Microarray data processing and Clinical samples
The microarray datasets of TNBC patients were extracted from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) and and the Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/). Three microarray gene expression datasets (GSE21653 , GSE31448  and GSE45827 ) were obtained from the GEO database. A total of 210 TNBC patients with complete clinicopathological and follow-up information were retrospectively reviewed from Fujian Medical University Union Hospital between June 2013 and February 2018. All patients received total mastectomy or breast conserving surgery without neoadjuvant chemotherapy or radiotherapy and should receive at least six cycles of adjuvant chemotherapy after surgery. Disease-free survival (DFS) was defined as the time from the date of diagnosis to the date of clinical relapse (with histopathology confirmation or radiological evidence of tumor recurrence). Overall survival (OS) was defined as the time from the date of diagnosis until death from any cause. The follow-up deadline was March 1, 2021. This procedure was approved by the Research Ethics Committee of Fujian Medical University Union Hospital and informed consent was obtained from each participant.
Immunohistochemistry (IHC) staining and evaluation
IHC staining analysis was performed on paraffin-embedded tissues to measure the protein expression of CTPS1 and YBX1 in all TNBC tissues and adjacent normal breast tissues according to the standard immunoperoxidase staining procedure. Briefly, slides were incubated with anti-CTPS1 (1:500; ab244492, Abcam) and anti-YBX1 (1:300, 20339-1-AP, Proteintech) according to the manufacturer’s instructions. The IHC staining scores of CTPS1 and YBX1 were evaluated by two independent pathologists blinded to the corresponding patients. The percentage of stained positive cells was scored from 1 to 4: 1, 0–25%; 2, 26–50%; 3, 51–75%; and 4, 75–100%. The staining intensity score was calculated from 0 to 3: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each patient. A score of 8–12 was defined as high expression level and a score of 0–7 was defined as low expression.
Cell culture and transfection
Human breast cancer cell lines (MDA-MB-231, HCC1937, BT-549, Hs578T, SKBR-3 and MCF-7) were purchased from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. All cell lines were cultured in DMEM (HyClone) supplemented with 10% FBS (Gibco) and 1% penicillin and streptomycin solution at 37°C under 5% CO2 conditions in a humidified incubator. Short hairpin RNA (shRNA) targeting CTPS1 were subcloned into GV115 and GV493 lentiviral shRNA vector (Genechem, Shanghai, China), respectively. For overexpressing YBX1, the construct was generated by subcloning PCR amplified full-length human YBX1 cDNA into the GV657 vector (Genechem, Shanghai, China). The constructed lentiviral vectors were packaged into the viruses in 293 T cells. Then, the harvested and concentrated viruses were added into cells and cultured for 72 h. The target sequences of the shRNA and negative control were as follows:
RNA isolation and qRT-PCR analysis
Total RNA was extracted with TRIzol reagent (Invitrogen). Complementary DNA was synthesized by PrimeScript RT Master Mix (Takara) and qRT-PCR was subsequently performed on a model 7500 Real-Time PCR System (Applied Biosystems) with SYBR Green kit (Takara) following the manufacturer’s instructions. GAPDH gene was detected for normalization of data. Fold changes of gene expression were calculated by the 2 − ΔΔct method, three independent replicates of all biological samples were assessed. The primers used in qRT-PCR were listed in Table S1.
Total protein was extracted by RIPA lysis buffer (Beyotime) and the protein concentrations were determined with BCA Protein Assay Kits (Beyotime). A total of 20 µg of protein was loaded for electrophoretic separation on SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membrane. Membranes were blotted with the following antibodies: anti-CTPS1 (#98287, Cell Signaling Technology), anti-YBX1 (#9744, Cell Signaling Technology) and anti-GAPDH (ab181602, Abcam). Binding of the primary antibody was detected by incubating the membranes with a horseradish peroxidase-conjugated secondary antibody, followed by visualization with the ECL reagent (Thermo Fisher Scientific, Inc.).
Cell proliferation assay
Cell growth and viability was detected by cell count kit-8 (CCK8) assay and colony formation assay. For CCK8 assay, cells were seeded into 96-well plates at the concentration of 2000 cells/well. Then, a 10 ul of Cell Counting Kit-8 (Dojindo, Japan) was added after 24, 48, 72, and 96 hour of incubation, respectively. After 2h, the absorbance was measured at 450 nm through a microplate reader. For colony formation assay, 1000 cells were seeded into a 6-well plate and continuously incubated for 14 days. The colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% of crystal violet solution (Sangon Bio, Inc.) for 15 min. Finally, the crystal violet stained colonies were counted to determine colony formation.
