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Modeling the initiation of Ewing sarcoma tumorigenesis in differentiating human embryonic stem cells.

Gordon DJ, Motwani M, Pellman D - Oncogene (2015)

Bottom Line: Here, we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells.The inducible expression of EWS-FLI1 in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation.Furthermore, these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth, which is a hallmark of Ewing sarcoma tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However, despite the well-established role of EWS-FLI1 in tumor initiation, the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here, we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation. These cell lines exhibit anchorage-independent growth, a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore, these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth, which is a hallmark of Ewing sarcoma tumors.

No MeSH data available.


Related in: MedlinePlus

The EF+ cells exhibit oncogene addiction. (A) RT-qPCR for the EWS-FLI1 fusion in the EF+ cells and the EF− cells after 2, 4, and 10 days of doxycycline removal. For comparison, expression of EWS-FLI1 in the EFFib cells and EF stem cells is also shown. Expression of EWS-FLI1 is shown relative to actin expression. Error bars indicate the standard error of the mean of three experiments (***, p<0.001). (B) Western blot of EWS-FLI1 in the EF+ cells and the EF− cells after 2, 4, 6, and 10 days of doxycycline removal. (C) Growth of EF+ cells and EF− cells. Cell growth was measured using trypan blue exclusion and a Vi-CELL Cell Viability Analyzer. Error bars indicate the standard error of the mean of three experiments. (D) Flow cytometry analysis of EdU incorporation by EF+ cells (red line) and EF− cells (black line) after four days without doxycycline. (E) Flow cytometry analysis of caspase-3 activation in EF+ cells (red line) and EF− cells (black line) after ten days without doxycycline. (F) Dose-response curves of EFFib, EF+, A673 (Ewing sarcoma), EW8 (Ewing sarcoma), U2OS (osteosarcoma) and BJ-fibroblast (non-transformed fibroblast) cells treated with olaparib for three days. Cell viability was measured using CellTiter-Glo luminescence. Data were log-transformed and normalized. Error bars indicate the standard error of the mean of three experiments.
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Figure 3: The EF+ cells exhibit oncogene addiction. (A) RT-qPCR for the EWS-FLI1 fusion in the EF+ cells and the EF− cells after 2, 4, and 10 days of doxycycline removal. For comparison, expression of EWS-FLI1 in the EFFib cells and EF stem cells is also shown. Expression of EWS-FLI1 is shown relative to actin expression. Error bars indicate the standard error of the mean of three experiments (***, p<0.001). (B) Western blot of EWS-FLI1 in the EF+ cells and the EF− cells after 2, 4, 6, and 10 days of doxycycline removal. (C) Growth of EF+ cells and EF− cells. Cell growth was measured using trypan blue exclusion and a Vi-CELL Cell Viability Analyzer. Error bars indicate the standard error of the mean of three experiments. (D) Flow cytometry analysis of EdU incorporation by EF+ cells (red line) and EF− cells (black line) after four days without doxycycline. (E) Flow cytometry analysis of caspase-3 activation in EF+ cells (red line) and EF− cells (black line) after ten days without doxycycline. (F) Dose-response curves of EFFib, EF+, A673 (Ewing sarcoma), EW8 (Ewing sarcoma), U2OS (osteosarcoma) and BJ-fibroblast (non-transformed fibroblast) cells treated with olaparib for three days. Cell viability was measured using CellTiter-Glo luminescence. Data were log-transformed and normalized. Error bars indicate the standard error of the mean of three experiments.

Mentions: We removed doxycycline from the EF+ cells to evaluate for dependency on continued EWS-FLI1 expression for growth, a feature of most Ewing sarcoma cells lines. RT-qPCR (Figure 3A) and western blotting (Figure 3B) were used to verify the rapid and complete depletion of EWS-FLI1 after drug removal. A morphological change was noted in the cells approximately three-to-five days after doxycycline removal, with the EF− cells exhibiting a larger, flatter and more spindle-shaped morphology (data not shown). After doxycycline removal, the EF− cells exhibit decreased proliferation (Figure 3C). Notably, EdU labeling demonstrated a complete lack of S-phase cells after four days of doxycycline removal (Figure 3D). Culture for additional days in the absence of doxycycline resulted in the loss of cell adhesion and cell death. This coincided with the expression of caspase-3, a marker of apoptosis, in the EF− cells (Figure 3E). Recent work has demonstrated that Ewing sarcoma cell lines exhibit sensitivity to PARP1 inhibitors, including olaparib31,32. The EF+ cells show enhanced sensitivity to olaparib treatment (IC50 2.37 μM; 95% confidence interval 2.1–2.7 μM) relative to the EFFib cells (IC50 54.2 μM; 95% confidence interval 23.6–124.4 μM) (Figure 3F). Similarly, the EF+ cells are also more sensitive than the EFFib cell to treatment with additional drugs with reported lethality toward Ewing sarcoma cell lines, including YK-4-279 (inhibitor of the interaction between RNA helicase A and EWS-FLI1), mithramycin (DNA-binding drug) and LY294002 (PI3K pathway inhibitor) (Supplemental Figure S5)33–35.


