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ΔNp63α enhances the oncogenic phenotype of osteosarcoma cells by inducing the expression of GLI2.

Ram Kumar RM, Betz MM, Robl B, Born W, Fuchs B - BMC Cancer (2014)

Bottom Line: ΔNp63, a splice variant of p63, is overexpressed and exhibits oncogenic activity in many cancers including pancreatic and breast cancer and promotes cell survival by inhibiting apoptosis.The comparative expression analyses identified ΔNp63α as the predominant p63 isoform expressed by invasive OS cell lines.Phenotypic analyses of SaOS-2-ΔNp63α cells in vitro indicate that ΔNp63α imparted tumorigenic attributes upon tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Orthopaedic Research, Department of Orthopaedics, Balgrist University Hospital, University of Zurich, Zurich, Switzerland. rkumar@research.balgrist.ch.

ABSTRACT

Background: ΔNp63, a splice variant of p63, is overexpressed and exhibits oncogenic activity in many cancers including pancreatic and breast cancer and promotes cell survival by inhibiting apoptosis. Despite its role in tumorigenesis, mechanistic activity of ΔNp63 mediated oncogenic function in osteosarcoma is poorly understood.

Methods: The expression levels of p63 isoforms in osteosarcoma cell lines were identified using quantitative techniques. Expression profiling using microarray, siRNA mediated loss-of-function, and chromatin immunoprecipitation assays were employed to identify novel ΔNp63α targets in p63- osteosarcoma SaOS-2 cells that were engineered to express ΔNp63α. The phenotype of SaOS-2-ΔNp63α cells was assessed using wound-healing, colony formation, and proliferation assays.

Results: The comparative expression analyses identified ΔNp63α as the predominant p63 isoform expressed by invasive OS cell lines. Phenotypic analyses of SaOS-2-ΔNp63α cells in vitro indicate that ΔNp63α imparted tumorigenic attributes upon tumor cells. Further, we show that in osteosarcoma cells ΔNp63α directly regulated the transcription factor GLI2, which is a component of the hedgehog signaling pathway, and that functional interactions between ΔNp63α and GLI2 confer oncogenic properties upon OS cells.

Conclusions: Here, we report that GLI2 is the novel target gene of ΔNp63α and that ΔNp63α-GLI2 crosstalk in osteosarcoma cells is a necessary event in osteosarcoma progression. Defining the exact mechanisms involved in this interaction that mediate the pathogenesis of osteosarcoma promises to identify targets for drug therapy.

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ΔNp63α activatesGLI2transcription. A. Real time PCR analysis of total RNA isolated from SaOS-2-ΔNp63α and SaOS-2-EV cells shows upregulation of GLI2 expression in SaOS-2-ΔNp63α cells. *P value < 0.05 (n = 3). B. Western blot analysis of cell lysates prepared from SaOS-2-EV and SaOS-2-ΔNp63α cells show the elevated expression of GLI2 in SaOS-2-ΔNp63α cells. C. Western blot analysis of ΔNp63α and GLI2 expression in lysates prepared from 143B and M132 cells after transfection with control or p63 siRNAs. D. Quantification of relative GLI2 expression is shown in panel D. *P value < 0.05 (n = 3). E. The GLI2 promoter and transcription initiation region and p63 consensus-binding site (essential sequences underlined) probed using chromatin immunoprecipitation (ChIP). F. ChIP analysis of occupancy of the GLI2 promoter by ΔNp63α using control IgG or an antibody against ΔNp63 (anti- ΔNp63) in SaOS-2-EV and SaOS-2-ΔNp63α cells as indicated. Amplification of the GLI2 and GAPDH promoter regions using GLI2-1 and GLI2-2 primer sets was analysed using agarose gel electrophoresis. Input control lanes, IgG and GAPDH controls are shown.
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Fig4: ΔNp63α activatesGLI2transcription. A. Real time PCR analysis of total RNA isolated from SaOS-2-ΔNp63α and SaOS-2-EV cells shows upregulation of GLI2 expression in SaOS-2-ΔNp63α cells. *P value < 0.05 (n = 3). B. Western blot analysis of cell lysates prepared from SaOS-2-EV and SaOS-2-ΔNp63α cells show the elevated expression of GLI2 in SaOS-2-ΔNp63α cells. C. Western blot analysis of ΔNp63α and GLI2 expression in lysates prepared from 143B and M132 cells after transfection with control or p63 siRNAs. D. Quantification of relative GLI2 expression is shown in panel D. *P value < 0.05 (n = 3). E. The GLI2 promoter and transcription initiation region and p63 consensus-binding site (essential sequences underlined) probed using chromatin immunoprecipitation (ChIP). F. ChIP analysis of occupancy of the GLI2 promoter by ΔNp63α using control IgG or an antibody against ΔNp63 (anti- ΔNp63) in SaOS-2-EV and SaOS-2-ΔNp63α cells as indicated. Amplification of the GLI2 and GAPDH promoter regions using GLI2-1 and GLI2-2 primer sets was analysed using agarose gel electrophoresis. Input control lanes, IgG and GAPDH controls are shown.

