NCAPH-YAP1 interaction promotes breast cancer stemness and tumor progression

Abstract

Rationale

Breast cancer (BC) is the most prevalent malignant tumor in women globally. Despite improved cure rates and survival for early- to mid-stage patients, around 30% still progress to metastatic BC due to cancer stem cells (CSCs), which drive tumor recurrence, progression, metastasis, and drug resistance.

Methods

This study used bioinformatics to analyze BC datasets from gene expression omnibus (GEO), cBioportal, and The Cancer Genome Atlas (TCGA), identifying NCAPH as a gene associated with breast cancer stem cells (BCSCs) characteristics. The mRNA stemness index algorithm was used to calculate tumor stemness scores. WGCNA, Lasso regression, and Kaplan–Meier analyses validated the link between NCAPH and BC prognosis. In vitro and in vivo experiments explored NCAPH's effects on BC cells. Transcriptomic sequencing and Gene set enrichment analysis (GSEA) analysis revealed Hippo-YAP1 pathways regulated by NCAPH. Co-immunoprecipitation and immunofluorescence co-localization experiments confirmed the interaction between NCAPH and YAP1, with functional rescue experiments using the YAP1 inhibitor Verteporfin.

Results

Results showed NCAPH was overexpressed in BC, linked to advanced tumor stages and poor prognosis. It enhanced CSCs properties, accelerated cell cycle progression, and promoted proliferation, migration, and invasion in vitro and in vivo. GSEA analysis suggested NCAPH regulates YAP1 in the Hippo signaling pathway. NCAPH promotes LATS1 and YAP1 expression, dephosphorylation, and nuclear translocation, enhancing BCSC traits and malignant phenotypes. Notably, Verteporfin reversed NCAPH-driven BCSC traits and malignant phenotypes.

Conclusion

This study identifies NCAPH as a novel oncogenic factor in BC. NCAPH interacts with YAP1, promoting its nuclear translocation and enhancing BCSC traits and malignancy. Critically, YAP1 inhibition reverses NCAPH-driven effects, validating the NCAPH as a promising therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-025-04648-0.

Introduction

Breast cancer (BC) is the most prevalent malignant tumor among women worldwide [1, 2]. Treatment modalities include local therapy and systemic adjuvant therapy. Although comprehensive therapy improves the cure rate and survival of patients with early- to mid-stage BC, nearly 30% of early-stage patients still progress to metastatic BC [3], with a median survival time of only 8–36 months [4]. This suggests that systemic adjuvant therapy may be insufficient to eliminate all tumor cells, particularly cancer stem cells (CSCs) [5]. CSCs possess self-renewal capabilities and can differentiate into tumor cells of diverse phenotypes, recapitulating the heterogeneity of the original tumor, and sustaining tumor initiation and growth. These properties make CSCs key drivers of tumor recurrence, progression, metastasis, and drug resistance [6–8]. Breast cancer stem cells (BCSCs) were first identified in 2003 [9].

Given the pivotal role of CSCs in BC pathogenesis and treatment resistance, targeting genes associated with CSCs phenotypes has emerged as a critical strategy in BC treatment. The Non-SMC Condensin I Complex Subunit H (NCAPH), a vital component of the condensin complex, is involved in chromosomal stability, morphology, DNA damage repair, and mitotic dynamics in normal cells [10, 11]. Emerging evidence links NCAPH to CSCs phenotypes. For instance, Xiong et al. reported that NCAPH is highly expressed in non-small cell lung cancer and regulates CSCs phenotypes via activation of the β-catenin signaling pathway [12]. Similarly, Lei et al. demonstrated that NCAPH, transcriptionally regulated by FOXM1, influences CSCs phenotypes and drug resistance in colon adenocarcinoma [13]. CSCs are also characterized by chemoresistance. Marina et al. found that NCAPH promotes drug resistance in Luminal-A BC. Additionally, the recurrence-risk prediction model named GSLA10, which incorporates NCAPH, effectively predicts relapse in Luminal-A BC patients [14]. However, the role of NCAPH in BCSCs and its underlying mechanisms remain unexplored.

The Hippo signaling pathway and its downstream effector YAP1 reveal critical insights into the interplay between stem cells, development, regeneration, and organ size regulation [15–17]. When the Hippo pathway is activated, YAP1 undergoes phosphorylation and inactivation [15, 17]. Phosphorylated YAP1 binds to the 14–3-3 protein complex, leading to its sequestration in the cytoplasm and subsequent degradation via the ubiquitin–proteasome pathway [18, 19]. Conversely, Hippo pathway inhibition results in nuclear translocation of unphosphorylated YAP1, where it acts as a transcriptional coactivator with TEAD family transcription factors to drive the expression of genes promoting tumor cell proliferation, migration, invasion, and stem-like properties [18]. Recent studies revealed that FOXM1, overexpressed in BC, enhances proliferation, migration, and stemness by suppressing YAP1 phosphorylation [20]. Conversely, RUNX1/3 inhibits YAP1-driven epithelial-mesenchymal transition (EMT), migration, and CSCs phenotypes in BC [21].

Through integrated bioinformatics analysis and experimental validation, this study identifies NCAPH as a critical oncogenic regulator of BCSCs and a significant prognostic biomarker in BC. We demonstrate that NCAPH expression levels correlate with malignant progression and drive tumorigenic phenotypes. Crucially, our findings establish a novel mechanism wherein NCAPH physically interacts with YAP1 to promote BCSC properties and tumor progression. This NCAPH-YAP1 axis represents a promising therapeutic target to overcome CSC-mediated resistance and recurrence in BC.

Methods

The methodological workflow of this study is schematically depicted in Fig. 1.

Fig. 1.

Schematic workflow of the experimental methodology

Bioinformatics analysis

1. Data Acquisition and Cohort Selection: Gene expression datasets GSE21653, GSE58812, and GSE76124 were retrieved from the GEO. From these, a combined cohort of 392 triple-negative breast cancer (TNBC) samples was curated: 87 TNBC cases from GSE21653 (selected from 266 total breast cancer samples), 107 from GSE58812, and 198 from GSE76124.

2. CSC Scoring: The mRNA stemness index (mRNAsi) algorithm [22] was applied to quantify the CSC characteristic score for each sample within the combined GEO-TNBC cohort.

3. Identification of mRNAsi-Associated Genes: Weighted Gene Co-expression Network Analysis (WGCNA) [23] was performed on the combined TNBC cohort to identify gene modules significantly correlated with mRNAsi scores. Genes exhibiting the highest correlation within key modules were prioritized for further investigation.

4. Prognostic Gene Screening: The Metabric-TNBC dataset (n = 276) was obtained. Genes identified as associated with mRNAsi in the GEO-TNBC cohort were subjected to analysis within this independent cohort. Lasso regression analysis was employed to select genes predictive of survival outcomes. Subsequently, Kaplan–Meier survival analysis was performed to evaluate the association between the expression levels of these candidate genes and overall survival (OS)/disease-free survival (DFS).

