Breast cancers with high DSS1 expression that potentially maintains BRCA2 stability have poor prognosis in the relapse-free survival

Breast tumor specimens from 289 female patients with invasive breast carcinoma, who were treated at Kumamoto University Hospital between 2001 and 2009, were included in this study. Among these patients, p53 immunohistochemical data were available for 227 (78.5%) patients. The patients were from a consecutive series; those with other malignancies or bilateral breast cancer were excluded. Samples were snap frozen in liquid nitrogen at the time of the pretherapeutic biopsy or surgical treatment and stored at -80°C until simultaneous total RNA extraction. The median age of the patients was 59 years (range, 21–93 years). Adjuvant treatment and neoadjuvant treatment were decided by risk evaluation according to tumor biology [estrogen receptor alpha (ERα), progesterone receptor (PgR), and HER2 but not Ki-67 status] and clinical staging, including sentinel lymph node biopsy, in accordance with the recommendations of the St. Gallen international expert consensus on the primary therapy of early breast cancer. In detail, neoadjuvant treatments were administered to 62 patients, 46 of whom received chemotherapy and 16 of whom received hormonal therapy. The breast-conserving rate was 68.2%, and most of these were treated with radiotherapy. Axillary lymph node dissection was carried out in 45.2% of cases; the others were spared dissection due to negative lymph node status by sentinel node exploration. A total of 208 patients were treated with hormone therapy, 106 patients were given chemotherapy, and 19 patients were treated with trastuzumab. The ethics committee of Graduate School of Medical Sciences, Kumamoto University approved the study protocol. Informed consent was obtained from all patients. Patients received follow-up studies every 3 months. The median follow-up period was 66 months (range 15–144 months).

Total RNA was isolated from tissue specimens using the Allprep DNA/RNA Mini Kit (Qiagen, Germantown, MD). Total RNA (0.5 μg) was reverse transcribed to cDNA using PrimeScript RT reagent Kit (Takara Bio Inc., Otsu, Japan), according to the manufacturer’s protocol. Each PCR was performed using 2 μl of cDNA and 0.2 μmol/l of each probe in a LightCycler System with SYBR Premix Dimer Eraser (Takara Bio Inc.). PCR primer sequences were as follows: for DSS1, forward 5′-GTTAGAGGAAGACGACGAGT-3′ and reverse 5′-GGATGCTATGAAGTCTCCAT-3′; for β-actin, forward 5′-TGGCACCCAGCACAATGAA-3′ and reverse 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′. Each reaction (20 μl samples) was performed under the following conditions: initialization for 10 sec at 95°C, and then 45 cycles of amplification, with 5 sec at 95°C for denaturation and 20 sec at 60°C for annealing and elongation. The expression level of DSS1 mRNA is given as relative copy numbers normalized to those of β-actin mRNA. In some experiments, qRT-PCR was performed using a LightCycler (Roche Diagnostics, Indianapolis, IN). Specific oligonucleotide primers and probes for DSS1 and gapdh were purchased (Nihon Gene Research Laboratories, Sendai, Japan). The specific primers for DSS1 were the same as above. Their sequences were as follows: dss1 donor probe, 5′-CCCAATTATCCTCCCAGACATGTGCATCTT-3′; dss1 acceptor probe, 5′-ATCTTCATCTAAGCCAGCCCAGTCTTCGG-3′; gapdh-F, 5-CAGCCTCAAGATCATCAGC-3′; gapdh-R, 5′-GGCCATCCACAGTCTTCT-3′; gapdh donor probe, 5′-GGTCATCCATGACAACTTTGGTATCGTGGAA-3′; gapdh acceptor probe, 5′-GACTCATGACCACAGTCCATGCCATCACTG-3′. The level of DSS1 mRNA expression was determined relative to gapdh.

