly secreted from human gastric cancer cell lines. Moreover, IL18 blockade either by a neutralizing anti-IL18 antibody or by CRISPR/Cas9-driven deletion of ASC augmented apoptosis in human gastric cancer cells. In clinical specimens of human gastric cancer tumors, we observed a significant positive correlation between elevated mature IL18 protein and ASC mRNA levels. Collectively, our findings reveal the ASC/IL18 signaling axis as a candidate therapeutic target in gastric cancer.Significance: Inflammasome activation that elevates IL18 helps drive gastric cancer by protecting cancer cells against apoptosis, with potential implications for new therapeutic strategies in this setting. Cancer Res; 78(5); 1293–307. ©2017 AACR.

Supplementary Table 1 from Induction of Prostaglandin E2 Pathway Promotes Gastric Hamartoma Development with Suppression of Bone Morphogenetic Protein Signaling

Supplementary Figure S5. Representative photographs of the whole intestine of X-ray irradiated wild-type and Tgfbr2ÃŽ?IEC mice.

Supplementary Figure 3 from Induction of Prostaglandin E2 Pathway Promotes Gastric Hamartoma Development with Suppression of Bone Morphogenetic Protein Signaling

Supplementary Figure 1 from Induction of Prostaglandin E2 Pathway Promotes Gastric Hamartoma Development with Suppression of Bone Morphogenetic Protein Signaling

Mutations in bone morphogenetic protein (BMP) receptor 1A (BMPR1A) are responsible for a subset of cases of juvenile polyposis (JP) syndrome that develops hamartomatous tumors in the gastrointestinal tract. Mouse genetic studies have shown that suppression of BMP signaling in the intestines causes JP-type hamartoma development. Here, we generated K19-Nog transgenic mice expressing noggin, a BMP antagonist, in gastric epithelium. However, inhibition of BMP signaling did not cause gastric phenotypes. We thus crossed K19-Nog with K19-C2mE mice that expressed Ptgs2 and Ptges in the stomach to generate compound transgenic mice. Expression of Ptgs2 and Ptges results in prostaglandin E2 (PGE2) biosynthesis, and both enzymes are induced in most human gastrointestinal tumors. Importantly, K19-Nog/C2mE compound mice developed gastric hamartomas that were morphologically similar to those found in JP with mucin-containing dilated cysts and inflammatory infiltration. Notably, treatment of K19-Nog/C2mE mice with a cyclooxygenase-2 inhibitor, celecoxib, significantly reduced tumor size with suppression of angiogenesis, suggesting that induction of the PGE2 pathway together with inhibition of BMP signaling is required for gastric hamartoma development. Moreover, microarray analyses revealed that canonical Wnt signaling target genes were not induced in K19-Nog/C2mE hamartomas, indicating that BMP inhibition and PGE2 induction lead to gastric hamartoma development independent of the Wnt/β-catenin pathway. These results, taken together, suggest that the PGE2 pathway is an effective preventive target against BMP-suppressed gastric hamartomas, as well as for Wnt/β-catenin–activated adenocarcinomas. [Cancer Res 2009;69(7):2729–33]

Supplementary Figure 2 from Induction of Prostaglandin E2 Pathway Promotes Gastric Hamartoma Development with Suppression of Bone Morphogenetic Protein Signaling

Supplementary Figure 4 from Induction of Prostaglandin E2 Pathway Promotes Gastric Hamartoma Development with Suppression of Bone Morphogenetic Protein Signaling

Legends for Supplementary Figures S1-S6.

Gastric cancer (GC) is strongly linked with chronic gastritis after Helicobacter pylori infection. Toll-like receptors (TLRs) are key innate immune pathogenic sensors that mediate chronic inflammatory and oncogenic responses. Here, we investigated the role of TLR9 in the pathogenesis of GC, including Helicobacter infection.TLR9 gene expression was profiled in gastric tissues from GC and gastritis patients and from the spontaneous gp130TLR9 expression was up-regulated in Helicobacter-infected gastric tissues from GC and gastritis patients and gp130Our data reveal that TLR9 promotes the initiating stages of GC and facilitates Helicobacter-induced gastric inflammation and hyperplasia, thus providing in vivo evidence for TLR9 as a candidate therapeutic target in GC.

