An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells.

The side population (SP) in human lung cancer cell lines and tumors is enriched with cancer stem cells. An endogenous inhibitor of angiogenesis known as tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), characterized for its ability to inhibit matrix metalloproteinases (MMPs), has been shown by several laboratories to impede tumor progression through MMP-dependent or -independent mechanisms. We recently reported that forced expression of TIMP-2, as well as the modified form Ala+TIMP-2 (that lacks MMP inhibitory activity) significantly blocks growth of A549 human lung cancer cells in vivo. However, the mechanisms underlying TIMP-2 antitumor effects are not fully characterized. Here, we examine the hypothesis that the TIMP-2 antitumor activity may involve regulation of the SP in human lung cancer cells. Indeed, using Hoechst dye efflux assay and flow cytometry, as well as quantitative reverse transcriptase-PCR analysis, we found that endogenous TIMP-2 mRNA levels showed a significant inverse correlation with SP fraction size in six non-small cell lung cancer cell lines. In A549 cells expressing increased levels of TIMP-2, a significant decrease in SP was observed, and this decrease was associated with lowered gene expression of ABCG2, ABCB1 and AKR1C1. Functional analysis of A549 cells showed that TIMP-2 overexpression increased chemosensitivity to cytotoxic drugs. The SP isolated from TIMP-2-overexpressing A549 cells also demonstrated impaired migratory capacity compared with the SP from empty vector control. More importantly, our data provide strong evidence that these TIMP-2 functions occur independent of MMP inhibition, as A549 cells overexpressing Ala+TIMP-2 exhibited identical behavior to those overexpressing TIMP-2 alone. Our findings provide the first indication that TIMP-2 modulates SP phenotype and function, and suggests that TIMP-2 may act as an endogenous suppressor of the SP in human lung cancer cells.

TIMP-2 upregulates RECK expression via dephosphorylation of paxillin tyrosine residues 31 and 118.

We previously demonstrated that TIMP-2 increases the association of Crk with C3G and via subsequent activation of Rap1 enhances the expression of RECK, a membrane-anchored MMP inhibitor. In the present study, we investigate the mechanism of how the TIMP-2 signal is transduced from the alpha3beta1 integrin receptor to the Crk-C3G-Rap1 molecular complex. TIMP-2 treatment of human microvascular endothelial cells (hMVECs) increased the phosphorylation levels of Src at Tyr-527, the negative regulatory site, through enhanced association of Src with Csk. This results in the reduction of Src kinase activity and dephosphorylation of paxillin at Tyr-31/118, the target sites for Src kinase phosphorylation and also the binding sites for the downstream effector Crk. Such TIMP-2 effects accompany the disassembly of paxillin-Crk-DOCK180 molecular complex and, in turn, Rac1 inactivation. On the contrary, levels of paxillin-Crk-C3G complex formation are not reduced, rather slightly increased, which is consistent with our previous finding. Therefore, TIMP-2-mediated inhibition of Src kinase activity leads to the signaling switch from Rac1 to Rap1, thereby leading to enhanced RECK expression.

Quantification of tissue inhibitor of metalloproteinases 2 in plasma from healthy donors and cancer patients.

Tissue inhibitor of metalloproteinases (TIMP)-2 is a highly conserved molecule, which binds both active and latent matrix metalloproteinase (MMP)-2. TIMP-2 is also involved in the activation of MMP-2 on the cell surface. A quantitative enzyme-linked immunosorbent assay (ELISA) was established and optimized for measurement of TIMP-2 in plasma. The capturing antibody in the ELISA was a monoclonal, while the detecting antibody was a chicken polyclonal antibody recognizing the native form of human TIMP-2. The levels of TIMP-2 were measured in ethylenediaminetetraacetic acid (EDTA) and citrate plasma from healthy donors. The median values were determined as 163 ng/ml (n = 186) with a range of 109-253 ng/ml for EDTA plasma and 139 ng/ml (n = 77) with a range of 95-223 ng/ml for citrate plasma. The TIMP-2 concentration in citrate plasma from 15 patients with advanced, stage IV breast cancer had a median value of 160 ng/ml, only slightly higher but statistically distinguishable from the level found in citrate plasma from the healthy donors. In addition, the TIMP-2 concentration in EDTA plasma from colorectal cancer patients revealed a significantly higher level in plasma from patients with Dukes stage A (P = 0.01) compared with patients with more advanced Dukes stages.

