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In the past few years, evidence has supported the role of plants as a valuable tool for the development of promising therapeutic support options for many diseases, including cancer. We recently discovered that the edible wild plant Crithmum maritimum L. effectively inhibits the growth of hepatocellular carcinoma (HCC) cells and we provide insights into the biological mechanisms involved. Here, we aimed to characterize the effect of ethyl acetate extract of Crithmum maritimum on the bioenergetic phenotype of HCC cells and if this is associated with the anti-tumour effect we previously described. Results show that Crithmum maritimum significantly increases cellular respiration and reduces lactic fermentation in HCC cells, and that this reduction of the fermentative glycolytic phenotype is linked to inhibition of HCC growth. These data provide new preclinical evidence supporting the role of Crithmum maritimum L. as a nutraceutical option to expand the therapeutic opportunities in the management of HCC.

Nowadays, a growing body of evidence supports the view that plants offer an extraordinary opportunity to discover and develop new promising therapeutic strategies for many diseases, including cancer. Here we tested the anticancer action against Hepatocellular carcinoma (HCC) of extracts obtained from two plants harvested in Apulia, namely Brassica oleracea L. and Crithmum maritimum L. B. oleracea was grown in biodynamical agriculture without any agrochemical input, instead C. maritimum was collected on Apulian coasts and is still commonly eaten in Apulia. HCC, one of the most frequent tumors worldwide, is estimated to become the third leading cause of cancer-related deaths in Western Countries by 2030. The approved synthetic drugs for the treatment of HCC are currently inadequate in terms of therapeutic results and tolerability. Hence, aim of the present study was to test the anticancer action against HCC of extracts obtained from Brassica oleracea L. and Crithmum maritimum L. We preliminary prepared extracts from both plants using four solvents with different polarity: hexane, ethyl acetate, methanol and ethanol. Then, we tested the effect of the different fractions in inhibiting HCC cell growth. Finally, we characterized the mechanism of action of the most effective fraction. We found that ethyl acetate fractions from both plants were the most effective in inhibiting HCC growth. In particular, we demonstrated that these fractions effectively reduce HCC growth by exerting, on one hand, a cytostatic effect through their action on the cell cycle, and on the other hand by triggering apoptosis and necrosis. Our findings support the notion that ethyl acetate fractions from Apulian B. oleracea and C. maritimum can be in perspective considered as promising tools to expand the opportunities to identify new and not toxic anticancer therapeutic approaches for HCC. Further pharmacological investigations will shed light on how this could be effectively achieved. Graphical Abstract Experimental workflow for the detection of the ethyl acetate extract of Brassica oleracea L. and Crithmum maritimum L. as an active fraction in inhibiting HCC cell growth.

Despite the increasing incidence of hepatocellular carcinoma (HCC) worldwide, current pharmacological treatments are still unsatisfactory. We have previously shown that lysophosphatidic acid receptor 6 (LPAR6) supports HCC growth and that 9-xanthenylacetic acid (XAA) acts as an LPAR6 antagonist inhibiting HCC growth without toxicity. Here, we synthesized four novel XAA derivatives, (±)-2-(9H-xanthen-9-yl)propanoic acid (compound 4 - MC9), (±)-2-(9H-xanthen-9-yl)butanoic acid (compound 5 - MC6), (±)-2-(9H-xanthen-9-yl)hexanoic acid (compound 7 - MC11), and (±)-2-(9H-xanthen-9-yl)octanoic acid (compound 8 - MC12, sodium salt) by introducing alkyl groups of increasing length at the acetic α-carbon atom. Two of these compounds were characterized by X-ray powder diffraction and quantum mechanical calculations, while molecular docking simulations suggested their enantioselectivity for LPAR6. Biological data showed anti-HCC activity for all XAA derivatives, with the maximum effect observed for MC11. Our findings support the view that increasing the length of the alkyl group improves the inhibitory action of XAA and that enantioselectivity can be exploited for designing novel and more effective XAA-based LPAR6 antagonists.

Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. Significance: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma.

