Nuevas terapias dirigidas para el tratamiento del cáncer

El cáncer es el término que se utiliza para englobar un conjunto de enfermedades que se caracterizan por el crecimiento descontrolado de células alteradas molecularmente por mutaciones o modificaciones epigenéticas.En la presente revisión describimos algunas terapias dirigidas que se están utilizando actualmente en clínic

Molecular Mechanisms Involved in Inflammatory Bowel Disease and Colorectal Cancer: Novel Therapeutic Strategies

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Abstract] Chapter One Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gastrointestinal tract and represents an important risk factor for the onset of colorectal cancer (CRC). Hakai is an E3 ubiquitin-ligase involved in tumour progression and metastasis of carcinomas through the degradation of E-cadherin. The loss of E-cadherin is the hallmark of EMT, a key process at early stages of carcinoma progression. In this work we investigated the expression of Hakai in all the different stages of CRC as well as its potential role in IBD. Our results show that Hakai is a potential biomarker for CRC progression and its expression is increased even at early TNM-stages of tumorigenesis. Interactome analysis of Hakai in CRC cells allowed us to identify a novel Hakai-interacting protein named FASN, a protein reported to be associated to IBD. We described that Hakai increases FASN ubiquitination and degradation, resulting in the regulation of FASN-mediated lipid accumulation. Moreover, we showed that Hakai is downregulated in inflammatory tissues by using different IBD mice models and that IFN-γ downregulates Hakai expression in CRC cells. Our findings open a new research field to better understand the mechanisms involved in intestinal inflammation and cancer bowel disease. Chapter Two CRC is one of leading causes of mortality in western countries and new strategies are required for its treatment. Natural products comprise a vast diversity of biologically active substances with high therapeutic potential including compounds widely used in anticancer chemotherapy. Polysaccharide-rich extracts from fungal origin, for example, have proven their antitumoral effects in different previous studies. In the present study, we aim to evaluate the possible antitumor effect on CRC cells of a series of fungal polysaccharide-rich extracts, obtained from autochthonous fungi of Galicia. We identified two extracts from Trametes versicolor (TV) and Grifola frondosa (GF) which exhibited inhibition of human colon cancer cell proliferation and induced cytotoxicity. Furthermore, both fungal extracts significantly inhibited oncogenic potential, cell migration and invasion in CRC cells as well as increased E-Cadherin expression and reduced metalloproteinase activity. Finally, our results also showed that the combination of the extracts with one the most clinical used agents for colorectal cancer, 5-fluorouracil, increases cell cytotoxicity, opening the door to further research for the use of fungal extracts as adjuvant treatment in cancer.[Resumen] Capítulo uno La enfermedad inflamatoria intestinal (EII) se caracteriza por una inflamación crónica del tracto gastrointestinal y representa un factor de riesgo importante para el desarrollo del cáncer colorrectal (CCR). Hakai es una E3 ubiquitina-ligasa involucrada en la progresión tumoral y metástasis de carcinomas a través de la degradación de E-cadherina. La pérdida de E-cadherina es el sello distintivo de la EMT, un proceso clave en las etapas más tempranas de la progresión de los carcinomas. En este trabajo se investiga la expresión de Hakai en todos los diferentes estadios del CCR, así como su potencial papel en la EII. Los resultados muestran que Hakai es un potencial biomarcador de la progresión del CCR con una expresión aumentada incluso en las primeras etapas de la carcinogénesis. El análisis del interactoma de Hakai en células de CCR permitió identificar una nueva proteína que interactúa con Hakai, FASN, una proteína asociada con el desarrollo de la EII. En este trabajo se describe que Hakai aumenta la ubiquitinación y degradación de FASN, ocasionado la regulación de la acumulación de lípidos mediada por FASN. Además, se demuestra que Hakai está regulado negativamente en los tejidos inflamatorios de diferentes modelos animales de EII y que IFN-γ regula negativamente la expresión de