HGF results in PI3K/Akt pathway phosphorylation and activates CXCR4 phosphorylation via PKCζ.

<p>(A). Detection of phosphorylated Akt or of the respective total Akt protein expression and Rac1-GTP or total Rac1 expression by western blot analysis. MDA-MB-436 cells were added with PBS or were exposed to HGF/SF (for 10 minutes at 50 ng/ml) after preincubation with the PI3-kinase inhibitors wortmannin (at 100 nM, for 60 min) or LY 294002 (at 10 µM, for 60 min) as indicated. The experiment was repeated twice with similar results. A representative study is shown. (B) Membrane CXCR4 expression in MDA-MB-436 cells cultured in the absence or presence of wortmannin (100 nM) or LY 294002 (50 µM, starting 1 hour prior to HGF/SF treatment) with or without HGF/SF treatment (for 16 hour at 50 ng/ml) as indicated. The flow cytometry analysis data are shown in arbitrary units (AU) as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (C) MDA-MB-436 cells were exposed to HGF with or without PI 3-kinase inhibitor LY294002 (30 µM) or Akt inhibitor III (50 µM) for various amounts of time, which resulted in the phosphorylation of PKCζ. Western blotting of total protein were performed in triplicate. A representative study is shown. (D). Cellular distribution of PKCζ and CXCR4 before and after 30 minutes of HGF stimulation. HGF, 50 ng/ml, for 10 minutes; LY294002, 30 µM, for 60 min. The experiment was repeated three times with similar results. A representative study is shown.</p

HGF upregulates CXCR4 expression and membrane presentation in human breast cancer cells.

<p>(A). qRT-PCR analysis of the total CXCR4 mRNA extracted from HGF- or SDF-1-treated MDA-MB-436 and MCF-7 cells. Results are presented as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (B). Western blot analysis of the total protein expression and phosphorylation levels of Met or CXCR4 in MDA-MB-436 and MCF-7 cells cultured for 24 hours with and without the presence of 50 ng/ml HGF or 20 ng/ml SDF-1. The experiment was repeated twice with similar results. A representative study is shown. (C). Time course of the relative extracted CXCR4 mRNA expression in MDA-MB-436 and MCF-7 cells following stimulation with 50 ng/ml HGF. Results are presented as the mean ± SD of three independent experiments. # P<0.01,* P<0.05 as compared to PBS. (D). Western blot analyses of CXCR4 protein expression in HGF-treated MDA-MB-436 and MCF-7 cells. The experiment was repeated three times with similar results. A representative study is shown. (E). Flow cytometric analysis of MDA-MB-436 cells stained for the expression of CXCR4 using a CXCR4 monoclonal antibody and isotype controls. The experiment was repeated three times with similar results. A representative study is shown. (F). Increased membrane and intracellular CXCR4 labeling in MDA-MB-436 cells incubated for 24 hours in the presence of 50 ng/ml HGF compared with untreated cells (PBS) taken as 1. Flow cytometry analysis data (mean±SD of three independent experiments) are shown. # P<0.01,* P<0.05 as compared to PBS. (G). Decreased internalization rate of anti-CXCR4-PE mAb in MDA-MB-436 cells treated for 24 hours with HGF compared with PBS. Results are presented as the mean ± SD of three independent experiments. * P<0.05 as compared to PBS at each time point.</p

Immunohistochemical staining of HGF, c-Met and CXCR4 in breast cancer specimens.

<p>Original magnification, 400×. (A) Representative micrographs of immunohistochemical results for negative HGF, c-Met or CXCR4 staining in breast benign diseases (an atypical hyperplasia or carcinoma in situ) and for positive HGF, c-Met or CXCR4 staining in an invasive breast carcinoma; (B) Representative micrographs of immunohistochemical results for positive HGF, c-Met and CXCR4 staining cell counts, where the intensity of immunohistochemical staining correlates with the histopathological grading of invasive breast carcinomas.</p

HGF enhances CXCR4 expression via PKCζ and promotes the invasion and metastasis of breast cancers in BALB/c-nu mice.

<p>(A) Micrographs of H&E-stained xenografts demonstrating the presence or absence of margin invasion by the MDA-MB-436 tumors untransduced (Un) or transduced with GFP-shRNA or PKCζ-shRNA (original magnification, 200×). (B) Representative micrographs of lung (upper) or liver (lower) tissue sections with H&E staining and PCNA immunohistochemical staining (original magnification, 200×). Each group from two independent experiments. (C) Mean ± SD wet lung weight in tumor-bearing mice. The number of mice in each group is indicated. * P<0.05 as compared to PBS-treated mice. (D) Expression of human HPRT mRNA relative to mouse 18S rRNA as determined by qRT-PCR in the lungs and livers of tumor-bearing mice. Data were normalized to PBS-treated mice. # P<0.01 as compared to PBS-treated mice. (E–F) Immunoblot of total PKCζ and p-PKCζ or total CXCR4 and p-CXCR4 protein, respectively, in breast tumor xenografts in female nude mice that were inoculated in the mammary fat pads with MDA-MB-436 cells treated as indicated. The experiment was repeated twice with similar results. A representative study is shown. (G) Representative microphotos of immunohistochemical results for p-PKCζ (middle) or p-CXCR4 (lower) expression in MDA-MB-436 tumors (original magnification, 400×).</p

HGF-induced increase in CXCR4 expression depends on PKCζ activity.

