Distinct roles of fibroblast growth factor receptor 1 and 2 in regulating cell survival and epithelial-mesenchymal transition

Two related receptor tyrosine kinases (RTKs), fibroblast growth factor receptor 1 and 2 (FGFR1 and FGFR2), exert distinct effects during carcinogenesis. To examine FGFR1 and FGFR2 signaling in polarized epithelia, we have developed an in vitro three-dimensional HC11 mouse mammary epithelial cell culture model combined with a chemically inducible FGFR (iFGFR) dimerization system. Although activation of both RTKs led to reinitiation of cell proliferation and loss of cell polarity, only iFGFR1 activation induced cell survival and epithelial to mesenchymal transition. In contrast, iFGFR2 activation induced cell apoptosis even in the cells in direct contact with the extracellular matrix. Activation of iFGFR2, but not iFGFR1, led to rapid receptor down-regulation and transient activation of downstream signaling, which were partially rescued by Cbl small interfering RNA knockdown or the proteasome inhibitor lactacystin. Importantly, inhibition of proteasome activity in iFGFR2-activated structures led to epithelial to mesenchymal transition and invasive phenotypes resembling those observed after iFGFR1 activation. These studies demonstrate, for the first time, that the duration of downstream signaling determines the distinct phenotypes mediated by very homologous RTKs in three-dimensional cultures.     

Opposing roles for Akt1 and Akt2 in Rac/Pak signaling and cell migration

The Akt/PKB isoforms have different roles in animals, with Akt2 primarily regulating metabolic signaling and Akt1 regulating growth and survival. Here we show distinct roles for Akt1 and Akt2 in mouse embryo fibroblast cell migration and regulation of the cytoskeleton. Akt1-deficient cells responded poorly to platelet-derived growth factor while Akt2-deficient cells had a dramatically enhanced response, resulting in a substantial increase in dorsal ruffling. Swapping domains between Akt1 and Akt2 demonstrated that the N-terminal region containing the pleckstrin homology domain and a linker region distinguishes the two isoforms, while the catalytic domains are interchangeable. Akt2 knock-out cells also migrated faster than wild-type cells, especially through extracellular matrix (ECM), while Akt1 knock-out cells migrated more slowly than wild-type cells. Consistently, Akt2 knock-out cells had elevated Pak1 and Rac activities, suggesting that Akt2 inhibits Rac and Pak1. Both Akt2 and Akt1 associated in complexes with Pak1, but only Akt2 inhibited Pak1 in kinase assays, suggesting an underlying molecular basis for the different cellular phenotypes. Together these data provide evidence for an unexpected functional link between Akt2 and Pak1 that opposes the actions of Akt1 on cell migration.     

Distinct roles for JNK1 and JNK2 in regulating JNK activity and c-Jun-dependent cell proliferation

Different c-Jun N-terminal kinases (JNKs) are activated by a plethora of signals and phosphorylate substrates such as c-Jun, which is required for efficient cell cycle progression. Although JNK1 and JNK2 were shown to differentially regulate fibroblast proliferation, the underlying mechanistic basis remains unclear. We found that Jnk2-/- fibroblasts exit G1 and enter S phase earlier than wild-type counterparts, while Jnk1-/- cells show the inverse phenotype. Moreover, Jnk2-/- erythroblasts also exhibit a proliferative advantage. JNK2 deficiency results in elevated c-Jun phosphorylation and stability, whereas the absence of JNK1 reduces c-Jun phosphorylation and stability. Re-expression of JNK2 in Jnk2-/- cells reverses the JNK2 null phenotype, whereas ectopic expression of JNK1 augments it. JNK2 is preferentially bound to c-Jun in unstimulated cells, thereby contributing to c-Jun degradation. In contrast, JNK1 becomes the major c-Jun interacting kinase after cell stimulation. These data provide mechanistic insights into the distinct roles of different JNK isoforms.     

