The CARM1–YIPF2 axis drives breast cancer progression and predicts poor prognosis

Cells and incubation conditions

In this study, four human breast cancer cell lines: MCF-7, MDA-MB-231, T47D, and BT-549 and one human normal breast cell line, MCF-10A, were obtained from the American Type Culture Collection (ATCC). All cell lines were cultured strictly according to ATCC-recommended protocols. Specifically, MCF-10A cells were maintained in DMEM/F12 supplemented with 5% horse serum, 20 ng/mL epidermal growth factor, 0.5 μg/mL hydrocortisone, 10 μg/mL insulin, and 100 ng/mL cholera toxin. MCF-7 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% FBS and 0.01 mg/mL human recombinant insulin. MDA-MB-231 cells were maintained in Leibovitz’s L-15 medium supplemented with 10% FBS at 37 °C in a free gas exchange with atmospheric air. T47D cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 0.2 U/mL bovine insulin (I6634, Sigma-Aldrich, USA). BT-549 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 0.023 IU/mL bovine insulin. Unless otherwise specified, cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. All media contained 1% penicillin/streptomycin.

Small interfering RNA (siRNA) and plasmid construction and transfection

CARM1 pre-designed siRNA (cat. No: HY-RS01926) was purchased from MCE Co., Ltd. (Shanghai, China). The YIPF2 overexpression plasmid (HG27094-CH) was obtained from Sino Biological Inc. (Beijing, China). Transfection was performed using the indicated complexation components: lipofectamine 3000 (L3000150, Invitrogen, USA), Opti-MEM (31,985,070, Gibco, USA), and siRNA (75 pmol) in 6-well plates, or plasmid DNA (0.25 μg) in 24-well plates. After 6 h of incubation, the cells were continuously cultured for 48 h at 5% CO₂ and 37 °C in complete DMEM.

CCK8 assay

To assess cell viability, 5000 cells were seeded into 96-well plates and cultured for 48 h. Then, 10 μL of CCK-8 reagent (GK10001, GlpBio, USA) was added and the cells were incubated for an additional 1 h. Absorbance (OD) was measured at 450 nm using a microplate reader (BioTek, Epoch 2).

5-Ethynyl-2′-deoxyuridine (EdU) assay

EdU detection was performed using the EdU Detection Kit (Abbkine, Wuhan, China). Cells were incubated with 10 μM EdU for 2 h under optimal conditions. Cells were then fixed with 4% neutral paraformaldehyde at room temperature for 15 min. After fixation, cells were permeabilized with PBS containing 0.5% Triton X-100 for 15 min, followed by three washes with washing buffer. The prepared Click-iT reaction mixture was added and incubated with cells in the dark for 30 min, followed by three washes with washing buffer. Cell nuclei were stained with 1× Hoechst 33,342 and visualized under a microscope.

qPCR

Total RNA from different groups was extracted using TRIzol reagent (12,183,555, Invitrogen, USA) according to the manufacturer’s protocol. Next, cDNA was synthesized using the QuantiTect Reverse Transcription Kit (205,311, QIAGEN, Germany) and amplified using the QuantiNOVA SYBR Green PCR Kit (208,052, QIAGEN, Germany) on the Sequence Detection System (ABI Q7 pro, Applied Biosystems). PCR cycling conditions were as follows: denaturation at 95 °C for 15 s, annealing at 60 °C for 10 s, and extension at 72 °C for 40 s, for a total of 40 cycles depending on the target genes. Primer sequences are listed below: CARM1 forward: 5′-TTCCAGTCACCACTGTTCGCCA-3′; reverse: 5′-CCAGGAGGTTACTGGACTTGGA-3′; β-actin forward: 5′-ATCATGTTTGAGACCTTCAACA-3′; reverse: 5′-CATCTCTT GCTCGAAGTCCA-3′. Relative expression levels were calculated using the 2^-ΔΔCT method.

Wound healing assay

For wound healing migration assays, 6×10⁵ cells were seeded into each well of a 6-well plate and allowed to attach for 24 h. A 200 μl pipette tip was then used to scratch a straight line across the cell monolayer. After gently washing with PBS, the wound closure percentages of different groups were quantified at 0, 24, and 48 h using a microscope. Migration ability was analyzed with ImageJ software.

Transwell assays

MDA-MB-231 and BT549 cells (5×10⁵ cells) were serum-starved for 24 h and then seeded into the upper chamber of a transwell system in a 24-well plate. The lower chamber was filled with 500 μl complete medium containing 10% FBS. For migration assays, cells were incubated at 37 °C with 5% CO₂ for 48 h. Migrated cells were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 10 min, and then photographed and counted under a microscope. For invasion assays, the upper chamber was pre-coated with Matrigel matrix gel (354,248, BD Biosciences, USA). After 48 h of incubation, cells that did not pass through the membrane were removed using cotton swabs. Invaded cells on the lower membrane surface were fixed, stained, photographed, and quantified under a microscope to assess the number of cells that penetrated the membrane.

