Integrins belong to a family of heterodimeric receptors composed of α and β subunits that form the transmembrane linkages between the ECM and actin cytoskeleton. The extracellular domain of integrins interacts with the ECM, while the cytoplasmic tail binds to cytoskeletal proteins. Aside from their structural role, integrins mediate cell adhesion to the ECM and migration, organize cytoskeletal structure and activate various intracellular signaling pathways. Expression of integrin α5 and β1 subunits has been found to be lower in HCC than the corresponding unaffected areas.7 On the contrary, expressions of α5 integrins and their extracellular ligands, fibronectin (FN) and vitronectin have been found to be more frequently associated with adverse histopathological prognostic parameters in HCC.14
Tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) can significantly increase the expression of α5 and stimulate cell adhesion and migration on vitronectin. TGF-β1 treatment can also enhance the expression of another integrin subunit, α5β1, subsequently resulting in stimulated cell adhesion onto FN and laminin matrix.15 Expression of α6β1 integrin has been shown to be necessary for focal adhesion kinase (FAK) phosphorylation and subsequent mitogen-activated protein kinases (MAPK) activation that enhance cell migration in HCC cells, indicating a functional role of integrins in regulating cellular movement.16 Retinoic acid treatment is able to increase the adhesiveness of HCC cells, and this is accompanied by an enhanced expression of β1 and α3 integrin subunits.17
Cooperation of FN/β1 integrin-mediated adhesion with actin polymerization and myosin light chain (MLC) phosphorylation (the latter through Rho activation) is necessary for phagokinetic motility and transcellular migration.18 Integrin α3β1 co-localizes with HAb18G/CD147 transmembrane glycoprotein, which is involved in metastasis of HCC cells. Indeed, integrin α3β1 antibodies are able to decrease the enhancing effect of CD147 on adhesion, invasion capacities and secretion of matrix metalloproteinases (MMP).19
Apart from cellular movement, α5β1 or β1 play a role in cell growth and apoptosis. Overexpression of α5β1 or β1 induces S-phase delay, an effect attributable to a decrease of protein kinase B phosphorylation and subsequent increases of p21 and p27.20 Treatment with antibody against integrin α5 or β1 subunits results in promotion of all-trans-retinoic acid (ATRA)-induced apoptosis. Proteolytic cleavage of integrin α5β1 has been linked to the early phase of ATRA-induced apoptosis.21
Integrin-linked kinase (ILK) was identified as an interacting protein of β1-integrin cytoplasmic domain.22 ILK functions as a scaffolding protein in a multiprotein complex at focal adhesions. Interaction with particularly interesting Cys-His-rich protein (PINCH) is crucial for the localization of ILK to focal adhesions.23 Besides interacting with integrin, ILK interacts with the actin-binding proteins, α-parvin, β-parvin and paxillin.24,25 ILK-associated phosphatase (ILKAP) is a serine/threonine phosphatase that interacts with and negatively regulates the kinase activity of ILK.26 On the other hand, ILK binds and is activated by phosphatidylinositol 3,4,5-triphosphate (PIP3).27 In this way, ILK is positively regulated in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Downstream targets of PI3K, for instance Akt and glycogen synthase kinase-3 (GSK3), are phosphorylated by ILK. Conversely, tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) dephosphorylates PIP3, resulting in negative regulation of ILK activity.28
Recent findings have indicated that ILK expression is increased in melanoma, ovarian, colon and prostate cancers.29–33 In normal liver, only weak ILK expression is detected in hepatocytes and bile duct epithelial cells. Conversely, ILK has been reported to be detected in 85% of cirrhotic livers and overexpressed in 100% of HCC.10,34 Overexpression of ILK significantly correlates with Akt phosphorylation in HCC as well as in cirrhosis. Accumulating evidence from in vitro and in vivo studies in cancer cells has shown that ILK is a putative oncogene and a potential therapeutic target. Currently, signaling pathways associated with ILK have not been clearly addressed in HCC. A number of signaling pathways are dysregulated in cancer cells in which ILK is overexpressed or is constitutively activated.35 TGF-β1 stimulated ILK expression in accordance with Akt phosphorylation in HCC cells in a dose-dependent manner.36 A gene microarray analysis showed that ILK was upregulated in HCC cells subjected to a hypoxic condition.37 Overexpression of ILK in epithelial cells induces epithelial–mesenchymal transition (EMT).38,39
In a recent study, ILK expression has been shown to correlate with the extent of EMT of HCC cell lines, as determined by the expressions of E-cadherin and vimentin.40 Functionally, kinase-inactive ILK transformants established with HCC cells display decreased ILK activity, activation of Akt, and increased sensitivity to epidermal growth factor receptor (EGFR) inhibitors. On the other hand, loss of ILK and FAK expression occurs when HCC cells are induced to apoptosis by chemical compounds.41 These findings suggest that adhesion molecules maintain cell–matrix interaction which is important for cell attachment and survival. Importantly, loss of adhesion molecules can induce apoptosis of HCC cells.
