Inactivation Of RUNX3 Research Articles

Frequent inactivation of RUNX3 in endometrial carcinoma

Objective Our objective was to determine whether RUNX3 tumor suppressor is inactivated in endometrial carcinoma. Methods We have investigated 24 endometrial carcinomas, 3 endometrial carcinoma cell lines, and 9 normal endometria for genetic and epigenetic alterations of RUNX3. Reverse-transcription PCR (RT-PCR), methylation-specific PCR (MS-PCR) analysis, and loss of heterozygosity (LOH) analysis were performed. We also tested RUNX3 protein expression by immunohistochemistry. Results Using RT-PCR technique, we observed a significant loss of RUNX3 mRNA expression in nine of 24 endometrial carcinomas (38%) and in all 3-cell lines (100%). In contrast, all nine of the normal endometria showed an abundant expression of RUNX3 mRNA. Methylation-specific PCR (MS-PCR) analysis of the CpG islands of RUNX3 showed the promoter region to be hypermethylated in 18 of 21 analyzed carcinomas (86%), whereas only two of nine normal endometria (22%) were methylated ( p < 0.01). By using two polymorphic microsatellite markers, D1S199 and D1S1676, we detected 1p36 LOH in 7 of 21 carcinomas (33%). We observed a significant relationship between the loss of RUNX3 mRNA expression and this regional LOH ( p < 0.01). Immunohistochemical staining showed that RUNX3 protein expression was lost in 12 of 21 endometrial carcinomas (57%). We observed a significantly more frequent loss of RUNX3 protein expression in the histologically higher-grade tumors (Grade 3) than in Grade 1 or 2 tumors ( p < 0.01). Conclusion These findings indicate that RUNX3 inactivation may play an important role in carcinogenesis of the endometrium, especially in high-grade endometrial carcinoma.

Downregulation of RUNX3 by protein mislocation and gene inactivation in human epithelial ovarian cancer cells

16545 Background: Runt-related transcription factor 3 (RUNX3) is mapped to 1p36, which is frequently deleted and has been recognized as a tumor suppressor gene in human gastric cancer and breast cancer, and its expression is frequently lost or down-regulated due to epigenetic gene silencing. Recent research also has revealed that RUNX3 is mislocalized to the cytoplasm and plays a more prominent role in gastric carcinogenesis than previously estimated. Methods: In this study, the expression and localization of RUNX3 protein in 63 primary epithelial ovarian cancer specimens were evaluated by immunohistochemistry using anti-RUNX3 monoclonal antibodies and correlation between these results and clinicopathological features was examined. Results: RUNX3 protein was expressed in cell nuclei in 21 of 63 (33.3%) ovarian cancer samples. RUNX3 was not detectable in 20.6% (13/63) of ovarian cancers and a further 46.0% (29/63) exhibited exclusive cytoplasmic localization, suggesting that RUNX3 is functionally inactive in 66.7% of epithelial ovarian cancers. The rate of inactivation was 51.7% (15/29) in serous cystadenocarcinoma, 100.0% (4/4) in endometrioid adenocarcinoma, 68.8% (11/16) in mucinous cystadenocarcinoma, 77.8% (7/9) in clear cell carcinoma and 60.0% (3/5) in the other subtypes. Inactivation of RUNX3 was not associated with the other clinicopathological features of breast cancer such as age, stage or survival. Conclusions: In conclusion, RUNX3 was found to be inactive in 66.7% of primary human ovarian cancers, primarily due either to protein mis-localization to the cytoplasm or gene inactivation. Our results suggest that RUNX3 may play a role in human epithelial ovarian cancer development and progression. No significant financial relationships to disclose.

Adverse prognosis of epigenetic inactivation in RUNX3 gene at 1p36 in human pancreatic cancer

Alteration in transforming growth factor-β signalling pathway is one of the main causes of pancreatic cancer. The human runt-related transcription factor 3 gene (RUNX3) is an important component of this pathway. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including pancreatic cancer. Therefore, we examined genetic and epigenetic alterations of RUNX3 in human pancreatic cancer. Thirty-two patients with pancreatic cancer were investigated in this study. We examined the methylation status of RUNX3 promoter region, loss of heterozygosity (LOH) at 1p36, and conducted a mutation analysis. The results were compared with clinicopathological data. Promoter hypermethylation was detected in 20 (62.5%) of 32 pancreatic cancer tissues, confirmed by sequence of bisulphite-treated DNA. Loss of heterozygosity was detected in 11 (34.3%) of 32 pancreatic cancers. In comparison with clinicopathological data, hypermethylation showed a relation with a worse prognosis (P=0.0143). Hypermethylation and LOH appear to be common mechanisms for inactivation of RUNX3 in pancreatic cancer. Therefore, RUNX3 may be an important tumour suppressor gene related to pancreatic cancer.

