Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.

Abstract Traditional clinicopathological factors are not accurate enough to predict the behavior of head and neck squamous cell carcinoma (HNSCC). The most powerful indicator of prognosis is the stage of the disease. New prognostic markers have, however, been searched for in order to better identify patient groups in need of different treatments or follow-up. Gelatinases (MMP-2, -9) are endopeptidases associated with tumor invasion and angiogenesis, and their tissue inhibitors (TIMP-1, -2) are also linked to cancer cell invasion and metastasis formation. In some cancer types they are even prognostic and relate with a more aggressive clinical course of the disease. In the present work the expression and the clinical significance of tumor tissue and circulating immunoreactive proteins for MMP-2, -9, TIMP-1 and -2 were assessed in HNSCC. The study group included 74 patients with HNSCC and 44 healthy controls. The expression of immunoreactive proteins was examined in paraffin-embedded tumor sections by immunohistochemical staining using specific antibodies, and the pretreatment serum levels of those proteins were quantitatively measured by ELISA assay. Immunohistochemical overexpression of MMP-9 in tumor was for the first time found to predict the prognosis for shortened survival in HNSCC, the cause-specific survival rates being 45% and 92% and relapse-free survival being 42% and 79% in MMP-9 positive or negative cases, respectively. Additionally, tissue TIMP-1, MMP-2 and TIMP-2 positivity were all also linked with poorer survival of patients with HNSCC. However, these differences remained less distinct than with MMP-9. The expression of gelatinases and their inhibitors in tumor tissue was also an indicator for later lymph node or hematogenic relapses in HNSCC patients. Circulating MMP-9 and TIMP-1 levels were significantly higher in HNSCC patients than in healthy controls. Further, the cause-specific and relapse-free survival rates were lower among HNSCC patients with high MMP-9 and TIMP-1 serum levels compared to patients with low levels of circulating MMP-9 and TIMP-1. A significant correlation was shown between circulating MMP-9 and MMP-9 immunohistochemical staining in the corresponding tumors. No correlation was found between tissue or circulating levels of gelatinases or their inhibitors and the traditional clinical or histopathological factors, except for the association between tissue and circulating TIMP-1 and the size of the primary tumor. Taken together, these results suggest that tissue expression of gelatinases and their inhibitors as well as pretreatment circulating MMP-9 and TIMP-1 levels could be prognostic in estimation of the clinical course of HNSCC. The results indicate further studies are needed with larger patient materials.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer 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.

Cancer is a major cause of morbidity and mortality with increasing incidence worldwide. Early detection of cancers and better treatments would improve the outcome for patients. The overall 5-year survival rates of head and neck squamous cell carcinoma have not improved in the past several decades due to diagnosis at advanced stages and recurrent disease. Early detection and improved chemotherapy drugs are two key areas that are required to help to improve the prognosis for this disease. This thesis focuses on the enzyme telomerase which is known to contribute to one of the hallmarks of cancer (immortality). Elevated telomerase activity has been observed in the majority of cancer cells but not in most normal human cells so there is an opportunity to use telomerase as a biomarker for disease. This first part of this study assessed telomerase activity in saliva and tissues of head and neck squamous cell cancer patients. The Telomerase PCR-ELISA kit was used to assess telomerase activity in the saliva of patients with confirmed oral carcinomas and its expression was analysed in paraffin embedded tissue using immunohistochemistry (IHC). Whilst telomerase was detected in cell lines, no telomerase activity was detected in saliva samples from patients but was detectable in IHC specimens. The second part of the study focused on the pharmacological evaluation of a series of small molecule G–quadruplex DNA binding agents as potential telomerase inhibitors. A total of 19 telomerase inhibitors were identified but of these, only 4 were specific inhibitors of telomerase. These compounds also caused toxicity to cell lines following a 2 hour drug exposure at doses that also inhibit telomerase activity. Further studies are required to explore these compounds further. In conclusion, the results of this study have demonstrated that detection of telomerase activity I the saliva of patients with oral cancers is unlikely to be useful in terms of detecting oral cancers before symptoms of the disease are clinically manifest. A series of novel and specific inhibitors of telomerase have been identified and further studies are required to develop these compounds further.