Focal adhesion kinase
Focal adhesion kinase is the most extensively studied focal adhesion protein in HCC (Fig. 2). It is a 125-kDa non-receptor cytoplasmic tyrosine kinase that has been implicated in cancer cell proliferation, survival, migration, invasion and metastasis.42–44 In human HCC, FAK mRNA and protein expressions have been consistently reported to be increased in HCC as compared to non-tumorous liver or chronic hepatitis.4–8,12,45,46 Moreover, FAK overexpression has been reported to correlate with hepatitis B virus (HBV) infection.6,45 Tyrosine phosphorylation of FAK at Y397 activates the kinase and recruits various structural and signaling molecules for focal adhesion assembly, as well as disassembly.47 Expression and phosphorylation (p-Y397) levels of FAK positively correlate with HCC cell motility, invasive ability and metastatic potential.15,48–51 Various viral or cellular proteins, chemical molecules and physical conditions can regulate the expression and activity of FAK and in turn affect the behavior of HCC cells. These observations strongly suggest a role for FAK in hepatocarcinogenesis, tumor progression, invasion and metastasis. The frequent correlation of FAK overexpression and poor prognosis in HCC4–6 warrant consideration of FAK expression as an independent prognostic factor in this cancer (Table 1).
Figure 2. Focal adhesion kinase (FAK) signaling pathways in hepatocellular carcinoma (HCC). FAK overexpression and enhanced FAK phosphorylation have been shown to correlate with HCC cell growth, survival, migration, invasion and metastasis. Regulators such as HBV X protein (HBx), CD147, stromal cell-derived factor 1 (SDF-1), chemokine (C-X-C motif) receptor 4 (CXCR4), 12-O-tetradecanoylphorbol-13-acetate (TPA), transforming growth factor-β1 (TGF-β1), peroxisome proliferator-activated receptor γ (PPARγ), suppressors of cytokine signaling (SOCS) and phosphatase and tensin homolog deleted from chromosome 10 (PTEN) have been shown to regulate the expression and activity of FAK and in turn affect the behavior of HCC cells. MAPK, mitogen-activated protein kinases.
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Chronic HBV infection significantly contributes to HCC development. The HBV X protein (HBx), encoded by the HBV genome, promotes FAK expression and phosphorylation, and activates Src tyrosine kinase and the downstream MAPK pathway. Interestingly, an in vitro study has shown that a phospho-defective Y397F mutant of FAK could dominantly inhibit the HBx-activated Src kinase signaling cascade, HBx-responsive nuclear factor-κB (NF-κB) and activated protein-1 (AP-1)-dependent transcription and HBV DNA replication.52 These findings highlight the cooperative effect of FAK in HBx-mediated signal transduction and transcriptional activation.
