Targeting ataxia telangiectasia-mutated- and Rad3-related kinase (ATR) in PTEN-deficient breast cancers for personalized therapy

The Nottingham Tenovus Primary Series is a well-characterized consecutive cohort of early-stage primary operable invasive BC patients from which the samples were used for the TMA construction [1, 2, 4, 21]. Patient demographics are summarized in Supplementary Table S1. Breast cancer specific survival (BCSS) was defined as the number of months from diagnosis to the occurrence of BC-related death. Survival was censored if the patient was still alive at the time of analysis, lost to follow-up, or died from other causes.

A mouse monoclonal PTEN antibody clone 6H2.1 (Dako, Denmark) was diluted in 1:20 dilution using Leica Antibody diluent. Antigen retrieval was performed as follows: the TMA slides were incubated at 98 °C (in water bath) for 25 min in target retrieval solution (pH 9.0; Leica Microsystems, Newcastle, UK) then the slides were incubated for 10 min at room temperature in warm Tris buffer saline (TBS, pH 7.6, 50 °C). Finally, slides were cooled by flooding in cold water (pH 6.0) for 5 min. The primary antibody was incubated for 1 h at room temperature. The Novocastra Novolink Max Polymer Detection Systems (RE7280-K: 1250 tests, Leica Biosystems) was used to visualise the staining. Briefly, the slides were heated at 60 °C for 10 min for dewaxing. Following cooling of samples to room temperature, the slides were rehydrated using a Leica Autostainer. After performing antigen retrieval, the slides were loaded onto Shandon Cover plates and fitted on sequenza plates for IHC. Initially, Endogenous peroxidase was blocked for 5 min, and then a protein block was applied for 5 min. The PTEN antibody was added, and the slides were incubated for 1 h at room temperature. After this, post-primary block was run for 30 min and then polymer was applied for further 30 min. The chromogen (DAB) was applied for 5 min, and tissues were counter-stained with haematoxylin for 6 min. Slides were dehydrated using Leica Autostainer and mounted with DPX. Negative (omission of the primary antibody) and positive controls were included according to manufacturer datasheet. ATR and pChk1 staining have been reported previously. Optimisation, specificity and cut-off points of ATR and pChk1 antibodies used in the current study have been reported previously [1]. A set of slides were incubated for 18 h at 4 °C with the primary mouse monoclonal anti-ATR antibody, clone 1E9 (H00000545-M03, Novus Biologicals, Cambridge, UK), at a dilution of 1:20. A further set of slides were incubated for 60 min with the primary rabbit polyclonal anti-phosphorylated Chk1 antibody (Ab58567, Abcam, Cambridge, UK), at a dilution of 1:140 [1].

The TMA slides were initially assessed through staining quality and specificity under light microscope. Slides were then scanned into high-resolution digital images (0.45 lm/pixel) using a NanoZoomer slide scanner (Hamamtsu Photonics, Welwyn Garden City, UK) and accessed using a web-based interface (Distiller, SlidePath Ltd, Dublin, Ireland). They were scored at ×20 magnification using a minimum of 2400 high-resolution screen (1920 × 1080). Assessment of staining was based on a semi-quantitative approach using a modified histochemical score (H-score) taking the intensity of staining and the percentage of stained cells into account. For the intensity, a score index of 0, 1, 2, and 3 was used, which corresponded to negative, weak, moderate, and strong staining intensity, respectively. The percentage of positive cells per intensity was estimated. PTEN staining in breast tumour cells was detected in the nuclei and cytoplasm of the breast cancer cells. Cut-off was used to classify the tumours with a nuclear-PTEN H-score equal to ‘0’ as negative, and those with an H-score more than 0 as positive. H-score > 90 was taken as the cut-off for high cytoplasmic-PTEN expression. Optimisation, specificity and cut-off points of ATR and pChk1 antibodies used in the current study have been reported previously [1]. H-score of ≥ 60 was taken as the cut-off for high ATR expression, H-score of ≥ 50 was taken as the cut-off for high cytoplasmic pChk1 expression and H-score of ≥ 50 was taken as the cut-off for high nuclear pChk1 expression.

Tumor marker prognostic studies (REMARK) criteria, recommended by McShane et al. [13], were followed throughout this study. Ethical approval was obtained from the Nottingham Research Ethics Committee (C202313).

Statistical analysis was performed using SPSS v 22 statistical software. Association with clinical and biological markers was assessed using Chi-squared test. Kaplan–Meier survival curve with log-rank test was plotted to determine the survival distribution of studied patients’ subgroups. Statistical significance was defined as p value ≤ 0.05. Due to multiple comparisons, the adjustment for p values of multiple testing was used according to Benjamini–Hochberg correction method.

