Hepatic stellate cells stimulate HCC cell migration via laminin-5 production.

Activated HSCs (hepatic stellate cells) are the main source of extracellular matrix proteins present in cirrhotic liver on which HCC (hepatocellular carcinoma) commonly develops. HCC cells behave differently according to differences in the surrounding microenvironment. In the present study, we have investigated a mechanism whereby HSCs modulate the migratory activity of HCC cells. We used primary cultures of human HSCs to investigate their effect on Hep3B, Alexander, HLE and HLF HCC cells. The expression of Ln-5 (laminin-5) was documented at transcript and protein levels both in vitro and in vivo. HCC cells strongly adhere, migrate and spread in the presence of HSC-conditioned medium and of co-culture. HSCs produce and secrete Ln-5 in the CM (conditioned medium). The electrophoretic pattern of secreted Ln-5 is consistent with that of a migratory substrate, showing the presence of the γ2x fragment. Blocking antibodies against Ln-5 inhibit HCC migration in the presence of HSC-CM. HCC cells migrate very poorly in the presence of Ln-5 immunodepleted HSC-CM. HCC migration in the presence of HSCs is dependent on the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway, but not the PI3K (phosphoinositide 3-kinase)/Akt pathway. HSC-CM, as well as Ln-5, activates the MEK/ERK but not the PI3K/Akt pathway. In human HCC tissues, Ln-5 is mainly distributed along α-SMA (smooth muscle actin)-positive cells, whereas in peritumoural tissues, Ln-5 is absent. HSCs stimulate HCC migration via the production and secretion of Ln-5.

Kinase activation profile associated with TGF-ß-dependent migration of HCC cells: a preclinical study.

PURPOSE: To identify the molecular mechanisms responsible for tumor cell migration is essential for developing agents that can prevent the relapse or the metastatic spread of hepatocellular carcinoma (HCC). METHODS: In this study, we investigated the effects of the transforming growth factor-ß receptor I inhibitor LY2109761 on two different human HCC cell lines, in vitro and in vivo. RESULTS: LY2109761 inhibits HCC migration in a dose-dependent manner. This inhibition is associated with the decreased phosphorylation of SMAD-2, FAK and ß1-integrin, and with increased levels of E-cadherin. By contrast, LY2109761 did not alter the phosphorylation pattern of p38MAPkinase. In a two- and a three-day time-course and in dose-titration experiments, LY2109761 inhibited HCC migration as well as phospho-SMAD-2 and the adhesion proteins. LY2109761 showed the best effect on day 2 at 1 nM and for 3 days at 100 nM concentration. This suggests that maximum effects were sustained for several days and were not dependent on excess concentrations. Finally, in a xenograft model of HCC, LY2109761 strongly inhibits tumor growth, intravasation and metastasis at the aforementioned lower concentrations. CONCLUSIONS: In conclusion, inhibition of transforming growth factor-ß (TGF-ß) appears to occur at low concentrations of LY2109761 that displays multiple effects on kinases that control HCC cell migration. These findings may help the design of future clinical trials with inhibitors of TGF-ß.

Inhibiting TGF-ß signaling in hepatocellular carcinoma.

One of the main complications in patients with liver fibrosis is the development of hepatocellular carcinoma (HCC). An understanding of the molecular mechanisms leading to HCC is important in order to be able to design new pharmacological agents serving either to prevent or mitigate the outcome of this malignancy. The transforming growth factor-beta (TGF-ß) cytokine and its isoforms initiate a signaling cascade which is closely linked to liver fibrosis, cirrhosis and subsequent progression to HCC. Because of its role in these stages of disease progression, TGF-ß appears to play a unique role in the molecular pathogenesis of HCC. Thus, it is a promising target for pharmacological treatment strategies. Recent studies have shown that inhibition of TGF-ß signaling results in multiple synergistic down-stream effects which will likely improve the clinical outcome in HCC. We also review a number of TGF-ß inhibitors, most of which are still in a preclinical stage of development, but may soon be available for trial in HCC patients. Hence, it is anticipated that there will soon be new agents available for clinical investigations to evaluate the role of the TGF-ß-associated signaling in this deadly cancer.

Down-regulation of connective tissue growth factor by inhibition of transforming growth factor beta blocks the tumor-stroma cross-talk and tumor progression in hepatocellular carcinoma.

