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AbstractBackgroundMethodsResultsConclusionsRUNX3 is hypermethylated in multiple cancers. TIMP2 also functions as a regulator of tumors. However, there are only very few reports on the association of methylation of RUNX3 and TIMP2 with lung cancer (LC) in peripheral blood.426 LC patients and 428 age- and sex-matched healthy controls were recruited. DNA methylation in blood was semi-quantitively assessed by mass spectrometry. For the association analysis, binary logistic regression analysis adjusted covariant was applied, and ORs were presented as per +10% methylation.Hypermethylation of CpG_1, CpG_5 and CpG_8 in RUNX3 was significantly associated with LC (ORs = 1.45, 1.35 and 1.35, respectively, adjusted p < 0.05), and even stage I LC. The association between the three RUNX3 CpG sites and LC was enhanced by increased age (> 55 years, ORs ranged from 1.43 to 1.75, adjusted p < 0.05), male gender (ORs ranged from 1.47 to 1.59, adjusted p < 0.05) and tumor stage (stage II&III&IV, ORs ranged from 1.86 to 3.03, adjusted p < 0.05).This study suggests a significant association between blood-based RUNX3 hypermethylation and LC, especially in elder people, in males and in LC patients with advanced stage. [ABSTRACT FROM AUTHOR]

Objective: Researchers are actively investigating new diagnostic and prognostic biomarkers that offer improved sensitivity and specificity for systemic lupus erythematosus (SLE). One area of interest is DNA methylation changes. Previous studies have shown a connection between the RUNX3 gene dysfunction and SLE. In this study, the focus was on examining the methylation level of the RUNX3 promoter in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy individuals. Methods: A total of 80 individuals diagnosed with SLE from Iran, along with 77 healthy individuals, were included. The methylation levels of the RUNX3 gene in the extracted DNA were evaluated using the MethyQESD method. To determine the diagnostic effectiveness of the RUNX3 promoter methylation level, a receiver operating characteristic (ROC) curve was generated. Results: The methylation of the RUNX3 promoter was found to be significantly higher in patients with SLE compared to healthy individuals (p <.001). This difference in methylation levels was observed between SLE patients and healthy individuals and between SLE patients with renal involvement and those without renal involvement (86.29 ± 10.30 vs 40.28 ± 24.21, p <.001). ROC analyses revealed that the methylation level of the RUNX3 promoter had a diagnostic power of 0.769 [95% CI (0.681–0.814)] for SLE. Additionally, there was a positive correlation between the RUNX3 methylation level and levels of creatinine and C4. Conclusion: The findings of this study emphasize the potential use of RUNX3 methylation levels in PBMCs of SLE patients as biomarkers for diagnosing the disease, predicting renal damage, and assessing disease activity. [ABSTRACT FROM AUTHOR]

Objectives: Previous reports have indicated that the methylation profile in peripheral blood mononuclear cells (PBMCs) in different genes and loci is altered in colorectal cancer (CRC). Regarding the high mortality rate and silent nature of CRC, screening and early detection can meaningfully reduce disease-related deaths. Therefore, for the first time, we aimed to evaluate the early non-invasive diagnosis of CRC via quantitative promoter methylation analysis of RUNX3 and RASSF1A genes in PBMCs. Materials and Methods: In the present study, we analyzed the methylation status of two important tumor suppressor genes including RUNX3 and RASSF1A in 70 CRC patients and 70 non-malignant subjects using methylation-quantification of endonuclease-resistant DNA (MethyQESD), and a bisulfite conversion-independent method. Results: RUNX3 was significantly hypermethylated in PBMCs of CRC patients compared to healthy controls(P < 0.001). By determining the efficient cutoff value, the sensitivity, and specificity of RUNX3 promoter methylation for CRC diagnosis reached 84.28% and 77.14%, respectively. The receiver operating characteristic (ROC) curve analyses demonstrated that RUNX3 promoter methylation has high accuracy (areas under the curve [AUC] = 0.840, P < 0.001) for discriminating CRC subjects from healthy individuals. Moreover, RUNX3 methylation levels in PBMCs progressively increased with the stage of the disease (P < 0.001). Although the amount of RASSF1A promoter methylation was not significantly different between CRC patients and controls as well as in different stages of the disease (P > 0.05). Conclusion: Our findings confirmed that PBMCs are reliable sources of methylation analysis for CRC screening, and RUNX3 promoter methylation can be used as a promising biomarker for early diagnosis of CRC. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy of adults. RUNX3 and p16 are tumor suppressor genes that may be inactivated by hypermethylation which is a key epigenetic mechanism that contributes to the initiation and progression of various types of human carcinomas including HCC. The aim of this study was to assess the association of hypermethylation of RUNX3 and p16 gene promoters with the incidence of HCC in Egyptian patients. The study included 120 subjects: 30 HCC patients, 30 patients with hepatitis C virus (HCV) without cirrhosis, 30 cirrhotic patients, and 30 healthy volunteers. Methylation-specific polymerase chain reaction (PCR) was done for detection of hypermethylated p16 and RUNX3. Serum levels of liver enzymes and albumin were detected spectrophotometrically and alpha fetoprotein (AFP) was measured in serum by ELISA. Results Methylation of RUNX3 and p16 was detected in 25/30 (83.3%) and 26/30 (86.7%) of HCC patients, respectively. The methylation state of both RUNX3 and p16 genes was significantly higher in HCC patients compared to the control subjects (P = 0.016, OR = 4.38) and (P = 0.014, OR = 4.97), respectively. The methylation of both promoters was associated with higher AFP levels in the serum of all patients. Conclusions Hypermethylation of RUNX3 and p16 is significantly associated with the development of HCC and may be implicated in its pathogenesis.