MDA-MB-231 human breast cancer cell line was purchased from American Type culture collection (ATCC, Manassas, USA) and was grown as per ATCC recommendations. MDA-MB-231 cells were cultured in RPMI-1640 medium (Sigma, St. Louis, MO, USA) and Minimum Essential Medium Eagle (Sigma-Aldrich) supplemented with 1% l-glutamine (200 mM) and 1% non-essential amino acids (0.1 mM), respectively. The breast cancer cell lines BT-549 and MDA-MB-468 (Cell Line Service, Eppelheim, Germany) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, Carlsbad, USA). All media were supplemented with 10% foetal bovine serum (Sigma) and 1% penicillin/streptomycin (10,000 units penicillin and 10 mg streptomycin/mL) (Sigma, Gillingham, UK). All cell lines were cultured as adherent cultures in a humidified 5% CO₂ incubator at 37 °C. ATR inhibitor (VE-821, 10 mM in 1 mL DMSO) was purchased from Selleckchem (Houston, TX, USA). DMSO (Sigma, Gillingham, UK) was used as solvent to dissolve VE-821 and was tested solely as a vehicle control (< 0.1% v/v).

Real-time PCR was performed using RT² Profiler DNA Repair PCR Array for 84 DNA repair genes in duplicates as described previously [3]. The data were analyzed as per manufacturer’s recommendations. GAPDH was used for normalization of the data. A twofold change or above in expression was considered significant. All experiments were performed in duplicate.

To evaluate the specificity of PTEN-antibody used for the immunohistochemical study, cell lysates were prepared and Western blot analysis was performed. The primary antibodies for PTEN, ATR, and β-actin used in this study were incubated at room temperature for 1 h (PTEN 1:100 dilution [Dako], ATR 1:1000 dilution [Cell signaling] and β-actin 1:5000 dilution [Sigma]). Infrared dye-labelled secondary antibodies (Li-Cor) [IRDye 800CW Donkey Anti-Rabbit IgG and IRDye 680CW Donkey Anti-Mouse IgG] were incubated at a dilution of 1:10,000 for 1 h. Membranes were scanned with a Li-Cor Odyssey machine (700 and 800 nm) to determine protein expression.

Cells were seeded at a density of 1000 cells per well in 96-well plates and allowed to adhere overnight. Inhibitory compounds were added to samples in plates at a range of concentrations after 16 h, followed by incubation of plates for total of 5 days. All steps of MTS assay were performed as per manufacturer’s recommendations. All experiments were performed in triplicate at least three times. Data analysis was performed in Microsoft Excel 2010 (Microsoft, Redmond, USA) and GraphPad Prism 6 (GraphPad, La Jolla, USA).

1 × 10⁵ cells were seeded in 6-well tissue culture plates, and were allowed to adhere overnight. VE-821, an ATR inhibitor, was added after 16 h at the concentration of 5 μM. Cells were harvested by trypsinisation and centrifuged at 1000 rpm for 5 min at 24 and 48 h post drug exposure. Cells pellets were then re-suspended in 1 mL of 70% ice-cold ethanol to fix the cells. Following this, samples were stored at 4 °C. Suspensions were centrifuged at 1000 rpm for 5 min followed by removal of the supernatant. Samples were processed using H2AX Phosphorylation Assay Kit (Merckmillipore, Nottingham, UK) as per manufacturer’s recommendations. At least 10,000 cells from each sample were analysed. All experiments were done in duplicate three times. Weasel (Victoria, Australia) flow cytometry analysis software was used for data analysis. Graphical representation and statistical analysis was performed in GraphPad Prism 6 (GraphPad, La Jolla, USA).

1 × 10⁵ cells were seeded into 6-well tissue culture plates and allowed to adhere overnight. VE-821 was added to tissue culture plates at a concentration of 5 μM after 16 h. 24 and 48 h after VE-821 administration. For detection of apoptosis, FITC-Annexin V flow cytometry was performed using the fluorescein isothiocyanate [FITC]-Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Jose, USA) as per manufacturer’s recommendations. At least 10,000 cells from each sample were analysed. Weasel (Victoria, Australia) flow cytometry analysis software was used for data analysis. The percentage of apoptotic cells (FITC-Annexin V positive, PI positive and FITC-Annexin V positive, PI negative) in the treated population was ascertained by comparing it to a control population of untreated samples. Statistical analysis was performed in GraphPad Prism 6 (GraphPad, La Jolla, USA). All experiments were done in duplicate three times.