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Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.
© 2017 Erdogan et al.
Vascular smooth muscle cells (VSMCs) represent important modulators of plaque stability in advanced lesions. We previously reported that loss of small proline-rich repeat protein 3 (Sprr3), leads to VSMC apoptosis in a PI3K/Akt-dependent manner and accelerates lesion progression. Here, we investigated the role of Sprr3 in modulating plaque stability in hyperlipidemic ApoE-/- mice. We show that loss of Sprr3 increased necrotic core size and reduced cap collagen content of atheromas in brachiocephalic arteries with evidence of plaque rupture and development of intraluminal thrombi. Moreover, Sprr3-/-ApoE-/- mice developed advanced coronary artery lesions accompanied by intraplaque hemorrhage and left ventricle microinfarcts. SPRR3 is known to reduce VSMC survival in lesions by promoting their apoptosis. In addition, we demonstrated that Sprr3-/- VSMCs displayed reduced expression of procollagen in a PI3K/Akt dependent manner. SPRR3 loss also increased MMP gelatinase activity in lesions, and increased MMP2 expression, migration and contraction of VSMCs independently of PI3K/Akt. Consequently, Sprr3 represents the first described VSMC modulator of each of the critical features of cap stability, including VSMC numbers, collagen type I synthesis, and protease activity through Akt dependent and independent pathways.
Apicobasolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of cancer. Integrin-mediated contact with the extracellular matrix defines the basal surface, setting in motion E-cadherin-mediated cell-cell contact, which establishes apicobasolateral polarity. Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We show that addition of an integrin β1-activating monoclonal antibody, P4G11, to invasive colorectal cancer cells in three-dimensional type 1 collagen reverts the invasive phenotype and restores apicobasolateral polarity. P4G11 induces clustering of integrin α5β1 at lateral, intercellular surfaces. This leads to deposition and polymerization of fibronectin and recruitment of paxillin to sites of lateral integrin α5β1 clustering and is followed by tight junction formation, as determined by ZO-1 localization. Inducible elimination of integrin α5 abrogates the epithelial-organizing effects of P4G11. In addition, polymerization of fibronectin is required for the effects of P4G11, and addition of polymerized superfibronectin is sufficient to induce tight junction formation and apicobasolateral polarization. In the normal human colon, we show that integrin α5 localizes to the lateral membrane of terminally differentiated colonocytes and that integrin α5 staining may be reduced in colorectal cancer. Thus we propose a novel role for integrin α5β1 in regulating epithelial morphogenesis.
© 2017 Starchenko et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Migration of cells toward chemical cues, or chemotaxis, is important for many biologic processes such as immune defense, wound healing and cancer metastasis. Although chemotaxis is thought to occur in cancer cells, it is less well characterized than chemotaxis of professional immune cells such as neutrophils. Here, we show that cancer cell chemotaxis relies on secretion of exosome-type extracellular vesicles. Migration of fibrosarcoma cells toward a gradient of exosome-depleted serum was diminished by knockdown of the exosome secretion regulator Rab27a. Rescue experiments in which chemotaxis chambers were coated with purified extracellular vesicles demonstrate that exosomes but not microvesicles affect both speed and directionality of migrating cells. Chamber coating with purified fibronectin and fibronectin-depleted exosomes demonstrates that the exosome cargo fibronectin promotes cell speed but cannot account for the role of exosomes in promoting directionality of fibrosarcoma cell movement during chemotaxis. These experiments indicate that exosomes contain multiple motility-promoting cargoes that contribute to different aspects of cell motility.
UNLABELLED - The early diagnosis and successful treatment of breast cancer (BC) is still a challenging task due to the diverse origin and functional heterogeneity of cancer cells. The heterogeneity of BC may likely to explained by molecular BC subtypes, comprises Luminal-A (LA), Luminal-B (LB), Triple-negative (TN) and HER2-positive (HP), which are governed by a variety of cancer associated pathways. To identify protein signatures in different BC subtypes, we performed isobaric tag for absolute and relative quantitation (iTRAQ) of enriched blood plasma samples of BC subtypes (N=32) and healthy subjects (N=8). After analyses of data, 58 proteins were found to be modulated in BC subtypes from healthy subjects (p<0.05) and among these; Fibronectin (FN1), Alpha-2-macroglobulin (A2M), and Complement component-4-binding protein-alpha (C4BPA) and Complement factor-B (CFB) were selected for validation in BC subtypes and healthy subjects in the independent set of blood plasma (N=100) and tissue samples (N=25). Statistical analysis showed the significant modulation of FN1 and C4BPA in LB, and A2M in TN patients in both plasma as well as tissues comparatively control (p<0.05). Further, FN1 and C4BPA in LB subtype revealed a good diagnostic accuracy in plasma level validation. The receiver operating characteristic (ROC) curve and regression analysis demonstrated that these proteins with associated criterion of expression could act as discriminating signatures among BC subtypes with diagnostic and prognostic relevance.
SIGNIFICANCE - The heterogeneity of breast cancer (BC) has gained many challenges for successful management of BC, thus, the delineating proteomic alterations BC subtypes may provide great clinical values in diagnostic, prognostic and therapeutics of BC. The findings from the present quantitative proteomic study have deciphered the altered proteomic patterns and their possible molecular interactions in each BC subtype. The study showed a strong association of FN1, A2M, C4BPA and CFB in molecular subtypes of BC, in which, C4BPA and A2M demonstrated a potent signature in blood plasma and tissue samples of LB and TN subtypes in BC patients, respectively. The findings also revealed the altered level expressions of these selected proteins could classify BC subtypes through plasma and tissue based expression analysis in patients and control samples. Hence, these proteins could have clinical importance for the diagnosis and prognosis purposes among molecular BC subtypes.
