Expression of alpha(v)beta6 integrin in oral leukoplakia

The distribution of alpha(v)beta6 integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins beta1, beta3, beta4, beta5, fibronectin and tenascin were also studied. The integrin alpha(v)beta6 was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. alpha(v)beta6 integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of beta1 integrins was localized in the basal layer, and that of the beta4 at the cell surface facing the basement membrane of all specimens. The integrins beta3 and beta5 were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both alpha(v)beta6-positive and alpha(v)beta6-negative tissues. A group of 28 leukoplakia patients were followed 1-4 years after first diagnosis. In this group, initially alpha(v)beta6 integrin-positive leukoplakia specimens had high tendency for disease progression while alpha(v)beta6-negative specimens did not progress. These results suggest that the expression of alpha(v)beta6 integrin could be associated in the malignant transformation of oral leukoplakias.     

Insulin-like growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by alphavbeta3 integrin signaling

Insulin-like growth factor 1 (IGF-1) has been implicated in promoting mitogenic, metastatic and antiapoptotic phenotypes in several types of cancer. But little is known about the signal interaction of IGF-1 and integrin in the regulation of cervical cancer development and progression. This study is to investigate the regulatory mechanism of IGF-1 receptor (IGF-1R) signaling and its importance in cervical cancer formation. The growth and invasiveness of cervical cancer cells (SiHa and CaSki) were dose-dependently stimulated by IGF-1, whereas those of normal cervical epithelial cells were not. The immunoblot showed that IGF-1R proteins were abundant in cervical cancer cell lines. In contrast, IGF-1R protein was nearly undetectable in normal cervical epithelial cells. IGF-1-stimulated invasion and proliferation were abolished by functional-blocking monoclonal antibody against IGF-1R, whereas these cellular functions were unaffected by either IgG or monoclonal antibody to insulin receptor. Functional-blocking monoclonal antibody against integrins alpha(v)beta3, but not alpha2 alpha3, alpha4 alpha6 beta1, beta4 or alpha2beta1, inhibited the IGF-1-stimulated invasion and proliferation in cervical cancer cells. alpha(v)beta3 integrin modulated IGF-1R phosphorylation by altering the rate of Src homology 2-containing phosphotyrosine phosphatase (SHP-2) recruitment to the activated IGF-1R. The modulation of alpha(v)beta3 occupancy also affected the activation of IGF-1R downstream-signaling elements, including activation of Akt and extracellular signal-regulated protein kinases 1/2 (Erk1/2). The treatment of blocking antibody of alpha(v)beta3 integrin or IGF-1R significantly inhibited tumor growth and caused tumor regression in SCID mice model. Immunoblots of tumor tissues confirmed that the phosphorylation of IGF-1R and downstream targets of Akt and Erk1/2 were remarkably decreased in SCID mice treated with blocking antibodies of alpha(v)beta3 or IGF-1R. Thus, these data suggest that the signal interaction between IGF-1R and alpha(v)beta3 integrin plays an important role in promoting the development and progression of cervical cancer.     

Activated K-ras is involved in regulation of integrin expression in human colon carcinoma cells

Integrins participate in controlling proliferation and migration. Therefore, changes in integrin expression might be responsible for unrestrained proliferation and invasiveness of tumor cells. Alterations of integrin subunit expression have been observed in human colon carcinoma, especially loss or reduction of the alpha5 subunit, which was observed consistently. The mechanisms responsible for reduction of alpha5 expression and alteration of expression of other integrins are not fully understood. Circumstantial evidence from previous investigations points to an involvement of activated ras oncogenes in repression of integrin expression. The K-ras protooncogene is activated by point mutation in 50% of human colon carcinomas. Thus, we choose an antisense approach for specific inactivation of activated K-ras in the human colon carcinoma cell line SW 480 in order to test whether activated K-ras contributes to changes in integrin expression on colon carcinoma cells. Cell surface expression of the alpha1 and the alpha5 subunit was increased in K-ras antisense transfected clones, cell surface expression of the alpha3 subunit and the alphav subunit was decreased. This shows, in a human system, that activated K-ras is involved in diminishing cell surface expression of the alpha1beta1 collagen/laminin receptor and the alpha5beta1 fibronectin receptor, both of which are implicated in maintenance of a non-transformed phenotype. Moreover, activated K-ras contributes to increased cell surface expression of the alpha3beta1 laminin/collagen/fibronectin receptor and the alphavbeta5 vitronectin receptor, which might play a role in metastatic behavior of tumor cells.     