Ulcerative colitis (UC) results from a complex interplay between the environment, gut microbiota, host genetics, and immunity. Runt-related transcription factor 3 (RUNX3) regulates Th1/Th2 balance and, thus, the synthesis of cytokines and inflammation. We aimed to analyze the dependence of RUNX3 promoter 2 (P2) methylation level on: age, sex, body mass index (BMI), C-reactive protein (CRP), serum albumin, disease duration, Pediatric Ulcerative Colitis Activity Index (PUCAI), the Paris classification, and exposure to medications. This multicenter, cross-sectional study recruited hospitalized children with UC. Methylation of RUNX3 P2 was measured with methylation-sensitive restriction enzymes in the whole blood DNA. Sixty-four children were enrolled, with a mean age of 14.5 ± 2.8 years. Half of them were female (51.6%), and the average BMI Z-score was −0.44 ± 1.14. The mean methylation of RUNX3 P2 was 54.1 ± 13.3%. The methylation level of RUNX3 P2 did not correlate with age, sex, nutritional status, CRP, albumin, PUCAI, or the extent of colitis (Paris E1–E4). RUNX3 P2 methylation did not differ between patients recruited within two and a half months of diagnosis and children who had UC for at least a year. Current or past exposure to biologics, immunosuppressants, or steroids was not associated with RUNX3 P2 methylation. Methylation of RUNX3 promoter 2 in whole blood DNA does not seem to be associated with the characteristics of UC in children. [ABSTRACT FROM AUTHOR]

Background: Hepatocellular carcinoma (HCC) is the most common primary hepatic malignancy of adults. RUNX3 and p16 are tumor suppressor genes that may be inactivated by hypermethylation which is a key epigenetic mechanism that contributes to the initiation and progression of various types of human carcinomas including HCC. The aim of this study was to assess the association of hypermethylation of RUNX3 and p16 gene promoters with the incidence of HCC in Egyptian patients. The study included 120 subjects: 30 HCC patients, 30 patients with hepatitis C virus (HCV) without cirrhosis, 30 cirrhotic patients, and 30 healthy volunteers. Methylation-specific polymerase chain reaction (PCR) was done for detection of hypermethylated p16 and RUNX3. Serum levels of liver enzymes and albumin were detected spectrophotometrically and alpha fetoprotein (AFP) was measured in serum by ELISA. Results: Methylation of RUNX3 and p16 was detected in 25/30 (83.3%) and 26/30 (86.7%) of HCC patients, respectively. The methylation state of both RUNX3 and p16 genes was significantly higher in HCC patients compared to the control subjects (P = 0.016, OR = 4.38) and (P = 0.014, OR = 4.97), respectively. The methylation of both promoters was associated with higher AFP levels in the serum of all patients. Conclusions: Hypermethylation of RUNX3 and p16 is significantly associated with the development of HCC and may be implicated in its pathogenesis. [ABSTRACT FROM AUTHOR]

Tianke Zhang,1,2 E Du,1 Yan Liu,1 Jun Cheng,2 Zhihong Zhang,1 Yong Xu,1 Shiyong Qi,1 Yue Chen1 1Department of Urology, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, Tianjin 300211, People’s Republic of China; 2Department of Anorectal Surgery, Tianjin Union Medical Center, Tianjin 300121, People’s Republic of ChinaCorrespondence: Yue Chen; Shiyong QiDepartment of Urology, The Second Hospital of Tianjin Medical University, Tianjin Institute of Urology, No. 23, Pingjiang Road, Hexi District, Tianjin 300211 Tel +86-18630913339; +86-022-88329692Email chenyue831106@126.com; yongshiqi@126.comPurpose: Zinc oxide nanoparticles (nZnO) have been widely used in the medicine field. Numerous mechanistic studies for nZnO’s anticancer effects are merely performed under high concentration exposure. However, possible anticancer mechanisms of epigenetic dysregulation induced by low doses of nZnO are unclear.Methods: nZnO were characterized and bladder cancer T24 cells were treated with nZnO for 48 hrs at different exposure concentrations. Cell cycle, apoptosis, cell migration and invasion were determined. We performed qRT-PCR, Western blot and chromatin immunoprecipitation to detect the mRNA and protein levels of signaling pathway cascades for histone modification.Results: In this study, we investigated the potential anticancer effects and mechanisms of nZnO on histone modifications in bladder cancer T24 cells upon low-dose exposure. Our findings showed that low concentrations of nZnO resulted in cell cycle arrest at S phase, facilitated cellular late apoptosis, repressed cell invasion and migration after 48 hrs exposure. These anticancer effects could be attributed to increased RUNX3 levels resulting from reduced H3K27me3 occupancy on the RUNX3 promoter, as well as decreased contents of histone methyltransferase EZH2 and the trimethylation of histone H3K27. Our findings reveal that nZnO are able to enter into the cytoplasm and nucleus of T24 cells. Additionally, both particles and ions from nZnO may jointly contribute to the alteration of histone methylation. Moreover, sublethal nZnO-conducted anticancer effects and epigenetic mechanisms were not associated with oxidative stress or DNA damage.Conclusion: We reveal a novel epigenetic mechanism for anticancer effects of nZnO in bladder cancer cells under low-dose exposure. This study will provide experimental basis for the toxicology and cancer therapy of nanomaterials.Keywords: zinc oxide nanoparticles, epigenetics, histone modification, methylation, EZH2, RUNX3

Background: To date, no predictive or prognostic molecular biomarkers except BRCA mutations are clinically established for epithelial ovarian cancer (EOC) despite being the deadliest gynecological malignancy. Aim of this biomarker study was the analysis of DNA methylation biomarkers for their prognostic value independent from clinical variables in a heterogeneous cohort of 203 EOC patients from two university medical centers. Results: The marker combination CAMK2N1/RUNX3 exhibited a significant prognostic value for progression-free (PFS) and overall survival (OS) of sporadic platinum-sensitive EOC (n = 188) both in univariate Kaplan–Meier (LogRank p < 0.05) and multivariate Cox regression analysis (p < 0.05; hazard ratio HR = 1.587). KRT86 methylation showed a prognostic value only in univariate analysis because of an association with FIGO staging (Fisher's exact test p < 0.01). Thus, it may represent a marker for EOC staging. Dichotomous prognostic values were observed for KATNAL2 methylation depending on BRCA aberrations. KATNAL2 methylation exhibited a negative prognostic value for PFS in sporadic EOC patients without BRCA1 methylation (HR 1.591, p = 0.012) but positive prognostic value in sporadic EOC with BRCA1 methylation (HR 0.332, p = 0.04) or BRCA-mutated EOC (HR 0.620, n.s.). Conclusion: The retrospective analysis of 188 sporadic platinum-sensitive EOC proved an independent prognostic value of the methylation marker combination CAMK2N1/RUNX3 for PFS and OS. If validated prospectively this combination may identify EOC patients with worse prognosis after standard therapy potentially benefiting from intensive follow-up, maintenance therapies or inclusion in therapeutic studies. The dichotomous prognostic value of KATNAL2 should be validated in larger sample sets of EOC. [ABSTRACT FROM AUTHOR]

Abstract Background Renal cell carcinoma accounts for 2–3% of all cancers and metastasis increased the malignancy of renal cancer. However, the role of methylation in metastasis of renal cancer is poorly understood. Methods We performed targeted gene array to compare the differential expressions of methylation regulated genes in metastatic and primary renal cancer tissues. Quantitative methylation specific PCR was performed to examine the CpG methylation levels of Runt related transcription factor 3 (RUNX3) and transforming growth factor (TGF)-β. Western blot was performed to detect the expression of target genes. Murine xenograft renal cancer model was established to assay gene expression, methylation level, tumor growth and animal survival in vivo. Results RUNX3 and TGF-β levels were decreased in metastatic renal cancer tissues as a result of their CpG methylation. Metastatic xenograft model displayed decreased expression levels of RUNX3 and TGF-β and higher CpG methylation levels, bigger tumor size and shorter survival time, all which were restored by treatment with a methylation inhibitor. Conclusions Hypermethylation in CpG islands promotes metastasis of renal cancer and is associated with TGF-β and RUNX3 inhibition.

