Les cancers des Voies Aérodigestives Supérieures sont classés parmi les dix cancers les plus agressifs du fait de leur radioresistance intrinsèque et leur forte probabilité de récurrence. L’objectif de ce travail a été d’étudier le potentiel radiosensibilisant de nanoparticules à base de gadolinium, AGuIX®, sur un modèle cellulaire de cancer des VADS. Après avoir déterminé et validé les conditions optimales de radiosensibilisation de notre modèle par les AGuIX®, leur localisation après internalisation ainsi que les conséquences biologiques générées à l’échelle subcellulaire ont été successivement étudiées. Enfin, une approche préliminaire protéomique a été initiée afin d’identifier des cibles moléculaires potentielles impliquées dans cette radiosensibilisation. Le traitement des cellules SQ20B avec 0.8mM Gd pendant 24h se sont révélées être optimales avec un DEF (dose enhancement factor) de 1.3. Les AGuIX® sont localisées presque exclusivement dans les lysosomes après internalisation. La radiosensibilisation est liée à une surproduction de radicaux libres oxygénés, minimisée toutefois par des défenses antioxydantes endogènes élevées. Le traitement combiné (AGuIX®+ irradiation) déclenche spécifiquement la mort cellulaire autophagique et s’accompagne d’une augmentation significative du nombre de cassures double brins résiduelles complexes. L’étude protéomique préliminaire a permis d’identifier une cible moléculaire potentiellement impliquée dans cette radiosensibilisation (la ribonucléotide réductase), cible qui fera l’objet d’une suite à ce travail. De plus, la prochaine étape sera de comprendre les mécanismes qui relient les AGuIX® internalisées dans les lysosomes avec l’augmentation de la mort cellulaire autophagique après irradiation / Head and Neck Squamous Cell Carcinoma is ranked among the top ten deadliest cancers due to its high radioresistance and recurrence. One radiosensitizing strategy is the use of high-Z metal nanoparticles. In this study, ultrasmall gadolinium-based nanoparticles, AGuIX®, were used for their potential as a radiosensitizing agent. The objectives of this work were to determine the radiosensitizing conditions of AGuIX® in an HNSCC cell model, their localization after uptake, and the biological consequences generated at the subcellular level after the combined treatment. A preliminary proteomic approach was initiated in order to identify potential molecular targets involved in radiosensitization. The treatment of SQ20B cells with 0.8mM Gd for 24h resulted in a dose enhancement factor (DEF) of 1.3. AGuIX® were predominantly localized in lysosomes. The overproduction of radical oxygen species following AGuIX® + radiation was intimately involved in the radiosensitization, although largely subdued by the high level of endogenous antioxidant defenses. Autophagy was specifically triggered after the combined treatment, while other irradiation-induced cell deaths remained unchanged. The number of complex, residual double strand breaks (DSBs) was specifically increased with AGuIX® combined to radiation. Lastly, our preliminary proteomic analysis allowed the isolation of potential molecular targets with great promise. Collectively, it seems that the radiosensitizing effect observed in this work may result from a combination of events.Future work is required to understand the mechanisms linking lysosomes-entrapped AGuIX® with the upregulation of autophagic cell death after radiation

