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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.

The CD63 tetraspanin is highly expressed in the early stages of melanoma and decreases in advanced lesions, suggesting it as a possible suppressor of tumor progression. We employed loss- and gain-of-gene-function approaches to investigate the role of CD63 in melanoma progression and acquisition of the epithelial-to-mesenchymal transition (EMT) program. We used two human melanoma cell lines derived from primary tumors and one primary human melanoma cell line isolated from a cutaneous metastasis, differing by levels of CD63 expression. CD63-silenced melanoma cells showed enhanced motility and invasiveness with downregulation of E-cadherin and upregulation of N-cadherin and Snail. In parallel experiments, transient and stable ectopic expression of CD63 resulted in a robust reduction of cell motility, invasiveness, and protease activities, which was proportional to the increase in CD63 protein level. Transfected cells overexpressing the highest level of CD63 when transplanted into immunodeficient mice showed a reduced incidence and rate of tumor growth. Moreover, these cells showed a reduction of N-cadherin, Vimentin, Zeb1, and a-SMA, and a significant resistance to undergo an EMT program both in basal condition and in the following stimulation with TGFβ. Thus, our results establish a previously unreported mechanistic link between the tetraspanin CD63 and EMT abrogation in melanoma.

Accumulating evidence implicates phosphoinositide 4-phosphate as a regulatory molecule in its own right recruiting specific effector proteins to cellular membranes. Here, we describe biochemical and immunocytochemical methods to evaluate tetraspanin-associated phosphoinositide-4 kinases activity in primary human hepatic stellate cells (hHSC) and neoplastic hepatoblastoma cells.

Evidence indicates that androgen-sensitive prostate cancer cells have a lower malignant potential. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells through modulation of alpha6beta4 expression. Treatment with the androgen further reduced adhesion and invasion of the cells without, however, modifying alpha6beta4. Here we investigated whether the androgen has a direct effect on alpha6beta4-EGF receptor (EGFR) interaction and signalling leading to invasion of these cells. Immunoconfocal microscopy demonstrated that in control cells (PC3-Neo), alpha6beta4 and EGFR colocalize and redistribute in response to epidermal growth factor (EGF). In PC3-AR cells colocalization and redistribution between the two molecules was reduced and abolished by pre-treatment with R1881. Co-immunoprecipitation studies demonstrated that tyrosine phosphorylation of beta4 in response to EGF was reduced in PC3-AR cells compared to PC3-Neo. Immunoconfocal and co-immunoprecipitation studies demonstrated colocalization at membrane level and co-immunoprecipitation of EGFR and AR, indicating an interaction between the two proteins. PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells in response to EGF and further reduced by treatment with R1881. EGFR internalization was strongly reduced in PC3-AR compared with PC3-Neo cells and was reduced by treatment with R1881. In conclusion, the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR--alpha6beta4 interaction and signalling leading to invasion through a mechanism involving an interaction between the classic AR and EGFR.

Background/Aims : Migration of activated hepatic stellate cells (HSC) is a key event in the progression of liver fibrosis. Little is known about transmembrane proteins involved in HSC motility. Tetraspanins (TM4SF) have been implicated in cell development, differentiation, motility and tumor cell invasion. We evaluated the expression and function of four TM4SF, namely CD9, CD81, CD63 and CD151, and their involvement in HSC migration, adhesion, and proliferation. Methods/Results : All TM4SF investigated were highly expressed at the human HSC surface with different patterns of intracellular distribution. Monoclonal antibodies directed against the four TM4SF inhibited HSC migration induced by extracellular matrix proteins in both wound healing and haptotaxis assays. This inhibition was independent of the ECM substrates employed (collagen type I or IV, laminin), and was comparable to that obtained by incubating the cells with an anti-β1 blocking mAb. Importantly, cell adhesion was unaffected by the incubation with the same antibodies. Co-immunoprecipitation studies revealed different patterns of association between the four TM4SF studied and β1 integrin. Finally, anti-TM4SF antibodies did not affect HSC growth. Conclusions : These findings provide the first characterization of tetraspanins expression and of their role in HSC migration, a key event in liver tissue wound healing and fibrogenesis.

