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1.
iScience ; 27(4): 109400, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38523777

RESUMO

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 ß-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.

2.
Oncogene ; 41(49): 5279-5288, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36316444

RESUMO

Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.


Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Miosina Tipo V , Animais , Camundongos , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Genes Supressores de Tumor , Camundongos Knockout , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Miosinas , Humanos
3.
Biomedicines ; 9(3)2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33809425

RESUMO

The global burden of chronic kidney disease (CKD) is increasing every year and represents a great cost for public healthcare systems, as the majority of these diseases are progressive. Therefore, there is an urgent need to develop new therapies. Oligonucleotide-based drugs are emerging as novel and promising alternatives to traditional drugs. Their expansion corresponds with new knowledge regarding the molecular basis underlying CKD, and they are already showing encouraging preclinical results, with two candidates being evaluated in clinical trials. However, despite recent technological advances, efficient kidney delivery remains challenging, and the presence of off-targets and side-effects precludes development and translation to the clinic. In this review, we provide an overview of the various oligotherapeutic strategies used preclinically, emphasizing the most recent findings in the field, together with the different strategies employed to achieve proper kidney delivery. The use of different nanotechnological platforms, including nanocarriers, nanoparticles, viral vectors or aptamers, and their potential for the development of more specific and effective treatments is also outlined.

4.
Mol Ther Nucleic Acids ; 21: 192-204, 2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32585627

RESUMO

Targeted therapeutics underwent a revolution with the entry of monoclonal antibodies in the medical toolkit. Oligonucleotide aptamers form another family of target agents that have been lagging behind in reaching the clinical arena in spite of their potential clinical translation. Some of the reasons for this might be related to the challenge in identifying aptamers with optimal in vivo specificity, and the nature of their pharmacokinetics. Aptamers usually show exquisite specificity, but they are also molecules that display dynamic structures subject to changing environments. Temperature, ion atmosphere, pH, and other variables are factors that could determine the affinity and specificity of aptamers. Thus, it is important to tune the aptamer selection process to the conditions in which you want your final aptamer to function; ideally, for in vivo applications, aptamers should be selected in an in vivo-like system or, ultimately, in a whole in vivo organism. In this review we recapitulate the implementations in systematic evolution of ligands by exponential enrichment (SELEX) to obtain aptamers with the best in vivo activity.

5.
Hepatology ; 72(1): 213-229, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31750554

RESUMO

BACKGROUND AND AIMS: Progressive familial intrahepatic cholestasis (PFIC) 6 has been associated with missense but not biallelic nonsense or frameshift mutations in MYO5B, encoding the motor protein myosin Vb (myoVb). This genotype-phenotype correlation and the mechanism through which MYO5B mutations give rise to PFIC are not understood. The aim of this study was to determine whether the loss of myoVb or expression of patient-specific myoVb mutants can be causally related to defects in canalicular protein localization and, if so, through which mechanism. APPROACH AND RESULTS: We demonstrate that the cholestasis-associated substitution of the proline at amino acid position 600 in the myoVb protein to a leucine (P660L) caused the intracellular accumulation of bile canalicular proteins in vesicular compartments. Remarkably, the knockout of MYO5B in vitro and in vivo produced no canalicular localization defects. In contrast, the expression of myoVb mutants consisting of only the tail domain phenocopied the effects of the Myo5b-P660L mutation. Using additional myoVb and rab11a mutants, we demonstrate that motor domain-deficient myoVb inhibited the formation of specialized apical recycling endosomes and that its disrupting effect on the localization of canalicular proteins was dependent on its interaction with active rab11a and occurred at the trans-Golgi Network/recycling endosome interface. CONCLUSIONS: Our results reveal a mechanism through which MYO5B motor domain mutations can cause the mislocalization of canalicular proteins in hepatocytes which, unexpectedly, does not involve myoVb loss-of-function but, as we propose, a rab11a-mediated gain-of-toxic function. The results explain why biallelic MYO5B mutations that affect the motor domain but not those that eliminate myoVb expression are associated with PFIC6.


Assuntos
Colestase Intra-Hepática/genética , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Genótipo , Humanos , Células Tumorais Cultivadas
6.
PLoS Biol ; 17(11): e3000531, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31682603

RESUMO

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome-associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome-associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel-sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture.


