Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

EPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

Tyrosine kinase; EPHA3; Colorectal cancerTirosina-cinasa; EPHA3; Càncer colorectalTirosina quinasa; EPHA3; Cáncer colorrectalEPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome

Mitigation of coupled wind-wave-earthquake responses of a 10 MW fixed-bottom offshore wind turbine

© 2020 Elsevier Ltd In this paper we present a study on the mitigation of dynamic responses of a 10 MW monopile offshore wind turbine under coupled wind-wave-earthquake excitations. We have developed and validated the generic seismic coupled analysis and structural control architecture tool to overcome the limitation of numerical tools when examining the wind-wave-earthquake coupling effects. We investigated the dynamic responses of a 10 MW monopile offshore wind turbine under different loading combinations and found that the earthquake loading increases the tower-top displacement and pile-cap moment by 47.6% and 95.1%, respectively, compared to the wind-wave-only condition. It is found that the earthquake-induced vibration in the fore-aft direction is mitigated by the wind and wave loadings due to the energy dissipated by the aerodynamic and hydrodynamic damping. In addition, the tower responses are dominated by the earthquake excitation. In order to alleviate the tower vibration induced by the earthquake, we implemented the structural control capability within the tool using tuned mass dampers. The tuned mass dampers with appropriately selected design parameters achieve a larger mitigation on the tower-top displacement for the earthquake-only condition compared to the coupled-loading scenario. The reason is that the tuned mass damper is only effective in mitigating tower vibration, and it is not capable of reducing the tower elastic deformation which is the major contribution of the tower displacement for the coupled-loading condition. In addition, we have found that a heavier tuned mass damper requires a lower tuned frequency to achieve a larger mitigation. A configuration for the mitigation control of the 10 MW offshore wind turbine is suggested by using a 5% mass ratio of the tuned mass damper

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

[EN] Background & Aims: Cholangiocarcinoma (CCA) is a neoplasia of the biliary tract driven by genetic, epigenetic and transcriptional mechanisms. Herein, we investigated the role of the transcription factor FOSL1, as well as its downstream transcriptional effectors, in the development and progression of CCA. Methods: FOSL1 was investigated in human CCA clinical samples. Genetic inhibition of FOSL1 in human and mouse CCA cell lines was performed in in vitro and in vivo models using constitutive and inducible short-hairpin RNAs. Conditional FOSL1 ablation was done using a genetically engineered mouse (GEM) model of CCA (mutant KRAS and Trp53 knockout). Followup RNA and chromatin immunoprecipitation (ChIP) sequencing analyses were carried out and downstream targets were validated using genetic and pharmacological inhibition. Results: An inter-species analysis of FOSL1 in CCA was conducted. First, FOSL1 was found to be highly upregulated in human and mouse CCA, and associated with poor patient survival. Pharmacological inhibition of different signalling pathways in CCA cells converged on the regulation of FOSL1 expression. Functional experiments showed that FOSL1 is required for cell proliferation and cell cycle progression in vitro, and for tumour growth and tumour maintenance in both orthotopic and subcutaneous xenograft models. Likewise, FOSL1 genetic abrogation in a GEM model of CCA extended mouse survival by decreasing the oncogenic potential of transformed cholangiocytes. RNA and ChIP sequencing studies identified direct and indirect transcriptional effectors such as HMGCS1 and AURKA, whose genetic and pharmacological inhibition phenocopied FOSL1 loss. Conclusions: Our data illustrate the functional and clinical relevance of FOSL1 in CCA and unveil potential targets amenable to pharmacological inhibition that could enable the implementation of novel therapeutic strategies. Lay summary: Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention.A.V. was supported by ADA of the University of Navarra, Spain, O.E. by FSE; MINECO; FJCI-2017-34233, Spain, R.E. by a donation from Mauge Burgos de la Iglesia’s family, Spain, and P. Olaizola by the Basque Government (PRE_2016_1_0269), Basque Country, Spain. M.J.P. was funded by ISCIII [FIS PI14; 00399, PI17; 00022] cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; Spanish Ministry of Economy and Competitiveness (MINECO: “Ramón y Cajal” Program RYC-2015-17755), Spain. M.A.A was funded by La Caixa Foundation, HEPACARE project, Spain, ISCIII FIS PI16/01126 cofinanced by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain, and “Fundación Científica de la Asociación Española Contra el Cáncer’’ (AECC Scientific Foundation) Rare Cancers 2017, Spain. J.M.B. was funded by the Spanish Carlos III Health Institute (ISCIII) (FIS PI15; 01132, PI18; 01075 and Miguel Servet Program CON14; 00129 and CPII19; 00008), Spain, co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER), Spain; “Euskadi RIS3” (2019222054) and BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15; CA; 016; BD), Basque Country, Spain; “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientific Foundation) Rare Cancers 2017, Spain. S.V. was supported by FEDER; MINECO (SAF2017-89944-R), Spain, by the Government of Navarra-Health Research Department (58; 2018), Navarra, Spain, by La Caixa and Caja Navarra Foundation-CIMA agreement, Spain. None of the funding sources were involved in the decision to submit the article for publication. This article is based upon work from COST Action CA18122 European Cholangiocarcinoma Network, supported by COST (European Cooperation in Science and Technology). COST (European Cooperation in Science and Technology) is a funding agency for research and innovation networks (www.cost.eu)