The biologic effects of growth factors are dependent on cell adhesion, and a cross talk occurs between growth factors and adhesion complexes. The aim of the present study was to evaluate the influence of cell adhesion on the major intracellular signaling pathways elicited by platelet-derived growth factor (PDGF) in hepatic stellate cells (HSC). PDGF signaling was investigated in an experimental condition characterized by lack of cell adhesion for different intervals of time. Basal and PDGF-induced focal adhesion kinase (FAK) tyrosine phosphorylation was maintained in a condition of cell suspension for 2, 4, and 6 hours, whereas it was completely lost after 12 and 24 hours. We examined MAP kinase activity at 2 and 24 hours, corresponding to the higher and lower levels of FAK phosphorylation. In these experiments, MAP kinase activity correlated with FAK phosphorylation. Stimulation with PDGF was able to cause Ras-GTP loading only in adherent cells. The ability of PDGF to induce phosphatidylinositol 3-kinase (PI 3-K) activity was abrogated in cells maintained in suspension. The Ser473 phosphorylation of Akt was only marginally affected by the lack of cell adhesion. We then evaluated the association of FAK with c-Src. This association was found to be cell adhesion dependent, and it did not appear to be dependent from phosphorylated FAK. These changes in PDGF-induced intracellular signaling were associated with a remarkable reduction of PDGF-proliferative potential in nonadherent cells, although no marked differences in the apoptotic rate were observed. In conclusion, these results suggest that cell adhesion differentially regulates major signaling pathways activated by PDGF in HSC.

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.

Background & Aims: Hepatocellular carcinoma is a common complication in liver cirrhosis. The integrin α6β1, a receptor for the laminin family of extracellular matrix proteins, has been found to be overexpressed in hepatocarcinoma. In an effort to further characterize the involvement of α6β1-integrin in hepatocarcinoma progression and to study α6β1-mediated functions, a human hepatocarcinoma cell line, HepG2, that express high surface levels of α6β1 and uses only this integrin to mediate adhesion on laminin was identified. Methods: To assess the role of α6β1 in these cells, a cytoplasmic domain deletion mutant of the β4-integrin subunit by complementary DNA transfection was expressed. The expression of the mutant β4 subunit in association with endogenous α6 showed a dominant-negative effect on α6β1 expression. Results: Stable transfectants of HepG2 that expressed the mutant β4 subunit showed a reduced ability to adhere and migrate on laminin matrices and to invade Matrigel. Furthermore, transfected cells showed significantly lower growth rates and reduced anchorage-independent growth compared with mock-transfected cells. Conclusions: These findings on the expression and function of α6β1 in hepatocarcinoma cells emphasize the potential contribution of this laminin receptor in the neoplastic transformation of hepatocytes. GASTROENTEROLOGY 1998;115:433-442

We investigated whether activation of integrin receptors could modulate the expression of monocyte chemotactic protein-1 (MCP-1) in human hepatic stellate cells (HSC), mesenchymal cells responsible for extracellular matrix synthesis within the liver. When compared to non-adherent cells, HSC plated on collagen types I or IV, or fibronectin, showed increased MCP-1 gene expression and protein secretion in the conditioned medium. Increased MCP-1 secretion was also observed when cells were plated on dishes coated with a monoclonal antibody directed against the β1-integrin subunit, demonstrating that ligation of β1-integrins is sufficient to stimulate MCP-1 expression. Conversely, integrin-independent cell adhesion on poly-l-lysine did not modify MCP-1 secretion. Disruption of the actin cytoskeleton by cytochalasin D blocked the collagen-dependent increase in MCP-1 secretion. Chemotactic assay of HSC-conditioned medium showed that HSC plated on collagen secrete higher amounts of chemotactic factors for lymphomonocytes, and that MCP-1 accounts for the great majority of this effect. These findings indicate a novel mechanism of MCP-1 regulation possibly relevant in those conditions where HSC interact with an altered extracellular matrix.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis. Integrin receptors contribute to the regulation cell adhesion and migration. The aim of this study was to evaluate the interaction between focal adhesion kinase (FAK) and phospholipase C gamma (PLC gamma) potentially involved in HSC integrin- mediated signaling pathways. METHODS: Interaction between FAK and PLC gamma was determined by immunoprecipitation and immunoblotting. HSC chemotactic activity was evaluated using the Boyden chamber technique. RESULTS: HSC adhesion to extracellular matrix components (collagen type I and IV, laminin, and fibronectin) and antibody-mediated beta 1 ligation elicited increased tyrosine phosphorylation of FAK. HSC adhesion to different extracellular matrix components did not result in PLC gamma tyrosine phosphorylation. However, HSC adhesion induced association between PLC gamma and FAK. All extracellular matrix components tested stimulated HSC chemotactic activity only at high concentrations. On the contrary, platelet-derived growth factor, homodimer BB (PDGF-BB), was able to stimulate HSC migration in a dose- dependent manner; this event, occurring in the presence of FAK phosphorylation, was associated to a dose-dependent PLC gamma tyrosine phosphorylation. CONCLUSIONS: These findings provide the first evidence that PLC gamma recruitment by FAK during HSC adhesion is an important process implicating a link between integrin and PDGF-mediated signaling pathways to regulate HSC adhesion and motility. (Gastroenterology 1997 Feb;112(2):522-31)