5. Expression and Clinical Correlation Analysis (TCGA-BRCA): The TCGA-BRCA dataset (n = 1231; comprising 1086 breast cancer tissues and 99 adjacent normal tissues) was utilized to assess NCAPH expression levels across breast cancer subtypes and normal tissue. Univariate logistic regression analysis was conducted to determine correlations between NCAPH expression and key clinical-pathological parameters. Results were visualized using forest plots.

6. Supplementary Database Interrogation: Publicly available online databases were leveraged to further validate NCAPH expression patterns, assess its clinical relevance across broader BC populations, and confirm its prognostic significance.

Clinical samples

Tumor tissues from 32 BC patients and 21 paired non-tumor tissues were collected at the First Affiliated Hospital of Guangxi Medical University between April 1 and May 31, 2023. All patients were preoperatively diagnosed with BC via needle biopsy and underwent modified radical mastectomy or breast-conserving surgery. Postoperative pathology confirmed invasive breast carcinoma. Tumor tissues were immediately collected during surgery, and normal breast tissues were sampled at least 5 cm from the tumor margin. Samples were stored on ice and transferred to a − 80 °C freezer. All patients provided informed consent. This study and its protocols were approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval No.: 2025-E0145). Inclusion criteria: (1) No prior chemotherapy or radiotherapy before surgery; (2) Complete clinical data for newly diagnosed BC patients; (3) Willingness to participate and sign informed consent; (4) Absence of other malignancies or immune system disorders; (5) No severe dysfunction of vital organs (heart, liver, kidneys); and (6) No preoperative radiotherapy or chemotherapy. Exclusion criteria: (1) Occult BC, male BC, or metastatic disease at initial diagnosis; (2) Patients who underwent prior tumor excision with no residual cancer tissue; and (3) Patients who withdrew consent during the study period.

RNA extraction and quantitative RT-PCR

Total RNA was isolated from tissues or cells using RNA Extraction Solution (Solarbio, China) following chloroform phase separation, isopropanol precipitation, and ethanol washing. Reverse transcription was performed with 5 × PrimeScript RT Master Mix (Takara Bio, Japan) using up to 500 ng total RNA in 10 μl reactions (37 °C for 15 min; 85 °C for 5 s).

Quantitative real-time PCR (qPCR) was conducted using TB Green Premix Ex Taq II (Takara Bio) in 20 μl reactions containing 0.8 μM of each primer (Genesys, China) and 2 μl cDNA template. Reactions were run in triplicate under the following conditions: 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 34 s; melt curve analysis (95 °C → 60 °C → 95 °C).

The relative expression of target genes was calculated using the 2^(−ΔΔCT) method normalized to ACTB.

Primer sequences:NCAPH: F 5′-CTGATGGAAGTGCTACTGAAATGG-3′, R 5′-TCTGAAACATGGGATCAATCTCAC-3′; ACTB: F 5′-TCACCATGGATGATGATATCGC-3′, R 5′-CCACATAGGAATCCTTCTGACC-3′.

Cell culture and culture conditions

The human normal mammary epithelial cell line MCF-10A and BC cell lines (MCF7, MDA-MB-231, SK-BR-3, HCC1143, MDA-MB-468) were purchased from Wuhan Procell Life Technology Co., Ltd. (China). BC cell lines were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MCF-10A cells were maintained in DMEM/F12 medium containing 20 ng/mL epidermal growth factor (EGF), hydrocortisone, insulin, non-essential amino acids (NEAA), 5% horse serum (HS), and 1% penicillin/streptomycin. All cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂, following the manufacturer’s recommended protocols. Additionally, all cell lines were regularly tested for mycoplasma contamination and were found to be mycoplasma-free.

Western blotting (WB) analysis

Cell lysates were prepared with RIPA buffer (Beyotime, China) containing protease and phosphatase inhibitors. After the addition of RIPA buffer, the protein samples were lysed for 15 min. Subsequently, ultrasonication was applied to further lyse the proteins. Following protein lysis, the BCA method was used to determine protein concentration. After quantification, an appropriate amount of loading buffer was added to the protein samples, which were then mixed thoroughly. The protein mixtures were heated at 100 ℃ for 5 min. After heating, the proteins could be stored at – 80 ℃ for later use or immediately subjected to SDS-PAGE for protein electrophoresis. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies overnight at 4℃. The next day, the PVDF membranes were taken out and washed three times with TBST, each time for 10 min. Then, the membranes were incubated with fluorescent secondary antibodies in the dark at room temperature for 1 h. After that, the PVDF membranes were removed and washed three times with TBST in the dark at room temperature, each time for 10 min. Finally, the membranes were imaged using an Amersham Typhoon™ laser scanner. The fluorescent secondary antibodies used were Multi-rAb™ CoraLite^® Plus 750-Goat Anti-Mouse Recombinant Secondary Antibody (H + L) and Multi-rAb™ CoraLite^® Plus 750-Goat Anti-Rabbit Recombinant Secondary Antibody (H + L), both diluted at 1:5000 and purchased from Proteintech (Rosemont, IL, USA). The primary antibodies used were against NCAPH (1:5000, Cat# 67,655–1-Ig), CD133 (1:5000, Cat#18,470–1-AP), CD44 (1:5000, Cat#60,224–1-Ig), YAP1 (1:5000, Cat# 13,584–1-AP), phospho-YAP1 (Ser127, 1:5000, Cat#29,018–1-AP), OCT4 (1:5000, Cat#60,242–1-Ig), SOX2 (1:2000, Cat#66,411–1-Ig), Nanog (1:2000, Cat#67,255–1-Ig), Cyclin D1 (1:5000, Cat#26,939–1-AP), CDK1 (1:5000, Cat#67,575–1-Ig), CDK2 (1:5000, Cat#10,122–1-AP), CDK4 (1:5000, Cat#66,950–1-Ig), β-actin (1:5000, Cat#66,009–1-Ig), Flag (1:5000, Cat#66,008–4-Ig), α-Tubulin (1:5000, Cat#66,031–1-Ig), and GAPDH (1:5000, Cat#10,494–1-AP), all sourced from Proteintech (Rosemont, IL, USA).