Mechanisms of inactivation of the tumour suppressor gene RHOA in colorectal cancer

Reduced RHOA signalling has been shown to increase the growth/metastatic potential of colorectal tumours. However, the mechanisms of inactivation of RHOA signalling in colon cancer have not been characterised. A panel of colorectal cancer cell lines and large cohorts of primary tumours were used to investigate the expression and activity of RHOA, as well as the presence of RHOA mutations/deletions and promoter methylation affecting RHOA. Changes in RHOA expression were assessed by western blotting and qPCR after modulation of microRNAs, SMAD4 and c-MYC. We show here that RHOA point mutations and promoter hypermethylation do not significantly contribute to the large variability of RHOA expression observed among colorectal tumours. However, RHOA copy number loss was observed in 16% of colorectal tumours and this was associated with reduced RHOA expression. Moreover, we show that miR-200a/b/429 downregulates RHOA in colorectal cancer cells. In addition, we found that TGF- β /SMAD4 upregulates the RHOA promoter. Conversely, RHOA expression is transcriptionally downregulated by canonical Wnt signalling through the Wnt target gene c-MYC that interferes with the binding of SP1 to the RHOA promoter in colon cancer cells. We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms

FOSL1 promotes cholangiocarcinoma via transcriptional effectors that could be therapeutically targeted

Understanding the molecular mechanisms involved in cholangiocarcinoma (bile duct cancer) development and progression stands as a critical step for the development of novel therapies. Through an inter-species approach, this study provides evidence of the clinical and functional role of the transcription factor FOSL1 in cholangiocarcinoma. Moreover, we report that downstream effectors of FOSL1 are susceptible to pharmacological inhibition, thus providing new opportunities for therapeutic intervention

