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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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Analysis of the expression of p63 isoforms in human OS cell lines. The human parental low metastatic (non-invasive) SaOS-2, MG63, HOS and HUO9 cell lines and the respective metastatic (invasive) LM5, M8, 134B and M132 sublines were analysed. A. mRNA levels of p63 splice variants in the indicated OS cancer cell lines were determined using semi quantitative PCR. BxPC3 cells served as the positive control and GAPDH mRNA levels were used as loading controls. M, molecular size marker lane. B. Protein levels of ΔNp63α in the indicated OS cell lines were detected using an antibody specific for ΔNp63.
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Fig1: Analysis of the expression of p63 isoforms in human OS cell lines. The human parental low metastatic (non-invasive) SaOS-2, MG63, HOS and HUO9 cell lines and the respective metastatic (invasive) LM5, M8, 134B and M132 sublines were analysed. A. mRNA levels of p63 splice variants in the indicated OS cancer cell lines were determined using semi quantitative PCR. BxPC3 cells served as the positive control and GAPDH mRNA levels were used as loading controls. M, molecular size marker lane. B. Protein levels of ΔNp63α in the indicated OS cell lines were detected using an antibody specific for ΔNp63.

Mentions: Human p63 generates the- α, β, and γ isoforms of TAp63 and ΔNp63 with distinct biological functions. To identify the p63 isoforms expressed by human OS cell lines, we conducted an expression analysis of various OS cell lines that represent heterogeneity of tumors in situ. Transcripts encoding TAp63α were only detected in the non-invasive human cell lines (HOS and HU09) and were undetectable in the invasive OS cell lines (LM5, M8, 143B, M132) (Figure 1A). In contrast, transcripts encoding ΔNp63α were expressed exclusively in the invasive OS cell lines. Transcripts encoding the β and γ isoforms of TAp63 and ΔNp63 were undetectable in the low and high metastatic cell lines.Figure 1


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 the expression of p63 isoforms in human OS cell lines. The human parental low metastatic (non-invasive) SaOS-2, MG63, HOS and HUO9 cell lines and the respective metastatic (invasive) LM5, M8, 134B and M132 sublines were analysed. A. mRNA levels of p63 splice variants in the indicated OS cancer cell lines were determined using semi quantitative PCR. BxPC3 cells served as the positive control and GAPDH mRNA levels were used as loading controls. M, molecular size marker lane. B. Protein levels of ΔNp63α in the indicated OS cell lines were detected using an antibody specific for ΔNp63.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Analysis of the expression of p63 isoforms in human OS cell lines. The human parental low metastatic (non-invasive) SaOS-2, MG63, HOS and HUO9 cell lines and the respective metastatic (invasive) LM5, M8, 134B and M132 sublines were analysed. A. mRNA levels of p63 splice variants in the indicated OS cancer cell lines were determined using semi quantitative PCR. BxPC3 cells served as the positive control and GAPDH mRNA levels were used as loading controls. M, molecular size marker lane. B. Protein levels of ΔNp63α in the indicated OS cell lines were detected using an antibody specific for ΔNp63.
Mentions: Human p63 generates the- α, β, and γ isoforms of TAp63 and ΔNp63 with distinct biological functions. To identify the p63 isoforms expressed by human OS cell lines, we conducted an expression analysis of various OS cell lines that represent heterogeneity of tumors in situ. Transcripts encoding TAp63α were only detected in the non-invasive human cell lines (HOS and HU09) and were undetectable in the invasive OS cell lines (LM5, M8, 143B, M132) (Figure 1A). In contrast, transcripts encoding ΔNp63α were expressed exclusively in the invasive OS cell lines. Transcripts encoding the β and γ isoforms of TAp63 and ΔNp63 were undetectable in the low and high metastatic cell lines.Figure 1

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