MELK 3-11-2016
Material and Methods:
Cell culture and cell lines
Breast cancer cells were propagated from frozen samples in cell culture media, and passaged when reaching confluence. Cell lines were chosen to include an appropriate representation of all molecular subtypes. All cell lines were purchased between 7/2012 and 8/2015 from ATCC (except the ACC cell lines) and the remainder (all ACC cell lines) from the Deutsche Sammlung von Mikroorganismens und Zellkulturen GmbH (DSMZ, Brunswick, Germany). All cell lines were authenticated and genotyped immediately prior to evaluation at the University of Michigan DNA Sequencing core facility by fragment analysis and ProfilerID utilizing the AmpFLSTR Identifier Plus PCR Kit (Life Technologies, Grand Island, NY, Cat #4322288) run on an Applied Biosystems AB 3730XL 96-capillary DNA analyzer. Sample fragments were compared against cell line standards provided by ATCC and DSMZ. ZR75-30, MDA-MB-231, MDA-MB-453, BT474, BT20, AU565, HCC 1954, HCC 1806, HCC38, HCC70, and HCC 1937 breast cancer cell lines were grown in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen) in a 5% CO2 cell culture incubator. ACC-231 cells were grown in 90% RPMI medium (Invitrogen) supplemented with 10% FBS (Invitrogen) in a 5% CO2 cell culture incubator. ACC-302 cells were grown in 80% DMEM (Invitrogen) supplemented with 20% FBS (Invitrogen) in a 5% CO2 cell culture incubator. ACC-422 cells were grown in 85% MEM (Invitrogen) supplemented with 15% FBS (Invitrogen) in a 5% CO2 cell culture incubator. BT549 and T47D cells were grown in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Invitrogen) and 0.023 IU/ml insulin in a 5% CO2 cell culture incubator. ACC-459, ACC-440, CAMA-1, were grown in DMEM (Invitrogen) supplemented with 10% FBS in a 5% CO2 cell culture incubator. MCF-7 cells were grown in modified MEM (Invitrogen) with 0.023 IU/ml insulin in a 5% CO2 cell culture incubator. All cultures were maintained with 50 units/ml of penicillin/streptomycin (Invitrogen).
RNA isolation and Quantitative RT-PCR (Q-RT-PCR):
Total RNA was isolated using TRIzol (Invitrogen) and an RNeasy kit (Qiagen) according to manufacturers' instruction. Total RNA was reverse transcribed into cDNA using SuperScript III and random primers (Invitrogen). Quantitative PCR (qPCR) was performed using SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems 7900HT Real-Time System. The relative quantity of the target gene was computed for each sample using the ΔΔCt method by comparing mean Ct of the gene to the mean Ct of the housekeeping gene GAPDH. All the primers were obtained from Integrated DNA Technologies (IDT). Sequences of all the primers used are listed in Supplementary Table S1B. Results were reported as average expression ± standard error of the mean. Primer sequences were as follows:
MELK forward #1: 5’ CT 3’ CCA ACA AAA TAT TCA TGG TTC TTG
MELK reverse #1: 5’ CT 3’ AGG CGA TCC TGG GAA ATT AT
Western blot analysis
For protein isolation from tissue culture cell lines, cells were washed once with ice-cold phosphate buffered saline (PBS) and lysed in protein lysis buffer consisting of 50mM HEPES pH7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 10% glycerol, 100mM NaF, Complete Mini protease inhibitors cocktail tablet (Roche), and phosphatase inhibitor cocktail I and II (Sigma-Aldrich). Protein concentration was determined using BCA Protein Assay Reagents (Pierce Biotechnology). Western blot analysis was performed as previously described (1). Briefly, aliquots of total protein (30μg) were resolved by electrophoresis in 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Amersham Biosciences). The membrane was blocked and incubated with primary antibody. After washing in TBST, the membrane was then incubated with horseradish peroxidase-conjugated secondary, washed again, and antigen-antibody complexes were detected using the ECL or ECL Plus chemiluminescent system (Amersham Bioscience). Primary antibodies specific for MELK were purchased from Sigma Prestige series (Catalog #HPA017214). Cleaved PARP (Asp214) (D64E10 Rabbit mAb) was purchased from Cell Signaling using the catalog #5625S. Total PARP antibody was also purchased from Cell Signaling using the catalog #9542S. Anti-rabbit secondary antibodies were also obtained from Sigma. Protein isolation from human tumor samples was identical to that of cells, except that samples were first homogenized using a 7 mm generator and a rotator-stator homogenizer (ProScientific). Samples were homogenized in protein lysis buffer and all isolation was done on ice.