Objectives Hepatocellular carcinoma (HCC) is one of the most frequent tumours worldwide and available drugs are inadequate for therapeutic results and tolerability. Hence, novel effective therapeutic tools with fewer side effects are of paramount importance. We have previously shown that Crithmum maritimum ethyl acetate extract exerts a cytostatic effect in HCC cells. Here, we tested whether C. maritimum ethyl acetate extract in combination with half sorafenib IC50 dose ameliorated efficacy and toxicity of sorafenib in inhibiting liver cancer cell growth. Moreover, we investigated the mechanisms involved. Methods Two HCC cell lines (Huh7 and HepG2) were treated with C. maritimum ethyl acetate extract and half IC50 sorafenib dose usually employed in vitro. Then, cell proliferation, growth kinetics and cell toxicity were analysed together with an investigation of the cellular mechanisms involved, focusing on cell cycle regulation and apoptosis. Key findings Results show that combined treatment with C. maritimum ethyl acetate extract and half IC50 sorafenib dose decreased cell proliferation comparably to full-dose sorafenib without increasing cell toxicity as confirmed by the effect on cell cycle regulation and apoptosis. Conclusions These results provide scientific support for the possibility of an effective integrative therapeutic approach for HCC with fewer side effects on patients.

Non-alcoholic fatty liver disease (NAFLD) is an emerging and threatening pathological condition, ranging from fatty liver (FL) to chronic steatohepatitis (NASH), liver cirrhosis, and eventually to hepatocellular carcinoma (HCC). Recent findings suggest that patients with NAFLD have a higher risk of cardiovascular events and thromboembolism and that this risk is independent of metabolic diseases that are frequently associated with NAFLD, such as diabetes, hyperlipidaemia, and obesity. The vascular involvement of NAFLD might be considered its systemic burden, conditioning higher mortality in patients affected by the disease. These clinical findings suggested the existence of a prothrombotic state in NAFLD, which is partially unexplored and whose underlying mechanisms are to date not completely understood. Here, we review the mechanisms involved in the pathogenesis of the prothrombotic state in NAFLD across the progression from the healthy liver through the different stages of the disease. We focused on the possible role of several metabolic features of NAFLD possibly leading to hypercoagulation other than endothelial and platelet activation, such as insulin-resistance, nitric oxide production regulation, and gut microbiota homeostasis. Also, we analysed the involvement of plasminogen activator inhibitor-1 (PAI-1) and thromboinflammation taking place in NAFLD. Finally, we described factors striking a prothrombotic imbalance in NASH cirrhosis, with a particular focus on the pathogenesis of portal vein thrombosis.

Genetic heterogeneity is a well-recognized feature of hepatocellular carcinoma (HCC). The coexistence of multiple genetic alterations in the same HCC nodule contributes to explain why gene-targeted therapy has largely failed. Targeting of early genetic alterations could theoretically be a more effective therapeutic strategy preventing HCC. However, the failure of most targeted therapies has raised much perplexity regarding the role of genetic alterations in driving cancer as the main paradigm. Here, we discuss the methodological and conceptual limitations of targeting genetic alterations and their products that may explain the limited success of the novel mechanism-based drugs in the treatment of HCC. In light of these limitations and despite the era of the so-called “precision medicine,” prevention and early diagnosis of conditions predisposing to HCC remain the gold standard approach to prevent the development of this type of cancer. Finally, a paradigm shift to a more systemic approach to cancer is required to find optimal therapeutic solutions to treat this disease.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide and the commonest liver cancer. It is expected to become the third leading cause of cancer-related deaths in Western countries by 2030. Effective pharmacological approaches for HCC are still unavailable, and the currently approved systemic treatments are unsatisfactory in terms of therapeutic results, showing many side effects. Thus, searching for new effective and nontoxic molecules for HCC treatment is of paramount importance. We previously demonstrated that lysophosphatidic acid (LPA) is an important contributor to the pathogenesis of HCC and that lysophosphatidic acid receptor 6 (LPAR6) actively supports HCC tumorigenicity. Here, we screened for novel LPAR6 antagonists and found that two compounds, 4-methylene-2-octyl-5-oxotetra-hydrofuran-3-carboxylic acid (C75) and 9-xanthenylacetic acid (XAA), efficiently inhibit HCC growth, both in vitro and in vitro, without displaying toxic effects at the effective doses. We further investigated the mechanisms of action of C75 and XAA and found that these compounds determine a G1-phase cell cycle arrest, without inducing apoptosis at the effective doses. Moreover, we discovered that both molecules act on mitochondrial homeostasis, by increasing mitochondrial biogenesis and reducing mitochondrial membrane potential. Overall, our results show two newly identified LPAR6 antagonists with a concrete potential to be translated into effective and side effect-free molecules for HCC therapy.