Altered surface expression and increased turnover of the alpha6beta4 integrin in an undifferentiated carcinoma

The integrin alpha6beta4, predominantly expressed on tissues of epithelial origin, is known to be variably expressed on carcinomas. The biochemical changes resulting in altered expression during tumor progression are unknown. We have analyzed the expression of alpha6beta4 in a multi-step mouse model of skin carcinogenesis representing normal keratinocyte, benign papilloma and malignant undifferentiated carcinoma. All cell lines expressed the alpha6 integrin exclusively as the alpha6beta4 integrin heterodimer. Analysis of this integrin by flow cytometry and immunoprecipitation of surface labeled proteins revealed that the undifferentiated carcinoma cells have an approximately 75% reduction in surface expression of the integrin as compared with the keratinocyte and papilloma cell lines. The alpha6beta4 integrin which remains expressed on the carcinoma cells is diffusely distributed in the membrane and has an approximately 2.5-fold increased biological turnover as compared with normal keratinocytes. The decreased biological half-life and the loss of polarized expression of alpha6beta4 on the carcinoma cells suggests an altered functional role for the alpha6beta4 integrin on carcinoma cells during tumor progression. These factors may contribute to the known supression of hemidesmosome structures and the increased migration phenotype associated with some epithelial carcinomas.     

Effect of retinoic acid on integrin receptors of B16F10 melanoma cells

The intriguing problem of metastasis requires the spreading of metastatic cells through the basement membrane barrier. The interaction of the basement membrane with the metastatic cell is a cell surface activity involving the function of integrin receptors. Integrins are a group of alpha,beta heterodimeric proteins responsible for transducing intracellular signals on binding to the extracellular matrix proteins present in the basement membrane. To understand the role of integrin receptors in tumor metastasis, the cell surface receptor functions were modulated by All Trans Retinoic Acid (ATRA) treatment in B16F10 tumor cells. Our experimental results clearly indicate that All Trans Retinoic Acid (ATRA) inhibit metastatic potential of highly metastatic B16F10 melanoma cells by 1) downregulating the cell surface integrin receptors against ECM proteins specially laminin and vitronectin and 2) by inhibiting the 72 kd collagenase activity.     

Engaged urokinase receptors enhance tumor breast cell migration and invasion by upregulating alpha(v)beta5 vitronectin receptor cell surface expression

We have previously shown that urokinase receptor physically and functionally interacts with alpha(v)beta5 vitronectin receptor, leading to tumor breast cell migration and invasion. Here, the link between these 2 receptors was further investigated by analyzing the expression levels of urokinase receptor and alpha(v)beta5 integrin in 35 human breast carcinomas and 5 benign breast lesions. The occurrence of a positive correlation between urokinase receptor and alpha(v)beta5 protein levels in benign and malignant tumor specimens prompted us to investigate whether engaged urokinase receptors might modulate alpha(v)beta5 expression. Here, we report the receptor-dependent ability of catalytically inactive urokinase to upregulate the alpha(v) and beta5 chains in MDA-MB-231 and MCF-7 breast carcinoma cell lines in a time- and concentration-dependent manner. This effect is dependent on protein kinase C activity and requires new protein synthesis. Accordingly, the availability of assembled alpha(v)beta5 receptors on the cell surface increases upon urokinase treatment, as shown by immunoprecipitation and immunocytochemical analyses. Exposure to urokinase leads to enhanced tumor cell migration and invasion, which is prevented by the "phosphorylation-like" urokinase receptor antagonist His-uPA(138E/303E), the DNA-binding drug mithramycin, the protein kinase C inhibitor calphostin C and anti-alpha(v)beta5 antibodies. Finally, urokinase enables benign breast MCF-10A cells to cross Matrigel in a alpha(v)beta5- and urokinase receptor-dependent manner, indicating that urokinase controls a regulatory circuitry crucial to breast tumor progression.     

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.     