Epidermal growth factor receptor signaling and the invasive phenotype of ovarian carcinoma cells.

BACKGROUND: Most (70%-100%) ovarian carcinomas express high levels of the epidermal growth factor receptor (EGFR). To examine the relationship between EGFR and the invasive phenotype, we assessed integrin expression, adhesion, matrix metalloproteinase (MMP) activity, and migration in ovarian cancer cells in which EGFR expression was modified. METHODS: NIH:OVCAR-8 human ovarian carcinoma cells were transfected with an expression vector containing the human EGFR complementary DNA in an antisense orientation (EGFR-antisense cells) or the vector alone (vector control cells). We compared vector control and EGFR-antisense cells for cell morphology and adhesion by light microscopy, expression of alpha(6)- and alpha(3)-integrin subunits by flow cytometry, MMP and tissue inhibitor of MMP (TIMP) activity by zymography, and migration by a wound migration assay. In some experiments, EGFR kinase activity in parental cells was inhibited by treatment with PD153035. All statistical tests were two-sided. RESULTS: EGFR-antisense cells were morphologically distinct from vector control cells and had a selective decrease in adhesion to laminin-1 that was not observed with vector control cells (P = .008) or on other extracellular matrix substrates. Compared with vector control cells, cell surface alpha(6)-integrin expression decreased by approximately 80% (difference = 78.7%; 95% confidence interval [CI] = 77.8% to 79.6), MMP-9 activity decreased by approximately 50%, and TIMP activity increased by approximately 50% in EGFR-antisense cells. Vector control cells were highly motile (5.51 arbitrary distance unit; 95% CI = 4.98 to 6.04), whereas the EGFR-antisense cells were not (0.99 arbitrary distance units; 95% CI = 0.38 to 1.60). The morphology and integrin profile of NIH:OVCAR-8 parental cells treated with PD153035 were similar to those of the EGFR-antisense cells. CONCLUSIONS: Reduced EGFR expression in ovarian carcinoma cells decreased their adhesion to laminin-1, expression of the alpha(6)-integrin subunit (a well-characterized laminin-1 receptor), and MMP-9 activity. These data support the hypothesis that EGFR overexpression in ovarian cancer cells results in multiple phenotypic changes that enhance the invasive phenotype.

Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.

Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

The role of matrix metalloproteinases in tumor invasion, metastasis, and angiogenesis.

Tumor invasion and metastasis formation are the hallmarks of malignant cancer. Metastatic spread of cancer cells is a result of a complex cascade of cellular events. This article discusses the matrix metalloproteinase family, the regulation of matrix metalloproteinase activity, and the functions of matrix metalloproteinases in tumor progression.

Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis.

Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells and EBV-infected B cells elicit humoral factors that inhibit tumor-induced angiogenesis, resulting in tumor necrosis and regression. Of the chemokine factors identified in association with this growth behavior, none have induced complete tumor regression. We have previously identified tissue inhibitors of metalloproteinase (TIMP)-1 in various B cell lymphoma cell lines. Here we show that induction of TIMP-1 expression in an EBV-negative Burkitt's lymphoma cell line results in a biphasic, in vivo tumor growth pattern in the nude mouse that is essentially identical to EBV-positive Burkitt's lymphoma cell lines. The initial effect of TIMP-1 is to enhance tumor growth, consistent with the reported anti-apoptotic effect of TIMP-1 on B cell growth. Tumor necrosis and regression then follow the initial period of rapid, increased tumor growth. Only microscopic foci of residual, proliferating tumor cells are observed on biopsy of the tumor site. This latter effect is mediated by TIMP-1 inhibition of an angiogenic response within the developing tumor mass, as demonstrated by immunostaining and microvessel counts. These findings suggest that TIMP-1 is an important mediator of the in vivo growth properties of EBV-positive Burkitt's lymphoma.

