K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3.

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.

IL18 Receptor Signaling Regulates Tumor-Reactive CD8+ T-cell Exhaustion via Activation of the IL2/STAT5/mTOR Pathway in a Pancreatic Cancer Model.

Intratumoral cytotoxic CD8+ T cells (CTL) enter a dysfunctional state characterized by expression of coinhibitory receptors, loss of effector function, and changes in the transcriptional landscape. Even though several regulators of T-cell exhaustion have been identified, the molecular mechanisms inducing T-cell exhaustion remain unclear. Here, we show that IL18 receptor (IL18R) signaling induces CD8+ T-cell exhaustion in a murine pancreatic cancer model. Adoptive transfer of Il18r-/- OT-1 CD8+ CTLs resulted in enhanced rejection of subcutaneous tumors expressing ovalbumin (OVA) as a model antigen (PancOVA), compared with wild-type OT-1 CTLs. Transferred intratumoral IL18R-deficient CTLs expressed higher levels of effector cytokines TNF and IFNγ and had reduced expression of coinhibitory receptors (PD-1, TIM-3, 2B4, LAG-3) and the transcription factors Eomes and TOX. Lower expression of coinhibitory receptors and TOX on IL18R-deficient versus IL18R-sufficient CD8+ T cells were confirmed in an orthotopic KPC model. IL18R-induced T-cell exhaustion was regulated by IL2/STAT5 and AKT/mTOR pathways, as demonstrated in an in vitro exhaustion assay. Concordantly, mice deficient in NLRP3, the molecular complex activating IL18, had decreased expression of coinhibitory receptors on intratumoral T cells and similar changes in signaling pathways at the transcriptome level. Thus, molecular pathways promoting T-cell exhaustion indicate an involvement of an NLRP3-expressing tumor microenvironment, which mediates IL18 release. The Cancer Genome Atlas analysis of patients with pancreatic carcinoma showed an association between NLRP3-mediated IL18 signaling and shorter survival. These findings indicate NLRP3-mediated IL18R signaling as a regulator of intratumoral T-cell exhaustion and a possible target for immunotherapy. See related Spotlight by Stromnes, p. 400.

Different signaling and functionality of Rac1 and Rac1b in the progression of lung adenocarcinoma.

Rac1 is a ubiquitously expressed Rho GTPase and an important regulator of the actin cytoskeleton. Its splice variant Rac1b exhibits a 19-amino acid (aa) in-frame insertion and is predominantly active. Both proteins were described in tumorigenesis or metastasis. We investigated the contribution of Rac1 and Rac1b to tumor progression of human non-small-cell lung adenocarcinoma (NSCLA). Rac1 protein was present in 8/8 NSCLA cell lines analyzed, whereas Rac1b was expressed in only 6/8. In wound-healing assays, enhanced green fluorescence protein (EGFP)-Rac1 slightly decreased cell migration, whereas proliferation was increased in both, Rac1- and Rac1b-expressing cells. In the in vivo chorioallantoic invasion model, EGFP-Rac1-expressing cells formed more invasive tumors compared to EGFP-Rac1b. This increased invasiveness correlated with enhanced phosphorylation of p38α, AKT and glycogen synthase kinase 3ß (GSK3ß), and activation of serum response- and Smad-dependent gene promoters by Rac1. In contrast, Rac1b solely activated the mitogen-activated protein kinase (MAPK) JNK2, together with TCF/LEF1- and nuclear factor kappa B (NFκB)-responsive gene reporters. Rac1b, as Rac1, phosphorylated p38α, AKT and GSK3ß. Knockdown of the splicing factor epithelial splicing regulatory protein 1 (ESRP1), which mediates out-splicing of exon 3b from Rac1 pre-messenger RNA, resulted in increased Rac1b messenger RNA (mRNA) and suppression of the epithelial-mesenchymal transition (EMT)-associated transcription factor ZEB1. Our data demonstrate different signaling and functional activities of Rac1 and Rac1b and an important role for Rac1 in lung cancer metastasis.

Galectin-8 binds to the Farnesylated C-terminus of K-Ras4B and Modifies Ras/ERK Signaling and Migration in Pancreatic and Lung Carcinoma Cells.