Expression of the alpha6beta4 integrin provides prognostic information in bladder cancer

Integrins are cell surface receptors for extracellular matrix components that may participate in metastatic processes. Normal urothelial tissues show a polarized expression of alpha6beta4 integrin on basal cells at their junction with the lamina propria. We have previously shown that bladder cancers frequently overexpress one member of the integrin family, the alpha6beta4 integrin. In this study, we evaluated the level of alpha6beta4 integrin expression in bladder cancer specimens from 57 patients and correlated the expression level with patient survival. Expression was evaluated by immunoperoxidase staining. Three patterns of alpha6beta4 expression were observed: negative (13 patients); strong overexpression throughout the tumor cells (21 patients); and weak expression that most closely resembled expression in normal urothelium (23 patients). Individuals with weak staining tumors had a statistically significantly better survival (p=0.041) than patients whose tumors exhibited either no expression or strong overexpression. These data indicate that evaluation of the expression of alpha6beta4 integrin may provide valuable prognostic information on clinical outcome in patients with bladder cancer.     

Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models

PURPOSE: Integrins alpha v beta 3 and alpha v beta 5 are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation. METHODS AND MATERIALS: Levels of alpha v beta 3 were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non-small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment. RESULTS: Human umbilical vein endothelial cells had the highest alpha v beta 3 level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of alpha v beta 3 in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment. CONCLUSIONS: We conclude that radiation induces expression of alpha v beta 3 integrin in endothelial and non-small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.     

The lipoxygenase metabolite 12(S)-HETE induces a cytoskeleton-dependent increase in surface expression of integrin alpha IIb beta 3 on melanoma cells

Integrin receptors are mediators of cell-extracellular matrix and cell-cell interactions. Biochemical and immunocytochemical evidence shows that the platelet integrin receptor alpha IIb beta 3 is present on the cell surface, at focal adhesion plaques and in the perinuclear region of metastatic B16a murine melanoma cells. Antibody to the fibronectin receptor alpha 5 beta i, inhibits basal adhesion by approx. 30%, whereas antibodies to alpha IIb beta 3 are ineffective. The surface immunoreactivity of tumor cells for alpha IIb beta 3 can be enhanced by pre-treatment (5 min) with a lipoxygenase metabolite of arachidonic acid [i.e. 12-(S)-HETE] in a dose-dependent manner (max. effect approx. 0.1 microM). Other lipoxygenase metabolites are ineffective. B16a cells possess a large intracellular pool of alpha IIb beta 3, from which the receptor complex translocates to the cell surface following 12-(S)-HETE pretreatment. This pre-treatment of tumor cells enhances their adhesion to fibronectin, which is mediated exclusively by alpha IIb beta 3 receptors. 12-(S)-HETE also facilitates the redistribution of alpha IIb beta 3 in the plasma membrane with localization at the focal adhesion plaques. The cytoskeleton of the B16a cell is characterized by an absence of distinct microtubules in interphase cells and the presence of prominent microfilaments and vimentin intermediate filaments. In B16a cells, the disruption of intermediate filaments and/or microfilaments prevents the 12-(S)-HETE-induced increase in plasma membrane alpha IIb beta 3 and enhanced tumor-cell adhesion to fibronectin. The microtubule-disrupting agent, colchicine, is ineffective in both respects. We conclude that the lipoxygenase metabolite of arachidonic acid, 12-(S)-HETE, regulates the surface expression and function of the alpha IIb beta 3 integrin in B16a cells. Further, these data support the hypothesis that microfilaments and intermediate filaments have a profound role in regulating the expression of a multifunctional integrin in B16a tumor cells.     

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.     

Expression of osteopontin and its receptors in ameloblastomas

This study used an immunohistochemical method to examine the expression of osteopontin (OPN) in 30 ameloblastomas and of integrin alpha(v) and CD44v6 in 20 of these 30 ameloblastomas. We found that the positive staining rate in ameloblastomas was 87% for OPN, 45% for integrin alpha(v), and 90% for CD44v6. Both ameloblast-like and stellate reticulum-like cells showed a high expression of OPN and CD44v6. There was also a strong integrin alpha(v) immunostaining on the cell membrane of osteoclasts. Binding of OPN to osteoclast cell membrane receptor integrin alpha(v) can activate the osteoclast and increase its osteolytic activity. In addition, binding of OPN to ameloblastoma tumor cell membrane receptor CD44v6 can enhance tumor cell migration, invasion and spread. Therefore, we suggest that the local aggressive behavior and high osteolytic ability of ameloblastomas in the jawbones can be explained by the high expression of OPN and CD44v6 in ameloblastoma cells and the high expression of integrin alpha(v) in osteoclasts.     

