Detailed information on the β-catenin activated reporter (BAR) has been previously described (20
). Briefly, the β-catenin activated reporter (pBAR) is a lentiviral plasmid that contains 12 TCF/LEF binding sites (5′-AGATCAAAGG-3′) each separated by distinct 5 base-pair linkers upstream of a minimal promoter and the firefly luciferase open reading frame. The reporter also contains a separate PGK promoter that constitutively drives the expression of a puromycin resistance gene for mammalian cell selection. Transient transfection of siRNA was performed with RNAiMAX, as directed by the manufacturer (13778-075, Invitrogen). siRNA sequences used are listed in Supplemental Table S1
. Protease (#11873580001) and phosphatase (#04906845001) inhibitor tablets were purchased from Roche (Indianapolis, IN). Con A Sepharose was purchased from GE Healthcare (Uppsala, Sweden #17-0440-03). U0126 was purchased from LC labs (Woburn, MA cat# U-6770). AZD6244 was purchased from Selleck Chemicals (Houston, TX cat# S1008). PLX4720 was purchased from Symansis (Australia cat# SY- PLX4720). CHIR99021 was purchased from Axon MedChem (Gronigen, Netherlands catalog #Axon1386). Z-VAD-FMK was purchased from R&D systems (Minneapolis, MN cat# FMKSP01). Anti-ERK (p42/44) (#9102), anti-phopho-ERK (p42/44) (#9101S), anti-phospho-β-catenin S33/37/T41 (#9561S), anti-BRAF (#9434), anti-cleaved CASP3 (#9661S), anti-cleaved PARP1 (#9541), anti-Bim (#2933), and anti-cleaved CASP3 Alexafluor 488 conjugate (#9669) antibodies were purchased from Cell Signaling (Cell Signaling, Beverly MA). Anti-β-tubulin (T7816) and anti-β-catenin (C2206) antibodies were purchased from Sigma Aldrich (Sigma Aldrich St. Louis, MO). Anti-phospho-GSK3 Y279/216 (05-413) was purchased from Upstate Biotechnology (Waltham, Massachusetts). The anti-AXIN1 (AF3287) antibody was purchased from R&D Systems (Minneapolis, MN). In Situ Cell Death Detection kit (cat# 12 156 792 910) was purchased from Roche (Indianapolis, IN).
The human melanoma cell lines A375, A2058 and MEL624 were a generous gift from Cassian Yee (Fred Hutchinson Cancer Research Institute; Seattle, WA). The human melanoma cell lines COLO-829, SKMEL28, SKMEL5 were purchased from ATCC (Manassas, VA). Human Epidermal Melanocytes, adult, lightly pigmented donor, (HEMa-LP) (C0245C) were purchased from Invitrogen (Carlsbad, CA). Stable BAR cell lines were generated as previously described(20
). BAR luciferase cell lines were also infected with a lentivirus carrying Renilla luciferase driven by a constitutive EF1alpha promoter.
The human melanoma lines A375, A2058 were cultured in DMEM supplemented with 5% FBS and 1% antibiotic. The human melanoma lines SK-MEL-5 and SK-MEL-28 were grown in EMEM supplemented with 10% FBS and 1% antibiotic. The human melanoma lines COLO-829 and MEL624 were grown in RPMI supplemented with 10% FBS and 1% antibiotic HEMa-LP cells were cultured in medium 254 supplemented with 1% HMGS and 1% antibiotic (Invitrogen Carlsbad, CA). Synthetic siRNAs were transfected into cultured cells at a final concentration of 20nM using RNAiMAX (Invitrogen; Grand Island, NY).