Assuntos
Endossomos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Fuso Acromático/metabolismo , Animais , Células CACO-2 , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Citocinese/genética , Citocinese/fisiologia , Endossomos/genética , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/fisiologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
7.
Mol Biol Cell ; 30(26): 3076-3089, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31664880

RESUMO

Loss-of-function mutations in the nonconventional myosin Vb (Myo5b) result in microvillus inclusion disease (MVID) and massive secretory diarrhea that often begins at birth. Myo5b mutations disrupt the apical recycling endosome (ARE) and membrane traffic, resulting in reduced surface expression of apical membrane proteins. ARE disruption also results in constitutive phosphoinositide-dependent kinase 1 gain of function. In MVID, decreased surface expression of apical anion channels involved in Cl- extrusion, such as cystic fibrosis transmembrane conductance regulator (CFTR), should reduce fluid secretion into the intestinal lumen. But the opposite phenotype is observed. To explain this contradiction and the onset of diarrhea, we hypothesized that signaling effects downstream from Myo5b loss of function synergize with higher levels of glucocorticoids to activate PKA and CFTR. Data from intestinal cell lines, human MVID, and Myo5b KO mouse intestine revealed changes in the subcellular redistribution of PKA activity to the apical pole, increased CFTR phosphorylation, and establishment of apical cAMP gradients in Myo5b-defective cells exposed to physiological levels of glucocorticoids. These cells also displayed net secretory fluid fluxes and transepithelial currents mainly from PKA-dependent Cl- secretion. We conclude that Myo5b defects result in PKA stimulation that activates residual channels on the surface when intestinal epithelia are exposed to glucocorticoids at birth.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Glucocorticoides/metabolismo , Miosina Tipo V/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Células CACO-2 , Linhagem Celular Tumoral , Canais de Cloreto/metabolismo , Diarreia/congênito , Diarreia/genética , Humanos , Síndromes de Malabsorção/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microvilosidades/genética , Microvilosidades/patologia , Mucolipidoses/genética
8.
Br J Cancer ; 118(1): 106-116, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29206819

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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-ß/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. CONCLUSIONS: We demonstrate a complex pattern of inactivation of the tumour suppressor gene RHOA in colon cancer cells through genetic, transcriptional and post-transcriptional mechanisms.


Assuntos
Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA , Regulação para Baixo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/genética , Metilação de DNA , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Ativação Transcricional , Via de Sinalização Wnt
9.
Sci Rep ; 7: 43702, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28262839

RESUMO

Although deregulation of EPHB signaling has been shown to be an important step in colorectal tumorigenesis, the role of EPHB6 in this process has not been investigated. We found here that manipulation of EPHB6 levels in colon cancer cell lines has no effect on their motility and growth on a solid substrate, soft agar or in a xenograft mouse model. We then used an EphB6 knockout mouse model to show that EphB6 inactivation does not efficiently initiate tumorigenesis in the intestinal tract. In addition, when intestinal tumors are initiated genetically or pharmacologically in EphB6+/+ and EphB6-/- mice, no differences were observed in animal survival, tumor multiplicity, size or histology, and proliferation of intestinal epithelial cells or tumor cells. However, reintroduction of EPHB6 into colon cancer cells significantly reduced the number of lung metastasis after tail-vein injection in immunodeficient mice, while EPHB6 knockdown in EPHB6-expressing cells increased their metastatic spread. Consistently, although EPHB6 protein expression in a series of 130 primary colorectal tumors was not associated with patient survival, EPHB6 expression was significantly lower in lymph node metastases compared to primary tumors. Our results indicate that the loss of EPHB6 contributes to the metastatic process of colorectal cancer.


Assuntos
Biomarcadores Tumorais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptores da Família Eph/deficiência , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo
10.
Sci Rep ; 7: 41576, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28169277

RESUMO

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.


Assuntos
Neoplasias Colorretais/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Expressão Gênica , Genótipo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Metástase Neoplásica , Receptores Proteína Tirosina Quinases/genética , Receptor EphA3 , Transdução de Sinais
11.
Sci Rep ; 5: 12312, 2015 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-26201991

RESUMO

Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and development of novel therapeutic approaches is hampered by the lack of animal models. In this study we describe the phenotype of a novel mouse model with targeted inactivation of Myo5b. Myo5b knockout mice show perinatal mortality, diarrhea and the characteristic mislocalization of apical and basolateral plasma membrane markers in enterocytes. Moreover, in transmission electron preparations, we observed microvillus atrophy and the presence of microvillus inclusion bodies. Importantly, Myo5b knockout embryos at day 20 of gestation already display all these structural defects, indicating that they are tissue autonomous rather than secondary to environmental cues, such as the long-term absence of nutrients in the intestine. Myo5b knockout mice closely resemble the phenotype of MVID patients and constitute a useful model to further investigate the underlying molecular mechanism of this disease and to preclinically assess the efficacy of novel therapeutic approaches.