siRNA transfection
For knockdown experiments, cells were seeded in 6-well plates and transfected with 100 nM ON-TARGET plus SMARTpool siRNA (ThemoScientific) targeting MELK or non-targeting control (Non-targeting Pool, catalogue no. D-001810-10-50, using oligofectamine (Invitrogen) according to the manufacturer’s instructions). The following are the catalogue numbers and the siRNA sequences: ON-TARGET plus Human MELK SMARTpool, catalogue no. J-004029-06, J-004029-09, set# LQ-004029-00-0002, target sequences, #1 GA and #4 GG. Cells were trypsinized 24 h post-transfection and used in clonogenic survival assays as well as for RNA extractions to determine the knockdown efficiency.
shRNA construct and stable clone generation
The pTRIPZ lentiviral system with MELK inducible shRNA transfection starter kit was purchased from ThermoScientific using catalog #RHS4696-200703132 and cat# RHS4696-200691582 for non-template control and shMELK. Stable cell lines were generated using lentiviral transduction. Lentiviral particles with shMELK or shControl were packaged and culture medium provided at the vector core facility of University Michigan. Stable cell lines were selected using complete medium containing puromycin. Clones were selected and screened for both RFP and MELK expression changes and were used as pools and as selected stable clones in all in vitro and in vivo experiments.
Clonogenic survival assays
Exponentially growing cells were treated with MELK knockdown and/or radiation at doses as indicated and then replated at cloning densities chosen to demonstrate the greatest dynamic range in the survival assays. Cells were grown for up to 14 days and then fixed and stained with methanol-acetic acid and crystal violet, respectively, and scored for colonies of 50 cells or more. Plating efficiency was corrected for in all experiments. Drug cytotoxicity was calculated as the ratio of surviving drug-treated cells relative to untreated control cells. Radiation survival data from MELK knockdown cells were corrected for siRNA transfection and gene knockdown cytotoxicity, as previously described (2). Cell survival curves were fitted using the linear-quadratic equation, and the mean inactivation dose calculated according to the method of Fertil and colleagues (3). The radiation enhancement ratio (EnhR) was calculated as the ratio of the mean inactivation dose under control conditions divided by the mean inactivation dose under gene knockdown conditions.
Irradiation
Irradiation was carried out using a Philips RT250 (Kimtron Medical) at a dose rate of ∼2 Gy/min in the University of Michigan Comprehensive Cancer Center Experimental Irradiation Core. Dosimetry was carried out using an ionization chamber connected to an electrometer system that is directly traceable to a National Institute of Standards and Technology calibration. For tumor irradiation, animals were anesthetized with isofluorane and positioned such that the apex of each flank tumor was at the center of a 2.4-cm aperture in the secondary collimator, with the rest of the mouse shielded from radiation.
Proliferation assays
Cells were plated in 48 well plates at various concentrations (15,000 cells/well for BT-549 and MCF-7; 10,000 cells /well for MDA-MB-231) and treated with the indicated conditions and placed in the Incucyte System (Incucyte ZOOM, Essen BioScience). Cell growth measurements were taken every 2 hrs. in the automated, non- invasive system for monitoring of live cells in culture with scanning every 2h for the indicated period of time (5-7 days, depending on the cell line). Automated image processing was accomplished by applying an appropriate processing definition and growth curves (proliferation) were generated from confluence measurements as noted above.
Flow Cytometry apoptosis assays
Cells were transfected with scrambled or MELK specific siRNAs or treated with MELK inhibitor OTS167 (1nM for 24 hours). Forty eight after transfection the cells were harvested and the apoptosis assay utilizing cleaved PARP was performed as detailed above. Apoptotic assays by flow cytometry were performed using ApoScreen Annexin V Apoptosis Kit (Southern Biotech #10010-02), per the manufacturer’s protocol. Briefly, cells were washed with cold PBS, suspended in cold 1× binding buffer, stained with Annexin V and propidium iodide, and subjected to flow cytometry by FACSAria Cell Sorter (BD Biosciences). Results were analyzed and plotted using Summit 6.0 Software (Beckman Coulter). A portion of cells was used to determine the knockdown efficiency using qRT-PCR.
