The RNA-binding protein Rbfox2: an essential regulator of EMT-driven alternative splicing and a mediator of cellular invasion.

The epithelial-mesenchymal transition (EMT), a prerequisite for cancer progression and metastasis formation, is regulated not only at the transcriptional but also at the post-transcriptional level, including at the level of alternative pre-mRNA splicing. Several recent studies have highlighted the involvement of splicing factors, including epithelial splicing regulatory proteins (Esrps) and RNA-binding Fox protein 2 (Rbfox2), in this process. Esrps regulate epithelial-specific splicing, and their expression is downregulated during EMT. By contrast, the role of Rbfox2 is controversial because Rbfox2 regulates epithelial as well as mesenchymal splicing events. Here, we have used several established cell culture models to investigate the functions of Rbfox2 during EMT. We demonstrate that induction of an EMT upregulates the expression of Rbfox2, which correlates with an increase in Rbfox2-regulated splicing events in the cortactin (Cttn), Pard3 and dynamin 2 (Dnm2) transcripts. At the same time, however, the epithelial-specific ability to splice the Enah, Slk and Tsc2 transcripts is either reduced or lost completely by Rbfox2, which might be due, in part, to downregulation of the expression of the Esrps cooperative factors. Depletion of Rbfox2 during EMT did not prevent the activation of transforming growth factor-ß signaling, the upregulation of mesenchymal markers or changes in cell morphology toward a mesenchymal phenotype. In addition, this depletion did not influence cell migration. However, depletion of Rbfox2 in cells that have completed an EMT significantly reduced their invasive potential. Taken together, our results suggest that during an EMT, Rbfox2-regulated splicing shifts from epithelial-to mesenchymal-specific events, leading to a higher degree of tissue invasiveness.

Hepatocyte growth factor protects hepatoblastoma cells from chemotherapy-induced apoptosis by AKT activation.

Hepatocyte growth factor/scatter factor (HGF) is a ubiquitously expressed molecule that elicits pleiotropic functions on epithelial cells, including mitogenic, motogenic, differentiating, angiogenic and morphogenic effects. In hepatoblastoma (HB), post-operative residual tumor growth and tumor recurrences are often associated with markedly elevated serum levels of HGF, suggesting a link between this molecule and tumor malignancy. Here, we demonstrate that HGF has no impact on overall cell viability and proliferation of HB cells, although signal transduction occurs downstream of HGF, such as c-Met phosphorylation, activation of phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/ERK-1/2 signaling. Instead of being mitogenic, HGF confers anti-apoptotic properties upon serum starvation and moreover protects HB cells against strong apoptotic inducers such as cisplatin and camptothecin, thereby contributing to chemotherapeutic resistance. This effect is mainly dependent on the PI3K/AKT signaling pathway, since inhibition by wortmannin resulted in abrogation of HGF-mediated survival, whereas inhibition of the MAPK pathway had no effect. Together, these findings highlight the importance of HGF in tumor cell survival and suggest that HGF and its cognate receptor c-Met should be considered as a candidate for combined therapeutic strategies of advanced pediatric liver tumors.

B-Raf is required for ERK activation and tumor progression in a mouse model of pancreatic beta-cell carcinogenesis.

Activation of the Raf/MEK/ERK pathway, often by gain-of-function mutations of RAS or RAF, is observed in many human cancers. The extracellular signal-regulated kinase (ERK) pathway is required for the proliferation of cancer cell lines harboring activating BRAF or, to a lesser extent, activating RAS mutations. It is still unclear, however, whether the pathway is required in vivo for tumor development, particularly in tumors in which B-Raf is not mutationally activated. During embryonic development, B-Raf is essential for angiogenesis in the placenta. To address the question of whether B-Raf contributed to tumor angiogenesis in vivo we conditionally ablated B-Raf in a model of pancreatic islet carcinoma driven by the functional inactivation of tumor suppressors (RIP1Tag2), which critically depends on angiogenesis for growth. We find that B-Raf is dispensable for the proliferation of tumor cells in culture, but necessary for ERK activation and for the expression of angiogenic factors by tumor cells in vivo and in vitro. In vivo, these defects result in the formation of hollow tumors with decreased vessel density and strongly reduced proliferation. The progression from adenoma to carcinoma is also significantly impaired. Thus, endogenous B-Raf contributes to the development of RIP1Tag2 tumors by supporting the stromal response and tumor progression.

