Integrin expression and ability to adhere to extracellular matrix proteins and endothelial cells in human lung cancer lines.

We examined the integrin expression in 19 human lung cancer cell lines with monoclonal antibodies to the integrin subunits alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, beta 1, beta 2, and beta 4. We measured their ability to adhere to the extracellular matrix (ECM) and human umbilical vein endothelial cells (HUVECs). Almost all lines expressed the beta 1 subunit and approximately half of the lines expressed the beta 4 subunit; by contrast, none expressed the beta 2 subunit. Subunits alpha 2, alpha 3, alpha 5 and alpha 6 were frequently expressed, whereas very few lines expressed alpha 1 and alpha 4. Most lines adhered strongly to ECM (type I collagen, laminin and fibronectin) in correspondence to their expression of integrins. Binding by most lines to fibronectin was completely inhibited by arginine-glycine-aspartic acid (RGD) peptide. Three lines that expressed few or no integrins had very weak ability to adhere to ECM. Strong binding to HUVECs was found in most lines, but the three lines had very little ability to adhere to HUVECs. Binding to HUVECs was strongly inhibited at 4 degrees C, under divalent cation-free conditions and by antibodies to the beta 1 subunit. These results suggest that lung cancer cells adhere to ECM and endothelial cells through integrins, especially the beta 1 subfamily.     

Integrin alpha6beta1 role in metastatic behavior of human pancreatic carcinoma cells

The factors that determine the metastatic behavior of pancreatic tumor cells are incompletely understood. In this study, we first demonstrate differences in adhesion properties, integrin expression and in vivo integrin function in the metastatic tumor cell line PaTu 8988s compared with the non-metastatic cell line PaTu 8988t. Both cell lines were derived from the same original tumor and exhibit identical genetic fingerprints. Using in vitro adhesion assays performed on purified extracellular matrix components, adhesion of PaTu 8988s cells was significantly increased on the basal membrane component laminin and decreased on the interstitial matrix protein fibronectin compared to PaTu 8988t cells. By immunocytochemistry and flow cytometry, and in correspondence with their adhesive properties, the metastatic PaTu 8988s cells did express a distinct pattern of integrin subunits. Laminin-binding integrins alpha6 and beta4 were overexpressed in PaTu 8988s cells. Fibronectin-binding alpha5 integrins were present at higher levels in the non-metastatic PaTu 8988t cells, whereas the beta1 subunit expression did not differ. Adhesion to laminin or fibronectin was specific and was mediated via integrins alpha6beta1 and alpha5beta1, respectively. In addition, metastasis formation in vivo after injection of cells into the tail vein of nude mice was inhibited by preincubation of PaTu 8988s cells with antibodies directed against the integrin alpha6 or beta1. We conclude that alpha6beta1 integrins are overexpressed and functionally active in metastatic human pancreatic carcinoma cells, and participate in metastasis formation probably through binding to the basal membrane component laminin.     

Tumor cell invasiveness correlates with changes in integrin expression and localization

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-beta (TGFbeta1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits beta1, alpha2 and alpha3, or alpha5 and alpha6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits beta1 and alpha6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and alpha5 at the entire plasma membrane. During EMT, EpRas cells formed an alpha5beta1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking alpha5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFbeta1-induced EMT in collagen gels, but failed to affect EpRas- or FibRas-derived structures. We conclude that functional alpha5beta1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the alpha6beta4 ligand laminin 5, suggesting that alpha6beta4 is no longer functional after EMT and replaced by mesenchymal integrins such as alpha5beta1.     

Interleukin-1alpha enhances integrin alpha(6)beta(1) expression and metastatic capability of human pancreatic cancer

