Hepatocytes convert to a fibroblastoid phenotype through the cooperation of TGF-β1 and Ha-Ras: Steps towards invasiveness
ABSTRACT In hepatocarcinogenesis, it is an open question whether transforming growth factor (TGF)-beta1 provides a tumor-suppressive or a tumor-promoting role. To address this question, we employed immortalized murine hepatocytes, which display a high degree of differentiation and, expectedly, arrest in the G1 phase under exposure to TGF-beta1. These hepatocytes maintain epithelial polarization upon expression of oncogenic Ha-Ras. However, Ras-transformed hepatocytes rapidly convert to a spindle-shaped, fibroblastoid morphology upon treatment with TGF-beta1, which no longer inhibits proliferation. This epithelial to fibroblastoid conversion (EFC) is accompanied by disruption of intercellular contacts and remodeling of the cytoskeletal framework. Fibroblastoid derivatives form elongated branching cords in collagen gels and grow to severely vascularized tumors in vivo, indicating their increased malignancy and even invasive phenotype. Additionally, fibroblastoid cells secrete strongly enhanced levels of TGF-beta1, suggesting an autocrine regulation of TGF-beta signaling. Expression profiling further revealed that the loss of the adhesion component E-cadherin correlates with the upregulation of its transcriptional repressor Snail in fibroblastoid cells. Moreover, the phosphoinositide 3-OH (PI3) kinase pathway was required for the maintenance of EFC, as inhibition of PI3 kinase reverted fibroblastoid cells to an epithelial-like phenotype. Taken together, these data indicate a dual role of TGF-beta1 in hepatocytes: it induces proliferation arrest but provides a crucial function in promoting late malignant events in collaboration with activated Ha-Ras.
- SourceAvailable from: Naoki Okumura
[Show abstract] [Hide abstract]
- "Multiple signaling cascades are activated when TGFβ binds to its cognate receptor. These include Smad , RhoA-related signals , mitogen-activated protein kinase (MAPK)-Erk-1 and −2 , stress kinases (i.e., c-Jun N-terminal kinase [JNK]) [20,21], p38 mitogen-actiated protein kinase (p38MAPK) [22,23], phosphatase 2A , and phosphoinositide 3-kinase/AKT (PI3K/AKT) [25,26]. The pathways involved in cellular differentiation or transformation are Smad, Rho proteins, and PI3-kinase. "
ABSTRACT: Our study examined the effect of a selective Rho kinase inhibitor, Y-27632, on corneal wound healing and potential stromal scarring after superficial keratectomy. Rabbit keratocytes were induced into myofibroblasts by transforming growth factor β1 (TGFβ1) either with or without Y-27632. Then α-smooth muscle actin (α-SMA) was examined by immunohistochemistry and western blotting, and the contractility of the seeded collagen gels was measured. Y-27632 eye drops (or vehicle only) were administered to eyes after a superficial keratectomy, and the tissue was examined by immunohistochemistry for α-SMA, collagen types I, II, and III, and keratan sulfate. Electron microscopy was conducted with and without histochemical contrasting of sulfated proteoglycans. Spindle-like cells in culture constituted 99.5±1.1% with TGFβ1 stimulation, but 3.5±1.0% after TGFβ1 and Y-27632 treatment (p<0.01, n=6). α-SMA was seen in 4% of TGFβ1-treated cells, but in only 0.3% of cells with Y-27632 added (p<0.01, n=6), which was confirmed by western blotting. Y-27632 also inhibited the TGFβ1-induced contraction of seeded collagen gels. After superficial keratectomies, collagen type I and keratan sulfate were unchanged by Y-27632 application. Collagen type II was not detected in Y-27632 or vehicle-only corneas. With Y-27632 treatment, α-SMA expression increased and the collagen type III signal became in the weaker subepithelial area. Interestingly, bundles of aligned and uniformly spaced collagen fibrils were more prevalent in keratocytes in Y-27632-treated corneas, which is reminiscent of fibripositor-like structures that have been proposed as a mechanism of matrix deposition in embryonic connective tissues. Y-27632 inhibits keratocyte-to-myofibroblast transition, and its topical application after a superficial lamellar keratectomy elicits an altered wound healing response, with evidence of an embryonic-type deposition of collagen fibrils.Molecular vision 06/2012; 18:1727-39. · 1.99 Impact Factor
[Show abstract] [Hide abstract]
- "Pre-malignant cells frequently gain their ability to proliferate and clonally expand due to constitutive activation of receptor tyrosine kinases and downstream effectors such as RAS. Several studies have demonstrated cooperation between growth factors and RAS signalling in the induction of EMT , ,  suggesting that coordinated activation of multiple pathways is essential for EMT to occur. "
ABSTRACT: Tumour invasion and metastasis is the most common cause of death from cancer. For epithelial cells to invade surrounding tissues and metastasise, an epithelial-mesenchymal transition (EMT) is required. We have demonstrated that FGFR1 expression is increased in bladder cancer and that activation of FGFR1 induces an EMT in urothelial carcinoma (UC) cell lines. Here, we created an in vitro FGFR1-inducible model of EMT, and used this model to identify regulators of urothelial EMT. FGFR1 activation promoted EMT over a period of 72 hours. Initially a rapid increase in actin stress fibres occurred, followed by an increase in cell size, altered morphology and increased migration and invasion. By using site-directed mutagenesis and small molecule inhibitors we demonstrated that combined activation of the mitogen activated protein kinase (MAPK) and phospholipase C gamma (PLCγ) pathways regulated this EMT. Actin stress fibre formation was regulated by PLCγ activation, and was also important for the increase in cell size, migration and altered morphology. MAPK activation regulated migration and E-cadherin expression, indicating that combined activation of PLCγ and MAPK is required for a full EMT. We used expression microarrays to assess changes in gene expression downstream of these signalling cascades. COX-2 was transcriptionally upregulated by FGFR1 and caused increased intracellular prostaglandin E(2) levels, which promoted migration. In conclusion, we have demonstrated that FGFR1 activation in UC cells lines promotes EMT via coordinated activation of multiple signalling pathways and by promoting activation of prostaglandin synthesis.PLoS ONE 06/2012; 7(6):e38972. DOI:10.1371/journal.pone.0038972 · 3.23 Impact Factor
[Show abstract] [Hide abstract]
- "Loss of E-cadherin expression is a hallmark of epithelial-mesenchymal transition (EMT), during which epithelial cells lose many of their epithelial characteristics and acquire certain properties of mesenchymal cells, conferring increased motility and invasiveness. Mounting evidence suggests that cancer cells exhibit a dynamic plasticity between epithelial and mesenchymal states that allows them to survive at distinct steps of metastasis , , , . While the consequence of the manipulation of EMT-related factors has been studied extensively, cycling of EMT has not been directly visualized in vitro or in vivo in real-time. "
ABSTRACT: The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters.PLoS ONE 01/2012; 7(1):e30177. DOI:10.1371/journal.pone.0030177 · 3.23 Impact Factor