Daniel M. Ramos

Synaptic and glial localization of the integrin αvβ8 in mouse and rat brain

Integrins are a large family of cell adhesion receptors mediating cell-extracellular matrix (ECM) interactions and are widely distributed in tissues. The beta8 integrin subunit mRNA has been shown to be expressed at higher levels in the central nervous system (CNS) than in other organs [M. Moyle, M.A. Napier, J.W. McLean, Cloning and expression of a divergent integrin subunit beta8, J. Biol. Chem. 266 (29) (1991) 19650-19658] but its cellular and subcellular localization in the CNS are unknown. In this report, we demonstrate that beta8 pairs exclusively with the alphav subunit in the CNS to form the alphavbeta8 heterodimer. Immunohistochemical analysis of the distribution of beta8 in adult mouse and rat brains revealed that the protein is expressed in several regions of the hippocampal formation and in the molecular layer and glomeruli of the granular cell layer of the cerebellum. Punctate and diffuse immunolabeling was observed occasionally surrounding neuronal pericarya and extensively throughout dendritic fields suggesting both pre- and post-synaptic localization and/or expression in non-neuronal cells. By immunoelectron microscopy, beta8 immunoreactivity was detected in dendritic spines where it was often localized at post-synaptic densities, occasionally in axon terminals and in glial processes. Association of beta8 with synaptic membranes was further supported by its enrichment in synaptosomal preparations as detected by immunoblotting. These results demonstrate that alphavbeta8 is present in mature synapses and therefore may play a role in synaptic function.

Expression of integrin β6 enhances invasive behavior in oral squamous cell carcinoma

Abstract Oral squamous cell carcinoma (SCC) is characterized by invasive growth and the propensity for distant metastasis. The expression of specific adhesion receptors promotes defined interactions with the specific components found within the extracellular matrix (ECM). We previously showed that the αvβ6 fibronectin receptor is highly expressed in oral SCC. Here we forced expression of the β6 subunit into poorly invasive SCC9 cells to establish the SCC9β6 cell line and compared these two cell lines in several independent assays. Whereas adhesion to fibronectin was unaffected by the expression of β6, migration on fibronectin and invasion through a reconstituted basement membrane (RBM) were both increased. Function-blocking antibodies to αvβ6 (10D5) reduced both migration on fibronectin and invasion through an RBM, whereas anti-α5 antibodies were effective only in suppressing migration on fibronectin, not invasion. Expression of β6 also promoted tumor growth and invasion in vivo and modulated fibronectin matrix deposition. When grown as a co-culture with SCC9 cells, peritumor fibroblasts (PTF) organized a dense fibronectin matrix. However, fibronectin matrix assembly was decreased in co-cultures of SCC9β6 cells and PTF and this decrease was reversed by the addition of function-blocking anti-αvβ6 antibodies. The expression of β6 also resulted in increased levels of matrix metalloproteinase 3. Addition of the general MMP inhibitor GM6001 to SCC9β6/PTF co-cultures dramatically increased fibronectin matrix assembly in a similar fashion as incubation with anti-αvβ6 antibodies. These results demonstrate that expression of β6 (1) increases oral SCC cell motility and growth in vitro and in vivo; (2) negatively affects fibronectin matrix assembly; and (3) stimulates the expression and activation of MMP3. We suggest that the integrin αvβ6 is a key component of oral SCC invasion and metastasis through modulation of MMP-3 activity.