Oncolytic viruses can be genetically modified to limit their replication in normal cells rendering them a cancer specific treatment. In addition, they can induce a 'danger signal' in the form of pathogen- and damage-associated molecular patterns leading to anti-tumour immunity. Furthermore, they can be armed with various immunomodulatory molecules to further enhance anti-tumour immunity. In this project I aim to exploit these qualities to develop a translatable cancer vaccine. Virus-infected cancer cells were injected subcutaneously in a prime/boost regimen. Dying cancer cells will release the required danger signal leading to dendritic cell activation and cross-presentation of tumour associated antigens to T cells to elicit an anti-tumour immune response. Our results in the murine pancreatic cancer model showed that vaccination with virusinfected DT6606 cells induced tumour specific immunity capable of protecting vaccinated animals against re-challenge with tumour cells. The highest level of interferon gamma production, a surrogate marker of anti-tumour immunity, was achieved when animals were primed with adenovirus-infected cells. There was no significant difference between various boost groups. To enhance the safety of the proposed protocol a secondary treatment was introduced to arrest the proliferation of tumour cells prior to injection. Our results confirmed that secondary treatment with mitomycin does not affect the induction of tumour specific immunity and it does not affect the release of pathogen-associated molecular patterns in the form of viral proteins and DNA. To test our vaccination regimen in head and neck squamous cell carcinoma (HNSCC) we develop a clinically relevant mouse model using SCC7, B4B8 and LY2 cells to replicate various clinical scenarios including locally advancing disease and post excision locoregional recurrence. Vaccinating mice with HNSCC cells pre-infected with our recently developed tumour-targeted triple-deleted adenovirus (AdTD) resulted in a cell-specific antitumour immune response. In addition, it resulted in an increase in effector memory T-cells of both CD4+ and CD8+ phenotypes. Efficacy studies showed our vaccination can significantly slow down the growth rate of tumours in locally advancing disease. This led to increase survival of the vaccinated mice although it did not reach statistical significance. To further enhance the efficacy of our vaccination regimen, we aimed to increase T cell trafficking to the tumour site. CCL25 is a gut homing chemokine. Priming T cells in the presence of CCL25 will lead to upregulation of the surface expression of α4β7 integrin. The latter is a ligand of MAdCAM-1, a cell adhesion molecule highly expressed in the gut and pancreatic tumours. The α4β7/MAdCAM-1 interaction results in preferential homing of activated T cells to these organs. We hypothesised that vaccinating mice with pancreatic tumour cells pre-infected with a CCL25-armed adenovirus will lead to increased T cell trafficking to pancreatic tumours leading to enhanced efficacy. Although we achieved encouraging results in our pilot experiment, we did not detect any significant increase in α4β7 expression once we added a secondary treatment to the vaccination protocol. Similarly, efficacy experiments in the pancreatic cancer transgenic KPC mice did not show any difference in survival between AdTD-CCL25 and the control virus although both groups showed a trend towards increased survival compared to naïve mice. In conclusion, Virus-infected cancer cell vaccine is a potentially promising immunotherapeutic strategy that can be combined with traditional cancer therapies to increase survival of HNSCC and pancreatic cancer patients.

HOXA7 belongs to a family of homeobox transcription factors that are master regulators of cell differentiation, morphogenesis during embryonic development and cell proliferation. Dysregulation and non-nuclear localization of these proteins play a role in a large number of solid tumours, with reports of significant upregulation of HOXA7 in oral dysplasia. It is unclear whether HOXA7 induction in solid tumours is causative or if it is a result of oncogenic changes. In this thesis we studied its effect on cell differentiation, growth, stemness, cell migration, EMT and cell senescence. The main hypothesis was that HOXA7 regulated keratinocyte differentiation through the regulation of activator protein 1 (AP-1), a keratinocyte specific activator of differentiation. We also hypothesised that HOXA7 increased the proliferation rate in keratinocytes. In an AP-1 reporter assay in HEK293 cells, HOXA7 was shown to decrease AP-1 activity significantly. The inactivation of AP-1 was not due to inactivation of PKC, as HOXA7 did not interfere with the activation of the kinases in HEK293. More specifically, we reported a very significant repression of c-Jun and JunD promoter activity in the presence of ectopic HOXA7 in HEK293 cells. We further showed that this mechanism might also be applicable in keratinocytes, as HOXA7 inhibited the transcription of AP-1 subunits of both the Jun and Fos family in skin keratinocytes. Furthermore, we showed transcriptional repression of four differentiation markers and a downregulation of K1 and FLG protein in transduced NEB-1 monolayers as well as K1 suppression in HaCaT cells. The organotypic cultures revealed a downregulation of K1, K10, and filaggrin in stratified HaCaT cells by HOXA7. There was however no downregulation in oral keratinocytes. These observations taken together suggested that HOXA7 repressed the synthesis of AP-1 units in skin keratinocytes, which would have resulted in reduced quantities of AP-1 and therefore lower activity. Contrary to previous reports, we observed no positive involvement of HOXA7 in keratinocyte proliferation, EMT or migration. There was however an indication of cell-type specific MET and induced cell senescence. Based on our results we propose a cell-type specific role of HOXA7 as an antagonist of AP-1 transcription in skin keratinocytes, and a possible direct binding of HOXA7 to c-Jun and JunD promoters.

