Overexpression of fucosyltransferase IV promotes A431 cell proliferation through activating MAPK and PI3K/Akt signaling pathways

Lewis Y (LeY) is a carbohydrate tumor‐asssociated antigen. The majority of cancer cells derived from epithelial tissue express LeY type difucosylated oligosaccharide. Fucosyltransferase IV (FUT4) is an essential enzyme that catalyzes the synthesis of LeY oligosaccharide. Our previous studies have shown that FUT4 overexpression promotes A431 cell proliferation, but the mechanism is still largely unknown. Herein, we investigated the role of the mitogen‐activated protein kinases (MAPKs) and phosphoinositide‐3 kinase (PI3K)/Akt signaling pathways on FUT4‐induced cell proliferation. Results show that overexpression of FUT4 increases the phosphorylation of ERK1/2, p38 MAPK, and PI3K/Akt. Inhibitors of PI3K (LY294002 and Wortmannin) prevented the phosphorylation of ERK1/2, p38 MAPK, and Akt PI3K). Moreover, phosphorylation of Akt is abolished by inhibitors of ERK1/2 (PD98059) and p38 MAPK (SB203580). These data suggested that FUT4 not only activates MAPK and PI3K/Akt signals, but also promotes the crosstalk among these signaling pathways. In addition, FUT4‐induced stimulation of cell proliferation correlates with increased cell cycle progression by promoting cells into S‐phase. The mechanism involves in increased expression of cyclin D1, cyclin E, CDK 2, CDK 4, and pRb, and decreased level of cyclin‐dependent kinases inhibitors p21 and p27, which are blocked by the inhibitors of upstream signal molecules, MAPK and PI3K/Akt. In conclusion, these studies suggest that FUT4 regulates A431 cell growth through controlling cell cycle progression via MAPK and PI3K/Akt signaling pathways. J. Cell. Physiol. 225: 612–619, 2010. © 2010 Wiley‐Liss, Inc.     

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho‐GDIα, Rac1, and p38 pathway

Cyclic strain is an important inducer of proliferation and migration of vascular smooth muscle cells (VSMCs) which are involved in vascular remodeling during hypertension. However, its mechanism remains to be elucidated. VSMCs of rat aorta were exposed to cyclic strains in vitro with defined parameters, the static, 5%‐strain (physiological) and 15%‐strain (pathological), at 1.25 Hz for 24 h respectively. Then the possible signaling molecules participated in strain‐induced VSMC migration and proliferation were investigated. The results showed that 15%‐strain significantly increased VSMC migration and proliferation in comparison with 5%‐strain. Expression of Rho GDP dissociation inhibitor alpha (Rho‐GDIα) was repressed by 15%‐strain, but expressions of phospho‐Rac1 and phospho‐p38 were increased. Expressions of phospho‐Akt and phospho‐ERK1/2 were similar between the static, 5%‐strain and 15%‐strain groups. Rho‐GDIα “knock‐down” by target siRNA transfection increased migration and proliferation of VSMCs, and up‐regulated phosphorylation of Rac1 and p38 in all groups. Rac1 “knock‐down” repressed migration and proliferation of VSMCs, down‐regulated phosphorylation of p38, but had no effect on Rho‐GDIα expression. When siRNAs of Rho‐GDIα and Rac1 were co‐transfected to VSMCs, the expressions of Rho‐GDIα and phospho‐Rac1 were both decreased, and the effects of Rho‐GDIα “knock‐down” were blocked. Rho‐GDIα “knock‐down” promoted while Rac1 “knock‐down” postponed the assembly of stress fibers and focal adhesions in static. The results demonstrate that the pathological cyclic strain might induce migration and proliferation of VSMCs via repressing expression of Rho‐GDIα, which subsequently verified phosphorylations of Rac1 and p38. J. Cell. Biochem. 109: 906–914, 2010. © 2010 Wiley‐Liss, Inc.     