Xiao-yun Song,1,* Bo-yan Li,2,* En-xiang Zhou,3 Feng-xia Wu4 1Department of Thoracic Surgery, Beijing Chest Hospital, Capital Medical University, Beijing, People’s Republic of China; 2Department of Breast Surgery, Inner Mongolia Forestry General Hospital, Inner Mongolia, People’s Republic of China; 3Department of General Surgery, the Second Xiangya Hospital of Central South University, Changsha, Hunan, People’s Republic of China; 4Department of Breast Surgery, Beijing Luhe Hospital, Capital Medical University, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Aberrant promoter methylation of RUNX3 has been reported in several tumors including human breast cancer (BC). However, the association between RUNX3 hypermethylation and incidence of BC remains elusive. In this study, a detailed literature search was performed in Medline and Google Scholar for related research publications. Analysis of pooled data were executed. Odds ratios with corresponding confidence intervals were determined and summarized, respectively. Finally, 13 studies were identified for the meta-analysis. Analysis of the pooled data showed that RUNX3 hypermethylation was significantly higher in both ductal carcinoma in situ and invasive ductal carcinoma (IDC) than in normal breast tissues. In addition, RUNX3 methylation was significantly higher in IDC than in benign tumor. However, RUNX3 methylation was not significantly higher in IDC than in ductal carcinoma in situ. We also determined that RUNX3 hypermethylation was significantly higher in ER positive BC than in ER negative BC. In addition, high RUNX3 mRNA expression was found to be correlated with better overall survival and relapse-free survival for all BC patients. Our results strongly support that RUNX3 hypermethylation may play an important role in BC incidence. RUNX3 methylation is a valuable early biomarker for the diagnosis of BC. Further large-scale studies will provide more insight into the role of RUNX3 hypermethylation in the carcinogenesis and clinical diagnosis of BC patients. Keywords: breast cancer, estrogen receptor, RUNX3, meta-analysis, methylation, odds ratio

Chronic exposure to aflatoxin B 1 (AFB 1 ) is linked to the development of hepatocellular carcinoma (HCC). To identify differentially methylated genes involved in AFB 1 -induced cell transformation, we analyzed DNA methylation patterns in immortal human hepatocyte L02 cells expressing an oncogenic H-Ras allele (L02R cells) and AFB 1 -transformed L02R (L02RT-AFB 1 ) cells by performing genome-wide methylation profiling. We treated L02R cells with 0.3 μM AFB 1 weekly and observed a transformed phenotype at the 17th week post-treatment. The transformed cells (L02RT-AFB 1 ) could grow in an anchorage independent fashion and form tumors in immunodeficient mice. qRT-PCR was performed to examine whether gene methylation led to a reduction in gene expression of methylated candidate genes. As a result, the expression of the following seven genes including JUNB , RUNX3 , NAV1 , CXCR4 , RARRES1 , INTS1 , and POLL was down-regulated in transformed L02RT-AFB 1 cells. The reduction of gene expression of these genes could be reversed by treatment of 5-azadeoxycytidine. The methylated CpG sites of RUNX3 genes were verified using bisulfite sequencing PCR (BSP) assay. Furthermore, a dynamic change in RUNX3 methylation was observed over the course of AFB 1 -induced cell transformation, which was corresponded to the alteration of gene expression and the extent of DNA damage. In vitro study showed that methylation of RUNX3 tended to abate in L02R cells treated with AFB 1 for a short-term period of time. Notably, hypermethylation of RUNX3 appeared in 70% (14/20) of human hepatocellular carcinomas. Moreover, LINE-1 hypomethylation and dynamic changes of DNMTs, TETs and MeCP2 expression were also observed during AFB 1 -induced transformation. Taken together, these observations suggest that aberrant methylation of RUNX3 and LINE-1 might be involved in AFB 1 -induced carcinogenesis. [ABSTRACT FROM AUTHOR]

Laryngeal squamous cell carcinoma (LSCC) is a highly aggressive malignant cancer and accounts for 1% to 2% of all malignancies diagnosed worldwide. Runt-related transcription factor 3 (RUNX3), an important tumor suppressor, is known to related to lymph node metastasis and the development of LSCC. However, the biological roles and potential mechanisms RUNX3 expression was not well understood. In this study, we reported that the RUNX3 was significantly downregulated and highly methylated in LSCC compared with their matched normal. The enforced expression of RUNX3 inhibited LSCC cell migration, invasion, and proliferation, whereas the inhibition of RUNX3 did the opposite. We identified that RUNX3 was regulated by miR-148a-3p and found that the expression level of miR-148-3p was significantly decreased and positively related with the expression of RUNX3 in LSCC. We also identified that DNA methyltransferase enzyme DNA (cytosine-5-)-methyltransferase 1 (DNMT1) was targeted by miR-148a-3p in LSCC. The knockdown of DNMT1 promoted the expression of RUNX3 and inhibited migration, invasion, and proliferation in LSCC cells. In summary, our study demonstrated that miR-148a-3p may regulate RUNX3 expression through the modulation of DNMT1-dependent DNA methylation in LSCC, providing a novel target and a potential therapeutic pathway against LSCC. LSCC is a highly aggressive malignant cancer and accounts for 1% to 2% of all malignancies diagnosed worldwide. In this study, we reported that RUNX3, an important tumor suppressor, was significantly downregulated and highly methylated in LSCC compared with their matched normal. The overexpression of RUNX3 inhibited LSCC cell migration, invasion, and proliferation, whereas the inhibition of RUNX3 did the opposite. Moreover, RUNX3 was regulated by miR-148a-3p, which targeted DNA methyltransferase enzyme DNMT1 in LSCC cells. Therefore, miR-148a-3p may regulate RUNX3 expression through the modulation of DNMT1-dependent DNA methylation in LSCC, providing a novel target and a potential therapeutic pathway against LSCC. [ABSTRACT FROM AUTHOR]