Role of myosin VI in colorectal cancer

La miosina VI (MYO6) es un motor molecular que puede anclar moléculas o proporcionar tráfico de corto alcance a lo largo del citoesqueleto de actina valiéndose de la energía obtenida de la hidrólisis de ATP. La miosina VI se mueve hacia el extremo negativo de los filamentos de actina, en dirección opuesta al resto de miosinas. Se ha demostrado que la función de transporte de la miosina VI está implicada en varios procesos celulares, como la endocitosis, el tráfico endocítico y de reciclaje de vesículas, la autofagia, la exocitosis y la transcripción nuclear; y su función de anclaje se ha visto involucrada en el mantenimiento de la estructura del complejo de Golgi, de las estereocilias de las células ciliadas cocleares, de la membrana de las microvellosidades de los enterocitos, y en el mantenimiento de las uniones adherentes. La miosina VI se expresa en la mayoría de los tejidos, pero sus niveles de expresión varían considerablemente entre ellos. Además, la miosina VI puede existir en varias isoformas que se expresan diferentemente dependiendo del tipo de tejido. Estas isoformas interactúan con diferentes moléculas y, por lo tanto, llevan a cabo diferentes funciones. La miosina VI está altamente expresada en las células epiteliales del intestino delgado y grueso, estando principalmente localizada en la membrana apical y en la basolateral, aunque también está presente de forma difusa en el citoplasma. La miosina VI ha sido relacionada con determinados tipos de cáncer. En el cáncer ovárico, el de próstata y en la leucemia linfoide, se ha visto que la miosina VI está sobre-expresada, donde se le atribuye un papel en la migración y la invasión. Otros estudios también han asociado la sobre-expresión de la miosina VI con una mayor proliferación celular en cánceres como el hepático, el de mama y el de pulmón. Sin embargo, se sabe poco sobre el papel de la miosina VI en el cáncer colorrectal, el tercer tipo más común de cáncer en el mundo. En este estudio, mediante el uso de un ‘tissue microarray’ (TMA) que contenía muestras de tejido normal de colon, tumores colorrectales primarios de estadio Dukes' C y muestras de metástasis de ganglios linfáticos, se observó que la expresión de MYO6 se reduce o se pierde con frecuencia en tumores primarios en comparación con la expresión de células epiteliales normales de colon; y que incluso aún se reduce más en las metástasis de los ganglios linfáticos, en comparación con los tumores primarios. Además, en pacientes con cáncer colorrectal localmente avanzado, la baja expresión de MYO6 en el tumor primario se asoció con una supervivencia general y una supervivencia libre de enfermedad más cortas. Para poder estudiar el posible papel de la miosina VI en el cáncer colorrectal, se diseñaron modelos celulares isogénicos inducibles por doxiciclina para reducir la expresión de MYO6. Los resultados mostraron que la inactivación de MYO6 no altera la capacidad de diferenciación/polarización de las células de cáncer de colon. Tampoco la capacidad de migración y de invasión in vitro, ni la habilidad de metastatizar en modelos de ratón, se vieron afectadas. Sin embargo, se observó que la reducción de la expresión de MYO6 incrementaba el crecimiento de las células de cáncer de colon en un modelo de xenoinjertos subcutáneos en ratones inmunodeficientes NOD/SCID, a pesar de no provocar ningún cambio en el crecimiento in vitro. Por otro lado, la ausencia de MYO6 en ratones ApcMin/+ o en los tratados con AOM, no aceleró la tumorigénesis intestinal. Colectivamente, estos resultados indican que la pérdida de MYO6 es importante para promover el crecimiento de las células de cáncer colon, lo que indica un posible papel supresor tumoral de MYO6 en el cáncer colorrectal.Myosin VI (MYO6) is a molecular motor that can provide short range trafficking along the actin cytoskeleton by using the energy from ATP hydrolysis or anchor cargoes to actin filaments. Myosin VI moves towards the minus-end of actin filaments, in the opposite direction to the rest of myosins. The trafficking function of myosin VI has been shown to be involved in several cellular processes, such as, endocytosis, endocytic trafficking and recycling of vesicles, autophagy, exocytosis and nuclear transcription; and its anchoring function has been shown to be involved in the maintenance of Golgi complex, cochlear hair-cell stereocilia, enterocytic brush border membrane and adherens-junctions. Myosin VI is widely expressed in most tissues, but its expression levels vary considerably among them. Moreover, myosin VI can exist in several isoforms that are expressed in a tissue-specific manner. The isoforms interact with different binding partners and thus, carry out different functions. Myosin VI is highly expressed in the epithelial cells of small and large intestine, being mainly localized to the apical and basolateral membranes, although it is also present diffusely in the cytoplasm. Myosin VI has been involved in certain types of cancer. In ovarian carcinoma, prostate cancer and lymphoid leukemia myosin VI is found overexpressed with a role in migration and invasion. Other studies have also associated MYO6 overexpression with an increased cell proliferation in cancers such as, hepatocellular carcinoma, breast cancer and lung cancer. However, little is known about the role of myosin VI in colorectal cancer, the third most common type of cancer worldwide. In this study, using a tissue microarray containing samples from normal colon tissues, primary Dukes’C colorectal tumors and lymph node metastases, MYO6 expression was observed to be frequently reduced or lost in primary tumors compared to normal colonic epithelial cells. Moreover, MYO6 levels were further reduced in regional lymph node metastases compared to primary tumors. Importantly, low tumor MYO6 expression was associated with shorter overall and disease-free survival in patients with locally advanced colorectal cancer. Therefore, to study the possible role of MYO6 in colorectal cancer, isogenic cell line systems with doxycycline-inducible MYO6 downregulation were engineered. The results showed that MYO6 inactivation does not alter the differentiation/polarization capability of colon cancer cells. The migration and invasion ability in vitro and the potential to metastasize in an in vivo mouse model were neither altered by MYO6 knockdown. Nevertheless, although no changes were observed in the growth in vitro, MYO6 inactivation increased the growth of colon cancer cells in a subcutaneous xenograft model in immunodeficient NOD/SCID mice. On the other hand, absence of MYO6 in ApcMin/+ or AOM-treated mice did not accelerate intestinal tumorigenesis. Collectively, our results indicate that loss of MYO6 provides a significant growth advantage to colon cancer cells, indicating a possible tumor suppressor role of MYO6 in colorectal cancer