K-Ras is the most prominent driver of oncogenesis and no effective K-Ras inhibitors have been established despite decades of intensive research. Identifying new K-Ras-binding proteins and their interaction domains offers the opportunity for defining new approaches in tackling oncogenic K-Ras. We have identified Galectin-8 as a novel, direct binding protein for K-Ras4B by mass spectrometry analyses and protein interaction studies. Galectin-8 is a tandem-repeat Galectin and it is widely expressed in lung and pancreatic carcinoma cells. siRNA-mediated depletion of Galectin-8 resulted in increased K-Ras4B content and ERK1/2 activity in lung and pancreatic carcinoma cells. Moreover, cell migration and cell proliferation were inhibited by the depletion of Galectin-8. The K-Ras4B-Galectin-8 interaction is indispensably associated with the farnesylation of K-Ras4B. The lysine-rich polybasic domain (PBD), a region that is unique for K-Ras4B as compared to H- and N-Ras, stabilizes the interaction and accounts for the specificity. Binding assays with the deletion mutants of Galectin-8, comprising either of the two carbohydrate recognition domains (CRD), revealed that K-Ras4B only interacts with the N-CRD, but not with the C-CRD. Structural modeling uncovers a potential binding pocket for the hydrophobic farnesyl chain of K-Ras4B and a cluster of negatively charged amino acids for interaction with the positively charged lysine residues in the N-CRD. Our results demonstrate that Galectin-8 is a new binding partner for K-Ras4B and it interacts via the N-CRD with the farnesylated PBD of K-Ras, thereby modulating the K-Ras effector pathways as well as cell proliferation and migration.

Covalent and density-controlled surface immobilization of E-cadherin for adhesion force spectroscopy.

E-cadherin is a key cell-cell adhesion molecule but the impact of receptor density and the precise contribution of individual cadherin ectodomains in promoting cell adhesion are only incompletely understood. Investigating these mechanisms would benefit from artificial adhesion substrates carrying different cadherin ectodomains at defined surface density. We therefore developed a quantitative E-cadherin surface immobilization protocol based on the SNAP-tag technique. Extracellular (EC) fragments of E-cadherin fused to the SNAP-tag were covalently bound to self-assembled monolayers (SAM) of thiols carrying benzylguanine (BG) head groups. The adhesive functionality of the different E-cadherin surfaces was then assessed using cell spreading assays and single-cell (SCSF) and single-molecule (SMSF) force spectroscopy. We demonstrate that an E-cadherin construct containing only the first and second outmost EC domain (E1-2) is not sufficient for mediating cell adhesion and yields only low single cadherin-cadherin adhesion forces. In contrast, a construct containing all five EC domains (E1-5) efficiently promotes cell spreading and generates strong single cadherin and cell adhesion forces. By varying the concentration of BG head groups within the SAM we determined a lateral distance of 5-11 nm for optimal E-cadherin functionality. Integrating the results from SCMS and SMSF experiments furthermore demonstrated that the dissolution of E-cadherin adhesion contacts involves a sequential unbinding of individual cadherin receptors rather than the sudden rupture of larger cadherin receptor clusters. Our method of covalent, oriented and density-controlled E-cadherin immobilization thus provides a novel and versatile platform to study molecular mechanisms underlying cadherin-mediated cell adhesion under defined experimental conditions.

Regulation of adherens junctions by Rho GTPases and p120-catenin.

The molecular mechanisms leading to tumor progression and acquisition of a metastatic phenotype are highly complex and only partially understood. The spatiotemporal regulation of E-cadherin-mediated adherens junctions is essential for normal epithelia function and tissue integrity. Perturbation of the E-cadherin complex assembly is a key event in epithelial-mesenchymal transition and is directed by a huge number of mechanisms that differ greatly with regard to cell types and tissues. The reduction in intercellular adhesion interferes with tissue integrity and allows cancer cells to disseminate from the primary tumor thereby initiating cancer metastasis. In the present review we will summarize the current findings about the influence of Rho GTPases on the formation and maintenance of adherens junction and will then proceed to discuss the involvement of p120-catenin on cell-cell adhesion and tumor cell migration.

Inflammation, regeneration, and transformation in the pancreas: results of the Collaborative Research Center 518 (SFB 518) at the University of Ulm.