UNLABELLED: Tumor-stroma interactions in hepatocellular carcinoma (HCC) are of key importance to tumor progression. In this study, we show that HCC invasive cells produce high levels of connective tissue growth factor (CTGF) and generate tumors with a high stromal component in a xenograft model. A transforming growth factor beta (TGF-beta) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF-beta-dependent down-regulation of CTGF diminished tumor growth, intravasation, and metastatic dissemination of HCC cells by inhibiting cancer-associated fibroblast proliferation. By contrast, noninvasive HCC cells were found to produce low levels of CTGF. Upon TGF-beta1 stimulation, noninvasive HCC cells form tumors with a high stromal content and CTGF expression, which is inhibited by treatment with LY2109761. In addition, the acquired intravasation and metastatic spread of noninvasive HCC cells after TGF-beta1 stimulation was blocked by LY2109761. LY2109761 interrupts the cross-talk between cancer cells and cancer-associated fibroblasts, leading to a significant reduction of HCC growth and dissemination. Interestingly, patients with high CTGF expression had poor prognosis, suggesting that treatment aimed at reducing TGF-beta-dependent CTGF expression may offer clinical benefits. CONCLUSION: Taken together, our preclinical results indicate that LY2109761 targets the cross-talk between HCC and the stroma and provide a rationale for future clinical trials.

Inhibition of transforming growth factor beta receptor I kinase blocks hepatocellular carcinoma growth through neo-angiogenesis regulation.

UNLABELLED: Curative therapies for patients with hepatocellular carcinoma (HCC) are mainly invasive, and with the exception of sorafenib, no medical treatments are available for advanced or metastatic stages of HCC. We investigated the antitumoral effect of blocking the transforming growth factor beta (TGF-beta) signaling pathway in HCC with LY2109761, a kinase inhibitor of TGF-beta receptor I kinase. The antitumor activity of LY2109761 was associated with inhibition of molecular pathways involved in neo-angiogenesis and tumor growth of HCC. This anti-angiogenic effect is more effective than that of bevacizumab, which specifically targets vascular endothelial growth factor (VEGF). We found that the paracrine cross-talk between HCC and endothelial cells is blocked by LY210976, inhibiting blood vessel formation. This effect was mediated by SMAD2/3 and affected the secretion of VEGF. Finally, LY2109761 does not show significant effects on physiological angiogenetic development. CONCLUSION: These data support the rationale for targeting TGF-beta signaling in patients with HCC.

HCC heterogeneity: molecular pathogenesis and clinical implications.

BACKGROUND: Hepatocellular carcinoma (HCC) poses a major challenge because of the extreme variability of the clinical outcome, which makes it difficult to properly stage the disease and thereby estimate the prognosis. There is growing evidence that this heterogeneous clinical behavior is attributable to several different biological pathways. A novel approach to mapping these differences is by investigating the epigenetics associated with certain clinical aspects. DESIGN: Herein, the relevance of these molecular differences in combination with the biological and molecular pathways regulating the clinical outcome will be discussed. Use of a mechanistic and pathogenic approach to clarify the natural history of HCC is not just an academic speculation but should help to develop new therapies and to tailor these therapies to each individual patient. CONCLUSION: New biological therapies targeting components of the tumoral or peritumoral microenvironment are crucial to the fight against HCC. However, biological redundancies and the presence of several growth factors, hormones, cytokines, etc., potentially involved in HCC tumor progression make it difficult to assess the best target. Sorafenib, a multi-tyrosine kinase inhibitor, blocks the functions of different growth factors present in the tissue microenvironment. The use of Sorafenib in patients with HCC offers a new approach to the therapy of this disease, stimulating research focusing on the development of drugs based on new molecular and pathogenic insights.

Tissue expression of Squamous Cellular Carcinoma Antigen (SCCA) is inversely correlated to tumor size in HCC.

BACKGROUND: This study aimed to investigate squamous cellular carcinoma antigen (SCCA) in serum and in tumoral and paired peritumoral tissues. We studied 27 patients with liver cirrhosis (LC) and 55 with HCC: 20 with a single nodule < 3 cm (s-HCC) and 35 with a single nodule > 3 cm or multifocal (l-HCC). METHODS: Serum SCCA was measured by the ELISA kit, and in frozen tissues by immunohistochemistry, quantified with appropriate imaging analysis software and expressed in square microns. Continuous variables are reported as means and 95% confidence intervals. Comparisons between independent groups were performed with a generalized linear model and Tukey grouping. Pearson's correlation coefficients were determined to evaluate relations between markers. Qualitative variables were summarized as count and percentage. Statistical significance was set at p-value < 0.05. RESULTS: Serum SCCA values in LC patients were 0.41 (0.31-0.55) ng/ml and statistically different from both HCC groups: 1.6 (1.0-2.6) ng/ml in s-HCC, 2.2 (1.28-2.74) ng/ml in l-HCC. SCCA in hepatic tissue was 263.8 (176.6-394.01) microm2 in LC patients, statistically different from values in s-HCC: 1163.2 (863.6-1566.8) microm2 and l-HCC: 625.8 (534.5-732.6). All pairwise comparisons between groups yielded statistically significant differences. Tumoral SCCA resulted linearly related with nodule size, showing a statistically significant inverse relation between the two variables (b = -0.099, p = 0.024). CONCLUSION: There was no statistically significant correlation between tissue and serum levels of SCCA. The significantly stronger expression of SCCA in smaller compared to larger HCC could be important for early HCC detection. However, the increased expression in peritumoral tissue could affect the significance of serological detection.