Proteases in invasion: matrix metalloproteinases.

The role of proteases in general, and the matrix metalloproteinases in particular, in tumor invasion and metastasis is well established. However, the classic view that these enzymes simply provide a mechanism for the breakdown of connective tissue barriers has been challenged. This overview summarizes recent evidence to support the changing view of the role of matrix metalloproteinases in cancer progression. First we briefly review the central role of cell invasion in cancer progression and also the matrix metalloproteinase family members. We then focus on the emerging roles for these enzymes in cancer progression, including the role of matrix metalloproteinases in cell proliferation and release of growth factors, cell migration and in modification of the extracellular matrix to reveal cryptic sites that alter cell behaviour.

Tissue inhibitor of metalloproteinases 1 regulation of interleukin-10 in B-cell differentiation and lymphomagenesis.

Tissue inhibitors of metalloproteinases (TIMPs), first described as specific inhibitors of matrix metalloproteinases, have recently been shown to exert growth factor activities. It was previously demonstrated that TIMP-1 inhibits apoptosis in germinal center B cells and induces further differentiation. Interleukin-10 (IL-10) is reported as a vital factor for the differentiation and survival of germinal center B cells and is also a negative prognostic factor in non-Hodgkin lymphoma (NHL). However, the mechanism of IL-10 activity in B cells and the regulation of its expression are not well understood. IL-10 has been shown to up-regulate TIMP-1 in tissue macrophages, monocytes, and prostate cancer cell lines, but IL-10 modulation of TIMP-1 in B cells and the effect of TIMP-1 on IL-10 expression has not been previously studied. It was found that TIMP-1 expression regulates IL-10 levels in B cells and that TIMP-1 mediates specific B-cell differentiation steps. TIMP-1 inhibition of apoptosis is not IL-10 dependent. TIMP-1 expression in B-cell NHL correlates closely with IL-10 expression and with high histologic grade. Thus, TIMP-1 regulates IL-10 expression in B-cell NHL and, through the inhibition of apoptosis, appears responsible for the negative prognosis associated with IL-10 expression in these tumors.

Monocyte membrane type 1-matrix metalloproteinase. Prostaglandin-dependent regulation and role in metalloproteinase-2 activation.

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs). Suppression of MT1-MMP by indomethacin and SQ 22536 was reversed by prostaglandin E(2) and dibutyryl cyclic AMP, respectively, demonstrating that induction of monocyte MT1-MMP is regulated through a prostaglandin-cAMP pathway. Functional analysis revealed that pro-MMP-2 in the supernatants from human bone marrow stromal fibroblasts, normal male-derived fibroblasts and melanoma cells (A2058) was converted to active MMP-2 when cultured with activated but not control monocytes. Antibodies against MT1-MMP blocked the activation of MMP-2. Tissue inhibitor of metalloproteinase-2 regulation of MMP-2 activation was shown through the addition of varying amounts of recombinant tissue inhibitor of metalloproteinase-2 with pro-MMP-2 to MT1-MMP-expressing monocytes. These findings demonstrate that activated monocytes express functionally active MT1-MMP that may play a significant role in the activation of MMP-2 produced by other cells and as such influence developmental and pathological conditions.

Inhibition of matrix metalloproteinases by over-expression of tissue inhibitor of metalloproteinase-2 inhibits the growth of experimental hemangiomas.

