Marta Sabariego Puig

Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts

Summary DNA methylation profiling of TAFs reveals global demethylation and a selective impact on the TGF-β pathway. Moreover, it suggests the fibrocyte origin of a fraction of TAFs, and identifies a novel prognostic biomarker in non-small cell lung cancer.

Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ∼1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Matrix Stiffening and β1 Integrin Drive Subtype-Specific Fibroblast Accumulation in Lung Cancer

The crucial role of tumor-associated fibroblasts (TAF) in cancer progression is now clear in non–small cell lung cancer (NSCLC). However, therapies against TAFs are limited due to a lack of understanding in the subtype-specific mechanisms underlying their accumulation. Here, the mechanical (i.e., matrix rigidity) and soluble mitogenic cues that drive the accumulation of TAFs from major NSCLC subtypes: adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were dissected. Fibroblasts were cultured on substrata engineered to exhibit normal- or tumor-like stiffnesses at different serum concentrations, and critical regulatory processes were elucidated. In control fibroblasts from nonmalignant tissue, matrix stiffening alone increased fibroblast accumulation, and this mechanical effect was dominant or comparable with that of soluble growth factors up to 0.5% serum. The stimulatory cues of matrix rigidity were driven by β1 integrin mechano-sensing through FAK (pY397), and were associated with a posttranscriptionally driven rise in β1 integrin expression. The latter mechano-regulatory circuit was also observed in TAFs but in a subtype-specific fashion, because SCC–TAFs exhibited higher FAK (pY397), β1 expression, and ERK1/2 (pT202/Y204) than ADC–TAFs. Moreover, matrix stiffening induced a larger TAF accumulation in SCC–TAFs (>50%) compared with ADC–TAFs (10%–20%). In contrast, SCC–TAFs were largely serum desensitized, whereas ADC–TAFs responded to high serum concentration only. These findings provide the first evidence of subtype-specific regulation of NSCLC–TAF accumulation. Furthermore, these data support that therapies aiming to restore normal lung elasticity and/or β1 integrin-dependent mechano regulation may be effective against SCC–TAFs, whereas inhibiting stromal growth factor signaling may be effective against ADC–TAFs. Implications: This study reveals distinct mechanisms underlying the abnormal accumulation of tumor-supporting fibroblasts in two major subtypes of lung cancer, which will assist the development of personalized therapies against these cells. Mol Cancer Res; 13(1); 161–73. ©2014 AACR.

Heterotypic paracrine signaling drives fibroblast senescence and tumor progression of large cell carcinoma of the lung

Senescence in cancer cells acts as a tumor suppressor, whereas in fibroblasts enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of cancer subtypes. However, the presence of senescent TAFs in lung cancer remains undefined. We examined senescence in TAFs from primary lung cancer and paired control fibroblasts from unaffected tissue in three major histologic subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Three independent senescence markers (senescence-associated beta-galactosidase, permanent growth arrest and spreading) were consistently observed in cultured LCC-TAFs only, revealing a selective premature senescence. Intriguingly, SCC-TAFs exhibited a poor growth response in the absence of senescence markers, indicating a dysfunctional phenotype rather than senescence. Co-culturing normal fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to render fibroblasts senescent through oxidative stress, indicating that senescence in LCC-TAFs is driven by heterotypic signaling. In addition, senescent fibroblasts provided selective growth and invasive advantages to LCC cells in culture compared to normal fibroblasts. Likewise, senescent fibroblasts enhanced tumor growth and lung dissemination of tumor cells when co-injected with LCC cells in nude mice beyond the effects induced by control fibroblasts. These results define the subtype-specific aberrant phenotypes of lung TAFs, thereby challenging the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless of their subtype. Importantly, because LCC often distinguishes itself in the clinic by its aggressive nature, we argue that senescent TAFs may contribute to the selective aggressive behavior of LCC tumors.