High Throughput Screening
Screening was performed at the Quellos high throughput screening facility at the University of Washington’s Institute for Stem Cells and Regenerative Medicine (Seattle, WA). A library of siRNAs targeting primarily the human kinome was screened in A375 melanoma cells stably expressing the β-catenin activated reporter (BAR). The kinome siRNA library was purchased from Sigma Aldrich (Sigma Aldrich St. Louis, MO) and resuspended in RNase free water. The library consists of a pool of three independent non-overlapping siRNAs for each mRNA target. siRNA pools were screened in quadruplicate at 9.5nM, 1.9nM, 0.38nM, and 0.08nM final concentration. Cell viability was assessed by adding resazurine (Sigma Aldrich St. Louis, MO) at a final concentration of 1.25ug/ml (PBS vehicle) and measuring fluorescence intensity (Ex=530nM Em=580nM) on an Envision multilabel plate reader (Perkin Elmer Waltham, MA). Luciferase activity was assessed by adding 5uL/well SteadyGlo (Promega Madison, WI) and measuring total luminescence on an Envision multilabel plate reader (Perkin Elmer Waltham, MA) The screen workflow was as follows:
On day 1, 1.5 uL of the appropriate concentration of siRNA was added to 28.5uL of Optimem (Invitrogen, Carlsbad, CA) containing 3.125uL/mL RNAiMAX (Invitrogen, Carlsbad, CA). 5uL of this mix was transferred to a 384 well plate containing 15uL of growth media (DMEM/5%FBS/1%PenStrep). 20uL of cells at 75 cells/uL was added to each well for a final cell number of 1500 cells/well. On day 3, 10uL of WNT3A conditioned media diluted 1:12.8 with growth media was added for a final dilution of 1:64. On Day 4, 10uL of 6X resazurine was added to each well, incubated at 37°C for three hours, and fluorescence intensity was measured. Immediately following, 5uL of SteadyGlo was added, incubated at room temperature for 10 minutes and total luminescence was measured. Data are represented as a ratio of BAR reporter activity (Luminescence) to cell viability (Resazurine fluorescence intensity).
Low throughput BAR reporter assays
Cells were plated in 96-well plates. 24 hours following plating, cells were treated with the indicated conditions and luciferase activity was measured 24 hours later with the a dual luciferase reporter assay kit (Promega; Madison, WI) and an Envision multi-label plate reader (Perkin Elmer, Waltham, MA) per manufactures suggestions. For BAR assays involving siRNAs, siRNAs were transfected 48 hours prior to treatment.
Low throughput siRNA transfections
Cells were reverse transfected with 20nM siRNA (final concentration) in 6-well plates using RNAiMAX reagent per manufactures suggestions (Invitrogen, Carlsbad, CA). Cells were incubated for 48 hours following transfection and then treated with the indicated conditions for the indicated amount of time.
Cytosolic and Nuclear β-catenin Fractionation
Cells were plated in 100mm dishes. 24 hours following plating, cells were treated with the indicated conditions for 24 hours. Cells were gently rinsed with PBS and harvested by scraping in 500uL of hypotonic lysis buffer (50mM HEPES pH 8.0, 1mM EDTA, 1mM DTT) containing protease and phosphatase inhibitors. Cells were swelled on ice for 30 minutes and then passed through a 27 gauge needle ten times and checked for complete lysis with a microscope. Lysates were centrifuged at 10,000xg for 20 minutes and supernatant was collected as the cytosolic fraction. Pelleted membranes were washed 5 times with hypotonic lysis buffer and then solubilized with solubilization buffer (50mM Tris pH 8.0, 150mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) containing protease and phosphatase inhibitors. After a 30 minute incubation on ice, lysates were centrifuged at 16,000xg for 20 minutes. The protein concentration of the cleared supernatant was determined by BCA analysis and an equal amount of protein and volume was then incubated with pre-washed Con A sepharose beads overnight at 4degree C. Supernatant was collected as the nuclear fraction.
RNA purification and qRT-PCR analysis
RNA was purified using the RNeasy kit following the manufacturer’s protocol (Qiagen; Maryland, MD). cDNA was synthesized using RevertAid™
M-MuLV Reverse Transcriptase (Fermentas; Ontario, CAN). Light Cycler FastStart DNA Master SYBR Green1 (Roche; Mannheim, Germany) was used for real-time PCR as previously described (50
). Quantitative PCR results presented in the manuscript are averages of a minimum of three biologic replicates.
Isobologram Analysis of Cell Viability
A375 melanoma cells were seeded in 96-well plates at a concentration of 8,000 cells per well in 100μl of growth media. 24 hours after plating, cells were treated with all combinations of 2-fold dilutions of WNT3A CM ranging from 20% to 0% and 2-fold dilutions of PLX4720 ranging from 5μM-0μM for 48 hours. 10uL of CellTiter-Glo (Promega Madison, WI) was added to each well and total luminescence was measured on an Envision multilabel plate reader (Perkin Elmer Waltham, MA). Each condition within an experiment was assayed in triplicate wells and three independent experiments were performed.