The pattern of metastasis of human melanoma to the central nervous system is not influenced by integrin alpha(v)beta(3) expression

We investigated the effect of integrin alpha(v)beta(3) expression on the metastatic pattern of human melanoma cells in the central nervous system (CNS). For this purpose, we developed a hematogenous CNS melanoma metastasis model in nude mice using a modified internal carotid artery infusion technique. This protocol revealed 2 different patterns of CNS metastasis. The integrin alpha(v)beta(3)-expressing melanoma lines Mel57 and Zkr nearly exclusively produced metastases in the brain parenchyma, whereas cells of the BLM and MV3 lines, devoid of integrin alpha(v)beta(3) expression, preferentially metastasized to dura mater and leptomeninges. Treatment with hyaluronidase to obtain single BLM cell suspensions did not influence the metastatic pattern, indicating that this was not simply the result of entrapment of tumor cell aggregates in large-sized leptomeningeal vessels. The role of integrin alpha(v)beta(3) expression in the process of metastasis was tested by transfection of BLM, but did not lead to an altered pattern of metastasis. We did observe, however, slower growth of the transfected tumors, although the in vitro growth rate was unaltered, indicating a reduction in tumorigenicity. We conclude from our findings that CNS metastasis of melanoma cells in the mouse xenograft model occurs in at least 2 different but very reproducible patterns. Although it is predicted that adhesion of tumor cells to endothelial cells plays a role in this phenomenon, tumor cell integrin alpha(v)beta(3) expression per se does not explain the difference in metastatic behavior in the CNS. We assume that other, as yet unknown factors, must be involved.     

alpha(v) integrins regulate cell proliferation through integrin-linked kinase (ILK) in ovarian cancer cells

Integrins regulate both adhesion and signaling processes involved in proliferation and survival. alpha(v)beta(3) and alpha(v)beta(5) integrins have been shown to mediate cell adhesion and migration. Here we used human ovarian cancer cell lines (IGROV1, SKOV-3) that express alpha(v)beta(3) and alpha(v)beta(5) to study their role in cell proliferation and the signaling pathways involved. We found that alpha(v) integrins regulate cell proliferation through activation of integrin-linked kinase (ILK). An anti-alpha(v)-blocking antibody specifically inhibits the growth of IGROV1 and SKOV-3. The inhibition of cell proliferation involves alpha(v)beta(3) in IGROV1 cells, and both alpha(v)beta(3) and alpha(v)beta(5) in SKOV-3 cells. The reduced growth rate induced by alpha(v) integrin blockade is linked in both cell lines to G1/S cell cycle arrest. alpha(v) integrin blockade by neutralizing antibody as well as cyclic-RGD peptide caused an inhibition of ILK activity and phosphorylation of PKB/Akt on serine-473 but not on threonine-308, and was accompanied by an increase in p27(Kip1) expression. Overexpression of wild-type ILK rescued the phosphorylation of PKB/Akt on serine-473 in cells treated with anti-alpha(v) antibody. Inhibition of ILK by a pharmacological inhibitor results in inhibition of cell proliferation, PKB/Akt phosphorylation and increase of p27(Kip1). These results demonstrate that alpha(v) integrins regulate ovarian cancer cell proliferation through ILK.     