The primary diseases of the pancreas include diabetes mellitus, acute and chronic pancreatitis, as well as pancreatic carcinoma. This review presents findings and emerging questions on the diseases of the pancreas obtained by the consortium of the Collaborative Research Center 518 (SFB 518), "Inflammation, Regeneration, and Transformation in the Pancreas" at the University of Ulm. During the last 12 years, the SFB 518 contributed considerably to the understanding of the cellular and molecular basis of pancreatic diseases and established the basis for the development of new strategies for prevention and causal therapy for diabetes, pancreatitis, and pancreatic cancer.

TGFß1 suppresses vascular smooth muscle cell motility by expression of N-cadherin.

Neointimal formation in atheromatous blood vessels is associated with both growth factor-induced differentiation of smooth muscle cells and endothelial-to-mesenchymal transition. Transforming growth factor beta (TGFß)-signaling is well known to play a critical role in the regulation of vessel remodeling as well as in atherosclerosis and restenosis. Here, we investigated the role of TGFß1 and N-cadherin on the differentiation and migration of human vascular smooth muscle cells (VSMC). TGFß1-treatment of cultured VSMC reduced their migratory activity as determined in cell migration assays. This reduced migration correlated with increased concentration of N-cadherin on mRNA and protein level. The TGFß1-induced increase of N-cadherin was sensitive against pharmacological inhibition of the ALK5 TGFß receptor and was accompanied by TGFß1-induced expression of the transcription factor snail1. Activation of N-cadherin by using a HAV-containing peptide of N-cadherin also decreased the migration of VSMC. N-cadherin-mediated suppression of VSMC migration was associated with an increased activity of RhoA, which is activated by binding of the HAV peptide to N-cadherin. Our results demonstrate that TGFß1 induces the differentiation of primary VSMC cells by Smad2/3-dependent up-regulation of the transcription factor snail1 and subsequently of N-cadherin, leading to inhibition of VSMC migration by RhoA-dependent modulation of the actin cytoskeleton.

Lef-1 isoforms regulate different target genes and reduce cellular adhesion.

The lymphoid enhancer factor 1 (Lef-1) belongs to the nuclear transducers of canonical Wnt-signalling in embryogenesis and cancer. Lef-1 acts, in cooperation with beta-catenin, as a context-dependent transcriptional activator or repressor, thereby influencing multiple cellular functions such as proliferation, differentiation and migration. Here we report that an increased Lef-1 expression in human pancreatic cancer correlates with advanced tumour stages. In pancreatic tumours, two different transcripts of Lef-1 have been detected in various stages, as demonstrated by RT-PCR analysis. One transcript was identified as the full length Lef-1 (Lef-1 FL), whereas the second, shorter transcript lacked exon VI (Lef-1 Deltaexon VI) compared to the published sequence. Comparative analysis of these two Lef-1 variants revealed that they exhibit different cellular effects after transient expression in pancreatic carcinoma cells. Forced expression of Lef-1 Deltaexon VI inhibited E-cadherin expression in a beta-catenin-independent way. Increased amounts of Lef-1 Deltaexon VI resulted in reduced cellular aggregation and increased cell migration. Expression of Lef-1 FL, but not the newly identified Lef-1 Deltaexon VI, induced the expression of the cell cycle regulating proteins c-myc and cyclin D1 in cooperation with beta-catenin and it enhanced cell proliferation. Our findings indicate that expression of alternatively spliced Lef-1 isoforms is involved in the determination of proliferative or migratory characteristics of pancreatic carcinoma cells.

Rac1 activation inhibits E-cadherin-mediated adherens junctions via binding to IQGAP1 in pancreatic carcinoma cells.

