Tumor-associated Endo180 requires stromal-derived LOX to promote metastatic prostate cancer cell migration on human ECM surfaces

The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded ‘amoeboid-like’ mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar ‘mesenchymal-like’ mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer

Survival outcome and EMT suppression mediated by a lectin domain interaction of Endo180 and CD147

Epithelial cell-cell contacts maintain normal glandular tissue homeostasis, and their breakage can trigger epithelial-to-mesenchymal transition (EMT), a fundamental step in the development of metastatic cancer. Despite the ability of C-type lectin domains (CTLD) to modulate cell-cell adhesion, it is not known if they modulate epithelial adhesion in EMT and tumor progression. Here, the multi-CTLD mannose receptor, Endo180 (MRC2/uPARAP), was shown using the Kaplan-Meier analysis to be predictive of survival outcome in men with early prostate cancer. A proteomic screen of novel interaction partners with the fourth CTLD (CTLD4) in Endo180 revealed that its complex with CD147 is indispensable for the stability of three-dimensional acini formed by nontransformed prostate epithelial cells (PEC). Mechanistic study using knockdown of Endo180 or CD147, and treatment with an Endo180 mAb targeting CTLD4 (clone 39.10), or a dominant-negative GST-CTLD4 chimeric protein, induced scattering of PECs associated with internalization of Endo180 into endosomes, loss of E-cadherin (CDH1/ECAD), and unzipping of cell-cell junctions. These findings are the first to demonstrate that a CTLD acts as a suppressor and regulatory switch for EMT; thus, positing that stabilization of Endo180-CD147 complex is a viable therapeutic strategy to improve rates of prostate cancer survival

How to study basement membrane stiffness as a biophysical trigger in prostate cancer and other age-related pathologies or metabolic diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement membrane (BM) during age-related pathologies and metabolic disorders (e.g. cancer, diabetes, microvascular disease, retinopathy, nephropathy and neuropathy). The premise of the model is non-enzymatic crosslinking of reconstituted BM (rBM) matrix by treatment with glycolaldehyde (GLA) to promote advanced glycation endproduct (AGE) generation via the Maillard reaction. Examples of laboratory techniques that can be used to confirm AGE generation, non-enzymatic crosslinking and increased stiffness in GLA treated rBM are outlined. These include preparation of native rBM (treated with phosphate-buffered saline, PBS) and stiff rBM (treated with GLA) for determination of: its AGE content by photometric analysis and immunofluorescent microscopy, its non-enzymatic crosslinking by ((sodium dodecyl sulfate polyacrylamide gel electrophoresis)) (SDS PAGE) as well as confocal microscopy, and its increased stiffness using rheometry. The procedure described here can be used to increase the rigidity (elastic moduli, E) of rBM up to 3.2-fold, consistent with measurements made in healthy versus diseased human prostate tissue. To recreate the biophysical microenvironment associated with the aging and diseased prostate gland three prostate cell types were introduced on to native rBM and stiff rBM: RWPE-1, prostate epithelial cells (PECs) derived from a normal prostate gland; BPH-1, PECs derived from a prostate gland affected by benign prostatic hyperplasia (BPH); and PC3, metastatic cells derived from a secondary bone tumor originating from prostate cancer. Multiple parameters can be measured, including the size, shape and invasive characteristics of the 3D glandular acini formed by RWPE-1 and BPH-1 on native versus stiff rBM, and average cell length, migratory velocity and persistence of cell movement of 3D spheroids formed by PC3 cells under the same conditions. Cell signaling pathways and the subcellular localization of proteins can also be assessed

