12 research outputs found

    Phosphorylation of LCRMP-1 at Thr-628 is required for filopodia formation.

    No full text
    <p>(A) Nonphosphorylated LCRMP-1 (T628A) mutant impairs filopodia formation. CL1-0 cells were transiently transfected with GFP-tagged LCRMP-1 (WT), LCRMP-1 (T628A), and LCRMP-1 (T628D). At 24 hours after transfection, these cells were fixed, permeabilized and then immunostained with rhodamine-conjugated phalloidin (red) for actin and DAPI (blue) for visualizing nuclei. Representative immunofluorescence images were visualized using fluorescence microscope. Scale bar, 10 µm. Insets show higher magnifications (400×). (B) Numbers of filopodia were counted with at least six cells per group. Data were presented as means ± SEM.</p

    LCRMP-1 and phosphorylated GSK3β expression in relation to clinical parameters and pathological characteristics<sup>*</sup>.

    No full text
    <p>*<i>P</i> values were calculated using a two-sided chi-squared test. Abbreviations: LCRMP-1, long-form collapsin response mediator protein-1; p-GSK3β, phosphorylated Glycogen synthase kinase-3β.</p>†<p>Adenosquamous carcinomas are not included.</p

    Phosphorylation of LCRMP-1 at Thr-628 is critical for cancer invasion and migration.

    No full text
    <p>(A) Protein expression levels of exogenous untagged LCRMP-1 (WT), LCRMP-1 (T628A), and LCRMP-1 (T628D) are confirmed by immunoblotting. After 48 hours post lentivirus infection, CL1-0 cells were stably expressing wild-type and mutant LCRMP-1. Lentivirus expressing GFP served as control. Cell lysates were harvested, following by assessing with immunoblotting using anti-LCRMP-1 antibody and anti-β-actin antibodies. (B) Nonphosphorylated LCRMP-1 (T628A) mutant lowers activity of cell invasion. The invasive capacity of these cells was determined with the modified Boyden chambers invasion assay <i>in vitro</i>. Percentage of invasive ability was normalized to GFP control. Data were shown as means ± SEM for three-independent experiments (n = 3). (C) Nonphosphorylated LCRMP-1 (T628A) mutant greatly suppressed cell migration tracks. Tract plots showed CL1-0 cells expressing GFP, GFP-LCRMP-1 (WT), GFP-LCRMP-1 (T628A), GFP-LCRMP-1 (T628D), respectively. Moving tracks of at least 10 representative cells at the start point all set to ‘0,0’ over a 20-hour period (different lines) showed the representative motility of cells. Scale bar, 100 µm. (D, E) Total migration distance (D) and cell migration velocity (E) were quantified from cell tracking assay for 20 hours. Data were presented as means ± SEM.</p

    GSK3β modulates ability of LCRMP-1-induced cancer cell invasion.

    No full text
    <p>(A) Lentivirus expressed GFP control, myc-tagged GSK3β (WT), GSK3β (CA), or GSK3β (KD) in CL1-0/LCRMP-1 (WT) overexpression cells (1015 and 1003). After 48 hours postinfection, these cells were lysed and subjected to immunoblotting analysis with using anti-Flag, anti-Myc, and anti-β-actin antibodies. (B) GSK3β activity affects LCRMP-1-induced cancer cell invasion. CL1-0/LCRMP-1 overexpression cells (1015) were infected with lentivirus expressing GFP control, myc-tagged GSK3β (CA) or GSK3β (KD). After 48 hours postinfection, these cells were subjected to the modified Boyden chambers invasion assay <i>in vitro</i>. Normalization to GFP control served as percentage of invasive ability.</p

    LCRMP-1 is a substrate of and phosphorylated by GSK3β at Thr-628.