BACKGROUND: Monomeric GTPases of the Rho family control a variety of cellular functions including actin cytoskeleton organisation, cell migration and cell adhesion. Defects in these regulatory processes are involved in tumour progression and metastasis. The development of metastatic carcinoma is accompanied by deregulation of adherens junctions, which are composed of E-cadherin/beta- and alpha-catenin complexes. RESULTS: Here, we show that the activity of the monomeric GTPase Rac1 contributes to inhibition of E-cadherin-mediated cell-cell adhesion in pancreatic carcinoma cells. Stable expression of constitutively active Rac1(V12) reduced the amount of E-cadherin on protein level in PANC-1 pancreatic carcinoma cells, whereas expression of dominant negative Rac1(N17) resulted in an increased amount of E-cadherin. Extraction of proteins associated with the actin cytoskeleton as well as coimmunoprecipitation analyses demonstrated markedly decreased amounts of E-cadherin/catenin complexes in Rac1(V12)-expressing cells, but increased amounts of functional E-cadherin/catenin complexes in cells expressing Rac1(N17). Cell aggregation and migration assays revealed, that cells containing less E-cadherin due to expression of Rac1(V12), exhibited reduced cell-cell adhesion and increased cell motility. The Rac/Cdc42 effector protein IQGAP1 has been implicated in regulating cell-cell adhesion. Coimmunoprecipitation studies showed a decrease in the association between IQGAP1 and beta-catenin in Rac1(V12)-expressing PANC-1 cells and an association of IQGAP1 with Rac1(V12). Elevated association of IQGAP1 with the E-cadherin adhesion complex via beta-catenin correlated with increased intercellular adhesion of PANC-1 cells. CONCLUSION: These results indicate that active Rac1 destabilises E-cadherin-mediated cell-cell adhesion in pancreatic carcinoma cells by interacting with IQGAP1 which is associated with a disassembly of E-cadherin-mediated adherens junctions. Inhibition of Rac1 activity induced increased E-cadherin-mediated cellular adhesion.

Specific induction of migration and invasion of pancreatic carcinoma cells by RhoC, which differs from RhoA in its localisation and activity.

RhoA and RhoC are highly related Rho GTPases, but differentially control cellular behaviour. We combined molecular, cellular, and biochemical experiments to characterise differences between these highly similar GTPases. Our findings demonstrate that enhanced expression of RhoC results in a striking increase in the migration and invasion of pancreatic carcinoma cells, whereas forced expression of RhoA decreases these actions. These isoform-specific functions correlate with differences in the cellular activity of RhoA and RhoC in human cells, with RhoC being more active than RhoA in activity assays and serum-response factor-dependent gene transcription. Subcellular localisation studies revealed that RhoC is predominantly localised in the membrane-containing fraction, whereas RhoA is mainly localised in the cytoplasmic fraction. These differences are not mediated by a different interaction with RhoGDIs. In vitro GTP/GDP binding analyses demonstrate different affinity of RhoC for GTP[S] and faster intrinsic and guanine nucleotide exchange factor (GEF)-stimulated GDP/GTP exchange rates compared to RhoA. Moreover, the catalytic domains of SopE and Dbs are efficacious GEFs for RhoC. mRNA expression of RhoC is markedly enhanced in advanced pancreatic cancer stages, and thus the differences discovered between RhoA and RhoC might provide explanations for their different influences on cell migration and tumour invasion.

Overexpression of manganese superoxide dismutase in human dermal fibroblasts enhances the contraction of free floating collagen lattice: implications for ageing and hyperplastic scar formation.

Cell-matrix interactions are of significant importance for tissue homeostasis of the skin and, if disturbed, may lead to ageing and hyperplastic scar formation. We have studied fibroblasts stably overexpressing manganese superoxide dismutase (MnSOD) with a defined capacity for the removal of superoxide anions and concomitant accumulation of hydrogen peroxide to evaluate the role of enhanced MnSOD activity on the dynamics of cell-matrix interactions in the three-dimensional collagen lattice contraction assay. MnSOD overexpressing fibroblast populated collagen lattices revealed a significantly enhanced contraction compared to collagen lattices populated with vector control cells. The enhanced collagen lattice contraction was in part due to an increase in active TGF-beta1 and the accumulation of H2O2 in MnSOD overexpressing fibroblasts populated collagen lattices. Inhibition of TGF-beta1 signalling by the ALK4,5,7 kinases' inhibitor SB431542 at least partly inhibited the enhanced collagen lattice contraction of MnSOD overexpressing fibroblasts populated lattices. In addition, supplementation of vector control fibroblast populated collagen lattices with recombinant TGF-beta1 concentration dependently enhanced the collagen lattice contraction. In the presence of the antioxidant Ebselen, a mimic of H2O2 and other hydroperoxides/peroxynitrite-detoxifying glutathione peroxidase, collagen lattice contraction and the activation of TGF-beta1 were significantly reduced in collagen lattices populated with MnSOD overexpressing fibroblasts. Collectively, these data suggest that H2O2 or other hydroperoxides or peroxynitrite or a combination thereof may function as important second messengers in collagen lattice contraction and act at least in part via TGF-beta1 activation.

Microenvironmental regulation of E-cadherin-mediated adherens junctions.