RGD肽在肿瘤治疗中的研究进展

RGD肽是一类含有精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp)的短肽,作为整合素和其配体相互作用的识别位点,介导细胞与细胞外基质及细胞之间的相互作用.肿瘤细胞或者新生血管可以特异表达某些整合素如αvβ3,能以一定的亲和力结合RGD肽,成为肿瘤治疗的新靶点.因此,RGD肽在肿瘤治疗中的应用已成为研究热点.放射性核素标记的RGD肽是一类很有前景的肿瘤显像剂.此外RGD肽可以诱导肿瘤细胞凋亡且作为载体在肿瘤靶向治疗中发挥重要作用.     

alpha(v)beta(3) Integrin-targeting radionuclide therapy and imaging with monomeric RGD peptide

The alpha(v)beta(3) integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress alpha(v)beta(3) integrin, as in tumor neovascularization, and alpha(v)beta(3) integrin expression in other microvascular beds and organs is limited. Therefore, alpha(v)beta(3) integrin is a suitable receptor for tumor-targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to alpha(v)beta(3) integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor-targeting properties and the therapeutic potential of 111In- and 90Y-labeled monomeric RGD peptides in BALB/c nude mice with SKOV-3 human ovarian carcinoma tumors. DOTA-c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to alpha(v)beta(3) integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In-DOTA-c(RGDfK) and 90Y-DOTA-c(RGDfK) showed high uptake in tumors (7.3 +/- 0.6% ID/g and 4.6 +/- 0.8% ID/g, respectively) and gradually decreased over time (2.3 +/- 0.4% ID/g and 1.5 +/- 0.5% ID/g at 24 hr, respectively). High tumor-to-blood and -muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple-dose administration of 90Y-DOTA-c(RGDfK) (3 x 11.1 MBq) suppressed tumor growth in comparison to the control group and a single-dose administration (11.1 MBq). Monomeric RGD peptides, 111In-DOTA-c(RGDfK) and (90)Y-DOTA-c(RGDfK), could be promising tracers for alpha(v)beta(3) integrin-targeting imaging and radiotherapy.     

Expression of integrin alpha(v)beta(3) correlates with activation of membrane-type matrix metalloproteinase-1 (MT1-MMP) and matrix metalloproteinase-2 (MMP-2) in human melanoma cells in vitro and in vivo

Activation of matrix metalloproteinase-2 (MMP-2) is mediated by binding to the complex of membrane-type matrix metalloproteinase-1 (MT1-MMP) with tissue inhibitor of MMP-2 (TIMP-2) on the cell surface. Binding of MMP-2 to integrin alpha(v)beta(3) has been implicated in presenting activated MMP-2 on the cell surface of invasive cells, but interactions with the MT1-MMP-TIMP-2 system have not been considered. Therefore, we studied the expression and interaction of MT1-MMP, MMP-2 and TIMP-2 in the alpha(v)beta(3)-negative melanoma cell line BLM and in its beta(3)-transfected, alpha(v)beta(3)-expressing counterpart BLM-beta(3), both on cell lines and in xenografts. Total expression levels of MMP-2, MT1-MMP and TIMP-2 did not differ markedly between the alpha(v)beta(3)-negative and alpha(v)beta(3)-positive cells. Remarkable differences, however, exist in the presence of active MMP-2 and MT1-MMP. Zymography on cell lysates revealed that active MMP-2 was restricted to alpha(v)beta(3)-positive cell line and clearly accumulated in xenografts derived from the BLM-beta(3) cells, confirming the relevance of this integrin for MMP-2 function. Western blotting of cell lysates showed that processing of proMT1-MMP to the activated form was enhanced in BLM-beta(3). The ratio of active and inactive MT1-MMP was 3-fold higher in the beta(3)-transfectants. Immunofluorescence double-labeling followed by confocal laser microscopy showed co-localization of MT1-MMP and alpha(v)beta(3) on BLM-beta(3) cells. In xenografts from BLM-beta(3) cells, active MT1-MMP was markedly increased. Our results demonstrate that expression of alpha(v)beta(3) in cell lines and xenografts was accompanied by an accumulation of active MT1-MMP and MMP-2. Furthermore, MT1-MMP and alpha(v)beta(3) are co-localized on the cell membrane of tumor cells. These findings suggest that activated MT1-MMP co-localized with alpha(v)beta(3) may be involved in activation of alpha(v)beta(3)-bound MMP-2.     

