Cell Lines and Culture Conditions
Head and neck squamous cell carcinomas HN5, HN30, HN31, UM-SCC-1, UM-SCC-25 and UM-SCC-47 were obtained from Dr. Jeffrey Myers (UT MD Anderson). HEK-293T, FaDu, UPCI:SCC-152, UPCI:SCC-154, NCI-H460, NCI-H1299 and Detroit562 cells were purchased from ATCC. At every new frozen batch generation, DNA fingerprinting and mycoplasma testing were performed by the Cancer Center Support Grant-funded Characterized Cell Line core at MD Anderson (CA016672). HN5, HN30, HN31 and UM-SCC-1 cells were cultured in DMEM/F-12 (Mediatech) supplemented with 10% heat-inactivated (56°C, 30 min) FBS (Sigma) and 1% Pen-Strep (Gibco). NCI-H460 and NCI-H1299 cells were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS and 1% Pen-Strep. UM-SCC-47 cells were cultured in DMEM (Gibco) supplemented with 10% heat-inactivated FBS, 1% Pen-Strep, 2% MEM vitamins (Gibco), 1% sodium pyruvate (Lonza), and 1% nonessential amino acids (Gibco). FaDu, Detroit562, UPCI:SCC-152 and UPCI:SCC-154 were grown in MEM (Gibco) with 10% heat-inactivated FBS, 1% Pen-Strep and 1% sodium pyruvate. All cells were incubated at 37oC, 5% carbon dioxide.
Clonogenic Survival Assays
Clonogenicity was tested following radiation using an X-RAD 320 biological irradiator (Precision X-Ray) as previously described(12). Briefly, single cells were plated into 6-well dishes and incubated overnight. The next day, the cells were irradiated and then returned to the incubator for 10-21 days until colonies formed. Colonies with more than 50 cells were counted. Survival curves were generated by extrapolation from radiation surviving fractions using alpha/beta analysis with GraphPad Prism. Each experiment was plated in triplicate and repeated at least three independent times. Error bars represent standard error.
Antibodies and reagents
USP7, RNF168, ARF-BP1 (HUWE1) and TRIP12 antibodies were purchased from Abcam, p16 antibody from BD Biosciences, BRCA1, alpha tubulin and HA from Santa Cruz, K48 and K63-linked ubiquitin and Aurora Kinase A from Cell Signaling Technology, and Actin from Millipore. MG132 was purchased from Cell Signaling Technology, and cells were treated with doses ranging from 5-10 µM for 5-12 h. Cycloheximide was purchased from Sigma-Aldrich, and cells were treated with 300 µg/ml for the times indicated. GNE-6640 was purchased from Sigma-Aldrich, and cells were treated with doses ranging from 0.1 to 10 µM for 6 to 48 h prior to irradiation and left on until collection or staining of cells. P5091 was purchased from Sigma-Aldrich, and cells were treated with doses ranging from 1-5 µM for 1 h prior to and 18 h post irradiation.
Western blot analysis
Following treatment, cells were lysed with extraction buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1 mM EDTA (pH 8.0), and 1 mM EGTA (pH 7.0). XPert protease and phosphatase inhibitors were added at a 1:100 dilution (GenDepot) and then sonicated. Equal amounts of protein were loaded into 4-15% gradient polyacrylamide gels (Bio-Rad) and then transferred to PVDF membranes for 10 min at 25 V using a Trans-Blot Turbo (Bio-Rad). Membranes were incubated in 5% dry milk for 1 h and then incubated with primary antibody overnight at 4°C. Immunoblots were detected using horseradish peroxidase-conjugated secondary antibodies (GE) and ECL2 chemiluminescent substrate (Pierce). Densitometry was measured using ImageJ. Western blots for TRIP12 were always run on the same day the cells were collected due to the instability of TRIP12 protein.