Hakai en células CCR. Nuestros hallazgos abren un nuevo campo de investigación para comprender mejor los mecanismos involucrados en la inflamación y el cáncer intestinales. Capitulo dos El CCR es una de las principales causas de mortalidad en los países occidentales y se requieren nuevas estrategias para su tratamiento. Los productos naturales comprenden una gran diversidad de sustancias biológicamente activas con un alto potencial terapéutico, incluidos compuestos ampliamente utilizados en la quimioterapia contra el cáncer. Los extractos ricos en polisacáridos de origen fúngico, por ejemplo, han demostrado sus efectos antitumorales en diferentes estudios previos. En el presente trabajo pretendemos evaluar el posible efecto antitumoral sobre las células de CCR de una serie de extractos fúngicos ricos en polisacáridos, obtenidos de hongos autóctonos de Galicia. Se identificaron dos extractos de Trametes versicolor (TV) y Grifola frondosa (GF) con capacidad citotóxica y de inhibición de la proliferación de células de CCR. Además, ambos extractos fueron capaces de inhibir significativamente el potencial oncogénico, la migración celular y la invasión en las células CRC, así como elevar los niveles de expresión de E-Cadherina y reducir la actividad de las metaloproteinasas. Finalmente, nuestros resultados también muestran que la combinación de los extractos con uno de los agentes más utilizados en la clínica para el cáncer colorrectal, el 5-fluorouracilo, aumenta la citotoxicidad celular, abriendo la puerta a nuevas investigaciones para el uso de extractos de hongos como tratamiento adyuvante en el cáncer.[Resumo] Capítulo Un A enfermidade inflamatoria intestinal (EII) caracterízase pola inflamación crónica do tracto gastrointestinal e representa un importante factor de risco para o desenvolvemento do cancro colorrectal (CRC). Hakai é unha E3 ubiquitina-ligasa implicada na progresión tumoral e metástase de carcinomas a través da degradación da E-cadherina. A perda de E-cadherina é o selo distintivo de TMS, un proceso clave nas primeiras fases da progresión dos carcinomas. Neste traballo, investígase a expresión de Hakai en todas as diferentes etapas do CRC, así como o seu potencial papel na EII. Os resultados mostran que Hakai é un biomarcador potencial da progresión do CRC cunha expresión aumentada incluso nas fases iniciais da carcinoxénese. A análise do interactoma de Hakai nas células CRC identificou unha nova proteína que interactúa con Hakai, FASN, unha proteína asociada ao desenvolvemento da EII. Neste traballo, descríbese que Hakai aumenta a ubiquitinación e degradación de FASN, provocando a regulación da acumulación de lípidos mediada por FASN. Ademais, móstrase que Hakai está regulado negativamente nos tecidos inflamatorios de diferentes modelos animais de IBD e que o IFN-γ regula negativamente a expresión de Hakai nas células CCR. Os nosos descubrimentos abren un novo campo de investigación para comprender mellor os mecanismos implicados na inflamación intestinal e no cancro. Capítulo dous O CRC é unha das principais causas de mortalidade nos países occidentais e son necesarias novas estratexias para o seu tratamento. Os produtos naturais comprenden unha gran variedade de substancias biolóxicamente activas con alto potencial terapéutico, incluídos compostos moi utilizados na quimioterapia contra o cancro. Extractos ricos en polisacáridos de orixe fúngica, por exemplo, demostraron os seus efectos antitumorais en diferentes estudos anteriores. No presente traballo pretendemos avaliar o posible efecto antitumoral sobre as células CRC dunha serie de extractos de fungos ricos en polisacáridos, obtidos a partir de fungos autóctonos de Galicia. Identificáronse dous extractos de Trametes versicolor (TV) e Grifola frondosa (GF) con capacidade citotóxica e de inhibición da proliferación de células CRC. Ademais, ambos extractos foron capaces de inhibir significativamente o potencial oncoxénico, a migración celular e a invasión das células CRC, así como aumentar os niveis de expresión da E-Cadherina e reducir a actividade das metaloproteinasas. Por último, os nosos resultados tamén mostran que a combinación dos extractos cun dos axentes máis empregados na clínica para o cancro colorrectal, o 5-fluorouracilo, aumenta a citotoxicidade celular, abrindo a porta a novas investigacións sobre o uso de extractos de fungos como tratamento adxuntivo no cancro

