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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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Microarray analysis to detect differential gene expression in SaOS-2- ΔNp63α cells. Hierarchical clustering of differentially expressed genes revealed by microarray analysis in SaOS-2- ΔNp63αcells compared with SaOS-2-EV cells. The heat maps depict the fold-change of up- (A) and down-regulated (C) genes in SaOS-2-ΔNp62α compared with SaOS-2-EV cells in descending order of 10 to 3. Quantitative RT-PCR validation of the microarray results obtained for indicated up (B) and down-regulated (D) genes. E. Transcript enrichment in SaOS-2-ΔNp63α cells grouped by functional related terms from the KEGG pathway analysis. F. Genes enriched in Hh signaling pathway are highlighted.
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Fig3: Microarray analysis to detect differential gene expression in SaOS-2- ΔNp63α cells. Hierarchical clustering of differentially expressed genes revealed by microarray analysis in SaOS-2- ΔNp63αcells compared with SaOS-2-EV cells. The heat maps depict the fold-change of up- (A) and down-regulated (C) genes in SaOS-2-ΔNp62α compared with SaOS-2-EV cells in descending order of 10 to 3. Quantitative RT-PCR validation of the microarray results obtained for indicated up (B) and down-regulated (D) genes. E. Transcript enrichment in SaOS-2-ΔNp63α cells grouped by functional related terms from the KEGG pathway analysis. F. Genes enriched in Hh signaling pathway are highlighted.

Mentions: Because ΔNp63α was specifically expressed in invasive OS cell lines and contributed to their oncogenic phenotypes, we analysed the ΔNp63α regulated transcriptome in OS cells to facilitate the identification of genes and signaling pathways that impart the ΔNp63α-mediated oncogenic phenotype of OS cells. Therefore, we compared the global gene expression pattern of SaOS-2-ΔNp63α and control SaOS-2-EV cells. Both cell lines were grown under similar conditions and total RNAs from exponentially growing cells were subjected to microarray analysis. The microarray analysis revealed major differences in the transcriptional profile of SaOS-2-ΔNp63α cells compared with that of the control cells. Thus, 196 and 230 genes were up- or down- regulated by factors of 3 respectively. A heat map showing the differential expression in descending order of 10 and 3 fold up and down-regulated genes respectively is shown in Figure 3A and C.Figure 3


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)

Microarray analysis to detect differential gene expression in SaOS-2- ΔNp63α cells. Hierarchical clustering of differentially expressed genes revealed by microarray analysis in SaOS-2- ΔNp63αcells compared with SaOS-2-EV cells. The heat maps depict the fold-change of up- (A) and down-regulated (C) genes in SaOS-2-ΔNp62α compared with SaOS-2-EV cells in descending order of 10 to 3. Quantitative RT-PCR validation of the microarray results obtained for indicated up (B) and down-regulated (D) genes. E. Transcript enrichment in SaOS-2-ΔNp63α cells grouped by functional related terms from the KEGG pathway analysis. F. Genes enriched in Hh signaling pathway are highlighted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Microarray analysis to detect differential gene expression in SaOS-2- ΔNp63α cells. Hierarchical clustering of differentially expressed genes revealed by microarray analysis in SaOS-2- ΔNp63αcells compared with SaOS-2-EV cells. The heat maps depict the fold-change of up- (A) and down-regulated (C) genes in SaOS-2-ΔNp62α compared with SaOS-2-EV cells in descending order of 10 to 3. Quantitative RT-PCR validation of the microarray results obtained for indicated up (B) and down-regulated (D) genes. E. Transcript enrichment in SaOS-2-ΔNp63α cells grouped by functional related terms from the KEGG pathway analysis. F. Genes enriched in Hh signaling pathway are highlighted.
Mentions: Because ΔNp63α was specifically expressed in invasive OS cell lines and contributed to their oncogenic phenotypes, we analysed the ΔNp63α regulated transcriptome in OS cells to facilitate the identification of genes and signaling pathways that impart the ΔNp63α-mediated oncogenic phenotype of OS cells. Therefore, we compared the global gene expression pattern of SaOS-2-ΔNp63α and control SaOS-2-EV cells. Both cell lines were grown under similar conditions and total RNAs from exponentially growing cells were subjected to microarray analysis. The microarray analysis revealed major differences in the transcriptional profile of SaOS-2-ΔNp63α cells compared with that of the control cells. Thus, 196 and 230 genes were up- or down- regulated by factors of 3 respectively. A heat map showing the differential expression in descending order of 10 and 3 fold up and down-regulated genes respectively is shown in Figure 3A and C.Figure 3

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