Targeting transforming growth factor (TGF)-betaRI inhibits activation of beta1 integrin and blocks vascular invasion in hepatocellular carcinoma.

UNLABELLED: Vascular invasion is one of the major negative prognostic factors in patients with hepatocellular carcinoma (HCC), leading to cancer recurrence. To invade, HCC cells must penetrate the vessel wall, consisting of endothelial cells and extracellular matrix components, including fibronectin and fibrinogen. Employing invasive and noninvasive HCC cells, we studied the mechanism underlying vascular invasion. We show that HCC cells invade blood vessels via alpha5beta1, that is equally expressed in invasive and noninvasive cells. However, in the former, the intracytoplasmic tail of beta1 integrin is constitutively phosphorylated at threonine 788-789 and the extracellular part is conformationally activated. In noninvasive cells, beta1 integrin is not activated. Transforming growth factor (TGF)-beta1 specifically phosphorylates beta1 integrin (threonine 788-789) via Smad-2 and Smad-3, causing a conformational change of the extracellular component with an inside-out mechanism. This leads noninvasive HCC cells to behave like invasive cells. A selective TGF-betaRI inhibitor inhibits phosphorylation of the beta1 integrin intracytoplasmic tail, and blocks invasion of HCC cells, both constitutively invasive and with acquired invasive properties. In human HCC tissues with microvascular invasion, phospho-beta1 integrin was detected as well as TGF-beta1, p-Smad-2, and E-cadherin. CONCLUSION: TGF-beta1 promotes vascular invasion by activating beta1 integrin. This suggests a rationale for targeting TGF-betaRI in future clinical trials.

Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells.

UNLABELLED: Hepatocellular carcinoma (HCC) treatment is challenging because the mechanisms underlying tumor progression are still largely unknown. Transforming growth factor (TGF)-beta1 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-beta signaling and its impact on HCC progression. Human HCC cell lines were treated with a TGF-beta receptor kinase inhibitor (LY2109761) whose selectivity was determined in a kinase assay. Exogenous TGF-beta1 phosphorylates the TGF-beta 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 microM. 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 10microM, 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 microM. 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-beta1 in the presence or absence of LY2109761. E-cadherin expression was reduced by TGF-beta1 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 up-regulating E-cadherin, suggesting that there could be a mechanistic use for this molecule in clinical trials.

Clinical validation of combined serological biomarkers for improved hepatocellular carcinoma diagnosis in 961 patients.

BACKGROUND: Alpha-fetoprotein (AFP), the only serological marker currently available for the detection of hepatocellular carcinoma (HCC), is unsatisfactory because of its poor sensitivity, as are other recently proposed markers. Therefore new biomarkers are badly needed. Squamous cell carcinoma antigen (SCCA), a serine protease inhibitor physiologically present in the skin, has recently been reported to be present in HCC patients, as also the immunocomplexed (IC) forms of SCCA and AFP: SCCAIC and AFPIC, respectively. METHODS: To determine the diagnostic accuracy of new serum biomarkers for the diagnosis of HCC a rapid, simple ELISA test was applied in 961 patients. Sensitivity and specificity were determined for each marker and for all the markers combined in detecting smaller and larger HCC versus liver cirrhosis. RESULTS: In smaller HCC, receiver operating characteristics analysis yielded the following AUC: AFP 0.714 (CI 95% 0.679-0.748), AFPIC 0.691 (CI95% 0.655-0.748), SCCA 0.703 (CI95% 0.667-0.736), SCCAIC 0.694 (CI 95% 0.659-0.728). SCCA was inversely correlated with size. The combined use of AFPIC, SCCA and SCCAIC in patients displaying low levels of AFP (<20 IU/mL) identified 25.6% HCC (186/725). CONCLUSION: This study suggests that the use of a combination of all these markers in clinical practice provides a non invasive and simple test that could increase the accuracy of HCC diagnosis.

