Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

Promoter hypermethylation mediated gene silencing is a frequent epigenetic finding in many cancers that affects genes known to have important roles in several aspects of cell biology. Hematological malignancies have been reported to harbor multiple genes aberrantly silenced by promoter hypermethylation and as a result, cytosine analogs known to inhibit the DNA methylation machinery are currently being evaluated in clinical trials. As such, the general goal of this thesis was to identify genes silenced by promoter hypermethylation in human acute myeloid leukemia (AML) and to study the mechanism of promoter hypermethylation mediated gene silencing. Interestingly, the cyclin dependent kinase inhibitor p15 was found to be methylated at a high frequency in AML patients and cell lines in association with a lack of detectable p15 mRNA. Treatment with the cytosine analog 5-Aza-2-deoxycytidine (5-Aza-dC) in vitro resulted in promoter demethylation and p15 mRNA re-expression, which was associated with a release of a transcriptionally repressive complex at the p15 promoter. Importantly, 5-Aza-dC treatment also reversed specific histone amino-terminal modifications at the p15 promoter which are normally associated with transcriptionally inactive chromatin regions, implicating chromatin remodeling in promoter hypermethylation mediated gene silencing. The recently discovered DNA methylation inhibitor, zebularine considered more stable than 5-Aza-dC was also able to reconstitute p15 mRNA in vitro in association with promoter demethylation, regional enrichment of histone acetylation, and growth inhibition. To identify novel genes silenced by promoter hypermethylation in AML, cDNA microarray analysis was employed following in vitro pharmacological inhibition of DNA methylation and histone deacetylation. Of note, four genes from the metallothionein family of cysteine rich small molecules were consistently upregulated following drug treatment and further evaluation identified the gene MT1H to be hypermethylated at a high frequency in AML patients and cell lines. Taken together, the data suggests that aberrant promoter hypermethylation mediated gene silencing occurs in multiple genes from different gene families during the molecular pathogenesis of human AML. Furthermore, the mechanism of promoter methylation mediated transcriptional silencing acts in concert with specific histone modifications which, importantly, can be reversed by treatment with pharmacological inhibitors of DNA methylation.

Protein product tumor suppressor PALB2 gene plays a major role in pathway of DNA repair of double-strand breaks throught the homologous recombination mechanism. Significance of its pathogenic variants in hereditary forms of breast cancer in BRCA1/2- negative patients in families with multiple breast cancers may be in the Czech Republic comparable with the BRCA2 gene. A role of the PALB2 gene in sporadic breast cancer occurence, which represent 90 - 95 % of all cancers, is still unknown. This thesis focuses on inactivation pathway of tumor suppressor PALB2 in the sporadic breast cancer by a mechanism of allelic loss detecting by loss of heterozygosity (LOH) of corresponding microsatellite markers and hypermethylation of promoter region as the most common mechanisms of inactivation tumor suppressors in early tumorigenesis. In a group of 51 nonselected patients with sporadic breast cancer we found four samples with PALB2 locus allelic loss. These samples were analyzed for somatic mutations. No mutation was found. There is no evidence of promotor hypermethylation in any of the samples. Our data suggest a role of the PALB2 gene inactivation in a minority group of sporadic breast cancers.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Colorectal cancer (CRC) remains a significant burden in contemporary society due to an aging population, unhealthy dietary choices and an increasingly sedentary lifestyle. While the underlying defects for many hereditary forms of CRC have been determined, many genetic and epigenetic changes promoting common sporadic CRCs have yet to be identified. The Mutated in Colorectal Cancer (MCC) gene, identified in 1991, was initially thought to be responsible for the hereditary form of CRC, familial adenomatous polyposis, before the discovery of the susceptibility gene Adenomatous Polyposis Coli (APC), which then became the focus of intense research. Recent data, however, suggests that MCC may also be important in the development of CRC. I have investigated the mechanism of MCC gene silencing, the putative structure, and multiple functions of MCC. MCC was frequently silenced by promoter hypermethylation in CRC cell lines and primary tumours. MCC methylation showed strong molecular and clinicopathological associations with hallmarks of the serrated neoplasia pathway. Furthermore, MCC methylation was more frequent in serrated precursor lesions compared with adenomas, thus occurring early during carcinogenesis. MCC is highly conserved in complex multicellular organisms. Re-introduction of MCC in CRC cell lines resulted in partial G1 to S phase, and G2/M phase cell cycle blocks, potentially by upregulating cell cycle inhibitor gene transcription and interfering with the process of mitotic checkpoints and division, respectively. Changes in MCC levels also modulated NF?B pathway signalling, the pathway required for maintaining cell viability and proliferation in colonic epithelial cells. In particular, MCC overexpression suppressed both TNF? and LPS-induced NF?B activation, decreasing both the magnitude and rate of cellular responses. Overexpression also resulted in downregulation of proteins involved in canonical NF?B pathway signalling, while increasing the transcription of non-canonical NF?B genes. Therefore, MCC may direct activation of this pathway to a specific subset of NF?B-regulated genes. These data provide a molecular basis for the role of MCC as a tumour suppressor gene in CRC. MCC may have multiple functions, regulating cell cycle progression and modulating NF?B pathway signalling, either through direct involvement in pathway signalling cascades, or by providing a scaffold on which signalling events can occur.

