ID4 Methylation Research Articles

CDC42 as an epigenetic regulator of ID4 in triple-negative breast tumors.

Triple-negative (TN) breast cancer represents a subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2). Clinically, it is characterized by high invasiveness, high metastatic potential, and poor prognosis. Inhibitor of DNA binding 4 (ID4) has been shown to be overexpressed in these tumors acting as an oncogene responsible for many of its aggressive features. CDC42, a plasma membrane-associated small GTPase, can downregulate ID4 gene expression through hypermethylation of its promoter in colorectal adenocarcinomas. Since ID4 acts as an oncogene and is hypomethylated in TN breast tumors, here we asked whether CDC42 could also epigenetically silence ID4 and in doing so revert aggressive features of this tumor type. Gene expression was retrieved from TCGA database using UCSC Xena. Association between overall survival (OS) and gene expression was assessed using Kaplan-Meier plotter. In vitro experiments involved ectopic expression of CDC42 in MDA-MB231and in MDA-MB468 breast cancer cell lines. Gene expression was analyzed by qPCR, western blot and inmunofluorescence assays and methylation by MSP, MS-MLPA, or ddMSP. Data mining analysis revealed that CDC42 expression varies among breast cancer subtypes that in the basal-like subtype there is an inverse correlation between CDC42 and ID4 expression and a positive correlation between CDC42 expression and ID4 methylation. In vitro experiments revealed that CDC42 overexpression induced ID4 methylation through the activation of the EZH2 pathway. ID4 silencing produced an increase in BRCA1 expression and a less aggressive phenotype in the tested cell line. We show that CDC42 silences ID4 through methylation in TN breast cancer. Given that ID4 acts as an oncogene in these tumors, we think that finding an epigenetic regulator of ID4 contributes to the research and clinical management of TN breast tumors.

UVB induces cutaneous squamous cell carcinoma progression by de novo ID4 methylation via methylation regulating enzymes.

BackgroundLittle is known about whether UVB can directly influence epigenetic regulatory pathways to induce cutaneous squamous cell carcinoma (CSCC). This study aimed to identify epigenetic-regulated signalling pathways through global methylation and gene expression profiling and to elucidate their function in CSCC development.MethodsGlobal DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) and genome-wide gene expression analysis by RNA sequencing (RNA-seq) in eight pairs of matched CSCC and adjacent normal skin tissues were used to investigate the potential candidate gene(s). Clinical samples, animal models, cell lines, and UVB irradiation were applied to validate the mechanism and function of the genes of interest.FindingsWe identified the downregulation of the TGF-β/BMP-SMAD-ID4 signalling pathway in CSCC and increased methylation of inhibitor of DNA binding/differentiation 4 (ID4). In normal human and mouse skin tissues and cutaneous cell lines, UVB exposure induced ID4 DNA methylation, upregulated DNMT1 and downregulated ten-eleven translocation (TETs). Similarly, we detected the upregulation of DNMT1 and downregulation of TETs accompanying ID4 DNA methylation in CSCC tissues. Silencing of DNMT1 and overexpression of TET1 and TET2 in A431 and Colo16 cells led to increased ID4 expression. Finally, we showed that overexpression of ID4 reduced cell proliferation, migration, and invasion, and increased apoptosis in CSCC cell lines and reduced tumourigenesis in mouse models.InterpretationThe results indicate that ID4 is downregulated by UVB irradiation via DNA methylation. ID4 acts as a tumour suppressor gene in CSCC development.FundingCAMS Innovation Fund for Medical Sciences (CIFMS) (2016-I2M-3-021, 2017-I2M-1-017), the Natural Science Foundation of Jiangsu Province (BK20191136), and the Fundamental Research Funds for the Central Universities (3332019104).

Epigenetic regulation of ID4 in breast cancer: tumor suppressor or oncogene?

