Search

The EMT (epithelial–mesenchymal transition) is involved in fibrosis and cancer, and is regulated by different signalling pathways mediated through soluble factors, actin reorganization and transcription factor actions. Because the tetraspan (also called tetraspanin) TM4SF5 (transmembrane 4 L6 family member 5) is highly expressed in hepatocellular carcinoma and induces EMT, understanding how TM4SF5 expression in hepatocytes is regulated is important. We explored the mechanisms that induce TM4SF5 expression and whether impaired signalling pathways for TM4SF5 expression inhibit the acquisition of mesenchymal cell features, using human and mouse normal hepatocytes. We found that TGFβ1 (transforming growth factor β1)-mediated Smad activation caused TM4SF5 expression and EMT, and activation of the EGFR [EGF (epidermal growth factor) receptor] pathway. Inhibition of EGFR activity following TGFβ1 treatment abolished acquisition of EMT, suggesting a link from Smads to EGFR for TM4SF5 expression. Further, TGFβ1-mediated EGFR activation and TM4SF5 expression were abolished by EGFR suppression or extracellular EGF depletion. Smad overexpression mediated EGFR activation and TM4SF5 expression in the absence of serum, and EGFR kinase inactivation or EGF depletion abolished Smad-overexpression-induced TM4SF5 and mesenchymal cell marker expression. Inhibition of Smad, EGFR or TM4SF5 using Smad7 or small compounds also blocked TM4SF5 expression and/or EMT. These results indicate that TGFβ1- and growth factor-mediated signalling activities mediate TM4SF5 expression leading to acquisition of mesenchymal cell features, suggesting that TM4SF5 induction may be involved in the development of liver pathologies.

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocarcinoma and causes epithelial–mesenchymal transition (EMT) of hepatocytes. We found that TM4SF5-expressing cells showed lower mRNA levels but maintained normal protein levels in certain gene cases, indicating that TM4SF5 mediates stabilization of proteins. In this study, we explored whether regulation of proteasome activity and TM4SF5 expression led to EMT. We observed that TM4SF5 expression caused inhibition of proteasome activity and proteasome subunit expression, causing morphological changes and loss of cell–cell contacts. shRNA against TM4SF5 recovered proteasome expression, with leading to blockade of proteasome inactivation and EMT. Altogether, TM4SF5 expression appeared to cause loss of cell–cell adhesions via proteasome suppression and thereby proteasome inhibition, leading to repression of cell–cell adhesion molecules, such as E-cadherin. J. Cell. Biochem. 112: 782–792, 2011. © 2010 Wiley-Liss, Inc.

Protein-protein interactions and/or signaling activities at focal adhesions, where integrin-mediated adhesion to extracellular matrix occurs, are critical for the regulation of adhesion-dependent cellular functions. Although the phosphorylation and activities of focal adhesion molecules have been intensively studied, the effects of the O-GlcNAc modification of their Ser/Thr residues on cellular functions have been largely unexplored. We investigated the effects of O-GlcNAc modification on actin reorganization and morphology of rat insulinoma INS-1 cells after glucosamine (GlcN) treatment. We found that paxillin, a key adaptor molecule in focal adhesions, could be modified by O-GlcNAc in INS-1 cells treated with GlcN and in pancreatic islets from mice treated with streptozotocin. Ser-84/85 in human paxillin appeared to be modified by O-GlcNAc, which was inversely correlated to Ser-85 phosphorylation (Ser-83 in rat paxillin). Integrin-mediated adhesion signaling inhibited the GlcN treatment-enhanced O-GlcNAc modification of paxillin. Adherent INS-1 cells treated with GlcN showed restricted protrusions, whereas untreated cells showed active protrusions for multiple-elongated morphologies. Upon GlcN treatment, expression of a triple mutation (S83A/S84A/S85A) resulted in no further restriction of protrusions. Together these observations suggest that murine pancreatic β cells may have restricted actin organization upon GlcN treatment by virtue of the O-GlcNAc modification of paxillin, which can be antagonized by a persistent cell adhesion process.

Overexpression of transmembrane 4 L six family member 5 (TM4SF5), a four-transmembrane L6 family member, causes aberrant cell proliferation and angiogenesis, but the roles of TM4SF5 in migration, invasion, and tumor metastasis remain unknown. Using in vitro hepatocarcinoma cells that ectopically or endogenously express TM4SF5 and in vivo mouse systems, roles of TM4SF5 in metastatic potentials were examined. We found that TM4SF5 expression facilitated migration, invadopodia formation, MMP activation, invasion, and eventually lung metastasis in nude mice, but suppression of TM4SF5 with its shRNA blocked the effects. Altogether, TM4SF5-mediated migration and invasion suggest that TM4SF5 may be therapeutically targeted to deal with TM4SF5-mediated hepatocellular cancers.

