Hiroshi Fukamachi

RUNX3 Attenuates β-Catenin/T Cell Factors in Intestinal Tumorigenesis

In intestinal epithelial cells, inactivation of APC, a key regulator of the Wnt pathway, activates beta-catenin to initiate tumorigenesis. However, other alterations may be involved in intestinal tumorigenesis. Here we found that RUNX3, a gastric tumor suppressor, forms a ternary complex with beta-catenin/TCF4 and attenuates Wnt signaling activity. A significant fraction of human sporadic colorectal adenomas and Runx3(+/-) mouse intestinal adenomas showed inactivation of RUNX3 without apparent beta-catenin accumulation, indicating that RUNX3 inactivation independently induces intestinal adenomas. In human colon cancers, RUNX3 is frequently inactivated with concomitant beta-catenin accumulation, suggesting that adenomas induced by inactivation of RUNX3 may progress to malignancy. Taken together, these data demonstrate that RUNX3 functions as a tumor suppressor by attenuating Wnt signaling.

miR‐212 is downregulated and suppresses methyl‐CpG‐binding protein MeCP2 in human gastric cancer

To clarify the role of micro (mi) RNAs in gastric carcinogenesis, we studied the expression and function of miRNAs in gastric carcinoma (GC) cells. Initially, we performed microarray analysis using total RNA from 3 human GC cell lines and noncancerous gastric tissue. Among the downregulated miRNAs in GC cells, miR‐212 expression was decreased in all 8 GC cell lines examined and a significant decrease of miR‐212 expression in human primary GC tissues was also observed in 6 of 11 cases. Transfection of the precursor miR‐212 molecule induced decreased growth of 3 GC cell lines. Using 3 different databases, methyl‐CpG‐binding protein MeCP2 was postulated to be a target of miR‐212. As seen on reporter assaying, miR‐212 repressed the construct with the MECP2 3′‐UTR. Ectopic expression of miR‐212 repressed expression of the MeCP2 protein but not the MECP2 mRNA level. These data suggest that downregulation of miR‐212 may be related to gastric carcinogenesis through its target genes, such as MECP2.

Stroma-Derived Matrix Metalloproteinase (MMP)-2 Promotes Membrane Type 1-MMP–Dependent Tumor Growth in Mice

Matrix metalloproteinase-2 (MMP-2) is a stroma-derived MMP belonging to the type IV collagenase family. It is believed to mediate tumor cell behavior by degrading deposits of type IV collagen, a major component of the basement membrane. The membrane type 1-MMP (MT1-MMP) is a highly potent activator of MMP-2 and is expressed in many tumor and stromal cells. However, the roles played by stromal MMP-2 in tumor progression in vivo remain poorly understood. We established a colon epithelial cell line from an Mt1-mmp(-/-) mouse strain and transfected these cells with an inducible expression system for MT1-MMP (MT1rev cells). Following s.c. implantation into Mmp-2(+/+) mice and induction of MT1-MMP expression, MT1rev cells grew rapidly, whereas they grew very slowly in Mmp-2(-/-) mice, even in the presence of MT1-MMP. This MT1-MMP-dependent tumor growth of MT1rev cells was enhanced in Mmp-2(-/-) mice as long as MMP-2 was supplied via transfection or coimplantation of MMP-2-positive fibroblasts. MT1rev cells cultured in vitro in a three-dimensional collagen gel matrix also required the MT1-MMP/MMP-2 axis for rapid proliferation. MT1rev cells deposit type IV collagen primarily at the cell-collagen interface, and these deposits seem scarce at sites of invasion and proliferation. These data suggest that cooperation between stroma-derived MMP-2 and tumor-derived MT1-MMP may play a role in tumor invasion and proliferation via remodeling of the tumor-associated basement membrane. To our knowledge, this is the first study demonstrating that MT1-MMP-dependent tumor growth in vivo requires stromal-derived MMP-2. It also suggests that MMP-2 represents a potential target for tumor therapeutics.

Claudin-1 Has Tumor Suppressive Activity and Is a Direct Target of RUNX3 in Gastric Epithelial Cells