Histological sections (4 μm) were deparaffinized and incubated for 10 min in methanol containing 0.3% hydrogen peroxide. Mouse monoclonal antibodies (mAbs) against ERα (SP1, Ventana Japan, Tokyo, Japan), PgR (1E2, Ventana Japan) and Ki67 (MIB1, Dako Japan, Tokyo, Japan), a polyclonal Ab against HER2 (Dako Japan), and a mouse mAb against p53 (DO7, Dako Japan) were used; staining was carried out in the NexES IHC Immunostainer (Ventana Medical Systems, Tucson, AZ), in accordance with the manufacturer's instructions. ERα and PgR status were evaluated based on the percentage of positively stained nuclei, and the status of a nucleus was considered positive when ≥1% of the nucleus was stained. HER2 was evaluated using the HercepTest method (Dako Japan), with membranous staining scored on a scale of 0 to 3+. Tumors with scores of ≥3 or with a ≥2.2-fold increase in HER2 gene amplification as determined by fluorescence in situ hybridization were considered to be positive for HER2 over-expression. Ki67 was scored as the percentage of nuclear-stained cells out of all cancer cells along the invasive front of the tumor in × 400 high-power fields; this gave the Ki67 labeling index. p53 was evaluated based on the percentage of positively stained nuclei, and the status of a nucleus was considered positive when ≥20% of the nucleus was stained.

The human breast cancer cell lines MCF7 and MDA-MB-231 were obtained from the American Type Culture Collection and maintained in RPMI-1640 (Life Technologies, Carlsbad, CA) supplemented with 10% heat-inactivated fetal calf serum (MP Biomedicals, Santa Ana, CA), 2 mM L-glutamine (Lonza, Allendale, NJ), and 5 × 10^-5 M 2-mercaptoethanol (Wako Pure Chemicals Industries, Osaka, Japan) with 5% CO₂ at 37°C. siRNA treatment was performed when the cells reached 50% confluence; cells were transfected with 10 nM of control (siCtrl) or DSS1 (siDSS1; Life Technologies) siRNA using Lipofectamine RNAiMAX (Life Technologies). The siRNA target sequences were as follows: siDSS1-(a), 5′-GACAAUGUAGAGGAUGACUUCUCUA-3′; siDSS1-(b), 5′-GCAGCCGGUAGACUUAGGUAUGUUA-3′; siCtrl-(a), 5′-GACGUAUAGGGUAAGUCCUUAACUA-3′; siCtrl-(b), 5′-GCAGGCGAUUCAGAUCUGGUGCUUA-3′. The results with siDSS1-(a) are shown in the figures as representatives.

A retroviral vector, designated pFB-DSS1-IRES-GFP, was transfected into PLAT-GP (Cellbiolabs, San Diego, CA) retrovirus packaging cells using FuGENE HD (Roche Diagnostics). MCF7 and MDA-MB-231 cells were infected with the retroviruses using polybrene (8 μg/ml; Sigma, St. Louis, MO) for 2 days. GFP-positive cells were sorted using a JSAN cell sorter (BayBioscience, Kobe, Japan).

In vitro cell proliferation was determined using an MTT assay performed with a Cell Proliferation Kit I (Roche Applied Science, Penzberg, Germany). Briefly, at 24 hr after siRNA treatment, 2.5 × 10³ cells per well were seeded and incubated with MTT labeling reagent (0.5 mg/ml) for 6 hr at 37°C. The soluble formazan product was quantified using an ELISA reader at 590 nm from day 1 to day 4.

DNA damage-inducing agents were added when the cells reached 80% confluence. CPT (Merck Millipore, Billerica, MA) or ETP (Merck Millipore) was introduced to the cells at a final concentration of 50 μM. After 24 to 72 hr, cells were harvested, washed with ice-cold PBS, resuspended in PBS with 250 μg/ml RNase A, and stained with 2× PI solution for 2 hr at 4°C. The cell cycle was analyzed using FACSCalibur (BD, Franklin Lakes, NJ) and CellQuest software.

Cells were washed twice with staining buffer (0.1 M Hepes pH 7.4, 1.4 M NaCl, 25 mM CaCl₂) and resuspended in FITC-conjugated Annexin XII (Abcam, Cambridge, UK) on ice for 10 min. Cells were then counterstained with 0.5 μg/ml PI on ice for 5 min, and an equal volume of staining buffer was added. Early apoptotic (Annexin XII⁺/PI^-) and late apoptotic/necrotic (Annexin XII⁺/PI⁺) cells are shown with merged color.