Focal adhesion kinase expression and phosphorylation have been shown to be regulated by CD147, and these then activate the FAK-mediated signaling pathways to confer an enhanced invasive capability to HCC cells. CD147 belongs to the immunoglobulin superfamily and can induce production of MMP in fibroblasts and endothelial cells. Several studies have demonstrated the positive regulatory effect of CD147 on FAK expression and phosphorylation.19,53,54 Downregulation of CD147 can suppress FAK expression via ERK1/2 and inhibit HCC cell invasion.54,55 In addition, CD147 can suppress the expression of other focal adhesion proteins, vinculin and paxillin, with reduction of the quantity of focal adhesions.19,53 On the other hand, Tang et al. have shown that enhanced expression and phosphorylation of FAK and paxillin were coupled with overexpression of CD147 in human HCC.19 CD147 interacts with α3β1 integrin, FAK-paxillin and FAK-PI3K-Ca2+ signaling pathways in promoting the metastatic potential of HCC cells.19
The positive correlation between FAK expression and invasive ability of HCC cell lines has also been demonstrated by Itoh et al.5 Expression of suppressors of cytokine signaling-3 (SOCS-3) is silenced in HCC cells as a result of promoter methylation. This abrogates cytokine and growth factor signaling by interacting with Janus kinase (JAK) to inhibit STAT activity.56,57 Niwa et al. have reported that SOCS-3 induced FAK degradation in HCC cells by interacting with phosphorylated FAK directly, thereby inhibiting cell motility. This inhibitory effect was suppressed by downregulation of SOCS-3; FAK phosphorylation was increased and cell migration was enhanced.48
Stromal cell-derived factor 1 (SDF-1) is a chemokine that mediates signals through chemokine (C-X-C motif) receptor 4 (CXCR4). FAK phosphorylation is induced in HCC cells that secrete SDF-1 and express CXCR4. It seems probable that this leads to cytoskeleton reorganization and contributes to the observed increase of cell motility and invasiveness.51
Activation and inhibition of peroxisome proliferator-activated receptor γ (PPARγ) also controls FAK phosphorylation and subsequently affects HCC cell adhesion, motility and survival under anchorage-independent conditions.49,58 PPARγ heterodimerizes with the 9-cis-retinoic acid receptor (RXR) and binds to specific PPAR-responsive elements (PPRE) to regulate gene transcription. The target genes are involved in lipid metabolism, inflammation and cell differentiation.59,60 Treatment with a PPARγ activator, rosiglitazone, has been shown to inhibit FAK phosphorylation and HCC cell migration without any effect on cell proliferation. This can be explained by upregulation of PTEN expression; PTEN dephosphorylates FAK and suppresses subsequent activation of the PI3K/Akt pathway.49,61 Overexpression of PTEN in HCC cells reduces the phosphorylation level of FAK, and there is an inverse correlation between FAK phosphorylation and PTEN protein expression observed in HCC cells.62 Suppressed PTEN expression in HCC is attributable to the upregulation of microRNA miR-21. In HCC cell lines, miR-21 promotes cell migration and invasion. Its expression correlates inversely with PTEN but was positively associated with FAK phosphorylation.50 Because PTEN suppresses MMP expression by dephosphorylating FAK in glioblastoma cells,63 it is postulated that the modulation of MMP-9 expression exerted by miR-21 is related to uninhibited FAK activity.50
Tumor cells encounter different microenvironments during metastasis and FAK are critical in determining the survival advantage of HCC cells in these adverse conditions. Treatment of HCC cells with PPARγ inhibitors reduced FAK activation and induced anoikis by preventing adhesion to ECM. These findings demonstrate the importance of FAK phosphorylation in anchorage-independent growth.58
Successful metastasis requires that disseminated tumor cells overcome the shear force of fluid flow within the microcirculation of metastatic target organs in order to establish stabilized adhesion under dynamic conditions. Von Sengbusch et al. have found that hydrodynamic shear forces induced FAK phosphorylation and favored dynamic cell adhesion. In support of this, their work has further demonstrated the importance of FAK phosphorylation for metastasis in vivo and the establishment of stabilized adhesive interactions.64 FAK phosphorylation and the subsequent activation of the PI3K/Akt/AP-1 pathway promote HCC cell proliferation upon exposure to hypo-osmotic stress.65 It is noted that actin cytoskeletal integrity is indispensable in the activation of FAK induced by osmotic stress as well as by TGF-β1 and the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA)—the latter through activation of protein kinase C (PKC).36,65,66 Thus, taken together, FAK phosphorylation is crucial in promoting HCC metastasis.