Plasmid extraction, lentiviral packaging, and stable cell line construction

The process of packaging lentiviruses encoding YAP1-Flag (pLV3-CMV-YAP1(human)-3 × FLAG-NEO), NCAPH-Flag (pLV3-CMV-NCAPH(human)-3 × FLAG-CopGFP-Puro), shNCAPH (pLV3-U6-NCAPH(human)-shRNA1-CopGFP-Puro), empty vector control (pLV3-CMV-MCS-3 × FLAG-CopGFP-Puro), and control shRNA (pLV3-U6-MCS-shRNA-CopGFP-Puro) began with plasmid extraction. The packaging plasmids (psPAX2 and PMD2.G), along with the target plasmids (YAP1-Flag, NCAPH-Flag, shNCAPH, empty vector, and control shRNA), were designed and synthesized by Miaoling Biotechnology (China). Glycerol stocks of plasmid-containing E. coli were streaked onto LB agar plates. Single colonies were then picked and cultured overnight in LB liquid medium at 37 °C with shaking. Subsequently, plasmids were purified using standard extraction protocols.

Lentiviral packaging was carried out by transfecting HEK293T cells with a plasmid mixture at a ratio of 4:3:1 (target plasmid: psPAX2: PMD2.G) using Lipo293 (Beyotime, China). Viral supernatants were collected at 48 h and 72 h post-transfection, concentrated through ultracentrifugation, and used to transduce target cells (SK-BR-3 and MDA-MB-231) seeded in 6-well plates.

Stable cell lines were generated by transducing SK-BR-3 and MDA-MB-231 cells with NCAPH/YAP1 overexpression lentivirus, empty vector control, NCAPH shRNA, or control shRNA. At 24 h post-transduction, cells were selected with 4 μg/mL puromycin (Meilun Bio, China) or 850 μg/mL G418 (Meilun Bio, China) for 2 weeks. Stable clones were maintained in medium containing 0.2 μg/mL puromycin or 8 μg/mL G418. Successful overexpression or knockdown of NCAPH/YAP1 was confirmed by WB.

Tumor sphere formation assay

Investigation into the impact of NCAPH overexpression or knockdown on the stemness of BC cells was conducted via a tumor sphere formation assay. Tumor stem cell culture medium was prepared by supplementing DMEM with 1 × B27, 20 ng/mL EGF, and 20 ng/mL bFGF. SK-BR-3 and MDA-MB-231 cells were seeded in ultra-low attachment 6-well plates at densities of 5000 and 10,000 cells per well, respectively. Each well was filled with 4 mL of the tumor stem cell medium. Tumor sphere formation was monitored under a microscope every 2–3 days. Once spheres reached approximately 50 μm in diameter, images of five randomly selected fields per well were captured for quantification. The number of spheres in each field was counted to evaluate stem cell-like properties.

Cell cycle analysis by flow cytometry

The effects of NCAPH overexpression or knockdown on BC cell cycle progression were analyzed using flow cytometry. Cells from the NCAPH overexpression group (NCAPH), empty vector control group (EV), NCAPH knockdown group (shNCAPH), and negative control group (NC) were collected and fixed in 75% ethanol at 4 °C for at least 1 h. Following fixation, cells were stained with PI/RNase staining buffer (Meilun, China) at 37 °C for 30 min in the dark. Cell cycle distribution (G0/G1, S, and G2/M phases) was analyzed using a BD FACSArray™ flow cytometer (BD Biosciences, USA). The Cell Cycle Detection Kit was obtained from Meilun Biotechnology (Dalian, China).

Cell proliferation assay (CCK-8)

The effects of NCAPH on cell proliferation were evaluated using a CCK-8 assay. MDA-MB-231 and SK-BR-3 cell lines with stably NCAPH overexpression or knockdown were seeded into 96-well plates at densities of 5000 and 2000 cells per well, respectively. Following a 12-h adhesion period, cell proliferation was measured at 24-h intervals over a 96-h timeframe using a CCK-8 kit (Meilun Biotechnology, China) according to the manufacturer’s protocol. Absorbance at 450 nm was recorded using a microplate reader (Tecan, Swiss) to quantify viable cells.

Colony formation assay

The effects of NCAPH on cell growth were evaluated using a colony formation assay. Briefly, stably NCAPH-overexpression or knockdown BC cells were plated into 6-well plates at a density of 1,000 cells per well. After culturing for 14 days, colonies were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet (Beyotime Biotechnology, Shanghai, China) for 15 min. Visible colonies were counted manually under a light microscope.

Wound healing, transwell migration, and invasion assays

In the wound healing assay, 4 × 10⁵ NCAPH-overexpression or knockdown BC cells were seeded in 6-well plates. To ensure observed wound closure reflected cell migration rather than proliferation, cells were treated with 10 μg/mL mitomycin B for 2 h upon reaching 100% confluence. After treatment, cells were washed twice with PBS to remove residual mitomycin B and cellular debris. Immediately after washing, a sterile 100 μL pipette tip was used to create a uniform scratch (“wound”) in the monolayer. Following this, cells were washed with PBS to eliminate debris and then cultured in serum-free medium. An inverted phase-contrast microscope (Olympus, Tokyo, Japan) was employed to monitor wound closure at 0 and 24 h. Subsequently, ImageJ software (v8.0) was used to quantify migration distances.

For the transwell migration assay, the upper chambers of 24-well Transwell inserts (8 μm pore size; JET BIOFIL, China) were seeded with 2 × 10⁴ cells/well in 200 μL serum-free medium. The lower chambers contained 600 μL medium with 10% FBS as a chemoattractant. After 24 h of incubation, non-migrated cells on the upper membrane surface were gently removed using a cotton swab. Migrated cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet (Beyotime Biotechnology, China), and then imaged under an inverted microscope (Olympus). Cell counts were conducted using ImageJ.

The transwell invasion assay was performed following the same protocol as the migration assay, with the exception that the upper chambers were pre-coated with 50 μL Matrigel (Corning, USA; diluted 1:8 in serum-free medium) and allowed to polymerize at 37 °C for 4 h. Invaded cells were quantified in the same manner as described above.

RNA sequencing and gene set enrichment analysis (GSEA)

Total RNA was isolated from NCAPH-overexpression and control MDA-MB-231 cells using TRIzol. Three biological replicates per group were sequenced (Illumina NovaSeq 6000, 150 bp paired-end) after quality verification (RIN > 7.0, NanoDrop and Agilent Bioanalyzer). Libraries were prepared with the NEBNext Ultra RNA Library Prep Kit.Raw reads were processed with Trimmomatic (adapter/low-quality removal) and aligned to GRCh38 using STAR. Differentially expressed genes (DEGs, p < 0.05, DESeq2) were functionally annotated via Kyoto Encyclopedia of Genes and Genomes (KEGG) and GSEA.

