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News Article | May 16, 2017
Site: www.eurekalert.org

A study published now on Nature Communications* shows that breast cancer cells undergo a stiffening state prior to acquiring malignant features and becoming invasive. The discovery made by a research team led by Florence Janody, from Instituto Gulbenkian de Ciencia (IGC; Portugal), identifies a new signal in tumor cells that can be further explored when designing cancer-targeting therapies. The progression of breast cancer disease takes several stages, from a benign lesion to an invasive carcinoma, possibly with metastasis. But actually, only 20 to 50% of benign tumors end up as invasive cancer. Predicting what lesions are within this group could result in a better use of therapeutics accordingly to the severity of the disease. Florence Janody's group has been looking for signals inside the cells that could help predicting benign tumors that will progress to invasive carcinoma. Their attention focus on the cell skeleton - the cytoskeleton --, an intricate network of fibers that can either exert or resist forces, and that may have an impact on tumor invasion and malignancy. These fibers can be organized into distinct architectures to confer cells a more rigid or soft structure. "Previously, it had been shown that cancer cell invasion requires cell softening. What we observed now is that prior to becoming invasive cells undergo a transient stiffening state caused by the accumulation of cytoskeleton fibers ", explains Sandra Tavares, first author of this study. The research team discovered that cell stiffening induces the activity of proteins that promote cell proliferation, driving the growth of benign tumors. Most importantly, this cell rigidity state also triggers the subsequent progression into invasive cancer. The proteins involved in this mechanism were identified by studies on a human breast cell line, which recapitulates the multistep development of breast cancer, and biopsies of breast cancers. The importance of these proteins for the formation of tumors was further confirmed in the fruit fly. Florence Janody says: "Our work adds an important piece to the intricate puzzle of breast tumor progression. Knowing what happens inside the cell before a cell becomes pre-invasive and acquires malignant features may help us predict, in the future, which tumors may result in metastasis. Also, it may help designing therapeutics better tailored for each type of lesion." This study was conducted at the IGC in collaboration with the Instituto de Investigacao e Inovacao em Saude (i3S), Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Faculty of Medicine of the University of Porto (Portugal), Biotechnology Center from the Technische Universitat Dresden (Germany), and Ophiomics - Precision Medicine (Portugal). Fundacao para a Ciencia e a Tecnologia, Liga Portuguesa contra o Cancro/Pfizer and Laco Grant in Breast Cancer 2015 funded this research. *Tavares, S., Vieira, A.F., Taubenberger, A.V., Araujo, M., Martins, N.P.S., Bras-Pereira, C., Polonia, A., Herbig, M., Barreto, C., Otto, O., Cardoso, J., Pereira-Leal, J.B., Guck, J., Paredes, J., Janody, F. (2017) Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells. Nature Communications. DOI: 10.1038/NCOMMS15237


Barros R.,IPATIMUP | Da Costa L.T.,IPATIMUP | Da Costa L.T.,University of Évora | Pinto-de-Sousa J.,Hospital S Joao | And 7 more authors.
Gut | Year: 2011

Background and aims: Intestinal metaplasia (IM) is a gastric preneoplastic lesion that appears following Helicobacter pylori infection and confers an increased risk for development of cancer. It is induced by gastric expression of the intestine-specific transcription factor CDX2. The regulatory mechanisms involved in triggering and maintaining gastric CDX2 expression have not been fully elucidated. The Cdx2+/- mouse develops intestinal polyps with gastric differentiation and total loss of Cdx2 expression in the absence of structural loss of the second allele, suggesting a regulatory defect. This putative haplo-insufficiency, together with the apparent stability of IM, led to the hypothesis that CDX2 regulates its own expression through an autoregulatory loop in both contexts. Methods: Gastrointestinal cell lines were co-transfected with wild-type or mutated Cdx2 promoter constructs and CDX2 expression vector for luciferase assays. Transfection experiments were also used to assess endogenous CDX2 autoregulation, evaluated by RT-PCR, qPCR and western blotting. Chromatin immunoprecipitation was performed in a cell line, mouse ileum and human IM. Results: CDX2 binds to and transactivates its own promoter and positively regulates its expression in gastrointestinal human carcinoma cell lines. Furthermore, CDX2 is bound to its promoter in the mouse ileum and in human gastric IM, providing a major contribution to understanding the relevance of this autoregulatory pathway in vivo. Conclusion: The results of this study demonstrate another layer of complexity in CDX2 regulation by an effective autoregulatory loop which may have a major impact on the stability of human IM, possibly resulting in the inevitable progression of the gastric carcinogenesis pathway.


