Kosei Ito

RUNX3, A Novel Tumor Suppressor, Is Frequently Inactivated in Gastric Cancer by Protein Mislocalization

Loss of RUNX3 expression is suggested to be causally related to gastric cancer as 45% to 60% of gastric cancers do not express RUNX3 mainly due to hypermethylation of the RUNX3 promoter. Here, we examined for other defects in the properties of RUNX3 in gastric cancers that express RUNX3. Ninety-seven gastric cancer tumor specimens and 21 gastric cancer cell lines were examined by immunohistochemistry using novel anti-RUNX3 monoclonal antibodies. In normal gastric mucosa, RUNX3 was expressed most strongly in the nuclei of chief cells as well as in surface epithelial cells. In chief cells, a significant portion of the protein was also found in the cytoplasm. RUNX3 was not detectable in 43 of 97 (44%) cases of gastric cancers tested and a further 38% showed exclusive cytoplasmic localization, whereas only 18% showed nuclear localization. Evidence is presented suggesting that transforming growth factor-β is an inducer of nuclear translocation of RUNX3, and RUNX3 in the cytoplasm of cancer cells is inactive as a tumor suppressor. RUNX3 was found to be inactive in 82% of gastric cancers through either gene silencing or protein mislocalization to the cytoplasm. In addition to the deregulation of mechanisms controlling gene expression, there would also seem to be at least one other mechanism controlling nuclear translocation of RUNX3 that is impaired frequently in gastric cancer.

RUNX3 Suppresses Gastric Epithelial Cell Growth by Inducing p21WAF1/Cip1 Expression in Cooperation with Transforming Growth Factor β-Activated SMAD

ABSTRACT RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor β1 (TGF-β1). It is known that TGF-β1 induces cell growth arrest by activating CDKN1A (p21 WAF1 /Cip1 ), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-β-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-β-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.

The RUNX3 Tumor Suppressor Upregulates Bim in Gastric Epithelial Cells Undergoing Transforming Growth Factor β-Induced Apoptosis

ABSTRACT Genes involved in the transforming growth factor β (TGF-β) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUNX3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3−/− gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-β. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids 1 to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3−/− mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim−/− gastric epithelium. We confirmed comparable expression of TGF-β1 and TGF-β receptors between wild-type and Runx3−/− gastric epithelia and reduction of Bim in TGF-β1−/− stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-β-induced apoptosis.

Genetic regulation of the RUNX transcription factor family has antitumor effects

Runt-related transcription factor 1 (RUNX1) is generally considered to function as a tumor suppressor in the development of leukemia, but a growing body of evidence suggests that it has pro-oncogenic properties in acute myeloid leukemia (AML). Here we have demonstrated that the antileukemic effect mediated by RUNX1 depletion is highly dependent on a functional p53-mediated cell death pathway. Increased expression of other RUNX family members, including RUNX2 and RUNX3, compensated for the antitumor effect elicited by RUNX1 silencing, and simultaneous attenuation of all RUNX family members as a cluster led to a much stronger antitumor effect relative to suppression of individual RUNX members. Switching off the RUNX cluster using alkylating agent–conjugated pyrrole-imidazole (PI) polyamides, which were designed to specifically bind to consensus RUNX-binding sequences, was highly effective against AML cells and against several poor-prognosis solid tumors in a xenograft mouse model of AML without notable adverse events. Taken together, these results identify a crucial role for the RUNX cluster in the maintenance and progression of cancer cells and suggest that modulation of the RUNX cluster using the PI polyamide gene-switch technology is a potential strategy to control malignancies.

Subnuclear targeting of the Runx3 tumor suppressor and its epigenetic association with mitotic chromosomes

Runx proteins are tissue‐specific transcriptional scaffolds that organize and assemble regulatory complexes at strategic sites of target gene promoters and at intranuclear foci to govern activation or repression. During interphase, fidelity of intranuclear targeting supports the biological activity of Runx1 and Runx2 proteins. Both factors regulate genes involved in cell cycle control and cell growth (e.g., rRNA genes), as well as lineage commitment. Here, we have examined the subcellular regulatory properties of the third Runx member, the tumor suppressor protein Runx3, during interphase and mitosis. Using in situ cellular and biochemical approaches we delineated a subnuclear targeting signal that directs Runx3 to discrete transcriptional foci that are nuclear matrix associated. Chromatin immunoprecipitation results show that Runx3 occupies rRNA promoters during interphase. We also find that Runx3 remains associated with chromosomes during mitosis and localizes with nucleolar organizing regions (NORs), reflecting an interaction with epigenetic potential. Taken together, our study establishes that common mechanisms control the subnuclear distribution and activities of Runx1, Runx2, and Runx3 proteins to support RNA polymerase I and II mediated gene expression during interphase and mitosis. J. Cell. Physiol. 218: 473–479, 2009.

