Co-expression analysis of pancreatic cancer proteome reveals biology and prognostic biomarkers.

PURPOSE: Despite extensive biological and clinical studies, including comprehensive genomic and transcriptomic profiling efforts, pancreatic ductal adenocarcinoma (PDAC) remains a devastating disease, with a poor survival and limited therapeutic options. The goal of this study was to assess co-expressed PDAC proteins and their associations with biological pathways and clinical parameters. METHODS: Correlation network analysis is emerging as a powerful approach to infer tumor biology from omics data and to prioritize candidate genes as biomarkers or drug targets. In this study, we applied a weighted gene co-expression network analysis (WGCNA) to the proteome of 20 surgically resected PDAC specimens (PXD015744) and confirmed its clinical value in 82 independent primary cases. RESULTS: Using WGCNA, we obtained twelve co-expressed clusters with a distinct biology. Notably, we found that one module enriched for metabolic processes and epithelial-mesenchymal-transition (EMT) was significantly associated with overall survival (p = 0.01) and disease-free survival (p = 0.03). The prognostic value of three proteins (SPTBN1, KHSRP and PYGL) belonging to this module was confirmed using immunohistochemistry in a cohort of 82 independent resected patients. Risk score evaluation of the prognostic signature confirmed its association with overall survival in multivariate analyses. Finally, immunofluorescence analysis confirmed co-expression of SPTBN1 and KHSRP in Hs766t PDAC cells. CONCLUSIONS: Our WGCNA analysis revealed a PDAC module enriched for metabolic and EMT-associated processes. In addition, we found that three of the proteins involved were associated with PDAC survival.

Mesoderm-independent regulation of gastrulation movements by the src tyrosine kinase in Xenopus embryo.

In vitro studies have demonstrated the involvement of Src kinases in several aspects of cell scattering, including cell dissociation and motility. We have therefore sought to explore their functions in the context of the whole organism. Loss-of-function microinjection studies indicate that the ubiquitous Src, Fyn, and Yes tyrosine kinases are specifically implicated in Xenopus gastrulation movements. Injection of mRNAs coding for dominant negative forms of the ubiquitous members of the Src family, namely Fyn, Src, and Yes, perturbs gastrulation movements, resulting in the inability to close the blastopore. Injection of mRNA coding for Csk, a natural inhibitor of Src kinase activity, produces the same phenotypic alterations. The ubiquitous Src kinases have redundant functions in gastrulation movements since overexpression of one member of the family can compensate for the inhibition of another. Interfering mutants of the Src family also inhibit activin-induced morphogenetic movements of animal cap explants isolated from injected embryos. In contrast, these mutants do not interfere with mesoderm induction, as inferred from the presence of mesoderm derivatives and from the expression of early mesodermal markers in injected embryos. In addition, Src kinase activity measured by an in vitro kinase assay is elevated in gastrulating embryos and in FGF- and activin-treated animal caps, confirming the implication of Src enzymatic activity during gastrulation. Altogether, our results demonstrate that Src kinases are essential components of the machinery that drives gastrulation movements independent of mesoderm induction and suggest that Src activity is primarily implicated in cellular movements that take place during the process of cell intercalation.

Induction and regulation of epithelial-mesenchymal transitions.

Herein we discuss the factors that bring about the transformation of epithelial cells into cells of fibroblastic phenotype. This type of transformation, referred to as epithelium-to-mesenchyme transition (EMT), allows cells to dissociate from the epithelial tissue from which they originate and to migrate freely. EMT is therefore thought to play a fundamental role during the early steps of invasion and metastasis of carcinoma cells. Among biological agents which have been identified as inducers of EMT are a number of cytokines and extracellular matrix macromolecules. The coordinated changes in cell morphology, associated with the induction of cell motility and the disruption of intercellular junctions, are the consequence of a signaling cascade emanating from the plasma membrane and leading to changes in gene expression. Understanding the mechanisms regulating EMT of normal and transformed epithelial cells may offer new perspectives for designing therapies for the treatment of metastatic cancers of epithelial origin.

Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells.

