VLA-4 Molecules on Tumor Cells Initiate an Adhesive Interaction with VCAM-1 Molecules on Endothelial Cell Surface

To elucidate the role of VLA-4 (α4β1 integrin) in tumor metastasis, we have transfected cDNA coding z4 subunit into human flbrosarcoma (HT1080) cells. VLA-4-overexpressing HT-VC1 cells exhibited increased ability to interact with known ligands for VLA-4, such as CS1 peptide and VCAM-1 (vascular cell adhesion molecule-1). In addition, the in vitro invasive ability of HT-VC1 cells was augmented and the mRNA for type IV collagenase was increased in HT-VC1 cells. The induction of VCAM-1 molecules on lung endothelial cells of nude mice by tumor necrosis factor-α treatment resulted in augmentation of in vivo HT-VC1 cell adhesion to the lung endothelial cells. Thus, the VLA-4 molecules on tumor cells initiate an adhesive interaction with VCAM-1 molecules on endothelial cells, that is important for hematogenous metastasis.     

VLA-4 molecules on tumor cells initiate an adhesive interaction with VCAM-1 molecules on endothelial cell surface.

To elucidate the role of VLA-4 (alpha 4 beta 1 integrin) in tumor metastasis, we have transfected cDNA coding alpha 4 subunit into human fibrosarcoma (HT1080) cells. VLA-4-overexpressing HT-VC1 cells exhibited increased ability to interact with known ligands for VLA-4, such as CS1 peptide and VCAM-1 (vascular cell adhesion molecule-1). In addition, the in vitro invasive ability of HT-VC1 cells was augmented and the mRNA for type IV collagenase was increased in HT-VC1 cells. The induction of VCAM-1 molecules on lung endothelial cells of nude mice by tumor necrosis factor-alpha treatment resulted in augmentation of in vivo HT-VC1 cell adhesion to the lung endothelial cells. Thus, the VLA-4 molecules on tumor cells initiate an adhesive interaction with VCAM-1 molecules on endothelial cells, that is important for hematogenous metastasis.     

Identification of integrins α6 and β7 as c‐Jun‐ and transformation‐relevant genes in highly invasive fibrosarcoma cells

Understanding the mechanisms of tumor cell invasion is essential for our attempts to prevent cancer deaths. We screened by DNAmicroarrays the c‐Jun‐ and transformation‐related gene expression changes in S‐adenosylmethionine decarboxylase (AdoMetDC)‐overexpressing mouse fibroblasts that are highly invasive in vivo, and their derivatives expressing a tetracycline‐inducible dominant‐negative mutant of c‐Jun (TAM67) or c‐Jun shRNA. Among the small set of target genes detected were integrins α6 and β7, cathepsin L and thymosin β4, all upregulated in the AdoMetDC‐transformed cells and downregulated upon reversal of transformation by TAM67 or c‐Jun shRNA. The upregulation of integrin α6 subunit, pairing with integrin β1, endowed the transformed cells with the capability to attach to basement membrane laminin and to spread. Further, inhibition of integrin α6 or β1 function with neutralizing antibodies blocked the invasiveness of AdoMetDC‐transformants and human HT‐1080 fibrosarcoma cells in three‐dimensional Matrigel. Moreover, immunohistochemical analyses showed strong integrin α6 staining in high‐grade human fibrosarcomas. Our data show that c‐Jun can regulate all three key steps of invasion: cell adhesion (integrin α6), basement membrane/extracellular matrix degradation (cathepsin L) and cell migration (thymosin β4). In addition, this is the first study to associate integrin β7, known as a leukocyte‐specific integrin binding to endothelial/epithelial cell adhesion molecules, with the transformed phenotype in cells of nonleukocyte origin. As tumor cell invasion is a prerequisite for metastasis, the observed critical role of integrin α6β1 in fibrosarcoma cell invasion/spreading allures testing antagonists to integrin α6β1, alone or combined with inhibitors of cathepsin L and thymosin β4, as chemotherapeutic agents. © 2009 UICC     

Inhibition of experimental metastasis of human breast-carcinoma cells in athymic nude-mice by anti-alpha(5)beta(1) fibronectin receptor integrin antibodies