Antifibrogenic effect of IFN-alpha2b on hepatic stellate cell activation by human hepatocytes.

Liver fibrosis commonly occurs in patients with hepatitis C virus (HCV) infection as a consequence of the chronic liver damage, thus leading to the development of liver cirrhosis. When hepatic stellate cells (HSCs) become active, they play an essential role in liver fibrogenesis. In this study, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), commonly elevated in chronic C hepatitis, stimulate the production of matrix metalloprotease-9 (MMP-9) by human hepatocytes at a transcriptional and translational level, but the addition of recombinant interferon-alpha2b (rIFN-alpha2b) hampers this effect. Furthermore, a human HSC line is activated in vitro by incubation with human MMP-9 in the presence of collagen I, and this effect is blocked by the MMP inhibitor BB94. A similar activation was observed when incubating HSCs with conditioned medium of hepatocytes previously stimulated with IL-1beta and TNF-alpha but not when using conditioned medium of hepatocytes costimulated with IL-1beta or TNF-alpha together with rIFN-alpha2b. In conclusion, our results show that hepatocytes stimulated by inflammatory cytokines participate in the activation of HSCs via MMP-9 production and that antiviral therapy modulates such activation.

Clinical role of serum and tissue matrix metalloprotease-9 expression in chronic HCV patients treated with pegylated IFN-alpha2b and ribavirin.

In chronic hepatitis C, the main goal of antiviral therapies is to block viral replication and to slow down the development of fibrosis. In this study, a decrease in matrix metalloprotease-9 (MMP-9) but not of MMP-2 and the tissue inhibitors of MMP (TIMP-1 and TMP-2) was observed in the plasma of chronic hepatitis C patients at the end of the follow-up period after ribavirin plus interferon-alpha2b (PEG-IFN-alpha2b) treatment in sustained virologic responders but not in nonresponders. Consistently, similar results are observed by immunofluorescence and real-time PCR in tissue specimens collected before and after therapy from the same patients in whom both Kupffer cells and hepatocytes express MMP-9. In conclusion, our results show that MMP-9 decreases in responder patients both in the serum and in the liver after therapy. Further studies are needed to investigate this new possible therapeutic activity of PEG-IFN-alpha2b.

Proteolytic imbalance is reversed after therapeutic surgery in breast cancer patients.

The occurrence of metastasis severely affects prognosis and survival of breast cancer patients. In order to metastasize, breast cancer cells need to cross the basement membrane (BM) tissue boundaries. Matrix metalloproteases (MMPs) are enzymes with proteolytic activity towards extracellular matrix components (ECM) of the BM, that are blocked by physiological tissue inhibitors (TIMPs). Cancer metastasis occurs as a result of an imbalance between MMPs, in particular MMP-2 and MMP-9, and TIMPs, in particular TIMP-2 and TIMP-1. This is the first study to report that pro-MMP-9 and TIMP-1 serum concentrations are inversely correlated in breast cancer patients. In the same patients, we determined the pro-MMP-9, the TIMP-1, the pro-MMP-2 and TIMP-2 before and after surgical eradication of the breast cancer. Our results show that after surgery, when the breast cancer tissue was removed, pro-MMP-9 concentrations dramatically decreased and TIMP-1 concentrations strongly increased, with statistically significant differences, so that a new balance was established. No statistically significant differences were observed regarding pro-MMP-2 and TIMP-2. Also, no correlation was found between pro-MMP-2, pro-MMP-9, TIMP-1 and TIMP-2 and a number of clinical and pathological parameters. In conclusion, our study suggests that pro-MMP-9 and TIMP-1 could be used as markers of disease during the follow-up of breast cancer patients and possibly as prognostic markers, although more studies are needed to address this issue.

Laminin-5 chains are expressed differentially in metastatic and nonmetastatic hepatocellular carcinoma.

