FoxM1 Promotes Glioma Cells Progression by Up-Regulating Anxa1 Expression

<div><p>Forkhead box M1 (FoxM1) is a member of the forkhead transcription factor family and is overexpression in malignant gliomas. However, the molecular mechanisms by which FoxM1lead to glioma carcinogenesis and progression are still not well known. In the present study, we show that Anxa1 was overexpression in gliomas and predicted the poor outcome. Furthermore, Anxa1 closely related to the FoxM1 expression and was a direct transcriptional target of FoxM1. Overexpression of FoxM1 up-regulated Anxa1 expression, whereas suppression of FoxM1 expression down-regulated Anxa1 expression in glioma cells. Finally, FoxM1 enhanced the proliferation, migration, and angiogenesis in Anxa1-dependent manner both <i>in vitro</i> and <i>in vivo</i>. Our findings provide both clinical and mechanistic evidences that FoxM1 contributes to glioma development by directly up-regulating Anxa1 expression.</p></div

Effect of FoxM1/Anxa1 expression on proliferation and migration of glioma cells <i>in vitro</i>.

<p><b>A,</b> RT-qPCR and Western blot analyses of FoxM1 and Anxa1 expression in stable pcDNA3.1-Anxa1-transfected U-87MG-RNAi cells (left) and Anxa1-shRNA-transfected SW0188-FoxM1 cells (right). <b>B,</b> Cells as in (A) were cultured in 96-well plates and analyzed by MTT assay. Cell proliferation curves were shown in 9 days. Three independent experiments were conducted. <b>C,</b> Cells as in (A) were examined for cell migration motility in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet (upper) and counted under a light microscope (lower). Three independent experiments were conducted. <b>D,</b> the angiogenic potential of glioma cells was determined by endothelial cell tube formation assay. Capillary tube formation in each group was photographed and quantified. *<i>P</i><0.05.</p

Expression of FoxM1 and Anxa1 in human normal brain and glioma tissues.

<p><b>A,</b> Anxa1 and FoxM1 mRNA expression by RT-qPCR. The mRNA expression was analyzed in 30 matched primary glioblastoma tissues and the adjacent normal brain tissues. B, FoxM1 expression levels correlated positively with Anxa1 expression levels in glioblastoma samples (Pearson’s correlation test r = 0.364; <i>P</i> = 0.048). <b>C,</b> Headmay of a glioblastoma microarray data set from ONCOMINE data showing the expression levels of FoxM1 and Anxa1. The FoxM1 expression is correlated with Anxa1 expression (r = 0.327, <i>P</i><0.0001). <b>D,</b> FoxM1 and Anxa1 protein expression levels in 4 matched primary glioblastoma tissues and the adjacent normal brain tissues by Western blot analysis. <b>E and F,</b> Kaplan-Meier estimates of overall survival time in patients who had a glioblastoma with different Anxa1 or FoxM1 expression.</p

Effect of FoxM1/Anxa1 expression on glioma growth in the brains of nude mice.

<p><b>A,</b> Glioma cells (1×10⁶) were implanted intracranially into nude mice. Mice were euthanized when they were moribund or on day 90. * Incidence: number of mice with tumor/number of mice injected. <b>B,</b> Kaplan-Meier estimates of overall survival time in nude injected with glioma cells (P<0.001). <b>C,</b> Morphologic alteration of the xenograft tumors was analyzed by H&E. D, CD31expression level in xenograft tumors was analyzed by IHC.</p

Effects of altered FoxM1 expression on Anxa1 expression in human glioma cell lines.

<p><b>A,</b> Determination of FoxM1 and Anxa1 expression in human glioma cell lines using RT-qPCR (lower) and Western blot (upper). <b>B,</b> Up-regulation of Anxa1 mRNA and protein expression by overexpressing FoxM1. FoxM1 and Anxa1 expression levels in parental, control, and Hs683-FoxM1 and SW1088-FoxM1 cells by RT-qPCR (lower) and Western blot (upper). <b>C,</b> Down-regulation of Anxa1 mRNA and protein expression by depletion of FoxM1 expression. FoxM1 and Anxa1 expression in parental, control, and LN-229-RNAi and U-87MG-RNAi cells by RT-qPCR (lower) and Western blot (upper).</p

The Anxa1 as a transcriptional target of FoxM1.

