An In Vivo Metastasis Model Using Genotype-Defined Tumor Organoids.

Recent cancer genome analyses have identified frequently mutated genes that are responsible for the development and malignant progression of cancers, including colorectal cancer (CRC). We previously constructed mouse models that carried major driver mutations of CRC, namely Apc, Kras, Tgfbr2, Trp53, and Fbxw7, in combinations. Comprehensive histological analyses of the models showed a link between mutation combinations and malignant phenotypes, such as invasion, epithelial-mesenchymal transition (EMT), and metastasis. The major cause of cancer-related death is metastasis, making it important to understand the mechanism underlying metastasis in order to develop novel therapeutic strategies. To this end, we have established intestinal tumor-derived organoids from different genotyped mice and generated liver metastasis models via transplantation of the organoids into the spleen. Through histological and imaging analyses of the transplantation models, we have determined that the combination of Apc, Kras, Tgfbr2, and Trp53 mutations promotes liver metastasis at a high incidence. We also demonstrated polyclonal metastasis of tumor cell clusters consisting of genetically and phenotypically distinct cells through our model analysis. These organoid transplantation models recapitulate human CRC metastasis, constituting a useful tool for basic and clinical cancer research as a preclinical model. We herein report the experimental protocols of the organoid culture and generation of metastasis models.

Neutral selection and clonal expansion during the development of colon cancer metastasis.

Intratumour heterogeneity has been shown to play a role in the malignant progression of cancer. The clonal evolution in primary cancer has been well studied, however, that in metastatic tumorigenesis is not fully understood. In this study, we established human colon cancer-derived organoids and investigated clonal dynamics during liver metastasis development by tracking barcode-labelled subclones. Long-term subclone co-cultures showed clonal drift, with a single subclone becoming dominant in the cell population. Interestingly, the selected subclones were not always the same, suggesting that clonal selection was not based on cell intrinsic properties. Furthermore, liver tumours developed by co-transplantation of organoid subclones into the immunodeficient mouse spleen showed a progressive drastic reduction in clonal diversity, and only one or two subclones predominated in the majority of large metastatic tumours. Importantly, selections were not limited to particular subclones but appeared to be random. A trend towards a reduction in clonal diversity was also found in liver metastases of multiple colour-labelled organoids of mouse intestinal tumours. Based on these results, we propose a novel mechanism of metastasis development, i.e. a subclone population of the disseminated tumour cells in the liver is selected by neutral selection during colonization and constitutes large metastatic tumours.

Gain-of-Function p53 Mutation Acts as a Genetic Switch for TGFß Signaling-Induced Epithelial-to-Mesenchymal Transition in Intestinal Tumors.

Signaling by TGFß family cytokines plays a tumor-suppressive role by inducing cell differentiation, while it promotes malignant progression through epithelial-to-mesenchymal transition (EMT). Identification of the mechanisms regulating the switch from tumor suppression to tumor promotion could identify strategies for cancer prevention and treatment. To identify the key genetic alterations that determine the outcome of TGFß signaling, we used mouse intestinal tumor-derived organoids carrying multiple driver mutations in various combinations to examine the relationship between genotypes and responses to the TGFß family cytokine activin A. KrasG12D mutation protected organoid cells from activin A-induced growth suppression by inhibiting p21 and p27 expression. Furthermore, Trp53R270H gain-of-function (GOF) mutation together with loss of wild-type Trp53 by loss of heterozygosity (LOH) promoted activin A-induced partial EMT with formation of multiple protrusions on the organoid surface, which was associated with increased metastatic incidence. Histologic analysis confirmed that tumor cells at the protrusions showed loss of apical-basal polarity and glandular structure. RNA sequencing analysis indicated that expression of Hmga2, encoding a cofactor of the SMAD complex that induces EMT transcription factors, was significantly upregulated in organoids with Trp53 GOF/LOH alterations. Importantly, loss of HMGA2 suppressed expression of Twist1 and blocked activin A-induced partial EMT and metastasis in Trp53 GOF/LOH organoids. These results indicate that TP53 GOF/LOH is a key genetic state that primes for TGFß family-induced partial EMT and malignant progression of colorectal cancer. Activin signaling may be an effective therapeutic target for colorectal cancer harboring TP53 GOF mutations. SIGNIFICANCE: KRAS and TP53 mutations shift activin-mediated signaling to overcome growth inhibition and promote partial EMT, identifying a subset of patients with colorectal cancer that could benefit from inhibition of TGFß signaling.