PURPOSE: The purpose of this work was to study the expression of the extracellular matrix protein laminin-5 (Ln-5) in hepatocellular carcinoma (HCC), which is the fifth most frequent cancer and the third most common cause of tumor-related death in the world. The occurrence of metastasis is the main problem in HCC patients. Ln-5 is an extracellular matrix component that promotes adhesion and migration; it is present at the basement membrane and has recently been associated with cancer metastasis. Although Ln-5 has been shown to promote motility and scatter of rat liver cells, it has never been found in the liver. EXPERIMENTAL DESIGN: We studied the expression and localization of the alpha3, beta3, and gamma2 chains of Ln-5 in 40 HCC patients. We analyzed tissue samples collected from the HCC primary nodule and from peritumoral and metastatic tissues. The presence of Ln-5 was investigated by immunohistochemistry, reverse transcription-PCR, and Northern blot analysis. The clinical outcome of the patients was evaluated over a 4-year follow-up period. RESULTS: This study provides the first report that Ln-5 is present in the HCC primary nodule, but not in normal or peritumoral cirrhotic tissues. In particular, the gamma2 chain is strongly associated with the occurrence of metastasis (96%; P < 0.001) and with worse prognosis. In peritumoral tissues, Ln-5 has been detected along the advancing edge of the metastatic nodule. CONCLUSIONS: Ln-5 is associated with a more metastatic phenotype of HCC, and its detection could be an important finding both as an unfavorable prognostic factor and as a diagnostic marker for detecting micrometastasis in peritumoral tissues.

Activated integrin alphavbeta3 cooperates with metalloproteinase MMP-9 in regulating migration of metastatic breast cancer cells.

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.

Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells.

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.

Proteolytic balance in patients with multiple sclerosis during interferon treatment.

Multiple sclerosis (MS) is a progressive, inflammatory, demyelinating disease. An altered cytokine network has been reported to occur during the disease, and its pathogenetic role has been hypothesized. To date, interferon-beta (IFN-beta) is the most effective and reliable therapy in the majority of MS patients, although the mechanisms underlying its therapeutic effects are not fully understood. Breakdown of the blood-brain barrier (BBB) with consequent extravasation of the T cells and their invasion of the brain parenchyma seems to be one of the most important steps in the pathogenesis of the disease. Matrix metalloproteinease-2 (MMP-2) and MMP-9 are enzymes with proteolytic activities toward extracellular matrix ECM components. They are physiologically balanced by the MMP tissue inhibitors TIMP-2 and TIMP-1, so that proteolysis occurs as the result of increased MMP or decreased TIMP levels. In 38 patients with MS, MMP-2 and TIMP-1 levels were similar before and after 9 months of IFN-beta therapy, whereas MMP-9 levels significantly decreased and TIMP-2 levels significantly increased in comparison to values obtained before treatment. These results suggest that IFN-beta modulates T cell activities, including MMP and TIMP production, thus contributing either to maintaining the integrity of the BBB or to slowing the progression of the disease.

Transforming growth factor-beta1 triggers hepatocellular carcinoma invasiveness via alpha3beta1 integrin.

Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express alpha3beta1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-beta1 stimulates alpha3-integrin expression at a transcriptional level in noninvasive HCC cells, causing transformation into a motile and invasive phenotype. Such activities are inhibited by neutralizing anti-alpha3- but not anti-alpha6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-beta1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-beta1. In this way, the cells express alpha3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-beta1-neutralizing antibody reduces alpha3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-beta1 serum concentrations and alpha3-integrin expression are strongly correlated. The integrin, absent in normal and peritumoral liver parenchyma, is abundantly expressed in HCC primary and metastatic tissue. In particular, patients with metastasis show higher levels of TGF-beta1 serum concentrations and stronger expression of TGF-beta1 and alpha3-integrin in HCC tissues. In conclusion, TGF-beta1 may play an important role in HCC invasiveness by stimulating alpha3-integrin expression, and could therefore be an important target for new therapies.

Gelatinase levels in male and female breast cancer.

Breast cancer is a common disease in females but very rare in males, in whom it shows a more metastatic behavior, and a worse prognosis. Matrix metalloprotease-2 (MMP-2) and MMP-9 are proteolytic enzymes balanced by tissue inhibitor of MMP-2 (TIMP-2), commonly involved in cancer metastasis. This is the first study on gelatinolytic activity in male breast cancer patients, compared to that in female patients. In cancer tissues, both gelatinases were more expressed than in normal samples, being and more concentrated in male than in female patients. TIMP-2 levels were slightly increased in normal compared to those in cancer tissues and more concentrated in males than in females. Immunostaining showed that in male cancer tissues MMP-2 and MMP-9 staining was more intense and diffuse than in female cancer tissues, while no differences were observed regarding TIMP-2. In conclusion, the increased expression of gelatinases in male breast cancer patients together with anatomical features might explain the high tendency toward metastasis and the worse prognosis.

Clinical role of MMP-2/TIMP-2 imbalance in hepatocellular carcinoma.

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.