Squamous cell carcinoma of the head and neck (HNSCC) is an extremely common and devastating disease with a bleak prognosis. Despite intensive research, survival rates have not improved over the past 30 years principally due to untreatable recurrent/metastasising disease. Feline oral squamous cell carcinoma (FOSCC) is an equally common disease in cats with an even less favourable prognosis than humans. Human and feline squamous cell carcinomas share similar etiopathogenesis, molecular markers, tumour biology and treatment thus making FOSCC an excellent model for HNSCC. Epithelial to mesenchymal transition (EMT), under the direction microRNAs (miRNAs/mirs) could be a key driver in oncogenic transformation and chemoresistance. The aim of this study was to induce resistance to characterise the EMT/resistance phenotype and to investigate whether common miRNA-mediated pathways are present in HNSCC and FOSCC that drive this phenomenon. We used epidermal growth factor (EGFR)-inhibitor gefitinib to induce resistance in HNSCC and FOSCC and investigated the associated EMT-related molecular changes. In vitro and in vivo invasive and migratory properties of both species were explored to determine whether resistance and/or EMT status conferred a functional advantage. We determined the miRNA expression pattern during acquisition of resistance to gefitinib in both species by next generation sequencing and screened candidate miRNAs as potential therapeutics. We found that gefitinib-resistance produced a previously unrecognised biphasic response that consisted of two distinct phenotypes, a highly invasive mesenchymal phenotype during early resistance, and a more epithelial phenotype associated with established resistance. The biphasic nature of this transition may prove critical in establishing effective therapeutic targets and the timing of treatment to overcome resistance or in preventing local invasion or metastatic spread of squamous cell carcinoma. We found that the major anti-apoptotic PI3K/AKT pathway was activated in transitioning and resistant cells of both species as demonstrated upregulation of AKT, pAKT and c-FLIP together with inactivation of PTEN by phosphorylation. This indicates that avoidance of apoptosis may be a major pathway in resistance that could be targeted therapeutically. We showed that three miRNAs were differentially expressed in both gefitinib-resistant human and feline cell lines: miR-107 was downregulated, and miR-551b and miR-574 were upregulated. These microRNAs provide potential therapeutic targets in the fight against drug resistance in head and neck cancer although much further research needs to be conducted to elucidate the complex network of interactions that may be affected by targeting these powerful regulatory molecules.

Carvacrol is a phenol monoterpene found in aromatic plants specially in Lamiaceae family, which has been evaluated in an experimental model of breast cancer. However, any proposed mechanism based on its antitumor effect has not been reported. In our previous study, carvacrol showed a protective effect on 7,12-dimethylbenz[α]anthracene- (DMBA-) induced breast cancer in female rats. The main objective in this research was to evaluate by using in silico study the carvacrol on HER2, PI3Kα, mTOR, hERα, PR, and EGFR receptors involved in breast cancer progression by docking analysis, molecular dynamic, and drug-likeness evaluation. A multilevel computational study to evaluate the antitumor potential of carvacrol focusing on the main targets involved in the breast cancer was carried out. The in silico study starts with protein-ligand docking of carvacrol followed by ligand pathway calculations, molecular dynamic simulations, and molecular mechanics energies combined with the Poisson–Boltzmann (MM/PBSA) calculation of the free energy of binding for carvacrol. As result, the in silico study led to the identification of carvacrol with strong binding affinity on mTOR receptor. Additionally, in silico drug-likeness index for carvacrol showed a good predicted therapeutic profile of druggability. Our findings suggest that mTOR signaling pathway could be responsible for its preventive effect in the breast cancer. / Revisión por pares