Over-expression and shRNA lentiviral infection
pLenti puro HA-Ubiquitin was a gift from Melina Fan (Addgene plasmid # 74218). Stable overexpression of USP7, p16 or rfp pLOC Turbo lentiviral vector (Precision LentiORF, Dharmacon) or stable shRNA knockdown of USP7 or p16 GIPZ lentiviral shRNA (Dharmacon) or GIPZ nonsilencial lentiviral shRNA control were cotransfected with lentiviral particles DR.8 and VSVG in HEK-293T cells for 48 h using Fugene (Promega) transfection reagent. Media plus lentivirus was then filtered through a 0.45-micron PES syringe filter and added to cells. Polybrene (5 µg/ml) was added, and the cells were transduced for 6 h. The transduction procedure was repeated for 2 consecutive days. Three days after initial transduction, stably expressing cells were selected with either 20 µg/ml blasticidin (overexpression) or 2 µg/ml puromycin (shRNA).
siRNA was transfected using Nucleofector II technology (Amaxa). Briefly, 1 million cells were resuspended in 100 µl Reagent T (Lonza) and 200 nM siRNA (Dharacon). Cells were electroporated with program T-001, plated in 6-well dishes containing complete media, and collected at the times indicated.
A single colony of the LentiCRISPRv2 plasmid (Addgene) was expanded in LB broth containing 100 µg/ml ampicillin, and plasmid DNA was isolated using the QIAfilter Plasmid Midi Kit (Qiagen). The plasmid was then linearized and dephosphorylated by BsmBl digestion and purified with a QIAquick Gel Extraction Kit (Qiagen). p16 guide RNA, sgCDKN2A CACCGTTCGGCTGACTGGCTGGCCA, and reverse compliment, AAACTGGCCAGCCAGTCAGCCGAAC (Sigma), were annealed by PCR. gRNA was ligated into the purified LentiCRISPRv2 plasmid and transformed into One shot Stbl3 Chemically Competent E. coli (Invitrogen). A single clone was then selected, propagated, and Sanger sequenced to confirm the insert. The sg-p16 CRISPR plasmid was then cotransfected with DR.8 lentiviral particles and VSVG in HEK-293T cells for 48 h using Fugene (Promega) transfection reagent. Media containing lentivirus was then filtered through a 0.45-micron PES syringe filter and added to cells. Polybrene (5 µg/ml) was added, and the cells were transduced for 6 h. The transduction procedure was repeated for 2 consecutive days. Three days after initial transduction, stably expressing cells were selected with 2 µg/ml puromycin.
Cells were collected and then lysed using a QIAshredder Kit (Qiagen). RNA extraction was performed using an RNeasy Kit (Qiagen), and RNA was quantified on a Take3 plate (BioTek) and read on an Epoch spectrophotometer (BioTek). Reverse transcription was performed using iScript Reverse Transcription Supermix (Bio-Rad) with 1 µg of total RNA/reaction. 50 ng of cDNA template was mixed with primers and SsoAdvanced Universal SYBR Green Supermix (BioRad). PrimePCR primer sets for GAPDH, HUWE1, TRIP12 or USP7 were purchased from BioRad. Real-time PCR was run on a CFX Connect Real-Time PCR system (BioRad). Data was normalized to GAPDH.
Cells were plated directly on a coverslip and allowed to adhere with overnight incubation. The following day, the cells were irradiated and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were then washed with PBS and permeabilized with 70% ethanol overnight at 4°C and for 20 minutes with 0.1% Igepal at room temperature. Cells were washed, blocked with 2% bovine serum albumin for 1 h and incubated overnight with 1:1000 Aurora Kinase A antibody (Cell Signaling Technology). Centrosomes were visualized by 1 h incubation with a 1:500 AlexaFluor 594 fluorochrome (Invitrogen). Cells were then incubated with 1:500 alpha tubulin (Santa Cruz) for 1 h at room temperature. Mitotic spindles were visualized by 1 h incubation with 1:600 FITC (Jackson Immuno), and pictures were captured with a Leica microscope. DNA was stained with 1 µg/ml DAPI (Sigma).
BRCA1 foci were visualized by incubating 1:500 BRCA1 antibody overnight (Santa Cruz) and 1:600 FITC (Jackson Immuno) for 45 minutes at room temperature.
HN5 cells stably expressing control or shUSP7 constructs were irradiated with 6 Gy and then incubated in medium containing 664 nM nocodazole (Sigma-Aldrich) for 4 h. At the end of nocodazole treatment, mitotic cells were harvested by gentle shaking and replated on coverslips in media without nocodazole for 24 h. Cells were then fixed and stained as described above.
