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. 2009 Apr 15;69(8):3589-96.
doi: 10.1158/0008-5472.CAN-08-4016. Epub 2009 Apr 7.

Defective repair of oxidative dna damage in triple-negative breast cancer confers sensitivity to inhibition of poly(ADP-ribose) polymerase

Elizabeth Alli  1 , Vandana B Sharma, Preethi Sunderesakumar, James M Ford
Affiliations

Defective repair of oxidative dna damage in triple-negative breast cancer confers sensitivity to inhibition of poly(ADP-ribose) polymerase

Elizabeth Alli et al. Cancer Res. .

Abstract

Subtypes of breast cancer that represent the two major types of epithelial cells in the breast (luminal and basal) carry distinct histopathologic profiles. Breast cancers of the basal-like subtype, which include the majority of hereditary breast cancers due to mutations in the breast cancer susceptibility gene 1 (BRCA1), frequently assume triple-negative status, i.e., they lack expression of estrogen receptor-alpha and progesterone receptor, and lack overexpression or amplification of the HER2/NEU oncogene. Defects in DNA damage response pathways result in genome instability and lead to carcinogenesis, but may also be exploited for therapeutic purposes. We analyzed repair of oxidative DNA damage by the base-excision repair (BER) pathway, which when aberrant leads to genomic instability and breast carcinogenesis, in cell lines that represent the different subtypes of breast cancer and in the presence of BRCA1 deficiency. We found that basal-like and BRCA1-mutated breast cancer cells were defective in BER of oxidative DNA damage, and that this defect conferred sensitivity to inhibition of poly(ADP-ribose) polymerase, a DNA repair enzyme. The defect may be attributed, at least in part, to a novel role for BRCA1 in the BER pathway. Overall, these data offer preventive, prognostic, and therapeutic usefulness.

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Figures

Figure 1
Figure 1. Increased ODD in basal-like and BRCA1-mutated breast cancer cell lines and in the presence of BRCA1 deficiency
(A) Analysis of H2O2 sensitivity, an indicator of ODD, by MTT assay in cell lines representing the normal breast and each subtype of breast cancer. Each box plot summarizes the data described in Table 1. For each subtype, the grey shaded area indicates the range of IC50 values, the dashed line indicates the average IC50 value, and the solid line indicates the median IC50 value. (B) Analysis of H2O2 sensitivity by MTT assay in BRCA1+/+ (-●-) and BRCA1-/- (-◇-) MMECs. The graph illustrates sensitivity following treatment with increasing concentration of oxidizing agent. The IC50 concentrations as determined by interpolation from the dose-response curves are indicated for each cell line. (C) Basal levels of ODD in BRCA1+/+ and BRCA1-/- MMECs. ODD was measured by the alkaline comet assay modified for detection of oxidized bases using logarithmically growing cells. ODD was visualized by fluorescent microscopy (top), and quantified by measuring the percentage of DNA in comet tails using comet software (bottom). White bars indicate typical comet tails.
Figure 2
Figure 2. Decreased BER activity in basal-like and BRCA1-mutated or deficient breast cancer cell lines
(A) BER activity in cell lines representing each subtype of breast cancer using a cell-based BER assay. GFP expression, i.e. an indicator of BER activity, was observed by fluorescent microscopy (top), and quantified by plate reading (bottom). Images were collected under 10X objective and are representative. (B) Relationship between H2O2 sensitivity and BER in breast cancer cell lines. Cell lines included: luminal formula image, basal-like formula image, and BRCA1-mutated formula image. The plot depicts linear regression of the average IC50 value obtained from MTT analysis of H2O2 sensitivity and GFP expression quantified following the BER assay. (C) BER activity in the SUM149PT breast cancer cell line (mutant BRCA1) transfected with wild-type BRCA1. In the top panel, Western blot illustrates nuclear expression of wild-type BRCA1. Tata-binding protein (TBP) was used as a nuclear loading control. In the bottom panel, each bar represents the expression of GFP as determined by the BER assay, which was quantified using a fluorescent plate reader and calculated relative to the vector control. (D) BER activity in the MDAMB361 breast cancer cell line (wild-type BRCA1) transfected with shRNA to BRCA1. Expression of BRCA1 (top) and GFP (bottom) were determined as in (C).
Figure 3
Figure 3. Decreased repair of ODD by BER in the presence of shRNA to hOGG1
(A) mRNA expression of hOGG1 by RTqPCR in isogenic cell lines stably expressing (two different) shRNA to hOGG1 or a non-targeting control shRNA. mRNA expression was calculated by ΔΔCT method. (B) H2O2 and (C) MMS sensitivity in shOGG1-expressing cell lines. Following treatment with increasing concentrations of DNA damaging agent, sensitivity was determined by MTT assay. Each bar represents the average IC50 value ± s.e.m. from at least three independent experiments. (D) BER activity in shOGG1-expressing cell lines. Each bar represents the average GFP expression ± s.e.m. from at least three independent experiments as determined by the BER assay. **, p<0.01; *, p<0.05.
Figure 4
Figure 4. Increased sensitivity of BER-compromised cells to PARP inhibition
(A) Sensitivity of cell lines expressing hOGG1 shRNA to IQD, an inhibitor of PARP. The graph illustrates sensitivity as determined by MTT assay following treatment with increasing concentration of drug. The IC50 concentration for each cell line was interpolated from the dose-response curve as indicated. MCF7CTRL (-◆-) is the control cell line containing non-targeting shRNA. MCF7shOGG1-A (-△-) and MCF7shOGG1-B (-○-) are two different isogenic cell lines stably expressing shRNA to hOGG1. Data are representative of three independent experiments. (B) Sensitivity of luminal and basal-like breast cancer cell lines to IQD. Each box plot summarizes the data described in Table 2. For each subtype, the grey shaded area indicates the range of IC50 values, the dashed line indicates the average IC50 value, and the solid line indicates the median IC50 value. (C) Relationship between PARP inhibitor and H2O2 sensitivity in breast cancer cell lines. Cell lines included: luminal (●) and basal-like (○). The plot depicts linear regression of the average IC50 values obtained from MTT analysis of IQD and H2O2 sensitivity.
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