<p><b>A,</b> transactivation of the Anxa1 promoter in SW1088-FoxM1 cells (left) and repression of the Anxa1 promoter in U-87MG-RNAi cells (right). Activation was calculated relative to SW1088 cells and inhibition was calculated as a percentage relative to U-87MG cells. Three independent experiments were conducted. <b>B,</b> Sequence and position of putative FoxM1 binding site on the Anxa1 promoter. <b>C,</b> ChIP assays were done with SW1088, SW1088-FoxM1, U-87MG and U-87MG-RNAi cells. Chromatin fragments of the cells were immunoprecipitated with anti-FoxM1 antibody (top) or negative control IgG (middle) and subjected to PCR. We subjected 1% of the total cell lysates to PCR before immunoprecipitation as inputs (bottom). <b>D,</b> schematic structure of the Anxa1 promoter. The sequence of the FoxM1 binding site is shown in both wild-type (WT) and mutant (Mut) forms. <b>E,</b> Luciferase activity with or without mutation in Anxa1 promoter. SW1088-FoxM1, U-87MG-RNAi, controls and parental cells were transfected with the wild-type Anxa1 promoter or its mutant. Three independent experiments were conducted. *<i>P</i><0.01.</p

Expression of FoxQ1 and NRXN3 in human normal brain and glioma tissues.

<p><b>A</b>, <i>NRXN3</i> and <i>FoxQ1</i> mRNA expression by RT-qPCR. The mRNA expression was analyzed in 30 matched primary glioblastoma tissues and the adjacent normal brain tissues. <b>B</b>, FoxQ1 expression levels correlated negatively with NRXN3 expression levels in glioblastoma samples (Pearson's correlation test <i>r</i> = −0.373; <i>P</i> = 0.042). <b>C,</b> Expression of FoxQ1 and NRXN3 protein in primary glioblastoma tissues and the adjacent normal brain tissues. Normal (N) and tumor (T) samples were analyzed by western blot. β-actin used as the loading control.</p

FoxQ1 suppress the NRXN3 expression in human glioma cell lines.

<p><b>A</b>, Determination of FoxQ1 and NRXN3 expression in human glioma cell lines and normal human astrocytes using RT-qPCR (lower) and Western blot (upper). <b>B</b>, Up-regulation of NRXN3 mRNA and protein expression by overexpressing FoxQ1. FoxQ1 and NRXN3 expression levels in parental, control, SW1088-FoxQ1 cells by RT-qPCR (lower) and Western blot (upper). <b>C</b>, Down-regulation of NRXN3 mRNA and protein expression by depletion of FoxQ1 expression. FoxQ1 and NRXN3 expression in parental, control, and U-87MG-RNAi cells by RT-qPCR (lower) and Western blot (upper). <b>E+F</b>, Effect of FoxQ1 on NRXN3 promoter activity. Repression of the NRXN3 promoter in SW1088-FoxQ1 cells (E) and transactivation of the NRXN3 promoter in U-87MG-RNAi cells (F). Inhibition was calculated as a percentage relative to U-87MG cells and activation was calculated relative to SW1088 cells. Three independent experiments were conducted.</p

Down-regulation of NRXN3 rescues the malignant phenotype of FoxQ1 down-regulated glioma cells <i>in vitro</i> and <i>in vivo</i>.

<p>A, Western blot (upper) and RT-qPCR (lower) analyses of FoxQ1 and NRXN3 expression in stable NRXN3-rescued U-87MG cells. B, Cells as in (A) were cultured in 96-well plates and analyzed by MTT assay. Cell proliferation curves were shown in 9 days. Three independent experiments were conducted. C, Cells as in (A) were examined for cell migration motility in 24-well plates with transwell chambers. Migrated cells were stained with crystal violet and counted under a light microscope. Three independent experiments were conducted. D, Glioma cells (1×10⁶) were implanted intracranially into nude mice. Mice were euthanized when they were moribund or on day 90. * Incidence: number of mice with tumor/number of mice injected. E, Kaplan-Meier estimates of overall survival time in nude injected with glioma cells. *<i>P</i><0.05.</p

The NRXN3 as a transcriptional target of FoxQ1.

<p><b>A</b>, Sequence and position of putative FoxQ1 binding sites on the NRXN3 promoter. <b>B</b>, ChIP assays were done with U-87MG cells. Chromatin fragments of the cells were immunoprecipitated with anti-FoxQ1 antibody or negative control IgG (middle) and subjected to PCR. We subjected 1% of the total cell lysates to PCR before immunoprecipitation as inputs. <b>C</b>, schematic structure of the NRXN3 promoter. The sequence of the FoxQ1 binding sites are shown in both wild-type (WT) and mutant (Mut) forms. <b>D+E</b>, Luciferase activity with or without mutations in NRXN3 promoter. U-87MG cells were transfected with the wild-type NRXN3 promoter or its mutants (D). SW1088 cells were co-transfected with the wild-type NRXN3 promoter or its mutants and pcDNA3.1-FoxQ1 (E). Luciferase activities were then determined. Three independent experiments were conducted. * <i>P</i><0.05</p