Dissertations / Theses on the topic 'Head and Neck Suqamous Cell Carcinoma (HNSCC)'

Background: HNSCC is a heterogeneous group of tumors caused mainly by environmental factors and human papillomavirus (HPV) infections. HPV- and HPV+ HNSCC are considered distinct entities, however, they are still treated with the same therapeutic strategies. HPV-induced tumorigenesis is mainly mediated by the E6/E7 oncoviral proteins, that, among all, alter the epigenetics of the host cells. Nevertheless, epigenetic profiles of HNSCC subtypes have not been clearly profiled. Results and Conclusion: hPTMs super-SILAC analysis of HNSCC cell lines and patients’ tissue samples revealed significant differences in the enrichment levels of some hPTM in HPV+ samples compared to HPV- ones and in tumoral tissues compared to normal ones. We focused on one of these identified hPTM and demonstrated that its levels are regulated by E6 and E7. We identified a histone modifier responsible for this hPTM whose levels are upregulated by E6/E7 and are higher in HPV+ compared to HPV- HNSCC cell lines and patients’ tissue samples, as is for the related hPTM. Silencing this enzyme through shRNA in HNSCC cell lines reduced proliferation and migration rates in both subtypes. It also downregulates the expression levels of some EMT mesenchymal makers and of a crucial oncogene involved in HNSCC. RNA-seq analysis revealed that other programs are instead specifically regulated according to the subtype: immune-response related genes are mainly activated in HPV- cell lines, while genes involved in cell differentiation in the HPV+ ones. Our research paves the way to novel lines of research and identifies a promising novel epigenetic target for HNSCC treatments.

To personalize treatment for cancer, correct staging of the primary tumor, nodal disease and metastatic disease is of essence. By targeting tumor specific receptors with radiolabeled antibodies, specificity and accuracy of imaging may be improved. Radio-immunodiagnostics can potentially detect small volume disease, occult metastasis and recurrent cancer in treated tissue. This thesis focuses on evaluation of radio-immunoconjugates directed towards CD44v6, which is a surface receptor overexpressed in many head and neck squamous cell carcinomas. At the outset, the monoclonal chimeric antibody cMab U36 and its cleavage products Fab’ and F(ab’)2 were labeled with 125I and assessed in vitro and in vivo (paper I). The best distribution pattern and tumor to organ ratio was achieved with F(ab’)2. Due to the immunological responses humans can develop towards chimeric antibodies, they are not optimal for clinical use, and subsequently fully human antibody fragments were developed. AbD15179, which is a monovalent fragment, was labeled with 111In and 125I and evaluated in vitro and in mice bearing CD44v6-expressing tumors. Tumor to organ ratios were improved compared to cMab U36 derived fragments, and 111In-AbD15179 displayed a more favorable distribution compared to 125I-AbD15179 (Paper II). A bivalent Fab-dHXL, AbD19384 derived from AbD15179, was then constructed and labeled with 125I and evaluated in cell- and biodistribution studies. Furthermore, an imaging study in a small animal PET was performed with 124I-AbD19384 (Paper III). Uptake in kidneys was reduced and liver uptake increased compared to AbD15179 reflecting the larger molecule. The high CD44v6 expressing tumor was clearly visualized with maximum uptake at 48 hours post injection.In paper IV human single chain fragments towards CD44v6v were selected, and the top candidates A11 and H12 were further evaluated in vitro and in vivo. Single chain fragments are small molecules exhibiting fast clearance and high affinity to the target. The study proved this by demonstrating superior tumor to blood ratios of radiolabeled A11 and H12 compared to previously studied molecules.

Head and neck cancer is the sixth leading type of cancer with 90 percent of head and neck cancer arising from squamous cell lining on the epithelium of the oral and nasal cavity, pharynx, and salivary gland. Even with tremendous achievements on chemotherapeutic drugs and therapies, the long-term prognosis of patients with advanced head and neck squamous cell carcinoma (HNSCC) has shown little improvement over the last three decades. Cisplatin is one of widely used chemotherapeutic drugs for multiple cancers, including head and neck cancer, but the prolonged use of this drug is limited by its toxicity and by the development of resistance. To overcome these major roadblocks to improved prognosis requires mechanism-based therapeutic strategies to maximize the antitumor effect of drugs while limiting their toxicities. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of BCL-2 family-dependent mitochondrial apoptosis. DNA damage activates a tumor suppressor p53 to induce apoptosis. One of its functions is to induce the expression of several pro-apoptotic proteins such as Noxa, which binds to an anti-apoptotic BCL-2 family protein, MCL-1 (myeloid leukemia cell-1) to inactivate its pro-survival function and induce apoptosis. We examined Noxa expression and apoptosis induced by cisplatin in p53-wild-type HN30 and HN31, p53-truncated and inactive HN4 and HN12, and p53-deleted HN22 and HN8 HNSCC cell lines. We found that Noxa was induced in HN30 and HN31 cells and down-regulation of Noxa by shRNA (short-hairpin RNA) decreased apoptosis, indicating Noxa contribution to cisplatin-induced apoptosis. Interestingly, cisplatin treatment induced Noxa and apoptosis even in p53-deleted HN22 and HN8 cells, suggesting the existence of the p53-independent pathways for the induction of Noxa. Based on these observations, we hypothesized that modulation of Noxa/MCL-1 axis could mimic cisplatin-induced cell death. We found that Noxa overexpression induced cell death in all cell lines tested regardless of p53 status. This finding could be applicable as a potential therapeutic strategy to treat head and neck cancer.

The primary focus of this research is the mechanisms of cell death in head and neck squamous cell carcinoma (HNSCC) treatment. These cancers typically originate in squamous cells that line the moist mucosal surfaces of head and neck. HNSCC is commonly treated with a platinum based agent, cisplatin. While the drug does offer strong antitumor effects, its prolonged use often results in tumor-acquired resistance, which limits treatment effectiveness. We have shown that cisplatin treatment induces the expression of a pro-apoptotic BCL-2 family member Noxa, which then initiates caspase- dependent apoptosis through its binding and sequestration of pro-survival protein MCL-1 for its inactivation. Without Noxa induction, cell death is significantly reduced when treating HNSCCs with cisplatin. The objectives of this study are (1) to determine the molecular mechanisms by which Noxa induces cell death in HNSCC cells; (2) to determine the molecular mechanisms of cisplatin-resistance in isogenic HNSCC cell lines. We observed an increase of apoptosis by ectopic expression of Noxa in all HNSCC cell lines tested, but not in immortalized human normal oral keratinocytes (NOK), suggesting that Noxa overexpression is sufficient to induce tumor-specific cell death. Noxa-induced cell death was mediated by BAX and BAK activation. BAK activation was mediated through Noxa binding to MCL-1, but not BCL-XL. Cisplatin- resistant cells induced less Noxa and apoptosis, supporting that Noxa induction is prerequisite for apoptosis induced by cisplatin. Taken together, Noxa induces tumor- specific cell death in HNSCC cells primarily through BAX and BAK activation, which suggests the therapeutic potential of this protein.

