Bioinformatic analysis
The mRNA expression levels of NUDT5 in patient samples were obtained from TCGA (20) data set via Oncomine platform (www.oncomine.org) (21) and METABRIC (22–24) data set via cBioPortal (https://www.cbioportal.org) (24). NUDT5 mRNA expression level was reported as Log2Median-centered intensity. The differences of NUDT5 expression levels between different subtypes of breast cancer were determined by one-way ANOVA with Bonferroni's multiple comparisons test. The mRNA expression levels of NUDT5 in breast cancer cell lines were obtained from the Cancer Cell Line Encyclopedia (25) and reported as Log2 (Fragments per kilobase million). The overexpressed phosphatases in TNBC vs ER-/HER2 + were obtained from Bonnefoi (26) data set, and TNBC vs non-TNBC were obtained from Bittner data set (Bittner et al. International Genomics Consortium (IGC) 2005) via Oncomine platform (21). The differences of NUDT5 expression levels between TNBC vs non-TNBC was determined by Student’s t-test. The survival data of breast cancer patients were obtained from METABRIC (22–24), Esserman (27), Kao (28) and Pawitan (29) data sets via Oncomine platform (www.oncomine.org) (21) with data retrieved in 2018. The Kaplan–Meier survival curves were generated by dichotomizing the patients at the mean expression level of NUDT5. The prognostic impact of NUDT5 expression is determined by Mantel-Cox log-rank analysis.
Cell line culture
Breast cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). MCF7, MDA-MB-361, MDA-MB-231, MDA-MB-436, BT20, T47D, ZR-75-1, MDA-MB-468, and HEK293T cell lines were passaged and cultured in Dulbecco’s modified Eagle medium (DMEM) and supplemented with 10% regular fetal bovine serum; MCF10A and MCF12A cell lines were cultured in DMEM/F12 medium with 5% horse serum, 20 ng/mL EGF, 0.5 mg/mL Hydrocortisone, 100 ng/mL cholera toxin, and 10µg/mL insulin (Cellgro, Mediatech, Inc., Manassas, VA). HCC1937 cells were cultured in Roswell Park Memorial Institute medium (RPMI) supplemented with 10% regular fetal bovine serum. Growth media for all cell lines was supplemented with Pen/Strept (100 mg/mL). For cell line identification, short tandem repeat DNA fingerprinting was confirmed as previously described (10). A Lonza Mycoplasma Detection Kit (LT07-418; Lonza Walkersville, Inc.,) was used according to manufacturer instructions to detect mycoplasma.
siRNA transfection
NUDT5-specific targeting siRNA oligos were purchased from Sigma-Aldrich (SASI_Hs01_00109215, SASI_Hs02_00345134, 3’UTR siRNA: 5' UGA AAG GGC UCU CCA GAU A 3'; St. Louis, MO). Cells were seeded at 50% confluency the day before siRNA transfection. A mixture of 20 nmol/L siRNA with DharmaFECT1 (T-2001-03; Dharmacon, Lafayette, CO) transfection reagent was added to the cells. Reagent volume was adjusted according to the manufacturer’s recommendation. Non-specific siLuc (SIC001; Sigma-Aldrich) was used as the negative control. Cells were harvested or reseeded for the next analysis after at least 48 hours of siRNA transfection.
Plasmids and lentiviral vectors
Lentiviral vectors were packaged in HEK 293T cells by cotransfection of viral helper plasmids (virus packaging plasmid VPR and VSV-G envelope gene) and target plasmid and mixed with X-treme-Gene9 transfection reagent (XTG9-RO; Roche, Basel, Switzerland) at a 1:1 ratio. The lentivirus supernatant was used to transduce the target cells. Inducible lentiviral shRNA of NUDT5 (pTRIPZ-shNUDT5: RHS4696-200756448 Clone Id: V3THS_357039 (ORF), RHS4696-200688778 Clone Id: V2THS_53654 (3'UTR)) was obtained from Dharmacon (Lafayette, CO). Doxycycline-inducible cas9 plasmid lenti-iCas9-neo was purchased from Addgene (#85400, Watertown, MA). Lenti-sgNUDT5 (HCP291504-LvSG03-3) or sgScramble control (LPPCCPCTR01L03-025) were obtained from GeneCopoeia (Rockville, MD). The doxycycline-inducible cas9 plasmid (30) was transfected into the MDA-MB-231 cells, and then the sgRNA plasmid was subsequently transfected into the icas9-MDA-MB-231 clones. Stable clones that contained both the inducible-cas9 gene and the sgRNA construct were selected as MDA-MB-231-icas9-sgNUDT5 or MDA-MB-231 icas9-sgScrambled control. After the integration of the viral RNA, cells were treated with or without doxycycline (2 µg/mL) for 4 days, cells were then harvested to detect the expression of target factors. NUDT5 ORF cDNA plasmid (OHu06714) and vector control (pcDNA3.1+/C-(K)-DYK) were purchased from GenScript (Piscataway, NJ).
Western blot analysis
Primary antibodies used were NUDT5 antibody (ab129172, 1:1000, Abcam, Cambridge, United Kingdom), vinculin antibody (V9131, 1:1000, Sigma-Aldrich). Secondary anti-mouse and anti-rabbit horseradish peroxidase antibodies were obtained from GE Healthcare Bio-Sciences Corp (Piscataway, NJ). All of the target proteins and loading controls were processed in parallel. Western blots were performed in triplicate, following previously published methods (10).
