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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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Related in: MedlinePlus

Analysis of ΔNp63α and GLI2 expression in OS cell lines. A. qRT PCR analysis of ΔNp63α and GLI2 mRNA expression in a panel of OS cell lines as indicated. GAPDH mRNA was used as an internal control. B. Immunocytochemical analysis of ΔNp63α and GLI2 expression in SaOS-2-ΔNp63α, SaOS-2-EV and 143B cells.
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Fig5: Analysis of ΔNp63α and GLI2 expression in OS cell lines. A. qRT PCR analysis of ΔNp63α and GLI2 mRNA expression in a panel of OS cell lines as indicated. GAPDH mRNA was used as an internal control. B. Immunocytochemical analysis of ΔNp63α and GLI2 expression in SaOS-2-ΔNp63α, SaOS-2-EV and 143B cells.

Mentions: The coordinate regulation of the expression of GLI2 and ΔNp63α in invasive and the cognate parental non- invasive OS cell lines was further confirmed by the results of qRT-PCR analysis (Figure 5A). The levels of mRNAs encoding ΔNp63α and GLI2 were up-regulated only in the invasive cell lines. To gain further insight into the functional correlation between ΔNp63α and GLI2, we analysed their expression in 143B cells that expressed endogenous ΔNp63α and GLI2 and in SaOS-2-ΔNp63α cells. Double-immunofluorescence assays detected ΔNp63α and GLI2 in both cell lines suggesting the presence of a ΔNp63α-GLI2 signaling axis in OS cells (Figure 5B). Neither protein was detected in SaOS-2-EV cells.Figure 5


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)

Analysis of ΔNp63α and GLI2 expression in OS cell lines. A. qRT PCR analysis of ΔNp63α and GLI2 mRNA expression in a panel of OS cell lines as indicated. GAPDH mRNA was used as an internal control. B. Immunocytochemical analysis of ΔNp63α and GLI2 expression in SaOS-2-ΔNp63α, SaOS-2-EV and 143B cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125704&req=5

Fig5: Analysis of ΔNp63α and GLI2 expression in OS cell lines. A. qRT PCR analysis of ΔNp63α and GLI2 mRNA expression in a panel of OS cell lines as indicated. GAPDH mRNA was used as an internal control. B. Immunocytochemical analysis of ΔNp63α and GLI2 expression in SaOS-2-ΔNp63α, SaOS-2-EV and 143B cells.
Mentions: The coordinate regulation of the expression of GLI2 and ΔNp63α in invasive and the cognate parental non- invasive OS cell lines was further confirmed by the results of qRT-PCR analysis (Figure 5A). The levels of mRNAs encoding ΔNp63α and GLI2 were up-regulated only in the invasive cell lines. To gain further insight into the functional correlation between ΔNp63α and GLI2, we analysed their expression in 143B cells that expressed endogenous ΔNp63α and GLI2 and in SaOS-2-ΔNp63α cells. Double-immunofluorescence assays detected ΔNp63α and GLI2 in both cell lines suggesting the presence of a ΔNp63α-GLI2 signaling axis in OS cells (Figure 5B). Neither protein was detected in SaOS-2-EV cells.Figure 5

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