Tumor progression induced by the loss of E-cadherin independent of beta-catenin/Tcf-mediated Wnt signaling.

E-cadherin-mediated cell-cell adhesion is frequently lost during the development of malignant epithelial cancers. Employing a transgenic mouse model of beta-cell carcinogenesis (Rip1Tag2) we have previously shown that the loss of E-cadherin is a rate-limiting step in the progression from adenoma to carcinoma. However, the mere loss of cell adhesion may not be sufficient and additional signals are required to cause tumor cells to permeate the basal membrane and to invade surrounding tissue. Besides being an important component of the E-cadherin cell-adhesion complex, beta-catenin plays a critical role in canonical Wnt signaling. We report here that beta-catenin-mediated Wnt signaling does not contribute to tumor progression in Rip1Tag2 mice. E-cadherin downregulates beta-catenin/Tcf-mediated transcriptional activity by sequestrating beta-catenin into E-cadherin cell-adhesion complexes even in the presence of activated Wnt signaling. Upon loss of E-cadherin expression, beta-catenin is degraded and Tcf/beta-catenin-mediated transcriptional activity is not induced. Moreover, forced expression of constitutive-active beta-catenin or genetic ablation of Tcf/beta-catenin transcriptional activity in tumor cells of Rip1Tag2 transgenic mice does not affect tumor progression. Together, the data indicate that signals other than beta-catenin/Tcf-mediated Wnt signaling are induced by the loss of E-cadherin during tumor progression in Rip1Tag2 transgenic mice.

The potential role of podoplanin in tumour invasion.

Podoplanin is a small mucin-like transmembrane protein, widely expressed in various specialised cell types throughout the body. Here, we revisit the mechanism of podoplanin-mediated tumour invasion. We compare molecular pathways leading to single and collective cell invasion and discuss novel distinct concepts of tumour cell invasion.

Conditional and specific NF-kappaB blockade protects pancreatic beta cells from diabetogenic agents.

Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta cells. Islet-infiltrating leukocytes secrete cytokines such as IL-1beta and IFN-gamma, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-kappaB is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-kappaB in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the DeltaNIkappaBalpha protein were resistant to the deleterious effects of IL-1beta and IFN-gamma, as assessed by reduced NO production and beta-cell apoptosis. This effect was even more striking in vivo, where nearly complete protection against multiple low-dose streptozocin-induced diabetes was observed, with reduced intraislet lymphocytic infiltration. Our results show in vivo that beta cell-specific activation of NF-kappaB is a key event in the progressive loss of beta cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.

Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment.

The fatality of cancer predominantly results from the dissemination of primary tumor cells to distant sites and the subsequent formation of metastases. During tumor progression, some of the primary tumor cells as well as the tumor microenvironment undergo characteristic molecular changes, which are essential for the metastatic dissemination of tumor cells. In this review, we will discuss recent insights into pro-metastatic events occurring in tumor cells themselves and in the tumor stroma. Tumor cell-intrinsic alterations include the loss of cell polarity and alterations in cell-cell and cell-matrix adhesion as well as deregulated receptor kinase signaling, which together support detachment, migration and invasion of tumor cells. On the other hand, the tumor stroma, including endothelial cells, fibroblasts and cells of the immune system, is engaged in an active molecular crosstalk within the tumor microenvironment. Subsequent activation of blood vessel and lymph vessel angiogenesis together with inflammatory and immune-suppressive responses further promotes cancer cell migration and invasion, as well as initiation of the metastatic process.

Correction of hyperglycemia in diabetic mice transplanted with reversibly immortalized pancreatic beta cells controlled by the tet-on regulatory system.

Pancreatic beta cell lines may offer an abundant source of cells for beta-cell replacement in type I diabetes. Using regulatory elements of the bacterial tetracycline (tet) operon for conditional expression of SV40 T antigen oncoprotein in transgenic mouse beta cells, we have shown that reversible immortalization is an efficient approach for regulated beta-cell expansion, accompanied by enhanced cell differentiation upon growth arrest. The original system employed the tet-off approach, in which the cells proliferate in the absence of tet ligands and undergo growth arrest in their presence. The disadvantage of this system is the need for continuous treatment with the ligand in vivo for maintaining growth arrest. Here we utilized the tet-on regulatory system to generate beta cell lines in which proliferation is regulated in reverse: these cells divide in the presence of tet ligands, and undergo growth arrest in their absence, as judged by [3H]thymidine and BrdU incorporation assays. These cell lines were derived from insulinomas, which heritably developed in transgenic mice continuously treated with the tet derivative doxycycline (dox). The cells produce and secrete high amounts of insulin, and can restore and maintain euglycemia in syngeneic streptozotocin-induced diabetic mice in the absence of dox. Such a system is more suitable for transplantation, compared with cells regulated by the tet-off approach, because ligand treatment is limited to cell expansion in culture and is not required for long-term maintenance of growth arrest in vivo.