Regulation of Epithelial–Mesenchymal Plasticity by the E3 Ubiquitin-Ligases in Cancer

The epithelial–mesenchymal plasticity (EMP) is a process by which epithelial cells acquire the ability to dynamically switch between epithelial and mesenchymal phenotypic cellular states. Epithelial cell plasticity in the context of an epithelial-to-mesenchymal transition (EMT) confers increased cell motility, invasiveness and the ability to disseminate to distant sites and form metastasis. The modulation of molecularly defined targets involved in this process has become an attractive therapeutic strategy against cancer. Protein degradation carried out by ubiquitination has gained attention as it can selectively degrade proteins of interest. In the ubiquitination reaction, the E3 ubiquitin-ligases are responsible for the specific binding of ubiquitin to a small subset of target proteins, and are considered promising anticancer drug targets. In this review, we summarize the role of the E3 ubiquitin-ligases that control targeted protein degradation in cancer-EMT, and we highlight the potential use of the E3 ubiquitin-ligases as drug targets for the development of small-molecule drugs against cancer

Regulation of Epithelial-Mesenchymal Plasticity by the E3 Ubiquitin-Ligases in Cancer

The epithelial-mesenchymal plasticity (EMP) is a process by which epithelial cells acquire the ability to dynamically switch between epithelial and mesenchymal phenotypic cellular states. Epithelial cell plasticity in the context of an epithelial-to-mesenchymal transition (EMT) confers increased cell motility, invasiveness and the ability to disseminate to distant sites and form metastasis. The modulation of molecularly defined targets involved in this process has become an attractive therapeutic strategy against cancer. Protein degradation carried out by ubiquitination has gained attention as it can selectively degrade proteins of interest. In the ubiquitination reaction, the E3 ubiquitin-ligases are responsible for the specific binding of ubiquitin to a small subset of target proteins, and are considered promising anticancer drug targets. In this review, we summarize the role of the E3 ubiquitin-ligases that control targeted protein degradation in cancer-EMT, and we highlight the potential use of the E3 ubiquitin-ligases as drug targets for the development of small-molecule drugs against cancer

Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression

Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90

Hakin-1, a New Specific Small-Molecule Inhibitor for the E3 Ubiquitin-Ligase Hakai, Inhibits Carcinoma Growth and Progression

The requirement of the E3 ubiquitin-ligase Hakai for the ubiquitination and subsequent degradation of E-cadherin has been associated with enhanced epithelial-to-mesenchymal transition (EMT), tumour progression and carcinoma metastasis. To date, most of the reported EMT-related inhibitors were not developed for anti-EMT purposes, but indirectly affect EMT. On the other hand, E3 ubiquitin-ligase enzymes have recently emerged as promising therapeutic targets, as their specific inhibition would prevent wider side effects. Given this background, a virtual screening was performed to identify novel specific inhibitors of Hakai, targeted against its phosphotyrosine-binding pocket, where phosphorylated-E-cadherin specifically binds. We selected a candidate inhibitor, Hakin-1, which showed an important effect on Hakai-induced ubiquitination. Hakin-1 also inhibited carcinoma growth and tumour progression both in vitro, in colorectal cancer cell lines, and in vivo, in a tumour xenograft mouse model, without apparent systemic toxicity in mice. Our results show for the first time that a small molecule putatively targeting the E3 ubiquitin-ligase Hakai inhibits Hakai-dependent ubiquitination of E-cadherin, having an impact on the EMT process. This represents an important step forward in a future development of an effective therapeutic drug to prevent or inhibit carcinoma tumour progression