Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis.

Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies

Effects of tobacco smoke on gene expression and cellular pathways in a cellular model of oral leukoplakia

Abstract In addition to being causally linked to the formation of multiple tumor types, tobacco use has been associated with decreased efficacy of anticancer treatment and reduced survival time. A detailed understanding of the cellular mechanisms that are affected by tobacco smoke (TS) should facilitate the development of improved preventive and therapeutic strategies. We have investigated the effects of a TS extract on the transcriptome of MSK-Leuk1 cells, a cellular model of oral leukoplakia. Using Affymetrix HGU133 Plus 2 arrays, 411 differentially expressed probe sets were identified. The observed transcriptome changes were grouped according to functional information and translated into molecular interaction network maps and signaling pathways. Pathways related to cellular proliferation, inflammation, apoptosis, and tissue injury seemed to be perturbed. Analysis of networks connecting the affected genes identified specific modulated molecular interactions, hubs, and key transcription regulators. Thus, TS was found to induce several epidermal growth factor receptor (EGFR) ligands forming an EGFR-centered molecular interaction network, as well as several aryl hydrocarbon receptor-dependent genes, including the xenobiotic metabolizing enzymes CYP1A1 and CYP1B1. Notably, the latter findings in vitro are consistent with our parallel finding that CYP1A1 and CYP1B1 levels were increased in oral mucosa of smokers. Collectively, these results offer insights into the mechanisms underlying the procarcinogenic effects of TS and raise the possibility that inhibitors of EGFR or aryl hydrocarbon receptor signaling will prevent or delay the development of TS-related tumors. Moreover, the inductive effects of TS on xenobiotic metabolizing enzymes may help explain the reduced efficacy of chemotherapy, and suggest targets for chemopreventive agents in smokers

Obesity-dependent changes in interstitial ECM mechanics promote breast tumorigenesis

Obesity and extracellular matrix (ECM) density are considered independent risk and prognostic factors for breast cancer. Whether they are functionally linked is uncertain. We investigated the hypothesis that obesity enhances local myofibroblast content in mammary adipose tissue and that these stromal changes increase malignant potential by enhancing interstitial ECM stiffness. Indeed, mammary fat of both diet- and genetically induced mouse models of obesity were enriched for myofibroblasts and stiffness-promoting ECM components. These differences were related to varied adipose stromal cell (ASC) characteristics because ASCs isolated from obese mice contained more myofibroblasts and deposited denser and stiffer ECMs relative to ASCs from lean control mice. Accordingly, decellularized matrices from obese ASCs stimulated mechanosignaling and thereby the malignant potential of breast cancer cells. Finally, the clinical relevance and translational potential of our findings were supported by analysis of patient specimens and the observation that caloric restriction in a mouse model reduces myofibroblast content in mammary fat. Collectively, these findings suggest that obesity-induced interstitial fibrosis promotes breast tumorigenesis by altering mammary ECM mechanics with important potential implications for anticancer therapies

Transforming growth factor‐β1 is a heparin‐binding protein: Identification of putative heparin‐binding regions and isolation of heparins with varying affinity for TGF‐β1

Previous studies indicated that a major factor in heparin\u27s ability to suppress the proliferation of vascular smooth muscle cells is an interaction with transforming growth factor‐β1 (TGF‐β1). Heparin appeared to bind directly to TGF‐β1 and to prevent the association of TGF‐β1 with α2‐macroglobulin (α2‐M). The present studies indicate that 20–70% of iodinated TGF‐β1 binds to heparin‐Sepharose and the retained fraction is eluted with ∼0.37 M NaCI. Native, unlabelled platelet TGF‐β1, however, is completely retained by heparin‐Sepharose and eluted with 0.9–1.2 M NaCI. Using synthetic peptides, the regions of TGF‐β1 that might be involved in the binding of heparin and other polyanions were examined. Sequence analysis of TGF‐β1 indicated three regions with a high concentration of basic residues. Two of these regions had the basic residues arranged in a pattern homologous to reported consensus heparin‐binding regions of other proteins. The third constituted a structurally novel pattern of basic residues. Synthetic peptides homologous to these three regions, but not to other regions of TGF‐β1, were found to bind to heparin‐Sepharose and were eluted with 0.15 M‐0.30 M NaCI. Only two of these regions were capable of blocking the binding of heparin to 125I‐TGF‐β. Immobilization of these peptides, followed by affinity purification of heparin, indicated that one peptide was capable of isolating subspecies of heparin with high and low affinity for authentic TGF‐β1. The ability of TGF‐β1 to bind to heparin or related proteoglycans under physiological conditions may be useful in understanding the biology of this pluripotent growth and metabolic signal. Conversely, a subspecies of heparin molecules with high affinity for TGF‐β1 may be a factor in some of the diverse biological actions of heparin. © 1992 Wiley‐Liss, Inc. Copyright © 1992 Wiley‐Liss, Inc

Translocation of protein kinase C-δ by PDGF in cultured vascular smooth muscle cells: Inhibition by TGF-β1