It has long been recognized that hepatocellular carcinoma heterogeneity arises from variation in the microenvironment or from genomic alteration. Only recently it has become clear that non-genetic alterations, such as cytoskeletal rearrangement, protein localization and formation of protein complexes, are also involved in generating phenotype variability. These proteome fluctuations cause genetically identical cells to vary significantly in their responsiveness to microenvironment stimuli. In the cirrhotic liver pre-malignant hepatocytes are continuously exposed to abnormal microenvironments, such as direct contact with activated hepatic stellate cells (HSCs) and extracellular matrix components. These abnormal environments can have pronounced influences on the epigenetic aspects of cells, translating into abnormal phenotypes. Here we discuss non-genetic causes of phenotypic heterogeneity of hepatocellular carcinoma, with an emphasis on variability of membrane protein complexes and transferred functions raising important implications for diagnosis and treatment.

The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence for a role of this phospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubated with progesterone and A23187, two known inducers of the exocytotic event. PAF synthesis (remodelling pathway) was assessed by [3H]acetate incorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resulted in an increase of [3H]acetate incorporation into PAF. Most of the newly synthesized [3H]PAF formed in response to acrosome reaction was found in the supernatant, suggesting a release of the phospholipid from spermatozoa. PAF-like material extracted from human spermatozoa was able to induce aggregation of rabbit platelets and showed identical retention time and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) activity in human spermatozoa was also studied and showed similar kinetic parameters to those described for other cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggesting an activation of phospholipase A. In conclusion, our results demonstrated increased production and release of PAF in human sperm following stimulation with progesterone and A23187 and suggest a role for this phospholipid in the activation of spermatozoa.

Myristoylated alanine-rich protein kinase c substrate (MARCKS) has been suggested to be implicated in cell adhesion, secretion, motility and mitogenesis through regulation of the actin cytoskeletal structure. In the present study, a possible link between MARCKS and the platelet-derived growth factor (PDGF) signaling pathway was investigated in activated human hepatic stellate cells (hHSC), critical regulators of hepatic fibrogenesis. PDGF-BB stimulation resulted in a bi-directional movement of MARCKS that coincided with the phosphorylation of MARCKS and the activation of both PKCepsilon and PKCalpha. Biochemical inhibition of PKC kinase activity and small interfering RNA (siRNA) against PKCepsilon demonstrated that PKCepsilon is indispensable for PDGF-BB-induced MARCKS phosphorylation and cell migration. Immunoprecipitation studies revealed an association between MARCKS and the PDGFbeta-receptor, while the PDGFbeta-receptor and PKCalpha associated with focal adhesion kinase (FAK). Transient transfection with MARCKS DNA plasmid remarkably reduced PDGF-BB stimulated cell motility. In contrast, siRNA against MARCKS increased cell migration in RNAi treated cells in comparison to the scrambled control cells. In conclusion, the present study indicates that MARCKS play a major key role in PDGF-BB-induced chemotaxis in activated hHSC.