DNA methylation profiling unveils TGF-ß hyperresponse in tumor associated fibroblasts from lung cancer patients

There is growing interest in defining the aberrant molecular differences between normal and tumor-associated fibroblasts (TAFs) that support tumor progression. For this purpose, we recently conducted a genome-wide DNA methylation profiling of TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer (NSCLC) patients, and reported a widespread hypomethylation concomitantly with focal gain of DNA methylation; in addition, we found evidence that a fraction of lung TAFs are fibrocytes in origin. Of note, the aberrant epigenome of lung TAFs had a global impact in gene expression and a selective impact on the TGF-s pathway. To get insights on the functional implications of the latter impact, we analyzed the response of lung TAFs to exogenous TGF-s1 in terms of activation and contractility. We found a larger expression of a panel of activation markers including a-SMA and collagen-I in TAFs compared to control fibroblasts. Likewise, TGF-s1 elicited a larger contractility in TAFs than in CFs as assessed by traction force microscopy. These findings reveal that lung TAFs are hyperresponsive to TGF-s1, which may underlie the expansion and/or maintenance of the tumor-promoting desmoplastic stroma in lung cancer.

El diálogo interreligioso en el espacio público: retos para los agentes socioeducativos en Cataluña

espanolEste articulo examina el concepto y los elementos que favorecen el dialogo intercultural e interreligioso, desde el punto de vista de expertos implicados en la gestion de la diversidad cultural en el espacio publico: profesionales de la Administracion, entidades sociales y mundo academico en Cataluna. Responde a un estudio descriptivo-comprensivo que sigue un enfoque cualitativo, y en el que se ha optado por la fundamentacion hermeneutica-interpretativa para obtener el relato de estos tres colectivos. Se han desarrollado once entrevistas semiestructuradas a participantes representativos de cada uno de ellos. Los resultados revelan definiciones diferenciadas pero complementarias sobre el dialogo interreligioso: relacion entre cultura y religion, dialogo como un primer paso de reconocimiento mutuo y los beneficios de este. Los participantes aportan elementos que facilitan este dialogo. Se concluye con un modelo de gestion de la diversidad religiosa en el espacio publico con propuestas comunitarias y educativas para orientar futuras politicas de prevencion, mediacion y cohesion social en Cataluna. EnglishThis article examines the concept and the elements that encourage intercultural and interreligious dialogue, from the point of view of experts involved in the management of religious diversity in the public space: policy-makers, associations and academics in Catalonia. It responds to a comprehensive-descriptive study with a qualitative approach and an hermeneutic-interpretative rationale as the most appropriate methodology to examine the accounts of the three aforementioned groups. Eleven individual semi-structured interviews have been conducted with representatives of each group. The results reveal different but complementary definitions of interreligious dialogue: the relationship between culture and religion, dialogue as a first step of mutual recognition and the benefits of it. The participants contribute elements that facilitate this dialogue. It concludes with a management model of religious diversity in the public space with community and educational proposals to guide future prevention, mediation and social cohesion policies in Catalonia.

Reflexiones sobre el proceso de integración de la juventud extranjera en Cataluña: un enfoque socioeducativo

This paper presents an analysis of the process of integration among migrant young people in Catalonia aged from 14 to 18. For this purpose, a study was made, using a survey and four discussion groups probing the points of view of both the migrant and native youth. Results are organised around a model of integration based on four core dimensions: structural, cognitive-cultural, social and of identity; and confirm that a society which is plural in its beliefs, convictions and forms should be reflected in democratic systems and social and educational policies based on a concept of integration as reciprocity and understood as a fundamental principle in the management of diversity.

The antigen-binding fragment of human gamma immunoglobulin prevents amyloid β-peptide folding into β-sheet to form oligomers

The amyloid beta-peptide (Aβ) plays a leading role in Alzheimers disease (AD) physiopathology. Even though monomeric forms of Aβ are harmless to cells, Aβ can aggregate into β-sheet oligomers and fibrils, which are both neurotoxic. Therefore, one of the main therapeutic approaches to cure or delay AD onset and progression is targeting Aβ aggregation. In the present study, we show that a pool of human gamma immunoglobulins (IgG) protected cortical neurons from the challenge with Aβ oligomers, as assayed by MTT reduction, caspase-3 activation and cytoskeleton integrity. In addition, we report the inhibitory effect of IgG on Aβ aggregation, as shown by Thioflavin T assay, size exclusion chromatography and atomic force microscopy. Similar results were obtained with Palivizumab, a human anti-sincitial virus antibody. In order to dissect the important domains, we cleaved the pool of human IgG with papain to obtain Fab and Fc fragments. Using these cleaved fragments, we functionally identified Fab as the immunoglobulin fragment inhibiting Aβ aggregation, a result that was further confirmed by an in silico structural model. Interestingly, bioinformatic tools show a highly conserved structure able to bind amyloid in the Fab region. Overall, our data strongly support the inhibitory effect of human IgG on Aβ aggregation and its neuroprotective role.

