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Multicenter Study
. 2010 Jul 27;107(30):13390-5.
doi: 10.1073/pnas.0910759107. Epub 2010 Jul 12.

"V体育平台登录" DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability

Fanny Lemée  1 , Valérie Bergoglio, Anne Fernandez-Vidal, Alice Machado-Silva, Marie-Jeanne Pillaire, Anne Bieth, Catherine Gentil, Lee Baker, Anne-Laure Martin, Claire Leduc, Elena Lam, Eddy Magdeleine, Thomas Filleron, Naïma Oumouhou, Bernd Kaina, Mineaki Seki, Fanny Grimal, Magali Lacroix-Triki, Alastair Thompson, Henri Roché, Jean-Christophe Bourdon, Richard D Wood, Jean-Sébastien Hoffmann, Christophe Cazaux
Affiliations
Multicenter Study

V体育2025版 - DNA polymerase theta up-regulation is associated with poor survival in breast cancer, perturbs DNA replication, and promotes genetic instability

Fanny Lemée et al. Proc Natl Acad Sci U S A. .

Abstract

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n=206; United Kingdom, n=117) of patients with previously untreated primary breast cancers. We report here that among the 13 human nuclear DNA polymerase genes, DNA Polymerase (POLQ) is the only one significantly up-regulated in breast cancer compared with normal breast tissues. Importantly, POLQ up-regulation significantly correlates with poor clinical outcome (4. 3-fold increased risk of death in patients with high POLQ expression), and this correlation is independent of Cyclin E expression or the number of positive nodes, which are currently considered as markers for poor outcome. POLQ expression provides thus an additional indicator for the survival outcome of patients with high Cyclin E tumor expression or high number of positive lymph nodes VSports手机版. Furthermore, to decipher the molecular consequences of POLQ up-regulation in breast cancer, we generated human MRC5-SV cell lines that stably overexpress POLQ. Strong POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage. Therefore, POLQ overexpression may be a promising genetic instability and prognostic marker for breast cancer. .

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Conflict of interest statement

The authors declare no conflict of interest.

VSports最新版本 - Figures

Fig. 1.
Fig. 1.
Relative expression of replicative and specialized DNA polymerases in breast tumors from set 1 of the French cohort (n = 101). Normalized mRNA expression ratios for tumor (T) and normal (N) breast samples (T/N) were calculated. T/N > 1 and < 1 indicate higher and lower expression levels in tumors compared with pooled normal tissues, respectively. The individual T/N ratios were normalized to the expression of HMBS and IPO8, the two most stable control genes. T/N expression ratios were transformed into binomial data: 0 if T/N < 1; 1 if T/N > 1. The new variables then followed a binomial distribution according to the parameters n and p0, which represent the number of patients and the probability that a gene is overexpressed, respectively. To test the significance of a gene over- or underexpression, we checked if the p0 parameter of this binomial distribution was different from 0.5 by using bilateral exact binomial tests (null hypothesis: p0 = 0.5). Because of multiple testing, the P values from these tests were compared with a significance level obtained by using the Benjamini et al. procedure (39) for an overall false discovery rate (FDR) of 0.05. The patients’ samples on the x axis are classified in the same order in each panel. + and – stand for higher and lower expression in the tumor (T) compared with the normal (N) tissues, respectively. For graph representation T/N values <1 were transformed into the inverse N/T values.
Fig. 2.
Fig. 2.
Relationship between POLQ expression level to cancer-specific survival. In the French cohort, POLQ expression levels ≤0.063 were defined as “low expression.” For the British cohort, POLQ expression ≤0.31 was defined as low expression. These cutoffs were defined in relation to survival to identify two statistically different populations of patients. ROC (Receiver Operating Characteristics) curves were used to determine the optimal cutoff point that best segregated patients in terms of survival. Cancer-specific survival was defined as the interval between the date of breast surgery and the date of death or of the last-follow-up (censored data). Patients who died from another cause were considered as censored observations. Survival rates were estimated according to the Kaplan–Meier method. (A and B) Kaplan–Meier survival curves of breast cancer patients according to the level of POLQ expression in the primary tumor. For these graphical representations, P values were given according to the log-rank test. A P value <0.05 was considered statistically significant. (A) Patients from the French cohort (n = 203 instead of 206, because 3 samples were discarded because POLQ expression could not be determined). (B) Patients from the British cohort (n = 117). (C and D) Kaplan–Meier survival curves of pairwise comparison between POLQ gene expression in the primary breast tumor and the number of positive lymph nodes. We used a multivariate Cox model adjusted for POLQ expression and the number of positive lymph nodes. This model detected an effect of POLQ adjusted for the number of lymph nodes on the survival in both the French (P = 0.0001) and the British cohort (P = 0.002). (C) Patients from the French cohort (n = 203). (D) Patients from the British cohort (n = 117). Ln stands for positive lymph node. A low number of metastatic lymph nodes was defined as a lymph node count equal to 1 or less, distinct from patients with 2 or more metastatic axillary nodes.
Fig. 3.
Fig. 3.
DNA damage and DNA damage response in POLQ overexpressing MRC5-SV clones. Immortalized human lung fibroblasts (MRC5-SV) were transfected with POLQ cDNA subcloned in the pcDNA3.1/Hygro(+) expression vector and three clones (Q1, Q2, and Q3) that show progressively increasing expression of POLQ were selected for stable expression. Average number of POLQ overexpressing cells positive for γ-H2AX (A) and PT68-CHK2 (B) immunolabeling. Control (CTL) clones (MRC5-SV cells stably transfected with empty pcDNA3.1 vector) treated with UV (10 J·m−2) were used as positive controls. (C) Representative confocal microscopy images of MRC5-SV cells overexpressing POLQ (Q1, Q2, Q3) show colocalization of γ-H2AX and PT68-CHK2 foci. (D) Western blots of whole-cell lysates prepared from POLQ overexpressing MRC5-SV cells (Q3 clone) transfected with either control Luciferase (C) or POLQ (Q) siRNAs at 24, 48, or 72 h after transfection. Quantification by Arrayscan analysis of γ-H2AX expression in control or Q3 MRC5-SV cells transfected with Luciferase or POLQ siRNAs. NT, nontransfected; *, aspecific signal. (E) Normalized real-time PCR quantification of POLQ expression in human mammary epithelial cell (HMEC) nontumoral controls and MCF7 breast cancer cells transfected with control Luciferase or POLQ siRNAs (n = 3). Western blotting did not allow detection of POLQ protein expression in MCF7 cells. Quantification by Arrayscan analysis of γ-H2AX signal. For each analysis, a minimum of 200 MRC5 or 1,000 MCF7 cells were analyzed in at least three independent experiments. MRC5 and MCF7 positive cells contained more than 1 and 5 foci, respectively. P values were calculated by using the Student’s t test.
Fig. 4.
Fig. 4.
DNA replication fork velocity in MRC5-SV cells overexpressing POLQ. (A) Representative image of combed and immunostained DNA fibers in control and Q2 and Q3 (which stably overexpress POLQ) MRC5-SV cells. (B) Replication fork velocity distribution in control (CTL2, cell stably transfected with empty vector), Q2, and Q3 MRC5-SV cells. (C) Total length (in megabases) is the sum of all DNA fibers that were studied for each clone; “n” is the number of tracks of IdU and CldU scored for each clone. The median value of the population is given in kilobases per min. The uncertainty of the median replication fork velocity is given in units of the median absolute deviation. The Mann–Whitney test was used to compare Q2 and Q3 data with the CTL2 control.
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