BackgroundInhibitor of differentiation protein 4 (ID4) is a dominant negative regulator of the basic helix-loop-helix (bHLH) family of transcription factors. During tumorigenesis, ID4 may act as a tumor suppressor or as an oncogene in different tumor types. However, the role of ID4 in breast cancer is not clear where both an oncogenic and a tumor suppressor function have been attributed. Here, we hypothesize that ID4 behaves as both, but its role in breast differs according to the estrogen receptor (ER) status of the tumor.MethodsID4 expression was retrieved from TCGA database using UCSC Xena. Association between overall survival (OS) and ID4 was assessed using Kaplan–Meier plotter. Correlation between methylation and expression was analyzed using the MEXPRESS tool. In vitro experiments involved ectopic expression of ID4 in MCF-7, T47D, and MDA-MB231 breast cancer cell lines. Migration and colony formation capacity were assessed after transfection treatments. Gene expression was analyzed by ddPCR and methylation by MSP, MS-MLPA, or ddMSP.ResultsData mining analysis revealed that ID4 expression is significantly lower in ER+ tumors with respect to ER− tumors or normal tissue. We also demonstrate that ID4 is significantly methylated in ER+ tumors. Kaplan–Meier analysis indicated that low ID4 expression levels were associated with poor overall survival in patients with ER+ tumors. In silico expression analysis indicated that ID4 was associated with the expression of key genes of the ER pathway only in ER+ tumors. In vitro experiments revealed that ID4 overexpression in ER+ cell lines resulted in decreased migration capacity and reduced number of colonies. ID4 overexpression induced a reduction in ER levels in ER+ cell lines, while estrogen deprivation with fulvestrant did not induce changes neither in ID4 methylation nor in ID4 expression.ConclusionsWe propose that ID4 is frequently silenced by promoter methylation in ER+ breast cancers and functions as a tumor suppressor gene in these tumors, probably due to its interaction with key genes of the ER pathway. Our present study contributes to the knowledge of the role of ID4 in breast cancer.

Epigenetic dysregulation of ID4 predicts disease progression and treatment outcome in myeloid malignancies.

Promoter hypermethylation‐mediated inactivation of ID4 plays a crucial role in the development of solid tumours. This study aimed to investigate ID4 methylation and its clinical relevance in myeloid malignancies. ID4 hypermethylation was associated with higher IPSS scores, but was not an independent prognostic biomarker affecting overall survival (OS) in myelodysplastic syndrome (MDS). However, ID4 hypermethylation correlated with shorter OS and leukaemia‐free survival (LFS) time and acted as an independent risk factor affecting OS in acute myeloid leukaemia (AML). Moreover, ID4 methylation was significantly decreased in the follow‐up paired AML patients who achieved complete remission (CR) after induction therapy. Importantly, ID4 methylation was increased during MDS progression to AML and chronic phase (CP) progression to blast crisis (BC) in chronic myeloid leukaemia (CML). Epigenetic studies showed that ID4 methylation might be one of the mechanisms silencing ID4 expression in myeloid leukaemia. Functional studies in vitro showed that restoration of ID4 expression could inhibit cell proliferation and promote apoptosis in both K562 and HL60 cells. These findings indicate that ID4 acts as a tumour suppressor in myeloid malignancies, and ID4 methylation is a potential biomarker in predicting disease progression and treatment outcome.

Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia.

Background:The diagnosis of myelodysplastic syndrome (MDS), especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA).Methods:The methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time methylation-specific PCR (MethyLight PCR) in 100 patients with MDS and 31 patients with AA.Results:The MDS group had a higher ID4 gene methylation positivity rate (22.22%) and higher methylation levels (0.21 [0–3.79]) than the AA group (P < 0.05). Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56]) than the use of genetic markers only (51.79% [29/56]).Conclusions:These results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

Clinical implications of the quantitative detection of ID4 gene methylation in myelodysplastic syndrome.

BackgroundMyelodysplastic syndrome (MDS) eventually transforms into acute leukemia (AL) in about 30% of patients. Hypermethylation of the inhibitor of DNA binding 4 (ID4) gene may play an important role in the initiation and development of MDS and AL. The aim of this study was to quantitatively assess ID4 gene methylation in MDS and to establish if it could be an effective method of evaluating MDS disease progression.MethodsWe examined 142 bone marrow samples from MDS patients, healthy donors and MDS-AL patients using bisulfite sequencing PCR and quantitative real-time methylation-specific PCR. The ID4 methylation rates and levels were assessed.ResultsID4 methylation occurred in 27 patients (27/100). ID4 gene methylation was more frequent and at higher levels in patients with advanced disease stages and in high-risk subgroups according to WHO (P < 0.001, P < 0.001, respectively) and International Prognostic Scoring System (IPSS) (P = 0.002, P = 0.007, respectively) classifications. ID4 methylation levels changed during disease progression. Both methylation rates and methylation levels were significantly different between healthy donor, MDS patients and patients with MDS-AL (P < 0.001, P < 0.001, respectively). Multivariate analysis indicated that the level of ID4 methylation was an independent factor influencing overall survival. Patients with MDS showed decreased survival time with increased ID4 methylation levels (P = 0.011, hazard ratio (HR) = 2.371). Patients with ID4 methylation had shorter survival time than those without ID4 methylation (P = 0.008).ConclusionsOur findings suggest that ID4 gene methylation might be a new biomarker for MDS monitoring and the detection of minimal residual disease.