Head and neck squamous cell carcinoma (HNSCC) is a type of cancer that begins in the epithelial cells that line the mucosal surfaces of the head and neck, including the oral cavity, pharynx, larynx, paranasal sinuses, nasal cavity, and salivary glands. Head and neck cancer is the sixth most common type of cancer with a 5-year survival rate of 60% for all cases. Over the past few years, a subset of cells with stem-like properties, called cancer stem cells, are believed to have tumor-initiation capabilities and are responsible for maintaining on-going tumor growth. Previous data from our lab suggested that cells grown in suspension, called spheroids, may have stem cell like properties. We employed a model system where a primary HNSCC cell line, HN4, was used to set up spheroids. We found that expression of EPS8 and its downstream targets, FOXM1 and CXCL5, was increased in HN4 spheroids. In addition, we measured the expression of Nanog, as it is a transcription factor involved in the self-renewal of human embryonic stem cells. We also used a metastatic HNSCC cell line, HN12, to see how it compared to spheroids. We wanted to investigate the hypothesis that activation of Src potentiates EPS8 function to deregulate downstream signaling pathways. We used a small molecule tyrosine kinase inhibitor, Dasatinib, on HN4 spheroids and HN12 cells. We found that when Src is inhibited, EPS8 expression is decreased in HN4 spheroids and it also interferes with spheroid formation. The results of the current study were also able to show that the proliferation capability of HN12 cells is greatly diminished when treated with Dasatinib, due to G1 arrest in the cell cycle. When we measured for FOXM1, which is a cell cycle regulator, we found the levels were reduced in Dasatinib treated cells, preventing the cells from completing mitosis. With all of the data taken together, it suggests that Src does in fact play a role in regulating the downstream signaling pathways of EPS8, and its inhibition leads to the loss of cell proliferation. Additional studies need to be performed to discover whether Src inhibition will stop the proliferation of cancer stem cells, which are believed to be more resistant to cytotoxic therapies.

Despite the incorporation of the epidermal growth factor receptor (EGFR) inhibitor cetuximab into the clinical management of recurrent and metastatic (R/M) head and neck squamous cell carcinoma (HNSCC), only a small subset of patients responds to cetuximab, despite EGFR overexpression in virtually all of their tumors. At this time, there is a lack of validated predictive biomarkers to predict which patients will respond to cetuximab. Our previous work suggests that cetuximab activates the interleukin-1 (IL-1) pathway via tumor release of IL-1 alpha (IL-1α), although the implications of activating this pathway are unclear. The IL-1 pathway plays a central role in immune response and displays both pro-tumor and anti-tumor activities. IL-1 may promote tumor growth by upregulating the secretion of pro-inflammatory mediators involved in angiogenesis and metastasis. On the other hand, IL-1 signaling may promote antitumor immunity via enhancement of natural killer (NK)-cell mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and T cell activity, which are important mechanisms of action of cetuximab. The goal of this work is to determine how modulation of the IL-1 pathway affects HNSCC tumor response to cetuximab and if IL-1 may serve as a predictive biomarker for patient response to cetuximab. Blockade of IL-1 signaling did not enhance the anti-tumor efficacy of cetuximab, while IL-1α overexpression and treatment with recombinant IL-1α and IL-1α nanoparticles increased HNSCC tumor response to cetuximab in immunodeficient and immunocompetent HNSCC mouse models. Mechanistically, these results appear to be due to activation of an anti-tumor NK and T cell-mediated immune response. Additionally, we found that both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and inducible nitric oxide synthase (iNOS) activity may be involved in the efficacy of IL-1-induced ADCC against cetuximab-coated HNSCC cells. Altogether, these results suggest that IL-1 signaling is necessary for HNSCC tumor response to cetuximab. Furthermore, we have shown that pre-treatment serum and tumor IL-1 ligands can predict progression-free survival of HNSCC patients treated with standard-of-care cetuximab and chemotherapy, cetuximab combined with other targeted therapies, and cetuximab monotherapy. Overall, we propose that IL-1α warrants further study as a novel therapeutic to enhance response to cetuximab and as a predictive biomarker for HNSCC response to cetuximab.

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of cancer in the western hemisphere with a five-year survival rate of only 50% for patients with a localized tumor, which decreases significantly to as low as 5% for those patients with tumors that have metastasized to distant sites of the body. It has been found that both mutant p53 and epidermal growth factor receptor (EGFR) signaling pathways function to increase the expression of CXCL5, which has been identified as a key mediator in the process of tumor metastasis. Previous data from our lab suggested that the p53 homolog, p63, may function as a negative regulator of CXCL5 and that mutant p53 may inhibit this molecule to elevate CXCL5 expression levels. In the current study we utilized an model system in which the H179L p53 mutant was expressed in HN4 cells to investigate the hypothesis that mutant p53 enhances expression of CXCL5 by both interfering with p63 function and cooperating with EGFR/EPS8 signaling, leading to increased cell proliferation and motility. The results of the current study indicate a role for mutant p53 in head and neck squamous cell carcinoma proliferation, migration and tumorigenicity, possibly through enhancement of CXCL5 expression. We were able to show that mutant p53 expression caused an increase in the expression of this chemokine in addition to increasing proliferation and migration of the cells compared to the vector control. Additionally, we showed that p63 protein is a negative regulator of CXCL5 that is downregulated in the cells expressing mutant p53, which suggests that through direct interaction, mutant p53 may function to inhibit p63 function as well as target it for degradation. These results support the hypothesis that GOF mutant p53 enhances expression of CXCL5 by interfering with p63 function in cancer cells. The results of the current study results also showed that upon treatment with EGF, HN4 cells expressing mutant p53 express elevated levels of CXCL5; and that the mutant p53-expressing HN4 cells cooperate with EGFR/EPS8 signaling to further deregulate chemokine expression. These data taken together suggest there are complex interactions taking place between mutant p53, p63, EGFR signaling, and CXCL5 to regulate the biological processes that promote tumor progression that could lead to metastasis. Additional studies are needed to further elucidate the molecules involved in the mutant p53 mechanism that promotes tumorigenesis.