Over the last decades, a better knowledge of the molecular machinery supervising the regulation of circadian clocks has been achieved, and numerous findings have helped in unravelling the outstanding significance of the molecular clock for the proper regulation of our physiologic and metabolic homeostasis. Non-alcoholic fatty liver disease (NAFLD) is currently considered as one of the emerging liver pathologies in the Western countries due to the modification of eating habits and lifestyle. Although NAFLD is considered a pretty benign condition, it can progress towards non-alcoholic steatohepatitis (NASH) and eventually hepatocellular carcinoma (HCC). The pathogenic mechanisms involved in NAFLD development are complex, since this disease is a multifactorial condition. Major metabolic deregulations along with a genetic background are believed to take part in this process. In this light, the aim of this review is to give a comprehensive description of how our circadian machinery is regulated and to describe to what extent our internal clock is involved in the regulation of hormonal and metabolic homeostasis, and by extension in the development and progression of NAFLD/NASH and eventually in the onset of HCC.

ABSTRACT Sorafenib is an oral multikinase inhibitor with anticancer activity against a wide spectrum of cancers. It is currently approved for the treatment of patients with hepatocellular carcinoma, advanced renal cell carcinoma or progressive, locally advanced or metastatic differentiated thyroid carcinoma. In this review, we present a number of studies that investigated the efficacy and safety of sorafenib in these settings. We also discuss the perspectives on the use of this molecule, including the role of sorafenib as comparator for the development of new drugs, the combination of sorafenib with additional therapies (such as transarterial chemoembolization for hepatocellular carcinoma) and the use of this treatment in several other advanced refractory solid tumors.

Despite the reducing incidence of chronic hepatitis infections, an unexpected increasing incidence of hepatocellular carcinoma (HCC) has being occurred. This may be explained by the increasing number of HCCs developing on steatosis (NAFLD) and steatohepatitis (NASH), related to metabolic risk factors (i.e. diabetes mellitus type II, obesity, metabolic syndrome), which are becoming emerging risk factors for HCC. This led to a growing scientific interest on the oncogenic mechanisms underlying the transition from NAFLD to HCC. However, patients with NASH receive significantly less HCC surveillance than patients with chronic hepatitis, and no specific preventive pharmacological treatments have recommended for NASH-related HCC. This review focuses on the pathogenic role of the emerging factors involved in the transition from NAFLD/NASH to HCC, including microbiota, insulin resistance, inflammation, lipid and bile acids metabolism. It will be emphasize their impact on the liver microenvironment, the implications in clinical practice and the future directions of research.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death in western countries. The major risk factors for HCC are hepatitis C or B viruses, alcohol and metabolic disorders. The increasing risk of HCC in patients with metabolic disorders (i.e. obesity, diabetes and non-alcoholic steatohepatitis/NASH) regardless of the presence of liver cirrhosis is becoming relevant. Nevertheless, molecular mechanisms linking these risk factors to liver oncogenesis are unclear. This review focuses on the pathogenic role of the lysophosphatidic acid (LPA) pathway in HCC, highlighting the implications of this bioactive phospholipid in liver cancer biology and metabolism and as potential therapeutic target.

Hepatocellular carcinoma, the most frequent solid tumor of the liver, has a very poor prognosis, being the second most common cause of death from cancer worldwide. The incidence and mortality of this liver tumor are increasing in most areas of the world as a consequence of aging and the emerging of new risk factors such as the metabolic syndrome, beside the recognized role of hepatitis B and C viral infections and alcohol abuse. Despite the increasing knowledge on the molecular mechanisms underlying hepatic carcinogenesis, effective therapeutic strategies are still an unmet clinical need. Efforts have been made to develop selective drugs as well as effective targeted drug delivery systems. The development of novel drug carriers for therapeutic molecules can indeed offer a valuable strategy to ameliorate the efficacy of HCC treatment. In this review, we discuss recent drug delivery strategies for HCC treatment based on the exploitation of targeted nanoparticles (NPs). Indeed, a few of these platforms have achieved an advanced stage of preclinical development. Here, we review the most promising drug nanovehicles based on both synthetic and natural polymers, including polysaccharides that have emerged for their biocompatibility and biodegradability. To maximize site-selectivity and therapeutic efficacy, drug delivery systems should be functionalized with ligands which can specifically recognize and bind targets expressed by HCC, namely cell membrane associated antigens, receptors or biotransporters. Cell surface and intracellular molecular targets are exploited either to selectively deliver drug-loaded nanovehicles or to design novel selective therapeutics. In conclusion, the combination of novel and safe drug delivery strategies based on site-specific targeted drug nanovehicles with therapeutic molecular targets may significantly improve the pharmacological efficacy for the treatment of HCC.