The interaction between tumor cells and the microenvironment has substantial effects on tumor cell behavior by influencing cell-cell as well as cell-matrix contacts. The underlying molecular mechanisms are only partially unraveled. In this review we focus on the influence of the stromal microenvironment, especially collagen type I and type III on cellular adhesion and epithelial to mesenchymal transition (EMT). Extensive studies have emphasized that components of the microenvironment such as fibrillar collagen or growth factors like transforming growth factor beta are involved in induction of dedifferentiation of epithelial cells accompanied by disruption of the E-cadherin adhesion complex and reduced E-cadherin concentrations. On the molecular level many different proteins have been identified which are involved in the regulation of EMT, such as activation of integrins, intracellular kinases such as Src, focal adhesion kinase (FAK) or phosphatidylinositol-3 kinase (PI3-kinase) and alteration of catenin phosphorylation. The reduced cellular adhesion influences the tissue integrity and allows tumor cells to disseminate from the primary tumor representing an early step in cancer metastasis.

Polysialylated NCAM represses E-cadherin-mediated cell-cell adhesion in pancreatic tumor cells.

BACKGROUND & AIMS: Inhibition of cell-cell adhesion between epithelial cells represents an early step during tumor metastasis. Down-regulation or perturbation of E-cadherin-mediated adherens junctions is an essential requirement in this process. METHODS: The interaction between polysialylated neural cell adhesion molecule (PSA-NCAM) and the E-cadherin adhesion complex was studied by coimmunoprecipitation assays. The presence of PSA-NCAM was correlated with tumor invasion by using cell-cell aggregation and cell migration assays. The importance of polysialic acid (PSA) in the interaction of NCAM with E-cadherin and inhibition of cell-cell adhesion was confirmed by enzymatic removal of PSA from NCAM and down-regulation of PSA-transferases by siRNA. RESULTS: Expression of oncogenic K-Ras(V12) in pancreatic carcinoma cells resulted in induction of PSA-NCAM expression and reduced E-cadherin-mediated cellular adhesion. The association of PSA-NCAM with the E-cadherin adhesion complex correlated with decreased cell-cell aggregation and elevated cell migration of pancreatic carcinoma cells. Enzymatic removal of PSA from NCAM or reduction of polysialyltransferase expression led to reduced association between NCAM and E-cadherin and subsequently increased E-cadherin-mediated cell-cell aggregation and reduced cell migration. CONCLUSIONS: Our data suggest the induction of PSA-NCAM by oncogenic K-Ras as a novel molecular mechanism by which E-cadherin-mediated cellular adhesion is reduced and dissemination of tumor cells is facilitated.

Smad4-independent TGF-beta signaling in tumor cell migration.

Transforming growth factor-beta (TGF-beta) belongs to a family of multifunctional growth factors that participates in the regulation of a variety of cellular activities. Beside induction of growth inhibition and differentiation of epithelial cells, TGF-beta has been shown to promote epithelial-mesenchymal transition in most epithelial tumors. While inhibition of epithelial cell proliferation in response to TGF-beta is mainly mediated by the well-characterized Smad pathway and subsequent inhibition of gene transcription, the molecular mechanism leading to TGF-beta-induced invasiveness and metastasis of epithelial tumors is less clear. Recent results from several groups suggest that the induction of tumorigenic activity by TGF-beta includes not only signaling by Smads, but also by Rho-GTPases and mitogen-activated protein kinases (MAP kinases). Activation of the MAP kinases extracellular signal-regulated kinases (ERK) 1 and 2 as well as c-jun N-terminal kinase (JNK) has been identified as important steps in TGF-beta-induced, Smad4-independent signal transduction in epithelial cells. Recent results identify a role of activated ERK and JNK and their association with focal complexes in TGF-beta-induced, Smad4-independent cell migration of breast carcinoma cells, and are reviewed here.

Signaling pathways involved in collagen-induced disruption of the E-cadherin complex during epithelial-mesenchymal transition.