Following treatment, cells were lysed with extraction buffer containing 20 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), and 1 mM EGTA (pH 7.0). XPert protease and phosphatase inhibitors were added at a 1:100 dilution (GenDepot) and then sonicated. 1 mg of cell lysate per sample was incubated with 5 μg of antibody of interest with rotation at 4°C overnight. Then, 30 μL of 100 mg/ml Protein-A Sepharose beads (GE Healthcare) were added to each sample and rotated at 4°C for 2 h. The beads were sedimented by centrifugation at 400 rcf, and the bead-bound samples were washed three times with 1 ml lysis buffer. The sample was eluted by heating the bead-bound sample with 25 μL 2X SDS Laemmli Sample Buffer (Bio-Rad) at 100°C for 7 min. After centrifugation, each sample was loaded into a 4-15% gradient polyacrylamide precast gel (Bio-Rad) and transferred to a PVDF membrane. The resulting sample was analyzed by immunoblot. Immunoprecipitations for TRIP12 were always run on the same day the cells were collected due to the instability of TRIP12 protein.
IP Mass Spectrometry
Following treatment, cells were lysed with extraction buffer containing 20 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mM EDTA (pH 8.0), and 1 mM EGTA (pH 7.0). XPert protease and phosphatase inhibitors were added at a 1:100 dilution (GenDepot) and then sonicated. 25 mg of cell lysate per sample were incubated with 25 μg of antibody of interest with rotation at 4°C overnight. Then, 100 μL of 100 mg/ml Protein-A Sepharose beads (GE Healthcare) were added to each sample and rotated at 4°C for 2 h. The samples were sedimented by centrifugation, and the bead-bound samples were washed three times in lysis buffer. Beads were then sent to the MD Anderson Proteomics core for mass spectrometry analysis.
Mouse experiments were carried out in the specific pathogen-free mouse colony of the Department of Experimental Radiation Oncology at MD Anderson Cancer Center and were approved by the American Association for Accreditation of Laboratory Animal Care, in accordance with current regulations and standards of the U.S. Department of Agriculture and the Department of Health and Human Services. HN5 cells were transfected with USP7 shRNA using lentiviral vectors as described above. 2 million cells suspended in 20 μL PBS were injected intramuscularly into the right hind leg of male Swiss Nu/Nu mice. When tumors reached 8 mm in diameter (range 7.7-8.3 mm), the animals were randomized into groups and treated with 4 Gy for 5 consecutive days using a 137 Cesium irradiator (dose rate 4 Gy/min). Mice were immobilized in a jig, and tumors were centered in a 3 cm diameter circular field for irradiation. The tumors were then measured every other day until they reached 14 mm in diameter. Animals were euthanized via CO2 inhalation followed by cervical dislocation. Following euthanasia, the tumors were excised, and a portion of each was snap frozen and formalin fixed.
The time for tumors to reach 12 mm in diameter was used to determine the dose enhancement factor (DEF). The growth curves for the four conditions shown are approximately linear (coefficient of t^2 not significant for any of the curves), so the enhancement ratio for radiosensitization by USP7 was estimated as the ratio of growth delays between shUSP7 and controls. The calculations were carried out for three diameters (11, 12, and 13 mm) and either for all times or times > 6 days to assess the effect of small nonlinearities at the start. Because the observations were not independent (the same tumors are measured at different times), we applied so-called mixture models with random and fixed effects. Linear models where intercept and slope were considered random effects were used in a bootstrapping procedure where data were sampled randomly 100 times and estimates with 95% CIs were obtained from the 2.5- and 97.5-centile distributions. DEF at 11 mm shown in Figure 3, all calculated DEFs had a lower limit of the 95% CI greater than 1 by at least 0.5 arbitrary units.
Clinical data analysis
For expression analysis, HUWE1 mRNA expression was examined for all available patients from The Cancer Genome Atlas (TCGA) Head and Neck cohort for which HPV status was available (n=519). For outcome analysis, data for which HUWE1 mutation status and/or mRNA expression, HPV status and disease-free status were available (n=392). Clinical characteristics (see Supplemental Table 1), outcomes and biologic data were accessed via cBioPortal. Disease-free survival (DFS) was analyzed using Cox regression analysis. Kaplan-Meier survival curves are shown with log rank statistics used to compare groups for statistical significance.