OBJECTIVE: To investigate the mechanisms of metastasis formation in metastatic human pancreatic cancer, we examined the enhancement in integrin expression, and adherence to and invasiveness into extracellular matrix (ECM) proteins of human pancreatic cancer cells after exposure to interleukin (IL)-1alpha. METHODS: Expression of IL-1 receptor type I (IL-1RI) and alterations in integrin subunits by IL-1alpha were examined by flow-cytometric analysis and by cellular enzyme-linked immunosorbent assay in four human pancreatic cancer cell lines (BxPC-3, PaCa-2, PANC-1, and SW1990), respectively. In addition, assays of cancer cell adhesion and invasion to ECM proteins were performed to investigate whether increased integrin expression affected the adhesive and invasive interaction between cancer cells and the putative integrin ECM ligands. Furthermore, immunohistochemistry was used to assess integrins and IL-1R1 expression in pancreatic tissues. RESULTS: In metastatic cancer cells, expression of the alpha(6) subunit was enhanced by IL-1alpha treatment. While metastatic cancer cells exhibited preferential adherence to and invasion into laminin, these properties were enhanced by IL-1alpha. The alpha(6) subunit and IL-1RI were strongly expressed in pancreatic tissues from pancreatic cancer patients with liver metastasis. CONCLUSIONS: In pancreatic cancer, IL-1alpha enhanced alpha(6)beta(1)-integrin expression, probably via increased IL-1RI levels. Our results indicated that alpha(6)beta(1)-integrin and IL-1RI expression may play important roles in metastasis formation.     

Modulation of integrin expression on mesotheliomas: the role of different histotypes in invasiveness.

The in vitro and in vivo integrin expression in human pleural malignant mesothelioma (MM) of three different histotypes was studied. Cell lines from MM of epithelioid (E1), fibrous (F1), byphasic histotype (B1) and normal mesothelial cells (NM) were analysed for the surface expression of alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, beta4 subunits and alphavbeta5 integrins. We found that alpha6, beta4 subunits and alphavbeta5, weakly detectable on NM cells, were expressed on MM cells. The beta3 subunit, well expressed on NM cells, was absent on MM cells. Differential expression among histotypes was observed, the MM-E1 was the least and the MM-B1 the most positive. Specimens for each MM histotype, were analysed by immunohistochemistry. The alpha6 and alphav subunits were more evident on the epithelioid histotype. Intense staining for beta3 and beta4 subunits, was found in all MM, particularly in invading cells, while the alpha5, and alphavbeta5 integrins were variously expressed. The different histotypes can affect the in vitro integrin expression and may indicate a preferential involvement of some subunits in vivo during MM tumor progression.     

alpha6beta1 integrin expression in hepatocarcinoma cells: regulation and role in cell adhesion and migration.

Liver carcinogenesis is associated with striking changes in the integrin repertoire of hepatocytes, including the overexpression of the laminin and collagen receptors alpha1beta1 and the de novo induction of the laminin receptor alpha6beta1. Our aim was to analyze the role of pro-inflammatory cytokines, interferons and fibrogenic cytokines TGF-beta and FGF2 in the regulation of the expression of beta1 integrins by neoplastic hepatocytes. The 2 human hepatocellular cell lines HepG2 and Hep3B were used as models. Integrin expression was assessed by qualitative methods (immunocytochemistry, Western blotting) and semi-quantitative techniques (FACS, cellular ELISA), before and after stimulation by TNFalpha, IL1-beta, TGF-beta, FGF2, interferon gamma and interferon alpha-2b. HepG2 and Hep3B constitutively expressed alpha1, alpha2, alpha6 and beta1 chains. A 24 to 48-hr stimulation with pro-inflammatory cytokines, TGF-beta and FGF2 induced a significant increase in the concentrations of all integrin chains. The maximum induction was registered for beta1 chain, which presented increases amounting up to 3, 4 and 7 times the control values in the presence of, respectively, TNF alpha/IL1-beta, TGF-beta and FGF2. Interferons had no direct effect on integrin expression and partially antagonized the effects of TNF alpha and TGF-beta. The increased concentrations of integrin chains were associated with an increased membrane expression of the corresponding dimers and with an increased adhesion of stimulated hepatocytes to laminin, which was antagonized by neutralizing anti-beta1 and anti-alpha6 antibodies. Finally, anti-alpha6 antibody inhibited the migration of HepG2 and Hep3B cells in reconstituted basement membrane. Our results suggest that the stimulation of alpha6beta1 integrin expression in hepatocarcinoma cells is essential for cell adhesion and migration.     

Aberrant expression of integrin and erbB subunits in breast cancer cell lines

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.     