In Vitro and In Vivo Models for Metastatic Intestinal Tumors Using Genotype-Defined Organoids.

It has been established that the accumulation of driver gene mutations causes malignant progression of colorectal cancer (CRC) through positive selection and clonal expansion, similar to Darwin's evolution. Following this multistep tumorigenesis concept, we previously showed the specific mutation patterns for each process of malignant progression, including submucosal invasion, epithelial mesenchymal transition (EMT), intravasation, and metastasis, using genetically engineered mouse and organoid models. However, we also found that certain populations of cancer-derived organoid cells lost malignant characteristics of metastatic ability, although driver mutations were not impaired, and such subpopulations were eliminated from the tumor tissues by negative selection. These organoid model studies have contributed to our understanding of the cancer evolution mechanism. We herein report the in vitro and in vivo experimental protocols to investigate the survival, growth, and metastatic ability of intestinal tumor-derived organoids. The model system will be useful for basic research as well as the development of clinical strategies.

Genetic and nongenetic mechanisms for colorectal cancer evolution.

The stepwise accumulation of key driver mutations is responsible for the development and malignant progression of colorectal cancer in primary sites. Genetic mouse model studies have revealed combinations of driver gene mutations that induce phenotypic changes in tumors toward malignancy. However, cancer evolution is regulated by not only genetic alterations but also nongenetic mechanisms. For example, certain populations of metastatic cancer cells show a loss of malignant characteristics even after the accumulation of driver mutations, and such cells are eliminated in a negative selection manner. Furthermore, a polyclonal metastasis model has recently been proposed, in which cell clusters consisting of genetically heterogeneous cells break off from the primary site, disseminate to distant organs, and develop into heterogenous metastatic tumors. Such nongenetic mechanisms for malignant progression have been elucidated using genetically engineered mouse models as well as organoid transplantation experiments. In this review article, we discuss the role of genetic alterations in the malignant progression of primary intestinal tumors and nongenetic mechanisms for negative selection and polyclonal metastasis, which we learned from model studies.

Frequent loss of metastatic ability in subclones of Apc, Kras, Tgfbr2, and Trp53 mutant intestinal tumor organoids.

Cancer evolution is explained by the accumulation of driver mutations and subsequent positive selection by acquired growth advantages, like Darwin's evolution theory. However, whether the negative selection of cells that have lost malignant properties contributes to cancer progression has not yet been fully investigated. Using intestinal metastatic tumor-derived organoids carrying Apc, Kras, Tgfbr2, and Trp53 quadruple mutations, we demonstrate here that approximately 30% of subclones of the organoids show loss of metastatic ability to the liver while keeping the driver mutations and oncogenic pathways. Notably, highly metastatic subclones also showed a gradual loss of metastatic ability during further passages. Such non-metastatic subclones revealed significantly decreased survival and proliferation ability in Matrigel and collagen gel culture conditions, which may cause elimination from the tumor tissues in vivo. RNA sequencing indicated that stemness-related genes, including Lgr5 and Myb, were significantly downregulated in non-metastatic subclones as well as subclones that lost metastatic ability during additional passages. Furthermore, a CGH analysis showed that non-metastatic subclones were derived from a minor population of parental organoid cells. These results indicate that metastatic ability is continuously lost with decreased stem cell property in certain subpopulations of malignant tumors, and such subpopulations are eliminated by negative selection. Therefore, it is possible that cancer evolution is regulated not only by positive selection but also by negative selection. The mechanism underlying the loss of metastatic ability will be important for the future development of therapeutic strategies against metastasis.

Mapping Nanomechanical Properties of Basal Surfaces in Metastatic Intestinal 3D Living Organoids with High-Speed Scanning Ion Conductance Microscopy.

