PRR15 deficiency facilitates malignant progression by mediating PI3K/Akt signaling and predicts clinical prognosis in triple-negative rather than non-triple-negative breast cancer

Cell culture and reagents

Non-cancerous mammary epithelium cell (MCF10A), breast cancer cells including luminal (MCF7, MDA-MB-361, T47D, and BT474), HER2amp (SKBR3), and triple-negative (MDA-MB-231, CAL51, BT20, and MDA-MB-468) subtypes, as well as human embryonic kidney 293T (HEK-293T) cells, were purchased from the American Type Culture Collection (Manassas, VA, USA). MDA-MB-231, CAL51, BT20, MDA-MB-468, MCF7, MDA-MB-361, and HEK-293T cells were cultured in Dulbecco’s Modified Eagle Medium (Hyclone, Logan, UT, USA) while T47D, BT474, and SKBR3 cells were grown in RPMI-1640 (Hyclone), both supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (Hyclone). MCF10A cells were maintained in mammary epithelial basal medium (Lonza, Walkersville, MD, USA) containing 100 ng/mL cholera toxin, 3 mg/mL bovine pituitary extract, 10 μg/mL hEGF, 0.5 mg/mL hydrocortisone, and 5 mg/mL insulin. All cells were incubated under a humidified atmosphere of 5% CO2 at 37 °C. Short tandem repeat profiling was performed by GENEWIZ (Suzhou, China), and mycoplasma tests were carried out using the MycoAlert Mycoplasma Detection Kit (Lonza).

The chemical reagents used in this study were purchased from the following providers: LY294002 from MedChemExpress (Shanghai, China, HY-10108), and the library containing 397 approved antineoplastics from TargetMol (Shanghai, China, L2110).

Cell proliferation assay

The proliferation of cells was detected by the IncuCyte Live-Cell Analysis System (Essen BioScience, Ann Arbor, MI, USA). Briefly, cells were seeded in a 96-well plate at a density of 3 × 103 cells per well. The plate was transferred into the IncuCyte platform after 24 h, and two sets of phase contrast images from different regions in each well were captured. Real-time proliferation was measured for 48 h or 72 h through the assessment of the percentage of cell confluence.

Cell viability was quantified by CCK8 assay. Cells were treated with the indicated agents and cultured in a 384-well plate for 72 h. CCK8 reagent (TargetMol) was added into each well and incubated for 1 h and the absorbance was measured at 450 nm using the microplate reader.

Wound healing assay

The wound healing assay was carried out using the IncuCyte platform (Essen BioScience). Cells were seeded in a 96-well plate until they reached confluent monolayers, and homogeneous linear scratches were created by the IncuCyte WoundMaker. Then, the monolayers were rinsed with PBS to remove cell residues, and sequential images were automatically taken to monitor wound closure.

Transwell migration and invasion assay

As regards the invasion assay, the 8 µm porous membrane (Corning, New York, NY, USA) of the transwell filter insert was coated with 0.5% Matrigel (Corning). Cells were seeded in the upper compartment containing serum-free medium at a density of 5 × 105 cells/mL, while medium with 20% FBS was placed in the lower chamber as a chemoattractant. The non-invading or non-migrated cells adherent to the top of the membrane were removed by scrubbing after 24 h or 20 h of incubation. The invading or migrated cells adherent to the opposite side of the membrane were fixed with methanol for 10 min, stained with 0.5% crystal violet for 5 min, and five randomly selected fields were imaged under a light microscope.

In vivo mouse experiments

Mice

The animal studies were performed in accordance with the protocol approved by the Ethical Committee for animal experiments of Cancer Hospital, Chinese Academy of Medical Science (CHCAMS). Four- to five-week-old female Balb/c-nude mice were purchased from HFK Bio-Technology (Beijing, China) and four-week-old female NOG mice were purchased from Charles River (Beijing, China). All mice were housed in pathogen-free, barrier-protected conditions at the CHCAMS animal facility, and were kept to acclimatize to the environment for one week prior to the experiments. Sample sizes for animal experiments were determined based upon pilot experiments. Mice were randomized into groups based on age and weight, and were not subjected to data blinding.

Xenograft

TNBC cells (MDA-MB-231 or CAL51, 3 × 106) and non-TNBC cells (MCF7, 3 × 106) with or without PRR15 knockdown were subcutaneously inoculated into the right flanks of 5-week-old Balb/c-nude mice and NOG mice, respectively. The tumor volume was measured by a caliper three times per week to assess the growth change. Mice were sacrificed at the end of the study or when the tumor grew enough to require euthanasia, and the tumors were extracted, weighed, and processed for further research. The volume was calculated as follows: 0.5 × length × width2.

