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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.

CDC73, also known as HRPT2, is a tumour suppressor gene whose expression is lost or downregulated in parathyroid, renal, breast, uterine and gastric cancers. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. As part of the Paf1 complex, it remains associated with ribonucleic acid (RNA) polymerase II and is involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly(A) length control and, coupling of transcriptional and posttranscriptional events. It has been reported to negatively regulate cellularproliferation by targeting oncogenes CCND1 (cyclin D1) and MYC (c-Myc). Moreover, it has also been indicated to inhibitβ-catenin-mediated transcription. Taken together, these findings strongly suggest that it contributes to the expression of genes whose products have an important role in the suppression of tumor development and cell death. In this study, we have attempted to study the transcriptional and posttranscriptional regulation of CDC73 and its role in OSCC. The main findings of the present study are listed below. 1. To begin with, the expression analysis of CDC73 was performed both at the RNA and the protein levels by qRT-PCR and IHC, respectively. As expected, a majority of the OSCC samples showed downregulation of CDC73 both at the RNA and the protein levels compared to their normal oral tissues. 2. Loss-of-heterozygosity (LOH), mutation and promoter methylation are the hallmarks of a tumor suppressor gene (TSG). Therefore, to characterize CDC73 as a TSG in OSCC and to look into the mechanisms that could be the cause of CDC73 downregulation in OSCC, LOH, mutation and promoter methylation of CDC73 were studied. The results showed that LOH, mutation and promoter methylation are not the major causes of CDC73 downregulation in OSCC. 3. To identify the alternate mechanisms as the cause of CDC73 downregulation in OSCC, a combination of bioinformatics and molecular approaches were used. The results showed that the upregulation of an inhibitory transcription factor WT1 (Wilms tumor protein 1) and an oncogenic microRNA-155 are the major causes of its downregulation in OSCC. 4. The luciferase reporter assay of SCC131 cells co-transfected with a WT1 construct and a CDC73 promoter construct showed that WT1 over expression represses CDC73 expression in a dose-dependent manner. 5. Due to the presence of zinc fingers in its C-terminal half, WT1 has been found to be a potent transcriptional regulator of genes. Therefore, to determine if WT1 down regulates CDC73 via binding its promoter, the chromatin immunoprecipitation (ChIP) assay was performed. The results showed the binding of WT1 to the CDC73 promoter in vivo. Binding of WT1 to the CDC73 promoter was further confirmed in vitro by the electrophoretic mobility shift assay (EMSA). 6. The 5-aza-2’-deoxycytidine (AZA) treatment of SCC131 cells led to upregulation of WT1 with a concomitant downregulation of CDC73. The COBRA technique demonstrated that the upregulation of WT1 upon the 5-AZA treatment was due to its promoter methylation. 7. To determine if the WT1-mediated reduction of CDC73 expression has a functional relevance in cell growth and proliferation, we knocked down CDC73 expression by transient over expression of WT1 in SCC131 cells and quantitated cell proliferation by the MTT assay. As expected, the results demonstrated that the reduced CDC73 level was associated with an increased cell proliferation. Cotransfection of CDC73 with WT1 in SCC131 cells attenuated the pro-oncogenic effect of WT1 by apoptosis induction. 8. After validating CDC73 as the target of WT1 by bioinformatics and in vitro assays, we quantitated the expression levels of WT1 and CDC73 by qRT-PCR in OSCC samples and their matched normal oral tissue samples. The results showed an inverse correlation between the expression levels of WT1 and CDC73 in a majority of the samples. To exclude the possibility of alternate mechanisms as the cause of CDC73 downregulation in OSCC, we selected a subset of OSCC samples with downregulated level of CDC73 and analysed them for LOH at the CDC73 locus and promoter methylation. Further, some of these OSCC samples were also analyzed for mutations in CDC73. The results showed that these OSCC samples did not have LOH, promoter methylation or any mutation, again validating the fact that CDC73 is a biological target of oncogenic WT1, and the transcriptional repression of CDC73 by WT1 could be a major mechanism for CDC73 downregulation in OSCC. 9. Recent studies have shown that a growing class of noncoding RNAs called microRNAs (miRNAs) is involved in posttranscriptional regulation of genes. There is a growing body of literature supporting the potential role of miRNAs in tumorigenesis. The importance of CDC73 in orchestration of several cellular functions and its role in tumorigenesis make it an attractive candidate for miRNA-mediated regulation of cell growth and proliferation. Using bioinformatics approaches, we identified an oncogenic microRNA-155 (miR-155) that could posttranscriptionally regulate CDC73 expression. 