Download Supplementary Materials and Methods

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
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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).
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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 antigenantibody 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.
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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# RHS4696200691582 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 linearquadratic 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
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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
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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
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breast cancer cells (0.5 × 10 cells) 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. MDAMB-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
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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
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(π/6) (L × W ), 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
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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 breastconserving 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.
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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
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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.
Statistical analysis: Results are presented as mean ± SD or SEM of at least three experiments as
indicated. Student's t test was used to assess the statistical significance of differences. A
significance level threshold of p < 0.05 was used. Correlation coefficients were calculated using
Pearson’s correlation methods. For xenograft studies, previously generated xenograft data was
used to determine the number of mice bearing tumors appropriate for each experiment. As an
example, preliminary data in MDA-MB-231 cancer xenografts indicate that after treatment with
RT alone, tumors are expected to grow approximately 5-fold in size over a 4-week period, with
coefficient of variation (CV) across xenografts of 30-50% at each time point (11). Using 10
xenografts/treatment, the statistical power to detect a mean 40% tumor volume reduction in the
MELK knockdown group is estimated to be 98.6% if the CV is 35% and, with even 10% of data
missing, the power is still > 80% if the CV increases to 50%, (Data projected using Student’s TTest on 5000 simulated datasets with each treatment group modeled as Gaussian random
variables). We used analysis of variance (ANOVA) to investigate the effect of all factors (RTsensitivity, MELK knockdown, and RT) individually and jointly. The F-test was used to test for
significance of factors. When statistically significant interactions were found, the Tukey Honest
Significant Difference method was used to compare differences in mean tumor volume for all
pairs of treatment groups. All tests were conducted at α=0.05. All other statistical analyses were
performed as described in the text. Kaplan-Meier curves were generated, and univariate and
multivariate analysis was performed using Cox regression. Univariable and Multivariable
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analyses were run using MedCalc® 15 software. All factors were inputted for univariate analysis
and those factors found to be significant were used in the multivariable cox proportional hazards
regression modeling. MELK was analyzed as a continuous variable. Analysis using MELK as an
ordinal value was also performed but not shown.
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