Papel de Hakai durante la progresión tumoral: implicaciones sobre la plasticidad epitelial

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumen] El cáncer es una enfermedad que presenta una elevada tasa de mortalidad a nivel mundial, y su incidencia se está incrementando progresivamente año tras año. El carcinoma, el más común de todos los cánceres, es el resultado de una serie de transformaciones en células epiteliales. Durante esta transformación, las células alteran varias características fenotípicas incluyendo un aumento de la proliferación, y una disminución de las adhesiones célula-célula y célula-sustrato adquiriendo capacidad motil e invasiva. La E-cadherina es el miembro prototipo mejor caracterizado de las uniones adherentes y con mayor expresión en células epiteliales. La pérdida de función o expresión de E-cadherina es un marcador de un proceso denominado transición epitelio-mesénquima lo que favorece la motilidad y la capacidad invasiva y es indicador de pronóstico malo. En 2002, se identificó la proteína Hakai como una E3 ubiquitina-ligasa, cuyo sustrato específico es Ecadherina, y actúa mediando su ubiquitinización y posterior degradación, debilitando así las uniones célula-célula y favoreciendo la motilidad celular. En este contexto, por un lado, se estudió el efecto de un alcaloide la vinca, la vinflunina, fármaco comúnmente utilizado en pacientes que padecen un carcinoma de vejiga metastásico en estadio avanzado, y que no responden al tratamiento con cisplatino. La vinflunina actúa inhibiendo la dinámica de microtúbulos, y dado que recientes publicaciones ponen de manifiesto la relación entre la dinámica de microtúbulos y las uniones adherentes se analizó mediante experimentos in vitro su efecto sobre las uniones adherentes dependientes de E-cadherina, y sobre Hakai. Se observó que la vinflunina es capaz de inducir diferenciación epitelial, y fortalecer las uniones célulacélula mediadas por E-cadherina, regulando a su vez la expresión de Hakai. Por otro lado, se analizó el papel de la proteína Hakai in vitro e in vivo. Para ello, se empleó en una línea celular epitelial no transformada (MDCK) a la que se le sobreexpresó de forma estable la proteína Hakai (Hakai-MDCK), y se corroboró que Hakai induce la transición epitelio-mesénquima, incrementa la proliferación y la invasividad celular, lo que se acompaña de una disminución de los sustratos específicos de dicha proteína, E-cadherina y Cortactina. Para el estudio in vivo, se utilizaron ratones atímicos desnudos a los que se inyectaron dichas células MDCK y Hakai-MDCK, y se confirmó que Hakai induce la formación de tumores, incrementa su tasa de proliferación, y regula los marcadores de transición epitelio-mesénquima in vivo, observándose tumores muy desdiferenciados, altamente agresivos, y con micrometástasis en pulmón.[Resumo] O cancro é unha enfermidade que presenta unha elevada tasa de mortalidade a nivel mundial, e a súa incidencia estase incrementando ano tras ano. O carcinoma, o máis común de todos os cancros, é o resultado dunha serie de transformacións en células epiteliais. Durante esta transformación, as células alteran varias características fenotípicas incluindo un incremento da proliferación, e unha diminución das unións célula-célula e célula-substrato adquirindo capacidade motil e invasiva. A E-cadherina é o membro prototipo mellor caracterizado das unións adherentes e con maior expresión en células epiteliais. A perda de función ou expresión de E-cadherina é un marcador dun proceso denominado transición epitelio-mesénquima, o que favorece a motilidade e a capacidade invasiva, e é indicador de pronóstico malo. En 2002, identificouse a proteína Hakai como unha E3 ubiquitina-ligasa, cuxo substrato específico é E-cadherina, e actúa mediando a súa ubiquitinización e posterior degradación, debilitando así as unións célula-célula e favorecendo a motilidade celular. Neste contexto, por un lado, estudouse o efecto dun alcaloide da vinca, a vinflunina, fármaco comúnmente utilizado en pacientes que padecen un carcinoma de vexiga metastásico en estadío avanzado, e que non responden ao tratamento con cisplatino. A vinflunina actúa inhibindo a dinámica de microtúbulos, e dado que recentes publicacións poñen de manifesto a relación entre a dinámica de microtúbulos e as unións adherentes, analizouse mediante experimentos in vitro o seu efecto sobre as unións adherentes dependentes de Ecadherina e sobre Hakai. Observouse que a vinflunina é capaz de inducir diferenciación epitelial, e fortalecer as unións célula-célula mediadas por Ecadherina, regulando á súa vez a expresión de Hakai. Por outro lado, analizouse o papel da proteína Hakai in vitro e in vivo. Para elo, empregouse unha línea celular epitelial non transformada (MDCK) á que se lle sobreexpresou de forma estable a proteína Hakai (Hakai-MDCK), e corroborouse que Hakai induce a transición epiteliomesénquima, incrementa a proliferación e a invasividade celular, o que se acompaña dunha diminución dos sustratos específicos de dita proteína, E-cadherina e Cortactina. Para o estudo in vivo utilizáronse ratóns atímicos desnudos aos que se lles inxectaron ditas células, MDCK e Hakai-MDCK, e confirmouse que Hakai induce a formación de tumores, incrementa a súa tasa de proliferación, e regula os marcadores de transición epitelio-mesénquima in vivo, observándose tumores moi desdiferenciados, altamente agresivos, e micrometástases en pulmón.[Abstract] Cancer is a disease with a high mortality rate worldwide, and its incidence is increasing steadily. Carcinoma, the most common type of cancers, is the result of transformations in epithelial cells. During this transformation, cells acquire various phenotypic characteristics including increased cell proliferation, loss of cell-cell and cell-substrate adhesions, increasing motility and invasiveness. Ecadherin, the best characterized and prototype member of the adherens junctions, is expressed in epithelial cells. Loss of function or expression of Ecadherin protein is considered as a hallmark of epithelial-to-mesenchymal transition, favouring cell motility and invasiveness and being a poor prognosis predictor. In 2002, Hakai protein was identified as an E3 ubiquitin-ligase for Ecadherin substrate. Hakai induces E-cadherin ubiquitination and subsequent degradation, thus altering cell-cell junctions and increasing cell motility. In this context, firstly it was studied the possible role of a vinca alkaloid, vinflunine, on cell-cell contacts. Vinflunine is commonly used in patients with advanced stage metastatic bladder carcinoma that do not respond to cisplatin treatment. Vinflunine is an anti-microtubule agent, and since recent publications show a relationship between microtubule dynamics and adherens junctions, vinflunine effect on E-cadherin-dependent junctions and on Hakai was analyzed by in vitro studies. It was observed that vinflunine induces epithelial differentiation, Ecadherin dependent cell-cell contacts. Moreover, it regulates Hakai protein expression. On the other hand, the role of the Hakai protein was analyzed in vitro and in vivo. For this purpose, it was used Hakai overexpressing MDCK cells (Hakai-MDCK) and it was corroborated that Hakai induces epithelial-tomesenchymal transition, increases proliferation and cellular invasiveness, which is accompanied by a decrease in its specific substrates, E-cadherin and Cortactin. For the in vivo study, an athymic nude mouse model was used by injecting MDCK and Hakai-MDCK cells into the flanks. Hakai induces tumor formation, increases its tumour growth, and regulates the epithelial-tomesenchymal transition markers in vivo. Undifferentiated and highly aggressive tumors are formed and micrometastasis are detected in lung of Hakai-MDCK injected mice