Expression of beta1 and beta4 integrins in normal arachnoid membrane and meningiomas

BACKGROUND: The aim of this work was to study the expression of alpha, beta1, and beta4 integrin subunits in meningiomas. METHODS: Seventeen atypical or anaplastic meningiomas were retrieved from the files of H?pital de Bellevue, Saint-Etienne, France. They were compared with 17 benign meningiomas consecutively examined in 1997 and 6 schwannomas. The tumors were classified according to standard histologic criteria. Frozen sections were immunostained for alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, beta1, and beta4 integrin subunits; collagen; laminin and fibronectin; cytokeratin; vimentin; neural cell adhesion molecule (NCAM); and MIB-1. RESULTS: The study included 7 fibrous meningiomas, 6 transitional meningiomas, 19 syncytial meningiomas, and 2 secretory meningiomas. The expression of alpha1, alpha3, alpha5, alpha6, and beta1 was constant. The expression of alpha1 was higher in fibrous meningiomas than in syncytial meningiomas. Only in transitional, syncytial, and secretory meningiomas was the expression of alpha2 detected. The expression of alpha2 and beta4 was associated with the expression of cytokeratin in the glandular structures of secretory meningiomas, whereas it was associated with NCAM expression in the whorls of meningothelial meningiomas. The expression of integrin receptors by tumor cells was strongly correlated with that of their respective ligands in the extracellular matrix. In invasive meningiomas, the expression of alpha3 and alpha6 by tumor cells was significantly lower. The higher the MIB-1 proliferation index, the lower the expression of alpha3. The 6 schwannomas expressed only alpha2, alpha3, alpha6, beta1, and beta4 integrins. CONCLUSIONS: Each histologic subtype of meningioma has a specific spectrum of integrin expression. The study of alpha3 and alpha6 may have prognostic value in the assessment of meningiomas. The study of the integrin profile is valuable for the differential diagnosis of fibrous meningiomas and schwannomas.     

Different phenotypes in human prostate cancer: alpha6 or alpha3 integrin in cell-extracellular adhesion sites

The distribution of alpha6/alpha3 integrin in adhesion complexes at the basal membrane in human normal and cancer prostate glands was analyzed in 135 biopsies from 61 patients. The levels of the polarized alpha6/alpha3 integrin expression at the basal membrane of prostate tumor glands were determined by quantitative immunohistochemistry. The alpha6/alpha3 integrin expression was compared with Gleason sum score, pathological stage, and preoperative serum prostate-specific antigen (PSA). The associations were assessed by statistical methods. Eighty percent of the tumors expressed the alpha6 or alpha3 integrin and 20% was integrin-negative. Gleason sum score, but not serum PSA, was associated with the integrin expression. Low Gleason sum score correlated with increased integrin expression, high Gleason sum score with low and negative integrin expression. Three prostate tumor phenotypes were distinguished based on differential integrin expression. Type I coexpressed both alpha6 and alpha3 subunits, type II exclusively expressed alpha6 integrin, and type III expressed alpha3 integrin only. Fifteen cases were further examined for the codistribution of vinculin, paxillin, and CD 151 on frozen serial sections using confocal laser scanning microscopy. The alpha6/alpha3 integrins, CD151, paxillin, and vinculin were present within normal glands. In prostate carcinoma, alpha6 integrin was colocalized with CD 151, but not with vinculin or paxillin. In tumor phenotype I, the alpha6 subunit did not colocalize with the alpha subunit indicating the existence of two different adhesion complexes. Human prostate tumors display on their cell surface the alpha6beta1 and/or alpha3beta1 integrins. Three tumor phenotypes associated with two different adhesion complexes were identified, suggesting a reorganization of cell adhesion structures in prostate cancer.     