Immunofluorescence (IF) staining

Cells were seeded onto coverslips the day before the experiment. The next day, the coverslips were removed from the incubator and rinsed quickly three times with PBS. Then, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT). After fixation, the cells were washed three times with PBS, each for 3 min. Then, they were permeabilized with 0.2% Triton X-100 for 10 min and washed again three times with PBS, each for 3 min. Subsequently, the cells were blocked with 5% bovine serum albumin (BSA) in PBS for 1 h and then washed three times with PBS, each for 3 min. Next, the cells were incubated with primary antibodies against NCAPH (1:100 dilution; Cat# 67,655–1-Ig, Proteintech, USA) and YAP1 (1:100 dilution; Cat# 13,584–1-AP, Proteintech) at 4 °C overnight. The next day, the cells were taken out and washed three times with 1 × TBST, each for 3 min. Then, they were incubated with Alexa Fluor 488- or 594-conjugated secondary antibodies (1:500 dilution; Beyotime, China) for 1 h at RT in the dark. After that, the cells were washed three times with TBST, each for 3 min. Then, the cell nuclei were stained with DAPI (1 μg/mL; Beyotime, China) for 10 min, followed by three more washes with TBST, each for 3 min. Finally, the coverslips were mounted onto slides using an anti-fade mounting medium. Fluorescence images were captured using a Zeiss confocal microscope (Carl Zeiss, Germany) and analyzed with ZEN 3.0 software.

Co-immunoprecipitation (Co-IP)

The interaction between NCAPH and YAP1 was verified by Co-IP using Flag-tagged stable NCAPH or YAP1 overexpression BC cell lines.

Cell lysis: BC cells (grown to 90% confluency in 10-cm dishes) were washed with PBS, lysed in 1 mL NP-40 lysis buffer containing 20 μL protease inhibitor cocktail, and then lysed on ice for 30 min. After lysis, the cell lysates were centrifuged at 12,000 rpm (4 ℃, 5 min) to collect the supernatants.

Beads preparation: Flag magnetic beads were pre-washed with PBST two times.

Immunoprecipitation: For the IP group, 400 μL of supernatant was incubated with 20 μL of Flag beads. For the IgG control group, 400 μL of supernatant was incubated with mouse or rabbit IgG. Both mixtures were rotated overnight at 4 °C.

Washing: Beads were washed 5 times with PBST.

Elution: Beads were resuspended in 50 μL of 1 × loading buffer, boiled at 100 °C for 10 min, and stored at − 80 °C for subsequent WB analysis.

Nuclear and cytoplasmic fractionation

Cells were washed twice with pre-cooled PBS (4 °C) and harvested using a cell scraper. The cell suspension was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 8000 ×g for 5 min at 4 °C. The supernatant was carefully aspirated, leaving the cell pellet. For every 20 μL of cell pellet volume, 200 μL of hypotonic lysis buffer (containing 1 mM PMSF) was added. The pellet was resuspended by vigorous vortexing for 15 s and incubated on ice for 15 min. Next, 10 μL of 10% NP-40 was added, followed by vortexing for 15 s. The mixture was centrifuged at 12,000 ×g for 5 min at 4 °C. The supernatant (cytoplasmic fraction) was carefully transferred to a new microcentrifuge tube, avoiding the nuclear pellet. The nuclear pellet was resuspended in 100 μL of RIPA lysis buffer (containing 1 mM PMSF) by vortexing for 15 s. Complete lysis was achieved by sonication (3 cycles of 10 s each at 30% amplitude). Both cytoplasmic and nuclear protein fractions were mixed with 4 × SDS-PAGE loading buffer, boiled at 100 °C for 10 min, and stored at − 80 °C until further analysis.

Animal studies

The NCAPH overexpression and empty vector control SK-BR-3 cells were harvested, counted, and resuspended in PBS. The cells (5 × 10⁶ cells/100 μl) were transplanted into the right axilla of 5-week-old male BALB/c nude mice (16–18 g, Experimental Animal Center of Guangxi Medical University). Mice were randomly assigned to NCAPH or control groups (4 mice/group) using a lottery method: each mouse was labeled with a unique number (1–8), and group allocation was determined by drawing labeled lots from a sealed container to eliminate selection bias.

Two cages (4 mice/cage) were used in this experiment. Mice were housed under specific pathogen-free (SPF) conditions with controlled temperature (22 ± 1 °C), humidity (50 ± 10%), and a 12 h light/dark cycle.

Tumor growth was monitored every 3 days. The experiment was terminated when tumors reached a maximum diameter of 1.5 cm to comply with humane endpoints. At the experimental endpoint, mice were anesthetized via intraperitoneal injection of pentobarbital sodium (40 mg/kg, 1% solution dissolved in sterile physiological saline). Anesthesia depth was confirmed by loss of corneal reflex and absence of pedal withdrawal response to toe pinch. Surgical procedures commenced within 5 min post-injection. Tumors were surgically excised using sterile scissors and forceps, immediately weighed on an analytical balance (0.1 mg precision), and measured with a vernier caliper. Tumor length (L) and width (W) were recorded. Tumor volume = (L × W²)/2.

All procedures were approved by the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval No. 2024-D179-01) and conducted in accordance with the ARRIVE 2.0 guidelines.

Statistical analysis

Each experiment included at least three biological replicates. All data were represented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 9.0 and R version 4.4.3. Differences in mRNA expression between breast cancer tissues and adjacent breast tissue were determined by Mann–Whitney U test. Comparisons between two independent groups were performed using an unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA). P-values were calculated using the built-in statistical functions of the software. A P value < 0.05 was considered statistically significant.

Results

NCAPH is identified as a BCSC-associated gene influencing BC prognosis and is aberrantly overexpressed in BC

Through mRNAsi-based stemness scoring of 392 TNBC samples from GEO datasets, WGCNA revealed that the turquoise module (MEturquoise) exhibited the highest correlation with tumor stemness (r = 0.86; Fig. 2A–E). Within this module, 41 genes were selected based on Module Membership > 0.8 and Gene Significance > 0.65 (Fig. 2E). Lasso regression analysis in the Metabric-TNBC cohort (n = 276) identified NCAPH as a key contributor to both overall survival (OS; coefficient = 0.554) and disease-free survival (DFS; coefficient = 0.178) (Fig. 2F–G, Tables 1–2). Kaplan–Meier analysis demonstrated that high NCAPH expression was significantly associated with poorer OS (Fig. 2H), whereas its association with DFS did not reach statistical significance (Fig. 2I).

Fig. 2.

NCAPH was identified through bioinformatics screening and experimentally validated as a differentially expressed gene. A–E WGCNA identified genes most correlated with mRNAsi scores. F, G LASSO regression screened survival-related prognostic genes. H Kaplan–Meier survival analysis of OS in the Metabric-TNBC dataset stratified by high/low NCAPH expression. I Kaplan–Meier survival analysis of DFS in the Metabric-TNBC dataset stratified by high/low NCAPH expression. J OS analysis for BC patients with high/low NCAPH expression using the KMplot database. K RFS analysis for BC patients with high/low NCAPH expression using the KMplot database. L Univariate logistic regression analysis of NCAPH expression with clinicopathological factors in the TCGA-BRCA dataset. M NCAPH expression in BRCA tumor vs. normal tissues from the GEPIA database. N ROC curve evaluating NCAPH as a diagnostic biomarker. O Protein expression levels of NCAPH across molecular subtypes of BC from the UALCAN database. P qPCR validation of NCAPH expression in breast tumor-adjacent tissues. Q WB analysis of NCAPH protein levels in breast cancer cell lines vs. normal mammary epithelial cells (Full-length gels are presented in Supplementary Fig. 1). Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Table 1.