We have investigated the role of the human alpha(5) beta(1) fibronectin receptor integrin in experimental metastasis. Treatment of human MDA-MB-231 breast carcinoma cells with monoclonal antibodies specific for alpha(5) or beta(1) integrin subunits prior to injection into the tail veins of 7 to 9 week old athymic nude mice significantly decreased the median number of lung colonies that were formed. In contrast, treatment of the cells with monoclonal antibodies specific for the alpha(2) subunit had no significant effect. In vitro, the anti-alpha(5) and the anti-beta(1) monoclonal antibodies both strongly inhibited breast carcinoma cell adhesion to fibronectin, while only the anti-beta(1) monoclonal antibody inhibited adhesion to laminin. In a Boyden chamber invasion assay, the anti-beta(1) antibody almost completely inhibited invasion of the breast carcinoma cells through an artificial Matrigel basement membrane. The anti-alpha(5) monoclonal antibody inhibited in vitro invasion approximately 30%, only if fibroblast conditioned medium was present as a chemoattractant. Cell migration on fibronectin could be inhibited by both the anti-alpha(5) and the anti-beta(1) monoclonal antibody. These results indicate that the alpha(5) beta(1) integrin fibronectin receptor on MDA-MB-231 human breast carcinoma cells plays an important role in experimental hematogenous metastasis and may function in this process by a combination of mechanisms, including tumor cell attachment to fibronectin and fibronectin-directed extravasation of tumor cells into the target organ.     

Breast cancer metastasis: Putative therapeutic role of vascular cell adhesion molecule-1

Background

Breast cancer is a notable cause of cancer-related death in women worldwide. Metastasis to distant organs is responsible for ~90% of this death. Breast cells convert to malignant cancer cells after acquiring the capacity of invasion/intravasation into surrounding tissues and, finally, extravasation/metastasis to distant organs (i.e., lymph nodes, lungs, bone, brain). Metastasis to distant organs depends on interactions between disseminated tumor cells (DTCs) and the endothelium of blood vessels present in the tumor microenvironment. Among several known endothelial adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) has been found to be involved in this process. It has been shown that VCAM-1 is aberrantly expressed in breast cancer cells and that it can bind to its natural ligand α4β1integrin, also denoted as very late antigen 4 (VLA-4). This binding appears to be responsible for the metastasis of breast cancer cells to lung, bone and brain. The α4β1 integrin - VCAM-1 interaction thus represents a potential therapeutic target for metastatic breast cancer cells. The development of inhibitors of this interaction may be instrumental for the clinical management of breast cancer patients.

Conclusions

This study focuses on recent progress on the role of VCAM-1, an important glycoprotein belonging to the immunoglobulin (Ig) superfamily of cell surface adhesion molecules in breast cancer angiogenesis, survival and metastasis. Targeting VCAM-1, expressed on the surface of breast cancer cells, and/or its specific ligand VLA-4/α4β1 integrin, expressed on cells at the site of metastasis, may be a useful strategy to reduce breast cancer cell invasion and metastasis. Various approaches to therapeutically target VCAM-1 and VLA-4 are also discussed.

     

Thymoquinone and cisplatin as a therapeutic combination in lung cancer: In vitro and in vivo

Background

Melanomas are highly malignant and have high metastatic potential; hence, there is a need for new therapeutic strategies to prevent cell metastasis. In the present study, we investigated whether statins inhibit tumor cell migration, invasion, adhesion, and metastasis in the B16BL6 mouse melanoma cell line.

Methods

The cytotoxicity of statins toward the B16BL6 cells were evaluated using a cell viability assay. As an experimental model, B16BL6 cells were intravenously injected into C57BL/6 mice. Cell migration and invasion were assessed using Boyden chamber assays. Cell adhesion analysis was performed using type I collagen-, type IV collagen-, fibronectin-, and laminin-coated plates. The mRNA levels, enzyme activities and protein levels of matrix metalloproteinases (MMPs) were determined using RT-PCR, activity assay kits, and Western blot analysis, respectively; the mRNA and protein levels of vary late antigens (VLAs) were also determined. The effects of statins on signal transduction molecules were determined by western blot analyses.