    No full text
    <p>(A) Protein sequence analysis showed the potential consensus site of LCRMP-1 for the phosphorylation by GSK3β. Protein sequences are aligned among CRMP2, CRMP-1, and LCRMP-1. The numbers represent amino acid sites and underline shows a potential phosphorylation site for Cdk5. (B) GSK3β phosphorylates LCRMP-1 (WT) <i>in vivo</i>. HEK293T cells were co-transfected with Flag-tagged LCRMP-1 and distinct activity of Flag-tagged GSK3β (WT, CA and KD form). Equal amount of plasmids were transfected into every condition by using empty vectors. Cells were lysed 30 hr post-transfection and protein extracts were analyzed by immunoblotting with anti-Flag antibodies. (C) GSK3β phosphorylates LCRMP-1 (WT) at Thr-628 <i>in vivo</i>. CL1-0 cells were co-transfected either Flag-tagged LCRMP-1(WT) or LCRMP-1 (T628A, T628D) mutants in the presence or absence of distinct activity of Flag-tagged GSK3β (CA and KD form). Empty vectors were used for supplement to equal amount of plasmids in transfection assay. Cell lysates were harvested 48 hr post-transfection and analyzed by immunoblotting with anti-Flag and anti-β-actin antibodies.</p

    MicroRNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma

    No full text
    <div><p>Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in <i>vivo</i> animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.</p></div

    IGF-1R and TGFBR1 mediate cell invasion through AKT signaling in lung cancer cell lines.

    No full text
    <p>(A) Measurement of the protein levels of receptors CL1-5 or A549 cell transfected with siEGFR, siIGF-1R or siTGFBR1. (B) Measurement of the cell invasion ability of CL1-5 or A549 cells transfected with siEGFR, siIGF-1R or siTGFBR1 for 48 hours. The number of invasive cells was counted 20 hours after the transfected cells were seeded and was presented relative to invasion of cells transfected with siCtl. (C) Measurement of the cell proliferation ability of CL1-5 or A549 cells transfected with siEGFR, siIGF-1R or siTGFBR1 for 72 hours. (D) Representative immunoblots show protein levels of pAKT (Ser473), AKT and β-actin in these cells.</p

    The miR-133a tumor suppressor modulates cell invasive capacity in lung cancer.

    No full text
    <p>(A) The expression levels of miR-133a in 434 tumor tissues and 46 normal tissues from the TCGA lung adenocarcinoma database were examined. (B) The expression level of endogenous miR-133a in BEAS-2B, EKVX, H23, H441, A549 and H1299 cell lines. MiR-133a expression level (C, left panel), as measured by real time RT-PCR, and cell morphology (C, right panel) in BEAS-2B cells were examined 72 hours after transfection of the anti-miR-133a inhibitor. (D)The number of invasive cells was counted 24 hours after seeding cells that post-treated anti-miR-133a inhibitor (100 nM) for 48 hours in a transwell containing matrigel. (E) The endogenous level of miR-133a in CL1-0, CL1-1, CL1-5 and CL1-5F4 cells. (F) Measurement of the invasion in CL1-5 and A549 cells transiently infected with the AS2-Neo (Ctl) or AS2-Neo-miR-133a-expressing viruses (right panel). The expression level of miR-133a was normalized to that of RNU48, which was used as an internal control (left panel) (G) 48 hours after transient transfection of the anti-miR-133a inhibitor, the invasive capacity of CL1-1 cells was determined (right panel). The expression level of miR-133a was normalized to that of RNU48, which was used as an internal control (left panel).</p

    MiR-133a expression decreases cancer metastasis <i>in vivo</i> and associates with longer survival rates in NSCLC patients.

    No full text
    <p>(A) Effects of miR-133a in metastasis <i>in vivo</i>. Left panel: histological examinations of the lung tumors by hematoxylin-eosin (H&E) staining. Representative lungs of mice intravenously (i.v.) injected with CL1-5/Ctl, CL1-5/miR-133a. White arrows indicate the lung tumors. Scale bars, 200 µm. Right panel: Numbers of metastatic tumor nodules were calculated 39 weeks after tail-vein injection (n = 9 per group). (B) The number of death/total (top panel) and survival curve (bottom panel) in each group at 39 days after i.v. injection were presented. Data were expressed as the mean ±SD by the log-rank (Mantel-Cox) test. P = 0.0164 (C) Kaplan-Meier plots of overall survival in 112 NSCLC patients in high- and low-risk groups based on miR-133a expression levels (P = 0.0409). (D) Model showing that miR-133a modulates the receptor-mediated cancer malignancy in NSCLC.</p
    corecore