Introduction: Le gène O6-méthylguanine-ADN méthyltransferase (MGMT) code pour une enzyme spécifique réparatrice de l’ADN qui protège les cellules de la toxicité des agents alkylants. Ainsi, l’activité du MGMT est un mécanisme majeur de résistance aux agents alkylants. Il a été démontré qu’une diminution de l’expression du gène MGMT par une hyperméthylation du promoteur résulte en une amélioration de la survie chez les patients avec certains types de tumeurs qui sont traitées avec des agents chimiothérapeuthique alkylants. Objectifs: Déterminer la prévalence de la méthylation du gène MGMT chez des patients avec des cancers épidermoïdes localement avancés de la sphère ORL traités avec chimioradiothérapie et évaluer l’impact de cette méthylation sur la survie. Méthodes: Sur 428 patients consécutifs, traités avec chimioradiothérapie à notre institution et suivis pour un période médiane de 37 mois, 199 spécimens chirurgicaux paraffinés ont été récupérés. L’ADN était extrait et modifié par le traitement au bisulfite. Une réaction en chaîne de la polymérase, spécifique à la méthylation était entreprise pour évaluer l’état de méthylation du promoteur du gène du MGMT. Les résultats de laboratoire étaient corrélés avec la réponse clinique. L’analyse statistique était exécutée à l’aide du test de Fisher pour les données catégoriques et à l’aide des courbes de Kaplan-Meier pour les échecs au traitement. Résultats : Des 199 extraits d’ADN initiaux, 173 (87%) étaient modifiés au bisulfite avec succès. Des ces spécimens modifiés, 71 (41%) ont démontré une hyperméthylation du MGMT. Pour les cas de méthylation et nonméthylation du MGMT, les caractéristiques des patients n’étaient pas significativement différentes. Les taux de réponse étaient 71 et 73% (p=NS) respectivement. Le contrôle locorégional était respectivement 87 et 77% (p=0.26), la survie sans maladie était 80 et 60% (p=0.38), la survie sans métastase à distance était 92 et 78% (p=0.08) et la survie globale était 64 et 62% (p=0.99) à 3 ans. Conclusions : L’état de méthylation du MGMT est fortement prévalent (41%) et semble avoir un possible impact bénéfique sur la survie quand la chimioradiothérapie est administrée aux patients avec des stades avancés de cancers tête et cou. / Background: The O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a specific DNA repair enzyme that protects cells from toxicity of alkylating agents. Thus, MGMT activity is a major mechanism of resistance to alkylating drugs. It has been shown that decreased MGMT gene expression by promoter hypermethylation results in improved survival in patients with certain types of tumors that are treated with alkylating chemotherapeutic agents. Objectives: To determine the prevalence of MGMT methylation in patients with locally advanced Head and Neck Squamous Cell Carcinoma (HNSCC) treated with chemoradiation therapy and to evaluate the impact of this methylation on survival. Methods: Out of 428 consecutive patients treated with chemoradiation therapy at our institution and followed for a median of 37 months, 199 paraffin embedded biopsy or surgical specimens were retrieved. DNA was extracted and subjected to bisulfite treatment. A methylation specific PCR (MSP) was conducted to assess the methylation status of the MGMT gene promoter. Laboratory data was correlated with clinical response. Statistical analysis was performed using Fisher’s test for categorical data and Kaplan-Meier’s curves and logrank statistics for failure times. Results: From the initial 199 DNA extracts, 173 (87%) were successfully modified with bisulfite. Out of these, 71 (41%) demonstrated hypermethylation of MGMT. For MGMT methylated cases and nonmethylated cases, patients characteristics were not significantly different. Response rates were 71 and 73% (p=NS), respectively. Local control rate (LCR) was respectively 87 and 77% (p=0.26), Disease-free survival (DFS) was 80 and 60% (p=0.38), distant metastasis free survival (DMFS) was 92 and 78% (p=0.08) and overall survival (OS) was 64 and 62% (p=0.99) at 3 years respectively. Conclusions: MGMT methylation status is highly prevalent (41%) and seems to have a possible beneficial impact on survival when chemoradiation therapy is given to patients with advanced stage HNSCC.

