3690

ITGB3

integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61);ITGB3;integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)

integrin beta-3|platelet membrane glycoprotein IIIa

17q21.32

protein-coding

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.

expression of adhesion receptor integrin alphavbeta3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that alphavbeta3 cooperates with matrix metalloproteinase type 9 (MMP-9) in breast cancer cell migration. This cooperation is regulated by the activation state of the integrin. expression of activated alphavbeta3 in metastatic variants of MDA-MB 435 human breast cancer cells and primary metastatic cells from breast cancer patients strongly enhanced migration toward vitronectin and fibrinogen. This enhancement was mediated by a soluble factor produced by breast cancer cells expressing activated alphavbeta3. When transferred, this factor also up-regulated alphavbeta3-dependent migration of breast cancer cells that express the nonactivated integrin. The factor was identified as metalloproteinase MMP-9. Whereas all tested breast cancer cell variants produced latent MMP-9, only those with activated alphavbeta3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced alphavbeta3-dependent breast cancer cell migration. The migratory response was inhibited by tissue inhibitors of metalloproteinase or when MMP-9 was depleted from the inducing supernatants. The results indicate a causal relationship between the expression of activated integrin alphavbeta3 and production of enzymatically active MMP-9 in metastatic breast cancer cells. These molecules cooperate to enhance breast cancer cell migration toward specific matrix proteins, and this may contribute to the strongly enhanced metastatic capacity of breast cancer cells that express activated alphavbeta3.

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of alphavbeta3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes alphavbeta3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of alphavbeta3 transcription to promote tumor metastasis.

AIM: To investigate integrin beta3 mRNA and vascular endothelial growth factor (VEGF) protein expression in gastric carcinoma, and its correlation with microvascular density, growth-pattern, invasion, metastasis and prognosis. METHODS: In situ hybridization(ISH) of integrin beta3 mRNA and immunohistochemistry of VEGF and CD34 protein were performed on samples from 118 patients with gastric cancer. RESULTS: The positive rate of integrin beta3 mRNA in non-tumor gastric mucosa (20%) was significantly lower than that of the gastric cancer tissue (52.5%, c2 = 10.20, P < 0.01). In patients of infiltrating type, stage T(3)-T(4), vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of integrin beta3 mRNA were significantly higher than those in patients of expanding type (P < 0.01), stage T(1)-T(2) (P < 0.01), non-vessel invasion (P < 0.01), without lymphatic metastasis (P < 0.01), without hepatic and peritoneal metastasis (P < 0.01), respectively. In patients of infiltrating type, stage T(3)-T(4), vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the positive expression rates of VEGF protein were significantly higher than those in patients of expanding type (P < 0.01), stage T(1)-T(2) (P < 0.01), non-vessel invasion (P < 0.01), without lymphatic metastasis (P < 0.01), without hepatic and peritoneal metastasis (P < 0.01), respectively. In patients of infiltrating type, stage T(3)-T(4), vessel invasion, lymphatic metastasis, hepatic or peritoneal metastasis, the mean MVD were significantly higher than those in patients of expanding type (P < 0.01), stage T(1)-T(2) (P < 0.01), non-vessel invasion (P < 0.01), without lymphatic metastasis (P < 0.01), without hepatic and peritoneal metastasis (P < 0.01), respectively. It was found that the positive expression rate of integrin beta3 mRNA was positively related to that of VEGF protein (P < 0.01) and MVD (P < 0.05), meanwhile the positive expression rate of VEGF protein was positively related to MVD (P < 0.05). The mean survival period in patients with positive expression of integrin beta3 mRNA and VEGF, and MVD > or = 54.9/mm(2) was significantly shorter than that in patients with negative expression of integrin beta3 mRNA (P < 0.05) and VEGF (P < 0.01), and MVD < 54.9/mm(2) (P < 0.01). Five-year survival rate in patients with positive expression of integrin beta3 mRNA and VEGF, and MVD > or = 54.9/mm(2) was significantly lower than those with negative expression of integrin beta3 mRNA (P < 0.05), VEGF (P < 0.05), and MVD < 54.9/mm(2) (P < 0.01). CONCLUSION: Integrin beta3 and VEGF expression can synergistically enhance tumor angiogenesis, and may play a crucial role in invasion and metastasis of gastric carcinoma. Therefore, they may be prognostic biomarkers and novel molecular therapeutic targets.