Effect of a microtubule-targeting drug on cell–cell contacts in bladder epithelial tumour cells

Póste

Clinical implications of epithelial cell plasticity in cancer progression

Mini-review[Abstract] In the last few years, the role of epithelial cell plasticity in cancer biology research has gained increasing attention. This concept refers to the ability of the epithelial cells to dynamically switch between different phenotypic cellular states. This programme is particularly relevant during the epithelial-to-mesenchymal transition (EMT) in cancer progression. During colonization, epithelial cells first activate the EMT programme to disseminate from a primary tumour to reach a distant tissue site. During this process, cells are transported into the circulation and are able to escape the immune system of the host. Then, a reverse process called mesenchymal-to-epithelial transition (MET) occurs on cells that settle in the distant organs. Although epithelial cell plasticity has an important impact on tumour biology, the clinical relevance of this concept remains to be recapitulated. In this review, we will update the current state of epithelial cell plasticity in cancer progression and its clinical implications for the design of therapeutic strategies, the acquisition of multidrug resistance, and future perspectives for the management of cancer patients.Instituto de Salud Carlos III; PI13/00250Xunta de Galicia; 10CSA916023PRXunta de Galicia; PS09/2

Descomposición de la capa de hojarasca de diferentes especies perennifolias y caducifolias

Las hojarascas de las especies caducifolias y perennifolias presentan procesos diferenciales de descomposición, debidos, en gran parte, a las diferencias en su composición químic

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

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

Hakai overexpression effectively induces tumour progression and metastasis in vivo.

At early stages of carcinoma progression, epithelial cells undergo a program named epithelial-to-mesenchymal transition characterized by the loss of the major component of the adherens junctions, E-cadherin, which in consequence causes the disruption of cell-cell contacts. Hakai is an E3 ubiquitin-ligase that binds to E-cadherin in a phosphorylated-dependent manner and induces its degradation; thus modulating cell adhesions. Here, we show that Hakai expression is gradually increased in adenoma and in different TNM stages (I-IV) from colon adenocarcinomas compared to human colon healthy tissues. Moreover, we confirm that Hakai overexpression in epithelial cells drives transformation in cells, a mesenchymal and invasive phenotype, accompanied by the downregulation of E-cadherin and the upregulation of N-cadherin, and an increased proliferation and an oncogenic potential. More importantly, for the first time, we have studied the role of Hakai during cancer progression in vivo. We show that Hakai-transformed MDCK cells dramatically induce tumour growth and local invasion in nude mice and tumour cells exhibit a mesenchymal phenotype. Furthermore, we have detected the presence of micrometastasis in the lung mice, further confirming Hakai role during tumour metastasis in vivo. These results lead to the consideration of Hakai as a potential new therapeutic target to block tumour development and metastasis