<p>(A). Time course of relative p-PKCζ levels as determined by immunoblot in MDA-MB-436 and MCF-7 cells following stimulation with 50 ng/ml HGF. The experiment was repeated three times with similar results. A representative study is shown. (B) and (C). 100 µM PKCζ-siRNA1 (si1) or PKCζ-siRNA2 (si2) transient transfections and siRNA-mediated PKCζ protein silencing in MDA-MB-436 cells. p-CXCR4 and p-PKCζ expression levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF or/and 100 µM PKCζ-siRNA1 (si1) or PKCζ-siRNA2 (si2). The experiment was repeated three times with similar results. A representative study is shown. (D). Western blot analysis of total CXCR4 expression and p-CXCR4 levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF, 2 µM cholesterol sulfate (Choles sul), or PBS. As indicated, 10 µM PS of PKCζ (PSζ), PKCε (PSε), or PKCα/β (PSα/β) or 25 µM NSC23766 (Rac1 inhibitor, Rac1 inh) was used. The experiment was repeated three times with similar results. A representative study is shown. (E). Membrane CXCR4 expression in MDA-MB-436 cells incubated for 24 hours with 50 ng/ml HGF, 2 µM Choles Sulfate, or PBS. Where indicated, 10 µM PS of PSζ, PSε, or PSα/β was used. The flow cytometry analysis data are shown in arbitrary units (AU) normalized to PBS as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (F). Western blot analysis of Rac1-GTP and total-Rac1 in MDA-MB-436 and MCF-7 cells treated with 50 ng/ml HGF with or without 25 µM NSC23766. The experiment was repeated three times with similar results. A representative study is shown. (G). Dose-dependent inhibition of HGF-induced p-PKCζ was achieved using NSC23766 in HGF-treated MDA-MB-436 cells; the cells were assayed by western blot. The experiment was repeated three times with similar results. A representative study is shown. (H). Western blot analysis of Rac1, PKCζ and CXCR4 expression in MDA-MB-436 cells cultured for 48 hours with 100 µM Rac1-siRNA. The experiment was repeated three times with similar results. A representative study is shown. (I). Cellular distribution of PKCζ (left panel) and CXCR4 (right panel) before and after HGF (50 ng/ml) or NSC23766 (50 µM) stimulation for 10 minutes. Representative of two independent experiments was shown.</p

Overexpressed CXCR4 in HGF-stimulated MDA-MB-436 cells is functional.

<p>(A–B). Confluent cells were grown in 0.5% FBS medium for 24 hours and were then wounded with a tip. The cells were washed, and the medium was replaced with or without addition of HGF, PSζ peptides or AMD3100. Representative micrographs of the wounds are shown together with the results of the migration quantification. Results are presented as the mean ± SD of 3 independent experiments. # P<0.01 as compared to PBS. Original magnification, 200×. (C).Dose and time-dependent response of HGF-induced MDA-MB-436 cell migration. (D).Dose-dependent response of HGF-induced MDA-MB-436 cell chemotaxis. (E).PKC and CXCR4 regulate HGF-mediated chemotaxis. MDA-MB-436 cells were incubated for 30 minutes with 10 µM chelerythrine chloride, an inhibitor of all PKC, or for 1 hour with 10 µM of PS-α/β, PS-ε, or PSζ peptide. (F). Boyden chamber assays were performed using the SDF-1 ligand for CXCR4 as a chemotactic attractive agent in the lower chamber. AMD3100, PSζ, PSα/β, PSε and NSC23766 (upper) or PKCζ-siRNA (lower) was added to the cell culture for the blocking assay. Data are shown as the mean ± SD of three experiments. A representative study is shown. (G). qRT-PCR analysis of the MT1-MMP mRNA extracted from MDA-MB-436 cells cultured for 24 hours with the indicated agents. Results are presented as the mean ± SD of three independent experiments. # P<0.01 as compared to PBS. (H). Western blot analysis of the total protein expression levels of MT1-MMP in MDA-MB-436 cells cultured for 24 hours with the indicated agents. The experiment was repeated three with similar results. A representative study is shown.</p

HOTAIR expression levels were upregulated in ESCC tissues compared with non-ESCC tissues as detected by qRT-PCR.

<p>Total RNA from 30 paired ESCC and non-ESCC specimens was extracted with the Trizol reagent, and HOTAIR expression levels were evaluated by qRT-PCR. The results are expressed as the means ± SD; * <i>P</i><0.001 compared with non-ESCC tissues.</p

Silencing of HOTAIR suppresses TE-1 cell growth and colony formation.

<p>A. Transfection with all siRNAs potently reduced the target mRNA levels. TE-1 cells were transfected with siRNAs against HOTAIR and GFP for 48 h. HOTAIR mRNA levels were evaluated by real-time quantitative PCR. GAPDH was used as an internal control. B. Silencing of HOTAIR suppressed the proliferation of TE-1 cells as determined by an MTT assay. TE-1 cells that were transfected with siRNAs against HOTAIR or GFP for the indicated times were subjected to an MTT assay. C. The effect of HOTAIR on the tumorigenesis of TE-1 cells was examined by a colony formation assay. The results are expressed as the means ± SD; n = 3, * <i>P</i><0.01 compared with the negative control.</p

Correlation between the clinicopathologic features and expression of HOTAIR.

<p>Correlation between the clinicopathologic features and expression of HOTAIR.</p

Silencing of HOTAIR suppresses the migratory capacity of TE-1 cells as determined by a transwell assay.

<p>TE-1 cells that were transfected with siRNAs against HOTAIR or GFP for 48 h were subjected to a transwell assay. The numbers of filtered cells were counted by microscopy. The results are expressed as the means ± SD; n = 3, * <i>P</i><0.01 compared with the negative control.</p