Gartanin induces cell cycle arrest and autophagy and suppresses migration involving PI3K/Akt/mTOR and MAPK signalling pathway in human glioma cells

In central nervous system, glioma is the most common primary brain tumour. The diffuse migration and rapid proliferation are main obstacles for successful treatment. Gartanin, a natural xanthone of mangosteen, suppressed proliferation, migration and colony formation in a time‐ and concentration‐dependent manner in T98G glioma cells but not in mouse normal neuronal HT22 cells. Gartanin, at low micromole, led to cell cycle arrest in G1 phase accompanied by inhibited expression level of G1 cell cycle regulatory proteins cyclin D1, while increased expression level of cyclin‐dependent kinase inhibitor p27Kip1. In addition, the secretion and activity of matrix metalloproteinases 2/9 (MMP‐2/‐9) were significantly suppressed in T98G cells treated with gartanin, and it might result from modulating mitogen‐activated protein kinases (MAPK) signalling pathway in T98G glioma cells. Moreover, gartanin significantly induced autophagy in T98G cells and increased GFP‐LC3 punctate fluorescence accompanied by the increased expression level of Beclin 1 and LC3‐II, while suppressed expression level of p62. Gartanin treatment resulted in obvious inhibition of PI3K/Akt/mTOR signalling pathway, which is important in modulating autophagy. Notably, gartanin‐mediated anti‐viability was significantly abrogated by autophagy inhibitors including 3‐methyladenine (3‐MA) and chloroquine (CQ). These results indicate that anti‐proliferation effect of gartanin in T98G cells is most likely via cell cycle arrest modulated by autophagy, which is regulated by PI3K/Akt/mTOR signalling pathway, while anti‐migration effect is most likely via suppression of MMP‐2/‐9 activity which is involved in MAPK signalling pathway.     

CFTR promotes malignant glioma development via up‐regulation of Akt/Bcl2‐mediated anti‐apoptosis pathway

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐activated Cl^‐ channel, is extensively expressed in the epithelial cells of various tissues and organs. Accumulating evidence indicates that aberrant expression or mutation of CFTR is related to carcinoma development. Malignant gliomas are the most common and aggressive intracranial tumours; however, the role of CFTR in the development of malignant gliomas is unclear. Here, we report that CFTR is expressed in malignant glioma cell lines. Suppression of CFTR channel function or knockdown of CFTR suppresses glioma cell viability whereas overexpression of CFTR promotes it. Additionally, overexpression of CFTR suppresses apoptosis and promotes glioma progression in both subcutaneous and orthotopic xenograft models. Cystic fibrosis transmembrane conductance regulator activates Akt/Bcl2 pathway, and suppression of PI3K/Akt pathway abolishes CFTR overexpression–induced up‐regulation of Bcl2 (MK‐2206 and LY294002) and cell viability (MK‐2206). More importantly, the protein expression level of CFTR is significantly increased in glioblastoma patient samples. Altogether, our study has revealed a mechanism by which CFTR promotes glioma progression via up‐regulation of Akt/Bcl2‐mediated anti‐apoptotic pathway, which warrants future studies into the potential of using CFTR as a therapeutic target for glioma treatment.     

HMGA1 positively regulates the microtubule-destabilizing protein stathmin promoting motility in TNBC cells and decreasing tumour sensitivity to paclitaxel

High Mobility Group A1 (HMGA1) is an architectural chromatin factor involved in the regulation of gene expression and a master regulator in Triple Negative Breast Cancer (TNBC). In TNBC, HMGA1 is overexpressed and coordinates a gene network that controls cellular processes involved in tumour development, progression, and metastasis formation. Here, we find that the expression of HMGA1 and of the microtubule-destabilizing protein stathmin correlates in breast cancer (BC) patients. We demonstrate that HMGA1 depletion leads to a downregulation of stathmin expression and activity on microtubules resulting in decreased TNBC cell motility. We show that this pathway is mediated by the cyclin-dependent kinase inhibitor p27^kip1 (p27). Indeed, the silencing of HMGA1 expression in TNBC cells results both in an increased p27 protein stability and p27-stathmin binding. When the expression of both HMGA1 and p27 is silenced, we observe a significant rescue in cell motility. These data, obtained in cellular models, were validated in BC patients. In fact, we find that patients with high levels of both HMGA1 and stathmin and low levels of p27 have a statistically significant lower survival probability in terms of relapse-free survival (RFS) and distant metastasis-free survival (DMFS) with respect to the patient group with low HMGA1, low stathmin, and high p27 expression levels. Finally, we show in an in vivo xenograft model that depletion of HMGA1 chemo-sensitizes tumour cells to paclitaxel, a drug that is commonly used in TNBC treatments. This study unveils a new interaction among HMGA1, p27, and stathmin that is critical in BC cell migration. Moreover, our data suggest that taxol-based treatments may be more effective in reducing the tumour burden when tumour cells express low levels of HMGA1.Subject terms: Breast cancer, Breast cancer     