Head and neck cancer is the sixth leading cancer worldwide. Head and neck squamous cell carcinoma (HNSCC) accounts for more than 90% of incident cases. Despite intense, multimodality treatment regimens for HNSCC including surgery, chemotherapy, and radiation, little progress has been made over the past 30 years in improving overall survival rates. Tumor cell death induced by both conventional and targeted chemotherapy is often mediated by the BCL-2 family-dependent mitochondrial apoptotic pathway. However, initiators of this apoptotic pathway, such as p53, are more than 50% of the time mutated or deleted in HNSCC rendering the disease refractory to treatment. To counter such resistance, direct therapeutic targeting of the BCL-2 family is conceptually appealing. For this purpose, we use three clinically-available drugs: cisplatin, fenretinide, and ABT-263 (navitoclax). Both cisplatin and fenretinide are known to induce a BH3-only pro-apoptotic protein, Noxa, which binds to and inactivates multi-domain anti-apoptotic protein MCL-1 and release from its interaction with multi-domain pro-apoptotic protein BAK, followed by the phosphorylation via CDK2 for the proteasome-mediated degradation. Activated BAK can now go through conformational change for the oligomerization at the outer membrane of the mitochondria to release cytochrome c into the cytosol and induce caspase-dependent apoptotic cell death. ABT-263 directly binds to multi-domain anti-apoptotic proteins, such as BCL-2 and BCL-XL, to inhibit their activity and leads to the activation of multi-domain pro-apoptotic protein BAX to induce apoptosis. We hypothesize that combining the Noxa-inducing drugs (cisplatin or fenretinide) along with ABT-263 can efficiently induce BAX and BAK activation and significantly increase cell death in HNSCC cells by simultaneously inhibiting the activity of MCL-1, BCL-2, and BCL-XL. Combination-induced treatments in four cell lines (HN8, HN30, HN31, and UMSCC1) tested led to significant increase in apoptotic cell death. Cisplatin and ABT-263 combined treatment is inducing the expression of Noxa and leading to increase in apoptosis in HN30, HN31, UMSCC1, but not HN8. Similarly, fenretinide and ABT-263 combined treatment is inducing the expression of Noxa in all four cell lines tested and is largely relying on expression of Noxa.

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

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck squamous cell carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) and is a key regulator of tumor cell growth and survival. Erlotinib, also known as tarceva, (a first-generation) and afatinib, also known as giotrif, (a second-generation) are tyrosine kinase inhibitors (TKIs) of EGFR. These TKIs are recognized therapeutic agents in these tumor types, as they inhibit EGFR signaling but show limited activity as single agents. Novel strategies will likely require EGFR-TKIs combination with an agent(s) that will enhance their therapeutic efficacy. Recently, we have demonstrated that combining statins, inhibitors of the mevalonate pathway, with erlotinib enhanced EGFR inhibition and induced synergistic cytotoxicity through the activation of cellular integrated stress response pathway (ISR) regulated by the induction of activating transcription factor 3 (ATF3). In our Phase I clinical trial, combining rosuvastatin with erlotinib, while demonstrating clinical activity, this treatment also showed statin-induced myopathies likely the result of diminished ubiquinone levels, which limited their utilization. Therefore, alternative strategies are warranted. Targeting geranylgeranyl diphosphate (GGPP) synthesis or its incorporation, a downstream mevalonate metabolite, represents such an approach with the potential to circumvent statin-associated toxicities but retain the efficacy in combination with EGFR inhibitors. In this project, we evaluated the effect of the combination of geranylgeranyl transferase-I inhibitor (GGTI-298) with the EGFR inhibitor, tarceva, (aim 1) and a GGPP synthase inhibitor, digeranyl bisphosphonate (DGBP), with the EGFR inhibitor, afatinib, (aim 2). For aim 1, we demonstrated that GGTI-298 treatment induced ATF3 expression in SCC9 and SCC25 cells and in a cohort of ex-vivo tumor tissues. Furthermore, GGTI-298 and tarceva induced synergistic cytotoxicity in SCC cells that was dependent on ATF3 expression, as ATF3 deficient murine embryonic fibroblasts (ATF3-/- MEFs) displayed attenuated cytotoxicity in response to GGTI-298 alone and in combination with tarceva. Similarly, SCC9 sub-lines that were selected as resistant to GGTI-298 through prolonged exposure to this agent also failed to demonstrate synergy with treatment of GGTI-298 in combination with tarceva. For aim 2, we demonstrated that the specific GGPP synthase inhibitor, DGBP, induced cytotoxicity in SCC cells. We further demonstrated this specificity as specific shRNA targeting of GGPP synthase as well as the inhibitor DGBP significantly enhanced the cytotoxic activity of the EGFR-TKI afatinib in SCC cells. DGBP as well as afatinib treatments induced ATF3 expression in SCC cells in vitro and in a cohort of ex-vivo tumor tissues. Co-administration of the downstream metabolite GGPP inhibits the induction of ATF3 and the cytotoxic and apoptotic effects associated with DGBP treatment. Furthermore, the synergistic cytotoxicity induced by the combination of DGBP and afatinib in SCC cells was also dependent on the expression of ATF3 through the induction of cellular stress response pathways. Taken together, these results suggest the potential clinical utility of combining downstream mevalonate inhibitors (GGTI-298 or DGBP) with EGFR inhibitors in HNSCC patients as a novel and more refined combination therapeutic approach.