Proline-rich tyrosine kinase 2
Proline-rich tyrosine kinase 2 (Pyk2) is a member of the FAK family. Similar to FAK, Pyk2 is activated upon tyrosine phosphorylation at Y402, thereby exposing a binding site for the SH2 domain of Src.44 Pyk2 is overexpressed in HCC and correlates positively with FAK expression and poor prognosis (Table 1).12 Overexpression of Pyk2 promotes the proliferation, migration, invasion and anchorage-independent growth of HCC cells, as it interacts with Src and activates the MAPK pathway.12,67 The pro-metastatic role of Pyk2 has been demonstrated by the positive staining found in infiltrative tumor cells as well as lung metastatic nodules.12 These events are kinase-dependent as the opposite behavior was observed upon introduction of PRNK (C-terminal non-kinase Pyk2) to HCC cells.67
Deleted in liver cancer (DLC) family
Deleted in liver cancer 1 (DLC1) is a candidate tumor suppressor gene on chromosome 8p21.3-22 and was first isolated from human HCC.68 It is widely expressed in normal human tissues. The frequent underexpression of DLC1 noted in HCC primary tissue and cell lines has been attributed to genomic deletion and promoter hypermethylation.2,69 DLC1 encodes a Rho GTPase-activating protein (RhoGAP) which co-localizes with vinculin at focal adhesions (Fig. 3).70 RhoGAP activity to RhoA and Cdc42 of DLC1 was first demonstrated by an in vitro assay.2 DLC1 negatively regulates the Rho/ROCK/MLC pathway and is implicated in its role in regulating formation of stress fibers and focal adhesions via RhoGAP activity.71–74 Introduction of DLC1 into HCC cells can also dephosphorylate other focal adhesion proteins, such as FAK, Crk-associated substrate (p130Cas) and paxillin.71
Figure 3. Functional role of deleted in liver cancer 1 (DLC1) in hepatocellular carcinoma (HCC). Residues Y442 and S440 are responsible for the focal adhesion targeting of DLC1. At the focal adhesions, DLC1 interacts with tensin and their interaction has been shown to be important for the growth suppression activity of DLC1. Introduction of DLC1 into HCC cells has also been shown to induce apoptosis, suppress migration and invasion, and inhibit stress fiber formation and focal adhesions.
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The importance of RhoGAP activity in the growth suppressive activity of DLC1 has been demonstrated by the loss of growth inhibitory activity in RhoGAP mutants.73 Ectopic expression of DLC1 in HCC cells suppresses cell growth and inhibits cell migration and invasion.69,71,73,74 Moreover, restoration of DLC1 expression induces apoptosis in HCC cells.75 In a murine model, knockdown of DLC1 cooperates with Myc in promoting hepatocarcinogenesis.76
Localization at focal adhesion is crucial to the growth suppressive ability of DLC1. Independent studies have identified tensin family members as the interacting partners of DLC1 and shown the ability of tensin proteins in mediating the focal adhesion localization, which in turn regulates the biological activities of DLC1. Mutations of focal adhesion targeting residues S440 and Y442 in DLC1 result in loss of focal adhesion localization and consequent ability to reduce cancer cell growth.77,78 Somatic mutation localized in the focal adhesion targeting region of DLC1 was first found in prostate cancer samples.79 These mutations in the focal adhesion targeting region of DLC1 can result in growth suppressive and RhoGAP activities. Somatic mutations of DLC1 in HCC and other cancers seem to be rare, but this awaits further investigation.2,80
Deleted in liver cancer 1 belongs to a family of tumor suppressor genes that contains two other members, DLC2 and DLC3. All family members are structurally similar, exhibit growth suppressive and RhoGAP activities, and localize to focal adhesions.70,81–83 DLC2 was mapped to chromosome 13q12.3, a region where a high frequency of allelic losses has been found.3,84 DLC2 exerts effects on suppression of cytoskeleton reorganization, cell growth, cell migration and transformation of HCC cells.83 Apart from localizing at focal adhesions, DLC2 also targets to mitochondria in HCC cells by its START domain.13
Tensins are a family of focal adhesion proteins that bind to the integrin cytoplasmic tail of β subunits. They share similar structural organization with two potential binding domains, Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains at the C-terminus. They also share sequence homology with PTEN.85 Tensin1 is the best characterized family member for its activities in organizing the actin cytoskeleton and mediating signal transduction.86,87 Recently, other structurally-related tensin family members have been identified.