MicroRNAs play an important regulatory role in carcinogenesis and cancer metastasis. Different members of let-7 family have been reported to be decreased in human lung tumors. However, the effect of specific let-7 member on metastasis of NSCLC remains undefined. Our current study detected the expression of let-7 members in 94 cases of NSCLC and a significant association was noticed between low levels of let-7c expression and metastasis, venous invasion, advanced TNM stages and poor survival of NSCLC patients. Consistently, ectopic expression of let-7c in relatively highly metastatic cells remarkably suppressed their migration and invasion. Inhibition of let-7c in cells with relatively low metastatic potential promoted their motility and invasion. We then analyzed the potential targets of let-7c and found that ITGB3 and MAP4K3 were directly repressed by let-7c. Upon restoring the expression of ITGB3 and MAP4K3, the effects of let-7c on tumor metastasis were partially reversed, and more importantly, the expression levels of ITGB3 and MAP4K3 were inversely correlated with let-7c in 64 NSCLC tissues. Collectively, our results suggest that let-7c, by degrading ITGB3 and MAP4K3, prevents NSCLC metastasis.

CD61 is a membrane glycoprotein that associates with CD41 (alphaIIb) to form the heterodimeric complex gpIIb/IIIa (CD41/CD61), predominantly expressed in platelets and megakariocytes. CD61 or beta3 integrin also associates with alpha v (CD51) to form the vitronectin receptor, which is expressed in many tissues. We have used a monoclonal antibody against the porcine gpIIIa or CD61 (JM2E5) to study the distribution of this molecule in different normal pig tissues. As in humans, CD61 was broadly expressed in all tissues examined. In the kidney, strong expression of CD61 was observed in epithelial cells from renal tubules. In the testis, CD61 expression was detected in the Leydig cells. However, in liver, CD61 was weak or not detected. Many integrins are particularly involved in tumogenicity and in tumor progression mediating cell-cell interaction. Immunofluorescence experiments using cultured human tumor HeLa cells showed nuclear and cytoplasmic staining of mAb JM2E5. Immunohistochemical analysis of human tumor sections from several organs showed a heterogeneus distribution in metastatic cases from colon and breast carcinoma. However, no staining was found in metastasis from melanoma.

Early metastasis is the primary cause of death in melanoma patients. The adhesion receptor integrin alpha v beta 3 contributes to tumor cell functions that are potentially involved in melanoma growth and metastasis. We tested whether integrin alpha v beta 3 supports metastasis of human melanoma cells when injected into the bloodstream of immune deficient mice. Comparing variants of the same melanoma cell type that expressed either alpha v beta 3, alpha IIb beta 3 or no beta 3 integrin, we found that only alpha v beta 3 strongly supported metastasis. Inhibition of tumor cell alpha v beta 3 function reduced melanoma metastasis significantly and prolonged animal survival. To understand mechanisms that allow alpha v beta 3, but not alpha IIb beta 3 to support melanoma metastasis, we analyzed proteolytic and migratory activities of the melanoma cell variants. Melanoma cells expressing alpha v beta 3, but not those expressing alpha IIb beta 3 or no beta 3 integrin, produced the active form of metalloproteinase MMP-2 and expressed elevated mRNA levels of MT1-MMP and TIMP-2. This indicates an association between alpha v beta 3 expression and protease processing. Furthermore, alpha v beta 3 expression was required for efficient melanoma cell migration toward the matrix proteins fibronectin and vitronectin. The results suggest that expression of integrin alpha v beta 3 promotes the metastatic phenotype in human melanoma by supporting specific adhesive, invasive and migratory properties of the tumor cells and that the related integrin alpha IIb beta 3 cannot substitute for alpha v beta 3 in this respect.

To clarify the function of integrin alpha(v)beta3 in the early stage of liver metastasis, we investigated the interactions of metastatic cells with their target organ under the actual blood flow by using positron emission tomography (PET). The cells used were CHO-K1 cells and their transfectants bearing human integrin alpha(v)beta3 cDNA (alpha(v)beta3-CHO-K1 cells). The liver accumulation of alpha(v)beta3-CHO-K1 cells was significantly higher than that of CHO-K1 cells after injection via the portal vein, whereas no significant difference was observed in the lung accumulation after tail vein injection, suggesting a specific interaction of alpha(v)beta3-CHO-K1 cells with the hepatic sinusoids. Furthermore, to clarify the precise location of each cell in the liver, i.e., to determine whether individual cells were intravascularly localized or had extravasated, we performed intravital fluorescence microscopy (IVM) on the liver by using stable transfectants bearing the green fluorescent protein (GFP) gene, namely, GFP-CHO-K1 and GFP-alpha(v)beta3-CHO-K1 cells. Both types of cells remained in the hepatic blood vessels 1 h after injection via the portal vein. On the other hand, expression of integrin alpha(v)beta3 promoted the cells to reach the extravascular region after 24 h. These results suggest the possibility that the specific accumulation of alpha(v)beta3-CHO-K1 cells in the liver is followed by migration of the cells into the extravascular region. Interestingly, the adhesion of the two types of cells to hepatic sinusoidal endothelial cells in vitro did not correspond to in vivo accumulation of these cells. Therefore, integrin alpha(v)beta3 may function to promote extravasation of integrin alpha(v)beta3-expressing tumor cells in liver through a process possibly mediated by vitronectin produced by this organ.