Age-related morphological, cellular and molecular changes in renal fibrosis

Miguel Alaga: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Paola Carzoglio: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Rodrigo Décima: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Ramiro Funes: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Cecilia Spiess: Estudiante de Medicina, Ciclo de Metodología Científica II, Facultad de Medicina, Universidad de la República. Uruguay. La contribución en la realización del trabajo fue equivalente a la de los demás estudiantes.-- Mercedes Rodriguez-Teja: Docente supervisor. Departamento de Genética, Facultad de Medicina, Universidad de la República, Uruguay. Contacto: Mercedes Rodríguez - Teja. E-mail: [email protected] los últimos 50 años se ha registrado un aumento gradual de la tasa de envejecimiento de la población uruguaya, reflejándose en un incremento de pacientes con enfermedad renal crónica mayores de 65 años. Este fenómeno plantea la interrogante de cómo el envejecimiento tisular afecta la función del riñón y, en particular cómo contribuye al desarrollo de la fibrosis renal. Con el envejecimiento se producen cambios morfológicos y funcionales en el riñón, tales como la esclerosis glomerular y la fibrosis intersticial. Estos cambios son consecuencia de alteraciones que ocurren a nivel celular. En este trabajo se profundizará en los mecanismos celulares que desencadenan la fibrosis intersticial y la gloméruloesclerosis, describiendo el proceso de inflamación sostenida, la transformación fenotípica de las células epiteliales a miofibroblastos, así como los mecanismos de producción de matriz extracelular y la perpetuación de la fibrosis renal. Además, detallaremos las cascadas moleculares involucradas en el proceso de fibrosis, poniendo énfasis en las cascadas reguladas por TGF-β1 y sus vías de interacción, que regulan la producción factores pro- y anti-fibróticos. También veremos como el TGF-β1 modula la expresión de ARN pequeños no-codificantes (microARNs), potentes inhibidores de la expresión génica, y como el gen anti-envejecimiento Klotho inhibe el avance de la fibrosis renal. Finalmente, discutiremos terapias para frenar o enlentecer el proceso de fibrosis renal, especialmente aquellas que tengan como blanco las cascadas de señalización activadas por TGF-β1, los microRNAs y posibles terapias de activación del gen Klotho para prevenir la progresión de esta patología.During the last 50 years the average age of the Uruguayan population has increased, coinciding with higher numbers of the population over 65 years old with chronic kidney disease. The pathological changes during ageing that affect kidney function are not very well understood. During ageing the kidney suffers morphological and functional changes, like interstitial fibrosis and glomerular sclerosis. These changes are the result of alterations that take place at the cellular level. Here, the cellular mechanisms that trigger interstitial fibrosis and glomerulosclerosis, including sustained inflammation, transition from epithelial to myofibroblastic cellular phenotype, and the mechanism involved in extracellular matrix production and progression of kidney fibrosis, will be discussed. We also consider the molecular cascades involved in fibrosis, with an emphasis on the transforming growth factor-beta1 (TGF-β1) signalling pathway that modulates the production of pro- and anti-fibrotic factors. We will also explore how TGF-β1 modulates non-coding small RNA (micro-RNA) expression, which is a powerful mechanism for gene regulation, and how the anti-ageing gene Klotho inhibits kidney fibrosis progression. Finally, alternative therapies that target pathways modulated by TGF-β1, micro-RNAs and activation of Klotho will be considered as precautionary measures to help to prevent age-related kidney disease

AGE-modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell–cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180ΔEx2–6/ΔEx2–6 mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors

Averting biodiversity collapse in tropical forest protected areas

The rapid disruption of tropical forests probably imperils global biodiversity more than any other contemporary phenomenon¹⁻³. With deforestation advancing quickly, protected areas are increasingly becoming final refuges for threatened species and natural ecosystem processes. However, many protected areas in the tropics are themselves vulnerable to human encroachment and other environmental stresses⁴⁻⁹. As pressures mount, it is vital to know whether existing reserves can sustain their biodiversity. A critical constraint in addressing this question has been that data describing a broad array of biodiversity groups have been unavailable for a sufficiently large and representative sample of reserves. Here we present a uniquely comprehensive data set on changes over the past 20 to 30 years in 31 functional groups of species and 21 potential drivers of environmental change, for 60 protected areas stratified across the world’s major tropical regions. Our analysis reveals great variation in reserve ‘health’: about half of all reserves have been effective or performed passably, but the rest are experiencing an erosion of biodiversity that is often alarmingly widespread taxonomically and functionally. Habitat disruption, hunting and forest-product exploitation were the strongest predictors of declining reserve health. Crucially, environmental changes immediately outside reserves seemed nearly as important as those inside in determining their ecological fate, with changes inside reserves strongly mirroring those occurring around them. These findings suggest that tropical protected areas are often intimately linked ecologically to their surrounding habitats, and that a failure to stem broad-scale loss and degradation of such habitats could sharply increase the likelihood of serious biodiversity declines.Keywords: Ecology, Environmental scienc