Integrin expression in malignant melanoma

Invasion of melanoma cells into the underlying interstitial stromal matrix is the initial step for subsequent local and distant metastasis. The invading tumor cell must interact with the extracellular matrix during the early stages of invasion and later during penetration of lymphatic and blood vessels. This interaction with different types of extracellular matrix predicts that the invasive cell must possess surface adhesion receptors with diverse ligand specificities, including the capacity to bind different types of collagens and adhesive glycoproteins. Metastatic melanoma cells do in fact express multiple adhesion receptors, including several of the receptors from the integrin family of heterodimers. The integrin receptors can be either extremely specific for a single ligand or capable of binding multiple ligands. It is likely that the tumor cells repertoire of adhesion receptors may influence not only its adhesive properties but its metastatic characteristics as well. There is evidence that normal melanocytes have an integrin profile distinct from that of melanoma cells. In particular, melanocytes adhere poorly to laminin while metastatic melanoma cells bind well to this ligand. This difference in adhesion between the two cell types appears to reflect the fact that melanoma cells express a melanoma-specific integrin (% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiGc9yrFr0xXdbba91rFfpec8Eeeu0x% Xdbba9frFj0-OqFfea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs% 0dXdbPYxe9vr0-vr0-vqpWqaaeaabiGaciaacaqabeaadaqaaqGaaO% qaaiaadggadaWgaaWcbaGaamODaaqabaGccqaHYoGydaWgaaWcbaGa% aG4maaqabaaaaa!40CF!\[a_7 \beta _1 \]) that binds laminin and is not detectable in normal melanocytes. The presence of increased laminin receptors and enhanced laminin binding in melanoma cells may contribute to the malignant phenotype.

Stromal fibroblasts influence oral squamous‐cell carcinoma cell interactions with tenascin‐C

In this study we identified tenascin‐C (TN‐C) and one of its integrin receptors, αvβ6, in oral squamous‐cell carcinoma (SCC) specimens. Neither TN‐C nor αvβ6 are expressed in normal oral mucosa. We also studied 2 human oral squamous‐cell carcinoma cell lines: the highly invasive HSC‐3 cells, and the poorly invasive SCC‐25 cells. We determined that adhesion of these cells to TN‐C involves both α2 and αv integrins. Migration on TN‐C by oral SCC cells required fibroblast‐conditioned medium and did not occur in its absence. This migration was blocked by anti‐α2 and anti‐αv antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor‐β1. When seeded on TN‐C, the poorly invasive SCC‐25 cells formed αvβ6‐positive focal contacts; the HSC‐3 cells did not. HSC‐3, SCC‐25 and PTF cells secrete TN‐C into the culture medium, as determined by Western blot. However, when HSC‐3 cells were inoculated into the floor of the mouth of nude mice, only murine TN‐C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC‐3 cells do not secrete TN‐C. Our results demonstrate that αvβ6 and tenascin‐C are neo‐expressed in oral squamous‐cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior. Int. J. Cancer 72:369–376, 1997.

Tenascin and β6 integrin are overexpressed in floor of mouth in situ carcinomas and invasive squamous cell carcinomas

Floor of the mouth squamous cell carcinomas exhibit many characteristics that suggest they represent a distinct biological subset within head and neck tumors. The features of preinvasive lateral intraepithelial spread, high rate of conversion of intraepithelial neoplasia to invasive carcinoma, and high incidence of occult metastases, suggest the importance of motility-associated proteins in the pathogenesis of these lesions. Two such proteins, tenascin and beta 6 integrin, are generally overexpressed in squamous carcinomas, and may play a central role in the invasive process of floor of the mouth lesions. The purpose of this study was to evaluate in situ and invasive squamous cell carcinomas from the floor of the mouth for the expression of tenascin and beta 6 integrin. Twenty lesions each of floor of the mouth in situ carcinomas and squamous cell carcinomas, and 10 normal controls were stained for tenascin and beta 6 using a standard immunohistochemical protocol for formalin-fixed specimens. Sections were assessed for staining intensity, pattern, and co-localization. Tenascin was highly expressed at the keratinocyte-connective tissue interface of both in situ and invasive carcinomas. beta 6 was expressed in basal keratinocytes of all in situ and invasive lesions, but was not evident in any of the control epithelia. There was no significant difference in staining of in situ and invasive carcinomas, but there was a significant difference in staining between these lesions and controls. Staining was colocalized in serial sections, supporting a receptor-ligand relationship. Both tenascin and beta 6 were weakly expressed in dysplastic areas adjacent to carcinomas suggesting that changes in the expression of these proteins occurs prior to the invasive phenotype. We conclude that tenascin and beta 6 are overexpressed in in situ and invasive floor of the mouth carcinomas, but that transgression of the basement membrane by neoplastic epithelial cells requires additional changes to the keratinocyte molecular profile.

