Felice Marinosci

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.

Transforming Growth Factor-β1 Triggers Hepatocellular Carcinoma Invasiveness via α3β1 Integrin

Metastasis occurrence in the course of hepatocellular carcinoma (HCC) severely affects prognosis and survival. We have shown that HCC invasive cells express α3β1-integrin whereas noninvasive cells do not. Here we show that transforming growth factor (TGF)-β1 stimulates α3-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-α3- but not anti-α6-integrin monoclonal antibodies. HCC invasive cells secrete abundant levels of active TGF-β1 in comparison with noninvasive cells, but in the latter, addition of active matrix metalloproteinases-2 increases the concentration of active TGF-β1. In this way, the cells express α3-integrin at a transcriptional level and acquire motility on Ln-5. By contrast, an anti-TGF-β1-neutralizing antibody reduces α3-integrin expression and the invasive ability of HCC invading cells. In HCC patients, TGF-β1 serum concentrations and α3-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-β1 serum concentrations and stronger expression of TGF-β1 and α3-integrin in HCC tissues. In conclusion, TGF-β1 may play an important role in HCC invasiveness by stimulating α3-integrin expression, and could therefore be an important target for new therapies.

Human Hepatocellular Carcinoma (HCC) Cells Require Both α3β1 Integrin and Matrix Metalloproteinases Activity for Migration and Invasion

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 α3β1 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 α3β1-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-α3β1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through Matrigel. In vivo, α3β1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both α3β1 integrin and gelatinase activity are required for HCC migration and invasion.

Clinical role of tissue and serum levels of SCCA antigen in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer in the world and a common occurrence in patients with liver cirrhosis in western and North American countries. Ultrasound screening is a powerful technique for HCC diagnosis, whereas the only available serologic test, alpha‐fetoprotein, has poor reliability. It has been reported that the squamous cell carcinoma antigen (SCCA) is overexpressed in HCC tissue. In our study, the expression of SCCA was investigated in tumoral and peritumoral tissues and in the serum of 52 HCC patients, as well as in the serum of 48 cirrhotic patients. The results show that SCCA expression is much stronger in the tumoral than in the peritumoral tissue of HCC. Moreover, it is also evident in metastatic nodules present in the peritumoral tissue. SCCA serum levels were significantly higher in HCC samples than in cirrhotic samples. However, no correlation was found between SCCA expression and the HCC histologic degree, nor did SCCA expression correlate with tumor size, presence of metastasis or clinical outcome. In conclusion, in HCC patients, the SCCA antigen could represent a useful marker for the detection of micro‐metastasis in the tissues and for large‐scale screening of serum in patients at risk.

SCCA antigen combined with alpha-fetoprotein as serologic markers of HCC

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Because of its increased incidence in the last decade and the estimated further increase in the next 2 decades, HCC is arousing great interest. In Europe and North America, it commonly develops on cirrhotic livers, and surveillance programs have therefore been suggested to identify early HCC, at a stage when it remains suitable for surgical therapy and has a better clinical outcome. The only serologic marker used in clinical practice is α‐fetoprotein (α‐FP), but its sensitivity is poor. In our study, 120 patients with HCC and 90 patients with liver cirrhosis were investigated. We report for the first time to our knowledge that as a marker of HCC, the squamous cell carcinoma (SCCA) antigen has high sensitivity (84.2%) but low specificity (48.9%). However, the combination of α‐FP and SCCA yielded a correct serologic diagnosis in 90.83% of the HCC patients. A small percentage of patients remain undetected, likely because of the low specificity of SCCA. In conclusion, the combined use of α‐FP and SCCA antigen represents a more powerful tool for the serologic detection of HCC.

Occurrence of portal vein tumor thrombus in hepatocellular carcinoma affects prognosis and survival. A retrospective clinical study of 150 cases

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Despite improvements in diagnostic and therapeutic interventions, prognosis and survival are still poor. To identify factors influencing survival, we retrospectively examined 150 consecutive patients with HCC from the time of first diagnosis of cirrhosis to death. In a multivariate analysis, we found that patients with larger HCC lesions had shorter survival, while other pathologic features had no predictive value. The most important and reliable prognostic factor was the occurrence of tumor thrombus of the portal vein (P<0.01). Childs stage of underlying liver disease was relevant only in the univariate, but not in the multivariate analysis. The survival of patients with HCC is mainly affected by the biological ability of cancer cells to invade surrounding tissue and vessels. More studies are needed to elucidate the mechanisms that modulate tumor cell motility, in order to design more effective therapies.

Gelatinase expression at positive patch test reactions

Matrix metalloproteases (MMPs) are proteolytic enzymes involved in tissue remodelling and extracellular matrix (ECM) turnover. They are secreted in a latent form and activated at the cellular surface by a membrane type‐1 MMP (MT1‐MMP) and a tissue inhibitor of MMP‐2 (TIMP‐2) that is also responsible for striking a balance between the proteolytic enzymes and TIMP‐2. In allergic contact dermatitis (ACD) patients, MMP‐2 and MMP‐9, two members of the MMPs family, were increased during the challenge phase, in involved but not uninvolved skin. In contrast, TIMP‐2 was more evident in uninvolved than involved skin, while no differences were observed with regard to MT1‐MMP staining. Comparing the serum of ACD patients with that of healthy subjects, these differences were not observed. These data suggest that MMP‐2 and MMP‐9 could play a role in the mechanisms inducing alterations of the epidermal architecture, and in the pathogenesis of the lesions.

Clinical outcomes of bosentan in pulmonary arterial hypertension do not correlate with levels of TIMPs

Background  Matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), are involved in tissue inflammation and fibrotic processes. Treatment with bosentan has been shown to improve the clinical outcome of patients with pulmonary arterial hypertension (PAH) with and without association with systemic sclerosis (SSc), and also to modulate the serum levels of matrix metalloproteases‐9. We measured TIMP‐1 and TIMP‐2 in the serum of patients with SSc with and without PAH treated with long‐term bosentan compared with healthy donors (HD).