Mouse xenograft experiments
We used a stable shMELK construct (described previously) that was inducible under doxycycline control. CB17-SCID mice were injected in the bilateral flank with MDA-MB-231 breast cancer cells (0.5 × 106cells) that had stably incorporated the shMELK construct under doxycycline control. After tumors reached 50-100 mm3, shMELK expression was induced by doxycycline in the experimental arm with the control mice receiving no doxycycline. MDA-MB-231 cells with stable incorporation of shScramble constructs were also used to control for doxycycline effects. The experiment was divided into four treatment groups that included a control group (with three internal controls—shMELK-dox, shScramble-dox, shScramble+dox), a MELK inhibition group [consisting of shMELK expression alone driven by doxycycline], a radiation therapy alone group [2 Gy daily for 6 days without doxycycline-mediated shMELK induction], and a combination MELK knock-down + radiation group [2 Gy daily for 6 days with doxycycline-mediated shMELK induction]. Each group contained 16-20 xenografts in each treatment arm. Growth in tumor volume was recorded 3 times per week after shaving of the bilateral flanks by using digital calipers and tumor volumes were calculated using the formula (π/6) (L × W2), where L = length of tumor and W = width. The fractional product method was used to determine additive versus synergistic effects as previously described (4). According to this method, the effects of two or more treatments, when combined, can be calculated by multiplying the fractional tumor volume by each single drug. If the effect of the treatments acting simultaneously is equal to, larger than, or smaller than that calculated (R value), additivity, synergism, or antagonism, respectively, is assumed. Efficacy of MELK knockdown in xenograft tumors was confirmed (as depicted). Loss of body weight during the course of the study was also monitored weekly. All procedures involving mice were approved by the University Committee on Use and Care of Animals (UCUCA) at the University of Michigan and conform to their relevant regulatory standards. Growth curve comparisons and statistical analysis was done using unpaired t test was used to calculate two-tailed P value to estimate statistical significance of differences between two treatment groups or as previously described in the statistical methods section.
Gamma H2AX foci formation: Analysis of γH2AX by flow cytometry was performed as previously described (5). Briefly, cells were harvested at varying time points after irradiation using 4 Gy (30 minutes, 4 hrs., 16 hrs., and 24 hrs.), washed with PBS, and fixed with 70% ethanol at 4º. Cells were resuspended in 100 ul of 1:500 diluted γH2AX antibody (Millipore, Cat#07-627). After overnight incubation at 4º, cells were washed, spun down, and resuspended in 1:50 fluorescein isothiocynate (FITC)-conjugated anti-mouse secondary (Sigma, Cat#F-0257) diluted 1:100 in PBT. Samples were then washed in PBT buffer and spun, resuspended in 500 ul of propidium iodine (BD Bioscience-0.33 mg/ml in 500 ul PBS), and analyzed by flow cytometry.
For cell staining and foci formation assays, cells were cultured on coverslips in 12-well plates and treated with siRNA oligonucleotides as indicated for 24 hours and then immediately exposed to 4 Gy radiation. Cells were collected at indicated time points (30 minutes, 4 hrs., 16 hrs., and 24 hrs.) and processed as previously described (6). Images were collected with a 60x objective lens using an Olympus DP70 camera fitted in an Olympus 1X-71 microscope. The H2AX foci were detected with mouse monoclonal antibodies phospho γH2AX (Millipore, #JBW301, Cat#05-636). For quantitation of γH2AX foci, at least 100 cells from each of three independent experiments were visually scored for each condition. Cells with ≥10 γH2AX foci were scored as positive and compared for statistical analyses.
Patient Cohorts
A publicly available clinical cohort with gene expression and local recurrence information was utilized for biomarker assessment (Servant). This multi-institutional training cohort consisted of 343 patients from the Netherlands and France with early stage breast cancer treated with breast-conserving surgery with post-op radiation (7). Gene expression from an additional dataset (Wang) consisting of patients with lymph-node negative breast cancer who were treated with breast conserving surgery or modified radical mastectomies from 1980–95 as previously described was also interrogated (8). These patients also received radiotherapy when indicated (87%). None of the patients in either dataset received any adjuvant systemic therapy. 219 patients had breast-conserving surgery and 67 had modified radical mastectomies. Radiotherapy was given to 248 patients (87%), and local recurrence-free survival was tracked in all patients. All patients from both datasets were used in the analysis and complete patient and cohort characteristics are included in the supplementary tables 2-3 for each dataset. Specimen characteristics and handling were described previously (7, 8). Data is presented in accordance with the REMARK guidelines and no patients from these studies were excluded from these analyses.
All appropriate IRB protocols were followed in the acquisition and analysis of the data from these clinical datasets. Please refer to the original cited publications for full details of the IRB approval.
Microarrays:
Normalized expression data for the cell lines was downloaded from the EMBL-EBI ArrayExpress website as described in the original publication (9). Normalized expression data for the Servant dataset was downloaded from the EMBL-EBI Array Express repository (http://www.ebi.ac.uk/arrayexpress/experiments/E-TABM-157/). All expression data was log transformed and median centered and scaled to the same minimum/maximum. Array expression data for the Wang dataset was obtained from oncomine.org by following the link to GSE2034 where the data has been reposited and made freely available (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2034) (8).
Bioinformatic analysis:
GSEA analysis:
Correlation was performed comparing MELK versus all other genes in the breast cancer samples from TCGA. RNAseq processing was described previously (10). The Spearmans correlation coefficient rho was generated by correlating MELK versus each other gene. This was put into GSEA using the pre-ranked algorithm and run with the C2:curated, C5:GO, and C6:oncogenic signatures gene sets.