Keratinocyte-specific onset of serine protease BSSP expression in experimental carcinogenesis.

Malignant transformation of mouse skin by chemical carcinogens and tumor promoters, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is a multistage process leading to the formation of squamous cell carcinomas. In an effort to identify target genes whose expression is associated with skin tumorigenesis we combined elements of suppression subtractive hybridization with differential screening to isolate genes that are differentially upregulated in mouse skin after short-term treatment with 12-O-tetradecanoylphorbol-13-acetate and that exhibit a high constitutive expression in squamous cell carcinomas. Here, we report the detailed analysis of one of these cDNAs encoding the serine protease BSSP in mouse skin. Phorbol ester application increases BSSP expression in keratinocytes of the epidermis and the hair follicle several-fold starting 4 h post- treatment. Transcriptional activation of BSSP by 12-O-tetradecanoylphorbol-13-acetate was found to be independent of c-Fos expression and resistant to downregulation by glucocorticoids. By monitoring BSSP expression throughout experimental skin carcinogenesis we found strong constitutive expression in hyperplastic epidermis as well as in proliferatively active keratinocytes of benign and malignant skin tumors. These results establish a novel link between expression of an as yet ill-defined serine protease and skin carcinogenesis.

N-CAM modulates tumour-cell adhesion to matrix by inducing FGF-receptor signalling.

Loss of expression of neural cell-adhesion molecule (N-CAM) is implicated in the progression of tumour metastasis. Here we show that N-CAM modulates neurite outgrowth and matrix adhesion of beta-cells from pancreatic tumours by assembling a fibroblast-growth-factor receptor-4 (FGFR-4) signalling complex, which consists of N-cadherin, FGFR-4, phospholipase C gamma (PLC-gamma), the adaptor protein FRS2, pp60(c-src), cortactin and growth-associated protein-43 (GAP-43). Dominant-negative FGFR-4, inhibitors of FGFR signalling and anti-beta(1)-integrin antibodies repress matrix adhesion induced by N-CAM. FGF ligands can replace N-CAM in promoting matrix adhesion but not neurite outgrowth. The results indicate that N-CAM stimulates beta1-integrin-mediated cell-matrix adhesion by activating FGFR signalling. This is a potential mechanism for preventing the dissemination of metastatic tumour cells.

Mammalian sprouty-1 and -2 are membrane-anchored phosphoprotein inhibitors of growth factor signaling in endothelial cells.

Growth factor-induced signaling by receptor tyrosine kinases (RTKs) plays a central role in embryonic development and in pathogenesis and, hence, is tightly controlled by several regulatory proteins. Recently, Sprouty, an inhibitor of Drosophila development-associated RTK signaling, has been discovered. Subsequently, four mammalian Sprouty homologues (Spry-1-4) have been identified. Here, we report the functional characterization of two of them, Spry-1 and -2, in endothelial cells. Overexpressed Spry-1 and -2 inhibit fibroblast growth factor- and vascular endothelial growth factor-induced proliferation and differentiation by repressing pathways leading to p42/44 mitogen-activating protein (MAP) kinase activation. In contrast, although epidermal growth factor-induced proliferation of endothelial cells was also inhibited by Spry-1 and -2, activation of p42/44 MAP kinase was not affected. Biochemical and immunofluorescence analysis of endogenous and overexpressed Spry-1 and -2 reveal that both Spry-1 and -2 are anchored to membranes by palmitoylation and associate with caveolin-1 in perinuclear and vesicular structures. They are phosphorylated on serine residues and, upon growth factor stimulation, a subset is recruited to the leading edge of the plasma membrane. The data indicate that mammalian Spry-1 and -2 are membrane-anchored proteins that negatively regulate angiogenesis-associated RTK signaling, possibly in a RTK-specific fashion.

Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.