Identification of signaling molecules that regulate cell migration is important for understanding fundamental processes in development and the origin of various pathological conditions. The migration of Nara Bladder Tumor II (NBT-II) cells was used to determine which signaling molecules are specifically involved in the collagen-mediated locomotion. We show here that paxillin is tyrosine phosphorylated after induction of motility on collagen. Overexpression of paxillin mutants in which tyrosine 31 and/or tyrosine 118 were replaced by phenylalanine effectively impaired cell motility. Moreover, stimulation of motility by collagen preferentially enhanced the association of paxillin with the SH2 domain of the adaptor protein CrkII. Mutations in both tyrosine 31 and 118 diminished the phosphotyrosine content of paxillin and prevented the formation of the paxillin-Crk complex, suggesting that this association is necessary for collagen-mediated NBT-II cell migration. Other responses to collagen, such as cell adhesion and spreading, were not affected by these mutations. Overexpression of wild-type paxillin or Crk could bypass the migration-deficient phenotype. Both the SH2 and the SH3 domains of CrkII are shown to play a critical role in this collagen-mediated migration. These results demonstrate the important role of the paxillin-Crk complex in the collagen-induced cell motility.

Characterization of the signaling pathways regulating alpha2beta1 integrin-mediated events by a pharmacological approach.

In certain instances of developing and adult organism, epithelial cells can change morphology and transform into mesenchymal-like type in order to move through the extracellular matrix. However, because of the multiplicity and complexity of signaling pathways that contribute to these processes, their molecular dissection has remained difficult. By using a pharmacological approach on the rat bladder carcinoma cell line NBT-II dispersion system, we have identified distinct signaling events for adhesion and motility in response to collagen, both activities depending on alpha2beta1 integrin. Treatment of cells with PKC inhibitors markedly impaired initial attachment on collagen without affecting the capacity of cells to move, suggesting that PKC activity is required for initial adhesion strength during cell translocation. Both adhesion and motility were diminished by tyrosine kinase inhibitors herbimycin and tyrphostin whereas tyrosine phosphatase inhibitors amplified cell scattering. The collagen-induced dispersion was insensitive to genistein which we previously showed to abrogate growth factor-induced scattering, thus demonstrating inducer specificity. Finally. Ras inhibitors and expression of a dominant negative form of Ras (N17Ras) while affecting initial cell attachment, did not prevent cell migration, and instead favored the dissociated state on collagen. The specific signaling pathways identified for adhesion and motility should help to understand the sequential processes associated with cell migration.

Alpha 2 beta 1 integrin is required for the collagen and FGF-1 induced cell dispersion in a rat bladder carcinoma cell line.

We have investigated the role of integrins in the epithelial to mesenchymal transition (EMT) induced by either collagen or fibroblast growth factor-1 (FGF-1) in the rat bladder carcinoma cell line NBT-II. The major collagen-binding receptor is the alpha 2 beta 1 integrin. An increase in expression of alpha 2 beta 1 integrin coincided with EMT induced by either collagen or FGF-1. When both inducers were present, a further increase in alpha 2 expression was observed which correlated with an enhancement in the speed of locomotion. Overexpression of human alpha 2 in NBT-II cells did not trigger EMT but rendered cells more sensitive to the dispersing effect of collagen and FGF-1. Anti-human alpha 2 blocking antibodies affected cell scattering and motility induced by either collagen or FGF-1. These data demonstrate that alpha 2 beta 1 integrin is the mediator of the cell scattering effect induced by collagen. They also indicate that a functional alpha 2 integrin is essential for the motile behavior of NBT-II cells during the FGF-1 induced EMT.

Model systems of epithelium-mesenchyme transitions.

This contribution discusses which factors bring about the transformation of epithelium to mesenchyme. Amongst biological agents which have this role are a number of cytokines (e.g. EGF, FGF-1, TGF-beta, HGF/SF) and extracellular matrix macromolecules, such as collagens. The coordinated changes in cell morphology, associated with the induction of cell motility and the loss of inter-cellular junctions, are under the control of signaling molecules that transduce the signal emanating from the plasma membrane, which ultimately lead to changes in gene expression.

pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line.

The NBT-II rat carcinoma cell line exhibits two mutually exclusive responses to FGF-1 and EGF, entering mitosis at cell confluency while undergoing an epithelium-to-mesenchyme transition (EMT) when cultured at subconfluency. EMT is characterized by acquisition of cell motility, modifications of cell morphology, and cell dissociation correlating with the loss of desmosomes from cellular cortex. The pleiotropic effects of EGF and FGF-1 on NBT-II cells suggest that multiple signaling pathways may be activated. We demonstrate here that growth factor activation is linked to at least two intracellular signaling pathways. One pathway leading to EMT involves an early and sustained stimulation of pp60c-src kinase activity, which is not observed during the growth factor-induced entry into the cell cycle. Overexpression of normal c-src causes a subpopulation of cells to undergo spontaneous EMT and sensitizes the rest of the population to the scattering activity of EGF and FGF-1 without affecting their mitogenic responsiveness. Addition of cholera toxin, a cAMP-elevating agent, severely perturbs growth factor induction of EMT without altering pp60c-src activation, therefore demonstrating that cAMP blockade takes place downstream or independently of pp60c-src. On the other hand, overexpression of a mutated, constitutively activated form of pp60c-src does not block cell dispersion while strongly inhibiting growth factor-induced entry into cell division. Moreover, stable transfection of a dominant negative mutant of c-src inhibits the scattering response without affecting mitogenesis induced by the growth factors. Altogether, these results suggest a role for pp60c-src in epithelial cell scattering and indicate that pp60c-src might contribute unequally to the two separate biological activities engendered by a single signal.