Aberrant promoter methylation of RUNX3 has been reported in several tumors including human breast cancer (BC). However, the association between RUNX3 hypermethylation and incidence of BC remains elusive. In this study, a detailed literature search was performed in Medline and Google Scholar for related research publications. Analysis of pooled data were executed. Odds ratios with corresponding confidence intervals were determined and summarized, respectively. Finally, 13 studies were identified for the meta-analysis. Analysis of the pooled data showed that RUNX3 hypermethylation was significantly higher in both ductal carcinoma in situ and invasive ductal carcinoma (IDC) than in normal breast tissues. In addition, RUNX3 methylation was significantly higher in IDC than in benign tumor. However, RUNX3 methylation was not significantly higher in IDC than in ductal carcinoma in situ. We also determined that RUNX3 hypermethylation was significantly higher in ER positive BC than in ER negative BC. In addition, high RUNX3 mRNA expression was found to be correlated with better overall survival and relapse-free survival for all BC patients. Our results strongly support that RUNX3 hypermethylation may play an important role in BC incidence. RUNX3 methylation is a valuable early biomarker for the diagnosis of BC. Further large-scale studies will provide more insight into the role of RUNX3 hypermethylation in the carcinogenesis and clinical diagnosis of BC patients. [ABSTRACT FROM AUTHOR]

In the present study, the methylation and protein expression of the runt-related transcription factor 3 (Runx3) gene was detected in sporadic colorectal cancer, colonic adenoma and normal colon tissue to evaluate their clinical significance in colorectal carcinogenesis. A total of 34 colonic cancer specimens, 34 colonic adenoma specimens and 34 normal colonic tissue specimens were used in the study. The CpG island methylation status of the Runx3 gene was detected by methylation-specific polymerase chain reaction and the protein expression of Runx3 was detected by immunohistochemistry. The results showed that the rates of methylation of the Runx3 gene in colonic cancer and colonic adenomas were significantly higher than that in the normal colonic tissue (23.5, 20.6 vs. 0.0%; P<0.05). There was no significant difference in the percentage of methylation of the Runx3 gene between colonic adenoma and colonic cancer (P>0.05). The positive percentage of Runx3 protein expression was significantly lower in colonic cancer compared with colonic adenoma and normal tissue (17.7 vs. 61.8, 76.5%; P<0.05). Methylation of the promoter CpG islands of the Runx3 gene is an important genetic event of colon carcinogenesis and may be associated with an altered protein level of Runx3. [ABSTRACT FROM AUTHOR]

Runt-related transcription factor 3 (RUNX3) has been reported to be a tumor-suppressing gene in hepatocellular carcinoma. Association between hepatocellular carcinoma and RUNX3 promoter methylation has been investigated in studies with specific ethnic backgrounds and small sample sizes. In this study, a meta-analysis was adopted to combine the data from 11 studies of association between RUNX3 promoter methylation and hepatocellular carcinoma. Pooled odds ratio for RUNX3 promoter methylation status in hepatocellular carcinoma versus control liver tissue was 24.37(95%CI: 12.14, 48.92), p < .00001, indicates a strong association between methylation of the RUNX3 promoter and hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that RUNX3 is a candidate tumor suppressor in several types of human tumors including hepatocellular carcinoma (HCC). However, the correlation between RUNX3 hypermethylation and incidence of HCC remains unclear. Here, we conducted a systematic review and meta-analysis aiming to comprehensively assess the potential role of RUNX3 hypermethylation in the pathogenesis of HCC. A detailed literature search was made from PubMed, EMBASE, and ISI web of knowledge to identify studies for related research publications. Methodological quality of the studies was also evaluated. The data were extracted and assessed by two reviewers independently. Analysis of pooled data was performed. Odds ratio (OR) was calculated and summarized, respectively. Final analysis of 821 HCC patients from 14 eligible studies was performed. We observed that RUNX3 hypermethylation was significantly higher in HCC than in normal liver tissue, the pooled OR from eight studies including 382 HCC and 161 normal liver tissue (OR = 39.32, 95 % confidence interval (CI) = 13.72-112.7, p < 0.00001). The pooled analysis showed significantly increased OR of RUNX3 hypermethylation (OR = 5.4, 95 % CI = 2.06-14.17, p < 0.00001) in HCC tissues and non-tumor liver tissues. In addition, statistically significant OR of RUNX3 hypermethylation was obtained from non-tumorous liver tissue of HCC patients and normal liver tissue (OR = 12.57, 95 % CI = 3.56-44.35, p < 0.0001). The results of this meta-analysis suggest that RUNX3 hypermethylation may be implicated in the pathogenesis of HCC. Thus, detection of RUNX3 hypermethylation may be a helpful and valuable biomarker for diagnosis of HCC. [ABSTRACT FROM AUTHOR]

The aim of this study is to investigate the possible roles of runt-related transcription factor 3 (RUNX3) and β-catenin in the carcinogenesis of sporadic colorectal tubular adenomas. The expression of the RUNX3 and β-catenin proteins was evaluated by immunohistochemistry in 23 normal colorectal mucosa (NCM), 81 sporadic colorectal tubular adenomas with different dysplasias (SCTA-D) (mild n = 33, moderate n = 23, and severe n = 25 dysplasia), and 48 sporadic colorectal tubular adenomas with cancerous changes (SCTA-Ca). RUNX3 methylation was assessed by methylation-specific polymerase chain reaction (MSP), combined with laser capture microdissection (LCM), in 17 NCM, 41 SCTA-D (mild n = 15, moderate n = 12, and severe n = 14 dysplasia), and 17 SCTA-Ca tissues. Compared to NCM (82.6 %), RUNX3 in SCTA-D (54.3 %) and SCTA-Ca (27.1 %) was significantly downregulated ( P < 0.05). In NCM, SCTA-D, and SCTA-Ca, the incidence of positive expression for β-catenin was 13.0, 60.5, and 79.2 %, respectively. A statistically significant difference was observed ( P < 0.05). RUNX3 levels were markedly higher in adenoma with mild dysplasia (75.8 %) and moderate dysplasia (60.9 %) than in adenoma with severe dysplasia (20.0 %) (both with P < 0.05). Likewise, the expression of β-catenin in severe dysplasia adenoma was 84.0 %, which was significantly higher than that in mild dysplasia adenoma (39.4 %). An inverse correlation was found between the protein expression of RUNX3 and β-catenin in SCTA-D and SCTA-Ca ( P < 0.05). MSP results showed that RUNX3 methylation in NCM, SCTA-D, and SCTA-Ca was 5.9, 17.1, and 41.2 %, respectively, with a statistically significant difference between NCM and SCTA-Ca ( P < 0.05). However, no significant difference of RUNX3 methylation was observed among different dysplasia groups. RUNX3 and β-catenin play important roles in the carcinogenesis of sporadic colorectal tubular adenomas. In addition, hypermethylation of RUNX3 can downregulate its expression. [ABSTRACT FROM AUTHOR]