10. Consistent with its oncogenic role, miR-155 was found dramatically upregulated in OSCC samples and was found to be another mechanism for downregulation of CDC73 in a panel of human cell lines and a subset of OSCC samples in the absence of LOH, mutations and promoter methylation. 11. miRNAs regulate posttranscriptional gene expression generally via binding to their cognate sites in the 3’UTR. Therefore, a luciferase reporter construct was made by cloning the 3’UTR of CDC73 downstream to the luciferase reporter gene and the reporter assay was performed. Our experiments clearly indicated that the mature miR-155 regulates CDC73 expression by interacting with its 3’UTR in a site specific manner. 12 Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells over expression miR-155 resulted in increased CDC73 level, decreased cell viability, increased apoptosis and marked regression of engrafts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3’UTR in CDC73. In nutshell, the knockdown of CDC73 expression due to over expression of WT1 and miR-155 not only adds a novelty to the list of mechanisms responsible for its downregulation in different tumors, but the restoration of CDC73 levels by the use of inhibitors to WT1 and antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, with a worldwide incidence of 275,000 new cases annually (Warnakulasuriya, 2009). Globally, the head and neck carcinoma represents a major cause of morbidity and mortality and is the sixth most commonly occurring cancer (Warnakulasuriya, 2009). A majority (>90%) of the head and neck cancers are squamous in origin and thus are linguistically referred to as head and neck squamous cell carcinoma (HNSCC) (Warnakulasuriya, 2009). HNSCC includes cancers of the oral cavity, larynx and pharynx; oral cancer being the most common (Warnakulasuriya, 2009). Although, HNSCC is the sixth most common cancer globally (Warnakulasuriya, 2009), the Indian scenario is graver. According to GLOBOCAN 2008 (http://globocan.iarc.fr), the worldwide age standardized incidence rate (ASR) for HNSCC (and thus OSCC) is 5.3 and 2.5 per 100,000 males and females respectively (Ferlay et al., 2010). In India, the ASR is 9.8 and 5.2 per 100,000 males and females respectively, clearly demonstrating a remarkably high incidence rate of OSCC (Ferlay et al., 2010; http://globocan.iarc.fr). OSCC is a peculiar cancer which is largely preventable and rarely presents as a familial disorder. The most common etiological factors associated with OSCC include tobacco and alcohol consumption (Johnson, 2001). Additionally, high risk human papillomaviruses (HPV strains 16 and 18) as well as genetic predispositions have been implicated. The treatment of OSCC mainly relies on surgical resection of the tumor. The site, size, depth of infiltration and proximity to the bone of the tumor determine whether a combination of surgery with radiation therapy or chemotherapy would be advised (Scully and Bagan, 2009). The concomitant chemo-radiation therapy is the most commonly used strategy in locally advanced cancer. Taxanes (e.g., paclitaxel and docetaxel) and platinum-based induction chemotherapy (e.g., cisplatin) are the options in the treatment of locally advanced cancer. Epidermal growth factor receptor (EGFR) targeted with cetuximab in combination with radiotherapy has been successfully tested in a large randomized trial and thus is currently a new option (Scully and Bagan, 2009). The success of cetuximab has paved the path for the development and implementation of molecules targeting various signaling pathways. Despite extensive research on oral squamous cell carcinoma (OSCC), the five-year survival rate has not changed in several decades with the exception of the targeted treatment strategies involving cetuximab as discussed above. The current chemotherapeutic approaches lack selectivity and are flagitious. Thus, effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, the present study took three distinct approaches in order to validate the use of existing targets and to reveal novel prognostic biomarkers and therapeutic targets. 1) Targeting the PI3K-AKT-MTOR pathway in OSCC and identification of determinants of its sensitivity. 