Inhibitors of proteases prevent tumor-associated matrix degradation, affecting tumor growth, angiogenesis and metastasis. Our study was designed to investigate the effect of inhibition of matrix metalloproteinases (MMPs) on the growth of experimental hemangiomas, using the model of murine endothelioma eEnd.1 cells. In nude mice, these cells generate hemangiomas, consisting mostly of host-recruited endothelial cells, whose growth requires the activity of MMPs. In vitro, eEnd.1 cells produce factors that recruit endothelial cells and stimulate them to release MMPs. Over-expression of TIMP-2, following retrovirus-mediated gene transfer, decreased tumor growth in vivo. The infected clone CR1, which produces high levels of TIMP-2 (as assessed by Northern blot, ELISA and reverse zymography), formed slow-growing tumors that did not grow beyond 0.4 g, while clone 1H, which produces little TIMP-2, grew not dissimilarly to mock-infected cells and parental e.End.1 cells. Histologically, control tumors presented the features of cavernous hemangiomas, while CR1 tumors had a more solid pattern, showing foci of apoptotic cells. In vitro, TIMP-2 over-expression had no autocrine anti-proliferative effect on endothelioma cells but reduced their ability to recruit endothelial cells. CR1 cells lacked the capacity of mock-infected or parental eEnd.1 cells to stimulate endothelial cell motility and invasiveness. Antibodies against TIMP-2 restored the ability of CR1 to induce endothelial cell invasion. We conclude that, in this model, genetic increase of TIMP-2 inhibits tumor growth, apparently by affecting the recruitment and organization of host endothelial cells by the transformed cells.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) suppresses TKR-growth factor signaling independent of metalloproteinase inhibition.

The tissue inhibitors of metalloproteinases (TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell proliferation, migration, and invasion that are associated with extracellular matrix (ECM) turnover. Here we demonstrate a direct role for TIMP-2 in regulating tyrosine kinase-type growth factor receptor activation. We show that TIMP-2 suppresses the mitogenic response to tyrosine kinase-type receptor growth factors in a fashion that is independent of MMP inhibition. The TIMP-2 suppression of mitogenesis is reversed by the adenylate cyclase inhibitor SQ22536, and implicates cAMP as the second messenger in these effects. TIMP-2 neither altered the release of transforming growth factor alpha from the cell surface, nor epidermal growth factor (EGF) binding to the cognate receptor, EGFR. TIMP-2 binds to the surface of A549 cells in a specific and saturable fashion (K(d) = 147 pm), that is not competed by the synthetic MMP inhibitor BB-94 and is independent of MT-1-MMP. TIMP-2 induces a decrease in phosphorylation of EGFR and a concomitant reduction in Grb-2 association. TIMP-2 prevents SH2-protein-tyrosine phosphatase-1 (SHP-1) dissociation from immunoprecipitable EGFR complex and a selective increase in total SHP-1 activity. These studies represent a new functional paradigm for TIMP-2 in which TIMP suppresses EGF-mediated mitogenic signaling by short-circuiting EGFR activation.

Validation of a model of colon cancer progression.

A unique feature of SW480 and SW620 colon carcinoma cell lines is that they are derived from primary and secondary tumours resected from a single patient. As such, they may represent a valuable resource for examining genetic changes late in colon cancer progression. In order to verify this, both cell lines have been characterized to determine whether phenotypic differences have been retained despite long-term cell culture in vitro. The primary tumour-derived SW480 cells have an epithelioid morphology in vitro, while metastasis-derived SW620 cells have a fibroblast-like appearance. Xenografts of SW480 cells form gland-like structures in vivo, while SW620 xenografts form solid sheets of tumour cells. SW620 cells have a higher BrdU labelling index than SW480 cells, and are more highly tumourigenic and metastatic. Furthermore, SW620 cells show less susceptibility to apoptosis induction by TNFalpha and anti-Fas monoclonal. Findings from these investigations therefore indicate that SW480 and SW620 cell lines do show appropriate phenotypic differences and represent an interesting model for studying the genetic events in the late stages of colon cancer progression.

Increased expression of tissue inhibitor of metalloproteinases type 1 (TIMP-1) in a more tumourigenic colon cancer cell line.