Role of myosin VI in colorectal cancer /

La miosina VI (MYO6) es un motor molecular que puede anclar moléculas o proporcionar tráfico de corto alcance a lo largo del citoesqueleto de actina valiéndose de la energía obtenida de la hidrólisis de ATP. La miosina VI se mueve hacia el extremo negativo de los filamentos de actina, en dirección opuesta al resto de miosinas. Se ha demostrado que la función de transporte de la miosina VI está implicada en varios procesos celulares, como la endocitosis, el tráfico endocítico y de reciclaje de vesículas, la autofagia, la exocitosis y la transcripción nuclear; y su función de anclaje se ha visto involucrada en el mantenimiento de la estructura del complejo de Golgi, de las estereocilias de las células ciliadas cocleares, de la membrana de las microvellosidades de los enterocitos, y en el mantenimiento de las uniones adherentes. La miosina VI se expresa en la mayoría de los tejidos, pero sus niveles de expresión varían considerablemente entre ellos. Además, la miosina VI puede existir en varias isoformas que se expresan diferentemente dependiendo del tipo de tejido. Estas isoformas interactúan con diferentes moléculas y, por lo tanto, llevan a cabo diferentes funciones. La miosina VI está altamente expresada en las células epiteliales del intestino delgado y grueso, estando principalmente localizada en la membrana apical y en la basolateral, aunque también está presente de forma difusa en el citoplasma. La miosina VI ha sido relacionada con determinados tipos de cáncer. En el cáncer ovárico, el de próstata y en la leucemia linfoide, se ha visto que la miosina VI está sobre-expresada, donde se le atribuye un papel en la migración y la invasión. Otros estudios también han asociado la sobre-expresión de la miosina VI con una mayor proliferación celular en cánceres como el hepático, el de mama y el de pulmón. Sin embargo, se sabe poco sobre el papel de la miosina VI en el cáncer colorrectal, el tercer tipo más común de cáncer en el mundo. En este estudio, mediante el uso de un 'tissue microarray' (TMA) que contenía muestras de tejido normal de colon, tumores colorrectales primarios de estadio Dukes' C y muestras de metástasis de ganglios linfáticos, se observó que la expresión de MYO6 se reduce o se pierde con frecuencia en tumores primarios en comparación con la expresión de células epiteliales normales de colon; y que incluso aún se reduce más en las metástasis de los ganglios linfáticos, en comparación con los tumores primarios. Además, en pacientes con cáncer colorrectal localmente avanzado, la baja expresión de MYO6 en el tumor primario se asoció con una supervivencia general y una supervivencia libre de enfermedad más cortas. Para poder estudiar el posible papel de la miosina VI en el cáncer colorrectal, se diseñaron modelos celulares isogénicos inducibles por doxiciclina para reducir la expresión de MYO6. Los resultados mostraron que la inactivación de MYO6 no altera la capacidad de diferenciación/polarización de las células de cáncer de colon. Tampoco la capacidad de migración y de invasión in vitro, ni la habilidad de metastatizar en modelos de ratón, se vieron afectadas. Sin embargo, se observó que la reducción de la expresión de MYO6 incrementaba el crecimiento de las células de cáncer de colon en un modelo de xenoinjertos subcutáneos en ratones inmunodeficientes NOD/SCID, a pesar de no provocar ningún cambio en el crecimiento in vitro. Por otro lado, la ausencia de MYO6 en ratones ApcMin/+ o en los tratados con AOM, no aceleró la tumorigénesis intestinal. Colectivamente, estos resultados indican que la pérdida de MYO6 es importante para promover el crecimiento de las células de cáncer colon, lo que indica un posible papel supresor tumoral de MYO6 en el cáncer colorrectal.Myosin VI (MYO6) is a molecular motor that can provide short range trafficking along the actin cytoskeleton by using the energy from ATP hydrolysis or anchor cargoes to actin filaments. Myosin VI moves towards the minus-end of actin filaments, in the opposite direction to the rest of myosins. The trafficking function of myosin VI has been shown to be involved in several cellular processes, such as, endocytosis, endocytic trafficking and recycling of vesicles, autophagy, exocytosis and nuclear transcription; and its anchoring function has been shown to be involved in the maintenance of Golgi complex, cochlear hair-cell stereocilia, enterocytic brush border membrane and adherens-junctions. Myosin VI is widely expressed in most tissues, but its expression levels vary considerably among them. Moreover, myosin VI can exist in several isoforms that are expressed in a tissue-specific manner. The isoforms interact with different binding partners and thus, carry out different functions. Myosin VI is highly expressed in the epithelial cells of small and large intestine, being mainly localized to the apical and basolateral membranes, although it is also present diffusely in the cytoplasm. Myosin VI has been involved in certain types of cancer. In ovarian carcinoma, prostate cancer and lymphoid leukemia myosin VI is found overexpressed with a role in migration and invasion. Other studies have also associated MYO6 overexpression with an increased cell proliferation in cancers such as, hepatocellular carcinoma, breast cancer and lung cancer. However, little is known about the role of myosin VI in colorectal cancer, the third most common type of cancer worldwide. In this study, using a tissue microarray containing samples from normal colon tissues, primary Dukes'C colorectal tumors and lymph node metastases, MYO6 expression was observed to be frequently reduced or lost in primary tumors compared to normal colonic epithelial cells. Moreover, MYO6 levels were further reduced in regional lymph node metastases compared to primary tumors. Importantly, low tumor MYO6 expression was associated with shorter overall and disease-free survival in patients with locally advanced colorectal cancer. Therefore, to study the possible role of MYO6 in colorectal cancer, isogenic cell line systems with doxycycline-inducible MYO6 downregulation were engineered. The results showed that MYO6 inactivation does not alter the differentiation/polarization capability of colon cancer cells. The migration and invasion ability in vitro and the potential to metastasize in an in vivo mouse model were neither altered by MYO6 knockdown. Nevertheless, although no changes were observed in the growth in vitro, MYO6 inactivation increased the growth of colon cancer cells in a subcutaneous xenograft model in immunodeficient NOD/SCID mice. On the other hand, absence of MYO6 in ApcMin/+ or AOM-treated mice did not accelerate intestinal tumorigenesis. Collectively, our results indicate that loss of MYO6 provides a significant growth advantage to colon cancer cells, indicating a possible tumor suppressor role of MYO6 in colorectal cancer

RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis

Rho GTPases are molecular switches regulating multiple cellular processes. To investigate the role of RhoA in normal intestinal physiology, we used a conditional mouse model overexpressing a dominant negative RhoA mutant (RhoAT19N) in the intestinal epithelium. Although RhoA inhibition did not cause an overt phenotype, increased levels of nuclear β-catenin were observed in the small intestinal epithelium of RhoAT19N mice, and the overexpression of multiple Wnt target genes revealed a chronic activation of Wnt signaling. Elevated Wnt signaling in RhoAT19N mice and intestinal organoids did not affect the proliferation of intestinal epithelial cells but significantly interfered with their differentiation. Importantly, 17-month-old RhoAT19N mice showed a significant increase in the number of spontaneous intestinal tumors. Altogether, our results indicate that RhoA regulates the differentiation of intestinal epithelial cells and inhibits tumor initiation, likely through the control of Wnt signaling, a key regulator of proliferation and differentiation in the intestine.</p

RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis

Rho GTPases are molecular switches regulating multiple cellular processes. To investigate the role of RhoA in normal intestinal physiology, we used a conditional mouse model overexpressing a dominant negative RhoA mutant (RhoAT19N) in the intestinal epithelium. Although RhoA inhibition did not cause an overt phenotype, increased levels of nuclear β-catenin were observed in the small intestinal epithelium of RhoAT19N mice, and the overexpression of multiple Wnt target genes revealed a chronic activation of Wnt signaling. Elevated Wnt signaling in RhoAT19N mice and intestinal organoids did not affect the proliferation of intestinal epithelial cells but significantly interfered with their differentiation. Importantly, 17-month-old RhoAT19N mice showed a significant increase in the number of spontaneous intestinal tumors. Altogether, our results indicate that RhoA regulates the differentiation of intestinal epithelial cells and inhibits tumor initiation, likely through the control of Wnt signaling, a key regulator of proliferation and differentiation in the intestine.</p