Chondrosarcoma is the second most common type of bone cancer. Loss of RUNX3 expression has been demonstrated in many other cancers. However, no studies have shown the relationship between RUNX3 expression and chondrosarcoma. In this study, we detected RUNX3 expression in the progression of chondrosarcoma. In patient samples, the levels of RUNX3 mRNA and protein were lower in cancer tissues than in normal tissues. Down-regulation of RUNX3 mRNA in tumor tissues was associated with an increase in RUNX3 promoter methylation. Loss of RUNX3 expression was significantly associated with more aggressive chondrosarcoma types and decreased survival time of patients. To examine the effects of exogenous expression of RUNX3 in vitro, chondrosarcoma cells were transfected with the pcDNA3.1-RUNX3 expression vector. Relative to control cells, RUNX3-expressing cells exhibited lower proliferation and higher apoptosis rates as assessed by colony formation and Annexin V-FITC/PI double staining, respectively. Taken together, these results suggest that RUNX3 acts a tumor suppressor in chondrosarcoma and that RUNX3 promoter methylation may be the molecular mechanism for its decreased expression. [ABSTRACT FROM AUTHOR]

Purpose: Repression of Runt-related transcription factor 3 (Runx3) gene, a tumor suppressor, has been known to be involved in Helicobacter pylori ( H. pylori)-associated gastric carcinogenesis and cancer development. The present study was undertaken to study the Runx3 intronic T/A polymorphism (rs760805) in H. pylori-infected patients and uninfected controls of Tamil Nadu region, South India. Also, Runx3 gene expression, HK alpha (H,K-ATPase) gene expression and the methylation status of the Runx3 CpG island was determined. Methods: In a prospective study, tissue biopsies were collected from 40 H. pylori-infected patients and 40 infection negative controls. Single nucleotide polymorphism analysis was carried out by polymerase chain reaction (PCR)-single-strand conformational polymorphism, and DNA sequencing. Gene expression analysis of Runx3 and HK alpha was carried out using semiquantitative reverse transcriptase-PCR. Methylation analysis of Runx3 CpG island was undertaken using methylation-specific restriction digestion-PCR Results: The homozygous TT genotype was found to be the predominant genotype in positive samples and negative controls. No significant variation in gene expression was observed in positive samples and controls with respect to Runx3 and Hk alpha genes. Methylation analysis suggested that there was a lack of methylation except in a few subjects studied. Conclusion: The present study, first from this region, could neither detect significant repression of the Runx3 and HK alpha genes nor methylation in positive patients studied suggesting a lack of involvement of this tumor suppressor as a risk factor in the H. pylori-associated gastric carcinogenesis in the South Indian population studied. [ABSTRACT FROM AUTHOR]

Runt-related transcription factor 3 (RUNX3) is a tumor suppressor gene, its inactivation due to hypermethylation related to carcinogenesis. The aim of this study was to investigate the effects of 5-aza-2'-deoxycytidine (5-Aza-CdR) on cell proliferation and apoptosis by demethylation of the promoter region and restoring the expression of RUNX3 in the breast cancer MCF-7 cell line. MCF-7 cells were cultured with different concentrations (0.4-102.4 μmol/L) of 5-Aza-CdR in vitro. MTT assay was used to determine the proliferation of MCF-7 cells. Flow cytometry and Hoechst staining were used for analyzing cell apoptosis. The methylation status and expression of RUNX3 in mRNA and protein levels were measured by methylation-specific polymerase chain reaction (PCR [MSP]), reverse transcription (RT)-PCR, and Western blot. It was shown that the RUNX3 gene downregulated and hypermethylated in MCF-7 cells. 5-Aza-CdR induced demethylation, upregulated the expression of RUNX3 on both mRNA and protein levels in cancer cells, and induced growth suppression and apoptosis in vitro in a dose- and time-dependent manner. The results demonstrate that RUNX3 downregulation in breast cancer is frequently due to hypermethylation, and that 5-Aza-CdR can inhibit cell proliferation and induce apoptosis by eliminating the methylation status of RUNX3 promoter and restoring its expression. [ABSTRACT FROM AUTHOR]

BACKGROUND: Helicobacter pylori has been recognized as a definite carcinogen for gastric cancer (GC); however, the pathogenesis of H. pylori infection remains unclear. Runt-related transcription factor 3 ( RUNX3) is a candidate tumor suppressor gene whose deficiency is causally related to GC. However, in H. pylori infection-associated GC, the role of RUNX3 has not been studied. METHODS: The authors used real-time methylation-specific polymerase chain reaction analysis to determine methylation status of the RUNX3 promoter in a spectrum of gastric lesions, including 220 samples of chronic atrophic gastritis, 196 samples of intestinal metaplasia, 134 samples of gastric adenoma, 102 samples of dysplasia, and 202 samples of GC with paired noncancerous mucosa tissues and corresponding blood specimens. The association of abnormal methylation with precancerous gastric lesions was evaluated along with the association between RUNX3 methylation and H. pylori infection, and the concordance of methylation levels was investigated between serum and tissues. RESULTS: The results indicated that increasing RUNX3 promoter methylation was correlated with distinct stages of GC progression. GC tissues had the highest methylation proportion (75.2%) compared with precancerous gastric lesions, including chronic atrophic gastritis (15.9%), intestinal metaplasia (36.7%), gastric adenoma (41.8%), and dysplasia (54.9%). H. pylori infection, a major risk factor for GC, contributed to the inactivation of RUNX3 in gastric epithelial cells through promoter hypermethylation. The levels of RUNX3 methylation in serum were in significant concordance with the methylation levels observed in GC tissues ( P = .887). CONCLUSIONS: The current findings supported RUNX3 methylation as a risk factors for the carcinogenesis of chronic atrophic gastritis with H. pylori infection and indicated that circulating RUNX3 methylation is a valuable biomarker for the detection of early GC. Cancer 2012. © 2012 American Cancer Society. [ABSTRACT FROM AUTHOR]