Studying mechanobiology is increasing of scientific interests in life science and nanotechnology since its impact on cell activities (e.g., adhesion, migration), physiology, and pathology. The role of apical surface (AS) and basal surface (BS) of cells played in mechanobiology is significant. The mechanical mapping and analysis of cells mainly focus on AS while little is known about BS. Here, high-speed scanning ion conductance microscope as a powerful tool is utilized to simultaneously reveal morphologies and local elastic modulus (E) of BS of genotype-defined metastatic intestinal organoids. A simple method is developed to prepare organoid samples allowing for long-term BS imaging. The multiple nano/microstructures, i.e., ridge-like, stress-fiber, and E distributions on BS are dynamically revealed. The statistic E analysis shows softness of BS derived from eight types of organoids following a ranking: malignant tumor cells > benign tumor cells > normal cells. Moreover, the correlation factor between morphology and E is demonstrated depending on cell types. This work as first example reveals the subcellular morphologies and E distributions of BS of cells. The results would provide a clue for correlating genotype of 3D cells to malignant phenotype reflected by E and offering a promising strategy for early-stage diagnosis of cancer.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells.

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

Genetic Alterations and Microenvironment that Drive Malignant Progression of Colorectal Cancer: Lessons from Mouse and Organoid Models.

Comprehensive genome analyses have identified frequently mutated genes in human colorectal cancers (CRC). These include APC, KRAS, SMAD4, TP53, and FBXW7. The biological functions of the respective gene products in cell proliferation and homeostasis have been intensively examined by in vitro experiments. However, how each gene mutation or combinations of specific mutations drive malignant progression of CRC in vivo has not been fully understood. Based on the genomic information, we generated mouse models that carry multiple mutations of CRC driver genes in various combinations, and we performed comprehensive histological analyses to link genetic alteration(s) and tumor phenotypes, including liver metastasis. In this review article, we summarize the phenotypes of the respective genetic models carrying major driver mutations and discuss a possible mechanism of mutations underlying malignant progression.

Characterization of RNF43 frameshift mutations that drive Wnt ligand- and R-spondin-dependent colon cancer.

Loss-of-function mutations in RNF43 induce activation of Wnt ligand-dependent Wnt/ß-catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)-type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC-derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI-high and BRAFV600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R-spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R-spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active ß-catenin levels in RNF43-mutant organoids were lower than those in APC two-hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43-mutant organoids with intestinal myofibroblasts accelerated the ß-catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43-mutant CRC cells. Sequencing of subcloned organoid cell-expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis-mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild-type RNF43 allele remaining, while the other organoid line carried two-hit biallelic RNF43 trans-mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second-hit RNF43 mutation may be advantageous in tumorigenesis compared with a single-hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43-mutant cell-derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation-associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43-mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Nano-scale physical properties characteristic to metastatic intestinal cancer cells identified by high-speed scanning ion conductance microscope.

Recent genetic studies have indicated relationships between gene mutations and colon cancer phenotypes. However, how physical properties of tumor cells are changed by genetic alterations has not been elucidated. We examined genotype-defined mouse intestinal tumor-derived cells using a high-speed scanning ion conductance microscope (HS-SICM) that can obtain high-resolution live images of nano-scale topography and stiffness. The tumor cells used in this study carried mutations in Apc (A), Kras (K), Tgfbr2 (T), Trp53 (P), and Fbxw7 (F) in various combinations. Notably, high-metastatic cancer-derived cells carrying AKT mutations (AKT, AKTP, and AKTPF) showed specific ridge-like morphology with active membrane volume change, which was not found in low-metastatic and adenoma-derived cells. Furthermore, the membrane was significantly softer in the metastatic AKT-type cancer cells than other genotype cells. Importantly, a principal component analysis using RNAseq data showed similar distributions of expression profiles and physical properties, indicating a link between genetic alterations and physical properties. Finally, the malignant cell-specific physical properties were confirmed by an HS-SICM using human colon cancer-derived cells. These results indicate that the HS-SICM analysis is useful as a novel diagnostic strategy for predicting the metastatic ability of cancer cells.

Pericentromeric noncoding RNA changes DNA binding of CTCF and inflammatory gene expression in senescence and cancer.

