The TGF-ß signaling pathway as a pharmacological target in a hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a cancer that usually develops on a liver already compromised by cirrhosis. Study of the underlying molecular mechanisms is essential so as to improve therapeutic strategies and to develop new pharmacological agents that may prevent or improve the course of this malignancy. Transforming growth factor-beta (TGF-ß) intervenes in the process of hepatic fibrogenesis and cirrhosis, two pathogenic preconditions for the formation and progression of HCC [1] [2]. In addition, TGF-ß plays a crucial role in the molecular pathogenesis of HCC and may, therefore, prove to be a promising drug target. We and other authors have recently demonstrated that inhibition of the TGF-ß signaling pathway results in a synergistic downstream action with an inhibitory effect on the progression of HCC. Several TGF-ß inhibitors have recently been developed, most of which are still in a preclinical phase, but they may soon be available for testing in patients with HCC. However, well-designed clinical trials will be needed to evaluate the effectiveness of these new agents prior to routine use in the clinic. Aim of this article is to make a brief review of the benefits and limitations of targeting the TGF-ß signaling pathway in HCC.

PI3K class IB controls the cell cycle checkpoint promoting cell proliferation in hepatocellular carcinoma.

Alterations of the cell cycle checkpoint frequently occur during hepatocarcinogenesis. Dysregulation of the phosphatidylinositol-3-kinases (PI3K) signaling pathway is believed to exert a potential oncogenic effect in hepatocellular carcinoma (HCC), ultimately promoting tumor cell proliferation. However, the impact of PI3K on cell cycle regulation remains unclear. We used a combined loss- and gain-of-function approach to address the involvement of p110γ in HCC cell proliferation, apoptosis and the cell cycle. We also investigated the correlation between p110γ and Ki-67 in 24 HCC patients. Finally, we analyzed the expression levels of p110γ and cell cycle regulators in HCC tissues. We found that PI3K class IB, but not class IA, is required for HCC cell proliferation. In particular, we found that knock-down of p110γ inhibits cell proliferation because of an arrest of the cell cycle in the G0-G1 phase. This effect is associated with an altered expression of proteins regulating the cell cycle progression, including p21, and with an increased apoptosis. By contrast, we found that ectopic expression of p110γ promotes HCC cell proliferation. Tissues analysis performed in HCC patients showed a positive correlation between the expression of p110γ and Ki-67, a marker of proliferation, and, even more importantly, that p21 expression is up-regulated in HCC patients with a lower p110γ expression. Our results emphasize the role of p110γ as a promoter of HCC proliferation and unveil an important cell cycle regulation function of this molecule.

PI3K functions in cancer progression, anticancer immunity and immune evasion by tumors.

The immunological surveillance of tumors relies on a specific recognition of cancer cells and their associate antigens by leucocytes of innate and adaptive immune responses. However, a dysregulated cytokine release can lead to, or be associated with, a failure in cell-cell recognition, thus, allowing cancer cells to evade the killing system. The phosphatidylinositol 3-kinase (PI3K) pathway regulates multiple cellular processes which underlie immune responses against pathogens or malignant cells. Conversely, there is accumulating evidence that the PI3K pathway is involved in the development of several malignant traits of cancer cells as well as their escape from immunity. Herein, we review the counteracting roles of PI3K not only in antitumor immune response but also in the mechanisms that cancer cells use to avoid leukocyte attack. In addition, we discuss, from antitumor immunological point of view, the potential benefits and disadvantages arising from use of anticancer pharmacological agents targeting the PI3K pathway.

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

UNLABELLED: 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.

TLRs innate immunereceptors and Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) CIDR1α-driven human polyclonal B-cell activation.