Os carcinomas de cabeça e pescoço (CECPs) compreendem um grupo de tumores que atingem vários sítios do trato aerodigestivo superior, incluindo cavidade oral, orofaringe, hipofaringe e laringe. Esses carcinomas são clinicamente heterogeneous e resultam de modificações cumulativas em genes que regulam proliferação, migração celular e apoptose. São estimados aproximadamente 500.000 novos casos de CECP anualmente no mundo. No Brasil, cerca de 14.000 novos casos são esperados em 2010, somente para cavidade oral. As taxas de morbidade e mortalidade e as limitações das estratégias terapêuticas enfatizam a necessidade de um melhor entendimento dos padrões moleculares envolvidos na iniciação e na progressão desses tumores, e de abordagens preventivas e terapêuticas efetivas. Infelizmente, apesar da intensa pesquisa nessa área, poucos marcadores moleculares são conhecidos que exibam sensibilidade e especificidade para diagnóstico e prognóstico de CECP. Em um estudo prévio, nós avaliamos dados de três bibliotecas SAGE de carcinoma de laringe com a finalidade de identificar eventos associados ao desenvolvimento e à agressividade de CECP. Utilizando abordagens estatísticas e de Bioinformática, nós identificamos 60 genes com expressão elevada ou reduzida em tumores metastáticos versus não-metastáticos e em ambos os grupos versus tecidos normais. O objetivo do presente estudo foi avaliar a expressão de quatro genes desta lista, os das queratinas 6A (KRT6A) e 19 (KRT19), da mesotelina (MSLN) e da calicreína 8 (KLK8), em um conjunto de 63 carcinomas primários de cabeça e pescoço e suas margens cirúrgicas e em quarto linhagens celulares (Hep-2, FaDu, SCC9 e UM-SSC-38) por RT-PCR em tempo real. Como amostra de referência para as linhagens, foram utilizados queratinócitos orais humanos normais, cultivados sobre uma camada de sustentação de fibroblastos irradiados. Todos os genes exibiram níveis de transcritos reduzidos ou ausentes nas linhagens celulares, exceto MSLN, que mostrou um padrão irregular de expressão. Em tumores primários, os genes KRT19 e MSLN apresentaram expressão diminuída em laringe, o mesmo sendo observado para o gene KLK8 em tumores de língua metastático. Além disso, foi detectada expressão elevada de MSLN e KLK8 em tumores não metastáticos de soalho de boca e expressão reduzida de KRT19 em tumores de soalho de boca e língua metastáticos. Os resultados levantam questões sobre o papel desses genes em processos biológicos associados com a tumorigênese de cabeça e pescoço e sobre sua participação no fenótipo neoplásico. / Head and neck squamous cell carcinomas (HNSCCs) encompass a group of tumors that affect a variety of sites in the upper aero-digestive tract, including oral cavity, oropharynx, hypopharynx and larynx. These carcinomas are clinically heterogeneous and result from cumulative changes in genes that regulate cell proliferation, migration and death. It is estimated that approximately 500,000 new cases of HNSCC are diagnosed worldwide each year. In Brazil, about 14,000 new cases are expected in the year 2010, only in oral cavity. The morbidity and mortality rates and the limitations of therapeutic strategies emphasize the need for a better understanding of the molecular pathways involved in the initiation and progression of these tumors and for effective preventive and therapeutic approaches. Unfortunately, despite intense research, few molecular markers are known to exhibit sensitivity and specificity for the diagnosis or prognosis of HNSCC. In a previous study, we evaluated data from three SAGE libraries of larynx carcinoma in order to identify events associated with the development and aggressiveness of HNSCCs. Using statistical and bioinformatic tools, we identified sixty top-up and 60 top-downregulated genes in metastatic versus non-metastatic tumors and in both these tumors versus normal tissues. The objective of the present study was to evaluate the expression of four genes from this list, keratin 6A (KRT6A), keratin 19 (KRT19), mesothelin (MSLN) and kallikrein 8 (KLK8), in a set of 63 primary carcinomas of head and neck and their surgical margins and in four cell lines (Hep-2, FaDu, SCC9 and UM-SSC-38) by real time RT-PCR. As a reference sample for cell lines, we used normal human oral keratinocytes grown on irradiated fibroblast feeder layer. All genes exhibited no or decreased levels of transcripts in the cell lines, except MSLN, which displayed an irregular pattern of expression. In primary tumors, KRT19 and MSLN genes were downregulated in larynx, and KLK8 in metastatic tongue tumors. In addition, MSLN and KLK8 were upregulated in non-metastatic floor of the mouth tumors and KRT19 was down regulated in metastatic floor of the mouth and tongue tumors. The results open questions about the role of these genes on biological processes related to head and neck tumorigenesis and on neoplastic phenotype.

