Cell lines and reagents
A human breast cancer cell line (T47D) and human renal TEC line (HEK293T) were purchased from the American Type Culture Collection and cultured following the manufacturer’s instructions. T47D cells were generated by three months of exposure to and selection with 1 μM abemaciclib. The cells with acquired CDK4/6i resistance were continuously cultured in normal medium supplemented with abemaciclib. T47D-resistant cells were passaged for no more than six months and were authenticated by a CCK-8 assay. No mycoplasma contamination was detected in any of the cell lines.
Antibodies and inhibitors
The antibodies and dilutions used were as follows: METTL14 (ab309096: immunoblotting, 1:2000) from Abcam; GAPDH (60,004–1-Ig: immunoblotting, 1:2000) from Proteintech; FLAG tag (66,008–4-Ig: immunoblotting, 1:20,000) from Proteintech; HA tag (51,064–2-AP: immunoblotting, 1:8000) from Proteintech; His tag (66,005–1-Ig: immunoblotting, 1:10,000) from Proteintech; SPOP (16,750–1-AP: immunoblotting, 1:2000) from Proteintech; METTL14 (6158–1-AP: immunohistochemistry, 1:50) from Proteintech; E2F1 (66,515–1-Ig: immunohistochemistry, 1:50; immunoblotting, 1:2000) from Proteintech; and F-actin (PF00001: immunofluorescence, 1:200) from Proteintech. The inhibitor was as follows: Abemaciclib (HY-16297A) was obtained from MedChemExpress.
Patient-derived organoid
Biopsy samples from clinical patients who received CDK4/6i therapy in the Department of General Surgery, Tangdu Hospital, Fourth Military Medical University (FMMU, Shaanxi, China) were obtained, and cancer organoids were generated according to a previously published protocol [2, 32]. Resistant organoids were established by treatment with a CDK4/6i at sequentially increasing concentrations.
Human CRISPR activation pooled library (SAM v2) screening
T47D cells were infected with lentivirus that over-expressed MS2-P65-HSF1 fusion protein and then selected with Hygromycin B (250 µg/mL) for 7 days. Then the stable strains were infected the lentiviral Human CRISPR Activation Pooled Library (SAM v2) (over 500× coverage) at an MOI of 0.3 to ensure that most cells took up only one stably short single guide RNA (sgRNA). Blasticidin (10 µg/mL) was added to the cells 48 h after infection, after which the cells were selected for 14 days. Then, the cells were split into two groups of equal densities. The cells were treated with vehicle or abemaciclib (1 μM) for 2 weeks. After drug selection, cells and tumors from each group were harvested, and genomic DNA was isolated using a DNA extraction kit (Omega, D3396). The sequences of the sgRNAs were amplified via PCR, and the products were purified prior to sequencing. Briefly, the read count of each sgRNA from each sample was normalized to adjust for the effects of library size and read count distribution. Resistance genes were subsequently identified by searching for genes whose corresponding sgRNAs were consistently ranked high using a robust rank aggregation (RRA) approach. Genes with lower RRA values ranked higher in the screen. Genes with adjusted P < 0.05 and fold change (FC) >1.2 were considered as DEGs, with the treatment group compared to the control group. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis and GO (Gene Ontology) enrichment analysis of the DEGs was then applied.
ScRNA-seq data processing
The clinical information of the GEO: GSE158724 datasets were obtained from their previous studies [33]. A cohort of twenty-seven patients with ERα-positive breast cancer who received CDK4/6 inhibitors. Among them, patients who met the criteria was identified and divided into two categories based on their response to CDK4/6 inhibitors: thirteen patients showed a favorable response, while fourteen did not. We utilized t-distributed stochastic neighbor embedding (t-SNE) analysis to visualize the differences between the two groups. Following the examination of differential gene expression, three predominant cell populations were distinguished: cancer cells (KRT19, CDH1), stromal cells (HTRA1, FAP), and immune cells (PTPRC). Genes with adjusted P < 0.05 and fold change (FC) >1.2 were considered as DEGs, with the nonresponder group compared to the responder group.