Metastasis is a frequent and lethal complication of cancer. Vascular endothelial growth factor-C (VEGF-C) is a recently described lymphangiogenic factor. Increased expression of VEGF-C in primary tumours correlates with dissemination of tumour cells to regional lymph nodes. However, a direct role for VEGF-C in tumour lymphangiogenesis and subsequent metastasis has yet to be demonstrated. Here we report the establishment of transgenic mice in which VEGF-C expression, driven by the rat insulin promoter (Rip), is targeted to beta-cells of the endocrine pancreas. In contrast to wild-type mice, which lack peri-insular lymphatics, RipVEGF-C transgenics develop an extensive network of lymphatics around the islets of Langerhans. These mice were crossed with Rip1Tag2 mice, which develop pancreatic beta-cell tumours that are neither lymphangiogenic nor metastatic. Double-transgenic mice formed tumours surrounded by well developed lymphatics, which frequently contained tumour cell masses of beta-cell origin. These mice frequently developed pancreatic lymph node metastases. Our findings demonstrate that VEGF-C-induced lymphangiogenesis mediates tumour cell dissemination and the formation of lymph node metastases.

Cell adhesion in tumor invasion and metastasis: loss of the glue is not enough.

Tumor cells often show a decrease in cell-cell and/or cell-matrix adhesion. An increasing body of evidence indicates that this reduction in cell adhesion correlates with tumor invasion and metastasis. Two main groups of adhesion molecules, cadherins and CAMs, have been implicated in tumor malignancy. However, the specific role that these proteins play in the context of tumor progression remains to be elucidated. In this review, we discuss recent data pointing to a causal relationship between the loss of cell adhesion molecules and tumor progression. In addition, the direct involvement of these molecules in specific signal transduction pathways will be considered, with particular emphasis on the alterations of such pathways in transformed cells. Finally, we review recent observations on the molecular mechanisms underlying metastatic dissemination. In many cases, spreading of tumor cells from the primary site to distant organs has been characterized as an active process involving the loss of cell-cell adhesion and gain of invasive properties. On the other hand, various examples of metastases exhibiting a relatively benign (i.e. not invasive) phenotype have been reported. Together with our recent results on a mouse tumor model, these findings indicate that 'passive' metastatic dissemination can occur, in particular as a consequence of impaired cell-matrix adhesion and of tumor tissue disaggregation.

Recent advances in research on multistage tumorigenesis.

Tumour development is a multi-step process during which genetic and epigenetic events determine the transition from a normal to a malignant cellular state. In the past decade, extensive effort has been made not only to define the molecular mechanisms underlying progression to malignancy but also to predict the development of the disease and to identify possible molecular targets for therapy. Common to most tumours, several regulatory circuits are altered during multistage tumour progression, most importantly, the control of proliferation, the balance between cell survival and programmed cell death (apoptosis), the communication with neighbouring cells and the extracellular matrix, the induction of tumour neovascularization (angiogenesis) and, finally, tumour cell migration, invasion and metastatic dissemination. De-regulation of each of these processes represents a rate-limiting step for tumour development and, hence, has to be achieved by tumour cells in a highly selective manner during tumour progression. In this review we summarize recent advances in cancer research that have provided new insights in the molecular mechanisms underlying the transition between one tumour stage and the next and into their concerted action during tumour progression. Cultured human tumour cell lines as well as transgenic and knock-out mouse models of tumorigenesis have been instrumental in these experimental approaches.

Fibroblast growth factors are required for efficient tumor angiogenesis.

Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF receptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and inhibited tumor angiogenesis in an ex vivo bioassay, in which endothelial cells were cocultured with angiogenic tumor biopsies in a collagen gel. Moreover, AdsFGFR repressed tumor angiogenesis and hence tumor growth in vivo in allograft transplantation experiments. Whereas adenoviral expression of a soluble form of VEGF receptor 1 (AdsFlt) predominantly affected the initiation of tumor angiogenesis, soluble FGF receptor (sFGFR) appeared to impair the maintenance of tumor angiogenesis. The combination of sFGFR and soluble Flt exhibited a synergistic effect in the repression of tumor growth. Finally, i.v. injection of AdsFGFR resulted in a dramatic repression of tumor growth in a transgenic mouse model of pancreatic beta cell carcinogenesis. Similar to control infections with AdsFlt, tumor-associated vessel density was decreased, indicating that the expression of sFGFR impaired tumor angiogenesis. These data indicate that FGFs play a critical role in tumor angiogenesis.