Abstract: Purpose: Previously, we reported a causal relationship between RUNX3 methylation and bladder tumor development. Thus, in order to clarify its role in tumorigenesis, this study aims to identify the function of RUNX3 methylation in normal adjacent urothelium of patients with non-muscle invasive bladder cancer (NMIBC). Methods: Tumor tissue and donor-matched normal adjacent tissue from 55 patients who underwent transurethral resection (TUR) were selected for the study, and RUNX3 promoter methylation was assessed using methylation-specific polymerase chain reaction (MS-PCR). Results: RUNX3 promoter methylation occurred more frequently in tumor samples than in histologically normal urothelium in patients with NMIBC (P = 0.02). The methylation rates for the RUNX3 promoter in normal adjacent urothelium and tumor tissue were 47% and 69%, respectively. Interestingly, RUNX3 methylation in normal adjacent urothelium was associated with tumor number (P = 0.022) and progression (P = 0.035). Kaplan–Meier estimates revealed that RUNX3 methylation in normal urothelium showed a significant association with time to progression (P = 0.017) in NMIBC patients. Stratifying the patients into ‘both methylation’, ‘one methylation’ and ‘no methylation’ groups for tumors and normal urothelium revealed that no progression occurred in the ‘no methylation’ group during follow-up. Multivariate Cox regression analysis demonstrated that RUNX3 methylation in normal urothelium [hazards ratio (HR): 5.692, P = 0.042] was an independent predictor of progression. Conclusions: RUNX3 methylation was associated with transition from normal urothelium to bladder tumor. More importantly, RUNX3 methylation in normal adjacent urothelium may predict progression in NMIBC patients who have undergone TUR. [Copyright &y& Elsevier]

This study sought to determine if runt-related transcription factor 3 (RUNX3) methylation could accurately predict the overall survival in patients with muscle invasive bladder cancer (MIBC). Forty-seven patients with a longer followup period (cohort 1; reported previously) were analyzed. Tumor samples (cohort 2; newly collected) were obtained from 139 MIBC patients. The prognostic significance of RUNX3 methylation was evaluated by Kaplan-Meier analysis and a multivariate Cox regression model. RUNX3 methylation affected the overall survival in cohort 1 (log-rank test; P=0.030). Among the patients in cohort 2, RUNX3 promoter methylation was observed in 93 of the 139 tumor samples (66.9%). Kaplan-Meier estimates showed a significant difference in overall survival according to RUNX3 methylation (P=0.020). By multivariate Cox regression analysis, positive RUNX3 methylation was an independent predictor of overall survival. These results suggest that RUNX3 promoter methylation is a significant prognostic factor for overall survival in MIBC patients. [ABSTRACT FROM AUTHOR]

Summary: We investigated the spreading pattern of runt-related transcription factor-3 (RUNX3) C-phosphate-G (CpG) island (3478 base pairs) methylation in salivary gland adenoid cystic carcinoma. The methylation status of multiple regions within the runt-related transcription factor-3 promoter CpG island (3478 base pairs) was detected by real-time methylation-specific polymerase chain reaction, and the runt-related transcription factor-3 protein was detected with a Western blot in 19 salivary gland adenoid cystic carcinoma samples and the corresponding nonneoplastic salivary glands. The risk ratio between runt-related transcription factor-3 CpG island methylation and salivary gland adenoid cystic carcinoma progression was analyzed by the logistic analysis of variance model. A possible association between runt-related transcription factor-3 methylation, clinicopathologic parameters, and runt-related transcription factor-3 protein was analyzed. Hypermethylation initially occurs the most at the 5′ region of runt-related transcription factor-3 CpG island before spreading to the transcription start site. The extent of methylation was found to be the highest in region nos. 1 and 2 among the successive 10 regions, which extend from the 5′ region to the transcription start site within the runt-related transcription factor-3 CpG island. The extent of methylation is lowest at the transcription start site, both in salivary gland adenoid cystic carcinoma and in normal salivary glands. No methylation in the transcription start site was found in normal salivary glands. Logistic analysis of variance model indicates that the transcription start site within the runt-related transcription factor-3 promoter CpG island is critical for gene silencing. Western blots revealed that levels of the runt-related transcription factor-3 protein in adenoid cystic carcinoma samples are significantly lower than those in normal salivary glands (P < .001). Methylation of the runt-related transcription factor-3 CpG island spreads the most from 5′ region to the transcription start site in adenoid cystic carcinoma tissues, and the transcription start site may be a critical region for the runt-related transcription factor-3 methylation. The spreading pattern of the runt-related transcription factor-3 methylation may play an a role in the progression of adenoid cystic carcinoma. [ABSTRACT FROM AUTHOR]

Subramaniam M M, Chan J Y, Omar M F M, Ito K, Ito Y, Yeoh K G, Salto-Tellez M & Putti T C (2010) Histopathology 57, 555-563 Lack of RUNX3 inactivation in columnar cell lesions of breast Aims: Ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) exhibit frequent RUNX3 inactivation by promoter hypermethylation and protein mislocalization. The aim of this study was to analyse columnar cell lesions (CCLs) to further characterize RUNX3 involvement in breast carcinogenesis. Methods and results: RUNX3 expression and methylation was analysed by immunohistochemistry and methylation-specific polymerase chain reaction (PCR), respectively, in 75 CCLs. Our previously reported DCIS and IDC data were also included. Consistent with terminal duct lobular units (TDLUs) (73 of 75, 97%), active nuclear RUNX3 protein was observed in 73 of 75 (97%) CCLs [columnar cell change, 46 of 48 (96%); columnar cell hyperplasia, 12 of 12 (100%) and flat epithelial atypia, 15 of 15 (100%). In contrast to matched TDLUs from cancer specimens [four of 40 (10%)] and CCLs, significantly inactivated RUNX3 expression was detected in DCIS [17 of 20 (85%)] and IDC [18 of 20 (90%)] (all P < 0.001). RUNX3 methylation was more frequent in DCIS [15 of 20 (75%)] and IDC [16 of 20 (80%)] than CCLs [(none of 20 (0%)] and matched TDLUs [one of 10 (10%)] from cancer patients (all P < 0.001). Conclusions: RUNX3 inactivation occurs specifically in DCIS and IDC cells. In addition, RUNX3 inactivation may not be a common association between CCLs and breast carcinomas. [ABSTRACT FROM AUTHOR]

