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Expression of adhesion receptor integrin ␣v␤3 in an activated functional form strongly promotes metastasis in human breast cancer cells. Here, we report that ␣v␤3 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 ␣v␤3 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 ␣v␤3. When transferred, this factor also up-regulated ␣v␤3-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 ␣v␤3 produced the mature form of this metalloproteinase. Recombinant mature MMP-9, but not latent MMP-9 or either form of MMP-2, enhanced ␣v␤3-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 ␣v␤3 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 ␣v␤3.M etastasis is the primary cause of death in breast cancer patients. Metastatic dissemination depends on tumor cell adhesion, migration, and invasion. These steps involve integrins, a family of transmembrane adhesion receptors, composed of noncovalently linked ␣ and ␤ subunits (1). Integrins are known to exist in distinct states of activation, and these determine integrin functionality and affinity for ligands (2). For example, integrin activation controls which ligands are recognized, whether an integrin can support cell arrest under dynamic flow conditions or only stationary adhesion, and whether cells can migrate on or toward specific substrates (3). The importance of integrin activation has long been appreciated in leukocytes and platelets, where it controls inflammatory responses and thrombus formation (4, 5). Recent findings indicate that other cell types, such as endothelial cells and tumor cells, can also regulate their interaction with extracellular matrix proteins by integrin activation (6-9). This regulation may help to control angiogenesis and tumor metastasis.In breast and ovarian cancer, as well as in melanoma and glioma, malignant progression is associated with expression of tumor cell integrin ␣v␤3 (10-14). We recently found that ␣v␤3 can exist in human breast cancer cells in an activated or a nonactivated functional state. Only the activated state supports breast cancer cell arrest du...

Hepatocellular carcinoma (HCC) treatment is challenging because the mechanisms underlying tumor progression are still largely unknown. Transforming growth factor (TGF)-␤1 is considered a crucial molecule in HCC tumorigenesis because increased levels of patients' serum and urine are associated with disease progression. The aim of the present study was to investigate the inhibition of TGF-␤ signaling and its impact on HCC progression. Human HCC cell lines were treated with a TGF-␤ receptor kinase inhibitor (LY2109761) whose selectivity was determined in a kinase assay. Exogenous TGF-␤1 phosphorylates the TGF-␤ receptor, consequently activating Smad-2, whereas the drug selectively blocks this effect and dephosphorylates autocrine p-Smad-2 at concentrations ranging from 0.001 to 0.1 M. A cytotoxic effect documented by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), trypan blue, and propidium iodide staining assays was observed at 10 M, whereas the drug inhibits (P < 0.001) the migration of HCC cells on fibronectin, laminin-5, and vitronectin and invasion through Matrigel (P < 0.001) at concentrations up to 0.1 M. LY2109761 up-regulates (P < 0.001) E-cadherin mRNA and protein levels. This increase was localized at the cellular membrane where E-cadherin mediates anchorage that is cell-cell dependent. Consistently, a functional monoclonal antibody that inhibits E-cadherin-dependent cell-cell contact restores the migratory and invasive activity. Finally, nonmetastatic HCC tissues from 7 patients were cultured with TGF-␤1 in the presence or absence of LY2109761. E-cadherin expression was reduced by TGF-␤1 and was significantly (P < 0.0001) increased by LY2109761 treatment, measured by quantitative real-time PCR on microdissected tissues and by immunohistochemistry on serial sections. In 72 patients, E-cadherin tissue expression was more weakly expressed in metastatic than in nonmetastatic HCC (P < 0.0001). Conclusion: LY2109761 blocks migration and invasion of HCC cells by upregulating E-cadherin, suggesting that there could be a mechanistic use for this molecule in clinical trials. (HEPATOLOGY 2008;47:1557-1566 H epatocellular carcinoma (HCC) is a highly malignant cancer that is the third most frequent cause of tumor-related death in the United States and Europe. 1 Current therapeutic options are invasive and aim to physically remove or destroy the tumor mass. However, later recurrence and/or metastatic spread are common and negatively affect survival. The overall prognosis is still unsatisfactory, and little progress has been made in finding new treatment options. However, a recent study with sorafenib suggests that targeting the vasculature may provide additional insights into how to develop future treatment options for patients with HCC. 2 Based on the recent clinical observations with sorafenib, the focus for developing new treatments in HCC has shifted from targeting the cancer to targeting the tissue microenvironment and its role in modulating the biological behavior of HCC. 3 Transforming grow...