Western blot

Total protein was extracted from each sample using RIPA lysis buffer (GK10023, GLPBIO, USA) supplemented with Phosphatase Inhibitor Cocktail II (GK10012, GLPBIO, USA). Protein concentration was measured using the BCA Protein Assay Kit (GK10009, GLPBIO, USA). Proteins were separated by SDS-PAGE (BL522A, Biosharp, China) and transferred onto PVDF membranes (0.45 μm, IPVH00010, Millipore, USA). Membranes were blocked with SuperBlock buffer for 10 min (37,515, Thermo Scientific, USA). The membranes were incubated overnight at 4 °C with the following primary antibodies: CARM1 (1:2000, ab245467, Abcam, USA), CDK4 (1:1000, ab68266, Abcam, UK), CDK6 (1:1000, ab151247, Abcam, UK), CCND1 (1:1000, 33–3500, Invitrogen, USA), BAX (1:1000, ab32503, Abcam, UK), cleaved caspase-3 (1:1000, abs132005, Absin, China), YIPF2 (1:1000, HPA019902, MERCK, Germany) and GAPDH (1:5000, ab8245, Abcam, UK), followed by three washes with TBST. Specific secondary antibodies (mouse or rabbit, 1:2000, ab97023/ab288151, Abcam, UK) were incubated at room temperature for 1 h. Bands were visualized using an Ultra High Sensitivity ECL Kit (GK10008, GLPBIO, USA), imaged with a Tanon-5200 system (Tanon Science & Technology, China), and quantified for protein expression using ImageJ software.

Immunohistochemistry (IHC) staining

All fresh tumor tissues were fixed in 4% formaldehyde, embedded in paraffin, and sectioned into 4 μm slices. Sections were deparaffinized with xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by incubating sections in sodium citrate buffer (10 mM sodium citrate and 0.05% Tween 20, pH 6.0) in a steam oven. Endogenous peroxidase activity was blocked using 0.3% H₂O₂. Routine blocking was performed with 5% FBS in PBS for 1 h at room temperature. Sections were incubated with primary antibodies against Ki67 (1:200, MA5–14,520, Invitrogen, USA) or CARM1 (1:1500, ab245467, Abcam, USA) for 1 h at 4 °C. After that, the sections were incubated with HRP-conjugated secondary antibodies and developed using an IHC Colorimetric kit (8800–6599–45, eBioscience, USA).

Flow cytometry apoptosis assay

Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Briefly, cells were harvested 48 h after transfection, washed twice with cold PBS, and resuspended in 1× binding buffer at a density of 1×10⁶ cells/mL. A 100 μL aliquot of cell suspension was incubated with 5 μL Annexin V-FITC and 5 μL PI in the dark at room temperature for 15 min. After adding 400 μL of binding buffer, cells were analyzed within 1 h using a flow cytometer (BD FACSCanto II, BD Biosciences, USA). Data were analyzed using FlowJo software (Tree Star, USA).

Co-immunoprecipitation (Co-IP)

Cells were lysed in IP lysis buffer (87,787, Thermo Scientific, USA) supplemented with protease inhibitor cocktail on ice for 30 min. The lysate was centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was collected. For immunoprecipitation, 500 μg of total protein was incubated with 2 μg of anti-CARM1 (ab245467, Abcam, USA), anti-YIPF2 (HPA019902, MERCK, Germany), or normal IgG (negative control) antibody overnight at 4 °C with gentle rotation. Protein A/G magnetic beads (88,802, Thermo Scientific, USA) were added and incubated for an additional 2 h at 4 °C. The beads were washed three times with IP lysis buffer, and bound proteins were eluted with 2× SDS loading buffer at 95 °C for 5 min. The immunoprecipitated proteins were analyzed by Western blotting as described above. For ADMA modification detection, immunoprecipitation was performed with anti-YIPF2 antibody, followed by Western blotting with anti-asymmetric dimethylarginine (ADMA) antibody (13,522, Cell Signaling Technology, USA).

Subcutaneous tumor models

All animal experiments were conducted under a protocol approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. Mice were anesthetized by inhalation of 3% isoflurane. Negative control shRNA or sh-CARM1-transfected BT549 cells (2×10⁶) were subcutaneously injected into BALB/c nude mice (female, 6–8 weeks old, n=3). To evaluate the anti-tumor effect of CARM1 knockdown, tumor volume was measured every three days and calculated as 1/2 × length × width² (mm³). After 28 days, mice were euthanized by cervical dislocation, and tumor tissues were collected and photographed. Tumor tissues from different groups were then processed for further histopathological staining.