Migration and invasion assays
Transwell assays were performed to detect cell migration and invasion. Cells were harvested, washed with PBS and suspended in DMEM without FBS at 1×105 cells/ml. The upper chamber of the Transwell (Corning, Inc.) was filled with 100 µl of cell suspension, and the lower chamber was filled with 600 µl of DMEM with 30% FBS. Following incubation for 24 h at 37°C, cells were fixed with 4% paraformaldehyde for 30 minute and stained with 0.5% crystal violet for 5 minute at room temperature. The chamber was then washed with PBS solution, and the cells on the surface of the chamber were wiped of with cotton swabs. The images of stained cells on the lower side were captured by a light microscope from 5 different randomly selected views under 200 × magnification. For invasion assays, transwell chambers precoated with Matrigel (BD Biosciences) for 2h at 37°C were utilized following a similar protocol as the cell migration assays.
An Annexin V-APC Apoptosis Detection Kit (eBioscience) was used for evaluation of cell apoptosis. Cells were seeded onto 6-well plates and grown to 70% confluence. After 72 h, the cells were harvested and washed following by 5 min of centrifugation at 1300 rpm. Cell pellets were subsequently resuspended and co-incubated with 10 ul of Annexin V-APC for 15 minutes in the dark at room temperature. Finally, the apoptosis rate was determined by a flow cytometer (Guava EasyCyte HT) .
Tumor Xenograft Model
Female BALB/c nude mice (4 weeks), weighing approximately 20 g, were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were kept in sterile cages with a controlled specific environment (22–25°C, 40–60% relative humidity and a 12:12 h day/night light cycle). MDA-MB-231 cells (1×107) stably transfected with shCTPS1 and negative control (shCtrl) were subcutaneously injected into the lower flank of the mice (n = 10 for each group). The size of tumor was measured twice a week for 4 weeks, and the tumor volume was calculated according to the formula: (length × width2)/2. After 4 weeks, all mice were sacrificed and the xenografts were dissected and weighed. The study protocol was approved by the Research Ethics Committee of Fujian Medical University Union Hospital. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals.
Dual-luciferase reporter assay
MDA-MB-231 cells were seeded in 24-well plates at an 60% concentration and transfected with relevant plasmid and the luciferase vector. After 48 h, the activities of firefly and Renilla luciferases were measured by a Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. The luminescence intensities of firefly and Renilla luciferases were recorded by a microplate reader. For data analysis, the luciferase activity was measured relative to Renilla to standardize the background signal.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was conducted in MDA-MB-231 cells with the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. The antibodies used in ChIP assay were anti-YBX1 (#9744, Cell Signaling Technology) and normal rabbit IgG (#2729, Cell Signaling Technology). The enriched DNA was analyzed by real-time PCR.
Gene set enrichment analysis (GSEA)
The “sva” R package was applied to remove batch effects of the three GEO datasets (GSE21653, GSE31448 and GSE45827). Following standardization, the three independent datasets were combined. TNBC patients obtained from three GEO datasets and TCGA database were divided into high and low CTPS1 group according to the median CTPS1 expression level, respectively. GSEA was performed with the “clusterprofler” R package. Gene sets with NOM p < 0.05 and False Discovery Rate (FDR) q < 0.05 were considered to be significant.
Weighted correlation network analysis (WGCNA)
The “WGCNA” R package was used to indicate the highly correlated genes and co-expression networks of CTPS1 in TNBC patients. Firstly, network topology was calculated to choose the most appropriate soft threshold. An adjacency matrix was then built with the co-expression similarity matrix and a topological overlap matrix (TOM) was constructed. Finally, network modules were identified by dynamic hierarchical tree clustering. The DAVID (http://david.abcc.ncifcrf.gov/) database was applied with gene KEGG pathway and GO functional enrichment analysis.
Statistical analyses were performed by SPSS 20.0 software (IBM, United States) and GraphPad Prism 7.0 (GraphPad Software). Each experiment was repeated 3 times and presented as the means ± SD (standard deviation). A student’s t-test was performed to compare variables between two groups. The Chi-Square test was used to examine the clinicopathological characteristics between CTPS1 high and CTPS1 low expressing patients. Correlation between the expression levels of YBX1 and CTPS1 was analyzed by Spearman rank correlation coefficients. Survival curves were plotted by Kaplan-Meier method and analyzed by log-rank test. Cox proportional hazard regression model was applied for univariate and multivariate survival analysis. A two-sided P value of less than 0.05 was considered statistically significant.