Background and Aims: Runx family transcription factors are integral components of transforming growth factor-β signaling pathways and have been implicated in cell cycle regulation, differentiation, apoptosis, and malignant transformation. The silencing of tumor suppressor genes by aberrant hypermethylation occurs frequently in human cancer. It has been noted previously that Runx3 is regarded as an important tumor suppressor gene. Methods: Reverse transcription polymerase chain reaction was used to measure Runx3 and the DNA methyltransferase 1 (Dnmt1) messenger RNA (mRNA) expression level of paired samples of primary gastric cancer and corresponding non-cancerous gastric mucosae, which were obtained from surgically resected specimens of 70 patients. Western blot was used to detect the expression of Runx3 at protein levels. The promoter methylation status was measured by using methylation-specific polymerase chain reaction. We used Annexin V-FITC/PI assay to detect cell apoptosis, and the cell cycle was also analyzed. In order to examine the cell cycle and/or apoptosis, we determined p27 and caspase 3 expression by immunohistological analysis. Results: Our results demonstrate a loss or substantial decrease of Runx3 expression in 70 cases of gastric tumors as compared with that in normal gastric mucosa (0.5749 ± 0.3580 vs 1.7252 ± 0.4085, P < 0.05). The protein levels of the Runx3 gene were significantly lower in gastric cancers than those in adjacent normal tissues. The hypermethylation of Runx3 was involved in 50% (28/56) of gastric cancer tissues, which had reduced Runx3 mRNA expression. The differences of the Dnmt1 mRNA level were significant between the methylated and unmethylated Runx3 cancerous groups. Runx3 methylation was significantly correlated with increased Dnmt1 ( r = 0.64, P < 0.01). Enforced restoration of Runx3 expression led to the induction of cell apoptosis and upregulation of p27 and caspase3 expression in vitro. Conclusions: Our results suggest that a decrease of Runx3 expression by DNA hypermethylation is frequently associated with the evolution of gastric cancer. Runx3 was an independent prognostic factor and a potential therapeutic target for gastric cancer. [ABSTRACT FROM AUTHOR]

Abstract: A major goal of molecular biology is to elucidate the mechanisms underlying cancer development and progression in order to achieve early detection, better diagnosis and staging and novel preventive and therapeutic strategies. We feel that an understanding of Runt-related transcription factor 3 (RUNX3)-regulated biological pathways will directly impact our knowledge of these areas of human carcinogenesis. The RUNX3 transcription factor is a downstream effector of the transforming growth factor-beta (TGF-β) signaling pathway, and has a critical role in the regulation of cell proliferation and cell death by apoptosis, and in angiogenesis, cell adhesion and invasion. We previously identified RUNX3 as a major gastric tumor suppressor by establishing a causal relationship between loss of function and gastric carcinogenesis. More recently, we showed that RUNX3 functions as a bona fide initiator of colonic carcinogenesis by linking the Wnt oncogenic and TGF-β tumor suppressive pathways. Apart from gastric and colorectal cancers, a multitude of epithelial cancers exhibit inactivation of RUNX3, thereby making it a putative tumor suppressor in human neoplasia. This review highlights our current understanding of the molecular mechanisms of RUNX3 inactivation in the context of cancer development and progression. [Copyright &y& Elsevier]

Abstract: Previous reports have indicated that Helicobacter pylori (H. pylori) causes epigenetic changes of certain genes such as cancer suppression genes, which may be associated with carcinogenesis. However, the mechanism by which it causes epigenetic changes in certain genes and not in others is unclear. Presently, we focused on a cancer suppression gene, runx3, and demonstrated the following: (1) H. pylori induces nitric oxide (NO) production in macrophages. (2) NO causes methylation of runx3 in epithelial cells. (3) H. pylori induces the methylation of epithelial cells in the presence of macrophages, which is reversed by an NO-specific inhibitor. These results indicate that H. pylori-induced methylation is mediated by NO, and suggest that NO may be a key to the mechanism of how H. pylori causes epigenetic changes in certain genes. Additionally, we demonstrated that lipopolysaccharide, as well as H. pylori, induces NO-mediated methylation, indicating that other inflammation inducers beside H. pylori might induce aberrant methylation of runx3. [Copyright &y& Elsevier]

Abstract: The DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5-Aza-CdR) has therapeutic value for the treatment of cancer. However, the mechanism by which 5-Aza-CdR induces antineoplastic activity is an important unresolved question. In this study, we found that 5-Aza-CdR at limited concentrations induced inhibition of colorectal cancer Lovo cell proliferation as well as increased apoptosis caused by DNA damage, which was independent of the caspase pathway. Regarding the mechanisms, for the first time, we examined that cytotoxicity against Lovo cells was regulated via down-regulation of DNA methyltransferase 3a, DNMT3b and then reactivated the expression of RUNX3. We therefore conclude that RUNX3 is a relevant target for methyltransferases dependent effects of 5-Aza-CdR on colorectal cancer Lovo cells. [Copyright &y& Elsevier]

Background 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. Methods 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. Results 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. Conclusion Our data suggest that RUNX3 inactivation by promoter hypermethylation and protein mislocalization constitute an early event in breast cancer progression. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Gastric cancer is the forth malignancy in frequency in the world. In the northern Brazil is the second neoplasia most frequent in males and the third most frequent in females. Genetic and epigenetic alterations are evolved on gastric carcinogenesis and DNA methylation is the epigenetic alteration better studied. We analyzed de novo DNA methyltransferases methylation pattern and its association with RUNX3 gene methylation pattern in Brazilian samples of intestinal-type and diffuse-type of gastric cancer. PCR methylation specific was used to evaluate DNA methylation pattern. Sixty-six samples were studied in this work. Only the gene RUNX3 presented altered methylation pattern, being methylated in 38.5% of gastric cancer intestinal-type samples and in 70% of gastric cancer diffuse-type samples and, by this reason, it should be evolved in the genesis of this neoplasia. There was a statistically significant difference among diffuse-type and intestinal-type samples (p=0.0418) and among normal and tumour tissues (p<0.0001) for RUNX3 gene but not to DNMT3A, DNMT3B e DNMT3 genes on CpG islands analyzed. Alteration of RUNX3 methylation pattern is not associated to de novo alteration of DNA methyltransferases methylation pattern on studied regions. Therefore, it becomes necessary a better comprehension of this phenomenon on gastric carcinogenesis. [ABSTRACT FROM AUTHOR]