Database screening

CARM1 or YIPF2 expression in patients was obtained from the Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov/) [16]. The prognostic value of CARM1 or YIPF2 in BRCA patients was analyzed using the Kaplan–Meier Plotter database (http://kmplot.com/analysis/) [17]. The association between CARM1 and YIPF2 was determined using the GEPIA database (http://gepia2.cancer-pku.cn/#analysis) [18].

Statistical analysis

All experiments were performed in triplicate. Data are presented as the mean ± SEM and analyzed using GraphPad Prism 10.0 software (GraphPad Software, San Diego, CA). Statistical differences between groups were assessed using Student’s t-test or one-way ANOVA. P < 0.05 was considered statistically significant. For Kaplan–Meier survival analysis, patients were stratified into high- and low-expression groups using the median expression value as the cutoff threshold. The log-rank test was used to compare survival curves between groups.

Overexpression of CARM1 is associated with poor survival outcomes in BRCA

In this study, we performed a comparative analysis to investigate the clinical relevance of CARM1. Specifically, we screened RNA-seq datasets from the TCGA database to assess CARM1 expression and conducted Kaplan–Meier survival analyses to correlate CARM1 expression with overall survival in BRCA patients. The results showed that CARM1 was significantly upregulated in breast tumor tissues compared with normal tissues (Fig. 1 A). Patients were stratified into high-expression (n = 552) and low-expression (n = 552) groups using the median CARM1 expression value (−0.1544, log₂ [RSEM normalized count + 1]) as the cutoff. Patients with higher CARM1 expression had significantly shorter survival than those with low CARM1 expression (P = 0.0024) (Fig. 1 B). The association between CARM1 expression and clinicopathological characteristics, including breast cancer molecular subtypes, is summarized in Table S1 and Figure S1A. Notably, even patients in the low-expression group exhibited significantly higher CARM1 levels compared with normal breast epithelium, indicating that CARM1 is broadly upregulated across breast tumors regardless of relative expression level. In breast cancer cell lines, MDA-MB-231 and BT-549 exhibited markedly increased CARM1 expression at both the mRNA and protein levels compared with the normal breast cell line MCF-10A (Fig. 1 C–D). Collectively, these TCGA data and our cohort suggest that CARM1 is overexpressed in BRCA tissues and cell lines and is associated with adverse malignant clinical outcomes in BRCA.

Silencing CARM1 inhibits BRCA cell proliferation, migration, and invasion

Next, we evaluated the oncogenic role of CARM1 in BRCA by transfecting MDA-MB-231 and BT-549 cells with CARM1 siRNA and confirming transfection efficiency by Western blot (Fig. 2 A). CARM1 knockdown strongly suppressed cell proliferation in MDA-MB-231 and BT-549 cells (Fig. 2B). Moreover, colony formation ability was significantly reduced in CARM1-knockdown cells compared with control cells (Fig. 2C). We further found that CARM1 knockdown markedly impaired the migration of BRCA cells (Fig. 2D). Consistently, knockdown of CARM1 significantly inhibited cell invasion (Fig. 2E). Taken together, these findings indicate that CARM1 promotes BRCA cell proliferation, migration, and invasion.To further validate the oncogenic role of CARM1, we performed gain-of-function experiments by overexpressing CARM1 in MCF-7 and T47D cells, which exhibited relatively lower endogenous CARM1 expression. CCK-8 assays demonstrated that CARM1 overexpression significantly promoted cell proliferation in both cell lines compared with control groups (Figure S2A), confirming that CARM1 functions as a tumor promoter in breast cancer.

Silencing CARM1 suppresses proliferation by inducing apoptosis and cell cycle arrest

To investigate the mechanism by which CARM1 regulates proliferation, we performed EdU staining assays. CARM1 knockdown significantly reduced the proliferation rate in both MDA-MB-231 and BT-549 cell lines (Fig. 3A). Consistently, pro-apoptotic proteins BAX and cleaved caspase-3 were increased in CARM1-knockdown cells (Fig. 3B). In addition, the expression of cell cycle-related proteins CDK4, CDK6, and CCND1 was suppressed following CARM1 knockdown compared with control cells (Fig. 3B). These results suggest that CARM1 contributes to the regulation of the cell cycle, proliferation, and apoptosis processes. To further corroborate the apoptotic effects of CARM1 knockdown, Annexin V/PI flow cytometry was performed. The results showed that CARM1 silencing significantly increased the apoptosis rate in both MDA-MB-231 and BT-549 cells compared with control groups (Figure S2B), consistent with the elevated expression of pro-apoptotic markers BAX and cleaved caspase-3.