Clinical significance of ID4 methylation detection by quantitative methylation-specific PCR in acute leukemia

The advances of treatment improved the prognosis of the patients with acute leukemia (AL) in the last decade, but the lack of general biomarker for predicting relapse in AL, which is one of the most important factors influencing the survival and prognosis. DNA methylation of ID4 gene promoter occurred frequently in patients with AL and was found to be highly related to the tumor progression. Based on the previous work of the setup of methylation-specific quantitative PCR system for ID4 gene, this study was designed to investigate the relation between the quantitative indicator of methylation density, percentage of methylation reference(PMR) value, and different disease status of AL. PMR of ID4 was detected by MS-PCR in bone marrow (BM) samples of 17 healthy persons and 54 AL patients in the status of newly diagnosis, complete remission and disease relapse. The results showed that at different disease status, PMR value in newly diagnosed group was significantly lower than that in complete remission group (P = 0.031). Among serial samples, PMR value remained very low at the status of patients with continuous complete remission (<1.5‰), and increased along with the accumulation of tumor cells at relapse. In 1 relapse case, the abnormal rise of PMR value occurred prior to morphological relapse. PMR value seemed to be related to body tumor cell load. It is concluded that the quantitative indicator of methylation density and PMR value may reflect the change of tumor cell load in acute leukemia patients. Dynamic monitoring of PMR maybe predict leukemia relapse.

Establishment of methylation-specific quantitative PCR system for ID4 gene in acute leukemia cells and its specificity and sensitivity

DNA methylation of ID4 gene promoter occurred frequently in patients with acute leukemia and was found to be highly related to the tumor progression. Due to lack of the appropriate methylation detection methods, the relation between the quantification of ID4 methylation and the states of acute leukemia is still unclear. This study purposed to set up a methylation-specific quantitative PCR system for ID4 and investigate the specificity and sensitivity of this methylation detection. The plasmids combined with target gene as well as with internal reference were constructed, and the standard curves were set up by using above mentioned plasmids. The specificity of this detection system in cell lines was verified through techniques of MSP and quantitative MSP. The sensitivity of this detection system was verified by mixing methylation-positive and negative cell lines in varying proportions and through amplification of qualitative MSP. The results showed that the standard curves were establish successfully. The results of quantitative MS-PCR in cell lines were consistent with those of MS-PCR, and as low as 1: 10(-5) of ID4 methylation positive cells could be detected by the new methylation detection assay. In newly diagnosed acute leukemia patients, the positive rate of quantitative MSP was higher. It is concluded that a complete quantitative MSP system for ID4 methylation detection has been established and this quantitative MSP method has good specificity and high sensitivity.

Effects of arsenic trioxide on Id4 methylation status in bone marrow mononuclear cells and its clinical efficacy for myelodysplastic syndrome

Effects of arsenic trioxide on Id4 methylation status in bone marrow mononuclear cells and its clinical efficacy for myelodysplastic syndrome

Epigenetic silencing of Id4 identifies a glioblastoma subgroup with a better prognosis as a consequence of an inhibition of angiogenesis