RUNX3 inactivation by frequent promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression

We had previously established that inactivation of RUNX3 occurs by frequent promoter hypermethylation and protein mislocalization in invasive ductal carcinomas (IDC) of breast. Here, we hypothesize that inactivation of RUNX3 occurring in ductal carcinoma in situ (DCIS) represent early event in breast carcinogenesis. The study cohort of 40 patients included 17 pure DCIS cases and 23 cases of DCIS with associated IDC (DCIS-IDC). The DCIS and IDC components of mixed cases were manually microdissected to permit separate evaluation. All the 63 samples including 17 pure DCIS, 23 samples each of DCIS and IDC of DCIS-IDC cases were analyzed for RUNX3 protein expression using R3-6E9 monoclonal antibody as well as promoter methylation status by methylation specific PCR. Compared to matched normal breast samples (4 of 40, 10%), DCIS (35 of 40, 88%) and IDC (21 of 23, 91%) exhibited significant RUNX3 inactivation (P<0.001) in the form of negative or weak nuclear staining. In contrast to normal breast tissues (1/10, 10%), promoter hypermethylation of RUNX3 was significantly higher in the neoplastic breast samples (46 of total 61, 75%) including 30 of 40 (75%) DCIS and 16 of 21 (76%) IDC samples (P=0.009). Overall, promoter hypermethylation correlated with RUNX3 inactivation in 42 of 46 (91%) methylated samples (P=0.03). Mislocalized cytoplasmic expression also accounted for RUNX3 inactivation in majority of DCIS (33/40, 83%) and IDC (20/23, 87%) samples independent of promoter hypermethylation. Our data suggest that RUNX3 inactivation by promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression.

The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells

Members of the Runx family of transcriptional regulators are required for the appropriate expression of CD4 and CD8 at discrete stages of T cell development. The roles of these factors in other aspects of T cell development are unknown. We used a strategy to conditionally inactivate the genes encoding Runx1 or Runx3 at different stages of thymocyte development, demonstrating that Runx1 regulates the transitions of developing thymocytes from the CD4−CD8− double-negative stage to the CD4+CD8+ double-positive (DP) stage and from the DP stage to the mature single-positive stage. Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages, respectively. Runx1-deficient CD4+ T cells had markedly reduced expression of the interleukin 7 receptor and exhibited shorter survival. In addition, inactivation of both Runx1 and Runx3 at the DP stages resulted in a severe block in development of CD8+ mature thymocytes. These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells.

The CpG Island Methylator Phenotype and Chromosomal Instability Are Inversely Correlated in Sporadic Colorectal Cancer

The CpG island methylator phenotype (CIMP) is one of the mechanisms involved in colorectal carcinogenesis (CRC). Although CIMP is probably the cause of high-frequency microsatellite instability (MSI-H) sporadic CRCs, its role in microsatellite stable (MSS) tumors is debated. The majority of MSS CRCs demonstrate chromosomal instability (CIN) with frequent loss of heterozygosity (LOH) at key tumor suppressor genes. We hypothesized that the majority of sporadic CRCs without CIN would be associated with CIMP. We tested 126 sporadic CRCs for MSI and LOH and categorized tumors into MSI, LOH, or MSI-/LOH- subgroups. Methylation status was evaluated using 6 CIMP-related markers (MINT1, MINT2, MINT31, p16(INK4alpha), p14(ARF), and hMLH1) and 6 tumor suppressor genes (PTEN, TIMP3, RUNX3, HIC1, APC, and RARbeta2). BRAF V600E mutation analysis was performed using allele-specific polymerase chain reaction and DNA sequencing. We observed frequent methylation at all 12 loci in all CRCs. BRAF V600E mutations correlated with the MSI (P < .0001) and MSI-/LOH- (P = .03) subgroups. MSI and MSI-/LOH- tumors exhibited more promoter methylation than CRCs with LOH (P < .0001). We also found an inverse correlation between the frequencies of methylation and LOH (rho = -0.36; P < .0001). The associations between methylation frequencies at CIMP-related markers and MSI or MSI-/LOH- sporadic CRCs suggest that the majority of these tumors evolve through CIMP. These findings suggest that CIN and CIMP represent 2 independent and inversely related mechanisms of genetic and epigenetic instability in sporadic CRCs and confirm that MSI cancers arise as a consequence of CIMP.

RUNX3 Is Frequently Inactivated by Dual Mechanisms of Protein Mislocalization and Promoter Hypermethylation in Breast Cancer

A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBF-beta, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment.