Four-transmembrane L6 family member 5 (TM4SF5) and its homolog L6, a tumor antigen, form a four-transmembrane L6 family. TM4SF5 expression causes uncontrolled cell proliferation and angiogenesis. Although other genuine transmembrane 4 superfamily (TM4SF) members co-operate with integrins for cell migration, roles of TM4SF5 in the cellular spreading and migration are unknown. Using hepatocarcinoma cell clones that ectopically express TM4SF5, we found that cross talks via an extracellular interaction between TM4SF5 and integrin alpha2 in collagen type I environment inhibited integrin alpha2 functions such as spreading on and migration toward collagen I, which were recovered by suppression of TM4SF5 or structural disturbance of its second extracellular loop using a peptide or mutagenesis. Altogether, the observations suggest that TM4SF5 in hepatocytes negatively regulates integrin alpha2 function via an interaction between the extracellular loop 2 of TM4SF5 and integrin alpha2 during cell spreading on and migration through collagen I environment.

Tetraspan TM4SF5 is highly expressed in a diverse number of tumor types. Here we explore the mechanistic roles of TM4SF5 in angiogenesis. We found that TM4SF5 overexpression correlates with vascular endothelial growth factor (VEGF) expression in SNU449 hepatocytes and with vessel formation in clinical hepatocarcinoma samples. Conditioned media from TM4SF5-expressing cells enhanced viability and tube formation of primary human umbilical vein endothelial cells, and outgrowth of endothelial cells from aorta ring segments, which was abolished by treatment with an anti-VEGF antibody. TM4SF5 retained integrin α5 on the cell surface for VEGF induction, and preincubation with anti–integrin α5 antibody abolished TM4SF5-mediated VEGF expression and secretion. TM4SF5-mediated effects required integrin α5, c-Src, and signal transducer and activator of transcription 3 (STAT3). In addition, tumors from nude mice injected with TM4SF5-expressing cells and from clinical human hepatocarcinoma tissues showed enhanced integrin α5 expression, vessel formation, and signaling activity, which were inhibited by administration of anti–integrin α5 or -VEGF antibody. This study suggests that TM4SF5 facilitates angiogenesis of neighboring endothelial cells through VEGF induction, mediated by cooperation between TM4SF5 and integrin α5 of epithelial cells.

We previously reported that the four-transmembrane L6 family member 5 (TM4SF5) was highly expressed in hepatocarcinoma, induced morphological elongation and epithelial-mesenchymal transition, and caused abnormal cell growth in multilayers in vitro and tumor formation in vivo. In this study, we identified a synthetic compound, 4′-(p-toluenesulfonylamido)-4-hydroxychalcone (TSAHC) that antagonized both the TM4SF5-mediated multilayer growth and TM4SF5-enhanced migration/invasion. TSAHC treatment induced multilayer-growing cells to grow in monolayers, recovering contact inhibition without accompanying apoptosis, and inhibited chemotactic migration and invasion. Tumor formation in nude mice injected with TM4SF5-expressing cells and the growth of cells expressing endogenous TM4SF5, but not of TM4SF5-null cells, was suppressed by treatment with TSAHC, but not by treatment with its analogs. The structure-activity relationship indicated the significance of 4′-p-toluenesulfonylamido and 4-hydroxy groups for the anti-TM4SF5 effects of TSAHC. Point mutations of the putative N-glycosylation sites abolished the TM4SF5-specific TSAHC responsiveness. Conclusion: These observations suggest that TM4SF5-enhanced tumorigenic proliferation and metastatic potential can be blocked by TSAHC, likely through targeting the extracellular region of TM4SF5, which is important for protein-protein interactions. (HEPATOLOGY 2009.)

