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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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Overexpression of ΔNp63α enhanced the malignant phenotype of SaOS-2 cells. A. Analysis of the motilities of SaOS-2- ΔNp63α, SaOS-2-EV and SaOS-2-TAp63α cells in a wound healing assay. Representative photomicrographs of scratch-wounds at the indicated time points after wounding. Arrows indicate the wound width. B. Quantitative analysis of wound closure in cultures of SaOS-2-EV, SaOS-2-ΔNp63α and SaOS-2-TAp63α cells. C. Analysis of anchorage-independent growth in soft agar of SaOS-2-ΔNp63α, SaOS-2-TAp63α and SaOS-2-EV cells. Representative images of colonies stained with 0.005% crystal violet. D. Quantitative analysis of colony formation in soft agar. The numbers of colonies formed by SaOS-2-EV cells were defined as 1. E. Analysis of proliferation of SaOS-2-ΔNp63α cells, SaOS-2-EV and SaOS-2-TAp63α cells using a WST assay. Data shown in 2 B, 2 D and 2E represent the mean ± SEM of three independent experiments ( *P < 0.05).
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Fig2: Overexpression of ΔNp63α enhanced the malignant phenotype of SaOS-2 cells. A. Analysis of the motilities of SaOS-2- ΔNp63α, SaOS-2-EV and SaOS-2-TAp63α cells in a wound healing assay. Representative photomicrographs of scratch-wounds at the indicated time points after wounding. Arrows indicate the wound width. B. Quantitative analysis of wound closure in cultures of SaOS-2-EV, SaOS-2-ΔNp63α and SaOS-2-TAp63α cells. C. Analysis of anchorage-independent growth in soft agar of SaOS-2-ΔNp63α, SaOS-2-TAp63α and SaOS-2-EV cells. Representative images of colonies stained with 0.005% crystal violet. D. Quantitative analysis of colony formation in soft agar. The numbers of colonies formed by SaOS-2-EV cells were defined as 1. E. Analysis of proliferation of SaOS-2-ΔNp63α cells, SaOS-2-EV and SaOS-2-TAp63α cells using a WST assay. Data shown in 2 B, 2 D and 2E represent the mean ± SEM of three independent experiments ( *P < 0.05).

Mentions: To investigate the effects of ΔNp63α expression in OS, we engineered the non-invasive human osteoblastic p63-/-//p53-/- SaOS-2 OS cell line to stably express ΔNp63α. Because SaOS-2 cells do not express p63 or p53, we asked whether ectopic expression of ΔNp63α in SaOS-2 cells (SaOS-2-ΔNp63α) would reveal its functional significance in OS. SaOS-2 cells transfected with empty vector (SaOS-2-EV) or the TAp63α expression construct (SaOS-2-TAp63α) served as controls. We tested whether ΔNp63α expression altered cell motility, proliferation, or both of non- invasive osteoblast cell lines. The result of the in vitro wound-healing assay demonstrated that ΔNp63α enhanced cell motility towards the wound area compared with SaOS-2-TAp63α or SaOS-2-EV cells. The motility of SaOS-2-ΔNp63α cells was significantly higher (P < 0.05) than that of SaOS-2-TAp63α and SaOS-2-EV cells (Figure 2A and B). Because anchorage-independent growth characterizes oncogenic transformation and metastatic potential is indicated by the ability of cells to form small colonies in soft agar, we asked whether ΔNp63α regulated anchorage- independent growth. The number of colonies formed by SaOS-2-ΔNp63α cells was higher by a factor of 2.6 (P < 0.05) compared with SaOS-2-TAp63α or SaOS-2-EV cells. This demonstrated that ectopic expression of ΔNp63α in SaOS-2 cells facilitated anchorage-independent growth (Figure 2C and D).Figure 2


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)

Overexpression of ΔNp63α enhanced the malignant phenotype of SaOS-2 cells. A. Analysis of the motilities of SaOS-2- ΔNp63α, SaOS-2-EV and SaOS-2-TAp63α cells in a wound healing assay. Representative photomicrographs of scratch-wounds at the indicated time points after wounding. Arrows indicate the wound width. B. Quantitative analysis of wound closure in cultures of SaOS-2-EV, SaOS-2-ΔNp63α and SaOS-2-TAp63α cells. C. Analysis of anchorage-independent growth in soft agar of SaOS-2-ΔNp63α, SaOS-2-TAp63α and SaOS-2-EV cells. Representative images of colonies stained with 0.005% crystal violet. D. Quantitative analysis of colony formation in soft agar. The numbers of colonies formed by SaOS-2-EV cells were defined as 1. E. Analysis of proliferation of SaOS-2-ΔNp63α cells, SaOS-2-EV and SaOS-2-TAp63α cells using a WST assay. Data shown in 2 B, 2 D and 2E represent the mean ± SEM of three independent experiments ( *P < 0.05).
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Fig2: Overexpression of ΔNp63α enhanced the malignant phenotype of SaOS-2 cells. A. Analysis of the motilities of SaOS-2- ΔNp63α, SaOS-2-EV and SaOS-2-TAp63α cells in a wound healing assay. Representative photomicrographs of scratch-wounds at the indicated time points after wounding. Arrows indicate the wound width. B. Quantitative analysis of wound closure in cultures of SaOS-2-EV, SaOS-2-ΔNp63α and SaOS-2-TAp63α cells. C. Analysis of anchorage-independent growth in soft agar of SaOS-2-ΔNp63α, SaOS-2-TAp63α and SaOS-2-EV cells. Representative images of colonies stained with 0.005% crystal violet. D. Quantitative analysis of colony formation in soft agar. The numbers of colonies formed by SaOS-2-EV cells were defined as 1. E. Analysis of proliferation of SaOS-2-ΔNp63α cells, SaOS-2-EV and SaOS-2-TAp63α cells using a WST assay. Data shown in 2 B, 2 D and 2E represent the mean ± SEM of three independent experiments ( *P < 0.05).
Mentions: To investigate the effects of ΔNp63α expression in OS, we engineered the non-invasive human osteoblastic p63-/-//p53-/- SaOS-2 OS cell line to stably express ΔNp63α. Because SaOS-2 cells do not express p63 or p53, we asked whether ectopic expression of ΔNp63α in SaOS-2 cells (SaOS-2-ΔNp63α) would reveal its functional significance in OS. SaOS-2 cells transfected with empty vector (SaOS-2-EV) or the TAp63α expression construct (SaOS-2-TAp63α) served as controls. We tested whether ΔNp63α expression altered cell motility, proliferation, or both of non- invasive osteoblast cell lines. The result of the in vitro wound-healing assay demonstrated that ΔNp63α enhanced cell motility towards the wound area compared with SaOS-2-TAp63α or SaOS-2-EV cells. The motility of SaOS-2-ΔNp63α cells was significantly higher (P < 0.05) than that of SaOS-2-TAp63α and SaOS-2-EV cells (Figure 2A and B). Because anchorage-independent growth characterizes oncogenic transformation and metastatic potential is indicated by the ability of cells to form small colonies in soft agar, we asked whether ΔNp63α regulated anchorage- independent growth. The number of colonies formed by SaOS-2-ΔNp63α cells was higher by a factor of 2.6 (P < 0.05) compared with SaOS-2-TAp63α or SaOS-2-EV cells. This demonstrated that ectopic expression of ΔNp63α in SaOS-2 cells facilitated anchorage-independent growth (Figure 2C and D).Figure 2

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