Graded Activity of Transcription Factor Runx3 Specifies the Laminar Termination Pattern of Sensory Axons in the Developing Spinal Cord

Different functional classes of dorsal root ganglion sensory neurons project their axons to distinct target zones within the developing spinal cord. To explore the mechanisms that link sensory neuron subtype identity and axonal projection pattern, we analyzed the roles of Runx and ETS transcription factors in the laminar targeting of sensory afferents. Gain- and loss-of-function studies in chick embryos reveal that the status of Runx3 expression is a major determinant of the dorso-ventral position of termination of proprioceptive and cutaneous sensory axons. In addition, the level of expression and/or activity of Runx3 in individual proprioceptive sensory neurons appears to specify whether their axons terminate in intermediate or ventral regions. Our findings suggest that the selectivity of Runx3 expression, and its level of activity, control sensory afferent targeting in the developing spinal cord.

RUNX3 Inactivation by Point Mutations and Aberrant DNA Methylation in Bladder Tumors

RUNX3 is inactivated at high frequency in many tumors. However, in most cases, inactivation is caused by silencing of the gene due to promoter hypermethylation. Because epigenetic silencing is known to affect many major tumor suppressor genes in cancer cells, it is not clear whether RUNX3 is primarily responsible for the induction of carcinogenesis in these cases, except for the gastric cancer cases that we reported previously. We investigated genetic and epigenetic alterations of RUNX3 in 124 bladder tumor cases and seven bladder tumor-derived cell lines. Here we show that RUNX3 is inactivated by aberrant DNA methylation in 73% (90 of 124) of primary bladder tumor specimens and 86% (six of seven) of bladder tumor cell lines. In contrast, the promoter regions of 20 normal bladder mucosae were unmethylated. Importantly, one patient bore missense mutations, each of which resulted in amino acid substitutions in the highly conserved Runt domain. The mutations abolished the DNA-binding ability of RUNX3. A second patient had a single nucleotide deletion within the Runt domain coding region that resulted in truncation of the protein. RUNX3 methylation was a significant risk factor for bladder tumor development, superficial bladder tumor recurrence, and subsequent tumor progression. These results strongly suggest that inactivation of RUNX3 may contribute to bladder tumor development and that promoter methylation and silencing of RUNX3 could be useful prognostic markers for both bladder tumor recurrence and progression.

Inactivation of p16, RUNX3, and HPP1 occurs early in Barrett's-associated neoplastic progression and predicts progression risk

Patients with Barrett's esophagus (BE) are at increased risk of developing esophageal adenocarcinoma (EAC). Clinical neoplastic progression risk factors, such as age and the length of the esophageal BE segment, have been identified. However, improved molecular biomarkers predicting increased progression risk are needed for improved risk assessment and stratification. Using real-time quantitative methylation-specific PCR, we screened 10 genes (HPP1, RUNX3, RIZ1, CRBP1, 3-OST-2, APC, TIMP3, p16, MGMT, p14) for promoter hypermethylation in 77 EAC, 93 BE, and 64 normal esophagus (NE) specimens. A subset of genes manifesting significant differences in methylation frequencies between BE and EAC was then analysed in 20 dysplastic specimens. All 10 genes except p14 were frequently methylated in EACs, with RUNX3, HPP1, CRBP1, RIZ1, and OST-2 representing novel methylation targets in EAC and/or BE. p16, RUNX3, and HPP1 displayed increasing methylation frequencies in BE vs EAC. Furthermore, these increases in methylation occurred early, at the interface between BE and low-grade dysplasia (LGD). To demonstrate the silencing effect of hypermethylation, we selected the EAC cells BIC1, in which the HPP1 promoter is natively methylated, and subjected them to 5-aza-2'-deoxycytidine (Aza-C) treatment. Real-time RT-PCR indicated increased HPP1 mRNA levels after 3 days of Aza-C treatment, as well as decreased levels of methylated HPP1 DNA. Hypermethylation of a subset of six genes (APC, TIMP3, CRBP1, p16, RUNX3, and HPP1) was then tested in a retrospective longitudinal study of 99 BE and nine LGD specimens obtained from 53 BE patients undergoing surveillance endoscopy. Only high-grade dysplasia (HGD) or EAC were defined as progression end points. Two patient groups were compared: eight progressors (P) and 45 nonprogressors (NP), using Cox proportional hazards models to determine the relative progression risks of age, BE segment length, and methylation events. Multivariate analyses revealed that only hypermethylation of p16 (odds ratio (OR) 1.74, 95% confidence interval (CI) 1.33-2.20), RUNX3 (OR 1.80, 95% CI 1.08-2.81), and HPP1 (OR 1.77, 95% CI 1.06-2.81) were independently associated with an increased risk of progression, whereas age, BE segment length, and hypermethylation of TIMP3, APC, or CRBP1 were not independent risk factors. In combined analyses, risk was detectable up to, but not earlier than, 2 years preceding neoplastic progression. Hypermethylation of p16, RUNX3, and HPP1 in BE or LGD may represent independent risk factors for the progression of BE to HGD or EAC. These findings have implications regarding risk stratification, early EAC detection, and the appropriate endoscopic surveillance interval for patients with BE.