LASSO regression analysis identified genes contributing to OS

Gene	Coef.
NCAPH	0.553966455034336
CDCA8	0.0992384210802551
TPX2	0.319379231877878
STIL	0.0477559135973922
NEK2	− 0.128994116282922
RAD51AP1	− 0.054591990122920
PTTG1	− 0.007781865281420
CCNA2	− 0.223134290364381
EZH2	− 0.297720348965471
CDC20	0.100655338549239
FOXM1	− 0.067757866008100

Table 2.

LASSO regression analysis identified genes contributing to DFS

Gene	Coef.
NCAPH	0.177936210317881
TPX2	0.0640759005993625

Univariate logistic regression using TCGA-BRCA data (n = 1,231) demonstrated that elevated NCAPH expression was significantly associated with TNBC subtype, higher pT stage, patients < 40 years old, advanced pathological stage (pStage), and invasive ductal carcinoma (Fig. 2L). Online database analyses via Kmplot and GEPIA further validated NCAPH’s prognostic relevance: high NCAPH expression predicted worse OS (HR = 1.65, p = 0.001) and relapse-free survival (RFS; HR = 1.43, p = 0.003) (Fig. 2J–K). GEPIA revealed marked NCAPH upregulation in BC tissues compared to normal breast tissues (Fig. 2M). Receiver operating characteristic (ROC) analysis (AUC = 0.93, Fig. 2N) confirmed NCAPH’s diagnostic utility. and UALCAN database shows that NCAPH protein expression levels ascending from normal tissues to Luminal, HER2 + , and TNBC subtypes (Fig. 2O).

Experimental validation by qPCR in 32 breast tumors and 21 adjacent tissues showed that NCAPH mRNA levels were 2.046-fold higher in tumors (Fig. 2P). WB analysis confirmed NCAPH protein overexpression in all five tested breast cancer cell lines relative to the normal mammary epithelial cell control (MCF-10A) (Fig. 2Q).

NCAPH promotes cancer stem cell properties in BC cells

Stable overexpression and knockdown of NCAPH in SK-BR-3 and MDA-MB-231 cell lines were validated by WB and elevated NCAPH expression in tumor spheres was confirmed (Fig. 3A). In tumor-sphere formation assays, stable NCAPH knockdown significantly impaired sphere-forming capacity, while stable NCAPH overexpression enhanced this capacity compared to empty vector controls (Fig. 3B). CSC properties were assessed by analyzing stemness markers in stable cell lines. Consistent with functional alterations, stable NCAPH overexpression upregulated CD133, CD44, SOX2, NANOG, and OCT4 protein expression, whereas knockdown downregulated them (Fig. 3C). Together, these data establish that stably manipulated NCAPH expression critically regulates breast cancer stemness.

Fig. 3.

Validation of stable NCAPH knockdown/overexpression in BC cell lines and its effects on CSC properties. A WB verification of successful overexpression and knockdown of NCAPH in breast cancer cell lines, as well as high expression of NCAPH in tumor spheres (Full-length gels are presented in Supplementary Fig. 1). B Tumor sphere formation assay demonstrating the impact of NCAPH overexpression/knockdown on sphere-forming ability in BC cell lines, with quantified statistical bar graphs. C WB analysis of CSC marker protein expression in BC cell lines after NCAPH knockdown/overexpression (Full-length gels are presented in Supplementary Fig. 2–3)

NCAPH promotes the acceleration of the BC cell cycle

Single-gene GSEA of NCAPH in TCGA-BRCA revealed significant enrichment in cell cycle-related pathways (Fig. 4A). Given this pathway association, we utilized flow cytometry to analyze cell cycle status in BC cells with NCAPH overexpression and knockdown. In MDA-MB-231 cells, NCAPH knockdown significantly increased the proportion of cells in G1 phase, with no significant change in G2/M phase compared to negative controls. Conversely, NCAPH overexpression reduced G1-phase cells, while increasing both S-phase and G2/M-phase populations (Fig. 4B). In SK-BR-3 cells, NCAPH knockdown induced G1-phase arrest, significantly increasing G1-phase cells while decreasing S-phase and G2-phase populations compared to negative controls. Conversely, NCAPH overexpression reduced G1-phase and S-phase cells, but increased the G2/M-phase population (Fig. 4C). WB further confirmed that NCAPH positively regulates core cell cycle regulators. In both SK-BR-3 and MDA-MB-231 cells, NCAPH overexpression upregulated CDK1, CDK2, CDK4, Cyclin B1 and Cyclin D1 protein expression while concomitantly downregulating p-CDK2 (Tyr15). Conversely, NCAPH knockdown suppressed these cell cycle regulators but elevated p-CDK2 (Tyr15) levels (Fig. 4D, E).

Fig. 4.

Effects of NCAPH on the cell cycle in BC cell lines. A GSEA analysis reveals that NCAPH influences the cell cycle phenotype in BC cell lines. B, C Flow cytometry analysis of the effects of NCAPH overexpression/knockdown on the cell cycle in BC cell lines, with quantified statistical bar graphs. D, E WB analysis of the effects of NCAPH knockdown/overexpression on the expression of cell cycle-related proteins in BC cell lines (Full-length gels are presented in Supplementary Fig. 4–5)

NCAPH promotes BC proliferation in vitro and in vivo

CCK-8 assays demonstrated that NCAPH overexpression significantly accelerated the proliferation of SK-BR-3 and MDA-MB-231 cells, whereas NCAPH knockdown suppressed cell growth (Fig. 5A).

Fig. 5.

Effects of NCAPH on BC cell proliferation in vivo and in vitro. A CCK-8 assay examining the effect of NCAPH on the proliferative capacity of BC cell lines. B Colony formation assay assessing the effect of NCAPH on the proliferative capacity of BC cell lines and the corresponding quantified statistical bar graph. C Xenograft tumor formation assay in nude mice evaluating the effect of NCAPH on BC cell proliferation in vivo and the corresponding quantified statistical bar graph. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Colony formation assays confirmed that NCAPH knockdown markedly impaired clonogenicity, while NCAPH overexpression enhanced colony-forming capacity (Fig. 5B).

In Vivo xenograft studies validated NCAPH's oncogenic role. Tumors derived from NCAPH-overexpressing cells exhibited significantly larger volumes and heavier weights compared to the negative control group (Fig. 5C). These findings collectively demonstrate that NCAPH drives tumor growth both in vitro and in vivo.