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SUMMARY:Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type-1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express ␣3␤1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2-cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC ␣3␤1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-␣3␤1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through Matrigel. In vivo, ␣3␤1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both ␣3␤1 integrin and gelatinase activity are required for HCC migration and invasion. (Lab Invest 2001, 81:613-627).

D rug-based treatments to cure patients with hepatocellular carcinoma (HCC) remain a highly desirable goal in drug development because socalled curative therapies are successful in only few patients. 1 The approval of sorafenib for the treatment of patients with advanced-stage disease has stimulated the investigation of new therapeutic strategies. 2 However, the development of new drugs is hampered by the heterogeneity of HCC and the underlying liver disease type, which limit the recognition of useful therapeutic targets. 3 Because HCC generally develops in fibrotic tissue, the growth of tumor cells is dependent on an intricate crosstalk between the tumor and the stroma. Due to this complexity and interaction with fibrotic liver, HCC cells develop a cross-talk with adjacent components of the tissue microenvironment, including extracellular matrix proteins and various growth factors. A growing body of evidence suggests that the cross-talk affects the malignant phenotype and is a key factor of poor clinical outcome. Cancer-associated fibroblasts (CAFs), a heterogeneous population of stromal cells, play a central role in this stroma-tumor interaction, involving the production of growth factors, which in turn may reduce the effect of chemotherapy and radiotherapy. 4,5 CAFs can form after stimulation with transforming growth factor ␤-1 (TGF-␤1), which plays an important role in HCC by stimulat-

H epatocellular carcinoma (HCC) poses a major health problem worldwide because of its constantly increasing incidence in developed countries and its poor prognosis. 1,2 The high mortality rate of HCC is attributable to late diagnosis, underlying liver disorders, and cancer recurrence. Only a minority (30% to 40%) of patients is diagnosed early and is eligible for the most effective treatment, including tumor resection, radiofrequency ablation, and, in limited cases, liver transplantation. 3 Nevertheless, even in these cases the prognosis and survival are not satisfactory because the recurrence rate is higher than 70% at 5 years after resection, and the currently available therapies have failed to reduce this rate. 4,5 In cases of HCC recurrence, therapeutic approaches are limited, due to multifocal and intrahepatic spread of the tumor in these advanced and more aggressive cancer forms with an unfavorable prognosis. 6-8 Literature reports show that the most reliable predictor of HCC recurrence is macroscopic or microscopic vascular invasion, which is also an adverse prognostic factor. 9-11 Furthermore, because an extensive histological examination is required to detect microscopic invasion, this problem is underestimated in patients undergoing radiofrequency ablation therapy.The molecular mechanisms underlying vascular invasion are unknown. Fibronectin (Fn), a major component of the blood vessel wall, is ubiquitously distributed. HCC cells need to engage with Fn to cross the wall, and with fibrinogen (Fg) to migrate into the vascular lumen. 12,13 This migration is mediated by a class of heterodimeric transmembrane receptors named integrins, formed by an Abbreviations: Fg, fibrinogen; Fn, fibronectin; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; TGF, transforming growth factor. From the

An imbalance between the proteolytic activity of matrix metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2 (TIMP-2) is responsible for degradation of extracellular matrix (ECM) components and plays a critical role in tumor invasion and in metastasis formation. The occurrence of intra-hepatic metastasis, which severely affects prognosis and long-term survival, is commonly observed in the course of hepatocellular carcinoma (HCC). We investigated the expression of MT1-MMP in tissues, whereas both MMP-2 and TIMP-2 were evaluated in the sera and tissues (primary and metastatic nodules) of HCC patients with and without metastasis, whose clinical outcome was followed over a 2-year period. MT1-MMP expression was similar among primary nodule tissues of patients with and without metastasis. Serum and tissue levels of MMP-2 were not statistically different between patients with and without metastasis, but MMP-2 was concentrated at the invasive edge of the metastatic tissue. On the contrary, serum and tissue levels of TIMP-2 were significantly higher in HCC patients without metastasis than in those with. This situation was not only observed in the primary HCC tissues, but also in the metastatic nodules. These results correlate with the clinical outcome, because more than 90% of the patients with high levels of TIMP-2 were still alive after 2 years, whereas less than 30% with low levels of TIMP-2 had survived. Furthermore, we found a strict correlation between tissue and serum levels of TIMP-2, this suggesting that a MMP-2/TIMP-2 imbalance and in particular TIMP-2 levels, could represent an important prognostic factor in patients with HCC.