Oxidation of Akt2 kinase promotes cell migration and regulates G1-S transition in the cell cycle

Phosphorylation has long been recognized as the key mediator of protein signaling. New modes of signaling regulation are emerging with the development of specific chemical probes and application of high-throughput mass spectrometry technologies. Using biotin-tagged chemical probes for protein oxidation, mass spectrometry and functional assays, our group has recently reported isoform-specific oxidation of Akt2 in response to PDGF signaling. The studies included here investigate the functional consequence of oxidation on Akt2-mediated cell migration and cell cycle. Akt2-KO MEFs transduced with WT and Cys124Ser Akt2 were used as the model system for these studies. The implications of these findings on disease pathology are discussed.Key words: oxidation, ROS, cell migration, cell cycle, wound healing, Akt2, starvation     

Oxidation of Akt2 kinase promotes cell migration and regulates G1-S transition in the cell cycle

Phosphorylation has long been recognized as the key mediator of protein signaling. New modes of signaling regulation are emerging with the development of specific chemical probes and application of high-throughput mass spectrometry technologies. Using biotin-tagged chemical probes for protein oxidation, mass spectrometry and functional assays, our group has recently reported isoform-specific oxidation of Akt2 in response to PDGF signaling. The studies included here investigate the functional consequence of oxidation on Akt2-mediated cell migration and cell cycle. Akt2-KO MEFs transduced with WT and Cys124Ser Akt2 were used as the model system for these studies. The implications of these findings on disease pathology are discussed.     

LncRNA NBR2 inhibits epithelial-mesenchymal transition by regulating Notch1 signaling in osteosarcoma cells

Long noncoding RNAs (lncRNAs) have been identified to have increasingly important roles in tumorigenesis, and they may serve as novel biomarkers for cancer therapy. Recent studies have demonstrated that lncRNA NBR2 (neighbor of BRCA1 gene 2), a novel identified lncRNA, is decreased in several cancers; however, the role of NBR2 in the development of osteosarcoma has not been elucidated. In our study, we found that NBR2 expression was downregulated in osteosarcoma tissues, and osteosarcoma cases with lower NBR2 expression exhibited a shorter overall survival time compared with those with higher NBR2 expression. NBR2 overexpression inhibited osteosarcoma cell proliferation, invasion, and migration but did not increase apoptosis. Furthermore, RNA-binding protein immunoprecipitation assays confirmed that NBR2 directly binds to Notch1 protein. Furthermore, overexpression of Notch1 in NBR2-overexpressing osteosarcoma cells reversed the effects of NBR2 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition. The in vivo results showed that NBR2 overexpression inhibited tumor growth in nude mice that were inoculated with osteosarcoma cells. NBR2 overexpression also suppressed the messenger RNA (mRNA) expression of Notch1, N-cadherin, and vimentin and increased the mRNA expression of E-cadherin in the tumor tissues. These data indicated that NBR2 served as a tumor suppressor gene in osteosarcoma and inhibited osteosarcoma cell proliferation, invasion, and migration. The current study provides a novel insight and treatment strategy for osteosarcoma.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Protease-activated receptor 2 induces migration and promotes Slug-mediated epithelial-mesenchymal transition in lung adenocarcinoma cells

Protease-activated receptor 2 (PAR2), a G protein-coupled receptor for trypsin, contributes to growth, anti-apoptosis, and migration in lung cancer. Given that PAR2 activation in airway epithelial cells compromises the airway epithelium barrier by disruption of E-cadherin adhesion, PAR2 may be involved in epithelial-mesenchymal transition (EMT) in lung adenocarcinoma cells. Although PAR2 is known to promote the migration of lung cancer cells, the detailed mechanism of this event is still not clear. Here, we found that PAR2 is highly expressed in several lung adenocarcinoma cell lines. In two lung adenocarcinoma cell lines, CL1-5 and H1299 cells, activation of PAR2 induces migration and Slug-mediated EMT. The underlying mechanisms involved in PAR2-induced migration and EMT in CL1-5 cells were further investigated. We showed that PAR2-induced migration of CL1-5 cells is mediated by the Src/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. β-arrestin 1, not G protein, is involved in this PAR2-mediated Src/p38 MAPK signaling pathway. PAR2-induced EMT in CL1-5 cells is dependent on the activation of extracellular-signal-regulated kinase 2 (ERK2). The activation of ERK2 further mediates Slug stabilization through suppressing the activity of glycogen synthase kinase 3β. In addition, a poor prognosis was observed in lung adenocarcinoma patients with a high expression of PAR2. Thus, PAR2 regulates migration through β-arrestin 1-dependent activation of p38 MAPK and EMT through ERK2-mediated stabilization of Slug in lung adenocarcinoma cells. Our finding also suggests that PAR2 might serve as a therapeutic target for metastatic lung adenocarcinoma and a potential biomarker for predicting the prognosis of lung adenocarcinoma.     

Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development

Background  

Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh) and Fibroblast Growth Factor (Fgf) families of secreted signaling proteins. Sonic hedgehog (Shh) directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb.     

Distinct roles of IkappaB proteins in regulating constitutive NF-kappaB activity

The inhibitor of NF-kappaB (IkappaB) family of proteins is believed to regulate NF-kappaB activity by cytoplasmic sequestration. We show that in cells depleted of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, a small fraction of p65 binds DNA and leads to constitutive activation of NF-kappaB target genes, even without stimulation, whereas most of the p65 remains cytoplasmic. These results indicate that although IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins could be dispensable for cytoplasmic retention of NF-kappaB, they are essential for preventing NF-kappaB-dependent gene expression in the basal state. We also show that in the absence of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, cytoplasmic retention of NF-kappaB by other cellular proteins renders the pathway unresponsive to activation.     

Distinct roles of the three Akt isoforms in lactogenic differentiation and involution

The three Akt isoforms differ in their ability to transduce oncogenic signals initiated by the Neu and PyMT oncogenes in mammary epithelia. As a result, ablation of Akt1 inhibits and ablation of Akt2 accelerates mammary tumor development by both oncogenes, while ablation of Akt3 is phenotypically almost neutral. Since the risk of breast cancer development in humans correlates with multiple late pregnancies, we embarked on a study to determine whether individual Akt isoforms also differ in their ability to transduce hormonal and growth factor signals during pregnancy, lactation and post-lactation involution. The results showed that the ablation of Akt1 delays the differentiation of the mammary epithelia during pregnancy and lactation, and that the ablation of Akt2 has the opposite effect. Finally, ablation of Akt3 results in minor defects, but its phenotype is closer to that of the wild type mice. Whereas the phenotype of the Akt1 ablation is cell autonomous, that of Akt2 is not. The ablation of Akt1 promotes apoptosis and accelerates involution, whereas the ablation of Akt2 inhibits apoptosis and delays involution. Mammary gland differentiation during pregnancy depends on the phosphorylation of Stat5a, which is induced by prolactin, a hormone that generates signals transduced via Akt. Here we show that the ablation of Akt1, but not the ablation of Akt2 or Akt3 interferes with the phosphorylation of Stat5a during late pregnancy and lactation. We conclude that the three Akt isoforms have different roles in mammary gland differentiation during pregnancy and this may reflect differences in hormonal signaling.     

Absent in melanoma 2 suppresses epithelial-mesenchymal transition via Akt and inflammasome pathways in human colorectal cancer cells

Absent in melanoma 2 (AIM2) is a critical component in natural immunity system and is closely related to cancer initiation and development. It has been shown that AIM2 inhibited colorectal cancer (CRC) development and cell proliferation. It remains unresolved how AIM2 acts on CRC metastasis. In this study, we assessed migration, invasion ability, and epithelial-mesenchymal transition (EMT) program upon AIM2 overexpression or knockdown in human CRC cells. Transwell assay demonstrated that upregulation of AIM2 reduced cell migration and invasion. Epithelial marker E-cadherin was augmented and mesenchymal markers vimentin, as well as Snail, were examined decreased by Western blot, real-time polymerase chain reaction, and immunofluorescence. Correspondingly, knockdown of AIM2 led to a reverse consequence. In addition, AIM2 regulated Akt phosphorylation and effects of AIM2 on cell invasion and EMT were recovered after administration of Akt inhibitor, suggesting that AIM2 suppressed EMT dependent on Akt pathway. In addition, caspase-1 inhibitor exposure indicated that AIM2 abrogated EMT through the inflammasome pathway as well. In summary, AIM2 suppressed EMT via Akt and inflammasome pathways in human CRC cells.     