Expression and function of CYR61, an angiogenic factor, in breast cancer cell lines and tumor biopsies

We have previously shown that expression of heregulin (HRG) is closely correlated with breast cancer progression. We have subsequently isolated Cyr61, a ligand for the alpha(v)beta3 integrin that is differentially expressed in HRG-positive cells, and have shown that it is expressed in all of the invasive and metastatic breast cancer cell lines tested. Preliminary evaluation of Cyr61 expression in breast tumor biopsies revealed expression of Cyr61 in about 30% of invasive breast carcinomas. Significantly, we demonstrated that Cyr61 is a downstream effector of HRG action, because a Cyr61-neutralizing antibody abolished the ability of HRG-expressing cells to migrate in vitro. Furthermore, we have shown that HRG-expressing cells denote higher levels of alpha(v)beta3 expression, and we have established that Cyr61 action is mediated, at least in part, through its receptor alpha(v)beta3, because a functional blocking antibody of the alpha(v)beta3 blocked the Matrigel outgrowth of HRG-expressing cells. These results strongly suggest that Cyr61 is necessary for HRG-mediated chemomigration and that Cyr61 plays a functional role in breast cancer progression, possibly through its interactions with the alpha(v)beta3 receptor.     

Fibronectin and fibronectin-receptors of the integrin type in normal colon mucosa, adenoma and carcinoma

Integrin alpha 3, alpha 4, alpha 5, and alpha v subunits heterodimerize with beta 1 and beta 3 chains to form functional fibronectin (FN) receptors. Two of them, alpha 4 beta 1 and alpha 5 beta 1, recognize FN as the only matrix molecule. Additional binding of laminin, collagen, and entactin is achieved by alpha 3 beta 1 while alpha v beta 1 and alpha v beta 3 also bind vitronectin. We investigated immunohistochemically the tissue distribution of FN and the expression of FN receptor alpha- and beta-chains in 20 normal colon tissues, 10 adenomas, 90 carcinomas, and 10 liver metastases derived thereof. In normal and adenomatous colon tissues FN was detected in association with reticular and fibrillar structures of the gut wall. The tumor stroma of carcinomas and liver metastases contained abnormally high amounts of FN which were detectable as chaotic deposits. Normal and adenomatous mucosa were alpha 3(+), alpha 4(-), alpha 5(-), alpha v(+/-), beta 1(+), beta 3(-) indicating that only promiscuous FN receptors were expressed. 21 of 90 carcinomas had a focal or complete loss of alpha 3 which was more often found in Dukes C/D tumors (p<0.005). The liver metastases more often had lower levels of alpha 3 expression compared to the primary tumor. The alpha v-expression was inconsistent and often weak. These losses of FN receptor constituents in carcinomas were not paralleled by any noticeable differences in stromal content or distribution pattern of FN, Enhanced expression/induction of FN receptors was found in only 2 of 90 carcinomas which focally neo-expressed the alpha 5-chain. This aberrant alpha 5 expression, however, was flow cytometrically found in 3/4 colon carcinoma cell lines which otherwise had receptor profiles corresponding to the in situ situation. FN-adherence of cell lines was variable. Anti-alpha 3 had no blocking effect on FN adherence of SW480, SW620, and COLO 205 but a minor effect on FN binding of HT-29. Anti-alpha 5 blocked FN adhesion whenever alpha 5 was expressed. Anti-av had no blocking effect while anti-beta 1 reduced FN adherence of all cell lines. The net biological effect(s) of aberrant expression of integrin type FN receptors in colon carcinomas is unpredictable as yet.     

Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography

The alpha(v)beta3 integrin is an important cell adhesion receptor involved in tumor-induced angiogenesis and tumor metastasis. Here we describe the 18F-labeling of the RGD-containing glycopeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys(sugar amino acid)-) with 4-nitrophenyl 2-[18F]fluoropropionate and the evaluation of this compound in vitro and in tumor mouse models. Binding assays with isolated immobilized alpha(v)beta3, alpha(v)beta5, and alpha(IIb)beta3 as well as in vivo studies using alpha(v)beta3-positive and -negative murine and xenotransplanted human tumors demonstrated receptor-specific binding of the radiolabeled glycopeptide yielding high tumor:background ratios (e.g., 120 min postinjection: tumor:blood, 27.5; tumor:muscle, 10.2). First imaging results using a small animal positron emission tomograph suggest that this compound is suitable for noninvasive determination of the alpha(v)beta3 integrin status and therapy monitoring.