The systemic evolutionary theory of cancer pathogenesis posits that cancer is generated by the de emergence of the eukaryotic cell system and by the re-emergence of its archaea (genetic material and cytoplasm) and prokaryotic (mitochondria) subsystems with an uncoordinated behavior. This decreased coordination can be caused by a change in the organization of the eukaryote environment (mainly chronic inflammation), damage to mitochondrial DNA and/or to its membrane composition by many agents (e.g. viruses, chemicals, hydrogenated fatty acids in foods) or damage to nuclear DNA that controls mitochondrial energy production or metabolic pathways, including glycolysis. Here, we postulate that the two subsystems (the evolutionarily inherited archaea and the prokaryote) in a eukaryotic differentiated cell are well integrated, and produce the amount of clean energy that is constantly required to maintain the differentiated status. Conversely, when protracted injuries impair cell or tissue organization, the amount of energy necessary to maintain cell differentiation can be restricted, and this may cause gradual de-differentiation of the eukaryotic cell over time. In cirrhotic liver, for example, this process can be favored by reduced oxygen availability to the organ due to an altered vasculature and the fibrotic barrier caused by the disease. Thus, hepatocarcinogenesis is an ideal example to support our hypothesis. When cancer arises, the pre-eukaryote subsystems become predominant, as shown by the metabolic alterations of cancer cells (anaerobic glycolysis and glutamine utilization), and by their capacity for proliferation and invasion, resembling the primitive symbiotic components of the eukaryotic cell. (C) 2016 Elsevier Ltd. All rights reserved.

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-β) intervenes in the process of hepatic fibrogenesis and cirrhosis, two pathogenic preconditions for the formation and progression of HCC [1] [2]. In addition, TGF-β 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-β signaling pathway results in a synergistic downstream action with an inhibitory effect on the progression of HCC. Several TGF-β 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-β signaling pathway in HCC.

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-β) 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-β 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-β signaling results in multiple synergistic down-stream effects which will likely improve the clinical outcome in HCC. We also review a number of TGF-β 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-β-associated signaling in this deadly cancer.

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 D isintegrin A nd M etalloproteases (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.

“It is likely that the development of new drugs acting on the RAS/RAF/MEK/ERK signaling pathway and the receptor crosstalk mechanisms may lead to a further improvement in the overall survival of patients with advanced hepatocellular carcinoma.”

As in many tumors, heterogeneity within the cell population is one of the main features of hepatocellular carcinoma (HCC). Heterogeneity results from the ability of tumor to produce multiple subpopulations of cells with diverse genetic, biochemical and immunological characteristics. Little is known about how heterogeneity emerges and how it is maintained. Fluctuations in single cells can be masked or completely misrepresented when cell populations are analyzed. It has become exceedingly apparent that the utility of measurement based on the analysis of bulk specimens is limited by intra-tumor genetic and epigenetic heterogeneity, as characteristics of the most abundant cell type might not necessarily predict the properties of cell populations. Yet, such non-uniformities often unveil molecular patterns that can represent mechanisms of tumor progression. Interestingly, variability among single cells in a population may arise from different responses to intrinsic and extrinsic perturbations mainly mediated by the plasma membrane. The association of certain proteins, including tetraspanins, and lipids in specific location on the plasma membrane constitutes specialized structure called tetraspanin-enriched microdomains (TEMs). TEMs organization in cancer may reveal essential clues for understanding pathogenic mechanisms underlying cancer progression. Along these lines, TEMs and HCC progression represent a valuable paradigm for gaining a deeper understanding of such mechanisms.

We investigated blocking the TGF-β signaling pathway in HCC using two small molecule inhibitors (LY2157299, LY2109761) and a neutralizing humanized antibody (D10) against TGF-βRII. LY2157299 and LY2109761 inhibited HCC cell migration on Laminin-5, Fibronectin, Vitronectin, Fibrinogen and Collagen-I and de novo phosphorylation of pSMAD2. LY2157299 inhibited HCC migration and cell growth independently of the expression levels of TGF-βRII. In contrast to LY2157299, D10 showed a reduction in pSMAD2 only after a short exposure. This study supports the use of LY2157299 in clinical trials, and presents new insights into TGF-β receptor cycling in cancer cells.

