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. 2015 Mar 16;10(3):e0119614.
doi: 10.1371/journal.pone.0119614. eCollection 2015.

Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer

Julie L Boerner  1 , Nicole Nechiporchik  1 , Kelly L Mueller  1 , Lisa Polin  1 , Lance Heilbrun  1 , Scott A Boerner  2 , Gina L Zoratti  1 , Karri Stark  1 , Patricia M LoRusso  2 , Angelika Burger  1
Affiliations

Protein expression of DNA damage repair proteins dictates response to topoisomerase and PARP inhibitors in triple-negative breast cancer

Julie L Boerner et al. PLoS One. .

Abstract

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. BRCA mutated TNBC cell lines express high levels of PARP1 and are sensitive to PARP inhibition.
(A) Cells were treated with increasing concentrations of ABT-888 over a 5 day incubation period. MTT assays were used to assess cell viability. The fraction of surviving cells was used to calculate the IC50 values for ABT-888 for each cell line by sigmoidal dose response curve analyses (GraphPad Prism). IC50 values calculated from three independent experiments performed in triplicate were graphed for each cell line. (B) PARP1 protein expression levels were evaluated from cell lysates collected from cells growing in log phase. Equal protein was separated by SDS-PAGE, transferred to PVDF, and immunoblotted using anti-PARP1 antibodies. β-actin protein levels were used as a loading control.
Fig 2
Fig 2. Response of BRCA mutated TNBC cell lines to topoisomerase inhibition correlates with ERCC1 protein expression.
(A) Cells were treated with increasing concentrations of CPT-11 over a 5 day incubation period. MTT assays were used to assess cell viability. The fraction of surviving cells was used to calculate the IC50 values for ABT-888 for each cell line by sigmoidal dose response curve analyses (GraphPad Prism). IC50 values calculated from three independent experiments performed in triplicate were graphed for each cell line. (B) ERCC1 protein expression levels were evaluated from cell lysates collected from cells growing in log phase. Equal protein was separated by SDS-PAGE, transferred to PVDF, and immunoblotted using anti-ERCC1 antibodies. β-actin protein levels were used as a loading control.
Fig 3
Fig 3. Knocking down ERCC1 protein expression sensitizes cells to CPT-11.
(A and B) MDA-MB-231 cells were transfected with 4 non-overlapping siRNA oligos targeting ERCC1 as well as a negative control siRNA using Dharmafect. (A) Forty-eight hours later, cell lysates were prepared, SDS-PAGE separated the lysates, and immunoblotting was performed using anti-ERCC1 antibodies. (B) Cells were placed in serum free media for 18 hours and pulsed with BrdU to measure DNA synthesis. Cells were fixed, permeabilized with 2N HCl, neutralized with borate buffer, and blocked with 20% goat serum. BrdU incorporation was detected using anti-BrdU alexa fluor 624. BrdU positive cells were counted as a fraction of 100 cells counted/each of four fields/coverslip. Each experiment was performed in duplicate at least three times. * p-value = 0.022.
Fig 4
Fig 4. The combination of PARP and topoisomerase inhibitors is an effective combination for TNBC in vivo.
MX-1 breast cancer xenografts were implanted between the front and hind leg of nude mice. Tumor volume (in mm3) was measured with calipers every two days, and body weight was taken bi-weekly. When tumors reached a measurable burden (~63 mm3), the indicated treatments were started. CPT-11 was given IV every 7 days in 5 doses for a total dosage of 225 mg/kg. ABT-888 was given PO twice a day from days 9 to 20 and again from days 23 to 28 for a total dosage of 240 mg/kg. Mean tumor volume (± standard error [SE] of the mean) is plotted over time, separately for each of the four drug treatment groups.
Fig 5
Fig 5. Measuring γH2AX phosphorylation as a biomarker for response to PARP and topoisomerase inhibitors.
Biopsies from MX-1 human tumor xenografts after 4 and 24 hours of CPT-11 (40mg/kg) and/or ABT-888 (5mg/kg) treatment were taken and centrifuged onto a glass slide. Cells were fixed, permeabilized, blocked overnight, and incubated with anti γ-H2AX antibody. Slides were washed with PBS followed by staining with FITC-conjugated secondary antibody. Following PBS washing, the slides were incubated with DAPI, washed in PBS, and mounted. The results were visualized and documented using the fluorescent setting of a Leica CTR5500 microscope and quantified using OpenLab software. Each experiment was repeated three times representing the bars in the graph.
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