After DSS1 knockdown, floating cells were centrifuged in microfuge tubes and then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.1% CaCl₂. After washing in the same buffer, embedding in 2% agarose and incubating post-fixation in 1% OsO₄ for 1 hr, the specimens were washed again, dehydrated in a series of ethanol baths, and embedded in Epon 812. Thin sections were cut with a Leica Ultracut UCT microtome, post-stained with 2% uranyl acetate and Reynolds lead citrate, and examined using a JEOL JEM 1400 operated at 80 kV. Two researchers (TS and ST) independently analyzed cells with apoptotic changes using digitized EM images.

DNA damage induced by DSBs and single-strand breaks were analyzed using a comet assay kit (Trevigen, Inc., Gaithersburg, MD), according to the manufacturer’s protocol. For quantification, the tail moment (an index of DNA damage calculated as the product of the tail length and the fraction of DNA in the comet tail) was evaluated using CometScore version 1.5 software (AutoComet.com). At least a hundred cells from each sample were scored.

The nonparametric Wilcoxon (for uni-variable), Kruskal-Wallis test (for multi-variables), and the χ² test were adopted for statistical analysis of the associations between the DSS1 mRNA status and various clinicopathological factors. RFS and BCSS curves were calculated according to the Kaplan-Meier method and verified by the log-rank test. All statistical significance was defined as P < 0.05. JMP software version 8.0.2 for Windows (SAS institute Japan, Tokyo, Japan) was used for all statistical analyses.

We examined the survival of breast cancer patients using the Kaplan-Meier method. We analyzed the expression level of DSS1 mRNA by qRT-PCR from the tumor samples obtained at surgery using primers for DSS1 mRNA (Additional file 1: Figure S1). The relative expression level was arbitrarily determined and used for classification of breast cancer cases into DSS1-high (DSS1 ^high) and DSS1-low (DSS1 ^low) groups. The DSS1 ^high group showed a significant shorter survival time (*P = 0.011) on the RFS curve, while the difference in the breast cancer specific survival (BCSS) curves was not significant (n.s.; P = 0.25) (Figure 1A). DSS1 physically interacts with the RPN3/S3 proteasomal subunit, which increases the degradation of ubiquitinated p53 [20]. In a large cohort, over-expression of p53 was significantly associated with poor prognosis in premenopausal women treated with tamoxifen after chemotherapy [21]. The p53 expression was used for classification by arbitrarily determined conditions (see Methods). We compared RFS among p53^low/DSS1 ^low, p53^high/DSS1 ^low, p53^low/DSS1 ^high, and p53^high/DSS1 ^high groups (Figure 1B). While the number of cases was small, no change of the survival curve was observed when p53 expression was low; however, the DSS1 ^high group showed a worse prognosis in comparison with the DSS1 ^low group in breast cancer cases with high p53 expression (*P = 0.045). Other parameters and markers were compared in the DSS1 ^high and DSS1 ^low groups (Table 1). No significant differences were observed in the groups of pre- or post-menopausal women, positive or negative nodal status, tumor size < 20 or ≥ 20 mm, nuclear grade I to III, positive or negative vessel invasion score, ERα status, and PgR status. However, DSS1 expression showed a significant difference (*P = 0.0255) based on HER2 status, which is presumably associated with effective treatment using trastuzumab as the standard regimen for HER2-positive breast cancer cases. Interestingly, DSS1 expression was markedly increased (55.6; median of DSS1 relative expression) in the group of breast cancer cases with low Ki67 labeling indices (< 20%) compared with the expression (36.0; median of DSS1 relative expression) in the group with high Ki67 labeling indices (≥ 20%).