Cellular senescence causes a dramatic alteration of chromatin organization and changes the gene expression profile of proinflammatory factors, thereby contributing to various age-related pathologies through the senescence-associated secretory phenotype (SASP). Chromatin organization and global gene expression are maintained by the CCCTC-binding factor (CTCF); however, the molecular mechanism underlying CTCF regulation and its association with SASP gene expression remains unclear. We discovered that noncoding RNA (ncRNA) derived from normally silenced pericentromeric repetitive sequences directly impairs the DNA binding of CTCF. This CTCF disturbance increases the accessibility of chromatin and activates the transcription of SASP-like inflammatory genes, promoting malignant transformation. Notably, pericentromeric ncRNA was transferred into surrounding cells via small extracellular vesicles acting as a tumorigenic SASP factor. Because CTCF blocks the expression of pericentromeric ncRNA in young cells, the down-regulation of CTCF during cellular senescence triggers the up-regulation of this ncRNA and SASP-related inflammatory gene expression. In this study, we show that pericentromeric ncRNA provokes chromosomal alteration by inhibiting CTCF, leading to a SASP-like inflammatory response in a cell-autonomous and non-cell-autonomous manner and thus may contribute to the risk of tumorigenesis during aging.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

Malignant subclone drives metastasis of genetically and phenotypically heterogenous cell clusters through fibrotic niche generation.

A concept of polyclonal metastasis has recently been proposed, wherein tumor cell clusters break off from the primary site and are disseminated. However, the involvement of driver mutations in such polyclonal mechanism is not fully understood. Here, we show that non-metastatic AP cells metastasize to the liver with metastatic AKTP cells after co-transplantation to the spleen. Furthermore, AKTP cell depletion after the development of metastases results in the continuous proliferation of the remaining AP cells, indicating a role of AKTP cells in the early step of polyclonal metastasis. Importantly, AKTP cells, but not AP cells, induce fibrotic niche generation when arrested in the sinusoid, and such fibrotic microenvironment promotes the colonization of AP cells. These results indicate that non-metastatic cells can metastasize via the polyclonal metastasis mechanism using the fibrotic niche induced by malignant cells. Thus, targeting the fibrotic niche is an effective strategy for halting polyclonal metastasis.

Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core.

Nuclear pore complex (NPC) is a gating nanomachine with a central selective barrier composed mainly of Nups, which contain intrinsically disordered (non-structured) regions (IDRs) with phenylalanine-glycine (FG) motifs (FG-NUPs). The NPC central FG network dynamics is poorly understood, as FG-NUPs liquid-liquid phase separation (LLPS) have evaded structural characterization. Moreover, the working mechanism of single FG-NUP-biofilaments residing at the central lumen is unknown. In general, flexible biofilaments are expected to be tangled and knotted during their motion and interaction. However, filament knotting visualization in real-time and space has yet to be visualized at the nanoscale. Here, we report a spatiotemporally tracking method for FG-NUP organization with nanoscale resolution, unveiling FG-NUP conformation in NPCs of colorectal cells and organoids at timescales of ~150 ms using high-speed atomic force microscopy (HS-AFM). Tracking of FG-NUP single filaments revealed that single filaments have a heterogeneous thickness in normal and cancer models which in turn affected the filament rotation and motion. Notably, FG-NUPs are overexpressed in various cancers. Using the FG-NUP inhibitor, trans-1,2-cyclohexanediol, we found that central plug size was significantly reduced and incompletely reversible back to filamentous structures in aggressive colon cancer cells and organoids. These data showed a model of FG-NUPs reversible self-assembly devolving into the central plug partial biogenesis. Taken together, HS-AFM enabled the tracking and manipulation of single filaments of native FG-NUPs which has remained evasive for decades.

Loss of wild-type p53 promotes mutant p53-driven metastasis through acquisition of survival and tumor-initiating properties.

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.

CRISPR-Cas9-mediated gene knockout in intestinal tumor organoids provides functional validation for colorectal cancer driver genes.

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 Beauty transposon-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 Arid2 could function as tumor suppressor genes (TSGs) in CRC and uncovered a role for Trp53 in tumor metastasis. We also showed that co-occurrent mutations in receptors for activin and transforming growth factor-ß (TGF-ß) 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.

Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

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.-Oshima, H., Kok, S.-Y., Nakayama, M., Murakami, K., Voon, D. C.-C., Kimura, T., Oshima, M. Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.

Interleukin 1 Up-regulates MicroRNA 135b to Promote Inflammation-Associated Gastric Carcinogenesis in Mice.

BACKGROUND & AIMS: Gastritis is associated with development of stomach cancer, but little is known about changes in microRNA expression patterns during gastric inflammation. Specific changes in gene expression in epithelial cells are difficult to monitor because of the heterogeneity of the tissue. We investigated epithelial cell-specific changes in microRNA expression during gastric inflammation and gastritis-associated carcinogenesis in mice. METHODS: We used laser microdissection to enrich epithelial cells from K19-C2mE transgenic mice, which spontaneously develop gastritis-associated hyperplasia, and Gan mice, which express activated prostaglandin E2 and Wnt in the gastric mucosa and develop gastric tumors. We measured expression of epithelial cell-enriched microRNAs and used bioinformatics analyses to integrate data from different systems to identify inflammation-associated microRNAs. We validated our findings in gastric tissues from mice and evaluated protein functions in gastric cell lines (SNU-719, SNU-601, SNU-638, AGS, and GIF-14) and knockout mice. Organoids were cultured from gastric corpus tissues of wild-type and miR-135b-knockout C57BL/6 mice. We measured levels of microRNAs in pairs of gastric tumors and nontumor mucosa from 28 patients in Japan. RESULTS: We found microRNA 135b (miR-135B) to be the most overexpressed microRNA in gastric tissues from K19-C2mE and Gan mice: levels increased during the early stages of gastritis-associated carcinogenesis. Levels of miR-135B were also increased in gastric tumor tissues from gp130F/F mice and patients compared with nontumor tissues. In gastric organoids and immortalized cell lines, expression of miR-135B was induced by interleukin 1 signaling. K19-C2mE mice with disruption of Mir-135b developed hyperplastic lesions that were 50% smaller than mice without Mir-135b disruption and had significant reductions in cell proliferation. Expression of miR-135B in gastric cancer cell lines increased their colony formation, migration, and sphere formation. We identified FOXN3 and RECK messenger RNAs (mRNAs) as targets of miR-135B; their knockdown reduced migration of gastric cancer cell lines. Levels of FOXN3 and RECK mRNAs correlated inversely with levels of miR-135B in human gastric tumors and in inflamed mucosa from K19-C2mE mice. CONCLUSIONS: We found expression of miR-135B to be up-regulated by interleukin L1 signaling in gastric cancer cells and organoids. miR-135B promotes invasiveness and stem-cell features of gastric cancer cells in culture by reducing FOXN3 and RECK messenger RNAs. Levels of these messenger RNA targets, which encode tumor suppressor, are reduced in human gastric tumors.

Mutant p53 in colon cancer.

The accumulation of genetic alterations in driver genes is responsible for the development and malignant progression of colorectal cancer. Comprehensive genome analyses have revealed the driver genes, including APC, KRAS, TGFBR2, and TP53, whose mutations are frequently found in human colorectal cancers. Among them, the p53 mutation is found in ~60% of colorectal cancers, and a majority of mutations are missense-type at 'hot spots', suggesting an oncogenic role of mutant p53 by 'gain-of-function' mechanisms. Mouse model studies have shown that one of these missense-type mutations, p53 R270H (corresponding to human R273H), causes submucosal invasion of intestinal tumors, while the loss of wild-type p53 has a limited effect on the invasion process. Furthermore, the same mutant p53 promotes metastasis when combined with Kras activation and TGF-ß suppression. Importantly, either missense-type p53 mutation or loss of wild-type p53 induces NF-κB activation by a variety of mechanisms, such as increasing promoter accessibility by chromatin remodeling, which may contribute to progression to epithelial-mesenchymal transition. These results indicate that missense-type p53 mutations together with loss of wild-type p53 accelerate the late stage of colorectal cancer progression through the activation of both oncogenic and inflammatory pathways. Accordingly, the suppression of the mutant p53 function via the inhibition of nuclear accumulation is expected to be an effective strategy against malignant progression of colorectal cancer.