Oncogene (2010) 29, 2605–2615 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 $32.00 www.nature.com/onc REVIEW RUNX3 is multifunctional in carcinogenesis of multiple solid tumors LSH Chuang1 and Y Ito1,2,3 1Cancer Biology Program, Cancer Science Institute of Singapore, National University of Singapore, Singapore; 2Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore and 3Institute of Molecular and Cell Biology, Singapore The study of RUNX3 in tumor pathogenesis is a rapidly Sgpp1, Ncam1, p21WAF1) (Wotton et al., 2008), the expanding area of cancer research. Functional inactiva- RUNX proteins have been shown to perform distinct tion of RUNX3—through mutation, epigenetic silencing, functions that are dependent on tissue type (Brady and or cytoplasmic mislocalization—is frequently observed in Farrell, 2009). This review explores the dualistic solid tumors of diverse origins. This alone indicates that associations of RUNX3 with cancer with emphasis on RUNX3 inactivation is a major risk factor in tumorigen- the emerging concept of RUNX3 as a multifunctional esis and that it occurs early during progression to suppressor of solid tumors. malignancy. Conversely, RUNX3 has also been described to have an oncogenic function in a subset of tumors. Although the mechanism of how RUNX3 switches from RUNX3 inactivation is prevalent in solid tumors tumor suppressive to oncogenic activity is unclear, this is of clinical relevance with implications for cancer detection Hemizygous deletion of RUNX3 gene and prognosis. Recent developments have significantly The location of RUNX3 at chromosome 1p36, a deletion contributed to our understanding of the pleiotropic tumor hotspot in diverse cancers of epithelial, hematopoietic, suppressive properties of RUNX3 that regulate major and neural origins (Bagchi and Mills, 2008), prompts signaling pathways. This review summarizes the important the following questions: How often is RUNX3 deleted? findings that link RUNX3 to tumor suppression. Does RUNX3 deletion confer any cancer growth Oncogene (2010) 29, 2605–2615; doi:10.1038/onc.2010.88; advantage? Examination of gastric cancer tissue speci- published online 29 March 2010 mens revealed hemizygous deletions of RUNX3 at Keywords: RUNX3; tumor suppressor; solid tumors; frequencies which increased with tumor grade (Li et al., functional inactivation; TGF-b; Wnt 2002). Likewise, RUNX3 hemizygous deletion and concomitant decrease in RUNX3 expression are also prevalent in gastric, bile duct, pancreatic, and lung cancer cell lines (Li et al., 2002; Wada et al., 2004; Introduction Yanada et al., 2005). This suggests that downregulation of RUNX3 expression is an early occurrence during RUNX family of transcription factors progression to malignancy, and thus, provides a strong The Runt-related transcription factors (RUNX) describe hint of RUNX3 tumor suppressive function. Moreover, a family of evolutionarily conserved metazoan proteins, it is noteworthy that genes mapping to human 1p36 which share the highly homologous N-terminal Runt show syntenic conservation with mouse chromosome 4, domain, a DNA-binding, and heterodimerization region lending weight to the theory that this region harbors a of about 120 amino acids. Initially discovered as a cluster of tumor suppressor genes, which work coopera- heterodimeric protein complex comprising RUNX and tively and when deleted would drive tumorigenesis cofactor PEBP2b/CBFb 20 years ago, RUNX proteins (Bagchi and Mills, 2008). The 1p36 tumor suppressor are rapidly gaining prominence as major players in genes include retinoblastoma protein-interacting zinc- cancer pathogenesis. In mammals, three RUNX family finger (RIZ)1, chromodomain helicase DNA-binding members have been identified. The RUNX genes are protein 5 (CHD5), secretory phospholipase PLA2G2A widely expressed, but show differential, tissue-specific (Modifier of Min-1) (Cormier et al., 1997), and TP73. expression. Although their strong homology indicates a Interestingly, RIZ1, also known as PRDM2,isa certain degree of redundancy as observed during mouse member of the nuclear histone/protein methyltransferase embryogenesis (Fukushima-Nakase et al., 2005) and superfamily that methylates histone 3 Lys 9 (H3K9) transcription regulation of various genes (for example residues, whereas CHD5 belongs to the SWI/SNF chromatin modifier family. As with all RUNX family Correspondence: Dr Y Ito, Institute of Molecular and Cell Biology, members, RUNX3 encodes a highly conserved Cancer Science Institute, National University of Singapore, 61 C-terminus motif VWRPY that was shown to bind Biopolis Drive, Proteos, Singapore 138673, Singapore. E-mail: [email protected] co-repressor proteins TLE1 and Sin3A, which in turn Received 4 November 