Mentions: The results of the transcriptional analysis presented above supports the conclusion that ΔNP63α specifically activates Hh signaling in OS cells. Moreover, GLI2 is implicated in OS and targeting GLI2 has been suggested for treating OS. We next investigated whether ΔNp63α regulates the transcription of GLI2. We conducted qRT-PCR analysis of SaOS-2-ΔNp63α cells and found that GLI2 mRNA levels were higher by a factor of 4.5 compared with SaOS-2-EV cells (Figure 4A), which is consistent with the microarray data and the western blotting analysis shown in Figure 4B. We next determined the effect of a p63 siRNA on the OS cell lines (143B and M132) that express high levels of endogenous ΔNp63α. The level of ΔNp63α protein was decreased by approximately 80% in p63 siRNA-transfected 143B and M132 cells compared with cells transfected with control siRNA (Figure 4C) and was accompanied by a decrease in the level of endogenous GLI2 (Figure 4C and D). In contrast, the level was unchanged in cells transfected with control siRNA. We determined whether TAp63α also regulate GLI2 expression by using siRNA approach to inhibit p63 expression in OS cells (HOS, HU09) that express endogenous TAp63. GLI2 levels did not change when TAp63 expression was inhibited as shown by the western blot (Additional file 5) thereby indicating that GLI2 is not regulated by TAp63.Figure 4


ΔNp63α enhances the oncogenic phenotype of osteosarcoma cells by inducing the expression of GLI2.

Ram Kumar RM, Betz MM, Robl B, Born W, Fuchs B - BMC Cancer (2014)