RNA isolation and quantitative real-time PCR (qPCR)
TRIzol reagent (Invitrogen) was used to extract total RNA from cells and tissues. After reverse transcription, RNA expression levels were measured by qPCR in triplicate on a Bio-Rad CFX96 system using a SYBR Green kit (Takara, RR420A). The sequences of the primers used are shown in Supplementary Table 6.
CellTiter-Glo 3D assay
To evaluate the cell viability in organoids, a CellTiter-Glo 3D assay (Promega, G9681) was performed. The culture medium of the organoids was discarded, and prewarmed detection reagent was added according to the manufacturer’s instructions. The contents were mixed for 5 min on an orbital shaker and then incubated for 25 min at room temperature, after which luminescence was measured with a BioTek plate reader.
Transfection and infection
The plasmids and siRNAs used were designed and synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). Transfection of plasmids and siRNAs was performed using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer’s instructions, and analysis was conducted 48–72 h later. The selected sequences are listed in Supplementary Table 6.
Western blot analysis
Total protein was extracted using RIPA lysis buffer supplemented with protease inhibitor cocktail. Protein concentrations were determined with a BCA kit (Thermo Fisher, USA). Proteins in the samples from each group were separated via SDS-PAGE before they were transferred to PVDF membranes (Millipore). The membranes were incubated with a primary antibody against each target protein at 4 °C overnight. Afterward, the membranes were incubated with the corresponding secondary antibody at room temperature for 1 h.
Immunohistochemical (IHC) staining
Paraffin-embedded tissues were subjected to IHC staining. Tissue sections on slides were deparaffinized in xylene and rehydrated through a graded ethanol series (dilutions of 100, 95, 85 and 75%). Endogenous peroxidase activity was blocked and antigen retrieval was performed before the sections were incubated with a primary antibody at 4 °C overnight. After the sections were incubated with the corresponding secondary antibody (HRP-conjugated) for 20 min at room temperature, they were stained with diaminobenzidine (DAB) substrate (Dako). Hematoxylin was used to stain the sections after DAB treatment. The staining intensity in each section was scored as 0 (no staining), 1 + (weak staining), 2 + (moderate staining), or 3 + (strong staining), and the percentage of positive cells was determined in different randomly selected regions. The H-score method (score range, 0–300) was used to semiquantify expression. An H-score of 0–200 was considered to indicate low expression, and an H-score of 201–300 was considered to indicate high expression.
Dot blot assay
An RNA m6A dot blot assay was used to measure the m6A content in poly(A)-tailed RNA. In brief, total RNA was isolated using TRIzol (Invitrogen, 15,596,018) following the manufacturer’s instructions. The RNA was purified with oligo-dT and mRNA was obtained (10ug per sample) and spotted onto nylon membranes (Sigma‒Aldrich, GERPN1210B). Then, the membranes were subjected to ultraviolet crosslinking, blocked in 5% nonfat milk and incubated with an anti-m6A antibody (Abcam) overnight. The membranes were finally incubated with the secondary antibody at room temperature for 1 h. Signals were detected. Then, a solution of 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2) was used to visualize the abundance of mRNA.
RNA immunoprecipitation
RIP was conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. In brief, T47D cells (1 × 107) were lysed in complete RIP lysis buffer. Magnetic beads coated with 5 g of mouse IgG (Millipore) or an antibody specific for IGF2BP2 (Abcam) were incubated with the prepared cell lysates at room temperature for 4 h. Then, the RNA‒protein complexes were washed six times and incubated with proteinase K digestion buffer to isolate the immunoprecipitated RNA. The relative interaction between E2F1 and IGF2BP2 was quantified via qPCR.
RNA stability assay
To assess E2F1 mRNA stability, cells were incubated with actinomycin D (5 g/mL) to terminate transcription. Samples were collected 0, 3, and 6 h after transcription termination. Total RNA was extracted, and the E2F1 level was measured via real-time PCR.