Cbfb regulates bone development by stabilizing Runx family proteins.

Runx family proteins, Runx1, Runx2, and Runx3, play important roles in skeletal development. Runx2 is required for osteoblast differentiation and chondrocyte maturation, and haplodeficiency of RUNX2 causes cleidocranial dysplasia, which is characterized by open fontanelles and sutures and hypoplastic clavicles. Cbfb forms a heterodimer with Runx family proteins and enhances their DNA‐binding capacity. Cbfb‐deficient (Cbfb−/−) mice die at midgestation because of the lack of fetal liver hematopoiesis. We previously reported that the partial rescue of hematopoiesis in Cbfb−/− mice revealed the requirement of Cbfb in skeletal development. However, the precise functions of Cbfb in skeletal development still remain to be clarified. We deleted Cbfb in mesenchymal cells giving rise to both chondrocyte and osteoblast lineages by mating Cbfbfl/fl mice with Dermo1 Cre knock‐in mice. Cbfbfl/fl/Cre mice showed dwarfism, both intramembranous and endochondral ossifications were retarded, and chondrocyte maturation and proliferation and osteoblast differentiation were inhibited. The differentiation of chondrocytes and osteoblasts were severely inhibited in vitro, and the reporter activities of Ihh, Col10a1, and Bglap2 promoter constructs were reduced in Cbfbfl/fl/Cre chondrocytes or osteoblasts. The proteins of Runx1, Runx2, and Runx3 were reduced in the cartilaginous limb skeletons and calvariae of Cbfbfl/fl/Cre embryos compared with the respective protein in the respective tissue of Cbfbfl/fl embryos at E15.5, although the reduction of Runx2 protein in calvariae was much milder than that in cartilaginous limb skeletons. All of the Runx family proteins were severely reduced in Cbfbfl/fl/Cre primary osteoblasts, and Runx2 protein was less stable in Cbfbfl/fl/Cre osteoblasts than Cbfbfl/fl osteoblasts. These findings indicate that Cbfb is required for skeletal development by regulating chondrocyte differentiation and proliferation and osteoblast differentiation; that Cbfb plays an important role in the stabilization of Runx family proteins; and that Runx2 protein stability is less dependent on Cbfb in calvariae than in cartilaginous limb skeletons.

Role of RUNX3 in Bone Morphogenetic Protein Signaling in Colorectal Cancer

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional cytokines regulating a broad spectrum of biological functions. Recent studies show the presence of BMP receptor 1a mutations in juvenile polyposis and frequent Smad4 mutations in colon cancer, suggesting that aberrations in BMP signaling play an important role in intestinal cancer pathogenesis. However, the exact molecular mechanisms remain poorly understood. The Runt domain transcription factor RUNX3 is an integral component of signaling pathways mediated by TGF-beta and BMPs. RUNX3 is a gastric and colon tumor suppressor, functioning downstream of TGF-beta. Recently, we showed the tumor-suppressive effects of RUNX3 by its ability to attenuate beta-catenin/T-cell factors (TCFs) transactivation in intestinal tumorigenesis. Here, we explore the molecular basis of the tumor-suppressive function of the BMP pathway through RUNX3 in colorectal carcinogenesis. BMP exerted a growth-suppressive effect in HT-29, a human colorectal cancer cell line. c-Myc oncogene was found to be downregulated by BMP and/or RUNX3. We show that upregulation of RUNX3 by BMP reduces c-Myc expression. Evidence is presented suggesting that RUNX3 downregulates c-Myc expression by two parallel pathways-directly at the transcriptional level and through attenuation of beta-catenin/TCFs, downstream of BMPs in colorectal cancer cells.

Overexpression of Cdk6 and Ccnd1 in chondrocytes inhibited chondrocyte maturation and caused p53-dependent apoptosis without enhancing proliferation