For molecular analysis, we used two breast cancer cell lines, MCF7 (ERα⁺PgR⁺ with wild type p53) and MDA-MB-231 (ERα^-PgR^- with p53 mutation). The effect of DSS1 over-expression on cell proliferation and cell cycle progression was examined in breast cancer cells that had been engineered to over-express DSS1 (MCF7^DSS1 or MDA-MB-231^DSS1) by retroviral transfection (Additional file 2: Figure S2). Stable transfectants that expressed high levels of DSS1 mRNA did not show any changes in cell cycle or cell proliferation compared with the control (GFP only) transfectants (Figure 2A). DSS1-over-expressing cells showed no abnormalities in proliferation. Trastuzumab is a first choice for treatment of HER2-positive breast cancer in our cohort, but camptothecin (CPT) is often chosen as a regular regimen for HER2-negative breast cancer cases (Table 1). The susceptibility of DSS1-over-expressing breast cancer cells to the DNA-damage inducing agents CPT and etoposide (ETP) was examined. DSS1 over-expression renders breast cancer cells more resistant to treatment with CPT, an inhibitor of type 1 topoisomerase (which cleaves one strand of double-stranded DNA), compared with the GFP-control MCF7 transfectants (Figure 2B), while no change was detected in DSS1 over-expressing MDA-MB-231 transfectants (Figure 2C). The effect of DSS1 over-expression was masked in both MCF7 and MDA-MB-231 cells upon exposure to ETP, an inhibitor of topoisomerase II (which cleaves two strands of double-stranded DNA and induces cell cycle arrest at G2/M) (Additional file 3: Figure S3A and B). Therefore, DNA damage was examined using the alkaline comet assay (Figure 3A and B). DSS1 over-expression reduced CPT-induced DNA damage in MCF7 and MDA-MB-231 cells, suggesting that DSS1 over-expression increases drug resistance in breast cancers.

Next, we examined cell growth in DSS1-depleted cells. Two siRNAs directed against two independent DSS1 mRNA sequences elicited marked reductions in DSS1 mRNA levels (Additional file 4: Figure S4A) and protein levels (Additional files 4: Figure S4B and 5); we therefore used one of these sequences in the following experiments. DSS1 knockdown (siDSS1) caused decreased cell growth, as observed in an MTT assay (Figure 4A: left) and in an assessment of cell number (right). At 3 and 5 days after siDSS1-treatment, the number of dead cells in the sub-G1 population increased compared to siCtrl-treated cells (Figure 4B). siDSS1-treated MCF7 cells experienced cell cycle arrest at M phase entry, with increases in G2/M phase populations at day 5 (27.0% vs. 6.20%). siDSS1 also markedly induced the number of sub-G1 (dead) cells in drug-resistant MDA-MB-231 (24.8%, compared with 3.99% for siCtrl).

Treatment of MCF7 cells with siDSS1 caused apoptosis, as detected by staining with Annexin XII and propidium iodide (PI). Representative Annexin XII⁺PI^- (early apoptotic) and Annexin XII⁺PI⁺ (late apoptotic/necrotic) cells are shown in Figure 5A. We further examined cell morphology at day 5 after siDSS1 treatment using electron microscopy (EM). siDSS1 treatment induced cell death in MCF7 cells, along with typical apoptotic changes including chromatin condensation, enlarged nuclei and smaller nuclei compared to the siCtrl-treated cells (Figure 5B; chromatin condensation marked with arrowheads). siDSS1 treatment also caused apoptotic cell death in MDA-MB-231, with condensed chromatin and smaller nuclei relative to the control cells (Figure 5B; marked with arrowheads); however, some of these cells also showed atypical cell death morphologies, with enlarged cell size accompanied by electron microscopically void nuclei containing finely condensed chromatin-like structures adjacent to the inner nuclear membrane (Figure 5B; marked with asterisks). Because MDA-MB-231 do not seem to arrest at M phase entry, presumably due to p53 mutation, the atypical cell death observed in siDSS1-treated MDA-MB-231 might represent cell death occurring at various cell cycle stages. Finally, while the cell morphologies were not identical, siDSS1-treated MCF7 and MDA-MB-231 cells showed similar extents of DNA damage in the alkaline comet assay (Figure 5C).

To confirm the effect of DSS1 on breast cancer cells, we investigated the effect of DSS1 knockdown on cell cycle and cell death in breast cancer cells during early stages of treatment with CPT or ETP. DSS1 knockdown markedly increased the number of cells in the sub-G1 apoptotic population in both MCF7 and MDA-MB-231 cells treated with CPT as early as day 2 (Figure 6A). Similarly, DSS1 knockdown increased the sub-G1 population in both breast cancer cell lines treated with ETP (Figure 6B). Because MDA-MB-231 cells, which are of mesenchymal origin and have a triple-negative phenotype [22], are well known to be resistant to several DNA-damaging agents, these data suggested that DSS1 depletion can increase chemosensitivity in drug-resistant breast cancer cells with either wild type or mutant p53.