Contexte : Peu d’études ont recherché le rôle des facteurs de risque professionnels dans la survenue des cancers des voies aérodigestives supérieures (VADS). Ces études ont été conduites principalement chez des hommes.Objectif : L’objectif de cette thèse était d’étudier les associations entre les cancers des VADS et les expositions professionnelles chez les femmes.Méthodes : Icare est une étude cas-témoins en population générale incluant 296 cas féminins de cancers épidermoïdes des VADS et 775 femmes témoins. Les historiques de carrières recueillis ont été codés et croisés avec les matrices emplois-expositions du programme Matgéné. Outre les intitulés d’emplois, les expositions à 5 solvants chlorés (chloroforme, chlorure de méthylène, perchloréthylène, trichloréthylène, tétrachlorure de carbone), 5 solvants oxygénés (éthylène glycol, tétrahydrofurane, éther éthylique, cétones, alcools), 5 solvants pétroliers (essences carburants, essences spéciales, gazole, benzène, white-spirit) et à 7 poussières et fibres (amiante, farine, cuir, fibres céramiques réfractaires, ciment, laines minérales, silice) ont été étudiées. Les odds-ratios et leurs intervalles de confiance à 95% ajustés sur l’âge, le département, les consommations de tabac et d’alcool ont été estimés par régressions logistiques non conditionnelles.Résultats :Plusieurs professions et secteurs d’activité associés à un risque élevé de cancer des VADS ont été identifiés. Certaines professions (ouvrières de l’alimentation et des boissons, monteuses en appareillage électrique ou électronique, soudeuses) peuvent être à l’origine d’expositions professionnelles aux solvants, aux métaux, aux fumées de soudage et à diverses poussières. Les analyses par nuisance ont mis en évidence des associations significatives entre le risque de cancer des VADS et l’exposition au perchloréthylène et au au trichloréthylène. Aucune association claire n’est observée avec les solvants pétroliers et oxygénés, certains largement utilisés par les femmes L’exposition aux poussières de farine augmente significativement le risque de cancer des VADS. Une exposition probable à l’amiante est associée à une augmentation modérée et non significative du risque. Les analyses par localisation de cancer (cavité orale, pharynx, larynx), limitées par des effectifs faibles, ne mettent pas en évidence d’association spécifique.Conclusion : Nos résultats suggèrent un rôle des expositions professionnelles au trichloréthylène, au perchloréthylène et aux poussières de farine dans la survenue des cancers des VADS chez les femmes. / Background : Few occupational studies have addressed head and neck cancer, and these studies have been predominantly conducted in men. Objective : Our objective was to investigate the associations between head and neck cancer and occupational exposures in women Population and methods : ICARE, a French population-based case–control study, included 296 squamous cell carcinomas of the head and neck (HNSCC) in women and 775 female controls. Lifelong occupational history was collected. Job-exposure matrices were used to assess exposure to five chlorinated solvents (carbon tetrachloride; chloroform; methylene chloride; perchloroethylene; trichloroethylene), 5 petroleum solvents (benzene; special petroleum product; gasoline; white-spirits and other light aromatic mixtures; diesel, fuels and kerosene), 5 oxygenated solvents (alcohols; ketones and esters; ethylene glycol; diethyl ether; tetrahydrofuran) and 7 fibers and dusts (asbestos, flour dust, leather dust, refractory ceramic fibers, cement dust, mineral wools and silica) . An analysis by job title was conducted, and then associations with specific occupational exposures were investigated.Odds ratio (ORs) and 95% confidence intervals (CI), adjusted for smoking, alcohol drinking, age and residence area, were estimated with logistic models. Results : Significantly increased HNSCC risks were found for several jobs and industries. Some of these occupations (food and beverage processors, electrical and electronic equipment assemblers, welders and flame cutters) may entail exposure to agents such as solvents, metals, welding fumes and various dusts. Analyses for specific occupational exposures showed a significantly elevated risk of HNSCC associated with exposure to trichloroethylene and perchloroethylene. There is no clear evidence that petroleum or oxygenated solvents, some of them commonly used by women, are risk factors for HNSCC. Exposure to flour dust increased significantly HNSCC risk. Probable exposure to asbestos was associated with a moderate, non-significant elevation in risk. Analyses by cancer site (oral cavity, pharynx, larynx) were hampered by small numbers and did to reveal any specific association.Conclusion : These findings suggest that occupational exposure to perchloroethylene, trichloroethylene and flour dust may increase the risk of HNSCC in women.

Purpose: The Hedgehog-signalling pathway (Hh) is frequently active in head and neck squamous cell carcinoma (HNSCC). Overexpressed Hh associates with poor prognosis. The Hh inhibitor vismodegib targets smoothened (SMO) and, based on molecular data, may prevent resistance to EGFR targeting. Methods: To elucidate potential roles of vismodegib in HNSCC therapy, its sole effects and those combined with cisplatin, docetaxel, and cetuximab on HNSCC cell lines were assessed by MTT metabolisation and BrdU incorporation. Colony formation (CF) of primary HNSCC cells was studied utilizing the FLAVINO-protocol. Combinatory effects were analysed regarding antagonism, additivity or synergism. Associations between the ex vivo detected mode of action of vismodegib with other treatments related to patient characteristics were assessed and progression-free survival (PFS) in patient groups compared using Kaplan-Meier curves. Results: Vismodegib suppressed BrdU incorporation significantly stronger than MTT turnover; CF was significantly inhibited at ≥20 µM vismodegib while concentrations <20 µM acted hormetic. Combining 20 µM vismodegib plus docetaxel (T), cisplatin (P), and cetuximab (E), additively enhanced antitumoral activity in HNSCC samples from patients with superior PFS highlighting a potential role for ex-vivo testing of this combination for use as a prognostic classifier. Conclusion: We provide ex-vivo evidence for vismodegib’s potential in HNSCC therapies especially if combined with cetuximab, cisplatin and docetaxel.:Abkürzungsverzeichnis 1 Einleitung 1.1 Kopf-Hals-Tumore 1.1.1 Therapie von Kopf-Hals-TumoreN 1.1.2 Limitationen der etablierten Therapien 1.2 Eingesetzte Chemotherapeutika 1.2.1 Cisplatin 1.2.2 Docetaxel 1.2.3 Cetuximab 1.3 Hedgehog-Signaltransduktionsweg 1.3.1 Hedgehog-Signalweg und Karzinogenese 1.3.2 Vermittlung von Tumortherapieresistenz durch den Hedgehog-Signalweg 1.3.3 Zielgerichtete Tumortherapie durch Blockade des Hedgehog-Signalweges 1.4 Vismodegib 1.5 Ex-Vivo-Chemoresponse-Testung mittels FLAVINO-Assay 1.6 Zusammenfassung der Rationale der Untersuchung 1.7 Aufgabenstellung der Promotionsarbeit 2 Publikation 2.1 Reduzierte Proliferation und Koloniebildung von Plattenepithelkarzinomen der Kopf Hals Region unter dualer Inhibition des EGFR- und Hedgehog-Signalweges 3 Zusammenfassung der Arbeit 4 Literaturverzeichnis 5 Anlagen 5.1 Darstellung des Eigenanteils 5.2 Erklärung über die eigenständige Abfassung der Arbeit 5.3 Lebenslauf 5.4 Publikationen 5.5 Danksagung

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno- LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.

Background: About five to 10% of cancers in the head and neck region are neck squamous cell carcinoma of unknown primary (NSCCUP). Their diagnosis and treatment are challenging given the risk of missing occult tumors and potential relapse. Recently, we described human papillomavirus (HPV)-related NSCCUP-patients (NSCCUP-P) as a subgroup with superior survival. However, standardized diagnostic workup, novel diagnostic procedures, decision-making in the multidisciplinary tumor board (MDTB) and multimodal therapy including surgery and post-operative radio-chemotherapy (PORCT) may also improve survival. Methods: For assessing the impact of standardized diagnostic processes simultaneously established with the MDTB on outcome, we split our sample of 115 NSCCUP-P into two cohorts treated with curative intent from 1988 to 2006 (cohort 1; n = 53) and 2007 to 2018 (cohort 2; n = 62). We compared diagnostic processes and utilized treatment modalities applying Chi-square tests, and outcome by Kaplan–Meier plots and Cox regression. Results: In cohort 2, the standardized processes (regular use of [18F]-FDG-PET-CT imaging followed by examination under anesthesia, EUA, bilateral tonsillectomy and neck dissection, ND, at least of the affected site) improved detection of primaries (P = 0.026) mostly located in the oropharynx (P = 0.001). From 66.0 to 87.1% increased ND frequency (P = 0.007) increased the detection of extracapsular extension of neck nodes (ECE+) forcing risk factor-adapted treatment by increased utilization of cisplatin-based PORCT that improved 5-years progression-free and overall survival from 60.4 and 45.3 to 67.7% (P = 0.411) and 66.1% (P = 0.025). Conclusions: Standardized diagnostic workup followed by ND and risk-factor adapted therapy improves survival of NSCCUP-P.

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.

No description available.

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.

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.

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