Informe final del proyecto: El papel de las células tumorales CD36+/Endo180+ en el cáncer de próstata metastásico

Si bien el principal factor de riesgo asociado al cáncer de próstata es la edad del paciente, se desconoce cómo un ambiente tisular envejecido contribuye con el desarrollo de esta patología. A lo largo de la vida la próstata sufre variaciones en sus propiedades visco-elásticas que acompañan a un progresivo crecimiento y endurecimiento del tejido glandular prostático. De hecho, el aumento en la rigidez del tejido prostático, conjuntamente con los valores en sangre del antígeno prostático específico, se utilizan como screening para la identificación de pacientes con cáncer. El progresivo endurecimiento del tejido prostático, entre otras cosas, es el resultado de la acumulación de glicotoxinas en el organismo, denominadas productos finales de glicación avanzada (AGEs, Avanced Glycation End-products). Las proteínas con vida media larga y poco recambio, como el colágeno que se encuentra en la matriz que rodea al acino prostático, son más susceptibles de ser modificadas por la acumulación de AGEs. Los AGEs entrecruzan los componentes de la matriz y generan una fuerza tensional en la superficie de la célula epitelial prostática. Estrés tensional es detectado por el mecano-receptor Endo180, desencadenando cambios en la célula epitelial y en la morfología acinar prostática que recuerdan a una lesión pre-maligna. Endo180 forma un complejo con la proteína CD36, cuya expresión es un marcador de células madre cancerosas resistentes a quimioterapias e iniciadoras de metástasis en diferentes tipos de cáncer. Un microambiente tisular rico en AGEs aumenta la expresión génica de Endo180 y CD36 en los cultivos de células madre de tumores prostáticos. Este aumento es acompañado de un incremento en la migración e invasión de las células madre tumorales prostáticos en presencia de AGEs. La caracterización de esta población de células madre de tumor Endo180+/CD36+ aporta a comprender el vínculo entre el envejecimiento tisular y la metástasis en cáncer de próstata. En el contexto de este proyecto se formaron dos estudiantes de grado y se realizó una tesis de Maestría.Agencia Nacional de Investigación e Innovació

Informe final del proyecto: El papel de las células tumorales CD36+/Endo180+ en el cáncer de próstata metastásico

Si bien el principal factor de riesgo asociado al cáncer de próstata es la edad del paciente, se desconoce cómo un ambiente tisular envejecido contribuye con el desarrollo de esta patología. A lo largo de la vida la próstata sufre variaciones en sus propiedades visco-elásticas que acompañan a un progresivo crecimiento y endurecimiento del tejido glandular prostático. De hecho, el aumento en la rigidez del tejido prostático, conjuntamente con los valores en sangre del antígeno prostático específico, se utilizan como screening para la identificación de pacientes con cáncer. El progresivo endurecimiento del tejido prostático, entre otras cosas, es el resultado de la acumulación de glicotoxinas en el organismo, denominadas productos finales de glicación avanzada (AGEs, Avanced Glycation End-products). Las proteínas con vida media larga y poco recambio, como el colágeno que se encuentra en la matriz que rodea al acino prostático, son más susceptibles de ser modificadas por la acumulación de AGEs. Los AGEs entrecruzan los componentes de la matriz y generan una fuerza tensional en la superficie de la célula epitelial prostática. Estrés tensional es detectado por el mecano-receptor Endo180, desencadenando cambios en la célula epitelial y en la morfología acinar prostática que recuerdan a una lesión pre-maligna. Endo180 forma un complejo con la proteína CD36, cuya expresión es un marcador de células madre cancerosas resistentes a quimioterapias e iniciadoras de metástasis en diferentes tipos de cáncer. Un microambiente tisular rico en AGEs aumenta la expresión génica de Endo180 y CD36 en los cultivos de células madre de tumores prostáticos. Este aumento es acompañado de un incremento en la migración e invasión de las células madre tumorales prostáticos en presencia de AGEs. La caracterización de esta población de células madre de tumor Endo180+/CD36+ aporta a comprender el vínculo entre el envejecimiento tisular y la metástasis en cáncer de próstata. En el contexto de este proyecto se formaron dos estudiantes de grado y se realizó una tesis de Maestría.Agencia Nacional de Investigación e Innovació