Results

We found that statins significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not have cytotoxic effects on B16BL6 cells. Statins also inhibited the mRNA expressions and enzymatic activities of matrix metalloproteinases (MMPs). Moreover, they suppressed the mRNA and protein expressions of integrin α₂, integrin α₄, and integrin α₅ and decreased the membrane localization of Rho, and phosphorylated LIM kinase (LIMK) and myosin light chain (MLC).

Conclusions

The results indicated that statins suppressed the Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion, and metastasis. Furthermore, they markedly inhibited clinically evident metastasis. Thus, these findings suggest that statins have potential clinical applications for the treatment of tumor cell metastasis.     

Cluster of differentiation 44 promotes osteosarcoma progression in mice lacking the tumor suppressor Merlin

Merlin is a versatile tumor suppressor protein encoded by the NF2 gene. Several lines of evidence suggest that Merlin exerts its tumor suppressor activity, at least in part, by forming an inhibitory complex with cluster of differentiation 44 (CD44). Consistently, numerous NF2 mutations in cancer patients are predicted to perturb the interaction of Merlin with CD44. We hypothesized that disruption of the Merlin-CD44 complex through loss of Merlin, unleashes putative tumor- or metastasis-promoting functions of CD44. To evaluate the relevance of the Merlin-CD44 interaction in vivo, we compared tumor growth and progression in Cd44-positive and Cd44-negative Nf2-mutant mice. Heterozygous Nf2-mutant mice were prone to developing highly metastatic osteosarcomas. Importantly, while the absence of the Cd44 gene had no effect on the frequency of primary osteosarcoma development, it strongly diminished osteosarcoma metastasis formation in the Nf2-mutant mice. In vitro assays identified transendothelial migration as the most prominent cellular phenotype dependent on CD44. Adhesion to endothelial cells was blocked by interfering with integrin α4β1 (very late antigen-4, VLA-4) on osteosarcoma cells and CD44 upregulated levels of integrin VLA-4 β1 subunit. Among other putative functions of CD44, which may contribute to the metastatic behavior, the passage through the endothelial cells also appears to be critical in vivo, as CD44 significantly promoted formation of lung metastasis upon intravenous injection of osteosarcoma cells into immunocompromised mice. Altogether, our results strongly suggest that CD44 plays a metastasis-promoting role in the absence of Merlin.     

Loss of VLA-2 Collagen Receptor in Breast Carcinoma, Facilitating Invasion and Metastasis

The integrin VLA-2 as a collagen receptor and VLA-5 as a fibronectin receptor were detected immunohistochemically in normal, benign tumor and carcinoma tissues of the breast. Both proteins were also detected by Western-blot analysis in some carcinoma cases. Epithelial and myoepitheiial cells of both normal breast and benign tumor were in all cases immunoreactive for VLA-2 in the plasma membrane. Carcinoma cells in the invasive component were not immunoreactive for VLA-2 in 31 (46%) of 67 cases. Carcinoma cells in the intraductal components were negative for VLA-2 in only 4 (11%) of 36 cases, while 20 cases (56%) showed weak expression and 12 cases (33%) showed strong expression. Metastatic carcinoma cells in the lymph nodes of 6 cases showed no immunoreactivity except in one case, whereas, again with the exception of one case, the carcinoma cells in the primary tumors did show VLA-2 expression. With regard to VLA-5, there was no difference in its expression in the invasive components and the intraductal components. These findings suggest that the loss of VLA-2 plays a role in the invasion and metastasis of breast carcinoma.     

Roles of VLA-2 and VLA-3 on the formation of peritoneal dissemination in gastric cancer

To clarify the mechanisms of the metastasis on the peritoneal surface from gastric cancer, a novel ex vivo co-culture system using human greater omentum and a highly metastatic cell line, MKN-45-P was developed. MKN-45-P was established from a gastric cancer cell line of MKN-45 by the progressive growing of the intraperitoneal passages. The differences of the expression of metastasis related genes between MKN-45 and MKN-45-P were examined by RT-PCR. Cancer cells adhered only to the naked area of the submesothelial basement membrane, which was exposed by the shrinkage and exfoliation of mesothelial cells of the omentum. The expressions of integrin alpha 2 and alpha 3 subunits in MKN-45-P were extremely elevated compared to those in MKN-45. Integrin beta 1 subunit expression did not change during the intraperitoneal passages. Anti-beta 1 integrin subunit antibody significantly inhibited the adherent number of MKN-45-P on the omentum. These results indicate that VLA-2 and VLA-3 may have an important role in the formation of the peritoneal dissemination from gastric cancer.     