Silencing CARM1 reduces YIPF2 expression

We next investigated the precise mechanisms by which knocking down CARM1-mediated BRCA inhibition occurs. First, the heatmap showed that CARM1 and YIPF2 were enriched in the same region, indicating that they may share similar functions (Fig. 4A). To confirm their relationship, MDA-MB-231 and BT549 cells were transfected with si-CARM1, and transfection efficiency was verified (Fig. 4B). Compared with control cells, siCARM1 significantly reduced YIPF2 protein levels in both cancer cell lines (Fig. 4C). According to the TCGA database, high expression of YIPF2 was observed in cases of BRCA tumor tissues, which was lower than in normal breast tissues (Fig. 4D). Patients with YIPF2 upregulation exhibited a shorter survival time, indicating a poorer prognosis compared with patients with low YIPF2 expression (P=0.0286) (Fig. 4E). Using the GEPIA database, we further analyzed the association between CARM1 and YIPF2, and the data revealed a positive co-expression of CARM1 and YIPF2 (P=1.43e-43). To further investigate the CARM1–YIPF2 relationship, we performed co-immunoprecipitation (Co-IP) experiments. Reciprocal Co-IP supported an association between CARM1 and YIPF2: CARM1 was detected in YIPF2 immunoprecipitates, and conversely, YIPF2 was detected in CARM1 immunoprecipitates (Figure S3A, S3B). Given the methyltransferase activity of CARM1, we examined whether CARM1 knockdown affected YIPF2-associated ADMA signals. Immunoprecipitation with anti-YIPF2 antibody followed by ADMA-specific Western blotting showed a reduced ADMA signal in CARM1-knockdown MDA-MB-231 cells compared with control cells (Figure S3C). These findings are consistent with CARM1-dependent YIPF2 methylation but do not establish direct enzymatic methylation of YIPF2 by CARM1.

CARM1 promotes BRCA progression via YIPF2

To further examine the functional relationship between CARM1 and YIPF2 during BRCA tumorigenesis, we transfected MDA-MB-231 and BT549 cells with si-CARM1 together with a YIPF2 overexpression plasmid (OE-YIPF2), and assessed cell proliferation, migration, and invasion. The results showed that the inhibitory effect of si-CARM1 on cell viability was reversed by YIPF2 overexpression; specifically, the OD value in the si-CARM1+OE-YIPF2 group was restored to the control level (Fig. 5A). Consistent with the CCK-8 assay, si-CARM1 suppressed colony formation, whereas OE-YIPF2 increased the number of colonies in both cell lines. These findings suggest that CARM1 knockdown inhibits cell proliferation by suppressing YIPF2 (Fig. 5B). Wound-healing and transwell assays were performed to determine whether the regulatory effects of CARM1 on metastatic capabilities were mediated by YIPF2. As shown in Fig. 5C, OE-YIPF2 significantly increased the number of invaded cells, while si-CARM1 partially inhibited cell movement to the lower chamber. After 48 h, si-CARM1-transfected cells migrated slowly, whereas OE-YIPF2-transfected cells migrated faster and almost completely covered the wound gap. In the combined group, the wound gap was partially recovered by OE-YIPF2 transfection (Fig. 5D). Taken together, YIPF2 overexpression partially attenuated the suppressive effects of CARM1 knockdown on cell proliferation, migration, and invasion.

Additionally, we investigated the roles of CARM1 and YIPF2 in cell proliferation using EdU staining assays. Cells were stained with EdU reagent. The results showed a significantly reduced proliferation rate in the si-CARM1 group. Compared with the si-CARM1 group, cells transfected with OE-YIPF2 exhibited the highest proliferation rate; this effect was reversed by CARM1 knockdown (Fig. 6A). Western blotting further revealed that, in the YIPF2 overexpression group, the expression of pro-apoptotic markers BAX and cleaved caspase-3 was decreased, while cell cycle markers CDK4, CDK6, and CCND1 were increased. Moreover, YIPF2 overexpression also reduced the effects of CARM1 knockdown on apoptosis- and cell cycle-related proteins (Fig. 6B). Consistent with these findings, Annexin V/PI flow cytometry analysis confirmed that YIPF2 overexpression partially reversed the CARM1 knockdown-induced increase in apoptosis rate (Figure S4A).

Knocking down CARM1 inhibits BRCA growth

In agreement with the in vitro findings, we established a subcutaneous tumor model using breast cancer BT-549/CARM1-deficient cells. Xenografted mice injected with BT-549/CARM1-deficient cells exhibited a marked reduction in tumor growth (Fig. 6C). The number of Ki-67- and CARM1-positive cells was significantly decreased in the sh-CARM1 group (Fig. 6D), suggesting that CARM1 downregulation suppresses tumor growth in breast cancer cells.