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Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

ABSTRACT: The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.

SUBMITTER: Saulnier A

PROVIDER: S-EPMC3422753 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

Saulnier Amandine A Vaissière Thomas T Yue Jiping J Siouda Maha M Malfroy Marine M Accardi Rosita R Creveaux Marion M Sebastian Sinto S Shahzad Naveed N Gheit Tarik T Hussain Ishraq I Torrente Mariela M Maffini Fausto Antonio FA Calabrese Luca L Chiesa Fausto F Cuenin Cyrille C Shukla Ruchi R Fathallah Ikbal I Matos Elena E Daudt Alexander A Koifman Sergio S Wünsch-Filho Victor V Menezes Ana M B AM Curado Maria-Paula MP Zaridze David D Boffetta Paolo P Brennan Paul P Tommasino Massimo M Herceg Zdenko Z Sylla Bakary S BS

International journal of cancer 20110922 11

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 w ...[more]

PMID: 21796618

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Project description:BackgroundAcute myeloid leukemia (AML) is the second most common form of leukemia in children. Aberrant DNA methylation patterns are a characteristic feature in various tumors, including AML. Metallothionein III (MT3) is a tumor suppresser reported to show promoter hypermethylated in various cancers. However, the expression and molecular function of MT3 in pediatric AML is unclear.MethodsEleven human leukemia cell lines and 41 pediatric AML samples and 20 NBM/ITP (Norma bone marrow/Idiopathic thrombocytopenic purpura) control samples were analyzed. Transcription levels of MT3 were evaluated by semi-quantitative and real-time PCR. MT3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BSG). The molecular mechanism of MT3 was investigated by apoptosis assays and PCR array analysis.ResultsThe MT3 promoter was hypermethylated in leukemia cell lines. More CpG's methylated of MT3 was observed 39.0% pediatric AML samples compared to 10.0% NBM controls. Transcription of MT3 was also significantly decreased in AML samples compared to NBM/ITP controls (P?<?0.001); patients with methylated MT3 exhibited lower levels of MT3 expression compared to those with unmethylated MT3 (P?=?0.049). After transfection with MT3 lentivirus, proliferation was significantly inhibited in AML cells in a dose-dependent manner (P?<?0.05). Annexin V assay showed that apoptosis was significantly upregulated MT3-overexpressing AML cells compared to controls. Real-time PCR array analysis revealed 34 dysregulated genes that may be implicated in MT3 overexpression and apoptosis in AML, including FOXO1.ConclusionMT3 may be a putative tumor suppressor gene in pediatric AML. Epigenetic inactivation of MT3 via promoter hypermethylation was observed in both AML cell lines and pediatric AML samples. Overexpression of MT3 may inhibit proliferation and induce apoptosis in AML cells. FOXO1 was dysregulated in MT3-overexpressing cells, offering an insight into the mechanism of MT3-induced apoptosis. However, further research is required to determine the underlying molecular details.

Project description:BackgroundLeucine-rich repeat C4 protein (LRRC4) is a new member of the leucine-rich repeat (LRR) superfamily. It is not only a brain-specific gene but also a novel candidate for tumor suppression. LRRC4 inactivation is commonly found in glioma cell lines and primary glioma biopsies. However, little is known about the mechanism controlling LRRC4 expression. In a previous study, we did not find any genetic alteration in LRRC4 in primary glioma, which led us to explore an alternative mechanism underlying this phenomenon.MethodsIn the present paper, we cloned the LRRC4 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the LRRC4 promoter region in glioma cell lines and primary gliomas was examined by methylation-specific PCR and bisulfite DNA sequencing. In order to demonstrate a functional association between LRRC4 promoter methylation and its gene inactivation, we performed DNA demethylation analysis with two human glioma cell lines using methylation-specific PCR and RT-PCR.ResultsThe sequence spanning positions -835 to -293 relative to the translation start site was identified as the LRRC4 promoter; this sequence is a TATA- and CAAT- less, high GC content region. It was found that LRRC4 promoter activity is strongly suppressed after treatment with SssI methylase in vitro. Furthermore, LRRC4 promoter methylation was observed by methylation-specific PCR in two glioma cell lines and all 30 primary glioma specimens, but not in normal brain tissue. Bisulfite DNA sequencing showed that most of the CpG sites were located around the LRRC4 promoter methylated in glioma cells and tissues, but not in normal brain tissue. In addition, the methylase inhibitor 5-Aza-2'-deoxycytidine could induce LRRC4 mRNA expression and LRRC4 promoter partial demethylation in SF126 and SF767 glioma cells.ConclusionMethylation-mediated inactivation of LRRC4 is a frequent and glioma-specific event, and it may be a potential biomarker for diagnosis or prognosis, or serve as a therapeutic target.

Project description:In attempt to discover novel aberrantly hypermethylated genes with putative tumor suppressor function in epithelial ovarian cancer (EOC), we applied expression profiling following pharmacologic inhibition of DNA methylation in EOC cell lines. Among the genes identified, one of particular interest was DOK1, or downstream of tyrosine kinase 1, previously recognized as a candidate tumor suppressor gene (TSG) for leukemia and other human malignancies. Using bisulfite sequencing, we determined that a 5'-non-coding DNA region (located at nt -1158 to -850, upstream of the DOK1 translation start codon) was extensively hypermethylated in primary serous EOC tumors compared with normal ovarian specimens; however, this hypermethylation was not associated with DOK1 suppression. On the contrary, DOK1 was found to be strongly overexpressed in serous EOC tumors as compared to normal tissue and importantly, DOK1 overexpression significantly correlated with improved progression-free survival (PFS) values of serous EOC patients. Ectopic modulation of DOK1 expression in EOC cells and consecutive functional analyses pointed toward association of DOK1 expression with increased EOC cell migration and proliferation, and better sensitivity to cisplatin treatment. Gene expression profiling and consecutive network and pathway analyses were also confirmative for DOK1 association with EOC cell migration and proliferation. These analyses were also indicative for DOK1 protective role in EOC tumorigenesis, linked to DOK1-mediated induction of some tumor suppressor factors and its suppression of pro-metastasis genes. Taken together, our findings are suggestive for a possible tumor suppressor role of DOK1 in EOC; however its implication in enhanced EOC cell migration and proliferation restrain us to conclude that DOK1 represents a true TSG in EOC. Further studies are needed to more completely elucidate the functional implications of DOK1 and other members of the DOK gene family in ovarian tumorigenesis.

Dataset Information

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

Publications

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

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