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CCAAT/enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4.

Aguilar-Morante D, Morales-Garcia JA, Santos A, Perez-Castillo A - Oncotarget (2015)

Bottom Line: In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion.Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells.By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, (CSIC-UAM), Departamento Modelos Experimentales de Enfermedades Humanas, Arturo Duperier, Madrid, Spain.

ABSTRACT
We have previously shown that decreased expression of CCAAT/enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells.

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Effect of C/EBPβ and S100A4 on GL261 cells motilityC1, I4 and I5 cells transfected with the S100A4 expression vector pIRES2-DsRed-Express or the corresponding control vector were grown until reach confluence. A linear scratch was performed with a plastic pipette tip. Images were taken with a phase contrast microscope at different times after wounding. (A) Representative phase-contrast images and (B) quantifications of the in vitro wound-healing assay are shown. Bar scale 100 μm. ***p < 0.001; *p < 0.05.
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Figure 4: Effect of C/EBPβ and S100A4 on GL261 cells motilityC1, I4 and I5 cells transfected with the S100A4 expression vector pIRES2-DsRed-Express or the corresponding control vector were grown until reach confluence. A linear scratch was performed with a plastic pipette tip. Images were taken with a phase contrast microscope at different times after wounding. (A) Representative phase-contrast images and (B) quantifications of the in vitro wound-healing assay are shown. Bar scale 100 μm. ***p < 0.001; *p < 0.05.

Mentions: Glioblastoma cells are characterized for their capacity to invade normal surrounding tissue. S100A4 is a well-known inductor of tumor cell motility and metastasis in different cancer cells [31], although its role in the invasiveness capacity of glioblastoma cells is not yet known. Previous data from our laboratory, using the “scratch-wound” assay showed that GL261 glioblastoma cells depleted of C/EBPβ presented a restricted cell motility [18]. Here we have analyzed the effect of S100A4 overexpression (Figure 3A) in invasion (transwell assay) and motility (scratch assay) of control and C/EBPβ-depleted GL261glioblastoma cells. As shown in Figure 3B, overexpression of S100A4 caused a clear increased in the invasion capacity of C1, I4 and I5, being more marked in the C/EBPβ-depleted cells. The I4 and I5 cells transfected with the pIRES2-DsRed-Express vector containing S100A4 cDNA showed an increase in their invasion capacity to levels similar to those found in the non-depleted cells (Figure 3B). Regarding cell motility, our results clearly show that the overexpression of S100A4 increased motility in all GL261 cells, C1, I4 and I5 (Figure 4A and 4B). These results are in accordance with the invasion results described above and suggest that S100A4 gene controls the motility of glioblastoma cells and that therefore could mediate the effects of C/EBPβ on motility and invasion.


CCAAT/enhancer binding protein β induces motility and invasion of glioblastoma cells through transcriptional regulation of the calcium binding protein S100A4.

Aguilar-Morante D, Morales-Garcia JA, Santos A, Perez-Castillo A - Oncotarget (2015)

Effect of C/EBPβ and S100A4 on GL261 cells motilityC1, I4 and I5 cells transfected with the S100A4 expression vector pIRES2-DsRed-Express or the corresponding control vector were grown until reach confluence. A linear scratch was performed with a plastic pipette tip. Images were taken with a phase contrast microscope at different times after wounding. (A) Representative phase-contrast images and (B) quantifications of the in vitro wound-healing assay are shown. Bar scale 100 μm. ***p < 0.001; *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414196&req=5

Figure 4: Effect of C/EBPβ and S100A4 on GL261 cells motilityC1, I4 and I5 cells transfected with the S100A4 expression vector pIRES2-DsRed-Express or the corresponding control vector were grown until reach confluence. A linear scratch was performed with a plastic pipette tip. Images were taken with a phase contrast microscope at different times after wounding. (A) Representative phase-contrast images and (B) quantifications of the in vitro wound-healing assay are shown. Bar scale 100 μm. ***p < 0.001; *p < 0.05.
Mentions: Glioblastoma cells are characterized for their capacity to invade normal surrounding tissue. S100A4 is a well-known inductor of tumor cell motility and metastasis in different cancer cells [31], although its role in the invasiveness capacity of glioblastoma cells is not yet known. Previous data from our laboratory, using the “scratch-wound” assay showed that GL261 glioblastoma cells depleted of C/EBPβ presented a restricted cell motility [18]. Here we have analyzed the effect of S100A4 overexpression (Figure 3A) in invasion (transwell assay) and motility (scratch assay) of control and C/EBPβ-depleted GL261glioblastoma cells. As shown in Figure 3B, overexpression of S100A4 caused a clear increased in the invasion capacity of C1, I4 and I5, being more marked in the C/EBPβ-depleted cells. The I4 and I5 cells transfected with the pIRES2-DsRed-Express vector containing S100A4 cDNA showed an increase in their invasion capacity to levels similar to those found in the non-depleted cells (Figure 3B). Regarding cell motility, our results clearly show that the overexpression of S100A4 increased motility in all GL261 cells, C1, I4 and I5 (Figure 4A and 4B). These results are in accordance with the invasion results described above and suggest that S100A4 gene controls the motility of glioblastoma cells and that therefore could mediate the effects of C/EBPβ on motility and invasion.

Bottom Line: In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion.Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells.By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, (CSIC-UAM), Departamento Modelos Experimentales de Enfermedades Humanas, Arturo Duperier, Madrid, Spain.

ABSTRACT
We have previously shown that decreased expression of CCAAT/enhancer binding protein β (C/EBPβ) inhibits the growth of glioblastoma cells and diminishes their transformation capacity and migration. In agreement with this, we showed that C/EBPβ depletion decreases the mRNA levels of different genes involved in metastasis and invasion. Among these, we found S100 calcium binding protein A4 (S100A4) to be almost undetectable in glioblastoma cells deficient in C/EBPβ. Here, we have evaluated the possible role of S100A4 in the observed effects of C/EBPβ in glioblastoma cells and the mechanism through which S100A4 levels are controlled by C/EBPβ. Our results show that C/EBPβ suppression significantly reduced the levels of S100A4 in murine GL261 and human T98G glioblastoma cells. By employing an S100A4-promoter reporter, we observed a significant induction in the transcriptional activation of the S100A4 gene by C/EBPβ. Furthermore, overexpression of S100A4 in C/EBPβ-depleted glioblastoma cells reverses the enhanced migration and motility induced by this transcription factor. Our data also point to a role of S100A4 in glioblastoma cell invasion and suggest that the C/EBPβ gene controls the invasive potential of GL261 and T98G cells through direct regulation of S100A4. Finally, this study indicates a role of C/EBPβ on the maintenance of the stem cell population present in GL261 glioblastoma cells.

Show MeSH
Related in: MedlinePlus