Distinct roles for the catalytic and hemopexin domains of membrane type 1-matrix metalloproteinase in substrate degradation and cell migration

Substrate degradation and cell migration are key steps in cancer metastasis. Membrane-type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Using the fluorescein isothiocyanate (FITC)-labeled fibronectin degradation assay combined with the phagokinetic cell migration assay, structure-function relationships of MT1-MMP were studied. Our data indicate that MT1-MMP initiates substrate degradation and enhances cell migration; cell migration occurs as a concurrent but independent event. Using recombinant DNA approaches, we demonstrated that the hemopexin-like domain and a nonenzymatic component of the catalytic domain of MT1-MMP are essential for MT1-MMP-mediated cell migration. Because the cytoplasmic domain of MT1-MMP was not required for MT1-MMP-mediated fibronectin degradation and cell migration, it is proposed that cross-talk between the hemopexin domain of MT1-MMP and adjacent cell surface molecules is responsible for outside-in signaling. Employing cDNAs encoding dominant negative mutations, we demonstrated that Rac1 participates in the MT1-MMP signal transduction pathway. These data demonstrated that each domain of MT1-MMP plays a distinct role in substrate degradation and cell migration.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

SETD8 induces stemness and epithelial-mesenchymal transition of pancreatic cancer cells by regulating ROR1 expression

Pancreatic cancer (PC) is one of the most deadly diseases,and its incidence is increasing year by year.The methyltransferase SETD8 has been demonstrated to play...     

Essential role of PDK1 in regulating endothelial cell migration

The serine/threonine protein kinase phosphoinositide-dependent kinase 1 (PDK1) plays a central role in cellular signaling by phosphorylating members of the AGC family of kinases, including PKB/Akt. We now present evidence showing that PDK1 is essential for the motility of vascular endothelial cells (ECs) and that it is involved in the regulation of their chemotaxis. ECs differentiated from mouse embryonic stem cells lacking PDK1 completely lost their ability to migrate in vitro in response to vascular endothelial growth factor-A (VEGF-A). In addition, PDK1(-/-) embryoid bodies exhibit evident developmental and vascular defects that can be attributed to a reduced cell migration. Moreover, the overexpression of PDK1 increased the EC migration induced by VEGF-A. We propose a model of spatial distribution of PDK1 and Akt in which the synthesis of phosphatidylinositol 3,4,5 triphosphate at plasma membrane by activation of phosphoinositide 3-kinase recruits both proteins at the leading edge of the polarized ECs and promotes cell chemotaxis. These findings establish a mechanism for the spatial localization of PDK1 and its substrate Akt to regulate directional migration.     

Galangin ameliorated pulmonary fibrosis in vivo and in vitro by regulating epithelial-mesenchymal transition

Pulmonary fibrosis (PF) is a disease that is characterized by abnormal epithelial-mesenchymal transition (EMT) and persistent inflammatory injury, with high mortality and poor prognosis, but the current therapies are accompanied by certain adverse side effects. In this study, we investigated the role of galangin (GA), an anti-inflammatory and anti-tumoral phytochemical extracted from galangal, in preventing and curing bleomycin (BLM)-induced pulmonary fibrosis and the underlying mechanism. Histopathological staining confirmed that GA dramatically moderated bleomycin-induced pulmonary fibrosis in mice. Compared with the vehicle treatment, GA treatment inhibited the expression of vimentin and increased the expression of E-cadherin. The expression of α-Smooth muscle actin (α-SMA), which is a myofibroblast marker, was also suppressed. In addition, GA diminished the increase in the numbers of CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells and dendritic cells induced by bleomycin, and reduced the residence of inflammatory cells in the lung tissues. Notably, GA inhibited the TGF-β1-induced EMT and fibroblast differentiation in vitro, which further confirmed the potential protective effect of GA on pulmonary fibrosis. Taken together, our results suggest that GA exerts a beneficial effect on bleomycin-induced pulmonary fibrosis by attenuating EMT and inflammatory damage and may have prevent potential of pulmonary fibrosis.