NCAPH enhances migration and invasion capacities of BC cells

Wound healing assays demonstrated that NCAPH knockdown significantly impaired migration in SK-BR-3 and MDA-MB-231 cells, while overexpression enhanced migration (Fig. 6A).

Fig. 6.

Effects of NCAPH on migration and invasion in BC cell lines. A Wound healing assay examining the effect of NCAPH on the migratory capacity of BC cell lines and the corresponding quantified bar graph. B Transwell migration and invasion assay evaluating the effect of NCAPH on the migratory and invasive capabilities of BC cell lines and the corresponding quantified bar graphs. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Furthermore, Transwell migration and invasion assays revealed that NCAPH knockdown reduced both migration and invasion capabilities in SK-BR-3 and MDA-MB-231 cells. Conversely, NCAPH overexpression promoted increases in migration and invasion (Fig. 6B). These findings collectively indicate that NCAPH drives metastatic potential in BC cells.

Transcriptome sequencing identifies YAP1 as a downstream pathway of NCAPH

Transcriptome sequencing was performed on MDA-MB-231 cells overexpressing NCAPH (NCAPH group) and empty vector controls (EV group). A heatmap of the top 50 differentially expressed genes (DEGs) between the two groups is shown in Fig. 7A. GSEA revealed that NCAPH overexpression predominantly altered pathways related to the Hippo signaling cascade (Fig. 7B). KEGG pathway analysis of DEGs further confirmed significant enrichment of Hippo pathway-associated genes in the NCAPH-overexpressing group (Fig. 7C).

Fig. 7.

Transcriptome sequencing analysis of BC cell lines after NCAPH overexpression. A Heatmap of DEGs following NCAPH overexpression. B GSEA analysis showing enrichment of the Hippo signaling pathway. C KEGG pathway analysis of DEGs. D KMplot database analysis of the impact of YAP1 expression levels on OS in BC patients

Using the HitPredict database, we identified YAP1—a core effector of the Hippo pathway—as a predicted interaction partner of NCAPH (Table 3). Survival analysis via the KMplot database demonstrated that BC patients with high YAP1 expression had significantly worse OS compared to those with low YAP1 expression (Fig. 7D).

Table 3.

HitPredict database-predicted proteins interacting with NCAPH

Name	Method score	Annotation score	interaction score	Confidence
CND3	0.95	1	0.972	High
SMC2	0.91	1	0.952	High
CND1	0.87	1	0.934	High
SMC4	0.86	1	0.927	High
ARC1A	0.74	0.6	0.667	High
G137B	0.74	0.55	0.638	High
STAU1	0.62	0.6	0.611	High
DPEP1	0.66	0.55	0.602	High
FZD10	0.66	0.55	0.602	High
TERA	0.62	0.55	0.585	High
EID3	0.62	0.55	0.585	High
YAP1	0.57	0.6	0.584	High

NCAPH promotes YAP1 expression and interacts with YAP1 to facilitate its nuclear translocation and pro-tumorigenic functions

WB analysis validated NCAPH-mediated regulation of LATS1 and YAP1 protein levels. In breast cancer cells, NCAPH overexpression significantly increased LATS1 and total YAP1 while reducing phosphorylated YAP1. Conversely, NCAPH knockdown decreased both LATS1 and total YAP1 as well as p-YAP1 (Fig. 8A). These findings indicate that NCAPH promotes YAP1 expression and activation via dephosphorylation.

Fig. 8.

NCAPH interacts with YAP1 and promotes YAP1 expression and nuclear translocation. A WB analysis examining the effects of NCAPH overexpression or knockdown on Hippo-YAP1 signaling (Full-length gels are presented in Supplementary Fig. 6). B WB analysis assessing the effect of NCAPH overexpression on YAP1 nuclear translocation (Full-length gels are presented in Supplementary Fig. 7). C IF showing co-localization of NCAPH and YAP1 proteins within cells. D Co-IP assay demonstrating the interaction between NCAPH and YAP1 (Full-length gels are presented in Supplementary Fig. 7)

Nuclear-cytoplasmic fractionation demonstrated NCAPH overexpression enhanced nuclear accumulation of YAP1, with concomitant reduction in cytoplasmic YAP1 (Fig. 8B). This confirms NCAPH drives YAP1 nuclear translocation.

Based on HitPredict’s predicted interaction, immunofluorescence revealed strong nuclear co-localization of NCAPH and YAP1 (Fig. 8C). Co-immunoprecipitation further confirmed their binding in breast cancer cells (Fig. 8D).

NCAPH promotes YAP1-mediated oncogenesis through a multi-step regulatory cascade: upregulating YAP1 expression, facilitating its dephosphorylation and enhancing nuclear translocation.

The pro-tumorigenic effects of NCAPH are reversed by the YAP1 inhibitor verteporfin

We assessed YAP1's functional contribution to NCAPH-mediated malignancy by treating NCAPH-overexpressing SK-BR-3 cells with the YAP1 inhibitor Verteporfin (VP). Comparative analyses were performed across three groups: empty vector control (EV), NCAPH overexpression (NCAPH), and NCAPH overexpression + VP (NCAPH + VP). Results demonstrated that VP inhibited YAP1 expression and reversed NCAPH-induced tumorsphere formation (Fig. 9A) as well as the upregulation of CSC markers (Fig. 9B). VP reversed NCAPH-stimulated cell growth (Fig. 9C). VP attenuated NCAPH-enhanced migration (Fig. 9D) and invasion (Fig. 9E).

Fig. 9.

The YAP1 inhibitor Verteporfin reverses NCAPH overexpression-induced enhancement of CSC properties, proliferation, migration, and invasion in BC cells. A Verteporfin inhibits YAP1 expression and also reverses the increase in tumorsphere formation induced by NCAPH overexpression (Full-length gels are presented in Supplementary Fig. 8). B WB experiments show that Verteporfin reverses CSC marker protein expression induced by NCAPH overexpression (Full-length gels are presented in Supplementary Fig. 8). C The CCK-8 assay shows that Verteporfin reverses the enhanced proliferative capacity in BC cell lines induced by NCAPH overexpression. D ​The wound healing assay indicates that Verteporfin reverses the increase in migratory capacity of BC cell lines induced by NCAPH overexpression. E The Transwell assay reveals that Verteporfin markedly reverses the enhancement of migratory and invasive capabilities in BC cell lines induced by NCAPH overexpression. Data are presented as mean ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Discussion

The findings of this study reveal the mechanism by which NCAPH influences BCSC phenotypes. Specifically, NCAPH upregulates LATS1 expression and interacts with YAP1, which collectively promotes YAP1 overexpression and nuclear translocation, thereby driving BCSC properties and BC progression.