The active form of Vitamin D (VD3) has been shown to induce pro-apoptotic and anti-proliferative effects in several mammalian cancer cell types. The molecular mechanisms of tumor suppression, however, are not clearly understood. Previous research has shown that head and neck squamous cell carcinoma (HNSCC) responds to VD3. This thesis used both in vivo and in vitro models to examine the effect of VD3 in HNSCC. Former work in the Albala laboratory showed that hamsters that received systemic VD3 and topical treatment of 7,12-dimethylbenz(a)anthracene (DMBA) to the buccal pouch showed no or delayed carcinogenesis over the 14-week study compared to DMBA-only treated hamsters. This research further investigated the effect of VD3 in this hamster model. Using immunohistochemical (IHC) and western blot analysis, we demonstrate that systemic application of VD3to hamsters downregulates Rad51 expression in the buccal pouch and hinders the onset of tumor formation. Rad51 is a protein that plays a critical role in cell proliferation and homologous recombinational DNA repair. In the in vitro model, we show that Rad51 expression decreased in response to 100nM VD3 in HNSCC cell lines. The dose and time-dependence of VD3 on these cells was also examined. Western blot analysis and comet assay investigations confirmed that the SCC25 cell line is most sensitive to 100nM VD3 than to other doses tested, and that VD3 impairs the DNA-damage response. SiRNA and co-immunoprecipitation studies examined the potential of Chk 1 and p38 MAPK as upstream regulators of Rad51. Rad51 protein expression was found to be associated with early carcinogenesis from HNSCC cancer patients using IHC studies of human carcinomas from the oral cavity. This study focused on further identifying the role of Rad51 in response to VD3 in HNSCC.

Malignancies of the upper aero-digestive tract are a major public health problem, especially in South Asia. The major risk factors in South Asia remain smoked/smokeless tobacco, areca nut, alcohol abuse and poor diet, with limited evidence for human papillomavirus (HPV). Although HPV-associated head and neck squamous cell carcinoma (HNSCC) is well documented in the western world, studies on South Asian populations are few and inconsistent. However, the incidence of HPV-associated head and neck cancer (HNC) has increased in recent years. Certain high-risk types of HPV infection are regarded as well-established risk factors for cervical cancer and a subset of HNSCC; however, their true role and importance in the progression of HNSCC remain unclear. Although HPV-associated HNC patients generally have a better prognosis than those with HPV-negative disease, current chemo- and radio-therapies are largely non-specific and have considerable toxicities. RNA interference (RNAi), which has shown great promise as a highly specific therapy for other diseases, has potential for treating HPV-associated HNC, especially if disease progression is dependent on the continual expression of HPV oncogenes.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Dentistry and Oral Health
Griffith Health
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AIM & OBJECTIVES: Human papilloma virus (HPV) has been related to cervical infection, however, its part in Head and Neck Squamous Cell Carcinoma (HNSCC) is still debatable and is easy to refute. Suspicion of HPV causation is heightened when carcinomas arise in patients that are young and have never smoked. The present UK based study undertaken at Northampton NHS Trust endeavoured to determine the extent to which HPV is an entity in HNSCC in the UK. Furthermore, the study investigated whether sexual behaviour (as measured by sexual health clinic (SHC) attendance) is linked the acquisition of HPV associated HNSCC in young age groups. HNSCC incidences and sexual trends in the UK were collected from publicly available databases to identify if there were any changes at a national level in sexual behaviours and their influence on HNSCC in young age groups. MATERIALS & METHODS: PCR was used to evaluate the presence of HPV in biopsy samples from of 99 patients diagnosed with HNSCC at Northampton Hospital from 2006 to 2014. Patient demographics on age, sex, smoking, alcohol use and SHC attendance were also collected. All HPV PCR positive biopsies were further genotyped using an ABI 3130xl genetic analyser. Databases in the UK; including GLOBOCAN, NATSAL and PHE were searched for data on HNSCC prevalence, sexual behaviour trends and vaccine uptake. Multinomial regression explored the relationship between HPV positivity and sex, age, smoking, drinking, race and SHC attendance. RESULTS: PCR showed that 25.2% (25/99) of biopsies tested were positive for HPV and were all obtained from white participants. Most specimens (23, 92%) were high-risk (HR) HPV 16 positive with a mean age of 56 for HPV positivity and 72% of the cases 50-60 years old. Smokers were 11% in total (11/99) with most 88.9% participants (88/99) being non-smokers. HPV positivity was strongly linked with non-smoking history (p < 0.001); no alcohol abuse (p < 0.001); male gender (p < 0.001); young age less than 60 years (p < 0.001) and SHC attendance (p < 0.001). A Kruskal-Wallis post hoc test affirmed the impact of age on HPV positivity (p= < 0.05). GLOBOCAN and Cancer Research demonstrated a rising UK HNSCC pattern of over 200% for both sexes from 1975 to 2011. The three NATSAL surveys undertaken in 1990-1991, 1999-2001 and 2010-2012 demonstrated an overall increase in opposite and same sex partners. The UK average of individuals engaging in oral sex was in the younger age groups of between 16 and 54 with at least 70% of males and 63% females of that age engaging in oral sex. Finally, NASTAL 1, 2 and 3 surveys reported 20 vs 15; 25 vs 55; 55 vs 65 of males and females respectively with more than 10 sexual partners to have attended the SHC. The UK immunization take-up was over 90% countrywide. CONCLUSION: Few research studies have been conducted to date on HPV as a cause of HNSCC in the UK. The present research showed 25.2% of HNSCC to be caused by HPV, with the high risk (HR) genotype 16 (the leading cause of cervical cancer) accounting for 92% (23/25) of the cases. These outcomes affirmed the high prevalence of HR-HPV in HNSCC, with a rate of 25.2% similar to those reported previously. Routine HPV testing in those aged below 60 is therefore warranted. Smoking and drinking showed negative correlation; the young age of below 60 and attendance of the SHC for both sexes showed a positive correlation with HPV positive HNSCC. NATSAL data showed increased sexually risky behaviour coupled with attending the SHC in younger ages for both sexes. Increased sexually risky behaviour as shown in NASTAL surveys may be the reason why young age and SHC attendance is positively correlated with HPV HNSCC. The study highlights a conceivable relationship between HPV positive HNSCC in those under 60 years with no smoking history who attended the SHC. Smoking and drinking are known risks for HNSCC in those past 65 years of age; the negative association with HPV HNSCC in the young in the present research revealed smoking and drinking to have reduced association with HPV HNSCC. The reported HR-HPV positive HNSCC in young age groups inform future vaccination strategies and consequently decrease the quantity of HPV HNSCC's.