791: The Causal Relationship Between RUNX3 Inactivation and Human Bladder Tumor

791: The Causal Relationship Between RUNX3 Inactivation and Human Bladder Tumor

Decreased expression and frequent allelic inactivation of the RUNX3 gene at 1p36 in human hepatocellular carcinoma

Alteration in transforming growth factor-beta signaling pathway is one of the main causes of hepatocellular carcinoma (HCC). The human runt-related transcription factor 3 gene (RUNX3) is an important component of this pathway. RUNX3 locus 1p36 is commonly deleted in a variety of human cancers, including HCC. Therefore, we examined genetic and epigenetic alterations of RUNX3 in human HCC. Five HCC cell lines and 41 patients with HCC were investigated in this study. We examined the expression of RUNX3 mRNA, methylation status of RUNX3 promoter region, loss of heterozygosity (LOH) at 1p36, and mutation analysis. These results were compared with clinicopathological data. Promoter hypermethylation was detected in four (80%) of five HCC cell lines and 31 (75.6%) of 41 HCC tissues, confirmed by sequence of bisulfite-treated DNA. LOH was detected in 14 (37.8%) of 37 HCC. By comparison with clinicopathological data, hypermethylation was more common in hepatitis C virus antibody and formation of capsule-positive cases, and decrease of expression was correlated strongly with advanced stage and LOH-detected cases. Hypermethylation and LOH appear to be common mechanisms for inactivation of RUNX3 in HCC. Therefore, RUNX3 may be an important tumor suppressor gene related to hepatocarcinogenesis.

Promoter hypermethylation downregulates RUNX3 gene expression in colorectal cancer cell lines.

It was recently reported that RUNX3 gene expression is significantly downregulated in human gastric cancer cells due to hypermethylation of its promoter region or hemizygous deletion (Cell, 109, 2002). To verify the genetic alterations and methylation status of the RUNX3 gene in colorectal carcinogenesis, we analysed for mutations, loss of heterozygosity (LOH), and RUNX3 gene promoter hypermethylation, in 32 colorectal cancer cell lines. RT-PCR analysis showed undetectable or low RUNX3 expression in 16 cell lines, and no mutations were found in the RUNX3 gene by PCR-SSCP analysis. Of these 16 cell lines, hypermethylation of the RUNX3 promoter was confirmed in 12. The following observations were made: (i) RUNX3 was re-expressed after 5-aza-2'-deoxycytidine treatment, (ii) the RUNX3 promoter was found to be methylated by MS-PCR, and (iii) hypermethylation of the RUNX3 promoter was confirmed by direct sequencing analysis after sodium bisulfite modification in the above 12 cell lines. RUNX3 was neither methylated nor expressed in four cell lines. Of these four, microsatellite instability (MSI) at the RUNX3 locus was found in three, SNU-61 (D1S246), SNU-769A, and SNU-769B (D1S199). This study suggests that transcriptional repression of RUNX3 is caused by promoter hypermethylation of the RUNX3 CpG island in colorectal cancer cell lines, and the results of these experiments may contribute to an understanding of the role of RUNX3 inactivation in the pathogenesis of colorectal cancers.

Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines.

RUNX3, a Runt domain transcription factor involved in TGF-beta signaling, is a candidate tumor-suppressor gene localized in 1p36, a region commonly deleted in a wide variety of human tumors, including those of the stomach, bile duct, and pancreas. Recently, frequent inactivation of RUNX3 has been demonstrated in human gastric carcinomas. In this study, to examine the involvement of RUNX3 abnormalities in tumorigenesis of bile duct as well as pancreatic cancers, we investigated not only the expression but also methylation status of RUNX3 in 10 human bile duct and 12 pancreatic cancer cell lines. Seven (70%) of the bile duct and nine (75%) of the pancreatic cancer cell lines exhibited no expression of RUNX3 by both Northern blot analysis and the reverse transcriptase polymerase chain reaction. All of the 16 cell lines that did not express RUNX3 also showed methylation of the promoter CpG island of the gene, whereas the six cell lines that showed RUNX3 expression were not methylated or only partially methylated in the RUNX3 promoter region. Moreover, treatment with the methylation inhibitor 5'-aza-2'-deoxycitidine activated RUNX3 mRNA expression in all of 16 cancer cell lines that originally lacked RUNX3 expression. Finally, hemizygous deletion of RUNX3, as detected by fluorescence in situ hybridization, was found in 15 of the 16 cancer cell lines that lacked RUNX3 expression. These data suggest that the inactivation of RUNX3 plays an important role in bile duct and pancreatic carcinogenesis, and that methylation is a common mechanism by which the gene is inactivated.