Chronic malaria severely affects the immune system and causes polyclonal B-cell activation, as evidenced by the presence of hypergammaglobulinemia, elevated levels of autoantibodies, loss of B-cell memory and the frequent occurrence of Burkitt's lymphomas (BL) in children living in malaria endemic areas. Previous studies have shown that the cysteine-rich interdomain region 1α (CIDR1α) of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) of the FCR3S1.2 strain, subsequently named CIDR1α, interacts with B cells partially through the binding to the B-cell receptor (BCR). This interaction leads to an activated phenotype, increased survival, and a low degree of proliferation. CIDR1α preferentially activates the memory B-cell compartment, therefore PfEMP1 is considered to act as a polyclonal B-cell activator and its role in memory maintenance has been suggested. In this report, we extend the analysis of the PfEMP1-CIDR1α B-cell interaction and demonstrate that PfEMP1-CIDR1α increases the expression of TLR7 and TLR10 mRNA transcripts and sensitizes B cells to TLR9 signalling via the MyD88 adaptor molecule. Furthermore, despite its ability to bind to surface Igs, PfEMP1-CIDR1α-induced B-cell activation does not seem to proceed through the BCR, since it does not induce Lyn and/or phospho-tyrosine mediated signalling pathways. Rather PfEMP1-CIDR1α induces the phosphorylation of downstream kinases, such as ERK1/2, p38 and IKBα, in human B cells. These findings indicate that PfEMP1-CIDR1α induces a persistent activation of B cells, which in turn can contribute to the exhaustion and impairment of B-cell functions during chronic malaria infection.

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.

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.

Quantitative determination of hepatitis C core antigen in therapy monitoring for chronic hepatitis C.

The correlation and kinetics of hepatitis C virus (HCV) RNA and HCV core antigen levels in chronic hepatitis C patients treated with pegylated interferon + ribavirin were evaluated in order to envision a combined use of the two assays in therapy monitoring. HCV core antigen levels by a chemiluminescent immunoassay (Abbott ARCHITECT) and HCV-RNA levels by branched DNA (bDNA) or real-time PCR have been evaluated on plasma specimens from 32 patients treated for chronic hepatitis C. An early virological response (undetectable levels of HCV-RNA 4 weeks after start of treatment) was found in 10/23 subjects (43.5%) followed up for 5 months or more. The response was linked to the HCV genotype (20% in genotype 1B vs. 61.5% in other genotypes; p < 0.05). HCV RNA and HCV antigen showed a good correlation (r = 0.814); HCV antigen was still detectable in 3 samples with undetectable (<615 IU/ml) RNA by bDNA, while no differences in clinical sensitivity were recorded in comparison with real-time PCR. These findings suggest that HCV-RNA and HCV antigen may be used at different time points in order to tailor therapy monitoring to individual needs.

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.

Impairment of TLR7-dependent signaling in dendritic cells from chronic hepatitis C virus (HCV)-infected non-responders to interferon/ribavirin therapy.

BACKGROUND AND AIM: Dendritic cell (DC) dysfunction has been suggested to play a role in the weak antiviral T-cell responsiveness observed during the course of chronic hepatitis C virus (HCV) infection. This study was undertaken to evaluate whether changes in DC functions might be related to a different therapeutic outcome in HCV-infected patients. METHODS: Peripheral blood DCs (PBDCs) or monocyte-derived DCs (MoDCs) were obtained from chronic HCV-infected patients, sustained virologic responders (SVR) or non-responders (NR) to interferon/ribavirin therapy, and from healthy controls (HC). The frequency of BDCA-1+, BDCA-3+ or CD16+ myeloid DCs (mDCs) and BDCA-2+ plasmacytoid DCs (pDCs), as well as the expression of the costimulatory molecule CD86 in each PBDC subset, were evaluated by flow cytometry. MoDCs from single individuals were stimulated with TLR2, TLR3, TLR4, and TLR7 ligands and analyzed for CD86, CD83, CD40, CD80, and CD209 expression. Finally, mitogen-activated protein kinase (MAPK) phosphorylation of TLR7-triggered MoDCs was assessed by Western blotting. RESULTS: NR exhibited a reduced percentage of BDCA-1+ mDCs, as well as lower levels of CD86+ cells, in both BDCA-1+ mDCs and pDCs as compared to SVR and HC. Furthermore, MoDCs from NR displayed a defective CD86 and CD83 increase and ERK1/2 or p38-MAPK phosphorylation upon TLR7-cell triggering. CONCLUSIONS: Our data suggest that a TLR7-dependent impairment of costimulatory molecule expression caused by HCV persistence may affect DC activity in NR patients.

Involvement of ADAMs in tumorigenesis and progression of hepatocellular carcinoma: Is it merely fortuitous or a real pathogenic link?