Growing evidence suggests that tensin proteins are not only structural proteins, but act as an important link between the ECM, actin cytoskeleton and signal transduction.88–93 Independent studies have reported the downregulation of tensin members in various human cancer cell lines and tissues, yet the mechanism underlying the role of tensins in cancers is far from clear. Tensin1 is underexpressed in human prostate and breast cancer cell lines.94 Cten, a COOH-terminal tensin-like member with restricted expression in prostate and placenta is downregulated in prostate cancer.95
Tensin2 was identified as the second family member of tensin.96 It was the first interacting partner of DLC1 identified in a yeast two-hybrid screen of a human liver cDNA library.70 Tensin2 is dominantly expressed in heart, kidney, skeletal muscle and liver. It localizes at the end of actin stress fibers and co-localizes with vinculin and tensin1 at the focal adhesions. Overexpression of tensin2 suppresses colony formation and induces apoptosis in HCC cells.70
Both tensin1 and tensin2 have been shown to play a role in regulating cell motility.96,97 Interestingly, a splicing variant of tensin2, variant 3, plays an opposing role in HCC.13 Clinicopathological analysis revealed that variant 3 overexpression in human HCC was associated with more aggressive and metastasis-related pathological features. Functionally, variant 3 promotes cell proliferation, migration and invasion in vitro, and enhances tumorigenicity and invasiveness of HCC cells in nude mice.13
Caveolae are flask-shaped invaginations in the plasma membrane that have been implicated in cellular transport processes and signal transduction.98 Caveolins are the major structural proteins of caveolae. They include Cav1, Cav2 and Cav3. Cav1 and Cav2 are abundant in diverse cell types, whereas Cav3 is muscle-specific.99 Caveolar Cav1 serves as a scaffold allowing key regulators of cell migration to associate with caveolae and interact in the intracellular compartment.100,101 Apart from localizing in caveolae, Cav1 is also found in the nucleus, cytoplasm, focal adhesions and the extracellular milieu. The non-caveolar Cav1 is involved in cell signaling and interacts with intracellular cytoskeleton complex, which in turn regulates cell adhesion and locomotion.
Cav1 was first isolated as a tyrosine phosphorylated substrate of v-Src.102 Cav1 was downregulated in oncogenically transformed cells and its diminution in NIH3T3 cells was sufficient to induce a transformed phenotype. It is therefore plausible to regard Cav1 as a tumor suppressor gene.103,104
There are conflicting data on Cav1 expression levels in various tumors or tumor cell lines, as compared to their normal tissue counterparts. For this reason, the role played by Cav1 in tumor progression has been ambiguous.105 In recent years, increasing evidence demonstrates that Cav1 is suppressed at the early stages of transformation and carcinogenesis, but upregulated and associated with cancer progression, metastasis and poor prognosis in later stages of various carcinomas.106–109 Cav1 is not detectable in non-neoplastic livers, but HCC (26%) show increased expression.110 In mouse HCC cell lines, cells with higher invasive ability have strong Cav1 expression.111
Cell migration is a critical component in the process of tumor metastasis; it requires cancer cells to undergo sequential morphological changes. Cav1 expression is essential for cell polarization and migration.112,113 Tyrosine phosphorylated Cav1 (pY14Cav1) has been identified to localize to focal adhesions and is implicated in such cell polarization and migration.114,115 pY14Cav1 is also involved in the regulation of Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion.116 Upregulation of Cav1 by FAK during EMT further demonstrates the importance of Cav1 in cell adhesion and migration.117
Other than FAK, small GTPases, Rac, Cdc42 and Rho, which are the key regulators of cytoskeletal rearrangement and focal adhesion formation, have been reported to interact with Cav1.118,119 Cav1-deficient mouse embryo fibroblasts (MEF) show aberrant activities of the Rho family small GTPases. Importantly, the altered basal activity of Rho might change morphological phenotypes in a way that encourages metastasis of the cancer cell.