Pulmonary metastatic model and treatment

PRR15-silenced MDA-MB-231 cells (5 × 105) or controls were injected into the tail vein of 5-week-old immunodeficient mice. The mice were randomly divided into 2 groups 2 weeks after tail vein injection, and were treated with an intraperitoneal administration of the PI3K inhibitor LY294002 (75 mg/kg) or vehicle twice per week. All mice were euthanized after 2 weeks of consecutive treatment, and lungs were collected, weighed, and subjected to subsequent histopathological analysis. Metastatic nodules were estimated by the gross examination of freshly resected lungs and histological evaluation of hematoxylin and eosin (H&E)-stained sections.

Constructs, transfection, and lentiviral infection

Stable knockdown of PRR15 was achieved in MDA-MB-231, CAL51, MCF7, and T47D cells by lentivirus-mediated RNA interference using validated shRNA sequences inserted in the lentivector pLKO.1. Randomized oligonucleotides were subcloned in the same vector and used as a control. Human DNA fragment encoding PRR15 was synthesized and incorporated into pCHD and pcDNA3.1 vectors to generate stable and transient overexpression plasmids, respectively, with the corresponding empty vectors used as controls. Details are available in Table S1.

As regards transient transfection, target cells were seeded in a 6-well plate and incubated overnight at 37 °C. Transfection mixes composed of 7.5 μL Lipofectamine 3000 (ThermoFisher, Waltham, MA, USA) in 125 μL Opti-MEM and 2.5 μg DNA together with 5 μL P3000 in 125 μL Opti-MEM (per well) were added to the wells according to the manufacturer’s instructions. As regards recombinant lentiviral particle production and infection, HEK-293T cells were co-transfected with knockdown or overexpression constructs and packaging plasmids (pMD2.G and psPAX) using Lipofectamine 3000 as described above. Virus-containing supernatants were collected after transfection, filtered, and supplemented with 5 μg/ml polybrene to infect target cells at 48 h and 72 h. After 24 h from the second infection, target cells were selected using 1.5 μg/mL puromycin dissolved in the medium for at least 7d.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from the suitably treated cells using Trizol (ThermoFisher) according to the provider’s protocol and quantified on the NanoDrop. cDNA was synthesized from 1 μg RNA using the high-capacity cDNA reverse transcription kit (ThermoFisher). The reaction was performed at 37 °C for 2 h and 85 °C for 5 min. RT-qPCR was performed using the Power SYBR Green master mix (ThermoFisher) under the following conditions: polymerase activation at 95 °C for 10 min, denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 60 s for a total of 40 cycles. The primers specific for PRR15, MKI67, PCNA, VIM, SNAI1, GAPDH, and ACTIN are listed in Table S1.

Immunoblotting

Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE gel and transferred by electrophoresis onto a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked with 5% bovine serum albumin or skim milk in phosphate-buffered saline containing 0.05% Tween-20 followed by hybridization with appropriate primary antibodies at the dilutions recommended by the suppliers and incubated overnight at 4 °C. Next, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, and the bands were visualized using enhanced chemiluminescence substrate (ThermoFisher, 32106). The primary antibodies were the following: PRR15 (RayBiotech, Norcross, GA, USA, 102-22108), PI3K (CST, Boston, MA, USA, 4292S), p-PI3K (Abcam, Cambridge, MA, USA, ab182651), Akt (CST, 4691S), p-Akt (CST, 4060S), mTOR (CST, 2972S), p-mTOR (CST, 2971S), N-cadherin (CST, 13116S), vimentin (Abclonal, Wuhan, China, A2584), Snail1(Invitrogen, Carlsbad, CA, USA, 14-9859-82), and β-actin (CST, 4967) used as a loading control. The intensity of the blots was quantified with software ImageJ and normalized to that of β-actin.

Tissue sample collection, histology, and immunohistochemistry (IHC)

Two batches of patient samples were used in this study. One batch was represented by breast cancer tissue microarray purchased from Shanghai Outdo Biotech (Shanghai, China), and the other batch was represented by paraffin-embedded specimens of TNBC patients that were obtained from the Department of Pathology at CHCAMS after surgical resection.

Mouse tumor and lung tissues were embedded in paraffin after fixation in 10% formalin. Five-μm sections were stained with DAKO Envision Kit following dewaxing, hydration, antigen retrieval, along with peroxidase activity quenching. Slides were blocked to prevent nonspecific staining, followed by incubation with the primary antibody and HRP-conjugated secondary antibody, color development with diaminobenzidine, and counterstained with hematoxylin. The average intensity and the proportion of cells positive for each marker were scored by experienced pathologists to assess protein expression.

The protocol regarding the collected human samples was approved by the Ethics Committee of CHCAMS and written informed consent was obtained from each enrolled patient.