Luciferase reporter assay
T47D cells were seeded in six-well plates and transfected with the pMIR-REPORT luciferase vector (Thermo Fisher Scientific, AM5795) containing either the wild-type or mutated E2F1 3′-UTR. All cells were harvested 48 h after transfection, and the firefly luciferase and Renilla luciferase activities in each well were measured with a dual-luciferase reporter assay system (Promega Corporation, E1910). The relative ratio of firefly luciferase activity to Renilla luciferase activity was determined. Each experiment was performed in triplicate.
MST measurement of binding affinity
The affinity of interactions between the peptide and the binding partners were measured in Monolith NT.115 Standard Treated Capillaries. The measurements were performed in PBST buffer. Before the MST measurements, the samples were centrifuged. The ligands for the binding studies were dissolved in target at double the concentration. The measurements were performed on a NanTemper Technologies Monolith® NT.115 instrument. The samples were measured at medium MST power with an LED power of 10%. The data were analyzed using MO. Affinity Analysis Software.
Measurement of binding affinity by SPR analysis
METTL14 was diluted in immobilization buffer (PBST, pH 7.4) supplemented with 10 mM sodium acetate, 4.0). The activator was prepared by mixing 400 mM EDC and 100 mM NHS immediately prior to injection. TFA was injected into the channel at a flow rate of 20 μL/min for an association phase of 240 s followed by a 360 s dissociation phase. The association and dissociation processes were all performed in running buffer. Cycles of analyte detection were repeated according to the analyte concentration in ascending order. After each cycle of interaction analysis, the sensor chip surface was regenerated completely with 10 mM glycine–HCl as the injection buffer delivered at a flow rate of 150 μL/min to remove the previous analyte. For the next analyte–TFA concentration cycle, the analyte injection and regeneration steps were repeated.
Coimmunoprecipitation (Co-IP)
Cells were lysed on ice using RIPA lysis buffer (Biosharp) supplemented with protease inhibitor cocktail (NCM Biotech), and the protein concentration in each sample was determined via a BCA assay kit (Thermo Fisher Scientific). Then, rProtein A/G MagPoly Beads (AM001-01, ACE) were incubated with 2 μg of an anti-SPOP antibody (16,750–1-AP, Proteintech) for 4 h at 4 °C, and the antibody-conjugated beads were then incubated overnight with 1000 μg of lysate samples, with rabbit IgG (B900610, Proteintech) serving as a negative control. The next day, proteins in the eluted samples were separated by SDS‒PAGE and analyzed by immunoblotting with an anti-METTL14 antibody (26,158–1-AP, Proteintech).
In-cell ubiquitination assay
T47D-Res cells were transfected with HA-SPOP, Flag-METTL14 and His-Ub plasmids via Lipofectamine 3000 Reagent (Invitrogen). 36 h post-transfection, the cells were lysed in pH 8.0 buffer A (6 M guanidine-HCl, 0.1 M Na2HPO4/NaH2PO4, and 10 mM imidazole) and subjected to sonication. After high-speed centrifugation, the supernatants were incubated with nickel-NTA beads (Ni–NTA) (QIAGEN) for 3 h at room temperature. The products were washed twice with buffer A, twice with buffer A/TI (1 volume of buffer A and 3 volumes of buffer TI), and one time with buffer TI (pH 6.8; 25 mM Tris–HCl and 20 mM imidazole). The precipitated proteins were separated by SDS-PAGE for immunoblot analysis.
In vitro ubiquitination assay
HEK293T cells were transfected with HA-SPOP (WT, Y87F, F102C, F133V) to purify various SPOP mutants by HA affinity precipitation. Then, 1 μg of bacterially purified Flag-METTL14 was incubated with the purified SPOP protein together with an E1 enzyme, an E2 enzyme (UbcH5a) and ubiquitin (UBbiotech) in reaction buffer. The reaction was performed at 37 °C for 4 h and stopped by the addition of 5 × SDS sample buffer; proteins were then separated by SDS–PAGE for immunoblotting.
