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DNA Damage and Repair

PARP Inhibition Induces Enrichment of DNA Repair–Proficient CD133 and CD117 Positive Ovarian Cancer Stem Cells

Chiara Bellio, Celeste DiGloria, Rosemary Foster, Kaitlyn James, Panagiotis A. Konstantinopoulos, Whitfield B. Growdon and Bo R. Rueda
Chiara Bellio
1Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
2Harvard Medical School, Boston, Massachusetts.
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Celeste DiGloria
1Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
2Harvard Medical School, Boston, Massachusetts.
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Rosemary Foster
1Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
2Harvard Medical School, Boston, Massachusetts.
3Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
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Kaitlyn James
4Deborah Kelly Center for Outcomes Research, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
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Panagiotis A. Konstantinopoulos
5Dana-Farber Cancer Institute, Boston, Massachusetts.
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Whitfield B. Growdon
1Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
2Harvard Medical School, Boston, Massachusetts.
3Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
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Bo R. Rueda
1Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
2Harvard Medical School, Boston, Massachusetts.
3Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.
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  • For correspondence: brueda@mgh.harvard.edu
DOI: 10.1158/1541-7786.MCR-18-0594 Published February 2019
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    Figure 1.

    Treatment with the PARPi olaparib in vitro induces an enrichment of CD133+ and CD117+ cells in ovarian serous cancer cell lines. A, Western analysis of PolyADP-Ribose (PAR) levels in cells treated in vitro with the indicated concentrations of olaparib for 7 days to confirm the dose-dependent on target effect of olaparib. B, Flow cytometric determination of the frequency of viable (left plots), CD133+ (center plots), and CD117+ (right plots) cells in the indicated cell lines 7 days following treatment with increasing concentrations of olaparib in vitro. C, Relative cell viability of CD133−CD117−, CD133+, CD117+, and CD133+CD117+ cells 7 days following in vitro treatment with the indicated concentrations of olaparib as determined by flow cytometry. The graph for each cell line shows either the mean percentage of expression ± SEM (B) or mean viable cell number ± SEM (C) calculated from three independent experiments; P value < 0.001.

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    Figure 2.

    PARPi olaparib induces enrichment of stemness phenotype and in vivo enrichment of CD133+ and CD117+ expression. A, ELDA of the indicated cell lines 7 days following treatment with 10 μmol/L olaparib treatment in vitro. The data are expressed as sphere-forming frequency of cells exposed to olaparib normalized to the corresponding vehicle control and suggest the relative frequency of CSCs in each sample. Three separate iterations of the analysis were performed with each cell line, and the numbers represent the mean sphere-forming frequency ± SEM (P value < 0.001). B, Mice harboring either OVCAR3-derived xenografts (left plots) or PEO1-derived xenografts (right plots) were treated with either vehicle or olaparib as described in Materials and Methods. CD133+ and CD117+ cell frequencies in tumors harvested at the end of the treatment period were determined by flow cytometry. Error bars represent SEM; *, P < 0.01.

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    Figure 3.

    CD133+CD117+ cells shift to G2–M after olaparib treatment in vitro. The cell-cycle distribution of CD133−CD117− and CD133+CD117+ cells treated with either vehicle or 10 μmol/L olaparib was assessed 72 hours after treatment by measurement of DNA content via DAPI staining. The figure shows the mean values from three different iterations of this analysis in each cell line. Measurement of G2–M phase after vehicle and olaparib treatment show statistical difference in CD133+CD117+ population (P < 0.0001) compared with the CD133−CD117− population, which is still significant but not as robust as CD133+CD117+ population.

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    Figure 4.

    Olaparib treatment induces an accumulation of RAD51 foci in CD133+ cells. A, Confocal images of RAD51 foci formation in UWB1.289 WT and UWB1.289 MUT cells 72 hours following treatment with 5 μmol/L carboplatin or 10 μmol/L olaparib. Graphs show RAD51 foci frequency (n = 3, mean ± SEM, P value < 0.0001). B, RAD51 foci formation in CD133+ and CD133− enriched fractions from vehicle- and olaparib-treated UWB1.289 WT and UWB1.289 MUT cells. The histogram for each cell line shows a statistically significant (P < 0.005) olaparib induced increase in foci formation in CD133+ cells (n = 3, mean ± SEM).

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    Figure 5.

    CD133+ cells show enhanced DNA repair efficiency. A, UWB1.289 WT (top plot0) and UWB1.289 MUT (bottom plot) cells were analyzed 96 hours after treatment with vehicle or 72 hours after treatment with 10 μmol/L olaparib followed by a 24-hour no treatment recovery period and then subjected to magnetic bead separation to isolate CD133+ and CD133− cells. DNA damage in the purified cell fractions was assessed by the comet assay. Representative results are shown. B, Quantitation of the comet assay results (see details in Materials and Methods) determined that the DNA repair efficiency of CD133+ cells is significantly enhanced compared with their CD133− counterparts (n = 3, mean ± SEM, P value < 0.0001).