Summary: Aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of the tumor suppressor and tumor-related genes. Some published studies suggest a relationship to exist between the methylation status of several genes and the prognosis in non-small cell lung cancer (NSCLC); hypermethylation of the specific genes may be expected to serve as a biomarker for the prognosis, after a curative resection of NSCLC. To determine the relationship between the methylation status of the tumor suppressor and the tumor-related genes, and the clinicopathologic characteristics, including the survival rate, in patients with NSCLC after a surgical resection, we studied methylation in 10 genes (DAPK, FHIT, H-cadherin, MGMT, p14, p16, RAR-β, RASSF1A, RUNX3, and TIMP-3) in 101 NSCLC cases by methylation-specific PCR (MSP). The methylation frequencies of the 10 genes examined in NSCLC were 26% for DAPK, 34% for FHIT, 26% for H-cadherin, 14% for MGMT, 8% for p14, 27% for p16, 38% for RAR-β, 42% for RASSF1A, 25% for RUNX3, and 12% for TIMP-3. Clinicopathologically, the patients with all stages of disease who had positive RASSF1A, RUNX3, or H-cadherin methylation status were found to have a significantly shorter duration of survival, as compared with the patients with a negative methylation status for those genes (RASSF1A:P =0.023, RUNX3:P =0.035, H-cadherin:P =0.039) in univariate analysis. Thereafter, while limiting our examination to patients with stage I disease, the patients who had a positive RASSF1A or RUNX3 methylation status were found to have a significantly shorter duration of survival, in comparison to the patients with negative methyaltion status for each of those genes (RASSF1A:P =0.022, RUNX3:P <0.01) in univariate analysis. Next, the histological differences were recognized that the patients with RUNX3 methylation had a shorter duration of survival in adenocarcinomas (ACs) (P =0.045), in contrast to those with RASSF1A methylation who had a shorter duration of survival in squamous cell carcinomas (SCCs) (P =0.021). In multivariate analysis, both positive RASSF1A methylation status, and positive RUNX3 methylation status were found to be independent prognostic factors (RASSF1A:P =0.031, RUNX3:P =0.028), as was TNM stage (P =0.004) and pleural involvement (P =0.037). In conclusion, the hypermethylation of RASSF1A or RUNX3 gene is therefore a useful biomarker to predict the prognosis in NSCLC, particularly RASSF1A due to SCCs and RUNX3 due to ACs. [Copyright &y& Elsevier]

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-β-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (P<0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-β and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2′-deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.Oncogene (2007) 26, 5927–5938. doi:10.1038/sj.onc.1210403; published online 26 March 2007 [ABSTRACT FROM AUTHOR]

Silencing of the RUNX3 gene by hypermethylation of its promoter CpG island plays a major role in gastric carcinogenesis. To quantitatively evaluate RUNX3 methylation, a fiber-type DNA microarray was used on which methylated and unmethylated sequence probes were mounted. After bisulfite modification, a part of the RUNX3 promoter CpG island, at which methylation is critical for gene silencing, was amplified by polymerase chain reaction using a Cy5 end-labeled primer. Methylation rates (MR) were calculated as the ratio of the fluorescence intensity of a methylated sequence probe to the total fluorescence intensity of methylated and unmethylated probes. Five gastric cancer cell lines were analyzed, as well as 26 primary gastric cancers and their corresponding non-neoplastic gastric epithelia. MR in four of the cancer cell lines that lost RUNX3 mRNA ranged from 99.0% to 99.7% (mean, 99.4%), whereas MR in the remaining cell line that expressed RUNX3 mRNA was 0.6%. In primary gastric cancers and their corresponding non-neoplastic gastric epithelia, MR ranged from 0.2% to 76.5% (mean, 22.7%) and from 0.7% to 25.1% (mean, 5.5%). Ten (38.5%) of the 26 gastric cancers and none of their corresponding non-neoplastic gastric epithelia had MR >30%. Most of the samples with MR >10% tested methylation-positive by conventional methylation-specific polymerase chain reaction (MSP). This microarray-based methylation assay is a promising method for the quantitative assessment of gene methylation. [ABSTRACT FROM AUTHOR]

The transforming growth factor beta (TGFbeta)-signalling pathway is deregulated in many cancers. We examined the role of gene silencing via aberrant methylation of DRM/Gremlin and HPP1, which inhibit TGFbeta signalling, and RUNX3, which facilitates TGFbeta-signalling, of all genes that are thought to be tumour suppressors, are aberrantly expressed, and are thus thought to have important role in human cancers. We examined DRM/Gremlin mRNA expression in 44 cell lines and the promoter methylation status of DRM/Gremlin, HPP1, and RUNX3 in 44 cell lines and 511 primary tumours. The loss of DRM/Gremlin mRNA expression in human cancer cell lines is associated with DNA methylation, and treatment with the methylation inhibitor-reactivated mRNA expression (n=13). Methylation percentages of the three genes ranged from 0-83% in adult tumours and 0-50% in paediatric tumours. Methylation of DRM/Gremlin was more frequent in lung tumours in smokers, and methylation of all three genes was inversely correlated with prognosis in patients with bladder or prostate cancer. Our results provide strong evidence that these TGFbeta-related genes are frequently deregulated through aberrant methylation in many human malignancies. [ABSTRACT FROM AUTHOR]

Recent analyses have revealed that RUNX family members play important roles in both normal developmental processes and carcinogenesis. Of the three known RUNX family members, RUNX3 has been shown to be involved in neurogenesis of the dorsal root ganglia, T-cell differentiation and tumorigenesis of gastric epithelium. Deletion of the Runx3 locus in mice resulted in hyperplasia of the gastric epithelium due to the stimulation of proliferation and suppression of apoptosis that was accompanied by a reduced sensitivity to TGF-β1. In primary human gastric cancer specimens, RUNX3 is frequently inactivated by allele loss or gene silencing due to promoter hypermethylation. The tumorigenicity of human gastric cancer cell lines in nude mice decreased as the level of RUNX3 expression increased, which indicates that RUNX3 is a bona fide tumor suppressor of gastric cancers. [ABSTRACT FROM AUTHOR]

RUNX3, a Runt domain transcription factor involved in TGF-ß 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.Oncogene (2004) 23, 2401-2407. doi:10.1038/sj.onc.1207395 Published online 26 January 2004 [ABSTRACT FROM AUTHOR]

RUNX family transcription factors are integral components of TGF-β signaling pathways and have been implicated in cell cycle regulation, differentiation, apoptosis, and malignant transformation. It was noted previously that allele loss and loss of expression of RUNX3 are causally involved in gastric carcinogenesis. Our results demonstrate that RUNX3 is inactivated by aberrant DNA methylation in approximately 19% of lung cancer cell lines and 24% of primary lung cancer specimens. RUNX3 methylation is tumor-specific, since it is not observed in surrounding normal lung tissues. Our results suggest that loss of RUNX3 expression by DNA hypermethylation is frequently associated with the evolution of lung cancer. [Copyright &y& Elsevier]