[ES] Los inhibidores de la tirosina quinasa (TKIs) son fármacos anticancerígenos que actúan atacando selectivamente a las células cancerosas, inhibiendo su crecimiento descontrolado y, por tanto, la consiguiente aparición de tumores. Sin embargo, muchos TKIs presentan un cromóforo en su estructura que les permite interaccionar con luz solar y, consecuentemente, pueden generar reacciones de fotosensibilización. A pesar de ello, existen pocos estudios sobre el mecanismo de reacción asociado a estos eventos. En este sentido, conocer la fotorreactividad de un fármaco en medio biológico es clave para entender dichos mecanismos y correlacionar el comportamiento fotofísico del fármaco con el daño fotobiológico que genera. La espectroscopía de fluorescencia y de absorción transitoria son técnicas altamente sensibles que permiten estudiar las especies transitorias generadas tras la absorción de luz por parte de un fármaco, las cuales podrían ser las responsables de las reacciones de fotosensibilización inducidas por éste. Habiendo estudiado recientemente la foto(geno)toxicidad asociada al TKI gefitinib (GFT) y a sus principales metabolitos fenólicos (GFT-M1 y GFT-M2), se ha llevado a cabo un estudio completo de la fotorreactividad de éstos tanto en disolución orgánica, utilizando disolventes de distintas polaridades, como en medio biológico, utilizando las proteínas transportadoras de origen humano albúmina sérica (HSA) y ¿1-glicoproteína ácida (HAG). Para ello, se han utilizado las técnicas espectroscópicas de absorción, fluorescencia y absorción transitoria, entre otras. Adicionalmente, se han llevado a cabo estudios teóricos de modelización molecular para investigar la unión ligando@proteína, así como las interacciones que tienen lugar en el complejo supramolecular tras la absorción de luz UV-A. Con este trabajo, se ha conseguido establecer una buena correlación entre el comportamiento fotofísico observado para gefitinib y sus dos metabolitos fenólicos con su potencial fototóxico. / [CA] Els inhibidors de la tirosina quinasa (TKIs) són fàrmacs anticancerígens que actuen atacant selectivament a les cèl·lules canceroses, inhibint el seu creixement descontrolat i, per tant, la consegüent aparició de tumors. No obstant això, molts TKIs presenten un cromòfor en la seua estructura que els permet interaccionar amb llum solar i, conseqüentment, poden generar reaccions de fotosensibilització. Malgrat això, existixen pocs estudis sobre el mecanisme de reacció associat a estos esdeveniments. En este sentit, conéixer la fotorreactivitat d'un fàrmac al mig biològic és clau per a entendre estos mecanismes i correlacionar el comportament fotofísic del fàrmac amb el dany fotobiològic que genera. La espectroscopia de fluorescència i d'absorció transitòria són tècniques altament sensibles que permeten estudiar les espècies transitòries generades després de l'absorció de llum per part d'un fàrmac, les quals podrien ser les responsables de les reaccions de fotosensibilització induïdes per aquest. Havent estudiat recentment la foto(geno)toxicitat associada al TKI gefitinib (GFT) i d'els seus principals metabòlits fenòlics (GFT-M1 i GFT-M2), s'ha dut a terme un estudi complet de la fotorreactivitat d'estos tant en dissolució orgànica, utilitzant dissolvents de diferents polaritats, com al mig biològic, utilitzant les proteïnes transportadores d'origen humà albúmina sèrica (HSA) i ¿1-glicoproteïna àcida (HAG). Per a això, s'han utilitzat les tècniques espectroscòpiques d'absorció, fluorescència i absorció transitòria, entre altres. Addicionalment, s'han dut a terme estudis teòrics de modelització molecular per a investigar la unió lligant@proteína, així com les interaccions que tenen lloc en el complex supramolecular després de l'absorció de llum UV-A. Amb este treball, s'ha aconseguit establir una bona correlació entre el comportament fotofísic observat per a gefitinib i els seu dos metabòlits fenòlics amb el seu potencial fototòxic. / [EN] Tyrosine kinase inhibitors (TKIs) are anticancer drugs that target cancer cells, inhibiting their uncontrolled growth and thus the subsequent development of tumours. However, many TKIs contain a chromophore in their structure that allows them to interact with sunlight and, consequently, to induce photosensitivity reactions. Despite this, there are few studies on the reaction mechanism associated with these events. In this regard, a better understanding on the photoreactivity of a drug in a biological environment is key to correlate the photophysical behaviour of the drug with the photoinduced damage it can induce to biomolecules. Fluorescence and transient absorption spectroscopies are very sensitive techniques that allow the study of the transient species generated after drug excitation, which could be responsible for drug-induced photosensitivity reactions. Having recently studied the photo(geno)toxicity associated with the TKI gefitinib (GFT) and its main phenolic metabolites (GFT-M1 and GFT-M2), a complete study of their photoreactivity has been carried out both in organic medium, using solvents of different polarities, and in a biological environment, using transport proteins such as human serum albumin (HSA) and human ¿1-acid glycoprotein (HAG). For this purpose, absorption, fluorescence and transient absorption techniques, among others, have been used. In addition, molecular dynamics simulation studies have been carried out to investigate the ligand@protein binding, as well as the interactions arising in the supramolecular complex after absorption of UV-A light. Therefore, a good correlation between the photophysical behaviour of gefitinib and its two phenolic metabolites and their phototoxic potential has been established in this research work. / Tamarit Mayo, L. (2024). Fotorreactividad de gefitinib y sus metabolitos fenólicos en disolución y en proteínas transportadoras [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/204145

Little is known about the signaling pathways that contribute to treatment response in advanced stage head and neck tumors. Increased expression of epidermal growth factor receptor (EGFR) and downstream pathways such as nuclear factor kappa B (NFκB) are implicated in aggressive tumor phenotypes and limited response to therapy. This study explored the rationale for combining the proteasome inhibitor bortezomib with the EGFR inhibitor gefitinib in a subset of head and neck squamous cell carcinomas with high EGFR gene amplification. Drug responses of gefitinib and bortezomib as single agents and in combination within head and neck squamous cell carcinoma cell lines were analyzed using MTS assays. The effects of gefitinib on the activation of EGFR and itsthree major downstream pathways, Akt, STAT3 and MAPK were determined by western blotting. The activation status of NFκB and the effects of bortezomib on the canonical pathway were assessed by DNA binding assays. Resistance to lower doses of gefitinib was associated with elevated EGFR and activated Akt expression. Gefitinib was able to effectively inhibit activation of STAT3, Akt and MAPK in HNSCC to varying degrees depending on EGFR expression status. Bortezomib treatment inhibited TNFα –induced nuclear NFκB/RelA expression but demonstrated variability in levels of baseline nuclear NFκB/RelA expression between sensitive and resistant cell lines. Bortezomib effectively suppresses NFκB/RelA nuclear activation but demonstrates additional modes of cellular toxicity beyond the NFκB pathway in sensitive cell lines. Further understanding of tumor response to the targeted inhibitors gefitinib and bortezomib may provide novel approaches in managing HNSCCs.