RNA sequencing (RNA-seq)

RNA samples (three replicates per group) were isolated as described above and processed for mRNA library establishment and sequencing at BGI (Wuhan, China). The sequencing platform was MGISEQ-2000 and the paired-end reads were 100/150 bp. As regards the downstream analysis, raw sequencing data were filtered using SOAPnuke (v1.5.2), and then stored in FASTQ format as clean reads. Then, they were mapped to the reference genome using HISAT2 (v2.0.4), the clean files were aligned to the reference coding gene set using Bowtie2 (v2.2.5), and the expression of genes was acquired using RSEM (v1.2.12). Subsequently, DEGs were identified by DESeq2(v1.4.5) with a Q value ≤0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the annotated DEGs were performed using Phyper, and a rigorous threshold of Q value ≤0.05 was used to correct the significant levels of terms and pathways by Bonferroni.

Drug screening

The CCK8 assay was used to evaluate the inhibition efficiency of the marketed anti-cancer drug library against the control and PRR15-knockdown MDA-MB-231 cells. In brief, 5 different concentrations were prepared for each compound, starting at 30 µM and diluted in a 5-fold gradient, with the working final concentration of 30, 6, 1.2, 0.24 and 0.048 µM. The positive control staurosporine was used at 10 µM. Additionally, wells without cells but containing medium were used as blank controls and wells with cells and containing vehicles were used as negative controls. The percentage of inhibition was calculated as follows: inhibition % = (1-(ODS-ODBLK)/(ODNC-ODBLK))×100, where ODS represents the absorbance of the samples, ODNC indicates the absorbance of the negative control, and ODBLK denotes the absorbance of the blank control.

Clinical data

A total of 113 medical records from female patients diagnosed with TNBC who underwent surgery were retrospectively reviewed and collected. The sample size was determined as the appropriate number of included patients that ensured adequate statistical power when the significance level was set at α = 0.05. These patients were histologically confirmed as ER-negative, PR-negative, and HER2-negative and harbored high-risk factors for recurrence, such as age <35 years, positive axillary lymph nodes, grade III disease, or intravascular cancer embolus. The critical demographic and clinicopathological characteristics were assessed along with follow-up information. Disease-free survival (DFS) was estimated as the interval from surgery to the first local/distant recurrence or death of any cause. Overall survival (OS) was defined as the period from surgery to death due to any cause. Participants without progression, recurrence, metastasis, and death were considered as censoring events at the end of the follow-up. Informed consent and ethical approval were provided, and all data presented in the research were deprived of patient-identifying parameters.

Bioinformatics

The transcriptomic data from The Cancer Genome Atlas (TCGA, cancergenome.nih.gov/), Oncomine (www.oncomine.org), and Genotype-Tissue Expression (GETx, gtexportal.org/home/) were used to analyze PRR15 expression in normal and tumor tissues of breast and other types of cancer. Gene Expression Profiling Interactive Analysis v2 (GEPIA2, gepia2.cancer-pku.cn) was used for pan-cancer survival analysis [16]. The information stored in Cancer Cell Line Encyclopedia (CCLE, portals.broadinstitute.org/ccle) was downloaded for the comparison of PRR15 expression in luminal and TNBC/basal breast cancer cells as well as for pan-cancer analysis according to previous reports [17]. Single-cell RNA sequencing (scRNA-seq) information stored in the Single Cell Portal database was used to investigate the abundance of single-cell PRR15 expression in different subtypes of breast cancer (singlecell.broadinstitute.org/single_cell) [18]. The number of cell types involved: Cancer Basal SC, 4312 points; Cancer Cycling, 5359 points; Cancer HER2 SC, 3708 points; Cancer LumA SC, 7742 points; Cancer LumB SC, 3368 points. The PRR15 genomic alteration in pan-cancer was detected via Gene Set Cancer Analysis (GSCA, bioinfo.life.hust.edu.cn/GSCA/#/). In addition to online tools, other bioinformatic analyses were performed using the R software v4.1.2.

Statistical analysis

Statistical analysis was performed using SPSS v23.0 or GraphPad Prism v7. Cellular and molecular experiments were carried out in triplicates and at least three independent repeats were performed. Results were presented as mean ± SEM for continuous variables, and number (%) for categorical variables, unless otherwise noted. Differences between two groups were determined by unpaired t-test, and one-way ANOVA was used for multiple comparisons. Continuous data that did not meet the assumptions of the parametric test were evaluated using appropriate non-parametric tests. Frequency tables were examined using the χ2 test, Fisher’s exact tests, or Wilcoxon rank-sum test, as appropriate. Kaplan–Meier survival curves were evaluated with log-rank test and correlations were calculated using Spearman correlation coefficients. All statistical tests were two-tailed and a value of P < 0.05 was considered statistically significant.

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