Title: Inhibition of interleukin-6 trans-signaling by sgp130Fc is anti-tumorigenic in head and neck squamous cell carcinoma. Background: Head and neck squamous cell carcinoma (HNSCC) is a highly inflammatory cancer type, and interleukin-6 (IL-6) is associated with this phenotype. Elevated expression of IL-6 is linked to tumor progression, recurrence, metastasis, and resistance to therapy in HNSCC. However, targeting IL-6 or IL-6 receptor (IL-6R) has demonstrated little to no clinical efficacy. IL-6 signals through a classical signaling pathway via membrane IL-6R or a trans-signaling pathway via soluble IL-6R (sIL-6R). Recent evidence suggests that classical signaling induces acute, transient inflammation, eventually resulting in homeostasis; whereas trans-signaling may induce chronic, pro-tumorigenic inflammation. Therefore we propose that IL-6 trans-signaling is associated with the pro-inflammatory phenotype observed in HNSCC. We wanted to determine whether inhibition of IL-6 trans-signaling by sgp130Fc would better demonstrate anti-tumor efficacy and increase HNSCC tumor response to radiation, chemotherapy, and targeted therapy (cetuximab) compared to global IL-6 pathway inhibition. Method/Results: Baseline levels of IL-6, IL-6R, sIL-6R, and sgp130 proteins in HNSCC cells were determined using ELISA and flow cytometry. Cisplatin, radiation, and cetuximab treatments each induced HNSCC cell secretion of IL-6 and sIL-6R in vitro, yet adding sgp130Fc to those treatments did not further reduce clonogenic survival. Sgp130Fc treatment significantly suppressed SQ20B tumor growth in nude mice, whereas global IL-6 pathway inhibition by IL-6R antagonist tocilizumab did not; however, cetuximab reduced the efficacy of sgp130Fc in this animal model. Sgp130Fc also sensitized SQ20B xenograft tumors to radiation and chemotherapy in nude mice and suppressed SCCVII tumor growth in male but not female C3H/HeJ mice. Conclusion: Inhibition of IL-6 trans-signaling by sgp130Fc displayed significant anti-tumor effects as a single therapy and sensitized resistant HNSCC tumors to radiation and chemotherapy in vivo; however, sgp130Fc did not reduce survival of HNSCC cells in vitro. These results suggest that the efficacy of sgp130Fc relies on targeting another part of the microenvironment instead of tumor cells directly. Sgp130Fc has promise both as a single therapy and potentially as combined therapy with radiation and chemotherapy in HNSCC.

<p>Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. </p><p>In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with ²¹¹At and ¹³¹I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using ¹²⁴I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies.</p><p>These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.</p>