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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

Non-specific chemical inhibition of the Fanconi anemia pathway sensitizes cancer cells to cisplatin

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Céline Jacquemont, Julian A Simon, Alan D D'Andrea, Toshiyasu Taniguchi
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-26) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no conflict of interest.

Authors’ contributions

CJ and TT carried out the experimental work and data analysis, CJ, ADA and TT designed the study, JS provided critical reagents, and CJ and TT wrote the manuscript, all authors edited and approved the manuscript.

Abstract

Background

Platinum compounds such as cisplatin and carboplatin are DNA crosslinking agents widely used for cancer chemotherapy. However, the effectiveness of platinum compounds is often tempered by the acquisition of cellular drug resistance. Until now, no pharmacological approach has successfully overcome cisplatin resistance in cancer treatment. Since the Fanconi anemia (FA) pathway is a DNA damage response pathway required for cellular resistance to DNA interstrand crosslinking agents, identification of small molecules that inhibit the FA pathway may reveal classes of chemicals that sensitize cancer cells to cisplatin.

Results

Through a cell-based screening assay of over 16,000 chemicals, we identified 26 small molecules that inhibit ionizing radiation and cisplatin-induced FANCD2 foci formation, a marker of FA pathway activity, in multiple human cell lines. Most of these small molecules also compromised ionizing radiation-induced RAD51 foci formation and homologous recombination repair, indicating that they are not selective toward the regulation of FANCD2. These compounds include known inhibitors of the proteasome, cathepsin B, lysosome, CHK1, HSP90, CDK and PKC, and several uncharacterized chemicals including a novel proteasome inhibitor (Chembridge compound 5929407).
Isobologram analyses demonstrated that half of the identified molecules sensitized ovarian cancer cells to cisplatin. Among them, 9 demonstrated increased efficiency toward FA pathway-proficient, cisplatin-resistant ovarian cancer cells. Six small molecules, including bortezomib (proteasome inhibitor), CA-074-Me (cathepsin B inhibitor) and 17-AAG (HSP90 inhibitor), synergized with cisplatin specifically in FA-proficient ovarian cancer cells (2008 + FANCF), but not in FA-deficient isogenic cells (2008). In addition, geldanamycin (HSP90 inhibitor) and two CHK1 inhibitors (UCN-01 and SB218078) exhibited a significantly stronger synergism with cisplatin in FA-proficient cells when compared to FA-deficient cells, suggesting a contribution of their FA pathway inhibitory activity to cisplatin sensitization.