Alvarenga C.A.,Institute Patologia Of Campinas | Paravidino P.I.,Institute Patologia Of Campinas | Alvarenga M.,Institute Patologia Of Campinas | Alvarenga M.,University of Campinas | And 5 more authors.
Journal of Clinical Pathology | Year: 2011

Background: The sentinel lymph node (SLN) is the first lymph node to receive the lymphatic drainage of a primary tumour; based on the knowledge that CK19 is positive in more than 95% of breast carcinomas, a molecular method for intraoperative diagnosis of SLN metastases (the one-step nucleic acid amplification (OSNA) assay) was developed. Aims: To evaluate CK19 immunoreactivity in a series of special histological types of breast carcinoma in order to verify whether the OSNA assay can be used in all breast cancer cases independently of the histological type. Methods: 116 samples of invasive breast carcinomas of special type were investigated for CK19 immunoreactivity in tissue microarrays (TMA); negative cases were evaluated in the entire tissue tumour tissue. Results: Of the 116 cases, 88.9% were positive CK19. Micropapillary and apocrine carcinomas were all positive for CK19 in TMAs. The tubular (93%), mucinous (86%), medullary typical and atypical (84%), mixed carcinomas (83%) increased the rate of positivity for this marker to 100% after repeating the immunostain in whole tissue of negative TMA cases, because the expression of those cases was focal. Conclusion: Most breast cancer cases were positive for CK19, independent of the histological type; therefore the OSNA assay can be used in all breast cancer cases with a potential low rate of false negative for CK19 detection of micrometastasis. There is an important variation between the positivity assessed on TMAs and the entire tissue; these findings can be clinically relevant because in some cases CK19 is evaluated on core-needle biopsy prior to surgery.


Coutinho M.F.,Research and Development Unit | Prata M.J.,IPATIMUP | Alves S.,Research and Development Unit
Molecular Genetics and Metabolism | Year: 2012

Lysosomal hydrolases are synthesized in the rough endoplasmic reticulum and specifically transported through the Golgi apparatus to the trans-Golgi network, from which transport vesicles bud to deliver them to the endosomal/lysosomal compartment.The explanation of how are the lysosomal enzymes accurately recognized and selected over many other proteins in the trans-Golgi network relies on being tagged with a unique marker: the mannose-6-phosphate (M6P) group, which is added exclusively to the N-linked oligosaccharides of lysosomal soluble hydrolases, as they pass through the cis-Golgi network. Generation of the M6P recognition marker depends on a reaction involving two different enzymes: UDP-N-acetylglucosamine 1-phosphotransferase and α-N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase.The M6P groups are then recognized by two independent transmembrane M6P receptors, present in the trans-Golgi network: the cation-independent M6P receptor and/or the cation-dependent M6P receptor. These proteins bind to lysosomal hydrolases on the lumenal side of the membrane and to adaptins in assembling clathrin coats on the cytosolic side. In this way, the M6P receptors help package the hydrolases into vesicles that bud from the trans-Golgi network to deliver their contents to endosomes that ultimately will develop into mature lysosomes, where recently-delivered hydrolases may start digesting the endocyted material.The above described process is known as the M6P-dependent pathway and is responsible for transporting most lysosomal enzymes.This review synthesizes the current knowledge on each of the major proteins involved in the M6P-dependent pathway. Impairments in this pathway will also be addressed, highlighting the lysosomal storage disorders associated to GlcNAc-1-phosphotransferase loss of function: mucolipidosis type II and III. © 2011 Elsevier Inc..


Coutinho M.F.,Research and Development Unit | Prata M.J.,IPATIMUP | Alves S.,Research and Development Unit
Molecular Genetics and Metabolism | Year: 2012

Lysosomal hydrolases have long been known to be responsible for the degradation of different substrates in the cell. These acid hydrolases are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the trans-Golgi network (TGN). From there, they are delivered to endosomal/lysosomal compartments, where they finally become active due to the acidic pH characteristic of the lysosomal compartment. The majority of the enzymes leave the TGN after modification with mannose-6-phosphate (M6P) residues, which are specifically recognized by M6P receptors (MPRs), ensuring their transport to the endosomal/lysosomal system.Although M6P receptors play a major role in the intracellular transport of newly synthesized lysosomal enzymes in mammalian cells, several lines of evidence suggest the existence of alternative processes of lysosomal targeting. Among them, the two that are mediated by the M6P alternative receptors, lysosomal integral membrane protein (LIMP-2) and sortilin, have gained unequivocal support. LIMP-2 was shown to be implicated in the delivery of beta-glucocerebrosidase (GCase) to the lysosomes, whereas sortilin has been suggested to be a multifunctional receptor capable of binding several different ligands, including neurotensin and receptor-associated protein (RAP), and of targeting several proteins to the lysosome, including sphingolipid activator proteins (prosaposin and GM2 activator protein), acid sphingomyelinase and cathepsins D and H.Here, we review the current knowledge on these two proteins: their discovery, study, structural features and cellular function, with special attention to their role as alternative receptors to lysosomal trafficking. Recent studies associating both LIMP2 and sortilin to disease are also extensively reviewed. © 2012 Elsevier Inc.