Flow cytometry for Active Caspase-3
Cells were seeded in a 6-well dish at a density to achieve 90–100% confluence at harvest. 24 hours after seeding, cells were treated with the indicated conditions for the indicated amount of time. At the time of collection, supernatants were collected and pooled with trypsinized cells. Cells were fixed with 4% paraformaldehyde and permeabilized according to vendor’s protocol for Cleaved Caspase-3 (Asp175) Antibody (AlexaFluor 488 Conjugate) (catalog # 9669) (Cell Signaling, Beverly MA). The antibody was used at a final dilution of 1:100. Flow was performed on a BD FACSCanto II, and data analysed with FlowJo 8.8.6 (Tree Star) software. Experiments were performed with biological triplicates and data are representative of at least three independent experiments.
For experiments involving siRNAs, cells were reverse transfected with 20nM siRNA in 6-well dishes in triplicate with RNAiMax according to manufacturer’s protocol. 48 hours following transfection, cells were treated with the indicated conditions for 24 hours and then harvested for analysis. Cells were harvested, stained, and analyzed as described above.
Glass coverslips were coated with poly-L-lysine in a 24-well dish, rinsed with PBS, and dried. Cells were seeded at a density to achieve 90–100% at harvest. 24 hours after seeding, cells were treated with the indicated conditions and incubated for 24 hours. TUNEL staining was performed according to vendor’s protocol (cat# 12 156 792 910) (Roche Indianapolis, IN). Briefly, media was gently aspirated and cells were fixed in 4% paraformaldehyde for 1 hour at room temperature. Cells were gently rinsed twice with PBS and permeabilized with 0.1% Triton X-100in 0.1% sodium citrate for 2 minutes on ice. Cells were rinsed twice with PBS, and 40uL of TUNEL reaction mixture was added directly on top of the slide and incubated for 1 hour at 37°C in a humidified incubator. Slips were rinsed 3 times and mounted on superfrost plus glass slides (cat# 48311-703 VWR West Chester, PA) with Prolong Gold anti-fade mounting media containing DAPI (cat# P36931 Invitrogen; Grand Island, NY). Images were obtained on a Nikon TiE inverted widefield high-resolution microscope (Nikon Melville, NY).
A375 cells were used for the spheroid assays. Spheroids were formed and implanted in collagen as previously described (51
). Spheroids were treated with indicated conditions 30 minutes after collagen polymerization. Images were obtained on a Nikon TiE inverted widefield high-resolution microscope (Nikon Melville, NY). For comparison of growth effects such as shown in , spheroids were imaged at 72 hours after spheroid implantation. For live-dead imaging assays such as shown in , imaging was performed at 24 hours after spheroid implantation.
NSG (NOD/SCID/IL2r-gamma (null)) mice were injected with 5×105 A375 cells stably expressing GFP or 5×105 A375 cells stably expressing WNT3A-IRES-GFP. Tumors were allowed to establish to approximately 100 mm3, after which mice where tumor size-matched and allocated to five per treatment group (vehicle or PLX4720). WNT3A-IRES-GFP tumors grew slower and therefore the first day of treatment was day 14 while GFP expressing tumors were first treated on day 9. Treatment was by oral gavage once daily with 5% DMSO in 1% carboxymethyl cellulose or 50mg/kg PLX4720 in 1% carboxymethyl cellulose (604mM PLX4720 in DMSO was diluted 1:20 in 1% carboxymethylcellulose). Tumor size was determined by caliper measurements of tumor length and width every 3 to 4 days. Tumor volume was then calculated using the following formula: volume = (width) 2 × length/2. Tumors were harvested 2 hours after the last dose and fixed in neutral-buffered formalin overnight at room temperature.
Hematoxylin- and eosin-stained tumor sections were scored for mitotic activity by a board-certified pathologist who was blinded to the treatment conditions. For each treatment condition, five tumors were evaluated and a range of 26–60 high-powered fields (hpf’s) per individual tumor were scored (average of 44 hpf’s per tumor). Areas with fixation artifact were excluded a priori from the final analysis, accounting for differences in the number of hpf’s per individual tumor. Analysis was performed using a one-way ANOVA followed by a post-test for linear trend.
Standard statistical analysis was performed using GraphPad Prizm (GraphPad Inc., LaJolla CA) version 5.01. Dose-effect analyses, including combination indices, dose reduction indices and median-effect analysis were performed using the method of Chou and Talay (52
) via the CalcuSyn software suite (Biosoft, Cambridge UK), version 2.1.