Heparanase antisense suppression of A-549 lung carcinoma invasion

Objective: Heparanase (HPSE), as the only enzyme which can degrade the extracellular matrix and heparin sulfate in basement membrane, plays an important role in invasion and metastasis of tumor cells. In this study, we evaluated the inhibitory effect of HPSE antisense oligoxydeonucleotide (ASODN) on lung carcinoma cell line A-549 invasion. Methods: Liposome-mediated ASODN was transfected into A-549 cells and expression of HPSE protein and mRNA were detected by flow cytometry and RT-PCR techniques. Matrigel invasion assays were employed to assess effects on invasiveness. Results: Lower expression of HPSE protein and mRNA and lower invasive ability to recombinate basal membrane were apparent after ASOND treatment (P<0.01). The inhibition rates of cell invasiveness were 55.6%, 82.3% and 91.2% treated by ASODN at final concentrations of 100, 200 and 400nmol/L, respectively. Conclusions: HPSE ASODN can downregulate the expression of HPSE protein and mRNA in the A-549 cell line and can obviously inhibit its invasive ability in a dose-dependent manner in vitro.     

Anti-α3 integrin antibody induces the activated form of matrix metalloprotease-2 (MMP-2) with concomitant stimulation of invasion through matrigel by human rhabdomyosarcoma cells

Proteolytic enzymes, such as matrix metalloproteases (MMPs), play a pivotal role in cancer invasion and metastasis. Invasive human rhabdomyosarcoma cells (RD) secreted proMMP-2 (72-kDa progelatinase). We found that anti-α3 and -α2 integrin antibodies induced the activated form of MMP-2 and enhanced proMMP-2 secretion by RD cells. The effect of anti-α2 integrin antibody was less prominent than that seen with anti-α3 integrin antibody. Moreover, we have found that anti-α3 and -α2 integrin antibodies enhanced RD-cell invasion through matrigel (reconstituted basement membrane) by 2.6-and 2.0-fold respectively this process was abrogated by neutralizing antibody to MMP-2. These data suggest that signaling events induced by anti-α3 integrin antibody may be involved in RD-cell invasion as a result of modulation of matrix-metalloprotease expression. © 1997 Wiley-Liss, Inc.     

整合素 α6β4 在肿瘤的侵袭和转移中的作用

整合素α6β4在肿瘤的侵袭和转移中的作用不仅依赖于它的黏附特性,而且还与它潜在的信号传递特性有关.本文对整合素α6β4在肿瘤的侵袭和转移中的作用进行综述.     

Expression of Type IV Collagen and Its Degrading Enzymes in Squamous Cell Carcinoma of Lung

We examined the in situ distribution of basement membrane collagen (Col IV), matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) by immunohisto-chemistry and their mRNA levels by Northern blot analysis in 14 cases of squamous cell carcinoma of the lung. Elevated mRNA levels of MMP-2 and Col IV were demonstrated in all the cases examined and were associated with in situ disruption of basement membranes around the tumor nests. In contrast, TIMP-1 mRNA levels were not altered. MMP-2, MMP-9 and TIMP-1 were localized in tumor cells, stromal fibroblasts and endothelial cells. There were no significant correlations between these parameters and clinical staging. The results suggest that the degrading enzymes of basement membrane collagen play an important role in the invasion and metastasis of human squamous cell carcinoma of the lung.     