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.

Implication of scatter/growth factors in tumor progression.

Several steps during cancer progression have been distinguished on the basis of anatomo-pathological observations and experimental data. The first step, which consists of the detachment of the cancer cells from the primary tumor prior to their migration, has received much attention. Several lines of evidence have indicated that inducer molecules of tumor cell dispersion are scatter factors which are similar or identical to some growth factors. Our studies have focused on the dispersing effect of growth factors, such as acidic FGF (aFGF) on a rat bladder carcinoma cell line. These studies demonstrated that specific extracellular matrix components might contribute to the scattering effect of soluble growth factors. Additionally, our results indicated that the dispersing action of aFGF is counterbalanced by its mitogenic effect, since these two functions of aFGF cannot be observed simultaneously for the same cell. Depending on its location in the cell collective, a given cell chooses to enter mitosis or to scatter in response to aFGF. The choice between the two responses is apparently driven by molecules belonging to the transducing pathways of aFGF signaling. Finally, our data indicated that aFGF-induced tumor cell scattering leads to increased in vitro invasiveness and in vivo metastasis. Interestingly, the presence of few aFGF-producing tumor cells in a population of non-producing cells dramatically enhances the growth rate and the metastatic properties of the whole tumor, suggesting that a low proportion of highly metastatic cells in a heterogeneous cell population might modify the behavior of the tumor mass.

Involvement of cell motility in tumor progression.

Since one crucial step in tumor progression consists of the acquisition of invasive and metastatic properties, it is important to analyze the mechanisms used by cancer cells to disperse. Among the possible mechanisms of cell dispersion, cell motility appears as a central phenomenon that still needs to be understood at the molecular level. Our experimental approach to the contribution of cell motility in carcinoma cell dissemination is based on the study of the NBT-II rat bladder carcinoma cell line. The epithelial cell line gives rise to isolated, actively migrating, fibroblast-like cells in response to specific stimuli (collagens and acidic fibroblast growth factor [aFGF]). Analysis of the scattering response indicates that the different stimuli can synergize, leading to increased motility and invasiveness. NBT-II cells have two types of response to aFGF: they can either proliferate or scatter. In addition, the two responses are mutually exclusive, suggesting that the cell status can dictate whether or not tumor cells will disperse after exposure to a scatter factor. Finally, recent studies on the involvement of epithelial-specific cadherins in the process of aFGF-induced cell scattering indicate that a sustained expression of E-cadherin is not sufficient to protect cells from dispersing. In conclusion, our experimental model offers the opportunity to dissect the molecular events leading to tumor cell dissemination.

Epithelial cell plasticity in neoplasia.

Tumor metastasis is associated with an increase in the plasticity of malignant cells, a phenomenon that is characterized by changes in cell morphology and a decrease in intercellular cohesiveness. The plasticity of cells is correlated with their motility. Therefore, factors that enhance plasticity promote the migration of malignant cells from a primary tumor. Several cytokines that induce the dissociation and dispersal of malignant cells have now been described. By inhibiting the activity of motogenic cytokines, it may be possible to design effective strategies for the treatment of patients with metastatic cancer.

Combined effects of extracellular matrix and growth factors on NBT-II rat bladder carcinoma cell dispersion.