BACKGROUND & AIMSnThe transcription factor RUNX3 is a gastric tumor suppressor. Tumorigenic Runx3(-/-) gastric epithelial cells attach weakly to each other, compared with nontumorigenic Runx3(+/+) cells. We aimed to identify RUNX3 target genes that promote cell-cell contact to improve our understanding of RUNX3s role in suppressing gastric carcinogenesis.nnnMETHODSnWe compared gene expression profiles of Runx3(+/+) and Runx3(-/-) cells and observed down-regulation of genes associated with cell-cell adhesion in Runx3(-/-) cells. Reporter, mobility shift, and chromatin immunoprecipitation assays were used to examine the regulation of these genes by RUNX3. Tumorigenesis assays and immunohistological analyses of human gastric tumors were performed to confirm the role of the candidate genes in gastric tumor development.nnnRESULTSnMobility shift and chromatin immunoprecipitation assays revealed that the promoter activity of the gene that encodes the tight junction protein claudin-1 was up-regulated via the binding of RUNX3 to the RUNX consensus sites. The tumorigenicity of gastric epithelial cells from Runx3(-/-) mice was significantly reduced by restoration of claudin-1 expression, whereas knockdown of claudin-1 increased the tumorigenicity of human gastric cancer cells. Concomitant expression of RUNX3 and claudin-1 was observed in human normal gastric epithelium and cancers.nnnCONCLUSIONSnThe tight junction protein claudin-1 has gastric tumor suppressive activity and is a direct transcriptional target of RUNX3. Claudin-1 is down-regulated during the epithelial-mesenchymal transition; RUNX3 might therefore act as a tumor suppressor to antagonize the epithelial-mesenchymal transition.

Loss of Runx3 Is a Key Event in Inducing Precancerous State of the Stomach

BACKGROUND & AIMSnRUNX3 is a tumor suppressor originally identified in gastric cancer. The mutation R122C in RUNX3 promotes gastric carcinogenesis by unclear mechanisms. We investigated how Runx3-deficiency contributes to distinct changes in the gastric epithelium that precede neoplasia.nnnMETHODSnRunx3-deficient (Runx3(-/-)) and wild-type BALB/c adult mice were subjected to histological analyses. Gastric cancer formation after administration of N-methyl-N-nitrosourea was evaluated. Runx3(+/+) and Runx3(-/-) gastric epithelial cell lines were used to investigate the molecular basis underlying Runx3 function.nnnRESULTSnThe gastric epithelia in Runx3(-)/(-) adult mice was hyperplastic, with loss of chief cells and development of mucin 6- and trefoil factor-2-expressing metaplasia. The gastric epithelium of Runx3(-)/(-) mice had an intestinal phenotype that expressed Cdx2. After addition of N-methyl-N-nitrosourea, Runx3- mice, unlike wild-type mice, consistently developed adenocarcinomas, indicating that Runx3-deficiency leads to premalignant changes in the gastric epithelia. RUNX3, but not the RUNX3 mutant R122C, repressed Cdx2 expression by attenuation of oncogenic beta(symbol)-catenin and Tcfs.nnnCONCLUSIONSnRunx3-deficiency leads to a precancerous state in the gastric epithelia of mice, characterized by loss of chief cells but not parietal cells; inflammation did not appear to be involved.

Runx3 Protects Gastric Epithelial Cells Against Epithelial-Mesenchymal Transition-Induced Cellular Plasticity and Tumorigenicity†‡§

The transcription factor RUNX3 functions as a tumor suppressor in the gastrointestinal epithelium, where its loss is an early event in carcinogenesis. While RUNX3 acts concurrently as a mediator of TGF‐β signaling and an antagonist of Wnt, the cellular changes that follow its loss and their contribution to tumorigenicity are not fully understood. Here, we report that the loss of Runx3 in gastric epithelial cells results in spontaneous epithelial‐mesenchymal transition (EMT). This produces a tumorigenic stem cell‐like subpopulation, which remarkably expresses the gastric stem cell marker Lgr5. This phenomenon is due to the compounding effects of the dysregulation of the TGF‐β and Wnt pathways. Specifically, Runx3−/−p53−/− gastric epithelial cells were unexpectedly sensitized for TGF‐β‐induced EMT, during which the resultant induction of Lgr5 was enhanced by an aberrantly activated Wnt pathway. These data demonstrate a protective role for RUNX3 in safeguarding gastric epithelial cells against aberrant growth factor signaling and the resultant cellular plasticity and stemness. STEM Cells2012;30:2088–2099

Transcription factor SOX2 up-regulates stomach-specific pepsinogen A gene expression

PurposeTranscription factor SOX2 is expressed in normal gastric mucosae but not in the normal colon. We aimed to clarify the role of SOX2 with reference to pepsinogen expression in the gastrointestinal epithelium.MethodsWe analyzed expression of SOX2 and pepsinogens, differentiation markers of the stomach, in ten gastric cancer (GC) and ten colorectal cancer (CRC) cell lines. The effects of over-expression and down-regulation of SOX2 on pepsinogen expression were also examined.ResultsSix GC and five CRC cell lines showed SOX2 expression on RT-PCR. Expression of pepsinogen A was detectable in eight GC and seven CRC cell lines, whereas the majority of the cell lines expressed pepsinogen C. Over-expression of SOX2 up-regulated expression of pepsinogen A but not that of pepsinogen C in 293T human embryonic kidney cells, and some GC and CRC cell lines. Moreover, pepsinogen A expression was significantly reduced by SOX2 RNA interference in two GC cell lines.ConclusionThese data suggest that SOX2 plays an important role in regulation of pepsinogen A, and ectopic expression of SOX2 may be associated with abnormal differentiation of colorectal cancer cells.