Tenascin-C matrix assembly in oral squamous cell carcinoma

We previously showed that the extracellular matrix component tenascin‐C (TN‐C) is upregulated in oral squamous cell carcinoma (SCC) compared with the normal oral mucosa. In this study we examined oral biopsy specimens of mild to moderate dysplasia or carcinoma in situ to study TN‐C expression. We found that carcinoma in situ is the stage at which TN‐C becomes widely expressed, suggesting it may be involved in the initial stages of tumor progression. To study TN‐C matrix production in vitro, we used an invasive oral SCC cell line (HSC‐3) and peri‐tumor fibroblasts (PTF). Neither cell type organized a TN‐C matrix when cultured alone; however, when co‐cultured with HSC‐3 cells, PTF were able to assemble a TN‐C matrix. PTF retained the ability to organize a TN‐C matrix when separated from the HSC‐3 cells by a semi‐permeable membrane, indicating that cell‐cell contact is not necessary for TN‐C matrix organization and suggesting that soluble factors may be involved. Moreover, PTF were induced to assemble TN‐C matrices when grown in medium conditioned by both the PTF and HSC‐3 cells. Antibodies to fibronectin (FN) and to the first FN type III repeat blocked both FN and TN‐C matrix assembly, indicating that TN‐C matrix organization is dependent on an FN template. Antibodies to α5, αv and β1 integrins also blocked TN‐C matrix formation. When seeded onto FN matrices, the co‐cultures were unaffected by the anti‐integrin and anti‐FN antibodies and were able to organize a TN‐C matrix. Our results suggest that progression of malignant oral SCC is accompanied by an alteration of the normal ECM to one rich in TN‐C, and that the organization of a TN‐C matrix is dependent on soluble cues provided by both the SCC cells and the PTF. Int. J. Cancer 75:680–687, 1998.© 1998 Wiley‐Liss, Inc.

Differential Expression of αv Integrins in K1735 Melanoma Cells

Tumor cell adherence to and migration on the extracellular matrix is an important aspect of cancer progression. This interaction with the extracellular matrix is mediated primarily through the integrin class of cell adhesion molecules. We identified a restricted expression of αvβ3 in highly metastatic K1735M2 and of αvβ5 in poorly metastatic K1735C23 murine melanoma cells. The highly metastatic cells were ten times more motile on vitronectin and fibronectin and approximately three times more invasive through a reconstituted basement membrane than the poorly metastatic cells. This motility was inhibited by addition of anti-β3 antibodies. Injection of the αvβ3-negative K1735C23 cells into syngeneic mice resulted in the generation of a metastatic variant (K1735C23PM) that neo expressed the αvβ3 complex, indicating that expression of αvβ3 is required for K1735 melanoma metastasis. Injection of highly metastatic K1735M2 cells in the presence of blocking antibody to β3 reduced tumor size by approximately 80%. Treatment of the K1735M2 cells with a retroviral antisense β3 construct significantly reduced their expression of αvβ3 and also reduced their motility on extracellular matrix ligands and their invasion through a reconstituted basement membrane. In contrast, when the K1735C23 cells were treated with a construct containing the full-length β3 cDNA, their motility on extracellular matrix proteins and invasion of a reconstituted basement membrane were significantly increased. These results indicate that αvβ3 is required for migration and invasion of K1735 melanoma cells in vitro and primary tumor growth and metastasis in vivo.

Development of an in vitro extracellular matrix assay for studies of brain tumor cell invasion.