ΔNp63α activatesGLI2transcription. A. Real time PCR analysis of total RNA isolated from SaOS-2-ΔNp63α and SaOS-2-EV cells shows upregulation of GLI2 expression in SaOS-2-ΔNp63α cells. *P value < 0.05 (n = 3). B. Western blot analysis of cell lysates prepared from SaOS-2-EV and SaOS-2-ΔNp63α cells show the elevated expression of GLI2 in SaOS-2-ΔNp63α cells. C. Western blot analysis of ΔNp63α and GLI2 expression in lysates prepared from 143B and M132 cells after transfection with control or p63 siRNAs. D. Quantification of relative GLI2 expression is shown in panel D. *P value < 0.05 (n = 3). E. The GLI2 promoter and transcription initiation region and p63 consensus-binding site (essential sequences underlined) probed using chromatin immunoprecipitation (ChIP). F. ChIP analysis of occupancy of the GLI2 promoter by ΔNp63α using control IgG or an antibody against ΔNp63 (anti- ΔNp63) in SaOS-2-EV and SaOS-2-ΔNp63α cells as indicated. Amplification of the GLI2 and GAPDH promoter regions using GLI2-1 and GLI2-2 primer sets was analysed using agarose gel electrophoresis. Input control lanes, IgG and GAPDH controls are shown.
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Fig4: ΔNp63α activatesGLI2transcription. A. Real time PCR analysis of total RNA isolated from SaOS-2-ΔNp63α and SaOS-2-EV cells shows upregulation of GLI2 expression in SaOS-2-ΔNp63α cells. *P value < 0.05 (n = 3). B. Western blot analysis of cell lysates prepared from SaOS-2-EV and SaOS-2-ΔNp63α cells show the elevated expression of GLI2 in SaOS-2-ΔNp63α cells. C. Western blot analysis of ΔNp63α and GLI2 expression in lysates prepared from 143B and M132 cells after transfection with control or p63 siRNAs. D. Quantification of relative GLI2 expression is shown in panel D. *P value < 0.05 (n = 3). E. The GLI2 promoter and transcription initiation region and p63 consensus-binding site (essential sequences underlined) probed using chromatin immunoprecipitation (ChIP). F. ChIP analysis of occupancy of the GLI2 promoter by ΔNp63α using control IgG or an antibody against ΔNp63 (anti- ΔNp63) in SaOS-2-EV and SaOS-2-ΔNp63α cells as indicated. Amplification of the GLI2 and GAPDH promoter regions using GLI2-1 and GLI2-2 primer sets was analysed using agarose gel electrophoresis. Input control lanes, IgG and GAPDH controls are shown.
Mentions: The results of the transcriptional analysis presented above supports the conclusion that ΔNP63α specifically activates Hh signaling in OS cells. Moreover, GLI2 is implicated in OS and targeting GLI2 has been suggested for treating OS. We next investigated whether ΔNp63α regulates the transcription of GLI2. We conducted qRT-PCR analysis of SaOS-2-ΔNp63α cells and found that GLI2 mRNA levels were higher by a factor of 4.5 compared with SaOS-2-EV cells (Figure 4A), which is consistent with the microarray data and the western blotting analysis shown in Figure 4B. We next determined the effect of a p63 siRNA on the OS cell lines (143B and M132) that express high levels of endogenous ΔNp63α. The level of ΔNp63α protein was decreased by approximately 80% in p63 siRNA-transfected 143B and M132 cells compared with cells transfected with control siRNA (Figure 4C) and was accompanied by a decrease in the level of endogenous GLI2 (Figure 4C and D). In contrast, the level was unchanged in cells transfected with control siRNA. We determined whether TAp63α also regulate GLI2 expression by using siRNA approach to inhibit p63 expression in OS cells (HOS, HU09) that express endogenous TAp63. GLI2 levels did not change when TAp63 expression was inhibited as shown by the western blot (Additional file 5) thereby indicating that GLI2 is not regulated by TAp63.Figure 4

Bottom Line: ΔNp63, a splice variant of p63, is overexpressed and exhibits oncogenic activity in many cancers including pancreatic and breast cancer and promotes cell survival by inhibiting apoptosis.The comparative expression analyses identified ΔNp63α as the predominant p63 isoform expressed by invasive OS cell lines.Phenotypic analyses of SaOS-2-ΔNp63α cells in vitro indicate that ΔNp63α imparted tumorigenic attributes upon tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Orthopaedic Research, Department of Orthopaedics, Balgrist University Hospital, University of Zurich, Zurich, Switzerland. rkumar@research.balgrist.ch.

ABSTRACT

Background: ΔNp63, a splice variant of p63, is overexpressed and exhibits oncogenic activity in many cancers including pancreatic and breast cancer and promotes cell survival by inhibiting apoptosis. Despite its role in tumorigenesis, mechanistic activity of ΔNp63 mediated oncogenic function in osteosarcoma is poorly understood.

Methods: The expression levels of p63 isoforms in osteosarcoma cell lines were identified using quantitative techniques. Expression profiling using microarray, siRNA mediated loss-of-function, and chromatin immunoprecipitation assays were employed to identify novel ΔNp63α targets in p63- osteosarcoma SaOS-2 cells that were engineered to express ΔNp63α. The phenotype of SaOS-2-ΔNp63α cells was assessed using wound-healing, colony formation, and proliferation assays.

Results: The comparative expression analyses identified ΔNp63α as the predominant p63 isoform expressed by invasive OS cell lines. Phenotypic analyses of SaOS-2-ΔNp63α cells in vitro indicate that ΔNp63α imparted tumorigenic attributes upon tumor cells. Further, we show that in osteosarcoma cells ΔNp63α directly regulated the transcription factor GLI2, which is a component of the hedgehog signaling pathway, and that functional interactions between ΔNp63α and GLI2 confer oncogenic properties upon OS cells.

Conclusions: Here, we report that GLI2 is the novel target gene of ΔNp63α and that ΔNp63α-GLI2 crosstalk in osteosarcoma cells is a necessary event in osteosarcoma progression. Defining the exact mechanisms involved in this interaction that mediate the pathogenesis of osteosarcoma promises to identify targets for drug therapy.

Show MeSH
Related in: MedlinePlus