Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli

Abstract Background Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-β1 and substrate stiffness affect fibroblast Fas expression are not well understood. Methods Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young’s moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-β1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. Results Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-β1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-β1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-β1. Conclusions Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.https://deepblue.lib.umich.edu/bitstream/2027.42/143539/1/12931_2018_Article_801.pd

Chemosensitization by phenothiazines in human lung cancer cells: impaired resolution of γH2AX and increased oxidative stress elicit apoptosis associated with lysosomal expansion and intense vacuolation

Chemotherapy resistance poses severe limitations on the efficacy of anti-cancer medications. Recently, the notion of using novel combinations of ‘old' drugs for new indications has garnered significant interest. The potential of using phenothiazines as chemosensitizers has been suggested earlier but so far our understanding of their molecular targets remains scant. The current study was designed to better define phenothiazine-sensitive cellular processes in relation to chemosensitivity. We found that phenothiazines shared the ability to delay γH2AX resolution in DNA-damaged human lung cancer cells. Accordingly, cells co-treated with chemotherapy and phenothiazines underwent protracted cell-cycle arrest followed by checkpoint escape that led to abnormal mitoses, secondary arrest and/or a form of apoptosis associated with increased endogenous oxidative stress and intense vacuolation. We provide evidence implicating lysosomal dysfunction as a key component of cell death in phenothiazine co-treated cells, which also exhibited more typical hallmarks of apoptosis including the activation of both caspase-dependent and -independent pathways. Finally, we demonstrated that vacuolation in phenothiazine co-treated cells could be reduced by ROS scavengers or the vacuolar ATPase inhibitor bafilomycin, leading to increased cell viability. Our data highlight the potential benefit of using phenothiazines as chemosensitizers in tumors that acquire molecular alterations rendering them insensitive to caspase-mediated apoptosis

Protective effect of geranylgeranylacetone, an inducer of heat shock protein 70, against drug-induced lung injury/fibrosis in an animal model

<p>Abstract</p> <p>Background</p> <p>To determine whether oral administration of geranylgeranylacetone (GGA), a nontoxic anti-ulcer drug that is an inducer of heat shock protein (HSP) 70, protects against drug-induced lung injury/fibrosis <it>in vivo</it>.</p> <p>Methods</p> <p>We used a bleomycin (BLM)-induced lung fibrosis model in which mice were treated with oral 600 mg/kg of GGA before and after BLM administration. Inflammation and fibrosis were evaluated by histological scoring, hydroxyproline content in the lung and inflammatory cell count, and quantification by ELISA of macrophage inflammatory protein-2 (MIP-2) in bronchoalveolar lavage fluid. Apoptosis was evaluated by the TUNEL method. The induction of HSP70 in the lung was examined with western blot analysis and its localization was determined by immunohistochemistry.</p> <p>Results</p> <p>We confirmed the presence of inflammation and fibrosis in the BLM-induced lung injury model and induction of HSP70 by oral administration of GGA. GGA prevented apoptosis of cellular constituents of lung tissue, such as epithelial cells, most likely related to the <it>de novo </it>induction of HSP70 in the lungs. GGA-treated mice also showed less fibrosis of the lungs, associated with the findings of suppression of both production of MIP-2 and inflammatory cell accumulation in the injured lung, compared with vehicle-treated mice.</p> <p>Conclusion</p> <p>GGA had a protective effect on drug-induced lung injury/fibrosis. Disease-modifying antirheumatic drugs such as methotrexate, which are indispensable for the treatment of rheumatoid arthritis, often cause interstitial lung diseases, an adverse event that currently cannot be prevented. Clinical use of GGA for drug-induced pulmonary fibrosis might be considered in the future.</p

CD44v4 Is a Major E-Selectin Ligand that Mediates Breast Cancer Cell Transendothelial Migration

BACKGROUND: Endothelial E-selectin has been shown to play a pivotal role in mediating cell-cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. However, the counterreceptor for E-selectin and its role in mediating breast cancer cell transendothelial migration remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: By assessing migration of various breast cancer cells across TNF-alpha pre-activated human umbilical vein endothelial cells (HUVECs), we found that breast cancer cells migrated across HUVEC monolayers differentially and that transmigration was E-selectin dependent. Cell surface labeling with the E-selectin extracellular domain/Fc chimera (exE-selectin/Fc) showed that the transmigration capacity of breast cancer cells was correlated to both the expression level and localization pattern of E-selectin binding protein(s) on the tumor cell surface. The exE-selectin/Fc strongly bound to metastatic MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells, but not non-metastatic MCF-7 and T47D cells. Binding of exE-selectin/Fc was abolished by removal of tumor cell surface sialyl lewis x (sLe(x)) moieties. Employing an exE-selectin/Fc affinity column, we further purified the counterreceptor of E-selectin from metastatic breast cancer cells. The N-terminal protein sequence and cDNA sequence identified this E-selectin ligand as a approximately 170 kD human CD44 variant 4 (CD44v4). Purified CD44v4 showed a high affinity for E-selectin via sLe(x) moieties and, as expected, MDA-MB-231 cell adhesion to and migration across HUVEC monolayers were significantly reduced by down-regulation of tumor cell CD44v4 via CD44v4-specific siRNA. CONCLUSIONS/SIGNIFICANCE: We demonstrated, for the first time, that breast cancer cell CD44v4 is a major E-selectin ligand in facilitating tumor cell migration across endothelial monolayers. This finding offers new insights into the molecular basis of E-selectin-dependent adhesive interactions that mediate breast cancer cell transendothelial metastasis

Exon-Level Transcriptome Profiling in Murine Breast Cancer Reveals Splicing Changes Specific to Tumors with Different Metastatic Abilities

In breast cancer patients, tumor metastases at distant sites are the main cause of death. However, the molecular mechanisms of metastasis of breast cancer remain unclear. It is thought that changes occurring at the level of RNA processing contribute to cancer. Alternative splicing (AS) of pre-mRNA, a key post-transcriptional mechanism allowing for the production of distinct proteins from a single gene, affects over 90% of human genes. Such splicing events are responsible for generating mRNAs that encode protein isoforms that can have very different biological properties and functions. A well-studied example is the BCL-X gene, whose two major transcript isoforms produce two proteins having antagonistic functions: the short form (BCL-XS) promotes apoptosis while the long form (BCL-XL) is anti-apoptotic. Moreover, overexpression of BCL-XL has been reported to enhance the metastatic potential of breast tumor cells in patients

Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms

Adhesion and migration are integrated cell functions that build, maintain and remodel the multicellular organism. In migrating cells, integrins are the main transmembrane receptors that provide dynamic interactions between extracellular ligands and actin cytoskeleton and signalling machineries. In parallel to integrins, other adhesion systems mediate adhesion and cytoskeletal coupling to the extracellular matrix (ECM). These include multifunctional cell surface receptors (syndecans and CD44) and discoidin domain receptors, which together coordinate ligand binding with direct or indirect cytoskeletal coupling and intracellular signalling. We review the way that the different adhesion systems for ECM components impact cell migration in two- and three-dimensional migration models. We further discuss the hierarchy of these concurrent adhesion systems, their specific tasks in cell migration and their contribution to migration in three-dimensional multi-ligand tissue environments

Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

Background: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. Methods: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. Results: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. Conclusions: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance