The integrin needle in the stromal haystack: emerging role in corneal physiology and pathology

Several studies have established the role of activated corneal keratocytes in the fibrosis of the cornea. However, the role of keratocytes in maintaining the structural integrity of a normal cornea is less appreciated. We focus on the probable functions of integrins in the eye and of the importance of integrin-mediated keratocyte interactions with stromal matrix in the maintenance of corneal integrity. We point out that further understanding of how keratocytes interact with their matrix could establish a novel direction in preventing corneal pathology including loss of structural integrity as in keratoconus or as in fibrosis of the corneal stroma.     

Laminin interactions with head and neck cancer cells under low fluid shear conditions lead to integrin activation and binding

Lymphatic metastasis of cancer cells involves movement from the primary tumor site to the lymph node, where the cells must be able to productively lodge and grow. It is there that tumor cells encounter cellular and non-cellular constituent elements that make up the lymph node parenchyma. Our work shows that head and neck squamous cell carcinoma (HNSCC) cell lines are able to bind to laminin, fibronectin, vitronectin, and hyaluronic acid, which are extracellular matrix elements within the lymph node parenchyma. HNSCC cell lines bound to laminin under lymphodynamic low shear stress (0.07 dynes/cm(2)), consistent with lymph flow via β1 integrins, including α2β1, α3β1, and α6β1. Binding occurred in the presence of shear stress and not in the absence of flow. Additionally, tumor cell binding to laminin under flow did result in calcium signaling. Our data indicate a novel role for β1 integrin-mediated binding of HNSCC cells to laminin under conditions of lymphodynamic flow that results in intracellular calcium signaling within the cancer cell.     

Invasion as target for therapy of glioblastoma multiforme

The survival of cancer patients suffering from glioblastoma multiforme is limited to just a few months even after treatment with the most advanced techniques. The indefinable borders of glioblastoma cell infiltration into the surrounding healthy tissue prevent complete surgical removal. In addition, genetic mutations, epigenetic modifications and microenvironmental heterogeneity cause resistance to radio- and chemotherapy altogether resulting in a hardly to overcome therapeutic scenario. Therefore, the development of efficient therapeutic strategies to combat these tumors requires a better knowledge of genetic and proteomic alterations as well as the infiltrative behavior of glioblastoma cells and how this can be targeted. Among many cell surface receptors, members of the integrin family are known to regulate glioblastoma cell invasion in concert with extracellular matrix degrading proteases. While preclinical and early clinical trials suggested specific integrin targeting as a promising therapeutic approach, clinical trials failed to deliver improved cure rates up to now. Little is known about glioblastoma cell motility, but switches in invasion modes and adaption to specific microenvironmental cues as a consequence of treatment may maintain tumor cell resistance to therapy. Thus, understanding the molecular basis of integrin and protease function for glioblastoma cell invasion in the context of radiochemotherapy is a pressing issue and may be beneficial for the design of efficient therapeutic approaches. This review article summarizes the latest findings on integrins and extracellular matrix in glioblastoma and adds some perspective thoughts on how this knowledge might be exploited for optimized multimodal therapy approaches.     

Shear stress modulates the interaction of platelet-secreted matrix proteins with tumor cells through the integrin alphavbeta3

Interaction of tumor cells with the vascular wall is required for metastasis from the bloodstream. The precise interaction among metastatic cells, circulating platelets, the vessel wall, and physiological flow conditions remains to be determined. In this study, we investigated the interaction of shear on metastatic cell lines adherent to lipopolysaccharide (LPS)-treated endothelium. Tumor cells were perfused over LPS-treated human umbilical vein endothelial cells (HUVECs) at incremental venous shear rates from 50 to 800 s^–1. At a venous shear rate of 400 s^–1, 3% of adherent tumor cells formed pseudopodia under shear, a process we termed shear-induced activation. Because platelets promote tumor dissemination, we then investigated the effect of pretreating tumor cells with platelet releasate collected from activated platelet concentrate. We found that in the presence of platelet releasate, the number of tumor cells adhering to HUVECs increased and tumor "activation" occurred at a significantly lower shear rate of 50 s^–1. This was inhibited with acetylsalicylic acid. Depletion of fibronectin or vitronectin from the platelet releasate resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s^–1. The integrin _v3 has been shown to mediate cell adhesion primarily through vitronectin and fibronectin proteins. Inhibition of _v3, followed by the addition of platelet releasate to the tumor cells, resulted in significantly less adhesion at higher venous shear rates of 600 and 800 s^–1. Collectively, our data suggest that _v3 promotes the metastatic phenotype of tumor cells through interactions with the secreted platelet proteins vitronectin and fibronectin under venous shear conditions. platelet releasate; vitronectin; fibronectin     

The role of alpha3beta1 integrin in determining the supramolecular organization of laminin-5 in the extracellular matrix of keratinocytes

Analyses of mice with targeted deletions in the genes for alpha3 and beta1 integrin suggest that the alpha3beta1 integrin heterodimer likely determines the organization of the extracellular matrix within the basement membrane of skin. Here we tested this hypothesis using keratinocytes derived from alpha3 integrin-null mice. We have compared the organizational state of laminin-5, a ligand of alpha3beta1 integrin, in the matrix of wild-type keratinocytes with that of laminin-5 in the matrix of alpha3 integrin-null cells. Laminin-5 distributes diffusely in arc structures in the matrix of wild-type mouse keratinocytes, whereas laminin-5 is organized into linear, spike-like arrays by the alpha3 integrin-null cells. The fact that alpha3 integrin-null cells are deficient in their ability to assemble a proper laminin-5 matrix is also shown by their failure to remodel laminin-5 when plated onto surfaces coated with purified laminin-5 protein. In sharp contrast, wild-type keratinocytes organize exogenously added laminin-5 into discrete ring-like organizations. These findings led us next to assess whether differences in laminin-5 organization in the matrix of the wild-type and alpha3 integrin-null cells impact cell behavior. Our results indicate that alpha3 integrin-null cells are more motile than their wild-type counterparts and leave extensive trails of laminin-5 over the surface on which they move. Moreover, HEK 293 cells migrate significantly more on the laminin-5-rich matrix derived from the alpha3 integrin-null cells than on the wild-type keratinocyte laminin-5 matrix. In addition, alpha3 integrin-null cells show low strength of adhesion to surfaces coated with purified laminin-5 compared to wild-type cells although both the wild type and the alpha3 integrin-null keratinocytes adhere equally strongly to laminin-5 that has been organized into arrays by other epithelial cells. These data suggest: (1) that alpha3beta1 integrin plays an important role in determining the incorporation of laminin-5 into its proper higher-order structure within the extracellular matrix of keratinocytes and (2) that the organizational state of laminin-5 has an influence on laminin-5 matrix function.     

alphaVbeta8 integrin is a Schwann cell receptor for fibrin

The interaction of Schwann cells with molecules in the extracellular environment following peripheral nerve injury is a critical aspect of nerve repair. A principal component of this material is fibrin, which derives from fibrinogen infiltrating into the nerve after the injury. This study was undertaken to identify cell surface receptor(s) that mediate the interaction of Schwann cells with fibrin. We found that adhesion of Schwann cells to fibrin could be effectively inhibited by low concentrations of RGD-containing peptides. Among RGD-sensitive integrins expressed by Schwann cell, alphaVbeta8, but not alpha5beta1, was found to bind to fibrin-Sepharose. In contrast, both of these integrins bound to fibronectin-Sepharose. We also found that alphaV, but not alpha5 or beta1 integrin subunit, accumulated in focal contacts of Schwann cell plated on fibrin. Taken together, these results strongly suggest that alphaVbeta8 integrin is a Schwann cell receptor for fibrin.     

Invadopodia: a guided tour

The controlled degradation of extracellular matrix is crucial in physiological and pathological cell invasion alike. In cultured cells, degradation occurs at specific sites where invasive cells make contact with the extracellular matrix via specialized plasma membrane protrusions termed invadopodia. Considerable progress has been made in recent years towards understanding the basic molecular components and the ultrastructural features of invadopodia. This current knowledge will be reviewed here together with some of the most important open questions in invadopodia biology. Considering the substantial interest and momentum in the field, the need for an operational framework to correctly define and identify invadopodia will also be discussed.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Laminin isoforms and their integrin receptors in glioma cell migration and invasiveness: Evidence for a role of alpha5-laminin(s) and alpha3beta1 integrin

Glioma cell infiltration of brain tissue often occurs along the basement membrane (BM) of blood vessels. In the present study we have investigated the role of laminins, major structural components of BMs and strong promoters of cell migration. Immunohistochemical studies of glioma tumor tissue demonstrated expression of alpha2-, alpha3-, alpha4- and alpha5-, but not alpha1-, laminins by the tumor vasculature. In functional assays, alpha3 (Lm-332/laminin-5)- and alpha5 (Lm-511/laminin-10)-laminins strongly promoted migration of all glioma cell lines tested. alpha1-Laminin (Lm-111/laminin-1) displayed lower activity, whereas alpha2 (Lm-211/laminin-2)- and alpha4 (Lm-411/laminin-8)-laminins were practically inactive. Global integrin phenotyping identified alpha3beta1 as the most abundant integrin in all the glioma cell lines, and this laminin-binding integrin exclusively or largely mediate the cell migration. Moreover, pretreatment of U251 glioma cells with blocking antibodies to alpha3beta1 integrin followed by intracerebral injection into nude mice inhibited invasion of the tumor cells into the brain tissue. The cell lines secreted Lm-211, Lm-411 and Lm-511, at different ratios. The results indicate that glioma cells secrete alpha2-, alpha4- and alpha5-laminins and that alpha3- and alpha5-laminins, found in brain vasculature, selectively promote glioma cell migration. They identify alpha3beta1 as the predominant integrin and laminin receptor in glioma cells, and as a brain invasion-mediating integrin.     

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and β1 integrin

We studied the induction of protease activity by the laminin alpha1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M1 cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Requirement of the conserved, hydrophobic C-terminus region for the activation of heparanase

Heparanase is an endo-β-d-glucuronidase responsible for the cleavage of heparan sulfate, participating in extracellular matrix degradation and remodeling. Heparanase activity is well correlated with the potential for metastasis and angiogenesis in a large number of tumor-derived cell types, directly implicating the involvement of heparanase in tumor progression. Here, we provide the first evidence that the hydrophobic C-terminus region of heparanase has specific roles in intracellular trafficking, secretion, activation, and heparanase-mediated tumor cell migration. Furthermore, partial deletion of this hydrophobic C-terminus region, substitution within the hydrophobic C-terminus region to hydrophilic amino acids, and experiments of single amino acid mutations further point out the importance of the hydrophobic C-terminus region. Therefore, our findings suggest that the hydrophobic C-terminus region of heparanase is a determinant for its intracellular trafficking to the Golgi apparatus, followed by secretion, activation, and tumor cell migration.     

Lymphangiogenesis in post-natal tissue remodeling: lymphatic endothelial cell connection with its environment

The main physiological function of the lymphatic vasculature is to maintain tissue fluid homeostasis. Lymphangiogenesis or de novo lymphatic formation is closely associated with tissue inflammation in adults (i.e. wound healing, allograft rejection, tumor metastasis). Until recently, research on lymphangiogenesis focused mainly on growth factor/growth factor-receptor pathways governing this process. One of the lymphatic vessel features is the incomplete or absence of basement membrane. This close association of endothelial cells with the underlying interstitial matrix suggests that cell–matrix interactions play an important role in lymphangiogenesis and lymphatic functions. However, the exploration of interaction between extracellular matrix (ECM) components and lymphatic endothelial cells is in its infancy. Herein, we describe ECM–cell and cell–cell interactions on lymphatic system function and their modification occurring in pathologies including cancer metastasis.     

Enhanced expression of Hab18g/CD147 and activation of integrin pathway in HCC cells under 3-D co-culture conditions

CD147 is reported to be correlated with the malignancy of some cancers, and its overexpression affects the progression of tumor. In the present study, we investigated the function of HAb18G/CD147, a member of CD147 family, on hepatocellular carcinoma (HCC) adhesion, invasion and metastasis in 3-dimensional (3-D) cell co-culture model. The results showed that the extracellular microenvironment could determine the cellular phenotypes and then affected the cellular functions. The expressions of HAb18G/CD147 in HCC cells and fibroblasts were both obviously elevated in 3-D co-culture model. The overexpression of HAb18G/CD147 increased MMPs' (MMP-2 and MMP-9) production (P < 0.01), and was obviously accompanied with enhanced expressions of paxillin, FAK and p-FAK in 3-D cell co-culture model. All the results suggest that HAb18G/CD147 plays an important role in HCC adhesion, invasion and metastasis mainly via modulating synthesis of MMPs and activating integrin signal pathways in fibroblasts and tumor cells themselves under the 3-D co-culture conditions.     

Engineered fibronectin type III domain with a RGDWXE sequence binds with enhanced affinity and specificity to human alphavbeta3 integrin

Fibronectin is an extracellular matrix protein with broad binding specificity to cell surface receptors, integrins. The tenth fibronectin type III domain (FNfn10) is a small, autonomous domain of fibronectin containing the RGE sequence that is directly involved in integrin binding. However, in isolation FNfn10 only weakly bind to integrins. We reasoned that high-affinity and high-specificity variants of FNfn10 to a particular integrin could be engineered by optimizing residues surrounding the integrin-binding RGD sequence in the flexible FG loop. Affinity maturation of FNfn10 to alphavbeta3 integrin, an integrin up-regulated in angiogenic endothelial cells and in some metastatic tumor cells, yielded alphavbeta3-binding FNfn10 mutants with a novel RGDWXE consensus sequence. We characterized one of the RGDWXE-modified clones, FNfn10-3JCLI4, as purified protein. FNfn10-3JCLI4 binds with high affinity and specificity to purified alphavbeta3 integrin. Alanine scanning mutagenesis suggested that both the tryptophan and glutamic acid residues following the RGD sequence are required for maximal affinity and specificity for alphavbeta3. FNfn10-3JCLI4 specifically stained alphavbeta3-positive cells as detected with flow cytometry and it inhibited alphavbeta3-dependent cell adhesion. As with the anti-alphavbeta3 antibody LM609, FNfn10-3JCLI4 can interfere with in vitro capillary formation. Taken together, these data show that FNfn10-3JCL14 is a specific, high-affinity alphavbeta3-binding protein that can inhibit alphavbeta3-dependent cellular processes similar to an anti-alphavbeta3 monoclonal antibody. These properties, combined with the small, monomeric, cysteine-free and highly stable structure of FNfn10-3JCLI4, may make this protein useful in future applications involving detection and targeting of alphavbeta3-positive cells.     

Modeling the tumor extracellular matrix: Tissue engineering tools repurposed towards new frontiers in cancer biology

Cancer progression is mediated by complex epigenetic, protein and structural influences. Critical among them are the biochemical, mechanical and architectural properties of the extracellular matrix (ECM). In recognition of the ECM's important role, cancer biologists have repurposed matrix mimetic culture systems first widely used by tissue engineers as new tools for in vitro study of tumor models. In this review we discuss the pathological changes in tumor ECM, the limitations of 2D culture on both traditional and polyacrylamide hydrogel surfaces in modeling these characteristics and advances in both naturally derived and synthetic scaffolds to facilitate more complex and controllable 3D cancer cell culture. Studies using naturally derived matrix materials like Matrigel and collagen have produced significant findings related to tumor morphogenesis and matrix invasion in a 3D environment and the mechanotransductive signaling that mediates key tumor–matrix interaction. However, lack of precise experimental control over important matrix factors in these matrices have increasingly led investigators to synthetic and semi-synthetic scaffolds that offer the engineering of specific ECM cues and the potential for more advanced experimental manipulations. Synthetic scaffolds composed of poly(ethylene glycol) (PEG), for example, facilitate highly biocompatible 3D culture, modular bioactive features like cell-mediated matrix degradation and complete independent control over matrix bioactivity and mechanics. Future work in PEG or similar reductionist synthetic matrix systems should enable the study of increasingly complex and dynamic tumor–ECM relationships in the hopes that accurate modeling of these relationships may reveal new cancer therapeutics targeting tumor progression and metastasis.