Clinicopathological and Functional Significance of RECQL1 Helicase in Sporadic Breast Cancers

Abstract

RECQL1, a key member of the RecQ family of DNA helicases, is required for DNA replication and DNA repair. Two recent studies have shown that germline RECQL1 mutations are associated with increased breast cancer susceptibility. Whether altered RECQL1 expression has clinicopathologic significance in sporadic breast cancers is unknown. We evaluated RECQL1 at the transcriptomic level (METABRIC cohort, n = 1,977) and at the protein level [cohort 1, n = 897; cohort 2, n = 252; cohort 3 (BRCA germline deficient), n = 74]. In RECQL1-depleted breast cancer cells, we investigated anthracycline sensitivity. High RECQL1 mRNA was associated with intClust.3 (P = 0.026), which is characterized by low genomic instability. On the other hand, low RECQL1 mRNA was linked to intClust.8 [luminal A estrogen receptor-positive (ER⁺) subgroup; P = 0.0455] and intClust.9 (luminal B ER⁺ subgroup; P = 0.0346) molecular phenotypes. Low RECQL1 expression was associated with shorter breast cancer-specific survival (P = 0.001). At the protein level, low nuclear RECQL1 level was associated with larger tumor size, lymph node positivity, high tumor grade, high mitotic index, pleomorphism, dedifferentiation, ER negativity, and HER-2 overexpression (P < 0.05). In ER⁺ tumors that received endocrine therapy, low RECQL1 was associated with poor survival (P = 0.008). However, in ER^- tumors that received anthracycline-based chemotherapy, high RECQL1 was associated with poor survival (P = 0.048). In RECQL1-depleted breast cancer cell lines, we confirmed doxorubicin sensitivity, which was associated with DNA double-strand breaks accumulation, S-phase cell-cycle arrest, and apoptosis. We conclude that RECQL1 has prognostic and predictive significance in breast cancers. Mol Cancer Ther; 16(1); 239-50. ©2016 AACR.

Figure 1. RECQL1 mRNA expression and breast cancer survival

A. RECQL1 mRNA expression and survival in the whole cohort. B. RECQL1 mRNA expression and survival in ER+ tumours. C. RECQL1 mRNA expression and survival in patients with ER+ tumours who received endocrine therapy. D. RECQL1 mRNA expression and survival in ER− tumours. E. RECQL1 mRNA expression and survival in patients with ER− tumours who received chemotherapy.

Figure 2. RECQL1 protein level and breast cancer survival

A. Photomicrographs of RECQL1 protein expression in breast cancers. B. RECQL1 protein level and survival in the whole cohort. C. RECQL1 protein level and survival in ER+ tumours. D. RECQL1 protein level and survival in patients with ER+ tumours who received endocrine therapy. E. RECQL1 protein level and survival in patients with ER− tumours who received anthracycline chemotherapy. [BCSS= breast cancer specific survival, DFS = Disease free survival].

Figure 3. RECQL1 depletion and doxorubicin sensitivity in breast cancer cell lines

A. RECQL1 mRNA expression in MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cell lines. B. RECQL1 protein level in MCF-7, MDA-MB-231 and MDA-MB-468 breast cancer cell lines. C. Transient RECQL1 depletion by siRNA (C1) and doxorubicin sensitivity in MDA-MB-468 (C2) [The graph shows the cellular surviving fractions measured at different doses of doxorubicin treatment in control and RECQL1-depleted cells. Surviving fraction values are the mean ± SEM from three independent experiments]. D. Transient RECQL1 depletion by siRNA (D1) and doxorubicin sensitivity in MDA-MB-231 (D2) [The graph shows the cellular surviving fractions measured at different doses of doxorubicin treatment in control and RECQL1-depleted cells. Surviving fraction values are the mean ± SEM from three independent experiments]. E. Stable RECQL1 depletion by shRNA (E1) and doxorubicin sensitivity in MDA-MB-231 (E2) [The graph shows the cellular surviving fractions measured at different doses of doxorubicin treatment in control and RECQL1-depleted cells. Surviving fraction values are the mean ± SEM from three independent experiments]. F. Representative immunofluorescence staining of γH2AX foci (green) and its merge with nuclear DNA stain DAPI (blue) in control and RECQL1-depleted MDA-MB-231 cells is shown here. G. Analysis of γH2AX foci in MDA-MB-231 cells stably transduced with control or RECQL1 shRNA. The percentage of cells exhibiting ≥5 γH2AX foci at indicated time points following recovery from doxorubicin treatment (0.1 μM for 4 h) was determined by immunofluorescence. Quantitative data shown represent the average from two independent experiments with associated SEMs.

Figure 4. RECQL1 depletion and cell cycle progression

A. Cell cycle distributions of MDA-MB-231 cells stably transduced with either control or RECQL1 shRNA at the indicated times following recovery from doxorubicin treatment (0.1 μM for 4 h). B. Data shown represent the average from two independent experiments with associated SEMs. Individual p values are summarized as a table here. C. Sub-G1 population in control and RECQL1-depleted MDA-MB-231 cells after doxorubicin treatment (0.1 μM) for indicated time. D. Data shown represent the average from two independent experiments with associated SEMs. Individual p values are summarized as a table here.