Modeling the initiation of Ewing sarcoma tumorigenesis in differentiating human embryonic stem cells.

Gordon DJ, Motwani M, Pellman D - Oncogene (2015)

The EF+ cells exhibit oncogene addiction. (A) RT-qPCR for the EWS-FLI1 fusion in the EF+ cells and the EF− cells after 2, 4, and 10 days of doxycycline removal. For comparison, expression of EWS-FLI1 in the EFFib cells and EF stem cells is also shown. Expression of EWS-FLI1 is shown relative to actin expression. Error bars indicate the standard error of the mean of three experiments (***, p<0.001). (B) Western blot of EWS-FLI1 in the EF+ cells and the EF− cells after 2, 4, 6, and 10 days of doxycycline removal. (C) Growth of EF+ cells and EF− cells. Cell growth was measured using trypan blue exclusion and a Vi-CELL Cell Viability Analyzer. Error bars indicate the standard error of the mean of three experiments. (D) Flow cytometry analysis of EdU incorporation by EF+ cells (red line) and EF− cells (black line) after four days without doxycycline. (E) Flow cytometry analysis of caspase-3 activation in EF+ cells (red line) and EF− cells (black line) after ten days without doxycycline. (F) Dose-response curves of EFFib, EF+, A673 (Ewing sarcoma), EW8 (Ewing sarcoma), U2OS (osteosarcoma) and BJ-fibroblast (non-transformed fibroblast) cells treated with olaparib for three days. Cell viability was measured using CellTiter-Glo luminescence. Data were log-transformed and normalized. Error bars indicate the standard error of the mean of three experiments.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: The EF+ cells exhibit oncogene addiction. (A) RT-qPCR for the EWS-FLI1 fusion in the EF+ cells and the EF− cells after 2, 4, and 10 days of doxycycline removal. For comparison, expression of EWS-FLI1 in the EFFib cells and EF stem cells is also shown. Expression of EWS-FLI1 is shown relative to actin expression. Error bars indicate the standard error of the mean of three experiments (***, p<0.001). (B) Western blot of EWS-FLI1 in the EF+ cells and the EF− cells after 2, 4, 6, and 10 days of doxycycline removal. (C) Growth of EF+ cells and EF− cells. Cell growth was measured using trypan blue exclusion and a Vi-CELL Cell Viability Analyzer. Error bars indicate the standard error of the mean of three experiments. (D) Flow cytometry analysis of EdU incorporation by EF+ cells (red line) and EF− cells (black line) after four days without doxycycline. (E) Flow cytometry analysis of caspase-3 activation in EF+ cells (red line) and EF− cells (black line) after ten days without doxycycline. (F) Dose-response curves of EFFib, EF+, A673 (Ewing sarcoma), EW8 (Ewing sarcoma), U2OS (osteosarcoma) and BJ-fibroblast (non-transformed fibroblast) cells treated with olaparib for three days. Cell viability was measured using CellTiter-Glo luminescence. Data were log-transformed and normalized. Error bars indicate the standard error of the mean of three experiments.
Mentions: We removed doxycycline from the EF+ cells to evaluate for dependency on continued EWS-FLI1 expression for growth, a feature of most Ewing sarcoma cells lines. RT-qPCR (Figure 3A) and western blotting (Figure 3B) were used to verify the rapid and complete depletion of EWS-FLI1 after drug removal. A morphological change was noted in the cells approximately three-to-five days after doxycycline removal, with the EF− cells exhibiting a larger, flatter and more spindle-shaped morphology (data not shown). After doxycycline removal, the EF− cells exhibit decreased proliferation (Figure 3C). Notably, EdU labeling demonstrated a complete lack of S-phase cells after four days of doxycycline removal (Figure 3D). Culture for additional days in the absence of doxycycline resulted in the loss of cell adhesion and cell death. This coincided with the expression of caspase-3, a marker of apoptosis, in the EF− cells (Figure 3E). Recent work has demonstrated that Ewing sarcoma cell lines exhibit sensitivity to PARP1 inhibitors, including olaparib31,32. The EF+ cells show enhanced sensitivity to olaparib treatment (IC50 2.37 μM; 95% confidence interval 2.1–2.7 μM) relative to the EFFib cells (IC50 54.2 μM; 95% confidence interval 23.6–124.4 μM) (Figure 3F). Similarly, the EF+ cells are also more sensitive than the EFFib cell to treatment with additional drugs with reported lethality toward Ewing sarcoma cell lines, including YK-4-279 (inhibitor of the interaction between RNA helicase A and EWS-FLI1), mithramycin (DNA-binding drug) and LY294002 (PI3K pathway inhibitor) (Supplemental Figure S5)33–35.

Bottom Line: Here, we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells.The inducible expression of EWS-FLI1 in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation.Furthermore, these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth, which is a hallmark of Ewing sarcoma tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

ABSTRACT
Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However, despite the well-established role of EWS-FLI1 in tumor initiation, the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here, we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation. These cell lines exhibit anchorage-independent growth, a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore, these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth, which is a hallmark of Ewing sarcoma tumors.

No MeSH data available.


Related in: MedlinePlus