Hepatocarcinogenesis is heavily influenced by chronic hepatitis B (HBV) and C (HCV) infection. Elevated levels of plasminogen activator inhibitor-1 (SERPINE1/PAI-1) have been reported in patients with hepatocellular carcinoma (HCC) associated with viral infection. The gene encoding SERPINE1 is highly polymorphic and the frequently associated 4/5 guanosine (4G/5G) polymorphism in the gene promoter may influence its expression. Here, we investigated the distribution of genotypes and the frequency of alleles of the 4G/5G polymorphism in patients with HCC, the influence of the 4G/5G polymorphism on plasma SERPINE1 levels and its association with viral infection. A total of 75 patients with HCC were enrolled: 32 (42.6%) were HBV(+)/HCV(+), 11 (14.6%) were only HCV(+), and 32 (42.6%) were negative for both viruses. A control group of healthy donors was also enrolled (n=50). SERPINE1 plasma concentrations were determined by ELISA and the detection of the promoter 4G/5G polymorphism was performed by an allele-specific PCR analysis. We found that the frequency of both the 4G/4G genotype (p=0.02) and the 4G allele (p=0.006) were significantly higher in patients with HCC compared to the control group, and particularly higher in patients with HCC co-infected with HBV(+)/HCV(+) than in those with no viral infection. We also found that patients with the 4G/4G genotype had significantly higher plasma SERPINE1 protein levels when compared with patients with the 4G/5G or 5G/5G genotype (p0.001). Differences in frequency of 4G allele and genetic variability of 4G/5G SERPINE1 polymorphism with a higher level of SERPINE1 protein in patients with HCC with HBV(+)/HCV(+) than those without infection, suggest the presence of two distinct pathogenic mechanisms in hepatocarcinogenesis, depending on the etiology.

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.

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;)

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).In this study, we investigated the effects of the transforming growth factor-β receptor I inhibitor LY2109761 on two different human HCC cell lines, in vitro and in vivo.LY2109761 inhibits HCC migration in a dose-dependent manner. This inhibition is associated with the decreased phosphorylation of SMAD-2, FAK and β1-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.In conclusion, inhibition of transforming growth factor-β (TGF-β) 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-β.

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.)

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.)

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) μm2 in LC patients, statistically different from values in s-HCC: 1163.2 (863.6–1566.8) μm2 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.

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.TGF-beta1 promotes vascular invasion by activating beta1 integrin. This suggests a rationale for targeting TGF-betaRI in future clinical trials.

Expression of the integrin, alpha6beta1, a receptor for laminins, is associated with the progression of hepatocellular carcinoma (HCC). The approach to investigating the alpha6beta1 integrin signaling in HCC cells was to express a deletion mutant of the beta4 integrin cytoplasmic domain (beta4-Deltacyt) in 2 HCC cell lines, HepG2 and Huh7. Expression of this mutant prevents formation of the alpha6beta1 heterodimer. As expected, adhesion of both the HepG2/beta4-Deltacyt and Huh7/beta4-Deltacyt transfectants to laminin, but not to collagen, was reduced compared with the mock transfectants. However, migration of the beta4-Deltacyt transfectants toward both collagen and laminin was inhibited, suggesting a role for alpha6beta1 in the signaling of migration. Migration of HCC cells requires mitogen-activated protein (MAP) kinase. The adhesion of the beta4-Deltacyt transfectants to collagen resulted in a substantial reduction in MAP kinase activation in comparison with the mock transfectants, although their ability to activate MAP kinase in response to epidermal growth factor (EGF) stimulation was not impaired. In addition, matrix adhesion of the beta4-Deltacyt transfectants did not stimulate the tyrosine phosphorylation of focal adhesion kinase (FAK), and this defect correlated with reduced binding of adaptor protein Grb2 to FAK. These results suggest that FAK tyrosine phosphorylation is dependent on alpha6beta1 expression, and that FAK-Grb2 association plays a central role in alpha6beta1-mediated activation of MAP kinase. Moreover, the expression of alpha6beta1 in HCC cells is necessary for FAK/MAP kinase-dependent migration.

Background & Aims: Human hepatocellular carcinoma (HCC) can invade the portal vein and metastasize to other parts of the liver. Currently, the molecular and cellular mechanisms underlying intrahepatic metastasis of HCC are poorly understood. Tumor invasiveness could be considered an aspect of dysregulated motility, and the mechanisms that inhibit cell movement are considered to counteract the spreading of cancer cells through the liver. Accumulating observations suggest that the CD81 tetraspanin may have an inhibitory effect on cell movement. Methods: In the present study using both loss- and gain-of-gene function approaches, we verified that the functional interaction of tetraspanin CD81 with type II phosphoinositide 4-kinase (PI4KII) suppressed HCC cell motility by promoting the formation of CD81-enriched vesicles, non-endosomal intracellular structures, that sequestered actinin-4 with consequent remodeling of actin cytoskeleton. Results: We reported that HCC cells expressing CD81 showed an inability to metastasize compared with HCC cells with undetectable levels of CD81. Conclusions: Taken together, these findings indicate that CD81 functions as a molecular organizer of membrane microdomains, whereby proteins such as PI4KII control actin remodeling and cell motility, establishing a role for these genes as negative modifiers of oncogenicity and HCC progression.