Dysregulated Collagen Homeostasis by Matrix Stiffening and TGF-β1 in Fibroblasts from Idiopathic Pulmonary Fibrosis Patients: Role of FAK/Akt

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease in which normal lung parenchyma is replaced by a stiff dysfunctional scar rich in activated fibroblasts and collagen-I. We examined how the mechanochemical pro-fibrotic microenvironment provided by matrix stiffening and TGF-β1 cooperates in the transcriptional control of collagen homeostasis in normal and fibrotic conditions. For this purpose we cultured fibroblasts from IPF patients or control donors on hydrogels with tunable elasticity, including 3D collagen-I gels and 2D polyacrylamide (PAA) gels. We found that TGF-β1 consistently increased COL1A1 while decreasing MMP1 mRNA levels in hydrogels exhibiting pre-fibrotic or fibrotic-like rigidities concomitantly with an enhanced activation of the FAK/Akt pathway, whereas FAK depletion was sufficient to abrogate these effects. We also demonstrate a synergy between matrix stiffening and TGF-β1 that was positive for COL1A1 and negative for MMP1. Remarkably, the COL1A1 expression upregulation elicited by TGF-β1 alone or synergistically with matrix stiffening were higher in IPF-fibroblasts compared to control fibroblasts in association with larger FAK and Akt activities in the former cells. These findings provide new insights on how matrix stiffening and TGF-β1 cooperate to elicit excessive collagen-I deposition in IPF, and support a major role of the FAK/Akt pathway in this cooperation.

Abstract 4103: Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Permanent cell cycle arrest through senescence has been previously regarded as a protective mechanism against tumor progression. In contrast, there is evidence that senescence in tumor-associated fibroblasts (TAFs) enhances tumor growth. Senescence has been reported in tumor associated fibroblasts (TAFs) from a growing list of selected cancer types. However, the presence of senescent TAFs in lung cancer remains undefined. To address this gap of knowledge, we examined common markers of senescence in primary TAFs from the 3 major non-small cell lung cancer (NSCLC) subtypes: adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large cell carcinoma (LCC). Given the difficulties in gathering LCC-TAFs owing to the lower prevalence of LCC compared to the other subtypes, primary fibroblasts from 2 independent cell collections were used. We found an enrichment of the myofibroblast-like phenotype in TAFs regardless their histologic subtype, yet senescence was observed in LCC-TAFs only. Likewise, co-culturing normal lung fibroblasts with LCC (but not ADC or SCC) cancer cells was sufficient to induce senescence, and this induction was prevented in the presence of an antioxidant, indicating that it is mediated through oxidative stress. Of note, senescent fibroblasts provided growth and invasive advantages to LCC cells in culture and in vivo beyond those provided by control (non-senescent) fibroblasts. These results expand recent evidence that challenges the common assumption that lung TAFs are a heterogeneous myofibroblast-like cell population regardless their histologic subtype. Of note, because LCC often distinguishes itself in the clinic by its aggressive nature, our findings support that senescent or senescent-like TAFs may contribute to the selective aggressive behavior of LCC tumors. Citation Format: Roberto Lugo, Marta Gabasa, Francesca Andriani, Marta Puig, Federica Facchinetti, Josep Ramirez, Abel Gomez-Caro, Ugo Pastorino, Pere Gascon, Albert Davalos, Noemi Reguart, Luca Roz, Jordi Alcaraz. Cancer cell-stromal cell crosstalk drives fibroblast senescence and tumor progression in large cell carcinoma of the lung in culture and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4103.