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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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GLI2 mediates the oncogenic potential of ΔNp63α in OS cells. A. Cell lysates of SaOS-2-EV and SaOS-2-ΔNp62α cells untreated or treated with 20 μM GANT61 were analysed using western blotting with an anti- GLI2 antibody. B. GANT61 (20 μM) reversed enhanced anchorage-independent growth in SaOS-2-ΔNp62α cells determined using a soft agar assay and (C) proliferation was assessed using a WST assay. Data represent the mean ± SE of three independent experiments. *P value < 0.05. D. SaOS-2-EV, SaOS-2-ΔNp62α and 143B cells were treated with 20 μM GANT61 and subjected to cell cycle analysis 24 h after drug treatment. SaOS-2-ΔNp62α and 143B cells exposed to GANT61 were arrested in G1 phase. E. Real time PCR analysis of mRNA expression of cell-cycle related genes in SaOS-2-EV and SaOS-2-ΔNp62α cells. 24 h treatment with GANT61 reduced the levels of CyclinD1 and SKPC1 but not n-MYC. *P value < 0.05 (n = 3).
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Fig6: GLI2 mediates the oncogenic potential of ΔNp63α in OS cells. A. Cell lysates of SaOS-2-EV and SaOS-2-ΔNp62α cells untreated or treated with 20 μM GANT61 were analysed using western blotting with an anti- GLI2 antibody. B. GANT61 (20 μM) reversed enhanced anchorage-independent growth in SaOS-2-ΔNp62α cells determined using a soft agar assay and (C) proliferation was assessed using a WST assay. Data represent the mean ± SE of three independent experiments. *P value < 0.05. D. SaOS-2-EV, SaOS-2-ΔNp62α and 143B cells were treated with 20 μM GANT61 and subjected to cell cycle analysis 24 h after drug treatment. SaOS-2-ΔNp62α and 143B cells exposed to GANT61 were arrested in G1 phase. E. Real time PCR analysis of mRNA expression of cell-cycle related genes in SaOS-2-EV and SaOS-2-ΔNp62α cells. 24 h treatment with GANT61 reduced the levels of CyclinD1 and SKPC1 but not n-MYC. *P value < 0.05 (n = 3).

Mentions: We next asked whether GLI2 mediated the oncogenic activities of ΔNp63α by treating SaOS-2-ΔNp63α cells with the small molecule GANT61 that specifically inhibit the binding of GLI1 and GLI2 to DNA and inhibits downstream signaling through the Hh pathway [23]. GLI2 expression was significantly inhibited in SaOS-2-ΔNp63α cells treated with 20 μM GANT61 (Figure 6A). In contrast, the levels of ΔNp63α were not affected (Additional file 8).


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)

GLI2 mediates the oncogenic potential of ΔNp63α in OS cells. A. Cell lysates of SaOS-2-EV and SaOS-2-ΔNp62α cells untreated or treated with 20 μM GANT61 were analysed using western blotting with an anti- GLI2 antibody. B. GANT61 (20 μM) reversed enhanced anchorage-independent growth in SaOS-2-ΔNp62α cells determined using a soft agar assay and (C) proliferation was assessed using a WST assay. Data represent the mean ± SE of three independent experiments. *P value < 0.05. D. SaOS-2-EV, SaOS-2-ΔNp62α and 143B cells were treated with 20 μM GANT61 and subjected to cell cycle analysis 24 h after drug treatment. SaOS-2-ΔNp62α and 143B cells exposed to GANT61 were arrested in G1 phase. E. Real time PCR analysis of mRNA expression of cell-cycle related genes in SaOS-2-EV and SaOS-2-ΔNp62α cells. 24 h treatment with GANT61 reduced the levels of CyclinD1 and SKPC1 but not n-MYC. *P value < 0.05 (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: GLI2 mediates the oncogenic potential of ΔNp63α in OS cells. A. Cell lysates of SaOS-2-EV and SaOS-2-ΔNp62α cells untreated or treated with 20 μM GANT61 were analysed using western blotting with an anti- GLI2 antibody. B. GANT61 (20 μM) reversed enhanced anchorage-independent growth in SaOS-2-ΔNp62α cells determined using a soft agar assay and (C) proliferation was assessed using a WST assay. Data represent the mean ± SE of three independent experiments. *P value < 0.05. D. SaOS-2-EV, SaOS-2-ΔNp62α and 143B cells were treated with 20 μM GANT61 and subjected to cell cycle analysis 24 h after drug treatment. SaOS-2-ΔNp62α and 143B cells exposed to GANT61 were arrested in G1 phase. E. Real time PCR analysis of mRNA expression of cell-cycle related genes in SaOS-2-EV and SaOS-2-ΔNp62α cells. 24 h treatment with GANT61 reduced the levels of CyclinD1 and SKPC1 but not n-MYC. *P value < 0.05 (n = 3).
Mentions: We next asked whether GLI2 mediated the oncogenic activities of ΔNp63α by treating SaOS-2-ΔNp63α cells with the small molecule GANT61 that specifically inhibit the binding of GLI1 and GLI2 to DNA and inhibits downstream signaling through the Hh pathway [23]. GLI2 expression was significantly inhibited in SaOS-2-ΔNp63α cells treated with 20 μM GANT61 (Figure 6A). In contrast, the levels of ΔNp63α were not affected (Additional file 8).

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