The growth of normal cells is arrested when they come in contact with each other, a process known as contact inhibition. Contact inhibition is lost during tumorigenesis, resulting in uncontrolled cell growth. Here, we investigated the role of the tetraspanin transmembrane 4 superfamily member 5 (TM4SF5) in contact inhibition and tumorigenesis. We found that TM4SF5 was overexpressed in human hepatocarcinoma tissue. TM4SF5 expression in clinical samples and in human hepatocellular carcinoma cell lines correlated with enhanced p27Kip1 expression and cytosolic stabilization as well as morphological elongation mediated by RhoA inactivation. These TM4SF5-mediated effects resulted in epithelial-mesenchymal transition (EMT) via loss of E-cadherin expression. The consequence of this was aberrant cell growth, as assessed by S-phase transition in confluent conditions, anchorage-independent growth, and tumor formation in nude mice. The TM4SF5-mediated effects were abolished by suppressing the expression of either TM4SF5 or cytosolic p27Kip1, as well as by reconstituting the expression of E-cadherin. Our observations have revealed a role for TM4SF5 in causing uncontrolled growth of human hepatocarcinoma cells through EMT.

Integrin-linked kinase (ILK) is involved in signal transduction by integrin-mediated cell adhesion that leads to dynamic actin reorganization. Actin (de)polymerization is regulated by cofilin, the Ser(3) phosphorylation (pS(3)cofilin) of which inhibits its actin-severing activity. To determine how ILK regulates pS(3)cofilin, we examined the effects of ILK on pS(3)cofilin using normal RIE1 cells. Compared with suspended cells, fibronectin-adherent cells showed enhanced pS(3)cofilin, depending on ILK expression and c-Src activity. The ILK-mediated pS(3)cofilin in RIE1 cells did not involve Rho-associated kinase, LIM kinase, or testicular protein kinases, which are known to be upstream of cofilin. The kinase domain of ILK, including proline-rich regions, appeared to interact physically with the Src homology 3 domain of c-Src. In vitro kinase assay revealed that ILK immunoprecipitates phosphorylated the recombinant glutathione S-transferase-cofilin, which was abolished by c-Src inhibition. Interestingly, epidermal growth factor treatment abolished the ILK effects, indicating that the linkage from ILK to cofilin is biologically responsive to extracellular cues. Altogether, this study provides evidence for a new signaling connection from ILK to cofilin for dynamic actin polymerization during cell adhesion, depending on the activity of ILK-associated c-Src.

Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-alpha5beta1-mediated actin reorganization. Ectopic expression of integrin alpha5 in integrin-alpha5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin alpha5 (hereafter referred to as alpha5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin alpha5 engagement with fibronectin phosphorylated Ser643 of PKCdelta, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKCdelta inactivation, dominant-negative PKCdelta adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of alpha5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKCdelta expression sensitized alpha5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin alpha5, but not alpha5/1, mediates PKCdelta phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.

CD98hc (SLC3A2) is the heavy chain component of the dimeric transmembrane glycoprotein CD98, which comprises the large neutral amino acid transporter LAT1 (SLC7A5) in cells. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor prognosis clinically, but its exact contributions to tumorigenesis are uncertain. In this study, we showed that genetic deficiency of CD98hc protects against Ras-driven skin carcinogenesis. Deleting CD98hc after tumor induction was also sufficient to cause regression of existing tumors. Investigations into the basis for these effects defined two new functions of CD98hc that contribute to epithelial cancer beyond an intrinsic effect of CD98hc on tumor cell proliferation. First, CD98hc increased the stiffness of the tumor microenvironment. Second, CD98hc amplified the capacity of cells to respond to matrix rigidity, an essential factor in tumor development. Mechanistically, CD98hc mediated this stiffness sensing by increasing Rho kinase (ROCK) activity, resulting in increased transcription mediated by YAP/TAZ, a nuclear relay for mechanical signals. Our results suggest that CD98hc contributes to carcinogenesis by amplifying a positive feedback loop, which increases both extracellular matrix stiffness and resulting cellular responses. This work supports a rationale to explore the use of CD98hc inhibitors as cancer therapeutics. Cancer Res; 74(23); 6878–89. ©2014 AACR.

TM4SF5 is a transmembrane glycoprotein of the transmembrane 4 L six family, a branch of the tetraspanin family and highly expressed in many types of cancers. TM4SF5 induces epithelial-mesenchymal transition (EMT) by morphological changes resulting from inactivation of RhoA mediated by stabilized cytosolic p27kip1. TM4SF5-mediated EMT can lead to loss of contact inhibition and enhanced migration/invasion, presumably depending on cross-talks between TM4SF5 and integrins. An anti-TM4SF5 agent appears to target the second extracellular domain of TM4SF5, which is important for cross-talk with integrins, leading to a blockade of TM4SF5-mediated multilayer growth and migration/invasion. In addition, TM4SF5 engages in cross-talk with integrin alpha5 to induce and secrete VEGF, which in turn causes activation of angiogenesis in endothelial cells. Therefore, TM4SF5 plays a central regulatory role in a wide variety of physiological processes through cross-talk with integrins. This review presents current knowledge from in vitro and in vivo observations of the roles of TM4SF5-integrin cooperation in hepatocellular carcinogenesis and discusses important areas for future investigation.