The Src oncogene belongs to a family of non-receptor tyrosine kinases. Its expression level has been shown to correlate with progression of diverse cancers.120 Src is elevated and active in HCC. Both total and active forms of Src have been detected in HCC but not non-tumorous surrounding livers or in normal livers.8 Expressions of total and active Src and FAK correlate with one another, and Src expression also correlates significantly with alpha-fetoprotein expression in HCC cells.8
Experimental studies have indicated that active Src in HCC confers resistance of HCC cells to TGF-β1-induced apoptosis and proliferation. In TGF-β1-sensitive HCC cells, TGF-β1 decreased Src activity and induced tyrosine dephosphorylation of Ras-GAP and hence inactivation of Ras. However, in TGF-β1-insensitive HCC cells, TGF-β1 enhanced both Src level and activity and Ras activation, the latter by Ras-GAP phosphorylation. Interestingly, HCC cells are sensitized to the effect of TGF-β1 by inhibiting Src activity.121 Another study has also shown that TGF-β1 enhances the transient activation of Src and its subsequent caspase-mediated degradation contributing to focal adhesion disassembly in HCC cells which are sensitive to TGF-β1. Inhibition of Src activity by specific inhibitors enhances TGF-β1-induced apoptosis, while overexpression of activated Src confers resistance to TGF-β1-induced apoptosis.122
Src also plays a role in promoting tumorigenicity of HCC cells. Upregulation of Src and Erk/MAPK pathways, together with interaction of Pyk2/Src, contributed to the promotion of proliferation and invasiveness of HCC cells.67 The Src kinase signaling cascade has also been shown to be involved in HBx activity. Thus, HBx elevated the expression and activity of FAK, a known regulator of Src family kinases. To the contrary, dominant-inhibiting FAK mutants are able to block HBx-activated Src kinases and downstream signal transduction.52
Paxillin acts as a scaffold that plays a pivotal role at focal adhesions. In response to adhesion stimuli and growth factors, paxillin is phosphorylated by diverse kinases, and such phosphorylation is crucial for paxillin to function as a docking site of other focal adhesion components and regulators of cell migration.123 One study suggests that paxillin phosphorylation at S178 by c-Jun NH2-terminal kinase (JNK) is important for Pak1-mediated migration of HCC cells. Treatment of metastatic HCC cells with hepatocyte growth factor also increases phosphorylation of JNK and paxillin.
Immunohistochemical staining of human HCC has revealed that expression of phosphorylated paxillin in Pak1-overexpressing human HCC is substantially increased.11 The integrin-mediated FAK–paxillin signaling pathway has also been implicated in the HAb18G/CD147-enhanced metastatic potential of human HCC cells.19 Paxillin and phospho-paxillin expression were increased in human HCC cells overexpressing HAb18G/CD147, while depletion of CD147 reduced the quantity of focal adhesions in these tumor cells.
ArfGAP with SH3 domain, ankyrin repeat and PH domain 3
ArfGAP with SH3 domain, ankyrin repeat and PH domain 3 (ASAP3), also known as upregulated in liver cancer 1 (UPLC1) or development and differentiation enhancing factor-like 1 (DDEFL1), is a subtype of Arf GTPase-activating proteins. It is involved with organization of the actin cytoskeleton and is upregulated in HCC.9 ASAP3 is localized in focal adhesions but not in invadopodia, and ASAP3 has no effect on invadopodia formation. However, downregulation of ASAP3 reduces cell invasion as well as proliferation; it is also speculated that ASAP3 participates in the Rho, Rac and Cdc42 pathways.1,9