Metastatic phenotype: growth factor dependence and integrin expression

Homogeneous subpopulations, which are endowed with low or high metastatic potential, were selected from Lewis lung carcinoma (3LL) in an attempt to correlate metastatic phenotype with specific properties of tumor cells. Since the growth of malignant cells at secondary sites could depend on their ability to respond to microenvironments, the growth factor dependence of 3LL variants has been studied. The ability of variant lines to grow in monolayer and in soft agar cultures, either in the presence or absence of different growth factors or serum, was analyzed and correlated with their metastatic potential. The reported results demonstrate that tumor cells expressing higher metastatic potential also exhibit higher capacity to grow and proliferate in all the culture conditions tested, independently of the addition of exogenous growth factors or serum. Moreover, since highly metastasizing cells express a significant amount of TGF-beta 1 mRNA, a pattern of autocrine growth is postulated for 3LL metastatic cells. One relevant aspect of the phenotype of transformed cells is their reduced adhesion to solid substrates; this phenomenon is thought to reflect the invasive and metastatic potential of tumor cells. Since the adhesion of the cells to substrata is mediated by molecules of the extracellular matrix, the expression of extracellular matrix receptors (integrins) was studied on 3LL metastatic variants. In particular, through immunochemical and biochemical studies we investigated the expression of the laminin receptor(alpha 6/beta 1) and of a novel receptor (integrin: alpha 6/beta 4), of unknown function. The receptors were quantitated on the cell surface of 3LL variants by the use of specific monoclonal antibodies which recognize, respectively, different epitopes of alpha 6, beta 4 or beta 1 subunits. Results demonstrate that the novel integrin alpha 6/beta 4, is specifically expressed in highly metastasizing 3LL cells, whereas the laminin receptor alpha 6/beta 1 is expressed in all 3LL variants. In conclusion, data presented demonstrate that 3LL cells endowed with higher metastatic potential are more independent of the microenvironmental conditions in that they possess a higher autocrine capacity than the lower metastasizing ones, and could acquire higher capacity to invade through the expression on their cell surface of specific receptors for cell adhesion (the novel integrin, defined as alpha 6/beta 4).     

Detection of a hypersialylated beta1 integrin endogenously expressed in the human astrocytoma cell line A172

Gliomas are the most common deadly brain tumors. Human cerebral tumors express high level of alpha5beta1 integrins. As a potential new target, alpha5beta1 was investigated here in two human astrocytoma cell lines, A172 and U87MG. We found that a hypersialylated beta1 integrin was endogenously expressed in A172 cells. It forms heterodimers with alpha5 subunits, localizes at the cell membrane and allows adhesion to fibronectin. This form of beta1 integrin was only recognized by the 9EG7 anti-beta1 antibody and appeared devoid of other specific antibody epitopes (12G10, TS2/16 and mAb13 shown here to be N-glycosylation sensitive). Overexpression of the beta1 integrin subunit in A172 cells not only increased the hypersialylated form but also led to the appearance of a non-hypersialylated beta1 form also addressed to the cell surface. Compared to wild-type A172 cells, beta1-A172 cells showed increased adhesion to fibronectin and decreased sensitivity to SJ749, a non-peptidic alpha5beta1 antagonist. In addition, beta1-A172 cells exhibited increased matrix dependence for normal cell cycling. Collectively, the data add new evidence for the role of beta1 glycosylation/sialylation in the regulation of integrin functions.     

Integrin receptors and ECM proteins involved in preferential adhesion of colon carcinoma cells to lung cells

To assess the putative role of extracellular matrix (ECM) proteins on lung cells interacting with integrin receptors on colon carcinoma cells, an in vitro adhesion assay was used to investigate these factors. Tumor necrosis factor (TNF)-alpha treatment of fetal lung cell line MRC 9, upregulated expression of ECM proteins and also supported enhanced adhesion of PTC colon carcinoma cells. Antibodies to ECM proteins significantly blocked this enhanced adhesion of PTC cells. Similarly, antibody blocking of beta1 and beta2 integrin receptors on PTC cells revealed the integrin receptors involved in this enhanced adhesion. beta1 integrin receptors like alpha2beta1, alpha4beta1 and alpha5beta1 on PTC cells were found interacting with their ECM ligands like fibronectin and laminin on TNF-alpha stimulated MRC 9 cells. Interestingly, PTC cells were found to constitutively express alphaLbeta2, which is normally expressed by leukocytes. The data from the present study indicates that expression of multiple beta1 and beta2 integrins by colon carcinoma cells putatively allows these cells to successfully adhere to secondary sites like lungs.     