Conclusion

Our findings suggest that, despite their lack of specificity, pharmaceutical inhibition of the FA pathway by bortezomib, CA-074-Me, CHK1 inhibitors or HSP90 inhibitors may be a promising strategy to sensitize cisplatin-resistant, FA pathway-proficient tumor cells to cisplatin. In addition, we identified four new small molecules which synergize with cisplatin. Further development of their analogs and evaluation of their combination with cisplatin may lead to the development of efficient cancer treatments.
Zusatzmaterial
Additional file 1 : Table S1. List of the chemicals scored as positives in the primary screening. The 43 compounds scored as FA pathway inhibitors in the primary screening are listed. The 15 compounds that wre verified in the secondary screenings are indicated with “X”. (DOCX 15 KB)
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Additional file 2 : Figure S1. Chemical structure of the Chembridge library compounds that inhibit DNA damage-induced FANCD2 foci formation. Compounds 5195243, 5373662, 5929407 and 5656325 were identified by the compound library screening. Compounds 5315179 and 7012246 were later selected upon their 2D analogy with 5656325. (TIFF 66 KB)
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Additional file 4 : Figure S2. Most FA pathway inhibitors inhibit HR. (A) Schematic of the HR assay performed using FA pathway inhibitors. (B) Flow cytometric analysis for HR efficiency using U2OS-DR-GFP cells and HA-tagged I-Sce1. Cells fixed and stained with anti-HA and APC-linked specific secondary antibodies were first gated in FCS/SSC scatter, then HA-positive population was gated in an APC/GFP dot plot and analyzed for GFP expression using a red/GFP dot plot. (C) The relative proportion of live cells compared to non-treated control cells in the HR assay in U2OS-DRGFP cells are shown (mean ± SEM (n=3 to 7)). Percentage of live cells was 95.0±1.2% in non-treated condition. (D) The relative proportion of cells expressing HA-tagged ISce1 compared to non-treated control cells in the HR assay in U2OS-DRGFP cells are shown (mean ± SEM (n=3 to 7)). An asterix (*) indicates significant decrease in proportion of HA-positive cells upon exposure to the indicated drug (p≤0.05, t test). Percentage of HA-positive cells was 39.2±2.5% in non-treated condition. (E) The relative proportion of GFP-positive cells in the HA-positive population, compared to non-treated control cells, is shown (mean ± SEM (n=3 to 7)). An asterix (*) indicates significant decrease in the proportion of GFP-positive/HA-positive cells upon exposure to the indicated drug (p≤0.05, t test). Percentage of GFP-positive/HA-positive cells was 9.5±0.9% in non-treated condition. These results are summarized in Figure 3. ( F) Examples of flow cytometric profiles obtained with U2OS-DR-GFP cells untreated and treated with geldanamycin (0.1μM) for 24 hours. (TIFF 729 KB)
Additional file 5 : Figure S3. Most FA pathway inhibitors do not significantly affect the proportion of cells in S+G2phases of the cell cycle. Cell cycle distribution quantified using BrdU/PI staining of U2OS-DR-GFP cells treated for 24 hours with the indicated drugs (A), or treated for 24 hours with the indicated drugs and irradiated with 10Gy 8 hours before the end of treatment (B) (n=3 to 4). An asterix (*) indicates significant decrease in proportion of S+G2 cells upon exposure to the indicated drug (p≤0.05, t test). Examples of flow cytometric profiles obtained with non-treated and FA pathway inhibitor-treated cells are shown. (TIFF 755 KB)
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Additional file 6 : Figure S4. Figure S2. Most FA pathway inhibitors inhibit HR. (A) Schematic of the HR assay performed using FA pathway inhibitors. (B) Flow cytometric analysis for HR efficiency using U2OS-DR-GFP cells and HA-tagged I-Sce1. Cells fixed and stained with anti-HA and APC-linked specific secondary antibodies were first gated in FCS/SSC scatter, then HA-positive population was gated in an APC/GFP dot plot and analyzed for GFP expression using a red/GFP dot plot. (C) The relative proportion of live cells compared to non-treated control cells in the HR assay in U2OS-DRGFP cells are shown (mean ± SEM (n=3 to 7)). Percentage of live cells was 95.0±1.2% in non-treated condition. (D) The relative proportion of cells expressing HA-tagged ISce1 compared to non-treated control cells in the HR assay in U2OS-DRGFP cells are shown (mean ± SEM (n=3 to 7)). An asterix (*) indicates significant decrease in proportion of HA-positive cells upon exposure to the indicated drug (p≤0.05, t test). Percentage of HA-positive cells was 39.2±2.5% in non-treated condition. (E) The relative proportion of GFP-positive cells in the HA-positive population, compared to non-treated control cells, is shown (mean ± SEM (n=3 to 7)). An asterix (*) indicates significant decrease in the proportion of GFP-positive/HA-positive cells upon exposure to the indicated drug (p≤0.05, t test). Percentage of GFP-positive/HA-positive cells was 9.5±0.9% in non-treated condition. These results are summarized in Figure  3. ( F) Examples of flow cytometric profiles obtained with U2OS-DR-GFP cells untreated and treated with geldanamycin (0.1μM) for 24 hours. (TIFF 1 MB)
Additional file 7 : Figure S6. Most FA pathway inhibitors significantly inhibit cisplatin-induced foci formation of FANCD2 in U2OS-DR-GFP cells. Relative proportion of U2OS-DR-GFP cells with more than 10 foci after 24hour incubation with 5μM cisplatin and the indicated drug, compared to controls (mean ± SEM (n=3)). An asterisk (*) indicates significant decrease in the proportion of foci-positive cells compared to controls (p ≤0.05, t test). In non-treated conditions, the percentage of FANCD2-positive cells was 83.13 ± 8.3%. Examples of immunofluorescence staining are also shown. Scale bar = 20μm. N.D. = not determined. (TIFF 904 KB)
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Additional file 8 : Table S2. 2008 and 2008+FANCF cells are equally sensitive to most FA pathway inhibitors. Lethal dose 50 (LD50) (mean ± SEM (n=3 to 6)) of the FA pathway inhibitors in 2008 and 2008+FANCF cells. An asterisk (*) indicates significant difference in sensitivity between 2008 and 2008+FANCF cells (p ≤0.05, paired t test). N.D. = not determined. Only two experiments were performed using lactacystin, because of the high concentration required to achieve 50% killing. (DOCX 15 KB)
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Additional file 9 : Figure S7. Isobologram analyses at 50% killing of the FA pathway inhibitors that did not sensitize 2008 or 2008+FANCF ovarian cancer cells to cisplatin. Isobolograms at the LD50 level are presented, showing the results obtained in at least 3 independent experiments. Results are summarized in Table 2. (TIFF 237 KB)
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Additional file 10 : Table S3. Drug interactions at 70% killing between cisplatin and the FA pathway inhibitors in FA pathway-deficient and -proficient ovarian cancer cells. Combination index (CI) at 70% killing values (mean ± SEM) calculated from isobologram at the LD70 level analyses of combination of cisplatin with all FA pathway inhibitor, performed in an FA-deficient (2008) and an FA-proficient (2008+FANCF) ovarian cancer cell lines. Synergism is indicated in bold text. (DOCX 15 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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