Anti-beta4 integrin antibodies enhance migratory and invasive abilities of human colon adenocarcinoma cells and their MMP-2 expression

Integrin-mediated adhesion of cells to extracellular matrix proteins has been shown to activate various intracellular signaling events. In the present study, we demonstrate that the addition of a monoclonal antibody raised against the beta4 integrin subunit in the culture medium of a clone derived from the colon adenocarcinoma cell line LoVo specifically results in stimulation of cell migration and invasion through reconstituted basement membrane matrices. Moreover, an increase in MMP-2 activity is observed. Conversely, monoclonal anti-alpha6 and anti-beta1 have no effect on MMP-2 expression. The s. c. co-injection of adenocarcinoma cells with antibodies raised against the beta4 integrin subunit to immunosuppressed newborn rats gives rise to tumors displaying altered and disorganized peri-tumoral basement membranes compared with tumors obtained when cells are injected with adenocarcinoma cells alone. Higher metastatic capacity of cells results when they are co-injected with antibodies to the beta4 integrin subunit. Our results suggest that the beta4 subunit of alpha6beta4 integrin, a laminin receptor in colon adenocarcinoma, may be responsible for the specific signals which stimulate cell motility, expression of MMP-2 and tumor invasion.     

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     

Melanoma cell adhesion to basement membrane mediated by integrin-related complexes

During invasion and metastasis, tumor cells use a variety of surface adhesion receptors to attach to and invade basement membranes and interstitial stroma. We examined the role of the cell surface integrin-like complex in the attachment of the invasive murine B16-BL6 melanoma cell line to basement membrane. Polyclonal antibodies prepared against integrin-related complexes isolated from hamster BHK cells (anti-ECMR) or mouse erythroleukemia cells (anti-mouse FnR) inhibited the attachment of B16 cells to complex basement membrane matrices and to substrates coated with purified extracellular matrix components (fibronectin, laminin, and type IV collagen). The expression of integrin-like receptors on the surface of B16 cells was confirmed by selective immunoprecipitation of radiolabeled and solubilized membrane proteins with the antibodies. Both antibodies also reacted with an integrin-related fibronectin-binding receptor complex purified by ligand affinity chromatography on fibronectin-Sepharose columns. The anti-integrin antibodies failed to react with the Mr 68,000 laminin-binding protein, suggesting that their inhibition of cell attachment to laminin and complex basement membrane was not due to contaminating antibodies against the Mr 68,000 laminin receptor. The results indicate that the integrin receptor complexes on B16-BL6 cells either interact directly with a diverse set of extracellular-matrix-associated components or somehow modulate the activity and function of other receptors. Thus integrins may have an important role in tumor cell invasion of tissue barriers.     

Reduction of lung metastasis,cell invasion,and adhesion in mouse melanoma by statin-induced blockade of the Rho/Rho-associated coiled-coil-containing protein kinase pathway

Background

Melanomas are highly malignant and have high metastatic potential; hence, there is a need for new therapeutic strategies to prevent cell metastasis. In the present study, we investigated whether statins inhibit tumor cell migration, invasion, adhesion, and metastasis in the B16BL6 mouse melanoma cell line.

Methods

The cytotoxicity of statins toward the B16BL6 cells were evaluated using a cell viability assay. As an experimental model, B16BL6 cells were intravenously injected into C57BL/6 mice. Cell migration and invasion were assessed using Boyden chamber assays. Cell adhesion analysis was performed using type I collagen-, type IV collagen-, fibronectin-, and laminin-coated plates. The mRNA levels, enzyme activities and protein levels of matrix metalloproteinases (MMPs) were determined using RT-PCR, activity assay kits, and Western blot analysis, respectively; the mRNA and protein levels of vary late antigens (VLAs) were also determined. The effects of statins on signal transduction molecules were determined by western blot analyses.

Results

We found that statins significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not have cytotoxic effects on B16BL6 cells. Statins also inhibited the mRNA expressions and enzymatic activities of matrix metalloproteinases (MMPs). Moreover, they suppressed the mRNA and protein expressions of integrin α₂, integrin α₄, and integrin α₅ and decreased the membrane localization of Rho, and phosphorylated LIM kinase (LIMK) and myosin light chain (MLC).

Conclusions

The results indicated that statins suppressed the Rho/Rho-associated coiled-coil-containing protein kinase (ROCK) pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion, and metastasis. Furthermore, they markedly inhibited clinically evident metastasis. Thus, these findings suggest that statins have potential clinical applications for the treatment of tumor cell metastasis.