Genetic changes occurring in the late stages of colonic tumour progression have received much less attention than those occurring in the early stages. As described in the accompanying paper, SW480 and SW620 cell lines provide a useful model for studying the advanced stages of progression for colon cancer. Comparison of the two cell lines by differential display reveals that SW620 cells express lower levels of the CC3 tumour suppressor gene and also lower levels of the tissue inhibitor of metalloproteinases-3 (TIMP-3) gene. Northern blot analysis for TIMP-3 confirms this finding and shows a similar difference in the expression of TIMP-2, which seems logical since TIMPs inhibit enzymes that play a role in tumour invasion. For this reason, it was surprising to find that TIMP-1 messenger RNA expression is markedly increased in SW620 cells. Consistent with this finding, western blot analysis shows a ten-fold increase in TIMP-1 protein secretion by SW620 cells. It is noteworthy that high TIMP-1 expression is associated with poor prognosis in colorectal cancer. This association between TIMP-1 expression and tumour progression may be related to additional growth factor-like effects described for TIMP-1 in some systems.

Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells.

Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothelioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24. 24) and MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggesting that stroma cells, particularly endothelial cells recruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. The increased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions.

Metalloproteinases and their inhibitors are known to play an important role in the extracellular matrix remodeling associated with preinvasive lesions and invasive carcinomas; however, little is known about their role in early lung carcinoma. Immunohistochemical studies were made of the reactivity of bronchial squamous preneoplastic lesions from cigarette smokers, including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma for matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen in 13 patients. Staining for type IV collagen disclosed discontinuities in basement membranes from basal cell hyperplasia to dysplasia, progressing to destruction in carcinoma in situ and invasive carcinoma. Reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma. In contrast, reactivity for MMP-1 was strong in basal cell hyperplasia and squamous metaplasia, decreasing in carcinoma in situ and invasive carcinoma. Some neoplastic cells in carcinoma in situ and invasive carcinoma were MMP-3 positive. Staining for MMP-2 and TIMP-1 was moderate to strong in all squamous preinvasive lesions. Confocal microscopy showed MMP-9-positive cells passing through fragmented basement membranes in which type IV collagen and MMP-9 were colocalized. Type IV collagen colocalized with MMP-2 in all lesions and with TIMP-1 in basal cell hyperplasia and squamous metaplasia. The inverse relationships between the reactivity for MMP-1 and MMP-9 with progression of bronchial squamous preinvasive lesions suggest important roles for these MMPs in basement membrane remodeling in these lesions.

MMP and TIMP gene expression in head and neck squamous cell carcinomas and adjacent tissues.

OBJECTIVE: To compare the frequency of gene expression of matrix metalloproteinases (MMP) stromelysins -1, -2 and -3 (MMP-3, -10, and -11), matrilysin (MMP-7), MTI-MMP (MMP-14), and of TIMPs (Tissue Inhibitors of MMPs) -1, -2, -3 and -4 in head and neck squamous cell carcinomas with those of matched adjacent normal tissues. MATERIALS AND METHODS: The present study included 20 surgically removed head and neck squamous cell carcinomas, seven of which were accompanied by matched adjacent oral mucosa excised from the border of the specimens outside the tumor area. RNA isolated from tumors and control samples was subjected to RT-PCR using primers specific for MMP-3, -7, -10, -11 and -14 and for TIMPs -1, -2, -3, and -4. RESULTS: Our findings demonstrate that each of the five MMP genes studied were expressed in essentially all the tumors, while the adjacent marginal tissue samples showed a more varied picture: while stromelysin-3 was located to a majority of the marginal samples, matrilysin was expressed in four of seven adjacent samples, stromelysin-1 and MTI-MMP genes were each expressed in three of these samples, and stromelysin-2 transcript was only expressed in two marginal tissue samples. Whereas TIMP-1 and TIMP-2 transcripts were identified in all tumor and adjacent tissue samples studied, TIMP-3 was expressed, albeit often at low levels, in 17 of 20 tumor samples but only in three of seven adjacent tissues. The novel TIMP-4 gene was not expressed at all. CONCLUSIONS: Specific MMP (MMP-3, -7, -10, -14) and TIMP-3 transcripts observed in head and neck squamous cell carcinomas compared to their frequency in specimens of matching tissues provide important information about expression of extracellular matrix degrading enzymes and their tissue inhibitors in head and neck carcinomas.

Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance.

We have previously reported the identification of the endogenous angiogenesis inhibitor angiostatin, a specific inhibitor of endothelial cell proliferation in vitro and angiogenesis in vivo. In our original studies, we demonstrated that a Lewis lung carcinoma (LLC-LM) primary tumor could suppress the growth of its metastases by generating angiostatin. Angiostatin, a 38-kDa internal fragment of plasminogen, was purified from the serum and urine of mice bearing LLC-LM, and its discovery provides the first proven mechanism for concomitant resistance (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M. A., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328). Subsequently, we have shown that systemic administration of angiostatin can regress a wide variety of malignant tumors in vivo. However, at the time of our initial discovery of angiostatin, the source of the protein was unclear. We hypothesized that the tumor or stromal cells might produce an enzyme that could cleave plasminogen sequestered by the primary tumor into angiostatin. Alternatively, we speculated that the tumor cells might express angiostatin. By Northern analysis, however, we have found no evidence that the tumor cells express angiostatin or other fragments of plasminogen (data not shown). We now report that gelatinase A (matrix metalloproteinase-2), produced directly by the LLC-LM cells, is responsible for the production of angiostatin, which suppresses the growth of metastases in our original model.

The von Hippel-Lindau tumor suppressor gene inhibits hepatocyte growth factor/scatter factor-induced invasion and branching morphogenesis in renal carcinoma cells.

Loss of function in the von Hippel-Lindau (VHL) tumor suppressor gene occurs in familial and most sporadic renal cell carcinomas (RCCs). VHL has been linked to the regulation of cell cycle cessation (G(0)) and to control of expression of various mRNAs such as for vascular endothelial growth factor. RCC cells express the Met receptor tyrosine kinase, and Met mediates invasion and branching morphogenesis in many cell types in response to hepatocyte growth factor/scatter factor (HGF/SF). We examined the HGF/SF responsiveness of RCC cells containing endogenous mutated (mut) forms of the VHL protein (VHL-negative RCC) with that of isogenic cells expressing exogenous wild-type (wt) VHL (VHL-positive RCC). We found that VHL-negative 786-0 and UOK-101 RCC cells were highly invasive through growth factor-reduced (GFR) Matrigel-coated filters and exhibited an extensive branching morphogenesis phenotype in response to HGF/SF in the three-dimensional (3D) GFR Matrigel cultures. In contrast, the phenotypes of A498 VHL-negative RCC cells were weaker, and isogenic RCC cells ectopically expressing wt VHL did not respond at all. We found that all VHL-negative RCC cells expressed reduced levels of tissue inhibitor of metalloproteinase 2 (TIMP-2) relative to the wt VHL-positive cells, implicating VHL in the regulation of this molecule. However, consistent with the more invasive phenotype of the 786-0 and UOK-101 VHL-negative RCC cells, the levels of TIMP-1 and TIMP-2 were reduced and levels of the matrix metalloproteinases 2 and 9 were elevated compared to the noninvasive VHL-positive RCC cells. Moreover, recombinant TIMPs completely blocked HGF/SF-mediated branching morphogenesis, while neutralizing antibodies to the TIMPs stimulated HGF/SF-mediated invasion in vitro. Thus, the loss of the VHL tumor suppressor gene is central to changes that control tissue invasiveness, and a more invasive phenotype requires additional genetic changes seen in some but not all RCC lines. These studies also demonstrate a synergy between the loss of VHL function and Met signaling.