Enhancement of integrins by interleukin-1alpha,and their relationship with metastatic and invasive behavior of human pancreatic ductal adenocarcinoma cells

BACKGROUND AND OBJECTIVES: Adhesion and invasion of tumor cells to extracellular matrix (ECM) proteins play an important role in tumor metastasis formation. We investigated the enhancement of adhesive and invasive behavior to ECM proteins of human pancreatic cancer cells by interleukin-1alpha (IL-1alpha) to examine the mechanism of adhesion and invasion of metastatic human pancreatic cancer cells to ECM proteins. METHODS: The enhancement of integrin subunits by IL-1alpha was examined by cellular enzyme-linked immunosorbent assay (CELISA) in two metastatic human pancreatic cancer cell lines (BxPC-3 and SW1990) and two nonmetastatic pancreatic cancer cell lines (PaCa-2 and PANC-1). In addition, assays of cancer cell adhesion and invasion to ECM proteins were performed to investigate whether increased integrin expression affected the invasive interaction between cancer cells and the putative integrin ECM ligands. RESULTS: Expression of the alpha6 subunit by metastatic cancer cells was enhanced by IL-1alpha. Metastatic cancer cells also exhibited preferential adherence and invasion to laminin compared with nonmetastatic cancer cells, and this was enhanced by IL-1alpha. CONCLUSIONS: The enhancement of alpha6beta1-integrin by Il-1alpha acting through IL-1RI, as well as the expression of alpha6beta1-integrin, plays an important role in metastasis formation in pancreatic cancer     

Thrombospondin-1 promotes alpha3beta1 integrin-mediated adhesion and neurite-like outgrowth and inhibits proliferation of small cell lung carcinoma cells

Although human small cell lung carcinoma (SCLC) cell lines are typically anchorage-independent and do not attach on most extracellular matrix proteins, OH-1, and several other SCLC cell lines attached on substrates coated with thrombospondin-1 (TSP1). SCLC cells grew long-term as adherent cells on a TSP1-coated substrate. Adhesion of SCLC cells on TSP1 was inhibited by heparin, function-blocking antibodies recognizing alpha3 or beta1 integrin subunits, and by soluble alpha3beta1 integrin ligands. SCLC cells extended neurite-like processes on a TSP1 substrate, which was also mediated by alpha3beta1 integrin. Process formation on a TSP1 substrate was specifically stimulated by epidermal growth factor and somatostatin. Adhesion on TSP1 weakly inhibited SCLC cell proliferation, but this inhibition was strongly enhanced in the presence of epidermal growth factor. TSP1 and an alpha3beta1 integrin-binding peptide from TSP1 also inhibited proliferation when added in solution. High-affinity binding of 125I-labeled TSP1 to OH-1 cells was heparin-dependent and may be mediated by sulfated glycolipids, which are the major sulfated glycoconjugates synthesized by these cells. Synthesis or secretion of TSP1 by SCLC cells could not be detected. On the basis of these results, the alpha3beta1 integrin and sulfated glycolipids cooperate to mediate adhesion of SCLC cells on TSP1. Interaction with TSP1 through this integrin inhibits growth and induces neurotypic differentiation, which suggests that this response to TSP1 may be exploited to inhibit the progression of SCLC.     

Overexpression of alpha(v)beta6 integrin in serous epithelial ovarian cancer regulates extracellular matrix degradation via the plasminogen activation cascade

Recent evidence suggests that integrins are involved in the multi-step process of tumour metastasis. The biological relevance of alpha(v) integrins and associated beta-subunits in ovarian cancer metastasis was examined by analysing the expression of these cell surface receptors in nine ovarian cancer cell lines and also in the primary human ovarian surface epithelial cell line (HOSE). beta1, beta3 and beta5 subunits were present in all ten ovarian cell lines. beta6 subunit was present at varying levels in eight out of nine cancer cell lines but was absent in the HOSE cell line. Immunohistochemical staining showed that beta6 was present in both non-invasive (borderline) and high-grade ovarian cancer tissues but was absent in benign and normal ovarian tissue. High alpha(v)beta6 integrin expressing ovarian cancer cell lines had high cell surface expression of uPA and uPAR. Ovarian cancer cell lines expressing high to moderate level of alpha(v)beta6 integrin demonstrated ligand-independent enhanced levels of high molecular weight (HMW)-uPA and pro-matrix metalloproteinase 2 and 9 (pro-MMP-2 and pro-MMP-9) expression in the tumour-conditioned medium. High and moderate expression of alpha(v)beta6 integrin correlated with increased plasminogen-dependent degradation of extracellular matrix which could be inhibited by inhibitors of plasmin, uPA and MMPs or by monoclonal antibody against uPA, MMP-9 or alpha(v)beta6 integrin. These results suggest that endogenous de novo expression of alpha(v)beta6 integrin in ovarian cancer cells may contribute to their invasive potential, and that alpha(v)beta6 expression may play a role in ovarian cancer progression and metastasis.     

Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.

Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.     

Different adhesion properties of highly and poorly metastatic HT-29 colon carcinoma cells with extracellular matrix components: role of integrin expression and cytoskeletal components.

Integrin-mediated tumour cell adhesion to extracellular matrix (ECM) components is an important step in the development of metastatic lesions. Thus, integrin expression and integrin-mediated adhesion of colon carcinoma cells to various ECM components was examined. Poorly (HT-29P) and highly (HT-29LMM) liver-metastatic colon carcinoma cells were used to study the rates of adhesion to collagen I (C I), collagen IV (C IV), laminin (LN), fibronectin (FN), or vitronectin (VN) in a static adhesion assay (10-120 min). Cells were untreated or treated with oligopeptides (RGD, GRGDS, YIGSR, RGES), anti-integrin antibodies, or colchicine, nocodazole, cycloheximide, acrylamide or cytochalasin D (to disrupt cytoskeletal structures). Both cell lines expressed similar patterns of integrin expression (alpha2, alpha3, ,alpha6, alphav, beta1, beta4, and beta5) by immunocytochemistry and immunoprecipitation. HT-29LMM cells showed significantly higher rates of adhesion to LN (P < 0.001) and FN (P < 0.001), but significantly poorer rates of adhesion to C I (P < 0.05) and C IV (P < 0.001) than HT-29P cells, respectively, adhesion to VN was insignificant. RGD and GRGDS inhibited HT-29LMM cell adhesion to FN only. Pretreatment with anti-beta, or anti-alpha2 integrin subunits suppressed adhesion to C I and C IV, and adhesion to LN was inhibited with anti-beta1 or anti-alpha6 integrin. Anti-beta1 or anti-alphav blocked adhesion to FN. Pretreatment of cells with cytochalasin D, cycloheximide or acrylamide inhibited adhesive interactions of both cell lines to the ECM components. In contrast, colchicine and nocodazole had no effect. The results demonstrate that adhesion of HT-29 cells to ECM is mediated, in part, by different integrins, depending on the substrate. Poorly and highly metastatic HT-29 cells possessed different patterns of adhesion to the various ECM substrates, but these differences were not due to different expression of integrin subunits. The results also suggested that the initial adhesion of poorly or highly metastatic HT-29 cells to ECM components requires, in part, the presence of native action and intermediate filaments, but not of microtubules. Thus the adhesion of tumour cells to ECM components may be dependent on signal transduction and assembly of microfilaments.     

Altered cell-matrix contact: a prerequisite for breast cancer metastasis?

The integrins are receptors that regulate interaction between epithelial cells and the extracellular matrix. Previous studies have shown that a reduction in the expression of the alpha2beta1, alpha3beta1, alpha6beta1, alpha(v)beta1 and alpha(v)beta5 integrins in primary breast cancer is associated with positive nodal status. In order to assess the functional significance of altered integrin expression, primary breast cancer cells were derived from individual patients with known tumour characteristics using immunomagnetic separation. Purified human fibronectin, vitronectin, laminin and type IV collagen were used to represent the principal extracellular matrix proteins in an in vitro adhesion assay. Primary breast cancer cells from lymph node-positive patients were significantly less adhesive to each of the matrix proteins studied (P<0.001, Mann-Whitney U-test). Matrix adhesion of primary breast cancer cells from node-negative patients was inhibited by appropriate integrin monoclonal antibodies (P<0.001, paired Wilcoxon test). Adhesion to fibronectin, vitronectin and laminin, but not type IV collagen, was influenced by the inhibitor arginine-glycine-aspartate, suggesting that breast cancer cell recognition of collagen IV is mediated through alternative epitopes. Weak matrix adhesion correlated with loss of integrin expression in tissue sections from corresponding patients assessed using immunohistochemistry. This study demonstrates a link between altered integrin expression and function in primary breast cancers predisposed to metastasize.