Gianluigi Giannelli

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.

Drug-loaded polyelectrolyte microcapsules for sustained targeting of cancer cells ☆

In this review we will overview novel nanotechnological nanocarrier systems for cancer therapy focusing on recent development in polyelectrolyte capsules for targeted delivery of antineoplastic drugs against cancer cells. Biodegradable polyelectrolyte microcapsules (PMCs) are supramolecular assemblies of particular interest for therapeutic purposes, as they can be enzymatically degraded into viable cells, under physiological conditions. Incorporation of small bioactive molecules into nano-to-microscale delivery systems may increase drugs bioavailability and therapeutic efficacy at single cell level giving desirable targeted therapy. Layer-by-layer (LbL) self-assembled PMCs are efficient microcarriers that maximize drugs exposure enhancing antitumor activity of neoplastic drug in cancer cells. They can be envisaged as novel multifunctional carriers for resistant or relapsed patients or for reducing dose escalation in clinical settings.

Transforming Growth Factor-β as a Therapeutic Target in Hepatocellular Carcinoma

Hepatocellular carcinoma arises in patients as a consequence of long-standing preexisting liver illnesses, including viral hepatitis, alcohol abuse, or metabolic disease. In such preexisting liver diseases, TGF-β plays an important role in orchestrating a favorable microenvironment for tumor cell growth and promoting epithelial-mesenchymal transition (EMT). TGF-β signaling promotes hepatocellular carcinoma progression by two mechanisms: first, via an intrinsic activity as an autocrine or paracrine growth factor and, second, via an extrinsic activity by inducing microenvironment changes, including cancer-associated fibroblasts, T regulatory cells, and inflammatory mediators. Although there is an increasing understanding on how TGF-β signaling is associated with tumor progression in hepatocellular carcinoma, it is not clear whether TGF-β signaling is limited to a certain subgroup of patients with hepatocellular carcinoma or is a key driver of hepatocellular carcinoma during the entire tumorigenesis of hepatocellular carcinoma. Inhibitors of the TGF-β signaling have been shown to block hepatocellular carcinoma growth and progression by modulating EMT in different experimental models, leading to the clinical investigation of the TGF-β inhibitor LY2157299 monohydrate in hepatocellular carcinoma. Preliminary results from a phase II clinical trial have shown improved clinical outcome and also changes consistent with a reduction of EMT.

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.)

AFP, PIVKAII, GP3, SCCA-1 and follisatin as surveillance biomarkers for hepatocellular cancer in non-alcoholic and alcoholic fatty liver disease

BackgroundThe incidence and mortality of hepatocellular cancer (HCC) complicating alcoholic and non-alcoholic fatty liver diseases (ALD and NAFLD) is rising in western societies. Despite knowing the at risk populations for HCC development, the lack of sensitive and specific means of surveillance hampers disease detection at curable stages. The most widely used serum HCC marker is alpha-fetoprotein (AFP), while PIVKA-II, glypican-3 (GP3) and Squamous Cell Carcinoma Antigen -1 (SCCA-1) have been proposed as new biomarkers. Assessment of these HCC biomarkers has largely been performed in patients with viral hepatitis. We conducted a cross sectional study assessing the value of these serum proteins, as well a novel candidate biomarker -follistatin – in patients with HCC arising on a background of ALD or NAFLD.MethodsPre-treatment serum samples from 50 patients with HCC arising on a background of ALD (n = 31) or NAFLD (n = 19) were assessed by specific ELISA assay for PIVKAII, Glypican-3, SCCA-1 and Follistatin. Results were compared and contrasted with a control patient group with biopsy proven steatohepatitis-related cirrhosis (n = 41). The diagnostic accuracy of each of the candidate biomarkers was evaluated using receiver operating characteristic (ROC) curve analysis, reporting the area under the curve (AUC) and its 95% confidence interval (CI). Performance was compared to that of the established biomarker, AFP.ResultsSerum levels of all proteins were assessed by specific ELISA assays. GP3, SCCA-1 and follistatin had no HCC surveillance benefit in these patients. AFP and PIVKAII were superior to the other markers, particularly in combination.ConclusionWe conclude that while novel means of surveillance are urgently required, the combination of AFP and PIVKAII for HCC is an improvement on AFP alone in ALD/NAFLD patients. Furthermore, our data in this homogenous subset of patients- particularly that confirming no role for SCCA-1 – suggests that the choice of optimal biomarkers for HCC surveillance may be determined by the aetiology of underlying chronic liver disease.

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.)