Molecular mechanisms of tumor angiogenesis and tumor progression.

The formation of new blood vessels (angiogenesis) is crucial for the growth and persistence of primary solid tumors and their metastases. Furthermore, angiogenesis is also required for metastatic dissemination, since an increase in vascular density will allow easier access of tumor cells to the circulation. Induction of angiogenesis precedes the formation of malignant tumors, and increased vascularization seems to correlate with the invasive properties of tumors and thus with the malignant tumor phenotype. In the last few years, the discovery and characterization of tumor-derived angiogenesis modulators greatly contributed to our understanding of how tumors regulate angiogenesis. However, although angiogenesis appears to be a rate-limiting event in tumor growth and metastatic dissemination, a direct connection between the induction of angiogenesis and the progression to tumor malignancy is less well understood. In this review, we discuss the most recent observations concerning the modulation of angiogenesis and their implications in tumor progression, as well as their potential impact on cancer therapy.

An insulin-like growth factor-mediated, phosphatidylinositol 3' kinase-independent survival signaling pathway in beta tumor cells.

Hyperproliferation of tumor cells usually coincides with increased tumor cell apoptosis. To overcome apoptosis, tumor cells frequently induce the expression of growth factors that mediate cell survival. In nontransformed cells, including fibroblasts and neurons, survival factor-mediated signal transduction involves the activation of phosphatidylinositol 3' kinase (PI-3K) and protein kinase B/c-Akt (PKB). Here we demonstrate that tumor cell lines derived from a transgenic mouse model of pancreatic beta cell carcinogenesis use insulin-like growth factors to repress apoptosis independently of PI-3K and PKB. The results indicate that tumor cells can use additional survival signal transduction pathways.

Formation of transformed endothelial cells in the absence of VEGFR-2/Flk-1 by Polyoma middle T oncogene.

The middle T antigen of murine Polyomavirus (PymT) rapidly transforms endothelial cells leading to vascular malformations reminiscent of endothelial tumors or hemangiomas. Flk-1, a receptor tyrosine kinase which is activated upon binding of its ligand VEGF, is predominantly expressed in endothelial cells and essential for the formation of blood vessels since absence of Flk-1 prevents the development of mature endothelial cells in mice and in ES-cell differentiation experiments. To investigate the role of Flk-1 in PymT-induced vascular tumor formation, we studied the expression of Flk-1 and VEGF in PymT-transformed endothelial cells (Endothelioma cells, END. cells). The receptor and its ligand were both expressed in END. cells suggesting that a VEGF/Flk-1 autocrine loop might be causally involved in the formation of vascular tumors. To test this hypothesis, ES cells lacking Flk-1 were generated and the transforming potential of PymT was analysed after in vitro differentiation. Flk-1(-/-) END. cell lines were established which are morphologically identical to flk-1(+/+) END. cells and which express several markers characteristic for endothelial cells. This result suggests that PymT functionally replaces the requirement of Flk-1 in expansion and/or survival of endothelial progenitor cells. Therefore, flk-1(-/-) END. cells provide a powerful tool to dissect the downstream signaling pathways of Flk-1.

An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis.

Simian virus 40 large T antigen transforms cells by sequestration and inactivation of the tumor suppressor proteins p53, retinoblastoma gene product (pRb), and the pRb-related proteins p107 and p130. Thus, the absence of functional p53 is expected to promote T antigen-mediated tumorigenesis. However, in a transgenic mouse model of T antigen-mediated beta cell carcinogenesis (Rip1Tag2), tumor volumes are significantly diminished when these mice are intercrossed with p53-deficient mice. Whereas the incidence of beta tumor cell apoptosis is unaffected, their proliferation rate is reduced in p53-deficient beta cell tumors in vivo and in cell lines established from these tumors in vitro. Biochemical analyses reveal higher levels of T antigen in wild-type tumor cells as compared to p53-deficient tumor cells. The data indicate that p53 stabilizes SV40 large T antigen, thereby augmenting its oncogenic potential as manifested by increased proliferation rates in wild-type beta tumor cells as compared to p53-deficient beta tumor cells.

The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene.

E-cadherin-mediated cell-cell adhesion is lost during the development of most epithelial cancers. Recent evidence indicates that the loss of E-cadherin function, besides causing loss of cell-cell adhesion, might also convey signals that actively induce tumour-cell invasion and metastasis.