Recent evidence indicates that androgen-sensitive prostate cancer cells have a less malignant phenotype characterized by reduced migration and invasion. We investigated whether the presence of the androgen receptor could affect EGFR-mediated signaling by evaluating autotransphosphorylation of the receptor as well as activation of the downstream signaling pathway PI3K/AKT. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signaling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. Our results suggest that the expression of androgen receptors by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signaling leading to invasion in response to EGF. We used the selective tyrosine kinase inhibitor of the EGFR gefitinib (also known as Iressa or ZD1839) to further investigate the role of EGFR in the invasion and growth of PC cells. We demonstrate that in the androgen-insensitive cell lines PC3 and DU145 this compound was able to decrease in vitro invasion of Matrigel by inhibiting EGFR autotransphosphorylation and subsequent PI3K activation. Gefitinib may be useful in the treatment of androgen-independent prostate cancer to limit not only the proliferation but also the invasion of these tumors.

The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage.

Sperm motility is regulated by a complex balance between kinases and phosphatases. Among them, phosphatidylinositol 3-kinase (PI 3-kinase) has been recently suggested to negatively regulate sperm motility (Luconi, M., Marra, F., Gandini, L., Lenzi, A., Filimberti, E., Forti, G. and Baldi, E. (2001). Hum. Reprod. 16, 1931-1937). We demonstrate the presence and activity of PI 3-kinase in human spermatozoa and have investigated the molecular mechanism(s) by which the PI 3-kinase inhibitor, LY294002, triggers an increase in sperm motility. PI 3-kinase inhibition results in an increase in intracellular cAMP levels and in tyrosine phosphorylation of the protein kinase A-anchoring protein AKAP3. These effects finally result in a stimulation of protein kinase A (PKA) binding to AKAP3 in sperm tails through the regulatory subunit RIIβ. The increased binding of RIIβ to AKAP3 induced by LY294002 is mainly due to tyrosine phosphorylation of AKAP3, since it is completely blocked by the tyrosine kinase inhibitor erbstatin, which also reverses the effects of LY294002 on motility and suppresses PKA-AKAP3 interaction. The requirement of PKA binding to AKAP3 for sperm motility is confirmed by the reduction of motility induced by an inhibitor of RIIβ-AKAP3 binding, Ht31, whose effects on sperm motility and PKA binding to AKAP3 are reversed by LY294002. These results demonstrate that PI 3-kinase negatively regulates sperm motility by interfering with AKAP3-PKA binding, providing the first evidence of a molecular mechanism by which PKA can be targeted to sperm tails by interaction with tyrosine phosphorylated form of AKAP3.

Tetraspanins is a large family of membrane proteins that are implicated in cell proliferation, differentiation and tumor invasion. Specifically, the tetraspanin CD81 has been involved in cell proliferation but the mechanism is unknown. Here, we show that CD81 clustering stimulates ERK/MAPKinase activity and tyrosine phosphorylation of the adapter protein Shc in Huh7 cancer cells. In addition, overexpression of CD81 in HepG2 cells, NIH3T3 cells, and murine fibroblasts GD25 lacking the beta1 family of integrins induces cell proliferation and ERK/MAPKinase activation. Linked with this event, we observed an increase in CD81-associated type II phosphatidylinositol 4-kinase activity. A mutant in the PTB domain of Shc failed to interact with phosphoinositides and localize to the plasma membrane thus blocking CD81-induced ERK/MAPKinase activation. Therefore, we conclude that CD81 stimulates synthesis of phosphoinositides with the recruitment of Shc to the plasma membrane via PTB domain, and this sequence of events induces activation of ERK/MAPKinase. These findings define a novel mechanism of ERK/MAPKinase activation and tumor cell proliferation.

