Salvatore Antonaci

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.

Biological and clinical relevance of Laminin-5 in cancer.

The occurrence of metastases is the hallmark of cancer. Development of metastasis severely affects prognosis and survival. It limits or discourages therapeutic interventions since no therapies are available to block or prevent cancer invasion. In order to invade, epithelial cancer cells need to penetrate through the basement membrane (BM) and remove extra-cellular matrix (ECM) tissue boundaries. In this context, proteases play a key role since they can either degrade or process the ECM components and thereby support cancer cell invasion. Laminin-5 (Ln-5) is an ECM protein, expressed predominantly in the BM structure, that promotes static adhesion and hemidesmosome formation. However, it also stimulates cell migration and/or invasion after having been cleaved by matrix metalloproteinases (MMPs) such as MMP-2 and MT1-MMP. Based on its dual functions, it would be intriguing to elucidate the role that Ln-5 plays in cancer cell motility and metastasis. One possibility is that MMPs, secreted by cancer cells or by neighbouring stromal cells, can cleave the γ2 chain of Ln-5 deposited along the advancing edge of tumors. Ln-5, and in particular its γ2 chain, has been found to be preferentially expressed in the cytoplasm of epithelial human cancer cells located at the advancing edge of the tumor. Such a distribution, which is restricted only to malignancies, suggests that the γ2 chain may be implicated in epithelial cancer growth and invasion. Although the clinical significance of this finding is not yet clear, it seems often to be associated with a more aggressive and invasive cancer phenotype. This article will review the current body of evidence implicating the Ln-5 molecule, and in particular its γ2 chain, as an important player in the tumor cascade and progression to metastatic disease. This will then be followed by a discussion of the presented data and its limitations. Finally, suggestions will be provided to improve the current state of knowledge in the field and future implications will be briefly discussed.

Down‐regulation of connective tissue growth factor by inhibition of transforming growth factor β blocks the tumor–stroma cross‐talk and tumor progression in hepatocellular carcinoma

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 β (TGF‐β) receptor inhibitor, LY2109761, inhibited the synthesis and release of CTGF, as well as reducing the stromal component of the tumors. In addition, the TGF‐β–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‐β1 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‐β1 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‐β–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. (HEPATOLOGY 2009.)

Non-specific immunity in aging: Deficiency of monocyte and polymorphonuclear cell-mediated functions

Peripheral blood monocytes obtained from 55 aged donors were evaluated for their chemotactic and phagocytic capacity. In the same subjects, polymorphonuclear cell-mediated functions were studied by chemotaxis, phagocytosis, nylon fiber adherence and nitroblue-tetrazolium reduction assay. Monocytes showed a normal chemotactic responsiveness to zymosan-activated serum, while the chemotactic activity induced by leukocyte-derived chemotactic factor and phagocytosis were rather depressed. A dramatic impairment of polymorphonuclear cell-mediated immune response was also observed. In fact, in spite of a normal nylon fiber adherence, chemotaxis, phagocytosis and nitroblue-tetrazolium reduction capacity were significantly depressed by the aging process. These data suggest that the deficiency of non-specific immunity may play an important role in the increased susceptibility to infections in aged donors.

Targeting transforming growth factor (TGF)-βRI inhibits activation of β1 integrin and blocks vascular invasion in hepatocellular carcinoma

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 α5β1, that is equally expressed in invasive and noninvasive cells. However, in the former, the intracytoplasmic tail of β1 integrin is constitutively phosphorylated at threonine 788‐789 and the extracellular part is conformationally activated. In noninvasive cells, β1 integrin is not activated. Transforming growth factor (TGF)‐β1 specifically phosphorylates β1 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‐βRI inhibitor inhibits phosphorylation of the β1 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‐β1 integrin was detected as well as TGF‐β1, p‐Smad‐2, and E‐cadherin. Conclusion: TGF‐β1 promotes vascular invasion by activating β1 integrin. This suggests a rationale for targeting TGF‐βRI in future clinical trials. (HEPATOLOGY 2009.)

Inhibition of transforming growth factor β receptor I kinase blocks hepatocellular carcinoma growth through neo‐angiogenesis regulation

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 β (TGF‐β) signaling pathway in HCC with LY2109761, a kinase inhibitor of TGF‐β 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 phsyiological angiogenetic development. Conclusion: These data support the rationale for targeting TGF‐β signaling in patients with HCC. (HEPATOLOGY 2009.)

Role of the α3β1 and α6β4 integrins in tumor invasion

Integrin receptors are well-known mediators of cell adhesion that also have a fundamental role in controlling the migration of cells through tissues. Among the numerous members of a still growing family, two particular molecular complexes have turned out to be of key importance in tumor cell invasion of basement membranes, the α3β1 and α6β4 integrins. In this Review, we will focus on the role of these two receptors and the mechanisms by which they influence the invasion process.

Tumor‐secreted lysophostatidic acid accelerates hepatocellular carcinoma progression by promoting differentiation of peritumoral fibroblasts in myofibroblasts

Hepatocellular carcinoma (HCC) occurs in fibrotic liver as a consequence of underlying cirrhosis. The goal of this study was to investigate how the interaction between HCC cells and stromal fibroblasts affects tumor progression. We isolated and characterized carcinoma‐associated fibroblasts (CAFs) and paired peritumoral tissue fibroblasts (PTFs) from 10 different patients with HCC and performed coculture experiments. We demonstrated a paracrine mechanism whereby HCC cells secrete lysophostatidic acid (LPA), which promotes transdifferentiation of PTFs to a CAF‐like myofibroblastic phenotype. This effect is mediated by up‐regulation of specific genes related to a myo/contractile phenotype. After transdifferentiation, PTFs expressed α‐smooth muscle actin (α‐SMA) and enhanced proliferation, migration, and invasion of HCC cells occur. A pan‐LPA inhibitor (α‐bromomethylene phosphonate [BrP]‐LPA), or autotaxin gene silencing, inhibited this PTF transdifferentiation and the consequent enhanced proliferation, migration, and invasion of HCC cells. In vivo, PTFs coinjected with HCC cells underwent transdifferentiation and promoted tumor progression. Treatment with BrP‐LPA blocked transdifferentiation of PTFs, down‐regulated myofibroblast‐related genes, and slowed HCC growth and progression. Patients with larger and metastatic HCC and shorter survival displayed higher serum levels of LPA. Analysis of microdissected tissues indicated that stroma is the main target of the LPA paracrine loop in HCC. As a consequence, α‐SMA–positive cells were more widespread in tumoral compared with paired peritumoral stroma. Conclusion: Our data indicate that LPA accelerates HCC progression by recruiting PTFs and promoting their transdifferentiation into myofibroblasts. Inhibition of LPA could prove effective in blocking transdifferentiation of myofibroblasts and tumor progression. (HEPATOLOGY 2011;)