Cell proliferation and differentiation are closely coupled. However, we previously showed that overexpression of cyclin-dependent kinase (Cdk6) blocks chondrocyte differentiation without affecting cell-cycle progression in vitro. To investigate whether Cdk6 inhibits chondrocyte differentiation in vivo, we generated chondrocyte-specific Cdk6 transgenic mice using Col2a1 promoter. Unexpectedly, differentiation and cell-cycle progression of chondrocytes in the Cdk6 transgenic mice were similar to those in wild-type mice. Then, we generated chondrocyte-specific Ccnd1 transgenic mice and Cdk6/Ccnd1 double transgenic mice to investigate the possibility that Cdk6 inhibits chondrocyte differentiation through E2f activation. Bromodeoxyuridine (BrdU)-positive chondrocytes and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive chondrocytes were increased in number, and chondrocyte maturation was inhibited only in Cdk6/Ccnd1 transgenic mice (K6H/D1H mice), which showed dwarfism. Retinoblastoma protein (pRb) was highly phosphorylated but p107 was upregulated, and the expression of E2f target genes was dysregulated as shown by upregulation of Cdc6 but downregulation of cyclin E, dihydrofolate reductase (dhfr), Cdc25a and B-Myb in chondrocytes of K6H/D1H mice. Similarly, overexpression of Cdk6/Ccnd1 in a chondrogenic cell line ATDC5 highly phosphorylated pRb, upregulated p107, induced apoptosis, upregulated Cdc6 and downregulated cyclin E, dhfr and B-Myb and p107 small interfering RNA reversed the expression of downregulated genes. Further, introduction of kinase-negative Cdk6 and cyclin D1 abolished all effects by Cdk6/cyclin D1 in ATDC5 cells, indicating the requirement of the kinase activity on these effects. p53 deletion partially restored the size of the skeleton and almost completely rescued chondrocyte apoptosis, but failed to enhance chondrocyte proliferation in K6H/D1H mice. These findings indicated that Cdk6/Ccnd1 overexpression inhibited chondrocyte maturation and enhanced G1/S cell-cycle transition by phosphorylating pRb, but the chondrocytes failed to accomplish the cell cycle, and underwent p53-dependent apoptosis probably due to the dysregulation of E2f target genes. Our findings also indicated that p53 deletion in addition to the inactivation of Rb was not sufficient to accelerate chondrocyte proliferation, suggesting the resistance of chondrocytes to sarcomagenesis.

Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells.

RUNX3 is a tumor suppressor for a variety of cancers. RUNX3 suppresses the canonical Wnt signaling pathway by binding to the TCF4/β‐catenin complex, resulting in the inhibition of binding of the complex to the Wnt target gene promoter. Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells. Notably, RUNX3 expression increased the ratio of the Wnt signaling‐high population in the KatoIII cells. although the maximum Wnt activation level of individual cells was similar to that in the control. As found previously, RUNX3 also binds to TCF4 and β‐catenin in KatoIII cells, suggesting that these molecules form a ternary complex. Moreover, the ChIP analyses revealed that TCF4, β‐catenin and RUNX3 bind the promoter region of the Wnt target genes, Axin2 and c‐Myc, and the occupancy of TCF4 and β‐catenin in these promoter regions is increased by the RUNX3 expression. These results suggest that RUNX3 stabilizes the TCF4/β‐catenin complex on the Wnt target gene promoter in KatoIII cells, leading to activation of Wnt signaling. Although RUNX3 increased the Wnt signaling activity, its expression resulted in suppression of tumorigenesis of KatoIII cells, indicating that RUNX3 plays a tumor‐suppressing role in KatoIII cells through a Wnt‐independent mechanism. These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/β‐catenin complex by cell context‐dependent mechanisms.

Cbfb2 Isoform Dominates More Potent Cbfb1 and Is Required for Skeletal Development.

Cbfb is a cotranscription factor that forms a heterodimer with Runx proteins Runx1, Runx2, and Runx3. It is required for fetal liver hematopoiesis and skeletal development. Cbfb has two functional isoforms, Cbfb1 and Cbfb2, which are formed by alternative splicing. To address the biological functions of these isoforms in skeletal development, we examined Cbfb1–/– and Cbfb2–/– mouse embryos. Intramembranous and endochondral ossification was retarded and chondrocyte and osteoblast differentiation was inhibited in Cbfb2–/– embryos but not in Cbfb1–/– embryos. Cbfb2 mRNA was upregulated in calvariae, limbs, livers, thymuses, and hearts of Cbfb1–/– embryos but Cbfb1 mRNA was not in those of Cbfb2–/– embryos, and the total amount of Cbfb1 and Cbfb2 mRNA in Cbfb1–/– embryos was similar to that in wild‐type embryos but was severely reduced in Cbfb2–/– embryos. The absolute numbers of Cbfb2 mRNA in calvariae, limbs, livers, thymuses, and brains in wild‐type embryos were about three times higher than those of Cbfb1 in the respective tissue. The levels of Runx proteins were reduced in calvariae, limbs, and primary osteoblasts from Cbfb2–/– embryos, but the reduction in Runx2 protein was very mild. Furthermore, the amounts of Runx proteins and Cbfb in Cbfb2–/– embryos differed similarly among skeletal tissues, livers, and thymuses, suggesting that Runx proteins and Cbfb are mutually required for their stability. Although Cbfb1–/– embryos developed normally, Cbfb1 induced chondrocyte and osteoblast differentiation and enhanced DNA binding of Runx2 more efficiently than Cbfb2. Our results indicate that modulations in the relative levels of the isoforms may adjust transcriptional activation by Runx2 to appropriate physiological levels. Cbfb2 was more abundant, but Cbfb1 was more potent for enhancing Runx2 activity. Although only Cbfb2 loss generated overt skeletal phenotypes, both may play major roles in skeletal development with functional redundancy.