SummaryInvasion of brain by tumor cells is an inherent feature of the malignant phenotype. Assays to quantitate invasiveness should provide a powerful tool to investigate this phenomenon. We have developed a modifiedin vitro assay to measure tumor cell invasion, attachment, and chemotaxis using a barrier of the complex basement membrane Matrigel on gelatin-coated filters. Within 5 hours, 7.8% of U251MGp and 2.6% of SF126 human malignant glioma cells invaded the Matrigel and filter, compared with 0.8% of normal human leptomeningeal cells. The extent of invasion was directly proportional to incubation time and filter pore size and inversely proportional to the Matrigel concentration. Cells from exponentially growing U251MGp cultures invaded more readily (10.9%) than cells from plateau-phase cultures (2.3%); however, labeling studies with bromodeoxyuridine showed that quiescent cells and rapidly dividing cells were equally capable of invading. This suggests that the mechanisms underlying invasion by malignant glioma cells are distinct from those underlying proliferation and indicates the need for therapy aimed specifically at invasive behavior. In a practical application of this assay to test a potential anti-invasive strategy, monoclonal antibodies to the β subunit of an integrin receptor mediating attachment to the extracellular matrix inhibited invasion by U251MGp cells in a dose-dependent manner. This assay should allow evaluation of the cellular and molecular basis of brain tumor progression and perhaps aid the development of rationally designed drugs that limit tumor invasion. It may also allow prediction of the clinical behavior of neoplasms in individual patients.

Wnt signaling regulates pulp volume and dentin thickness.

Odontoblasts, cementoblasts, ameloblasts, and osteoblasts all form mineralized tissues in the craniofacial complex, and all these cell types exhibit active Wnt signaling during postnatal life. We set out to understand the functions of this Wnt signaling, by evaluating the phenotypes of mice in which the essential Wnt chaperone protein, Wntless was eliminated. The deletion of Wls was restricted to cells expressing Osteocalcin (OCN), which in addition to osteoblasts includes odontoblasts, cementoblasts, and ameloblasts. Dentin, cementum, enamel, and bone all formed in OCN‐Cre;Wlsfl/fl mice but their homeostasis was dramatically affected. The most notable feature was a significant increase in dentin volume and density. We attribute this gain in dentin volume to a Wnt‐mediated misregulation of Runx2. Normally, Wnt signaling stimulates Runx2, which in turn inhibits dentin sialoprotein (DSP); this inhibition must be relieved for odontoblasts to differentiate. In OCN‐Cre;Wlsfl/fl mice, Wnt pathway activation is reduced and Runx2 levels decline. The Runx2‐mediated repression of DSP is relieved and odontoblast differentiation is accordingly enhanced. This study demonstrates the importance of Wnt signaling in the homeostasis of mineralized tissues of the craniofacial complex.

Soluble fibronectin promotes migration of oral squamous‐cell carcinoma cells

We have shown that a fibronectin (FN) matrix is required for the organization of tenascin‐C (TN‐C) matrices by peritumor fibroblasts (PTF) cultured from tissue surrounding oral squamous‐cell carcinoma (SCC). In the present study, we detected alternatively spliced FN containing both the EDA and EDB domains decorating the reactive stroma adjacent to the invading tumor nests in oral SCC biopsies. In vitro, PTF cells organized an extensive FN matrix rich in the EDA domain and containing a small amount of EDB. In contrast, normal human fibroblasts deposited a FN matrix which expressed only the EDA domain. PTF‐conditioned medium (CM), shown to enhance migration of oral SCC cells on TN‐C, was found to enhance their migration on FN and invasion of a reconstituted basement membrane. Addition of antibodies to FN to the PTF‐CM inhibited SCC‐cell migration on TN‐C, and depletion of FN from the PTF‐CM abolished its ability to induce migration or invasion by oral SCC cells, suggesting that FN promotes the migration and invasion of oral SCC cells. Western blots of the PTF‐CM identified FN containing the EDA but not the EDB domain. When soluble FN was added to the control medium in the lower chamber of the Transwell system, SCC‐cell migration increased significantly. These results demonstrate that both the EDA and the EDB domains of FN are expressed in the extracellular matrix of oral SCC in vivo and PTF in vitro and indicate that FN is the probable chemotactic factor in the PTF‐conditioned medium. Int. J. Cancer 78:261–267, 1998.© 1998 Wiley‐Liss, Inc.