Copyright © 2016 Elsevier B.V. All rights reserved.
The extracellular matrix protein fibronectin (FN) contributes to the structural integrity of tissues as well as the adhesive and migratory functions of cells. While FN is abundantly expressed in adult tissues, the expression of several alternatively spliced FN isoforms is restricted to embryonic development, tissue remodeling and cancer. These FN isoforms, designated ED-A and ED-B, are frequently expressed by cancer cells, tumor-associated fibroblasts and newly forming blood vessels. Using a highly sensitive collagen-based indirect ELISA, we evaluated the correlation of urinary ED-A and ED-B at time of cystectomy with overall survival in patients with high-grade bladder cancer (BCa). Detectable levels of total FN as well as ED-A and ED-B were found in urine from 85, 73 and 51 % of BCa patients, respectively. The presence of urinary ED-A was a significant independent predictor of 2-year overall survival (OS) after adjusting for age, tumor stage, lymph node stage, and urinary creatinine by multivariable Logistic Regression (p = 0.029, OR = 4.26, 95 % CI 1.16-15.71) and improved accuracy by 3.6 %. Furthermore, detection of ED-A in the urine was a significant discriminator of survival specifically in BCa patients with negative lymph node status (Log-Rank, p = 0.006; HR = 5.78, 95 % CI 1.39-24.13). Lastly, multivariable Cox proportional hazards analysis revealed that urinary ED-A was an independent prognostic indicator of 5-year OS rate for patients with BCa (p = 0.04, HR = 2.20, 95 % CI 1.04-4.69). Together, these data suggest that cancer-derived, alternatively spliced FN isoforms can act as prognostic indicators and that additional studies are warranted to assess the clinical utility of ED-A in BCa.
Directional cell movement through tissues is critical for multiple biological processes and requires maintenance of polarity in the face of complex environmental cues. Here we use intravital imaging to demonstrate that secretion of exosomes from late endosomes is required for directionally persistent and efficient in vivo movement of cancer cells. Inhibiting exosome secretion or biogenesis leads to defective tumour cell migration associated with increased formation of unstable protrusions and excessive directional switching. In vitro rescue experiments with purified exosomes and matrix coating identify adhesion assembly as a critical exosome function that promotes efficient cell motility. Live-cell imaging reveals that exosome secretion directly precedes and promotes adhesion assembly. Fibronectin is found to be a critical motility-promoting cargo whose sorting into exosomes depends on binding to integrins. We propose that autocrine secretion of exosomes powerfully promotes directionally persistent and effective cell motility by reinforcing otherwise transient polarization states and promoting adhesion assembly.
Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.
The filtration unit of the kidney is the glomerulus, a capillary network supported by mesangial cells and extracellular matrix (ECM). Glomerular function is compromised in diabetic nephropathy (DN) by uncontrolled buildup of ECM, especially type IV collagen, which progressively occludes the capillaries. Increased levels of the ECM protein fibronectin (FN) are also present; however, its role in DN is unknown. Mesangial cells cultured under high glucose conditions provide a model system for studying the effect of elevated glucose on deposition of FN and collagen IV. Imaging of mesangial cell cultures and analysis of detergent-insoluble matrix show that, under high glucose conditions, mesangial cells assembled significantly more FN matrix, independent of FN protein levels. High glucose conditions induced protein kinase C-dependent β1 integrin activation, and FN assembly in normal glucose was increased by stimulation of integrin activity with Mn(2+). Collagen IV incorporation into the matrix was also increased under high glucose conditions and colocalized with FN fibrils. An inhibitor of FN matrix assembly prevented collagen IV deposition, demonstrating dependence of collagen IV on FN matrix. We conclude that high glucose induces FN assembly, which contributes to collagen IV accumulation. Enhanced assembly of FN might facilitate dysregulated ECM accumulation in DN.
© 2014 Miller et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
During tumorigenesis, matrix rigidity can drive oncogenic transformation via altered cellular proliferation and migration. Cells sense extracellular matrix (ECM) mechanical properties with intracellular tensile forces generated by actomyosin contractility. These contractile forces are transmitted to the matrix surface as traction stresses, which mediate mechanical interactions with the ECM. Matrix rigidity has been shown to increase proteolytic ECM degradation by cytoskeletal structures known as invadopodia that are critical for cancer progression, suggesting that cellular contractility promotes invasive behavior. However, both increases and decreases in traction stresses have been associated with metastatic behavior. Therefore, the role of cellular contractility in invasive migration leading to metastasis is unclear. To determine the relationship between cellular traction stresses and invadopodia activity, we characterized the invasive and contractile properties of an aggressive carcinoma cell line utilizing polyacrylamide gels of different rigidities. We found that ECM degradation and traction stresses were linear functions of matrix rigidity. Using calyculin A to augment myosin contractility, we also found that traction stresses were strongly predictive of ECM degradation. Overall, our data suggest that cellular force generation may play an important part in invasion and metastasis by mediating invadopodia activity in response to the mechanical properties of the tumor microenvironment.
Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.