碩士
國立陽明大學
臨床醫學研究所
107
Neuroendocrine tumors (NETs) of the head and neck (HNC) are rare neoplasms, compared with common HNC, the NETs have neuroendocrine property. The mechanism of NED in HNC is unclear. In previous study the mechanism of prostate cancer neuroendocrine differentiation (NED) have already discuss, In castration-resistant prostate neuroendocrine cancer patients elevated serum levels of IL-6 have high expression and IL-6 is play an important role in prostate NED cancer. IL-6 is one of the key molecules that has been widely studied and implicated in poor clinical outcomes in HNSCC patients. IL-6 is a pleiotropic cytokine which plays an important role in a number of cellular processes including proliferation, survival, differentiation, migration and invasion. IL-6 mediates its downstream effects by activating a number of signaling pathways. IL-6 also regulates tumor progression and tumor metastasis by modulating tumor angiogenesis and tumor lymphangiogenesis, in HNSCC patients serum also find high level IL-6. We therefore hypothesize that in head and neck neuroendocrine tumor IL-6 may induce HNC neuroendocrine differentiation. First, we found IL-6 induce head and neck squamous cells (HNSCC) NED. Therefore we want know the source of IL-6. In tumor microenvironment, macrophages generally play a pro-tumor role and can stimulate angiogenesis and enhance tumor cell invasion and motility. Cancer stem cells (CSCs) enhance tumor selfrenew property. Both of two cells secreted IL-6. In Clinical experiment, we used human HNSCC patients tissue to prove in NED type patients also have higher M1 macrophage than none NED type patients. Finally, we used big database The Cancer Genome Atlas (TCGA) to analysis human HNSCC patients IL-6 and NED correlation. In high level of IL-6 and NED marker patients group the survival rate is lower than short of IL-6 and NED marker patients group. These result show that in HNSCC NED the IL-6 play an important role, the source may from M1 macrophage and CSCs. The regulating mechanism of NETs in HNC needs to be further investigated.

碩士
國立臺灣大學
藥理學研究所
105
Head and neck squamous cell carcinoma (HNSCC) has high metastasis and recurrent rates and causes high cancer-related mortality in Taiwan. However, there is still no effective treatment for HNSCC. We therefore aimed to identify novel therapeutic targets to prevent HNSCC metastasis. 　　Mutations and amplification of FGFR (Fibroblast growth factor receptor) genes were reported to be important in cell proliferation, differentiation and migration of many cancers, including breast cancer, lung cancer and melanoma. FGFRs were also reported to be associated with epithelial–mesenchymal transition, angiogenesis and regulation of cell migration. Therefore, our question is: Does FGFR participate in HNSCC metastasis? Could it be a potential targret in curing HNSCC? To answer this question, we used shRNAs targeting FGFRs to systemically examine the effect of FGFR on HNSCC cell migration. The screen result revealed that FGFR2 knockdown significantly reduced HNSCC motility. Therefore, we proposed that FGFR2 may promote HNSCC cell migration. 　　To validate our hypothesis, we examined the expression level of FGFR2 in 53 tumor samples from HNSCC patients of various stages. We found that FGFR2 expression was decreased in late-stage HNSCC. To further understand the role of FGFR2 in cancer cell migration, we knocked-down, overexpressed and rescued FGFR2 levels in SAS cells, and measured how such treatments affected HNSCC cell migration. We found that FGFR2 knockdown significant decreased cell migration, probably through impairing directionality and cell-cell coordination. These results implied that FGFR2 might promote HNSCC cell migration. Interesingly, FGFR2IIIb or IIIc overexpression slightly decreased cell migration. These results suggested that there might be some interaction between FGFR2IIIb and FGFR2IIIc. Based on our findings, FGFR2 may play an important role in HNSCC metastasis. Further investigations are currently under the way to clarify the mechanism how FGFR2 changes HNSCC migration.

Squamous cell carcinoma of the head and neck region (HNSCC) is an aggressive malignancy with generally poor prognosis and high mortality. The Special AT-rich binding protein 1 (SATB1) is a genome organizer protein that participates in regulating gene expression by acting as a trans-acting element as well as by recruiting chromatin remodeling complexes and enzymes. SATB1 is often overexpressed in cancer, and its possible role in tumour progression has been explored in several types of cancers and also suggested in HNSCC. However, its influence on molecular and cellular processes in HNSCC has not been examined, and, using primary cell lines, provided the basis of this thesis. This is a comprehensive study of molecular and cellular processes being affected upon siRNA-mediated SATB1 knockdown in vitro and in vivo. 15 HNSCC primary cell lines were obtained from the University of Turku and screened for SATB1 mRNA levels. The comparison of SATB1 mRNA levels with location, lymph node metastasis, disease staging (TNM) or SATB2 mRNA levels revealed no association. Hence, for deeper analysis 7 primary cell lines were selected based on growth inhibitory effects upon transient SATB1 knockdown, rather than their initial SATB1 mRNA levels. Growth inhibition upon SATB1 depletion was shown in monolayer (viable cell quantitation and colony forming ability) as well as non-adherent (spheroid assay) culture conditions. In some cell lines, cell death induced by apoptosis or retardation of cell cycle progression was observed as well. Parallel to this, using the FLAVINO assay, colony forming abilities of tumour cells from patient biopsies obtained from the University Hospital of Leipzig (Department of Otorhinolaryngology, Head and Neck Surgery) were tested post SATB1 knockdown. For molecular analysis, effects of SATB1 knockdown on transcription rates of selected oncogenes were analyzed. Among EMT markers, N-cadherin and beta catenin levels were found reduced upon SATB1 knockdown. The transcription of HER3 and its ligands Heregulin α & β was attenuated in all the seven primary cell lines, irrespectively of the growth inhibitory effects of SATB1 knockdown. These results demonstrated the role of SATB1 in the process of EMT and in autocrine signalling. Effects of HER3 inhibition on transcription rates of SATB1 were tested as well. HER3 inhibition was achieved by Patritumab, a novel monoclonal antibody against HER3. While SATB1 transcription rates remained unchanged upon HER3 inhibition, growth inhibition assays (2D and 3D) revealed that the combined use of HER1 and HER3 inhibitory antibodies provides better tumour cell inhibition over the single treatment. Finally, antitumor effects of SATB1 knockdown were monitored in vivo in two xenograft models (UT-SCC-14 and UT-SCC-42B). Treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles revealed reduced tumour growth, based on the knockdown of SATB1 as demonstrated on the protein level. Taken together, in this work SATB1 knockdown is demonstrated to mediate growth inhibition, induction of apoptosis, cell cycle retardation, negative impact on EMT and autocrine signaling and in vivo anti-tumour effects, thus highlighting the relevance of SATB1 in HNSCC.:156 pages