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    Figure 6.

    PARPi olaparib induces overexpression of DNA repair genes in CD133+ cells. CD133+ cells and CD133− CD117− cells were FACS purified from UWB1.289 WT and UWB1.289 MUT 72 hours following treatment with either vehicle or 10 μmol/L olaparib. Expression of DSB repair genes was assessed by RT-PCR array analysis. DMC1 expression is upregulated in response to olaparib treatment in CD133+ cells (A). DMC1 induction in CD133−CD117− cells (B) was lower than that observed in CD133+ cells. C, Comparison of DMC1 gene expression in UWB1.289 WT (left) and UWB1.289 MUT (right) cell lines showing the mean fold change of expression ± SEM relative to the vehicle samples. D, Representative confocal images of nuclear RAD51 (green) and DMC1 (red) immunofluorescence in UWB1.289 WT and UWB1.289 MUT-derived CD133− and CD133+ cells following treatment with vehicle or olaparib. Nuclei were stained with DAPI. Histograms showing the mean frequency ± SEM of DMC1 foci from three individual experiments in CD133− and CD133+ cells from UWB1.289 WT (left) and UWB1.289 MUT (right), normalized to the vehicle CD133− samples (n = 3, P value < 0.0001). E, Analysis and quantification of DMC1 foci formation 72 hours following treatment with vehicle, rucaparib, or carboplatin were carried out as described in A (n = 3, P value < 0.0001).

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    Figure 7.

    A, Western analysis of DMC1 protein levels in UWB1.289 WT (left plot) and UWB1.289 MUT (right plot) cells transfected with either a DMC1 expression vector (DMC1) or the corresponding control vector (CTRL). Exogenous DMC1 expression was analyzed 2 days after transfection and again 3 days after the transfected cells had undergone treatment with vehicle or olaparib (5-day samples). The relative DMC1 protein level in each sample was determined by comparing DMC1 expression to β-tubulin level. Results were normalized to the DMC1 level in cells transfected with the control plasmid. B, UWB1.289 WT (left plot) and UWB1.289 MUT (right plot) cells were assessed 96 hours after treated with either vehicle or 72 hours after treatment with 10 μmol/L olaparib followed by a 24-hour recovery period in the absence of olaparib. CD133− cells were then isolated by magnetic bead separation. DNA damage in the purified cell fractions was assessed by the comet assay. Representative results are shown. Quantitation of the comet assay results (see details in Materials and Methods) determined that the DNA repair efficiency of CD133− cells is significantly enhanced in the DMC1-transfected cells compared with the CTRL-transfected cells (n = 3, mean ± SEM, P value < 0.0001). C, CD133+ and CD117+ cells show enhanced DNA repair efficiency following DSB DNA damage. Schematic representation of homologous recombination assay. Exogenous expression of SceI in cells transfected with pDR-GFP results in a DSB in GFP and disruption of the open reading frame. The efficiency of inherent DNA repair in the transfected cells can be assessed by flow cytometric analysis of restored GFP fluorescence. D, Level of GFP expression in UWB1.829 WT- and UWB1.289 MUT-gated CD133+, CD117+, CD133−, and CD117− cell fractions (n = 3, mean ± SEM, P value < 0.05).

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    • Supplementary Data - Supplemental methods and supplementary figure legends
    • Supplementary Figures - Supplementary figures: SA, SB, SC, SD, SE, SF, SG and SH
    • Supplementary Table S1 - Supplementary Table S1: p-values of RT-PCR analysis of PROM1 (CD133), KIT (CD117), SOX2 and POU5F1 (OCT4) genes (Supplementary Figure SC).
    • Supplementary Table S2 - Supplementary Table S2: p-values of RT-PCR analysis of RAD51 gene family in CD133+ cells and CD133- CD117- cells FACS purified (Figure 6A and 6B).
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Molecular Cancer Research: 17 (2)
February 2019
Volume 17, Issue 2
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PARP Inhibition Induces Enrichment of DNA Repair–Proficient CD133 and CD117 Positive Ovarian Cancer Stem Cells
Chiara Bellio, Celeste DiGloria, Rosemary Foster, Kaitlyn James, Panagiotis A. Konstantinopoulos, Whitfield B. Growdon and Bo R. Rueda
Mol Cancer Res February 1 2019 (17) (2) 431-445; DOI: 10.1158/1541-7786.MCR-18-0594

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PARP Inhibition Induces Enrichment of DNA Repair–Proficient CD133 and CD117 Positive Ovarian Cancer Stem Cells
Chiara Bellio, Celeste DiGloria, Rosemary Foster, Kaitlyn James, Panagiotis A. Konstantinopoulos, Whitfield B. Growdon and Bo R. Rueda
Mol Cancer Res February 1 2019 (17) (2) 431-445; DOI: 10.1158/1541-7786.MCR-18-0594
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