Inhibitors of DNA binding/differentiation (Id1 to Id4) are a family of helix-loop-helix transcription factors, which are highly expressed during embryogenesis and at lower levels in mature tissues. Id4 plays an important role in neuronal stem cell differentiation, and its deregulation has been implicated in glial neoplasia. The methylation status of Id4 was analyzed by methylation-specific polymerase chain reaction (PCR) in 62 glioblastoma (GBM) cases and in 20 normal brain tissues. Methylation status of Id4 was confirmed by sequencing after subcloning and messenger RNA (mRNA) and protein expression. We also evaluated the mRNA expression of MGP (matrix GLA protein), TGF-β1 (transforming growth factor beta 1), and VEGF (vascular endothelial growth factor) by real-time PCR analysis. Clinical and histological assessment of tumor angiogenesis was performed by evaluating the relative enhancing tumor ratio on magnetic resonance imaging and microvessel density on von Willebrand factor-stained sections, respectively. The promoter of Id4 was methylated in 23 of 62 (37%) GBMs. In methylated GBMs, Id4 mRNA was significantly reduced, compared with unmethylated GBMs (P = .0002). A significant reduction of protein expression was detected in all hypermethylated cases. GBMs with methylated Id4 showed a significant reduction of MGP, TGF-β1, and VEGF mRNA expression and had significantly lower relative enhancing tumor ratio (P = .0108) and microvessel density (P = .0241) values with respect to unmethylated GBMs. Finally, Id4 methylation was significantly associated with a favorable clinical outcome (P = .0006). These data suggest that methylation of Id4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment throughout MGP-mediated neoangiogenesis.

Abstract A24: Epigenetic silencing of ID4 (inhibitor of DNA binding 4) is associated with prostate cancer

Abstract Introduction: Inhibitor of DNA binding/differentiation protein 4 (Id4) is dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Id4 shares the homology of HLH domain with other Ids and lack the basic DNA binding region. Previous studies from our lab had shown that unlike Id1, Id2 and Id3, Id4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promote apoptosis. Consistent with these observations Id4 is epigenetically silenced in Du145 prostate cancer cell line. In this study we investigated whether Id4 is also epigenetically silenced in prostate cancer. We also examined association between Id4 promoter hypermethylation and its expression in prostate cancer cell lines. Experimental procedures: Id4 protein expression was analyzed on human prostate adenocarcinoma samples by Immunhistochemistry. Id4 promoter methylation pattern on prostate cancer cell lines (C33, C81, DU145, LNCaP, DU145+ID4, and PC3) was examined. In addition, we performed Laser capture microdisection (LCMD) on the human prostate tissues and methylation specific PCR to correlate our cell line studies with clinical studies. Results: IHC demonstrated decreased Id4 protein expression in human prostate tissue samples whereas higher nuclear Id4 expression was found in normal prostate tissues.Id4 methylation specific PCR (MSP) on prostate cancer cell lines, showed Id4 methylation in DU145 cell and but not in LNCaP and C33 cells. In C81 and PC3 cells it showed partial methylation. C81 are derivatives of LNCaP cell lines and are Androgen independent and are more metastatic. Increased Id4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with Id4 promoter hypermethylation showed distinct loss of Id4 expression. Conclusions: Id4 appears as a potential tumor suppressor gene that is epigenetically silenced during cancer development.Id4 promoter hypermethylation had been reported in breast, colorectal cancer and chronic lymphocytic leukemia Thus, loss of Id4 expression due to promoter hypermethylation. Id4 methylation status could serve as a prognostic biomarker in human prostate cancer. Acknowledgement: The research was supported by NIH/NCI-RO1CA128914 and in part by NIH/NCRR/RCMI G12RR03062. Citation Format: Pankaj Sharma, Swathi Chinaranagari, Divya Patel, Jaideep Chaudhary. Epigenetic silencing of ID4 (inhibitor of DNA binding 4) is associated with prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A24.

ID4 is frequently downregulated and partially hypermethylated in prostate cancer

The candidate tumor suppressor ID4 is downregulated in various cancers by DNA hypermethylation. We have performed the first systematic analysis of ID4 expression and methylation in prostate cancer. ID4 mRNA expression was analyzed by quantitative RT-PCR in 47 carcinoma and 13 benign prostatic tissues obtained by prostatectomy. Methylation was analyzed in an extended series of samples by methylation-specific MS-PCR and pyrosequencing, controlled by bisulfite sequencing. ID4 expression was significantly decreased in prostate cancers, especially in cases with adverse clinical and histopathological features and earlier recurrence. Hypermethylation in carcinomas was detected by MS-PCR and pyrosequencing, but the results of the two techniques were not fully concordant. The difference was created by generally partial and heterogeneous methylation. Weak methylation was also detected in benign prostatic tissue samples. ID4 downregulation may contribute to prostate cancer pathogenesis and is often accompanied by DNA hypermethylation. The case of ID4 illustrates exemplarily the limits and pitfalls of techniques for the detection of methylation changes in prostate cancer tissues.