IQGAP2 inactivation through aberrant promoter methylation and promotion of invasion in gastric cancer cells

Invasion and metastases of cancer cells are the main causes of treatment failure in cancer. IQ motif‐containing GTPase activating protein 1 (IQGAP1), plays pivotal roles in intercellular adhesion, migration, invasion and metastases in various cancer cells. However, the role of another family member, IQGAP2, in carcinogenesis remains unknown. Here, we investigated IQGAP2 functions in gastric cancers. We found that IQGAP2 protein expression was lost in 5 of the 9 gastric cancer cell lines. Through analysis by the methylation‐specific PCR, aberrant IQGAP2 methylation was detected in 3 gastric cancer cell lines. IQGAP2 mRNA was found to be activated after 5‐aza‐2′‐deoxycytidine treatment of the methylation‐positive cells. Moreover, IQGAP2 methylation was detected in 28 of the 59 (47%) primary gastric cancer tissues, but not in 12 normal gastric mucosa samples. Immunohistochemical staining revealed that 7 of the 8 (88%) gastric cancer tissues without methylation signals displayed IQGAP2 expression, whereas among 10 with methylation signals none expressed IQGAP2 (p = 0.0002), indicating that IQGAP2 methylation is highly associated with loss of the IQGAP2 expression in the primary gastric cancer tissues as well as gastric cancer cell lines. Furthermore, IQGAP2 methylation was also associated with tumor invasion and a poor prognosis. IQGAP2 knockdown with small interfering RNA increased the invasive capacity of a gastric cancer cell line. These results suggest that silencing of IQGAP2 by promoter methylation may contribute to gastric cancer development.

Human Gsα mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle

Pseudohypoparathyroidism type Ia (PHP-Ia) results from the loss of one allele of Gsα, causing resistance to parathyroid hormone and other hormones that transduce signals via Gs. Most Gsαmutations cause the complete loss of protein expression, but some cause loss of function only, and these have provided valuable insights into the normal function of G proteins. Here we have analyzed a mutant Gsα (αs-AVDT) harboring AVDT amino acid repeats within its GDP/GTP binding site, which was identified in unique patients with PHP-Ia accompanied by neonatal diarrhea. Biochemical and intact cell analyses showed that αs-AVDT is unstable but constitutively active as a result of rapid GDP release and reduced GTP hydrolysis. This instability underlies the PHP-Ia phenotype. αs-AVDT is predominantly localized in the cytosol, but in rat and mouse small intestine epithelial cells (IEC-6 and DIF-12 cells) αs-AVDT was found to be localized predominantly in the membrane where adenylyl cyclase is present and constitutive increases in cAMP accumulation occur in parallel. The likely cause of this membrane localization is the inhibition of an activation-dependent decrease in αs palmitoylation. Upon the overexpression of acyl-protein thioesterase 1, however, αs-AVDT translocates from the membrane to the cytosol, and the constitutive accumulation of cAMP becomes attenuated. These results suggest that PHP-Ia results from the instability of αs-AVDT and that the accompanying neonatal diarrhea may result from its enhanced constitutive activity in the intestine. Hence, palmitoylation may control the activity and localization of Gsα in a cell-specific manner.

CD133 is a marker of gland‐forming cells in gastric tumors and Sox17 is involved in its regulation

CD133 is a universal marker of tissue stem/progenitor cells as well as cancer stem cells, but its physiological significance remains to be elucidated. Here we examined the relationship between expression of CD133 and features of gastric epithelial cells, and found that CD133‐positive (CD133[+]) tumor cell lines formed well‐differentiated tumors while CD133‐negative (CD133[−]) lines formed poorly differentiated ones when subcutaneously injected into nude mice. We also found that CD133(+) and CD133(−) cell populations co‐existed in some cell lines. FACS analysis showed that CD133(+) cells were mother cells because CD133(+) cells formed both CD133(+) and CD133(−) cells, but CD133(−) cells did not form CD133(+) cells. In these cell lines, CD133(+) cells formed well‐differentiated tumors while CD133(−) cells formed poorly differentiated ones. In human gastric cancers, CD133 was exclusively expressed on the luminal surface membrane of gland‐forming cells, and it was never found on poorly differentiated diffuse‐type cells. Considering that poorly differentiated tumors often develop from well‐differentiated tumors during tumor progression, these results suggest that loss of expression of CD133 might be related to gastric tumor progression. Microarray analysis showed that CD133(+) cells specifically expressed Sox17, a tumor suppressor in gastric carcinogenesis. Forced expression of SOX17 induced expression of CD133 in CD133(−) cells, and reduction of SOX17 caused by siRNA in CD133(+) cells induced a reduction in the level of CD133. These results indicate that Sox17 might be a key transcription factor controlling CD133 expression, and that it might also play a role in the control of gastric tumor progression. (Cancer Sci 2011; 102: 1313–1321)