博士
國立臺灣大學
藥理學研究所
102
The mechanism of dasatinib-induced apoptosis of head and neck squamous cell carcinoma (HNSCC) cells was investigated in this study. HNSCC is a worldwide disease with aggressive course and dismal outcome. The expression of epidermal growth factor receptor (EGFR) promotes cell growth and proliferation and is associated with clinical poor outcome of HNSCC. Estrogen receptor is a nuclear receptor which can exert both genomic and non-genomic actions. In HNSCC, estrogen receptor signaling is found to work in concert with EGFR to enhance cell growth and invasion. In the era of molecular targeted therapy, the emergence of cetuximab, an monoclonal antibody against EGFR, has led to a progress in HNSCC, but the efficacy is modest. Thus, new therapies are needed. Dasatinib is a Bcr-abl and Src inhibitor which has been approved for chronic myeloid leukemia and is expected to have activity against solid tumors. However, few patients benefit from dasatinib in clinical trials despite consistent Src inhibition, implicating that there are mechanisms beyond Src inhibition responsible for the efficacy of dasatinib. The first part of our study disclosed that EGFR degradation was the key event to mediate dasatinib-induced apoptosis in HNSCC cells. This event was through lysosome degradation. In addition, estrogen receptor was found to be associated with EGFR in dasatinib-induced apoptosis. Furthermore, xenograft model showed that dasatinib inhibited HNSCC tumor growth through in vivo down-regulation of EGFR and estrogen receptor. In the second part of the study, we further investigated the mechanism of dasatinib-induced EGFR degradation and showed that AMPK-dependent endoplasmic reticulum (ER) stress is responsible for this event. Dasatinib induced ER stress which mediated EGFR degradation in a c-cbl-dependent manner. AMPK activation induced by dasatinib might be due to ATP decrease through the up-regulation of pyruvate dehydrogenase kinase 4 (PDK4). Furthermore, activation of AMPK by metformin sensitized dasatinib-induced in vitro and in vivo anti-cancer effect. The correlation of AMPK activation and EGFR expression was seen in HNSCC cells and human tumor specimens. Our results disclose that AMPK-dependent ER stress-mediated EGFR degradation plays a crucial role in the anti-cancer effect of dasatinib in HNSCC. Activation of AMPK by metformin might enhance dasatinib efficacy in HNSCC treatment.

Yes
Background - Previous studies have demonstrated that a number of HOX genes, a family of transcription factors with key roles in early development, are up-regulated in head and neck squamous cell carcinoma (HNSCC) and other cancers. The loci of several Homeobox (HOX) genes also contain microRNAs (miRs), including miR-196a. Methods - Global miR expression and expression of all 39 HOX genes in normal oral keratinocytes (NOKs), oral pre-malignant (OPM) and HNSCC cells was assessed by expression microarray and qPCR and in tissues by immunohistochemistry (IHC) and qPCR of laser microdissected (LCM) tissues. Expression of miR196a and HOXB9 was reduced using anti-miR-196a and siRNA, respectively. Expression microarray profiles of anti-miR196a and pre-miR196a transfected cells were compared to parental cells in order to identify novel targets of miR- 196a. Putative miR196a targets were validated by qPCR and were confirmed as binding to the 3’UTR of miR196a by a dual luciferase reporter assay combined with mutational analysis of the miR-196a binding site. Results - miR-196a and HOXB9 are highly expressed in HNSCC compared to NOKs, a pattern also seen in HNSCC tissues by HOXB9 IHC and qPCR of miR-196a in LCM tissue. Knock-down of miR-196a expression decreased HNSCC cell migration, invasion and adhesion to fibronectin, but had no effect on proliferation. Furthermore, knock-down of HOXB9 expression decreased migration, invasion and proliferation but did not alter adhesion. We identified a novel primary mRNA transcript containing HOXB9 and miR196a-1 as predicted from in-silico analysis. Expression array analysis identified a number of miR196a targets, including MAMDC2 and HOXC8. We confirmed that MAMDC2 is a novel miR-196a target using a dual luciferase reporter assay with the effect abolished on mutation of the binding site. Conclusions - These results show that miR-196a and HOXB9 are overexpressed, perhaps co-ordinately, as HNSCC develops and exert a pro-tumourigenic phenotype in HNSCC and OPM cells.