2) Gene expression analysis of ectopically overexpressed TSC2 to identify new therapeutic targets and prognostic biomarkers as well as to elucidate the genes regulated by it. 3) Expression profiling of CYP1B1 in order to validate the use of CYP1B1 based prodrug therapy in OSCC. Investigations pertaining to the changes in gene and protein expression profiles in malignant as well as pre-malignant lesions have documented the deregulation of the PI3K-AKT-MTOR (phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin) and EGFR (epidermal growth factor receptor) pathways in OSCC which are being widely targeted in many therapeutic strategies (Molinolo et al., 2007; Chakraborty et al., 2008; Matta and Ralhan, 2009; Molinolo et al., 2009; Stransky et al., 2011). The PI3K-AKT-MTOR pathway is a central hub for controlling cellular proliferation and growth in response to various intracellular as well as extracellular stimuli. Crucial signaling cascades including WNT, RAS, HIF-1α and AMPK cross-talk with the PI3K-AKT-MTOR pathway at a variety of molecular junctions. Thus, making this pathway sensitive to perceiving various growth modulatory conditions, ranging from the presence of growth factors to hypoxia and nutrient deprivation (Sengupta et al., 2010; Yang and Guan, 2007). The aberrant expression of the PI3K-AKT-MTOR pathway in OSCC advocated the targeting of this coveted pathway (Chakraborty et al., 2008). In various cancers, the monotherapeutic treatments with inhibitors like LY294002 (PI3K inhibitor) and rapamycin (MTOR inhibitor) demonstrated reduced efficacies. Such reduced efficacies were attributed to the drug toxicity and non-specific action of LY294002 (Davies et al., 2000; Sun et al., 2005; Ikezoe et al., 2007; Wang et al., 2008; Liu et al., 2009), or the ablation of a feedback inhibition loop leading to the reactivation of the PI3K-AKT-MTOR pathway by rapamycin (O'Reilly et al., 2006; Carracedo et al., 2008). Thus, rapamycin or its analogues demonstrated mediocre efficacy due to cytostatic effects in clinical trials, primarily due to the paradoxical activation of major survival kinases namely MAPK and AKT (O'Reilly et al., 2006; Carracedo et al., 2008). The present study aimed at increasing the efficacy of these drugs by incorporating a combinatorial approach. The MTT assay demonstrated that prolonged monotherapeutic treatments with rapamycin led to a modest growth inhibition in three OSCC (KB, SCC131 and SCC084) and HeLa cell lines. Western blot analysis of the phosphorylation status of AKT and RPS6KB1 revealed that monotherapeutic treatments with rapamycin for 96 hr led to the reactivation of the PI3K-AKT-MTOR pathway. Thus, the modest growth inhibitory effect of rapamycin was attributed to the reactivation of the PI3K-AKT-MTOR pathway. A combinatorial treatment approach was hence believed to circumvent this problem in order to increase the efficacy of targeting the PI3K-AKT-MTOR pathway. The PI3K inhibitor LY294002 was used combinatorially with rapamycin. This prolonged dual combinatorial treatment regime was distinctly more efficacious than either of the drugs alone and led to a reduction in cellular viability accompanied by increased sub-G1 population, indicating marked cell death that was characterized as caspase-3 dependent apoptosis. The differential sensitivity of the cell lines towards this combinatorial treatment revealed a novel determinant of the sensitivity, the transactivation of EGFR. The cell lines (SCC131 and SCC084) that were capable of transactivating EGFR were relatively resistant to the dual targeting of PI3K and MTOR in comparison to cell lines that did not transactivate EGFR (HeLa and KB). Further, targeting PI3K, MTOR and EGFR simultaneously was more efficacious in the presence of EGFR transactivation than dually targeting PI3K and MTOR. The results conclusively proved that the combinatorial therapeutic approach dually targeting PI3K and MTOR is a promising treatment strategy as compared to a monotherapeutic treatment and a major factor determining the sensitivity towards this treatment is the status of autophosphorylation of EGFR (Tyr1173) which governs the potential for EGFR transactivation by the combinatorial treatment. Thus, this study demonstrated that the status of EGFR autophosphorylation (Tyr1173) can be used as a biomarker to predict the sensitivity towards the combinatorial targeting of PI3K and MTOR in OSCC. The PI3K-AKT-MTOR pathway is negatively regulated by TSC2 (tuberous sclerosis complex 2; tuberin) (Tee et al., 2002). The importance of the TSC2 gene in the regulation of cell growth and proliferation is irrefutable. TSC2 facilitates the crosstalk between a variety of cellular signals, making it a crucial hub where many cellular networks integrate like AKT, MAPK and AMPK (Clements et al., 2007; Rosner et al., 2007; Rosner et al., 2008). It is a tumor suppressor gene and is downregulated in many cancers including OSCC (Chakraborty et al., 2008). In order to identify the genes regulated by TSC2 in OSCC, we stably overexpressed TSC2 in KB cells and the changes in the gene expression profiles caused by this ectopic overexpression were observed using a whole genome expression microarray. The results showed differential regulation of 268 genes (107 genes were upregulated and 161 genes were downregulated, p<0.05, fold change ≥ 1.5). A majority of these genes were functionally associated with transcription, cell growth and proliferation, apoptosis, cell cycle and neurogenesis. Functional annotation and network analysis was performed by using the DAVID v6.7 and IPA version 8.7 softwares. The microarray data revealed a novel aspect in the crosstalk between WNT signaling and TSC2, namely the transcriptional regulation of WNT signaling by TSC2. Further, in the context of therapeutic applications, the