Farnesyltransferase inhibitors (FTIs) block the growth of tumor cells in vitro and in vivo with minimal toxicity toward normal cells. In general, inhibition of protein farnesylation results in G0/G1 cell cycle block, G2/M cell cycle arrest, or has no effect on cell cycle progression. One aspect of FTI biology that is poorly understood is the ability of these drugs to induce cancer cell growth arrest at the G2/M phase of cell cycle. In the present study, we investigated the effects of the farnesyltransferase inhibitor FTI-277 on two human liver cancer cell lines, HepG2 and Huh7. Treatment of these cells with FTI-277 inhibited Ras farnesylation in a dose-dependent manner. Both HepG2 and Huh7 cell growth was inhibited by FTI-277 and cells accumulated at the G2/M phase of the cell cycle. In HepG2 and Huh7 cells, FTI-277 induced an up-regulation of the cyclin-dependent kinase inhibitor p27(Kip1) without affecting the cellular levels of p53 and p21(Waf1). This event correlated with reduced activity of the cyclin-dependent kinase 2 and cyclin-dependent kinase 1. Moreover, increased expression of Bcl-2 protein was observed in HepG2 and Huh7 cells treated with FTI-277, and this was coincidental with reduced association between Raf-1 and Bcl-2. Finally, transient transfection of a dominant-negative Ras allele induced Bcl-2 expression and reduced Bcl-2/Raf-1 association demonstrating a requirement for Ras. Taken together, these findings show that increased expression of p27(Kip1) and Bcl-2 is concomitant with altered association between Ras, Raf-1 and Bcl-2 and suggest that this is responsible for the growth-inhibitory properties of FTI-277.

BACKGROUND & AIMS: Human hepatic stellate cells (HSCs), liver-specific pericytes, are currently considered major producers of extracellular matrix (ECM) components and key elements in the development of liver fibrogenesis. However, little is known about the possible functional interactions between HSCs and the various ECM components. Therefore, the present study was designed to evaluate the expression of integrins, the major family of extracellular matrix receptors. METHODS: Integrin expression was evaluated by immunoprecipitation and confirmed by immunocytochemistry and flow cytometry. RESULTS: Human HSCs were shown to express alpha1beta1, alpha2beta1, alpha(v)beta1. Adhesion to type IV collagen, type I collagen, fibronectin, and laminin 1 was inhibited by anti-beta1 antibody identifying beta1-containing integrins as possible receptors for these components. In addition, we showed that HSCs express alpha6beta4, a heterodimer known to mediate adhesion of epithelial cells to laminin and not previously characterized in mesenchymal cells. Adhesion to laminin 1 was not inhibited by antibodies specific for alpha6 or beta4, thus establishing that laminin 1 is not a ligand for alpha6beta4 in this cell type. CONCLUSIONS: These findings represent the first description of integrin receptors in HSC and provide an attempt to cover the gap of information in the field of HSC-ECM interactions.

Hepatic stellate cells (HSC), perisinusoidal mesenchymal elements with possible pericytic functions within the liver, have recently been shown to play multiple physiological and pathophysiological roles. In particular, several in vivo and in vitro studies have clearly indicated that HSC play a relevant role in the progression of liver fibrogenesis. More recently, the attention has been focused on the mechanisms leading to HSC activation, proliferation and synthesis of extracellular matrix components. Among other soluble factors potentially involved in these processes, transforming growth factor-sl and platelet-derived growth factor have been shown to act in a paracrine, and possibly autocrine, fashion on HSC, thus perpetuating their activated state.

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.

Hepatic stellate cells (HSC) are presently regarded as one of the key cell types involved in the progression of liver fibrosis and in the related pathophysiological and clinical complications. Following acute or chronic liver tissue damage, HSC undergo a process of activation towards a phenotype characterised by increased proliferation, motility, contractility and synthesis of extracellular matrix (ECM) components. Several factors have been shown to play a key role in the promotion of the full-blown picture of activated HSC. These include extensive changes in the composition and organisation of the ECM, the secretion of several growth factors, cytokines, chemokines, products of oxidative stress and other soluble factors. It is evident that each cellular response to extracellular stimuli must be framed in a scenario where different forces modulate one another and result in a prevalent biological effect. Along these lines, the identification and characterisation of intracellular signalling pathways activated by different stimuli in HSC represent a mandatory step. In this review article we have made an attempt to summarise recent acquisitions to our knowledge of the involvement of different intracellular signalling pathways in key aspects of HSC biology.