Using the rat bladder carcinoma cell line NBT-II we showed that collagens but not laminin and fibronectin were able to induce cell scattering. Acidic fibroblast growth factor and transforming growth factor alpha also promoted NBT-II cell dispersion on glass or tissue culture plastic. We have now further analysed the scatter response to these two growth factors in the presence of extracellular matrix molecules. In the presence of growth factors, no peripheral single-cell dispersion occurred on fibronectin and laminin, although time-lapse video analyses revealed intense cell mingling and motility inside the monolayer forming around NBT-II aggregates. Patterns of strings or files of cells protruding from the monolayer were often observed. The presence of a scattering activity in the complex acellular extracellular matrix deposited by NBT-II cells themselves strongly suggested that substratum conditioning was responsible for this effect. On the other hand, the two growth factors accelerated collagen-mediated NBT-II individual cell dispersion and locomotion in a reversible way. As a marker of cell dissociation, we studied desmosome distribution in aggregate cultures: desmosomes were present in aggregates formed in suspension even in the presence of growth factors, whereas internalization occurred after cell-to-substratum contact. On laminin or fibronectin and in the presence of growth factors, peripheral cells inside the halo of NBT-II aggregates did not exhibit desmosome linkages. These observations suggest that scatter effects per se are dependent on the composition of the extracellular matrix. In particular, on a substratum nonpermissive for direct cell translocation, individual cell dispersion can be replaced by en bloc patterns of migration following substratum conditioning by the cells.

Amplification of invasiveness in organotypic cultures after NBT-II rat bladder carcinoma stimulation with in vitro scattering factors.

Acidic fibroblast growth factor (aFGF) or transforming growth factor-alpha (TGF-alpha), in addition to being mitogenic, induce individual scattering of NBT-II rat bladder carcinoma cell clusters on tissue culture dishes, suggesting that they may contribute to tumor cell dissemination. To assay their scattering potential and their effect on cell invasiveness in a more complex and physiologically relevant model, we analyzed the behavior of NBT-II spheroids confronted with urinary bladder in organotypic cultures. NBT-II spheroids progressively replaced the urothelium at the site of contact with the bladder explant. In the absence of aFGF or TGF-alpha, inserted cells grew in a pattern suggestive of local hyperplasia, with occasional invasive cell protrusions. Exogenous scattering growth factors elicited a more rapid appearance of these protrusions, which were also more numerous. NBT-II cells transfected with cDNA constructs bearing the gene of aFGF, TGF-alpha or the oncogene hst/KFGF were also used. After exogenous or autocrine stimulation of NBT-II cells with the growth factors, a deeper penetration of the bladder wall in the form of nodular outgrowths and clusters of infiltrating cells was always observed. Altogether these observations suggest that the stimulation of NBT-II clusters by scattering/growth factors can promote cell shedding and amplify invasiveness in the complex extracellular environment of bladder tissues.

[Modulation of epithelial differentiation of cultured rat bladder carcinoma]. / Modulation de la différenciation épithéliale d'un carcinome vésical de rat en culture.

Epithelium-to-mesenchyme transformation plays a key role in tissue remodelling in embryos since it allows cells from the primitive epithelia to migrate to other sites where they participate in the formation of new structures. A similar phenomenon may be involved in the detachment of malignant cells from neighboring primary tumor cells, which is a prerequisite to the invasion of neighboring tissues or the development of metastases. To test this hypothesis, an in vitro model using a rat bladder carcinoma cell line was developed. Cells exhibited epithelial features under standard culture conditions. After exposure to a soluble inducer (acidic FGF) or the specific extracellular matrix components (collagens), the cells acquired a fibroblastic phenotype, separated from one another, and started to move freely on the substrate. Inducers were found to act synergistically on the fibroblastic transformation of carcinoma cells and to promote the penetration of these cells into collagen gels.

Alternative patterns of mitogenesis and cell scattering induced by acidic FGF as a function of cell density in a rat bladder carcinoma cell line.

The dual function exerted by acidic fibroblast growth factor (aFGF) in a rat bladder carcinoma cell line has been explored under two different conditions of culture density. At low cell density, aFGF promotes the epithelium-to-mesenchyme transition of NBT-II cells characterized by cell dissociation, morphological changes toward a fibroblastic-like phenotype, and acquisition of cell motility. Under these conditions, NBT-II cells are unresponsive to the growth-promoting effect of aFGF. At high cell density, aFGF is a potent mitogenic factor, but its scattering activity is essentially abrogated. Slight modifications in the binding of aFGF to its specific receptors were observed at high cell density; these changes correlated with a downregulation of receptors with no apparent change in their molecular form. NBT-II cells located at the edge of artificial wounds mimicked the behavior of subconfluent cells, because they did not proliferate upon aFGF treatment. Furthermore, in large-sized NBT-II colonies, peripheral cells were the first to dissociate in response to aFGF. Altogether, our results suggest that the cellular response to multifunctional growth factors might depend on the localization within the responding cell population.