CSCs play a central role in tumor heterogeneity and malignant progression. Through bioinformatics analysis, we identified NCAPH as a BCSC regulator, a role previously unreported in BC. Existing studies have shown that NCAPH is aberrantly overexpressed in multiple malignancies [11, 24] and is associated with CSC properties in colon cancer, non-small cell lung cancer, gastric cancer, and endometrial cancer [12, 13, 25, 26]. Our experiments yielded similar results, which showed that NCAPH expression levels were higher in breast cancer tissues than in non-breast cancer tissues, and NCAPH expression levels were higher in tumor spheres than in non-tumor spheres.

Drug resistance is a hallmark phenotype of CSCs. Shimomura et al. [27] reported that NCAPH influences the sensitivity of oral squamous cell carcinoma to platinum-based chemotherapeutic drugs, and silencing NCAPH restores drug sensitivity in these cells. Chen et al. further demonstrated that NCAPH reduces β-catenin degradation in clear cell renal cell carcinoma, thereby enhancing PD-L1 expression, promoting glycolysis and immune tolerance, and conferring resistance to immunotherapy [28]. Collectively, studies across diverse tumor types indicate that NCAPH is closely linked to CSC properties and CSC-specific drug resistance. Through WB and tumor sphere formation assays, we similarly demonstrated that NCAPH is associated with BCSC properties. However, we have not yet investigated whether NCAPH influences drug resistance in breast cancer cells.

CSCs additionally exhibit characteristics of EMT. During EMT, epithelial cells lose key epithelial markers, transform into cells expressing mesenchymal markers, and acquire enhanced migratory and invasive capacities, anti-apoptotic ability, and extracellular matrix degradation properties [29]. Studies have demonstrated that NCAPH overexpression in liver cancer reduces E-cadherin levels and elevates N-cadherin expression, thereby promoting hepatocellular carcinoma invasion and migration [30]. In cervical cancer, NCAPH knockdown increases ZO-1 protein expression, significantly decreases Vimentin levels, and suppresses migratory and invasive capacities of cervical cancer cells in Transwell assays. Moreover, cervical cancer patients with high NCAPH expression exhibit increased lymph node metastasis, suggesting that NCAPH may drive cervical cancer invasion and metastasis by inducing EMT [31]. In ovarian cancer, NCAPH knockdown elevates E-cadherin expression while reducing N-cadherin and Vimentin levels, thereby impairing ovarian cancer cell migration and invasion [32]. Although our study did not investigate whether NCAPH regulates the expression levels of EMT-related proteins, our phenotypic assays demonstrated a correlation between NCAPH and the migration and invasion capabilities of breast cancer cells. This observation aligns with the research findings detailed above.

As a subunit of the condensin complex, NCAPH directly participates in chromosomal condensation during the cell cycle. Studies indicate that NCAPH promotes tumor cell mitosis, thereby accelerating tumor proliferation. In pancreatic cancer, NCAPH knockdown leads to significant upregulation of cyclin A and phosphorylated histone H3 (Ser10), resulting in failure of mature chromosome condensation, cell cycle arrest in the S and G2/M phases, and marked suppression of pancreatic cancer cell proliferation [33]. In cervical cancer, the HPV virus elevates the transcription factor E2F1 by producing the E7 protein. E2F1 binds to the NCAPH promoter, increasing NCAPH transcription. NCAPH further shifts the AP-1 complex from Fra-1/c-Jun to c-Fos/c-Jun, accelerating E7 transcription and forming a positive feedback loop that enhances cervical cancer cell proliferation [31]. In NSCLC, NCAPH knockdown reduces tumor cell proliferation, induces cell cycle arrest in the G0/G1 phase, and significantly decreases the expression of cell cycle regulators such as CDK2, CDK4, and CDK6 [12]. Furthermore, NCAPH has been implicated in regulating proliferation, migration, and invasion across cancer types via diverse mechanisms. For instance, NCAPH regulates cervical cancer, BC, and glioma through the PI3K/AKT pathway; bladder cancer via the MEK/ERK pathway; and lung cancer through p65 and β-catenin [12, 34–36]. NCAPH can also act as a downstream gene modulated by other proteins, such as FOXM1, TRIM21, ZCCHC3, and OCT1, to exert its pro-tumorigenic effects [13, 37–39]. Our study yielded similar findings regarding NCAPH’s role. Based on CCK-8 and colony formation assay results, NCAPH was found to be associated with breast cancer cell proliferation. Flow cytometry and WB experiments further demonstrated that NCAPH correlates with accelerated cell cycle progression. Specifically, NCAPH overexpression was associated with increased expression levels of CDK1, CDK2, CDK4, Cyclin D1, and Cyclin B1, concomitant with decreased expression of p-CDK2(Tyr15) in breast cancer cells. Importantly, p-CDK2(Tyr15) is a phosphorylation form associated with functional inhibition of CDK2 [40].

In order to explore the mechanism underlying NCAPH’s oncogenic role, transcriptome sequencing and pathway analyses (KEGG, GSEA) revealed enrichment of Hippo signaling-related genes. HitPredict database analysis predicted an interaction between NCAPH and YAP1, a core Hippo pathway effector. YAP1 integrates mechanical, metabolic, and signaling cues to regulate development, tissue homeostasis, fibrosis, inflammation, and tumorigenesis [15, 41]. In BC, YAP1 drives tumor growth, metastasis, drug resistance, microenvironment remodeling, angiogenesis, and CSC maintenance [42]. For instance, Lin et al. demonstrated that rigid microenvironments activate YAP1 to confer lapatinib resistance in HER2 + BC, which is reversed by YAP1 knockdown or Verteporfin-mediated inhibition of YAP1-TEAD interaction [43]. Similarly, YAP1 upregulation in trastuzumab-resistant BC cells restores drug sensitivity upon YAP1 suppression [44]. Knight et al. reported that KIBRA downregulation activates RHOA and promotes YAP1 nuclear translocation, enhancing metastasis and CSC phenotypes [45]. Additionally, YAP1 promotes drug resistance in BC. Studies have shown that the VEGF-NRP2 signaling pathway confers chemotherapy resistance in TNBC by promoting RAD51 expression to repair chemotherapy-induced DNA damage. RAD51 transcription depends on activation by the YAP-TEAD complex, highlighting YAP1’s role in driving tumor cell drug resistance [46]. Zhang et al. reported that chemotherapy-resistant BC cells exhibit enhanced CSC properties and upregulated ROR1 expression, which further activates YAP1 to induce drug resistance [47]. Our study similarly established that the oncogenic effects of NCAPH are mediated via the Hippo-YAP1 signaling pathway. KMplot survival analysis confirmed that high YAP1 expression predicts worse OS in BC, mirroring the adverse prognostic impact of high NCAPH expression and reinforcing evidence that YAP1 acts as a downstream effector of NCAPH. Experimental validation demonstrated that NCAPH upregulates LATS1 protein levels, increases total YAP1 protein, reduces phosphorylated YAP1 (the inactive form), and promotes YAP1 nuclear accumulation. Immunofluorescence (IF) and co-immunoprecipitation (Co-IP) assays confirmed a direct physical interaction between NCAPH and YAP1, predominantly localized within the nucleus. Critically, rescue experiments using the YAP1-TEAD inhibitor Verteporfin (VP) effectively reversed the NCAPH-induced enhancement of CSC properties, proliferation, migration, and invasion, confirming YAP1 as the essential downstream mediator of NCAPH’s oncogenic functions.