In this study, significantly higher expression of Gal3ST-2 (Gal: 3-O-sulfotransferase-2) and 3'-sulfated glycoconjugates were observed in highly metastastic cancer cells and in larynx cancer tissues with lymph node metastasis than those in lowly metastatic cancer cells and larynx cancer tissues without metastasis (P<0.01, n=42). These results indicated that there was a marked correlation between the expression of Gal3ST-2 and tumor metastasis potential. After RNAi transfection, a striking morphological change of SMMC7721 hepatoma cells from polygon to shuttle shape and significant decrease in adhesion to sL-selectin and HUVEC were observed. Interestingly, the expression of integrin subunit alphaV was markedly downregulated and 3'-sulfated subunit alphaV almost disappeared in the transfectants, but integrin subunit beta3 almost had no change. These results suggested that Gal3ST-2 was involved in tumor metastasis process by regulation of adhesion ability to selectins and expression of integrins.

Transendothelial migration is a key step in the extravasation of tumor cells during metastasis formation. Here, we have classified 45 human tumor cell lines derived from various tissues according to their capacity for transendothelial migration in vitro. We could distinguish cell lines showing strong transmigration (TEM+ cell lines) from others that did not transmigrate (TEM- cell lines). By DNA microarray analysis we could cluster TEM+ and TEM- cell lines according to their gene expression pattern and identify genes differentially expressed between the 2 groups. Among these we found the integrin beta3 subunit to be highly expressed in TEM+ cell lines as compared to TEM- cell lines. Cell surface localization of alphavbeta3 integrin receptors was exclusively found in TEM+ cell lines. Transendothelial migration of TEM+ cells but not their adhesion to the endothelial cells, or invasion into collagen gels could be blocked with an antibody against alphavbeta3 integrin and by RNAi mediated knock-down of the integrin beta3 subunit. These data establishes alphavbeta3 integrin as one key component of the transendothelial migration process of tumor cells, and as a potential target for anti-metastatic therapy. Our gene expression analysis of a defined collection of tumor cell lines can be used as a starting point to identify further genes functionally involved in transendothelial migration.

Alternatively spliced integrins may play an important role in integrin mediated tumor cell adhesion, spreading, and migration. Here we report in human tumor cells a naturally occurring alternatively spliced variant of the beta3 integrin [i.e., truncated (tr) beta3] that lacked a cytoplasmic and a transmembrane domain. The presence of trbeta3 was demonstrated at the mRNA level by RT-PCR, cloning, and sequencing; at the protein level by immunohistochemistry and Western Blotting. The alternately spliced beta3 integrin was detected in human prostate carcinomas, breast carcinomas, and melanoma cells. expression in vivo was confirmed by immunohistochemistry with an antibody to trbeta3 that does not recognize wild type beta3. tumor cells secreted this protein and deposited it on the extracellular matrix. Secreted trbeta3 inhibited adhesion of melanoma and prostate cancer cells to fibronectin and vitronectin, which was partially reversed by adsorption of trbeta3 from the media. Confocal microscopy and time lapse live cell microscopy demonstrated that trbeta3 distributed to the trailing edge of migrating cells, which may represent an alternative cell detachment mechanism in these cells. Results suggest that trbeta3 may act as an anti-integrin and play a crucial role in cell migration, which is an important process in tumor invasion and metastasis.