Transmembrane 4 L six family member 5 (TM4SF5) causes epithelial–mesenchymal transition (EMT) for aberrant cell proliferation. However, the effects of TM4SF5 expression on cell cycle are unknown so far. In this study, using hepatocytes that either ectopically or endogenously express TM4SF5 and human hepatocarcinoma tissues, the role of TM4SF5 in G1/S phase progression was examined. We found that TM4SF5 expression accelerated G1/S phase progression with facilitated cyclin D1 and E expression and Rb phosphorylation. Furthermore, TM4SF5 enhanced trafficking of CDK4 and cyclin D1 into the nucleus and induced complex formation between them. However, TM4SF5-facilitated G1/S phase progression was blocked by silencing of p27 Kip1 using siRNA or by infection of active RhoA. Pharmacological inhibition of ROCK accelerated the G1/S phase progression of control TM4SF5-unexpressing cells. Altogether, these observations suggest that TM4SF5 accelerates G1/S phase progression with facilitated CDK4/cyclin D1 entry into the nucleus, which might be supported by TM4SF5-mediated actin reorganization through cytosolic p27 Kip1 expression and Rho GTPase activity.

Cell adhesion to the extracellular matrix (ECM) can activate signaling via focal adhesion kinase (FAK) leading to dynamic regulation of cellular morphology. Mechanistic basis for the lack of effective intracellular signaling by non-attached epithelial cells is poorly understood. To examine whether signaling in suspended cells is regulated by Fer cytoplasmic tyrosine kinase, we investigated the effect of ectopic Fer expression on signaling in suspended or adherent hepatocytes. We found that ectopic Fer expression in Huh7 hepatocytes in suspension or on non-permissive poly-lysine caused significant phosphorylation of FAK Tyr577, Tyr861, or Tyr925, but not Tyr397 or Tyr576. Fer-mediated FAK phosphorylation in suspended cells was independent of c-Src activity or growth factor stimulation, but dependent of cortactin expression. Consistent with these results, complex formation between FAK, Fer, and cortactin was observed in suspended cells. The Fer-mediated effect correlated with multiple membrane protrusions, even on poly-lysine. Together, these observations suggest that Fer may allow a bypass of anchorage-dependency for intracellular signal transduction in hepatocytes.

O -GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues with O -GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O -GlcNAc. However, the nature and significance of the Akt1 O -GlcNAc modification is unknown. The relationship of O -GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine β-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O -GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O -GlcNAcase revealed an inverse correlation between O -GlcNAc modification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOF mass spectrometry analysis of Akt1 immunoprecipitates from glucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified with O -GlcNAc. Furthermore, in vitro O -GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O -GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O -GlcNAc modification, and the balance between these may regulate murine β-pancreatic cell fate.

Cell–cell contacts play important roles in the homeostasis of normal epithelium and in the steps of metastasis of tumor cells, although signaling mechanisms to regulate cell–cell contacts are unclear. In this study, we observed that phenotype of no cell–cell contacts in rat intestinal epithelial cell subline (RIE1-Sca) correlated with increased Erk1/2 signaling activity, compared to that of parental RIE1 cells growing in colonies. Furthermore, cell–cell contacts between RIE1-Sca cells were reformed by treatment with a specific MEK inhibitor (U0126), with translocation of ZO1 and β-catenin to cell–cell contacts, without changes of their expression levels. U0126 treatment also increased EGFR phosphorylation in a ligand-independent manner. Pretreatment with EGFR kinase inhibitor abolished U0126 treatment-mediated EGFR phosphorylation, and expression of dominant negative H-Ras N17 allowed EGFR phosphorylation and cell–cell contacts even without U0126 treatment. Furthermore, the expression of a nonphosphorylatable EGFR Y5F mutant abolished U0126-mediated cell–cell contacts. U0126 treatment also caused less efficient wound healing by keeping monolayer integrity intact, compared to control untreated cells. This U0126-mediated reduction in wound healing was further altered either by pretreatment of EGFR kinase inhibitor or expression of H-Ras N17 or EGFR Y5F. Taken together, this study supports a unique mechanism of cell–cell contact formation through MEK/Erks inhibition-mediated EGFR phosphorylation.