TSG101 promotes the proliferation,migration and invasion of hepatocellular carcinoma cells by regulating the PEG10

The tumour susceptibility gene 101 (TSG101) is reported to play important roles in the development and progression of several human cancers. However, its potential roles and underlined mechanisms in human hepatocellular carcinoma (HCC) are still needed to be further clarified. In the present study, we reported that knock down of TSG101 suppressed the proliferation, migration and invasion of HCC cells, while overexpression of TSG101 facilitated them. Molecularly, the results revealed that knock down of TSG101 significantly decreased the cell cycle related regulatory factor p53 and p21. In another point, knock down of TSG101 also obviously decreased the level of metallopeptidase inhibitor TIMP1 (Tissue inhibitors of metalloproteinases 1), which results in inhibition of MMP2, MMP7 and MMP9. In contrast, overexpression of TSG101 had opposite effects. The iTRAQ proteomics analysis identified that oncogenic protein PEG10 (Paternally expressed gene 10) might be a potential downstream target of TSG101. Further investigation showed that TSG101 interacted with PEG10 and protected it from proteasomal degradation thereby regulating the expression of p53, p21 and MMPs. Finally, we found that both TSG101 and PEG10 proteins are up‐regulated and presented a direct correlation in HCC patients. In conclusion, these results suggest that TSG101 is up‐regulated in human HCC patients, which may accelerate the proliferation, migration and invasion of HCC cells through regulating PEG10.     

PI3K/Akt/FOXO3a pathway contributes to thrombopoietin-induced proliferation of primary megakaryocytes in vitro and in vivo via modulation of p27Kip1

Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. We previously demonstrated that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in primary MKs in a PI3K dependent fashion. UT-7/TPO, a megakaryocytic cell line, stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to reduce p27Kip1 expression after TPO stimulation, and fail to induce p27Kip1 expression following TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In addition, the number of MKs is significantly increased in bone marrow from Foxo3a-deficient mice. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings indicate that the PI3K/Akt/FOXO3a/p27Kip1 pathway contributes to normal TPO-induced MK proliferation.     

Non‐canonical Raf‐1/p70S6K signalling in non–small‐cell lung cancer

Lung cancer is the leading cause of cancer‐related death globally, with non–small‐cell lung cancer (NSCLC) being the predominant subtype. Overall survival remains low for NSCLC patients, and novel targets are needed to improve outcome. Raf‐1 is a key component of the Ras/Raf/MEK signalling pathway, but its role and downstream targets in NSCLC are not completely understood. Our previous study indicated a possible correlation between Raf‐1 levels and ribosomal protein S6 kinase (p70S6K) function. In this study, we aimed to investigate whether p70S6K is a downstream target of Raf‐1 in NSCLC. Raf‐1 was silenced in NSCLC cell lines by using small hairpin RNA, and Raf‐1 and p70S6K protein levels were measured via Western blot. p70S6K was then overexpressed following Raf‐1 knock‐down; then, cell proliferation, apoptosis and the cell cycle in NSCLC cell lines were examined. Tumour xenografts with NSCLC cells were then transplanted for in vivo study. Tumours were measured and weighed, and Raf‐1 and p70S6K expression, cell proliferation and apoptosis were examined in tumour tissues by Western blot, Ki‐67 staining and TUNEL staining, respectively. When Raf‐1 was silenced, p70S6K protein levels were markedly decreased in the A549 and H1299 NSCLC cell lines. A significant decrease in NSCLC cell proliferation, a profound increase in apoptosis and cell cycle arrest were observed in vitro following Raf‐1 knock‐down. Overexpression of p70S6K after Raf‐1 depletion effectively reversed these effects. Xenograft studies confirmed these results in vivo. In conclusion, Raf‐1 targets p70S6K as its downstream effector to regulate NSCLC tumorigenicity, making Raf‐1/p70S6K signalling a promising target for NSCLC treatment.     