Cell growth assays
Cells were treated either with siRNA for 48 hours or induced with doxycycline (2 µg/mL) for 4 days. Then, cells were counted with a Countess automated cell counter (Invitrogen, Waltham MA) and re-seeded on 96-well plates at 1000 cells per well. Cells were grown in indicated media for 7 days. 4′,6-diamidino-2-phenylindole (DAPI)–stained cell nuclei were imaged at days 1, 3, 5, and 7 using an ImageXpress Pico microscope (Molecular Devices, San Jose, CA) and analyzed with CellReporterXpress image acquisition and analysis software. Cells were treated either with DMSO or 10 µM TH5427 and cell nuclei were stained with 20 µM Hoechst (Thermo Fisher Scientific, Hoechst 33342 Solution, Waltham MA) for imaging at days 1, 3, 5, and 7. The half maximal inhibitory concentration (IC50) of TH5427 was calculated using the 4 parameter logistic regression models by Prism 9.1 (GraphPad). The cells were individually plated into each well as biological quadruplicates, and cell numbers were reported as average cell count ± SD.
Xenograft growth
This study was conducted following animal protocols approved by The University of Texas M.D. Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC). Female nude mice (The Jackson Laboratory, Bar Harbor ME, 4–6 weeks old) were used in the experiments, and 1 million MDA-MB-231 cells were injected subcutaneously into the 2nd mammary fat pad of the nude mice. Mice were divided randomly into different treatment groups when tumor size reached 50 mm3. Mice received an intraperitoneal (i.p.) injection of vehicle (water) or TH5427 (50 mg/kg) 5 times per week. Xenograft tumors were measured twice per week. Tumor volumes were calculated with the formula V = 0.5(width2 × length). Individual tumor growth rates were calculated by log-transformed linear regression slopes. Growth rates were compared between the slopes of the vehicle and treatment groups using a Student’s t-test.
H&E and Immunohistochemistry
Mouse tumor samples were processed with 1:10 formalin and embedded in paraffin. Hematoxylin-eosin staining (H&E) and immunohistochemical staining (IHC) of tumor tissue slides was performed as previously described (22). For IHC staining, tissues were incubated with Ki67 primary antibody (Lab Vision, 1:1000).
Proliferation, death, and apoptosis assays
Cells were seeded into 96-welll plates at a density of 5,000 cells/well. Lovastatin (10µM) was used to synchronize the cell cycle for 48 hours, and 1 M mevalonate was used to release cells for 24 hours. We used a bromodeoxyuridine (BrdU) detection kit (Roche Cell Proliferation ELISA, BrdU (chemiluminescent) kit) according to the manufacturer's instructions. DRAQ7™ (Abcam, ab109202) was added to the cells at a final concentration of 3 µM according to the manufacturer’s recommendation. Hoechst (Thermo Fisher Scientific™, Hoechst 33342 Solution, Waltham MA) was used at 20 µM according to the manufacturer’s recommendation to counter stain nuclei. Stained cells were imaged using an ImageXpress® Pico microscope (Molecular Devices, San Jose, CA) and analyzed with CellReporterXpress image acquisition and analysis software. The Annexin V-PI (Invitrogen, Annexin V-FITC Conjugates, Waltham MA) apoptosis assay was used according to the manufacturer’s recommendation, and samples were analyzed by flow cytometry with the assistance of the Flow Cytometry and Cellular Imaging Core Facility North Campus at The University of Texas MD Anderson Cancer Center. Experimental data points were performed in biological triplicates, and results were reported as average ± SD.
DNA fiber assay
After 48 hours of siRNA treatment, 10,000 MDA-MB-231 cells were seeded into 6-well plates. The next day, cells were treated with 50 µM 5-Iodo-2'-Deoxyuridine (IdU) for 30 minutes, washed with phosphate-buffered saline, and treated with 100 µM 5-chloro-2′-deoxyuridine (CIdU) for 30 minutes. The DNA fiber assay was performed as described (31). First, samples were incubated with a primary antibody mix of rat monoclonal anti-BrdU antibody (Abcam, ab6326, 1:500) and mouse monoclonal anti-BrdU antibody (BD Biosciences, 347580, 1:500, San Jose, CA) in a blocking solution overnight at 4°C. Samples were then incubated with a secondary antibody mix of Alexa Fluor 594 goat anti-rat IgG (ThermoFisher Scientific, A-11007) and Alexa Fluor 488 goat anti-mouse IgG (ThermoFisher Scientific, A-11001). Experimental data points were performed in triplicate, and results were reported as average ± SD. Statistical differences of the mean fiber length were compared using a Student’s t-test.
Immunofluorescence staining
After 72 hours of siRNA treatment, 5,000 cells were seeded into 96-well plates. Cells were fixed with cold methanol after 4 days for 8oxoG staining and 7 days for γH2AX staining. Samples were incubated with 8-oxoG (Santa Cruz Biotechnology, sc-130914, 1:500) and γH2AX (Millipore Sigma, 05-636, 1:1000) antibodies at 4°C overnight. Samples were then incubated with the secondary antibodies Alexa Fluor 488 (Invitrogen, A28175, 1:1000) and Alexa Fluor 594 (Invitrogen, A-11032, 1:1000) for 1 hour at room temperature. The image was obtained using the ImageXpress® Pico microscope (Molecular Devices, San Jose, CA) and analyzed with CellReporterXpress image acquisition and analysis software. Experimental data points were performed in quadruplicate, and results were reported as average ± SD. 8-oxoG intensity and γH2AX positivity were compared between siLuc and siNUDT5 using the Student's t-test.
Statistics
All graphs were presented as mean ± standard deviation. Two-tailed Student’s t-tests were used to determine the statistical significance between two different groups, and one-way ANOVA tests were used to determine the statistical significance among multiple groups. p-values less than 0.05 were considered statistically significant. * represents p ≤ 0.05, ** represents p ≤ 0.01, *** represents p ≤ 0.001, **** represents p ≤ 0.0001.