Aufgrund der heterogenen Tumorbiologie variiert der Therapieerfolg bei lokal fortgeschrittenen Plattenepithelkarzinomen stark, woraus ein mittleres 5-Jahres-Überleben dieser Patienten von etwa 50% resultiert. Um die Therapie besser an die Tumoreigenschaften anzupassen, muss die Therapieresistenz der Tumoren vor der Behandlung bestimmt werden. In dieser Dissertationsschrift werden Methoden aus dem Bereich des maschinellen Lernens angewandt um Genexpressionsdaten zu analysieren, um so Signaturen und Modelle zu erzeugen die eine Klassifizierung der Tumoren in verschiedene Risikogruppen bezüglich der loko-regionären Tumorkontrolle erlauben. Für Patienten, die mit postoperativer Radiochemotherapie behandelt wurden, konnte eine 7-Gen Signatur entwickelt und erfolgreich validiert werden. Außerdem konnte gezeigt werden, dass verschiedene Signaturen ähnlich gut zur Patientenklassifizierung geeignet sein können. Daher wurde eine Methode vorgeschlagen, die es erlaubt verschiedene prognostiche Modelle zu kombinieren. Weiterhin wurden verschiedene genbasierte Biomarker zwischen verschiedenen Genexpressionsmessmethoden verglichen. In den resultierenden Patienteneinteilungen zeigten Biomarker, die auf Signaturen basieren, eine geringere Variabilität als Biomarker, die auf einzelnen Genen basieren.:Abbreviations VII Figures IX Tables XII 1 Introduction 1 2 Biological & Statistical Background 4 2.1 Head and Neck Squamous Cell Carcinoma 4 2.1.1 Tumorigenesis 4 2.1.2 Biomarkers 8 2.2 Statistics 14 2.2.1 Survival analysis 14 2.2.2 Model and data evaluation 18 2.2.3 Data sampling methods 22 2.3 Machine learning algorithms 23 2.3.1 Feature selection algorithms 24 2.3.2 Prognostic models 27 2.4 Gene expression measurement methods 30 2.4.1 Real-time polymerase chain reaction (RT-PCR) 31 2.4.2 nCounter® gene expression 32 2.4.3 In situ-synthesized oligonucleotide microarrays 32 3 Material and methods 35 3.1 Patient cohorts 35 3.1.1 Primary radiochemotherapy (pRCTx) cohorts 35 3.1.2 Postoperative radio(chemo)therapy (PORT-C) cohorts 36 3.1.3 Clinical endpoints 38 3.2 Gene expression analyses 39 3.2.1 HPV status 39 3.2.2 Immunohistochemical staining 39 3.2.3 RT-PCR measurements 40 3.2.4 nCounter® measurements 40 3.2.5 GeneChip® analyses (only training cohorts) 41 3.3 Machine learning framework 41 3.3.1 Pre-processing of gene expression data 41 3.3.2 Determination of the ensemble gene signature 42 3.3.3 Expanding the ensemble signature by highly correlated genes 43 3.3.4 Independent validation and patient stratification 45 4 Identification of gene expression signatures as prognostic biomarkers 46 4.1 Hypoxia classification 46 4.2 nCounter® gene expression based signatures 50 4.2.1 Patients treated with primary radiochemotherapy 50 4.2.2 Clinical Features 55 4.2.3 Signature extension using clinical features 64 4.2.4 Patients treated with postoperative radiochemotherapy 65 4.2.5 Signature extension using clinical features – Port-C 72 4.3 GeneChip® gene expression-based signatures 78 4.3.1 Pre-selection 78 4.3.2 Patients treated with primary radiochemotherapy 79 4.3.3 Patients treated with postoperative radiochemotherapy 87 4.4 Combined models for PORT-C 91 4.4.1 Creation of a consensus model 92 4.4.2 Consensus model based on 2 models 93 4.4.3 Consensus model based on more than 2 models 97 4.4.4 Discussion and summary of model combination 101 5 Stability of gene expression-based biomarkers 102 5.1 Reproducibility depending on time of nCounter® 102 5.2 Comparison of nCounter® and GeneChip® gene expression 106 5.2.1 Introduction 106 5.2.2 Correlation analyses 106 5.2.3 Model and biomarker transfer 108 6 Conclusion and outlook 123 Zusammenfassung 125 Summary 128 Appendix 130 A. Supplementary Figures 130 B. Supplementary Tables 133 Bibliography 147 Acknowledgements 188 Erklärungen 189
Due to heterogeneous tumour biology, the treatment response of locally advanced head and neck squamous cell carcinoma differs largely between patients, resulting in a mean 5-year survival of about 50%. In order to adapt the treatment to the properties of the tumour, the therapy resistance of the tumours must be assessed before treatment. In this thesis, gene expression data were analysed to identify novel gene signatures and models that allow for stratifying patients into risk groups with low and high risk of loco-regional tumour recurrence. To identify those signatures, methods from the field of machine learning were applied. For patients treated with postoperative radiochemotherapy, a 7-gene signature was developed and successfully validated. Furthermore, it was shown that several models based on different gene signatures may be equally suitable for patient stratification. A method is presented that combines those distinct prognostic models. In addition, gene-expression-based biomarkers were transferred between different gene expressions measurement methods with the result that signatures showed less variability in patient stratification than single-gene biomarkers.:Abbreviations VII Figures IX Tables XII 1 Introduction 1 2 Biological & Statistical Background 4 2.1 Head and Neck Squamous Cell Carcinoma 4 2.1.1 Tumorigenesis 4 2.1.2 Biomarkers 8 2.2 Statistics 14 2.2.1 Survival analysis 14 2.2.2 Model and data evaluation 18 2.2.3 Data sampling methods 22 2.3 Machine learning algorithms 23 2.3.1 Feature selection algorithms 24 2.3.2 Prognostic models 27 2.4 Gene expression measurement methods 30 2.4.1 Real-time polymerase chain reaction (RT-PCR) 31 2.4.2 nCounter® gene expression 32 2.4.3 In situ-synthesized oligonucleotide microarrays 32 3 Material and methods 35 3.1 Patient cohorts 35 3.1.1 Primary radiochemotherapy (pRCTx) cohorts 35 3.1.2 Postoperative radio(chemo)therapy (PORT-C) cohorts 36 3.1.3 Clinical endpoints 38 3.2 Gene expression analyses 39 3.2.1 HPV status 39 3.2.2 Immunohistochemical staining 39 3.2.3 RT-PCR measurements 40 3.2.4 nCounter® measurements 40 3.2.5 GeneChip® analyses (only training cohorts) 41 3.3 Machine learning framework 41 3.3.1 Pre-processing of gene expression data 41 3.3.2 Determination of the ensemble gene signature 42 3.3.3 Expanding the ensemble signature by highly correlated genes 43 3.3.4 Independent validation and patient stratification 45 4 Identification of gene expression signatures as prognostic biomarkers 46 4.1 Hypoxia classification 46 4.2 nCounter® gene expression based signatures 50 4.2.1 Patients treated with primary radiochemotherapy 50 4.2.2 Clinical Features 55 4.2.3 Signature extension using clinical features 64 4.2.4 Patients treated with postoperative radiochemotherapy 65 4.2.5 Signature extension using clinical features – Port-C 72 4.3 GeneChip® gene expression-based signatures 78 4.3.1 Pre-selection 78 4.3.2 Patients treated with primary radiochemotherapy 79 4.3.3 Patients treated with postoperative radiochemotherapy 87 4.4 Combined models for PORT-C 91 4.4.1 Creation of a consensus model 92 4.4.2 Consensus model based on 2 models 93 4.4.3 Consensus model based on more than 2 models 97 4.4.4 Discussion and summary of model combination 101 5 Stability of gene expression-based biomarkers 102 5.1 Reproducibility depending on time of nCounter® 102 5.2 Comparison of nCounter® and GeneChip® gene expression 106 5.2.1 Introduction 106 5.2.2 Correlation analyses 106 5.2.3 Model and biomarker transfer 108 6 Conclusion and outlook 123 Zusammenfassung 125 Summary 128 Appendix 130 A. Supplementary Figures 130 B. Supplementary Tables 133 Bibliography 147 Acknowledgements 188 Erklärungen 189