Assuntos
Diarreia/patologia , Diarreia/fisiopatologia , Modelos Animais de Doenças , Síndromes de Malabsorção/patologia , Síndromes de Malabsorção/fisiopatologia , Microvilosidades/patologia , Mucolipidoses/patologia , Mucolipidoses/fisiopatologia , Miosina Tipo V/genética , Animais , Diarreia/etiologia , Feminino , Síndromes de Malabsorção/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucolipidoses/complicações , Miosina Tipo V/metabolismo
12.
Clin Cancer Res ; 21(16): 3695-704, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25944804

RESUMO

PURPOSE: The clinical management of colorectal cancer patients has significantly improved because of the identification of novel therapeutic targets such as EGFR and VEGF. Because rapid tumor proliferation is associated with poor patient prognosis, here we characterized the transcriptional signature of rapidly proliferating colorectal cancer cells in an attempt to identify novel candidate therapeutic targets. EXPERIMENTAL DESIGN: The doubling time of 52 colorectal cancer cell lines was determined and genome-wide expression profiling of a subset of these lines was assessed by microarray analysis. We then investigated the potential of genes highly expressed in cancer cells with faster growth as new therapeutic targets. RESULTS: Faster proliferation rates were associated with microsatellite instability and poorly differentiated histology. The expression of 1,290 genes was significantly correlated with the growth rates of colorectal cancer cells. These included genes involved in cell cycle, RNA processing/splicing, and protein transport. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protoporphyrinogen oxidase (PPOX) were shown to have higher expression in faster growing cell lines and primary tumors. Pharmacologic or siRNA-based inhibition of GAPDH or PPOX reduced the growth of colon cancer cells in vitro. Moreover, using a mouse xenograft model, we show that treatment with the specific PPOX inhibitor acifluorfen significantly reduced the growth of three of the seven (42.8%) colon cancer lines investigated. CONCLUSIONS: We have characterized at the transcriptomic level the differences between colorectal cancer cells that vary in their growth rates, and identified novel candidate chemotherapeutic targets for the treatment of colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Flavoproteínas/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Proteínas Mitocondriais/biossíntese , Proteínas de Neoplasias/biossíntese , Protoporfirinogênio Oxidase/biossíntese , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Feminino , Flavoproteínas/antagonistas & inibidores , Flavoproteínas/genética , Regulação Neoplásica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Células HCT116 , Humanos , Masculino , Camundongos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Terapia de Alvo Molecular , Proteínas de Neoplasias/genética , Nitrobenzoatos/administração & dosagem , Transporte Proteico/genética , Protoporfirinogênio Oxidase/antagonistas & inibidores , Protoporfirinogênio Oxidase/genética , Splicing de RNA/genética , RNA Interferente Pequeno , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Nat Commun ; 5: 5458, 2014 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-25413277

RESUMO

Activation of the small GTPase RHOA has strong oncogenic effects in many tumour types, although its role in colorectal cancer remains unclear. Here we show that RHOA inactivation contributes to colorectal cancer progression/metastasis, largely through the activation of Wnt/ß-catenin signalling. RhoA inactivation in the murine intestine accelerates the tumorigenic process and in human colon cancer cells leads to the redistribution of ß-catenin from the membrane to the nucleus and enhanced Wnt/ß-catenin signalling, resulting in increased proliferation, invasion and de-differentiation. In mice, RHOA inactivation contributes to colon cancer metastasis and reduced RHOA levels were observed at metastatic sites compared with primary human colon tumours. Therefore, we have identified a new mechanism of activation of Wnt/ß-catenin signalling and characterized the role of RHOA as a novel tumour suppressor in colorectal cancer. These results constitute a shift from the current paradigm and demonstrate that RHO GTPases can suppress tumour progression and metastasis.


Assuntos
Neoplasias do Colo/enzimologia , Inativação Gênica , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
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