microarray analysis revealed multiple genes that were functionally categorized to be involved in response to radiation, UV and drugs (e.g., SERPINB13 and IL1B). Future studies on the regulation of such genes that are involved in responses to drugs and radiation may give insights into the role of TSC2 in resistance or sensitivity towards chemotherapy and radiation therapy. Moreover, EREG, a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in tuberous sclerosis lesions and its association with poor clinical prognosis in OSCC patients (Li et al., 2008; Shigeishi et al., 2008), making its regulatory aspects intriguing. Additionally, published data on the transcriptional functions of TSC2 instigated us to analyze the role of TSC2 in the regulation of EREG. TSC2 has been shown to modulate the transcription mediated by members of the steroid receptor superfamily of genes (Henry et al., 1998) and was shown to bind specifically to ERα and inhibit estrogen induced proliferation (Finlay et al., 2004). Also, TSC2 has been shown to possess C-terminal transcriptional activation domains (Tsuchiya et al., 1996). We have therefore attempted to investigate the transcription related functional aspects of TSC2 by exploiting the observed transcriptional repression of EREG. The physiological roles of TSC1 and TSC2 that are independent of the PI3K-AKT-MTOR pathway have been termed as ‘non-canonical’ (Neuman and Henske, 2011). The repression of EREG by TSC2 was observed to be insensitive to rapamycin, suggesting that it was independent of MTORC1 and thus a non-canonical function of TSC2. To determine whether the repression in EREG was at the level of the promoter, we performed a dual luciferase reporter assay. The results showed that the EREG promoter was repressed by stable as well as transient overexpression of TSC2. In order to elucidate the mechanism of transcriptional regulation by TSC2, we performed the ChIP analysis to observe the in vivo binding of TSC2 to the EREG promoter. In the ChIP analysis with the anti-TSC2 antibody, we observed that TSC2 did not bind to the EREG promoter between the regions -857 bp to -302 bp or -325 bp to +165 bp. Further, in silico analysis revealed an interesting trend among the transcription factors that were differentially regulated by TSC2 and had putative binding sites on the EREG promoter. A majority of these transcription factors (17/21) were downregulated by the overexpression of TSC2. This observation suggested that the repression of EREG could be an indirect effect due to repression of transcription factors caused by overexpression of TSC2. On the whole, this study revealed novel functions of TSC2 in OSCC with implications in determining novel biomarkers and therapeutic targets. As discussed previously, OSCC has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy (Rooseboom et al., 2004). CYP1B1, a member of the cytochrome P450 family, has been implicated in chemical carcinogenesis (Bandiera et al., 2005; Sliwinski et al., 2010). There exists a general accordance that this protein is overexpressed in a variety of cancers (e.g., colon, lung, renal, bladder, prostate, breast, endometrial and esophageal cancers), making it an ideal candidate for a prodrug therapy (McFadyen et al., 1999; Murray et al., 2001; McFadyen et al., 2004; Sissung et al., 2006; Wen and Walle, 2007; Sliwinski et al., 2010). The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits (Rooseboom et al., 2004). This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. Counterintuitively, CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This clearly demonstrated the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers. Since CYP1B1 has been shown to be involved in the activation of pro-carcinogens (Murray et al., 2001; Bandiera et al., 2005; Sissung et al., 2006), its inhibition could facilitate the development of a prophylactic therapy for oral cancer. Overall, this study has identified the transactivation of EGFR as a determinant of sensitivity towards combinatorial targeting of PI3K and MTOR in OSCC and has demonstrated that the autophosphorylation of EGFR (Tyr1173) can be used as a marker to judge the sensitivity towards this treatment. In the clinical perspective, the identification of such markers would aid in predicting the efficacy of targeted therapies. Such investigations would enable the strategic treatment of OSCC patients, thus decreasing the time lost in trial and errors for determining the appropriate treatment. Additionally, this study elucidated a novel role of TSC2 in the transcriptional repression of EREG, a prognostic biomarker for OSCC. Further, the study revealed potential prognostic biomarkers as well as therapeutic targets that are regulated by TSC2 by using a whole genome expression microarray. Moreover, the counterintuitive downregulation of CYP1B1 in OSCC tumors suggested the possibility of a prophylactic therapy for oral cancer but also advised a precautionary note for the application of prodrug treatments based on CYP1B1 overexpression in OSCC.