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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase is required for glial cell line-derived neurotrophic factor-induced migration and invasion of pancreatic carcinoma cells.

Pancreatic carcinoma cells exhibit a pronounced tendency to invade along and into intra- and extrapancreatic nerves, even at early stages of the disease. The neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) has been shown to promote pancreatic cancer cell invasion. Here, we demonstrate that pancreatic carcinoma cell lines, such as PANC-1, expressed the RET and GDNF family receptor alpha receptor components for GDNF and that primary pancreatic tumor samples, derived from carcinomas with regional lymph node metastasis, exhibited marked expression of the mRNA encoding the RET51 isoform. Moreover, GDNF was an efficacious and potent chemoattractant for pancreatic carcinoma cells as examined in in vitro and in vivo model systems. Treatment of PANC-1 cells with GDNF resulted in activation of the monomeric GTPases N-Ras, Rac1, and RhoA, in activation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) and in activation of the phosphatidylinositol 3-kinase/Akt pathway. Both inhibition of the Ras-Raf-MEK (mitogen-activated protein/ERK kinase)-ERK cascade by either stable expression of dominant-negative H-Ras(N17) or addition of the MEK1 inhibitor PD98059 as well as inhibition of the phosphatidylinositol 3-kinase pathway by LY294002 prevented GDNF-induced migration and invasion of PANC-1 cells. These results demonstrate that pancreatic tumor cell migration and possibly perineural invasion in response to GDNF is critically controlled by activation of the Ras-Raf-MEK-ERK and the phosphatidylinositol 3-kinase pathway.

The role of metastasis-associated protein 1 in prostate cancer progression.

Distinguishing aggressive prostate cancer from indolent disease represents an important clinical challenge, as current therapy requires over treating men with prostate cancer to prevent the progression of a few cases. Expression of the metastasis-associated protein 1 (MTA1) has previously been found to be associated with progression to the metastatic state in various cancers. Analyzing DNA microarray data, we found MTA1 to be selectively overexpressed in metastatic prostate cancer compared with clinically localized prostate cancer and benign prostate tissue. These results were validated by demonstrating overexpression of MTA1 in metastatic prostate cancer by immunoblot analysis. MTA1 protein expression was evaluated by immunohistochemistry in a broad spectrum of prostate tumors with tissue microarrays containing 1940 tissue cores from 300 cases. Metastatic prostate cancer demonstrated significantly higher mean MTA1 protein expression intensity (score = 3.4/4) and percentage of tissue cores staining positive for MTA1 (83%) compared with clinically localized prostate cancer (score = 2.8/4, 63% positive cores) or benign prostate tissue (score = 1.5/4, 25% positive cores) with a mean difference of 0.54 and 1.84, respectively (P < 0.00001 for both). Paradoxically, for localized disease, higher MTA1 protein expression was associated with lower rates of prostate specific antigen recurrence after radical prostatectomy for localized disease. In summary, this study identified an association of MTA1 expression and prostate cancer progression.

Claudin-4 expression decreases invasiveness and metastatic potential of pancreatic cancer.

Claudin-4 has been identified as an integral constituent of tight junctions and has been found to be highly expressed in pancreatic cancer. The aim of the present study was to elucidate the effect of claudin-4 on growth and metastatic potential in pancreatic cancer cells, as well as the regulation of claudin-4 by oncogenic pathways. Claudin-4 was stably overexpressed in SUIT-2 pancreatic cancer cells, and its effect on invasion and growth in vitro was examined by using two-chamber invasion assays, soft agar assays, and fluorescence-activated cell sorter analysis. Claudin-4 localization was characterized by light and electron microscopy, and pulmonary colonization was analyzed in vivo after injection of claudin-4 overexpressing cells into the tail vein of nude mice. Overexpression of claudin-4 was associated with significantly reduced invasive potential in vitro and inhibited colony formation in soft agar assays. In vivo, tail vein-injected claudin-4 overexpressing cells formed significantly less pulmonary metastases in comparison with mock-transfected cells. These effects were not caused by changes in proliferation, cell cycle progression, or matrix metalloproteinase gelatinolytic activity, but were paralleled by increased cell contact formation. Moreover, proinvasive transforming growth factor beta was able to down-regulate claudin-4 in PANC-1 cells. Inhibition of Ras signaling by using dominant-negative Ras and specific inhibitors of both downstream effectors mitogen-activated protein/extracellular signal-regulated kinase kinase and phosphatidylinositol 3'-kinase also decreased claudin-4 expression. Our findings identify claudin-4 as a potent inhibitor of the invasiveness and metastatic phenotype of pancreatic cancer cells, and as a target of the transforming growth factor beta and Ras/Raf/extracellular signal-regulated kinase pathways.

E- and N-cadherin differ with respect to their associated p120ctn isoforms and their ability to suppress invasive growth in pancreatic cancer cells.

E-cadherin functions as suppressor of invasion in epithelial cells and its loss is described in many invasive carcinomas. In some tumours, the disappearance of E-cadherin has been correlated with upregulation of other classical cadherins, such as N- or P-cadherin. To analyse the different cellular functions of cadherin molecules, we stably expressed E-cadherin or N-cadherin in the E- and N-cadherin-deficient pancreatic tumour cell line MIA PaCa-2. Only E-cadherin was able to induce a mesenchymal-epithelial transition and suppressed invasion of MIA PaCa-2 cells. Furthermore, only re-expression of E-cadherin resulted in an upregulation of alpha- and beta-catenin mRNAs and protein concentrations. Ectopically expressed N-cadherin failed to assemble cadherin/catenin adhesion complexes and failed to inhibit invasion. Analysis of p120(ctn), which was associated with both cadherins, demonstrated that E-cadherin was linked to a shorter isoform of p120(ctn). In contrast, N-cadherin was associated with the long, 120 kDa p120(ctn) isoforms. In addition, p120(ctn) connected with N-cadherin was phosphorylated at tyrosine residues, whereas the isoform linked to E-cadherin was not phosphorylated. Thus, the differences between E- and N-cadherin in recruiting different phosphorylated isoforms of p120(ctn) to the membrane might be responsible for the inability of N-cadherin to replace E-cadherin as suppressor of invasion in pancreatic carcinoma cells.

TGFbeta-induced fibrogenesis of the pancreas.

The biological cause of fibrosis is the accumulation of excessive amounts of extracellular matrix (ECM) which leads to tissue dysfunction and organ failure. A strong correlation can be found between pancreatic diseases and fibrotic processes, in particular chronic pancreatitis and pancreatic cancer. There is growing evidence that pancreatic fibrosis represents a dysregulation of the normal repair processes after injury. This concept is based on the findings that fibrosis and tissue repair involve similar biological reactions regulated by the same group of molecules. The best characterized example for these regulatory molecules are the members of the transforming growth factor beta family (TGFbeta). TGFbeta1 represents the prototype of this family of highly similar growth factors, with the unique ability to stimulate the expression and deposition of extracellular matrix and to inhibit its degradation. Growth factor-induced fibrotic events are targeted by a myofibroblast-like cell called pancreatic stellate cell (PSC). These cells show enhanced expression of all-important ECM proteins after TGFbeta stimulation including collagen, fibronectin and proteoglycans. At the same time TGFbeta inhibits the degradation of ECM by blocking the secretion of proteases and stimulating the production of naturally occurring protease inhibitors.