The migration and proliferation of vascular smooth muscle cells (SMC) into the neointima are important early events in the development of atherosclerosis and post-angioplasty restenosis. The stimulation of SMC migration by platelet derived growth factor (PDGF) involves the induction of protein kinase C activity. Using immunoblot techniques, we demonstrated that rat aortic SMC express a pattern of PKC isoforms which includes PKC-α, δ, ε, ζ, and η. Upon exposure to PDGF-BB, a fraction of PKC-δ was rapidly translocated from the cytosol to the post-nuclear particulate fraction at 15 seconds and reached an apparent maximum at 30 minutes. In contrast, PKC-α and ζ were not translocated by PDGF-BB. TGF-β1, which inhibits PDGF- induced DNA synthesis and chemotaxis, reduced the immunoreactive levels of PKC-δ and blocked the PDGF-induced translocation of PKC-δ to the particulate fraction. This suggests that the activation of PKC-δ by translocation to the particulate fraction may play an important role in the control of vascular smooth muscle cell migration by PDGF and TGF-β1

Protection of transforming growth factor β activity by heparin and fucoidan

The transforming growth factor‐β (TGF‐β) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF‐β activity in vitro or in vivo. Our previous work indicated that (1) TGF‐β1 has strong heparin‐binding properties that were not previously recognized because of neutralization by iodination, and (2) heparin, and certain other polyanions, could block the binding of TGF‐β1 to α2‐macroglobulin (α2‐M). The present studies investigated the influence of heparin‐like molecules on the stability of the TGF‐β1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L‐fucose polymer, suppress the formation of an initial non‐covalent interaction between 125I‐TGF‐β1 and activated α2‐M. Electrophoresis of 125I‐TGF‐β1 showed that fucoidan protects TGF‐β1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF‐β derived from vascular smooth muscle cells (SMC), plasmin degraded acid‐activated TGF‐β, and purified TGF‐β1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half‐life of 125I‐TGF‐β1 and doubled the amount of cell‐associated 125I‐TGF‐β1. Consistent with this protective effect, heparin‐ and fucoidan‐treated SMC demonstrated elevated levels of active, but not latent, TGF‐β activity. © 1994 wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc

Genomic profiling of acquired resistance to apoptosis in cells derived from human atherosclerotic lesions: Potential role of STATs, cyclinD1, BAD, and Bcl-X\u3csub\u3eL\u3c/sub\u3e

Current theories suggest that atherosclerosis, plaque rupture, stroke, and restenosis after angioplasty may involve defective apoptotic mechanisms in vascular cells. Prior work has demonstrated that cells from human atherosclerotic lesions, and cells from the aorta of aged rats, exhibit functional resistance to apoptosis induced by TGF-β and glucocorticoids. The present studies demonstrate that human lesion-derived cells (LDC) are also resistant to apoptosis induced by fas ligation compared to cells derived from the adjacent media, and that in vitro expansion of LDC causes acquired resistance to apoptosis. Microarray profiling of fas-resistant versus sensitive cells identified a set of genes including STATs, caspase 1, cyclin D1, Bcl-xL, VDAC2, and BAD. The STAT proteins have been implicated in resistance to apoptosis, potentially via their ability to modulate caspase 1 (ICE), Bcl-xL, and cyclin D1 expression. Western blot analysis of sensitive and resistant LDC clonal lines confirmed increases in cyclin D1, STAT6, Bcl-xL, and BAD, with decreased expression of caspase 1. Thus, transcript profiling has identified a potential pathway of apoptotic regulation in subsets of lesion cells. The resistant phenotype may contribute to plaque stability and excessive vascular repair, while sensitive cells may be involved in plaque rupture and infarction. The data suggests both genetic interventions and novel small-molecule inhibitors that may be effective modulators of apoptosis in atherosclerosis, angina, and in-stent restenosis. © 2005 Elsevier Ltd. All rights reserved

Elevated Egr-1 in human atherosclerotic cells transcriptionally represses the transforming growth factor-β Type II receptor

Atherosclerotic lesions may progress due to a failure to die by vascular repair cells. Egr-1, a zinc finger transcription factor, is elevated more than 5-fold in human carotid lesions relative to the adjacent tunica media. Lesion cells in vitro also express 2-3-fold higher Egr-1 mRNA and protein levels but express much lower levels of the transforming growth factor-β (TGF-β) Type II receptor (TβR-2) and are functionally resistant to the antiproliferative effects of TGF-β. Lesion cells fail to express a TβR-2 promoter/chloramphenicol acetyltransferase (CAT) construct but overexpress an Egr-1-inducible platelet-derived growth factor-A promoter/CAT construct. Transfection of Egr-1 cDNA represses TβR-2/CAT constructs but induces PDGF-A/CAT. Egr-1 transfection reduces the levels of TβR-2 and confers resistance to the antiproliferative effect of TGF-β1. Egr-1 can interact directly with both the -143 Sp1 site and the positive regulatory element 2 (PRE2) (ERT/ets) region of the TβR-2 promoter. Thus, although activating a family of stress-responsive genes, Egr-1 also transcriptionally represses one of the major inhibitory pathways that restrains vascular repair