Introduction: Le gène O6-méthylguanine-ADN méthyltransferase (MGMT) code pour une enzyme spécifique réparatrice de l’ADN qui protège les cellules de la toxicité des agents alkylants. Ainsi, l’activité du MGMT est un mécanisme majeur de résistance aux agents alkylants. Il a été démontré qu’une diminution de l’expression du gène MGMT par une hyperméthylation du promoteur résulte en une amélioration de la survie chez les patients avec certains types de tumeurs qui sont traitées avec des agents chimiothérapeuthique alkylants. Objectifs: Déterminer la prévalence de la méthylation du gène MGMT chez des patients avec des cancers épidermoïdes localement avancés de la sphère ORL traités avec chimioradiothérapie et évaluer l’impact de cette méthylation sur la survie. Méthodes: Sur 428 patients consécutifs, traités avec chimioradiothérapie à notre institution et suivis pour un période médiane de 37 mois, 199 spécimens chirurgicaux paraffinés ont été récupérés. L’ADN était extrait et modifié par le traitement au bisulfite. Une réaction en chaîne de la polymérase, spécifique à la méthylation était entreprise pour évaluer l’état de méthylation du promoteur du gène du MGMT. Les résultats de laboratoire étaient corrélés avec la réponse clinique. L’analyse statistique était exécutée à l’aide du test de Fisher pour les données catégoriques et à l’aide des courbes de Kaplan-Meier pour les échecs au traitement. Résultats : Des 199 extraits d’ADN initiaux, 173 (87%) étaient modifiés au bisulfite avec succès. Des ces spécimens modifiés, 71 (41%) ont démontré une hyperméthylation du MGMT. Pour les cas de méthylation et nonméthylation du MGMT, les caractéristiques des patients n’étaient pas significativement différentes. Les taux de réponse étaient 71 et 73% (p=NS) respectivement. Le contrôle locorégional était respectivement 87 et 77% (p=0.26), la survie sans maladie était 80 et 60% (p=0.38), la survie sans métastase à distance était 92 et 78% (p=0.08) et la survie globale était 64 et 62% (p=0.99) à 3 ans. Conclusions : L’état de méthylation du MGMT est fortement prévalent (41%) et semble avoir un possible impact bénéfique sur la survie quand la chimioradiothérapie est administrée aux patients avec des stades avancés de cancers tête et cou. / Background: The O6-methylguanine-DNA methyltransferase (MGMT) gene encodes a specific DNA repair enzyme that protects cells from toxicity of alkylating agents. Thus, MGMT activity is a major mechanism of resistance to alkylating drugs. It has been shown that decreased MGMT gene expression by promoter hypermethylation results in improved survival in patients with certain types of tumors that are treated with alkylating chemotherapeutic agents. Objectives: To determine the prevalence of MGMT methylation in patients with locally advanced Head and Neck Squamous Cell Carcinoma (HNSCC) treated with chemoradiation therapy and to evaluate the impact of this methylation on survival. Methods: Out of 428 consecutive patients treated with chemoradiation therapy at our institution and followed for a median of 37 months, 199 paraffin embedded biopsy or surgical specimens were retrieved. DNA was extracted and subjected to bisulfite treatment. A methylation specific PCR (MSP) was conducted to assess the methylation status of the MGMT gene promoter. Laboratory data was correlated with clinical response. Statistical analysis was performed using Fisher’s test for categorical data and Kaplan-Meier’s curves and logrank statistics for failure times. Results: From the initial 199 DNA extracts, 173 (87%) were successfully modified with bisulfite. Out of these, 71 (41%) demonstrated hypermethylation of MGMT. For MGMT methylated cases and nonmethylated cases, patients characteristics were not significantly different. Response rates were 71 and 73% (p=NS), respectively. Local control rate (LCR) was respectively 87 and 77% (p=0.26), Disease-free survival (DFS) was 80 and 60% (p=0.38), distant metastasis free survival (DMFS) was 92 and 78% (p=0.08) and overall survival (OS) was 64 and 62% (p=0.99) at 3 years respectively. Conclusions: MGMT methylation status is highly prevalent (41%) and seems to have a possible beneficial impact on survival when chemoradiation therapy is given to patients with advanced stage HNSCC.