One of most challenging issues in tumor immunology is a better understanding of the dynamics in the accumulation of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TIME), as this would lead to the development of new cancer therapeutics. Here, we show that translationally controlled tumor protein (TCTP) released by dying tumor cells is an immunomodulator crucial to full-blown MDSC accumulation in the TIME. We provide evidence that extracellular TCTP mediates recruitment of the polymorphonuclear MDSC (PMN-MDSC) population in the TIME via activation of Toll-like receptor-2. As further proof of principle, we show that inhibition of TCTP suppresses PMN-MDSC accumulation and tumor growth. In human cancers, we find an elevation of TCTP and an inverse correlation of TCTP gene dosage with antitumor immune signatures and clinical prognosis. This study reveals the hitherto poorly understood mechanism of the MDSC dynamics in the TIME, offering a new rationale for cancer immunotherapy. Cell death in the context of cancer therapies is often associated with immunogenicity. Taniguchi and colleagues instead find that release of the cellular protein TCTP following cell death triggers an immunosuppressive pathway in the tumor microenvironment.

FOXO3 is a member of the FOXO transcription factors thought to play a tumor-suppressor role in gastrointestinal cancer, while tumor-promoting function of FOXO3 has also been reported. These results suggest a context-dependent function of FOXO3 in tumor development. However, the relationship between the FOXO3 expression pattern and its role in tumorigenesis has not been elucidated. We examined the FOXO3 expression in 65 human primary gastric cancer and patient-derived xenograft tissues by immunohistochemistry and identified three subtypes according to subcellular localization: FOXO3-nuclear accumulated (FOXO3-Nuc), FOXO3-nuclear/cytoplasmic or cytoplasmic distributed (FOXO3-Cyt), and FOXO3-negative. In the FOXO3-Cyt gastric cancer cells, the expression of the constitutive active mutant FOXO3 (Act-ER FOXO3) induced the nuclear accumulation of FOXO3 and significantly suppressed colony formation and proliferation. The inhibition of the PI3K-AKT pathway by inhibitor treatment also suppressed the proliferation of FOXO3-Cyt gastric cancer cells, which was associated with the nuclear accumulation of endogenous FOXO3. Furthermore, the expression of Act-ER FOXO3 by an endogenous promoter significantly suppressed gastric tumorigenesis in Gan mice, a model of gastric cancer. Finally, treatment of FOXO3-Cyt human gastric cancer-derived organoids with an AKT inhibitor significantly suppressed the survival and proliferation. These results indicate that FOXO3 is a latent tumor suppressor for FOXO3-Cyt-type gastric cancer cells and that activation of the PI3K-AKT pathway protects this type of gastric cancer cell from FOXO3-mediated growth suppression via constitutive nuclear export. Thus, the inhibition of the PI3K-AKT pathway and nuclear translocation of endogenous FOXO3 may have therapeutic applications in the treatment of FOXO3-positive and cytoplasmic-type gastric cancer.

Protein phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and they correlate with poor prognosis in glioblastoma. This raises a question about how PP6 protein levels are regulated in normal and transformed cells. In this study, we show that PP6 protein levels increase in response to pharmacologic and genetic inhibition of autophagy. PP6 associates with autophagic adaptor protein p62/SQSTM1 and is degraded in a p62‐dependent manner. Accordingly, protein levels of PP6 and p62 fluctuate in concert under different physiological and pathophysiological conditions. Our data reveal that PP6 is regulated by p62‐dependent autophagy and suggest that accumulation of PP6 protein in tumor tissues is caused at least partially by deficiency in autophagy., Protein phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and correlate with poor prognosis in glioblastoma. Our data reveal that PP6 is regulated by p62‐dependent autophagy and suggest that accumulation of PP6 protein in tumor tissues is caused at least partially by deficiency in autophagy.

Missense-type mutant p53 plays a tumor-promoting role through gain-of-function (GOF) mechanism. In addition, the loss of wild-type TP53 through loss of heterozygosity (LOH) is widely found in cancer cells. However, malignant progression induced by cooperation of TP53 GOF mutation and LOH remains poorly understood. Here, we show that mouse intestinal tumors carrying Trp53 GOF mutation with LOH (AKTPM/LOH) are enriched in metastatic lesions when heterozygous Trp53 mutant cells (AKTP+/M) are transplanted. We show that Trp53 LOH is required for dormant cell survival and clonal expansion of cancer cells. Moreover, AKTPM/LOH cells show an increased in vivo tumor-initiating ability compared with AKTPNull and AKTP+/M cells. RNAseq analyses reveal that inflammatory and growth factor/MAPK pathways are specifically activated in AKTPM/LOH cells, while the stem cell signature is upregulated in both AKTPM/LOH and AKTPNull cells. These results indicate that TP53/Trp53 LOH promotes TP53/Trp53 GOF mutation-driven metastasis through the activation of distinct pathway combination., Both gain-of-function of mutant p53 (GOF) and loss of wild-type p53 (LOH) are independently associated with cancer. Here, the authors show that LOH promotes GOF tumourigenesis by increasing tumor-initiating ability and metastasis.

Comprehensive genome analyses have identified frequently mutated genes in human colorectal cancers (CRC). These include