Due to the pivotal role of YAP1 in the pathogenesis and progression of breast cancer, YAP1-targeted therapy has emerged as a promising approach. However, YAP1 is also essential for normal tissue development and regeneration. Consequently, therapies solely targeting YAP1 may incur significant toxic side effects [41]. Since YAP1 lacks a DNA-binding domain, it primarily exerts its context-specific transcriptional functions by collaborating with other DNA-binding proteins [48, 49]. In the majority of cancers, YAP1 acts as an oncogene by forming a transcriptional complex with proteins of the TEAD family [50]. Therefore, disrupting the formation of the YAP1-TEAD complex or directly inhibiting TEAD offers a strategy to block YAP1’s pro-tumorigenic activity.

Verteporfin, an FDA-approved compound for treating macular degeneration, was the first drug identified to block the interaction between YAP1 and TEAD. It inhibits the growth and metastasis of breast cancer in vitro and in vivo [51].

Like Verteporfin, numerous other compounds—such as CA3 and CPD3.1 [52–54]—have subsequently been identified as inhibitors of YAP1-TEAD-mediated transcriptional activity, thereby suppressing tumor cell growth. However, their target specificity and selectivity require further validation. However, their target specificity and selectivity require further validation. Furthermore, VGLL4 has been recognized as a tumor suppressor in human cancers by directly competing with YAP1 for TEAD binding. This led to the development of a VGLL4-mimicking peptide named “super-TDU”, which has demonstrated anti-tumor efficacy in both in vitro and in vivo models [55, 56]. Additionally, the palmitoylation of TEAD transcription factors is an essential modification for their stability and transcriptional function within the Hippo pathway [57, 58]. Consequently, several drugs targeting TEAD palmitoylation have been developed and shown to possess anti-tumor properties in preclinical models [59, 60]. Targeting TEAD palmitoylation thus represents another potential strategy for treating YAP1-driven tumor progression. At least four TEAD palmitoylation inhibitors have advanced to clinical trials: VT3989 (NCT04665206), ISM6331 (NCT06566079), SW-682 (NCT06251310), and IK-930 (NCT05228015) [61].

Numerous studies highlight the involvement of the Hippo-YAP1 pathway in regulating breast cancer progression. Concurrently, a growing number of upstream regulators of this pathway are being identified. Targeting these upstream regulators to suppress YAP1 activity therefore constitutes a promising therapeutic approach [48, 49]. Our research reveals that NCAPH promotes the expression of LATS1, a key upstream kinase in the Hippo pathway, while simultaneously increasing YAP1 protein levels, decreasing YAP1 phosphorylation, and physically interacting with YAP1. These findings indicate that NCAPH functions as an upstream regulator of the Hippo pathway and also interacts with YAP1 downstream. Therefore, combining inhibition of NCAPH expression with existing TEAD-targeting drugs may effectively isolate YAP1 and offer a “dual-inhibition” strategy against tumor progression.

These findings position NCAPH as a promising therapeutic target for disrupting CSC dynamics in BC, offering potential clinical benefits for patients. Our study elucidates the molecular mechanisms through which NCAPH functions as a tumor promoter in BC, revealing its previously unrecognized role in regulating CSC properties, cell cycle progression, and Hippo-YAP1 signaling. This work establishes a foundational framework for developing NCAPH-targeted therapies to combat BC aggressiveness and therapeutic resistance.

Conclusion

Our integrated findings demonstrate that NCAPH levels strongly correlate with the CSCs phenotype, proliferative capacity, and metastatic potential of BC cells. This association coincides with NCAPH’s interaction with YAP1 and increased nuclear translocation of YAP1, suggesting that the NCAPH-YAP1 axis may play a key role in sustaining oncogenic activity. These insights identify NCAPH as a novel and promising oncogenic factor in BC pathogenesis, meriting investigation as a therapeutic target. While our study delineates this association and proposes a YAP1-dependent pathway, direct causal links and the precise molecular mechanisms require further validation. Moreover, the broader functional landscape of NCAPH—including its potential involvement in metabolic reprogramming, immune evasion, or therapy-induced senescence—remains to be fully elucidated. Future investigations aimed at causally validating this pathway and exploring the pan-cancer regulatory networks orchestrated by NCAPH will be crucial for informing comprehensive therapeutic strategies.

Author contributions

Caixin Qiu played a pivotal role in the development of this manuscript, contributing significantly through the conception and design of the study, the acquisition and assembly of data, and the initial drafting of the article. Yansha Wei was instrumental in the creation of this article, participating actively in the study's design, data collection, and manuscript preparation. Jiehua Li contributed extensively to this paper, engaging in the initial design of the research, gathering and analyzing the data, and contributing to the drafting process.

Funding

This study was supported by Guangxi Natural Science Foundation (NO.2023GXNSFAA026037).

Data availability

The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA010836) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the Declaration of Helsinki and received ethical approval from the Medical Ethics Committee of First Affiliated Hospital of Guangxi Medical University (Approval No. 2025-E0145). Written informed consent was obtained from all individual participants prior to their inclusion in the study. (1) Title of the approved project: NCAPH-YAP1 Interaction Promotes Breast Cancer Stemness and Tumor Progression; (2) Name of the institutional approval committee: Medical Ethics Committee of First Affiliated Hospital of Guangxi Medical University; (3) Approval number: 2025-E0145; (4) Date of approval: February 28, 2025. The animal experiments conducted under the protocol titled ​​"NCAPH-YAP1 Interaction Promotes Breast Cancer Stemness and Tumor Progression"​​ were approved by ​the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University​ (Approval No. 2024-D179-01; Date: June 1, 2024). All procedures adhered to the ​AVMA Guidelines for the Euthanasia of Animals (2020)​​ and complied with institutional and national standards for animal welfare. We had institutional approval for performing experiments using human cells. MCF10A, MCF7, MDA-MB-231, SK-BR-3, HCC1143 and MDA-MB-468 were obtained from Procell Life Science&Tech (Wuhan, China). Procell Life Science&Tech Company has confirmed that there was initial ethical approval for collection of human cells, and that the donors had signed informed consent.

Artificial intelligence (AI)

The authors declare that they have not use AI-generated work in this manuscript.

Consent to publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.