Prognostic relevance of promoter methylation-associated loss of ID4 expression in glioblastoma multiforme.

2076 Background: Inhibitors of DNA binding/differentiation (ID1-4) are a family of helix-loop-helix transcription factor, highly expressed during embryogenesis and at lower levels in mature tissues. Expression of ID proteins have related to features of malignancies, including cellular transformation, angiogenesis and metastasis. ID4 plays an important role in neuronal stem cell differentiation and its deregulation has been implicated in glial neoplasia. Recent works have shown that hypermethylation of ID4 can determine the silencing of this protein in several human cancers. Methods: We analyzed the methylation status of ID4 by methylation-specific-PCR in 85 glioblastoma multiforme (GBM) cases and in 20 normal brain tissues. After surgical treatment, all GBMs were subjected to a cycle of radiotherapy (2 Gy per fraction, once a day, 5 days a week, 60 Gy total dose) with concomitant administration of temozolomide (75 mg per square meter of body surface area per day) for 7 days a week from the first to the last day of radiotherapy, followed by 5 cycles of adjuvant temozolomide (at 200 mg per square meter of body surface area on days 1 through 5) given at four-week intervals. Methylation status was confirmed by sequencing after subcloning and RNA and protein expression was assessed by real-time PCR and immunohistochemistry. The relationship between ID4 methylation and clinical outcome was investigated. Results: The promoter of ID4 was methylated in 17/85 GBMs. After subcloning, the methylation was confirmed by sequencing in over 95% of GpC dinucleotides. In methylated GBMs, ID4 mRNA expression was reduced by 5 folds as compared with unmethylated GBMs. A significant reduction of protein expression was detected in all hypermethylated cases. ID4 methylation was significantly associated with a favourable clinical outcome (p=0.0033). No significant differences were found between patients with and without ID4 methylation with respect to age, sex, p53 mutation, Ki67 labeling index and extent of surgical resection. Conclusions: Our data suggest that methylation of ID4 may be involved in the pathogenesis of GBM and in the resistance of this neoplasm to conventional treatment.

ID4 methylation predicts high risk of leukemic transformation in patients with myelodysplastic syndrome

Epigenetic gene silencing due to promoter methylation is observed in human cancers like acute myeloid leukemia (AML). Little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a ∼30% risk of transformation into secondary AML. Recent evidence demonstrated that ID4, a negative regulator of transcription, may act as a tumor-suppressor gene. To clarify the role of ID4 in MDS, we employed methylation-specific PCR (MSP) to examine the methylation status of ID4 in 144 adult de novo MDS patients. We found that ID4 methylation was present in 35.4% ( n = 51) of these MDS patients and methylaiton was correlated significantly with World Health Organization (WHO) subtypes and International Prognostic Scoring System (IPSS) risk groups. Patients with advanced stages of WHO subtypes (45.8% vs. 21.3%, P = 0.002) and higher risk IPSS subgroups (45.7% vs. 26.0%, P = 0.014) exhibited a significantly higher frequency of ID4 methylation. The median survival of patients with ID4 methylation was shorter than patients without ID4 methylation (12.2 months vs. 26.9 months, P = 0.005). Multivariate analysis indicated that ID4 methylation status was the independent factor that impacted leukemia-free survival (LFS). Disease in patients with ID4 methylation progressed more rapidly than those without ID4 methylation ( P = 0.047, HR = 2.11). Our results suggest that ID4 may be a therapeutic target in MDS.

Promoter methylation-associated loss of ID4expression is a marker of tumour recurrence in human breast cancer

BackgroundInhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. MethodsID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. ResultsFrequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level. Interestingly, ID4 promoter methylation was a factor for unfavourable recurrence-free survival (P=0.036) and increased risk for lymph node metastasis (P=0.030). ConclusionID4 is indeed a novel tumour suppressor gene in normal human breast tissue and is epigenetically silenced during cancer development, indicating increased risk for tumour relapse. Thus, ID4 methylation status could serve as a prognostic biomarker in human breast cancer.

Epigenetic Inactivation of ID4 in Colorectal Carcinomas Correlates with Poor Differentiation and Unfavorable Prognosis

ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed. In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen. CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza-cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066). ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.