博士
國立成功大學
生物科技與產業科學系
107
Most head and neck cancers are squamous cell types, also called head and neck squamous cell carcinoma (HNSCC). About 75% of head and neck cancer is caused by the use of alcohol or tobacco. Other risk factors such as betel quid, some types of human papillomavirus, radiation exposure, specific workplace exposures, and the Epstein-Barr virus. Early period HNSCC is treated comparatively well with a single-modality therapy (either surgery or radiation alone). However, almost 66% of patients current with advanced sickness, and less than 30% of these patients can be cured. The treatment of advanced HNSCC frequently requires multimodality therapy and involves significant toxicity. HNSCC can spread and metastasize to other parts of the body, for example the lymph nodes or lungs. Metastasis is an complex sequential process that demands a discrete population of tumor cells to have the capacity to intravasate from the original tumor into systemic circulation, exist and survive in circulation, extravasate at a distant site, growth spurt and proliferate in a foreign hostile environment. Once HNSCC spreads, patients with metastatic disease have very poor prognosis with a survival rate of fewer than a year. Metastasis remains a main cause of decease in patients with HNSCC. There are many molecular markers that could be linchpin players in important procedures of HNSCC progression, including EGF, EGFR, BCL-2, BAX, JUN, VEGFA, MMP2 and MMP9. Most notably, epidermal growth factor receptor (EGFR), also known as ErbB1/HER1, is a member of the ErbB family of receptor tyrosine kinases. EGFR is overexpressed in several epithelial malignancies, including HNSCC, which exhibits EGFR overexpression in up to 90% of tumors. EGFR act a pivotal role in HNSCC growth, invasion, metastasis, and angiogenesis. EGFR ligands such as epidermal growth factor (EGF) play a major role in the malignant transformation of HNSCC. EGFR contributes to a metastatic HNSCC tumor cell phenotype through regulation of invasion, migration, and anoikis. ANGPTL4 (Angiopoietin Like Protein 4), which is also called (ARP4, FIAF, HFARP, NL2, PGAR, pp1158) is a protein coding gene. It is a secreted protein with a coiled-coil Nterminal domain and a fibrinogen-like C-terminal domain. The ANGPTL4 protein belongs to a superfamily of secreted proteins structurally related to factors modulating angiogenesis known as angiopoietins that may play as a regulator of angiogenesis and modulate tumorigenesis. Initially, ANGPTL4 was evaluated as an adipokine exclusive involved in lipid metabolism because of its widespread expression in liver and adipose tissue. ANGPTL4 is an endogenous inhibitor of lipoprotein lipase that modulates lipid standards, coronary atherosclerosis risk, and nutrient partitioning. Hypoxia-induced ANGPTL4 protein expression in endothelial cells. Studies show that ANGPTL4 mediates the cross talk between metabolic syndromes, for example diabetes, obesity, and cancer, through regulation of its expression by PPARs, and studies have also demonstrated that ANGPTL4 may be involved in cancer progression and metastasis. For example, ANGPTL4 boosts the malice phenotype of primary melanomas with risky of metastasizing to the brain, and it could be also promoted metastsis and curb the apoptosis of colorectal cancer cells. The induction of ANGPTL4 by the cytokine TGFβ via the Smad signaling pathway in the breast tumor microenvironment trigger cancer cells for metastasis to the lungs. HNSCC is an invasive life-threatening disease related to high mortality rates. Efforts have been made to probe the molecular mechanisms that promoted the initiation and progression of HNSCC. However, the functional role and regulatory mechanism of ANGPTL4 in the development of EGF-enhanced HNSCC metastasis is still unclear. We confirm that EGF stimulates the protein secretion of ANGPTL4 in HNSCC. In this study, we demonstrate that ANGPTL4 expression enhances EGF-promoted head and neck cancer cell anoikis resistance. In addition to EGF-, ANGPTL4 enhances HNSCC malignant behavior including migration, invasion and anoikis resistance. Furthermore, we demonstrated that ANGPTL4 expression and secretion by EGF is involved in EGF-promoted HNSCC migration, invasion, and transendothelial invasion. ANGPTL4 alone can also influence HNSCC migration, invasion, and transendothelial invasion. In addition, EGF-stimulated anoikis-resistant tumor cell interaction with endothelial cells is significantly repressed by abolishing the expression of ANGPTL4. Down-regulation of ANGPTL4 has been shown to block EGF-induced protein expression and enzyme activation of matrix metalloproteinases-1 (MMP-1) involved in head and neck cancer cell migration, invasion, and metastasis. Furthermore, ANGPTL4 activates the expression of c-Jun affect activator protein-1 (AP-1) binding to the MMP-1 promoter via the integrin β1 (ITGβ1) signal pathway. The c-Jun signaling pathway is essential for ANGPTL4-regulated expression of MMP-1 and is involved in the EGF/ANGPTL4/MMP-1 axis in HNSCC. EGF-induced ANGPTL4 autocrine production stimulates tumor-endothelial cell interactions, which then facilitates the ability of tumor cells to infiltrate into blood vessels. The repression of ANGPTL4 significantly suppresses EGF-stimulated HNSCC both in terms of extravasation and metastatic seeding into the lungs. All in all, these results indicate that ANGPTL4 facilitates EGF-promoted cancer metastasis via increasing the expression of MMP-1 in HNSCC. Both the protein secretion and autocrine production of ANGPTL4 are important markers in epidermal growth factor receptor (EGFR)-mediated HNSCC metastasis.

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.

Thesis (Ph.D.)--State University of New York at Buffalo, 2005.
Title from PDF title page (viewed on Dec. 6, 2005) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Richard Mazurchuk, Youcef M. Rustum. Includes bibliographical references.

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.

It is estimated that cancer will cause 9.6 million deaths and 18.1 million new patients diagnosed during 2018. Within this number, over 350 000 have oral tumors with tobacco and alcohol consumption identified as the biggest risk factors. Cancer research is still missing a comprehensive model that mimics human cancer as a whole. Therefore, the aim of this study was to analyse an experimental model that accurately mimics human cancer. To this end we treated mice with the carcinogen 4-nitroquinoline-1-oxide in the drinking water for over 16 weeks. This allowed us to induce differently graded tumors in mice tongue oral cavity. We performed whole-exome sequencing of the tumors and the analysis confirmed similarities with human oral cancer genomic landscape. This study allowed us to gain new insight on the genomic progression of oral cancer and to explore an animal model that mimics not only the histological changes but also the genetic alterations observed in human oral cancer. Previous work has shown that knockout mice for keratin 76 are more susceptible to develop oral cancer due to increased and over-suppressive regulatory T cells in the absence of keratin 76. However, the link between the loss of keratin 76 and these changes in the immune system remains unknown. Keratin 76 is progressively more expressed in mice thymus with aging and there is a parallel with the Hassall’s corpuscles in human thymus. We showed that mice lacking keratin 76 present bigger thymic medullary regions and hypothesise one of the targets in the thymus to be Aire since its expression is reduced in the knockout. This study suggested an important role for keratin 76 in regulating the immune system.