Targeting PRMT5/Akt signalling axis prevents human lung cancer cell growth

The emerging evidence reveals that protein arginine methyltransferase 5 (PRMT5) is involved in regulation of tumour cell proliferation and cancer development. Nevertheless, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remains largely obscure. Here, we showed that PRMT5 was highly expressed in human lung cancer cells and lung cancer tissues. Furthermore, we generated PRMT5 stable knockdown cell lines (A549 and H1299 cells) and explored the functions of PRMT5 in lung cancer cell proliferation. We found that the down‐regulation of PRMT5 by shRNA or the inhibition of PRMT5 by specific inhibitor GSK591 dramatically suppressed cyclin E1 and cyclin D1 expression and cell proliferation. Moreover, we uncovered that PRMT5 promoted lung cancer cell proliferation via regulation of Akt activation. PRMT5 was directly co‐localized and interacted with Akt, but not PTEN and mTOR. Down‐regulation or inhibition of PRMT5 markedly reduced Akt phosphorylation at Thr308 and Ser473, whereas the expression of PTEN and mTOR phosphorylation was unchanged, indicating that PRMT5 was an important upstream regulator of Akt and induced lung cancer cell proliferation. Altogether, our results indicate that PRMT5 promotes human lung cancer cell proliferation through direct interaction with Akt and regulation of Akt activity. Our findings also suggest that targeting PRMT5 may have therapeutic potential for treatment of human lung cancer.     

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.     

Genetic characterization of p27kip1 and stathmin in controlling cell proliferation in vivo

The CDK inhibitor p27^kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27^kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27^kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27^kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27^kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27^kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27^kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.     

Overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type‐dependent manner

Objectives

Human CAP10‐like protein 46 kDa (hCLP46), also known as Poglut1, has been shown to be an essential regulator of Notch signalling. hCLP46 is overexpressed in primary acute myelogenous leukaemia, T‐acute lymphoblastic leukaemia samples and other leukaemia cell lines. However, effects of hCLP46 overexpression, up to now, have remained unknown.

Materials and methods

In this study, we established stable 293TRex cell lines inducibly overexpressing hCLP46, and knocked down hCLP6 with a specific small interfering RNA to explore function of the protein in Notch signalling and cell proliferation.

Results

hCLP46 overexpression enhanced Notch1 activation in 293Trex cells in a ligand‐dependent manner, with increased Notch signalling enhancing Hes1 expression. We further verified that overexpression of hCLP46 inhibited proliferation of 293TRexs and was correlated with increases in cyclin dependent kinase inhibitors p21 and p27, whereas reduced hCLP46 expression moderately increased cell proliferation. In addition, p21 and p27 protein levels were higher when Notch signalling was activated by EDTA treatment.

Conclusions

Taken together, hCLP46 enhanced Notch activation and inhibited 293TRex cell proliferation through CDKI signalling.

     

LncRNA MALAT1/miR‐129 axis promotes glioma tumorigenesis by targeting SOX2

We aimed to explore the interaction among lncRNA MALAT1, miR‐129 and SOX2. Besides, we would investigate the effect of MALAT1 on the proliferation of glioma stem cells and glioma tumorigenesis. Differentially expressed lncRNAs in glioma cells and glioma stem cells were screened out with microarray analysis. The targeting relationship between miR‐129 and MALAT1 or SOX2 was validated by dual‐luciferase reporter assay. The expressions of MALAT1, miR‐129 and SOX2mRNA in both glioma non‐stem cells and glioma stem cells were examined by qRT‐PCR assay. The impact of MALAT1 and miR‐129 on glioma stem cell proliferation was observed by CCK‐8 assay, EdU assay and sphere formation assay. The protein expression of SOX2 was determined by western blot. The effects of MALAT1 and miR‐129 on glioma tumour growth were further confirmed using xenograft mouse model. The mRNA expression of MALAT1 was significantly up‐regulated in glioma stem cells compared with non‐stem cells, while miR‐129 was significantly down‐regulated in glioma stem cells. MALAT1 knockdown inhibited glioma stem cell proliferation via miR‐129 enhancement. Meanwhile, miR‐129 directly targeted at SOX2 and suppressed cell viability and proliferation of glioma stem cells by suppressing SOX2 expression. The down‐regulation of MALAT1 and miR‐129 overexpression both suppressed glioma tumour growth via SOX2 expression promotion in vivo. MALAT1 enhanced glioma stem cell viability and proliferation abilities and promoted glioma tumorigenesis through suppressing miR‐129 and facilitating SOX2 expressions.