Nogueira C.,National Institute of Health | Barros J.,Hospital Santo Antonio | Barros J.,Abel Salazar Biomedical Sciences Institute | Sa M.J.,Hospital Sao Joao | And 13 more authors.
Neurogenetics | Year: 2013

Complex III of the mitochondrial respiratory chain (CIII) catalyzes transfer of electrons from reduced coenzyme Q to cytochrome c. Low biochemical activity of CIII is not a frequent etiology in disorders of oxidative metabolism and is genetically heterogeneous. Recently, mutations in the human tetratricopeptide 19 gene (TTC19) have been involved in the etiology of CIII deficiency through impaired assembly of the holocomplex. We investigated a consanguineous Portuguese family where four siblings had reduced enzymatic activity of CIII in muscle and harbored a novel homozygous mutation in TTC19. The clinical phenotype in the four sibs was consistent with severe olivo-ponto-cerebellar atrophy, although their age at onset differed slightly. Interestingly, three patients also presented progressive psychosis. The mutation resulted in almost complete absence of TTC19 protein, defective assembly of CIII in muscle, and enhanced production of reactive oxygen species in cultured skin fibroblasts. Our findings add to the array of mutations in TTC19, corroborate the notion of genotype/phenotype variability in mitochondrial encephalomyopathies even within a single family, and indicate that psychiatric manifestations are a further presentation of low CIII. © 2013 Springer-Verlag Berlin Heidelberg.


De Pace R.,University of Hamburg | Coutinho M.F.,Research and Development Unit | Koch-Nolte F.,University of Hamburg | Haag F.,University of Hamburg | And 4 more authors.
Human Mutation | Year: 2014

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β-subunit precursor protein of GlcNAc-1-phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α-subunit-specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild-type and mutant α/β-subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β-subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc-1-phosphotransferase that may help to explain the clinical phenotype of MLII patients. © 2013 Wiley Periodicals, Inc.


Ortiz S.,Centro Hospitalar Lisbon Norte | Tortosa F.,Centro Hospitalar Lisbon Norte | Sobrinho Simoes M.,Ipatimup
Endocrine Pathology | Year: 2015

Secondary tumours of the thyroid gland account for 1.25 to 3 % in clinical series and reach 24 % in autopsy series. Chondrosarcoma is a rare malignant mesenchymal tumour of chondrogenic nature; the mesenchymal variant represents less than 3 % of all chondrosarcomas, being therefore extremely rare. A mesenchymal chondrosarcoma metastasis in the thyroid is exceptional; to our knowledge, only three previous cases of chondrosarcoma metastasis in the thyroid have been reported to date but none of such cases corresponded to a mesenchymal chondrosarcoma. We present the first of such a case in a 27-year-old woman with a 4-year history of mesenchymal chondrosarcoma of the sacrum that was treated by surgery and chemotherapy. At the present admission, head and neck computed tomography revealed a well-defined nodule in the thyroid gland. The diagnosis of metastasis from the mesenchymal chondrosarcoma was made in the right lobectomy specimen. © 2014, Springer Science+Business Media New York.


van Asch B.,IPATIMUP
Proceedings. Biological sciences / The Royal Society | Year: 2013

Dogs were present in pre-Columbian America, presumably brought by early human migrants from Asia. Studies of free-ranging village/street dogs have indicated almost total replacement of these original dogs by European dogs, but the extent to which Arctic, North and South American breeds are descendants of the original population remains to be assessed. Using a comprehensive phylogeographic analysis, we traced the origin of the mitochondrial DNA lineages for Inuit, Eskimo and Greenland dogs, Alaskan Malamute, Chihuahua, xoloitzcuintli and perro sín pelo del Peru, by comparing to extensive samples of East Asian (n = 984) and European dogs (n = 639), and previously published pre-Columbian sequences. Evidence for a pre-Columbian origin was found for all these breeds, except Alaskan Malamute for which results were ambigous. No European influence was indicated for the Arctic breeds Inuit, Eskimo and Greenland dog, and North/South American breeds had at most 30% European female lineages, suggesting marginal replacement by European dogs. Genetic continuity through time was shown by the sharing of a unique haplotype between the Mexican breed Chihuahua and ancient Mexican samples. We also analysed free-ranging dogs, confirming limited pre-Columbian ancestry overall, but also identifying pockets of remaining populations with high proportion of indigenous ancestry, and we provide the first DNA-based evidence that the Carolina dog, a free-ranging population in the USA, may have an ancient Asian origin.


Aggarwal S.,Nizam's Institute of Medical Sciences | Aggarwal S.,DNA Diagnostics Center | Coutinho M.F.,DNA Diagnostics Center | Dalal A.B.,DNA Diagnostics Center | And 3 more authors.
Gene | Year: 2014

We report a neonate who was diagnosed as a case of skeletal dysplasia during pregnancy, and was subsequently diagnosed as a case of MLII alpha/beta on the basis of clinical and radiological findings and molecular testing of the parents. A novel GNPTAB mutation c.1701delC [p.F566L. fsX5] was identified in the father. The case reiterates the severe prenatal phenotype of MLII alpha/beta which mimics skeletal dysplasia and illustrates the utility of molecular genetic analysis in confirmation of diagnosis and subsequent genetic counselling. © 2014 Elsevier B.V.

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