2009; revised 12 February 2010; accepted 16 recruit histone deacetylase (Stifani et al., 1992; Aronson February 2010; published online 29 March 2010 et al., 1997; Imai et al., 1998; Levanon et al., 1998). RUNX3 and carcinogenesis of solid tumors LSH Chuang and Y Ito 2606 As part of this multiprotein complex, RUNX3 can is required for stem cell pluripotency and self-renewal. repress gene transcription through modulation of It is targeted to gene promoters of important develop- chromatin structure. It is tempting, therefore, to mental regulators, including RUNX3 (Lee et al., 2006), speculate that collaboration of RUNX3 with these in which it methylates histone H3 at the Lys27 (H3K27) other 1p36 chromatin modifiers suppresses transcription residue to repress transcription. Interestingly, EZH2 can that contributes to tumor development. direct DNA methylation by recruitment of DNMTs to target gene promoters, suggesting that it may be a significant causative factor of aberrant methylation in Epigenetic silencing of RUNX3 cancer (Vire et al., 2006). In addition, H3K27 methylation In tumor cells, mutation of one allele of a gene is has been shown to mark genes for de novo methylation in frequently accompanied by hypermethylation of the cancer (Schlesinger et al., 2007). A recent study found other allele, resulting in complete functional inactivation that frequent overexpression of EZH2 in aggressive of the gene (Jones and Baylin, 2002). Besides deletion of solid tumors is largely due to the loss of its regulator the RUNX3 gene, recent years have seen a surge in microRNA-101 (Varambally et al., 2008). More speci- reports of other modes of RUNX3 inactivation—be it fically, reintroduction of miR-101 or knockdown of through promoter hypermethylation or protein mislo- EZH2 expression was sufficient to reduce H3K27 calization—in solid tumor tissues. To date, hypermethy- trimethylation and increase RUNX3 mRNA levels. In lation of the CpG island at the RUNX3 promoter is the same vein, Fujii et al. (2008) found that EZH2 binds one of the most common aberrant methylation events to the RUNX3 promoter, resulting in upregulation of in cancer, suggesting that RUNX3 inactivation is a H3K27 methylation and concomitant downregulation significant risk factor for tumorigenesis. Diverse tumor of RUNX3 expression (Fujii et al., 2008). Recently, Lee tissues have been reported to exhibit RUNX3 hyper- et al. (2009) extended this finding by showing that methylation and inactivation. These include tissues hypoxia-induced upregulation of G9a histone methyl- and cell lines that originated from gastric (Li et al., transferase and HDAC1 expression also cause epige- 2002; Oshimo et al., 2004), bladder (Kim et al., 2005; netic RUNX3 silencing through H3K9 methylation and Wolff et al., 2008), colorectal (Ahlquist et al., 2008; decreased H3 acetylation at RUNX3 promoter. As Soong et al., 2009; Subramaniam et al., 2009), breast hypoxia is commonly associated with solid tumors of (Lau et al., 2006; Jiang et al., 2008), lung (Sato et al., 41mm3, this suggests that RUNX3 inactivation may be 2006), pancreatic (Wada et al., 2004), brain cancers associated with increased tumor size (Lee et al., 2009). (Mueller et al., 2007), and hepatocellular carcinoma Helicobacter pylori, a major causative factor of gastric (Kim et al., 2004). Notably, RUNX3 methylation status cancer, was also reported to alter RUNX3 methylation is one of the five markers used to classify colorectal status through enhanced production of nitric oxide in tumors associated with very high frequencies of CpG macrophages (Katayama et al., 2009). Moreover, the island methylation (CIMP), microsatellite instability, same group showed that lipopolysaccharide can also and BRAF mutation (Weisenberger et al., 2006). More- induce methylation changes, again through nitric oxide over, hypermethylation of RUNX3 is associated with a production, suggesting that inflammation may be 100-fold increase risk of developing bladder cancer another cause of RUNX3 hypermethylation in gastric (Kim et al., 2005). RUNX3 methylation is not only cancer. Estrogen exposure is an important risk factor in acquired early during tumorigenesis, but also increases breast cancer pathogenesis. It induces neoplastic trans- with age: RUNX3 is thus an attractive candidate for formation in epithelial cells that were derived from cancer detection and prognosis (Wolff et al., 2008). mammospheres isolated from healthy breast tissues. Conversely, RUNX3 overexpression correlates with Interestingly, exposure of these mammosphere-derived reduced invasive potential of breast cancer cells cells to estrogen was sufficient to induce hypermethyla- (Chen et al., 2007). Similarly, RUNX3 expression tion of RUNX3 (Cheng et al.,