There is substantial interest in the influence of the microenvironment on tumor cells. Cell-cell as well as cell-matrix interactions have been correlated with the control of different processes such as tumor cell proliferation, differentiation, survival and migration. In this review, we focus on the influence of collagen types I and III expressed in carcinomata on the E-cadherin-mediated adhesion between epithelial tumor cells. Recently published studies described the ability of fibrillar collagen to reduce E-cadherin gene expression and to induce disruption of the E-cadherin adhesion complex. The reduced cellular adhesion influences tissue integrity and has been correlated with elevated cell migration and invasion of different carcinoma cells. Altered tyrosine phosphorylation of the intracellular, cadherin-associated catenins was identified as an important regulator of collagen-induced disassembly of the E-cadherin adhesion complex. The molecular mechanisms involved in collagen-induced cell transformation include activation of integrins, activation and translocation of the focal adhesion kinase to the E-cadherin/catenin complex as well as inhibition of the phosphatase PTEN.

Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells.

Pancreatic cancer is characterized by its invasiveness, early metastasis, and the production of large amounts of extracellular matrix (ECM). We analyzed the influence of type I collagen and fibronectin on the regulation of cellular adhesion in pancreatic cancer cell lines to characterize the role of ECM proteins in the development of pancreatic cancer. We show that collagen type I is able to initiate a disruption of the E-cadherin adhesion complex in pancreatic carcinoma cells. This is due to the increased tyrosine phosphorylation of the complex protein beta-catenin, which correlates with collagen type I-dependent activation of the focal adhesion kinase and its association with the E-cadherin complex. The activation and recruitment of focal adhesion kinase to the E-cadherin complex depends on the interaction of type I collagen with beta1-containing integrins and an integrin-mediated activation of the cellular kinase Src. The disassembly of the E-cadherin adhesion complex correlates with the nuclear translocation of beta-catenin, which leads to an increasing expression of the beta-catenin-Lef/Tcf target genes, cyclin D1 and c-myc. In addition to that, cells grown on collagen type I show enhanced cell proliferation. We show that components of the ECM, produced by the tumor, contribute to invasiveness and metastasis by reducing E-cadherin-mediated cell-cell adhesion and enhance proliferation in pancreatic tumor cells.

TGFbeta-induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN.

Transforming growth factor beta (TGFbeta) has profound growth-suppressive effects on normal epithelial cells, but supports metastasis formation in many tumour types. In most epithelial tumour cells TGFbeta(1) treatment results in epithelial dedifferentiation with reduced cell aggregation and enhanced cellular migration. Here we show that the epithelial dedifferentiation, accompanied by dissociation of the E-cadherin adhesion complex, induced by TGFbeta(1) depended on phosphatidylinositol 3-kinase (PI3-kinase) and the phosphatase PTEN as analysed in PANC-1 and Smad4-deficient BxPC-3 pancreatic carcinoma cells. TGFbeta(1) treatment enhanced tyrosine phosphorylation of alpha- and beta-catenin, which resulted in dissociation of the E-cadherin/catenin complex from the actin cytoskeleton and reduced cell-cell adhesion. The PI3-kinase and PTEN were found associated with the E-cadherin/catenin complex via beta-catenin. TGFbeta(1) treatment reduced the amount of PTEN bound to beta-catenin and markedly increased the tyrosine phosphorylation of beta-catenin. By contrast, forced expression of PTEN clearly reduced the TGFbeta(1)-induced phosphorylation of beta-catenin. The TGFbeta(1)-induced beta-catenin phosphorylation was also dependent on PI3-kinase and Ras activity. The described effects of TGFbeta(1) were independent of Smad4, which is homozygous deleted in BxPC-3 cells. Collectively, these data show that the TGFbeta(1)-induced destabilisation of E-cadherin-mediated cell-cell adhesion involves phosphorylation of beta-catenin, which is regulated by E-cadherin adhesion complex-associated PI3-kinase and PTEN.

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation.

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.

CUTL1 is a target of TGF(beta) signaling that enhances cancer cell motility and invasiveness.

CUTL1, also known as CDP, Cut, or Cux-1, is a homeodomain transcriptional regulator known to be involved in development and cell cycle progression. Here we report that CUTL1 activity is associated with increased migration and invasiveness in numerous tumor cell lines, both in vitro and in vivo. Furthermore, we identify CUTL1 as a transcriptional target of transforming growth factor beta and a mediator of its promigratory effects. CUTL1 activates a transcriptional program regulating genes involved in cell motility, invasion, and extracellular matrix composition. CUTL1 expression is significantly increased in high-grade carcinomas and is inversely correlated with survival in breast cancer. This suggests that CUTL1 plays a central role in coordinating a gene expression program associated with cell motility and tumor progression.