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the most frequent types of cancer worldwide. It normally develops in patients with chronic liver disease, especially cirrhosis, although some cases without an apparent underlying liver disease have been reported. The pathogenesis of HCC is multi-factorial and complex. Hepatitis viruses are the main factors favoring the development of HCC. In fact, chronic inflammation associated with hepatitis C or B virus infection can lead to progressive liver fibrosis, cirrhosis and ultimately HCC. Chronic inflammation and liver fibrosis cause a continuous remodeling of the extracellular matrix (ECM), a dynamic process that involves several molecules including integrins and matrix processing enzymes. An increasing body of evidence indicates that ADAMs are involved in promoting tumor formation and progression of HCC. A Disintegrin And Metalloproteases (ADAMs) are a group of proteins belonging to the zinc protease superfamily. ADAMs are usually transmembrane proteins that contain disintegrin and metalloprotease domains and are, therefore, able to carry out both cell adhesion and protease activities. Soluble isoforms of ADAMs have also been discovered and characterized. In this review, we focus on the contribution of ADAM proteins to HCC tumorigenesis and cancer progression. The potential role of ADAMs as key modulators of tumor-stroma interactions during tumor progression, by means of the activities of their constituent domains, is also discussed.

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.

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.

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.

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.

[An unusual case of transient loss of consciousness: the Fahr's syndrome]. / Uno strano caso di perdita transitoria di coscienza: la sindrome di Fahr.

A 69-year-old male was admitted to our Division because of repeated episodes of transient loss of consciousness and minimal cognitive and behavioural disturbances. Brain CT showed Fahr-type bilateral calcifications. Laboratory findings showed a phospho-calcium metabolism disorder consistent with the diagnosis of pseudohypoparathyroidism, whose correction led to a significant clinical improvement. This is the first case of Fahr's syndrome associated with syncope-type episodes. This observation suggests that a brain CT, as well as the search for a dysparathyroidism condition, should always carried out in all patients with unexplained recurrent episodes of loss of consciousness.

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.

EGFR and VEGFR as potential target for biological therapies in HCC cells.

Hepatocellular carcinoma (HCC) is a highly malignant cancer with poor prognosis. Inhibitors of EGFR and VEGFR for HCC treatment are currently under investigation. Gefitinib and vandetanib inhibit migration of HCC cells on Laminin-5 and Fibronectin, and invasion through matrigel. Both drugs inhibit p-EGFR after short time, while their efficacy on p-Erk1/2 and p-Akt is progressive and stable over time. PI3K/Akt and MEK/Erk1/2 inhibitors, inhibit migration and invasion as well as inducing de-phosphorylation of downstream effectors. Finally, both inhibitors, vandetanib and gefitinib down-regulated the secretion of matrix metalloproteases MMP-2 and MMP-9. All these biological effects seem to depend on the activity of gefitinib and vandetanib blocking activity towards p-EGFR mediated pathways.

Laminin-5 stimulates hepatocellular carcinoma growth through a different function of alpha6beta4 and alpha3beta1 integrins.

UNLABELLED: Hepatocellular carcinoma (HCC) growth severely affects prognosis. Ki-67, a known marker of cell proliferation, is a negative prognostic factor in HCC. Growth factors such as the epidermal growth factor (EGF) induce HCC cell proliferation but do not explain the great heterogeneity of HCC growth. Laminin-5 (Ln-5) is an extracellular matrix protein (ECM) present in the tissue microenvironment of HCC. The two main receptors for Ln-5, integrins alpha3beta1 and alpha6beta4, are expressed on the cell surface of HCC cells. The aim of this study is to investigate an alternative mechanism of HCC growth whereby Ln-5 promotes HCC cell proliferation through alpha3beta1 and alpha6beta4. HCC tissues containing Ln-5 display a larger diameter and higher number of positive cells for Ki-67, a well known proliferative index, as determined by double immunofluorescence staining and real-time PCR on microdissected tissues. In vitro, Ln-5, but not collagen I, collagen IV or fibronectin, induces proliferation as much as EGF does, via Erk phosphorylation as a consequence of beta4 integrin phosphorylation. However, the two HCC cell lines do not proliferate in presence of Ln-5 despite beta4 integrin and Erk1/2 activation. After transfection with alpha3 integrin, in the presence of Ln-5 one of these HCC cell lines acquires a proliferative activity whereas one of the proliferative HCC cell lines, knocked-down for alpha3 integrin, loses its proliferative activity. CONCLUSIONS: Our study suggests a new mechanism of HCC growth whereby Ln-5 stimulates proliferation via a different function of alpha6beta4 and alpha3beta1.