The incidence of brain metastasis is rising and poses a severe clinical problem, as we lack effective therapies and knowledge of mechanisms that control metastatic growth in the brain. Here we demonstrate a crucial role for high-affinity tumor cell integrin alpha(v)beta(3) in brain metastatic growth and recruitment of blood vessels. Although alpha(v)beta(3) is frequently up-regulated in primary brain tumors and metastatic lesions of brain homing cancers, we show that it is the alpha(v)beta(3) activation state that is critical for brain lesion growth. Activated, but not non-activated, tumor cell alpha(v)beta(3) supports efficient brain metastatic growth through continuous up-regulation of vascular endothelial growth factor (VEGF) protein under normoxic conditions. In metastatic brain lesions carrying activated alpha(v)beta(3), VEGF expression is controlled at the post-transcriptional level and involves phosphorylation and inhibition of translational respressor 4E-binding protein (4E-BP1). In contrast, tumor cells with non-activated alpha(v)beta(3) depend on hypoxia for VEGF induction, resulting in reduced angiogenesis, tumor cell apoptosis, and inefficient intracranial growth. Importantly, the microenvironment critically influences the effects that activated tumor cell alpha(v)beta(3) exerts on tumor cell growth. Although it strongly promoted intracranial growth, the activation state of the receptor did not influence tumor growth in the mammary fat pad as a primary site. Thus, we identified a mechanism by which metastatic cells thrive in the brain microenvironment and use the high-affinity form of an adhesion receptor to grow and secure host support for proliferation. Targeting this molecular mechanism could prove valuable for the inhibition of brain metastasis.

INTRODUCTION: expression of adhesion molecules such as alphavbeta3 integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether alphavbeta3 expression correlated with the metastatic potential of human osteosarcoma cells. MATERIALS AND METHODS: We developed a series of sublines (LM2-LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6-8 weeks. We quantified alphavbeta3 integrin expression using flow cytometry. RESULTS: alphavbeta3 expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of alphavbeta3. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that alphavbeta3 was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. CONCLUSIONS: alphavbeta3 integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. Alphavbeta3 integrin may therefore be a potential new target for osteosarcoma.

The growth of metastatic prostate cancer cells in the bone involves an intimate interaction between the tumor cells and various elements of the bone microenvironment, resulting in increased rate of bone turnover and rapid tumor growth. The alpha(v)beta3 integrin has been shown to play an important role in tumor growth and angiogenesis, and is known to be critical to osteoclast formation and activity. This study was designed to examine the role of alpha(v)beta3 expressed by cells native to the bone in the growth and pathogenesis of prostate cancer bone metastases. Human prostate cancer cells which do not express alpha(v)beta3 or alpha(IIb)beta3 integrins were injected directly into human bone fragments previously implanted subcutaneously in SCID mice (SCID-human-bone model). At the same time treatment with anti-beta3 antibody fragment (m7E3 F(ab')2) i.p. at 300 microg/dose 3 x per week was initiated and continued for 2 weeks. In this system, m7E3 F(ab')2 only recognizes human bone-derived alpha(v)beta3. Antibody inhibition of alpha(v)beta3 integrin in vivo resulted in a specific reduction in the proportion of antigenically-human blood vessels within tumor-bearing bone implants (from 73.5% +/- 3.93 in controls to 17.74% +/- 5.64 in treated animals). Proliferation of the alpha(v)beta3-negative tumor cells was also reduced, although the overall vessel density was maintained by compensating mouse vasculature. Blockage of human bone-derived alpha(v)beta3 also significantly reduced the recruitment of osteoclasts in response to tumor cells, as well as degradation of calcified bone tissue. Together these observations confirm the importance of alpha(v)beta3 in bone metabolism and angiogenesis, and point to the role of these processes in controlling growth of metastatic prostate cancer cells in the bone.

BACKGROUND: Stromal cell-derived factor-1 (SDF-1 or CXCL12) and CXCR4 are key elements in the metastasis of prostate cancer cells to bone--but the mechanisms as to how it localizes to the marrow remains unclear. METHODS: Prostate cancer cell lines were stimulated with SDF-1 and evaluated for alterations in the expression of adhesion molecules using microarrays, FACs, and Western blotting to identify alpha(v)beta(3) receptors. Cell-cell adhesion and invasion assays were used to verify that activation of the receptor is responsive to SDF-1. RESULTS: We demonstrate that SDF-1 transiently regulates the number and affinity of alpha(v)beta(3) receptors by prostate cancer cells to enhance their metastatic behavior by increasing adhesiveness and invasiveness. SDF-1 transiently increased the expression of beta(3) receptor subunit and increased its phosphorylation in metastatic but not nonmetastatic cells. CONCLUSIONS: The transition from a locally invasive phenotype to a metastatic phenotype may be primed by the elevated expression of alpha(v)beta(3) receptors. Activation and increased expression of alpha(v)beta(3) within SDF-1-rich organs may participate in metastatic localization.