MMTV-PyMT±-METTL14± heterozygous mice
MMTV-PyMT± mice were maintained in our laboratory [2], while METTL14± mice were obtained from Gene and Peace Company (China). Subsequently, the aforementioned mice were crossed to generate male MMTV-PyMT±-METTL14± mice and female MMTV-PyMT−/−-METTL14± mice. From the progeny resulting from crossing male MMTV-PyMT±-METTL14± mice with female MMTV-PyMT−/−-METTL14± mice, female MMTV-PyMT±-METTL14± mice (METTL14± group) and female MMTV-PyMT±-METTL14+/+ mice (METTL14+/+ group) were identified through genomic DNA analysis and subsequently evaluated separately.
Liposome preparation
Soyabean lecithin (SPC), cholesterol, DSPE-PEG-folate (MW 2000), and drugs were dissolved in chloroform, evaporated in a sample bottle under reduced pressure, and added with polypeptide aqueous solution. After the treatment with ultrasound and liposome extruder through a 200 nm pore size polycarbonate membrane, liposomes were then dialyzed with a nano-dialysis device (polycarbonate membrane, pore size 50 nm) to remove unloaded peptides and inhibitors. Finally, liposomes were added with freeze-dried protective agent and were freeze-dried.
Patients and tissue samples
The clinical characteristics of the breast cancer patients are documented in Supplementary Table 2 and Supplementary Table 3. Breast cancer tissue samples were obtained from the Department of General Surgery, Tangdu Hospital, Fourth Military Medical University (FMMU, Shaanxi, China). The Ethics Committee of The Fourth Military Medical University approved the study protocol (Approval Number: TD20230903). The experiments were conducted after obtaining informed consent from each participant. All procedures strictly adhered to the principles delineated in the Declaration of Helsinki.
Animal study
Female nude mice aged six weeks were used for establishment of the orthotopic BC model and the PDX model. This study was conducted in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee (Air Force Medical University, Shanxi, China). Six-week-old female BALB/c nude mice were obtained from GemPharmatech (Nanjing, China). For orthotopic implantation, 5 × 106 T47D cells stably expressing luciferase were inoculated into the mammary fat pads of nude mice. The volume of each mixed cell suspension was 150 µL. An IVIS Lumina II imaging system was used to monitor in vivo tumor growth via bioluminescence imaging. Mice were randomized to the study groups (n = 5 per group) two weeks after inoculation, and different treatments were commenced. In the CDK4/6i treatment group, CDK4/6i (90 mg/kg) was administered once every day. In the CDK4/6i and WKYMVM combination treatment group, CDK4/6i was administered via the same dosing schedule, and WKYMVM was intraperitoneally administered (50 mg/kg) every two days. For the PDX model, fresh lesion tissues were obtained from patients with clinically diagnosed ERα + breast cancer, and primary tumor cells were isolated and subsequently inoculated into mice after expansion. Once the tumors reached the desired size, they were excised and digested again, and this process was repeated until third-generation tumors had formed to establish stable tumor proliferation. The stably proliferating tumors were implanted into the dorsal region of mice, which were subjected to treatment with a CDK4/6i to allow the development of the largest tumor, indicative of heightened resistance to the CDK4/6i. Subsequently, the tumor was excised and subjected to re-digestion to establish the CDK4/6i-resistant PDX model. Treatment began 10 days after inoculation via the same dosing schedule. Liposomes (1 g/kg, i.v.) were administered via injection at three-day intervals for 2 weeks. Then, the tumor-bearing mice were sacrificed, and the tumors were excised for immunohistochemical staining and HE staining.
Statistical analysis
All the statistical analyses were performed using SPSS version 23.0. GraphPad Prism 8.0 was used to generate the figures. All the data were tested for a normal distribution using the Shapiro‒Wilk method, and normally distributed quantitative data are reported as the means ± SDs. Independent sample t tests and one-way ANOVA followed by Tukey’s post hoc test were used to examine the significance of differences in the means among three or more groups, respectively. A P value of < 0.05 was considered to indicate statistical significance.
