BUB1 kinase inhibition slows endometrial cancer growth in lab study

A multi-omics and experimental study published in Future Journal of Pharmaceutical Sciences reports that the mitotic checkpoint kinase BUB1 is significantly overexpressed in uterine corpus endometrial carcinoma (UCEC), where its elevated levels are associated with advanced disease stage and reduced overall survival. Combining transcriptomic data from The Cancer Genome Atlas (TCGA) and an independent GEO validation dataset with laboratory experiments in a single endometrial cancer cell line, the researchers position BUB1 as both an unfavorable prognostic biomarker and a candidate therapeutic target whose pharmacological inhibition reduced cell viability and migration in vitro.

The analysis began with 545 UCEC tumor samples from TCGA and 94 samples from the GEO dataset GSE106191. By intersecting 5,527 differentially expressed genes with a curated list of 600 anoikis-related genes from the GeneCards database, the team identified 28 differentially expressed anoikis-related genes, with BUB1 standing out as prominently upregulated in tumors. Expression analysis showed significantly elevated BUB1 levels in tumors compared with normal endometrium (p < 0.001), particularly in high-grade and advanced-stage cases, and expression increased progressively from early- to late-stage tumors.

Survival analyses strengthened the prognostic case. Kaplan–Meier curves indicated that high BUB1 expression was associated with significantly worse overall survival (hazard ratio 2.14, 95% confidence interval 1.46–3.12, p = 0.0002) and worse disease-free survival (HR 1.89, 95% CI 1.32–2.71, p = 0.0008). Univariate and multivariate Cox regression, the latter adjusting for age, tumor stage, and histological grade, identified BUB1 as an independent prognostic marker alongside stage and grade. These are observational associations from retrospective cohort data, so they show correlation with outcome rather than proof that BUB1 drives mortality.

Functional enrichment analyses linked high BUB1 expression to mitotic checkpoint regulation, spindle assembly, DNA damage response, and cell cycle pathways (adjusted p < 0.01), with gene set enrichment analysis also showing a strong association with PI3K-AKT signaling (adjusted p = 0.0015) and enrichment of anoikis resistance pathways, including ERK1/2 activation and G2/M checkpoint control. Anoikis resistance—the ability of cancer cells to survive detachment from the extracellular matrix—is a recognized mechanism facilitating metastasis, and the authors suggest BUB1 may help UCEC cells evade detachment-induced apoptosis. Protein–protein interaction mapping using STRING placed BUB1 at a central node with MAD2L1, BUB3, and BUBR1, core components of the spindle assembly checkpoint, and TIMER-based validation confirmed high expression of these partners in UCEC samples.

The study also explored why BUB1 might be overexpressed. Mutation profiling through the Gene Set Cancer Analysis database identified frequent copy number amplifications and missense single-nucleotide variants in BUB1, which correlated with reduced patient survival. Methylation analysis revealed hypomethylation of the BUB1 promoter region, inversely associated with mRNA expression (p < 0.01), supporting the hypothesis that epigenetic de-repression, together with genomic amplification, contributes to the elevated transcript levels observed in tumors.

Immune infiltration analysis using three independent deconvolution algorithms—CIBERSORT, TIMER2.0, and TIDE—revealed a significant positive correlation between BUB1 expression and the abundance of neutrophils, tumor-associated macrophages, CD4-positive T cells, and dendritic cells. Because these immune populations are often associated with immunosuppression and tumor-promoting inflammation, the authors propose that BUB1 may help shape an immune-suppressive tumor microenvironment. They are careful, however, to frame this as a correlation-based hypothesis: proposed mechanisms involving cGAS-STING signaling, NF-κB-driven cytokine transcription, and immune checkpoint upregulation remain to be experimentally validated in UCEC.

On the therapeutic side, in silico drug sensitivity analysis using Genomics of Drug Sensitivity in Cancer data indicated that high BUB1 expression was associated with resistance to 17-AAG and (5Z)-7-oxozeaenol, while BUB1-high tumors were predicted to be more responsive to 5-fluorouracil, GW843682X, 2OH-BNPP1, and foretinib. These are computational predictions from correlative drug response data, not clinical trial findings, but they guided the choice of 2OH-BNPP1, a low-micromolar BUB1 kinase inhibitor, for experimental validation.

In the laboratory, HEC-1-B endometrial carcinoma cells treated with 2OH-BNPP1 showed a dose-dependent decline in viability, with significant cytotoxicity at concentrations of 5 µM and above (p < 0.01), alongside morphological changes consistent with apoptosis. Quantitative PCR showed that a 5 µM treatment reduced BUB1 expression by more than 50% relative to untreated controls (p < 0.01), and wound healing assays demonstrated markedly delayed migration at 24 and 48 hours compared with mock and vehicle controls (p < 0.0001). All in vitro experiments were performed in at least three independent biological replicates with appropriate statistical corrections.

The authors acknowledge important limitations. The inhibitor has known off-target activity against PDGFRβ, CSF1R, and VEGFR2/3 at sub-micromolar concentrations, and although the authors report no unexpected cytotoxicity attributable to off-targets at the tested doses, they recommend next-generation inhibitors such as BAY-320 (IC50 0.56 µM) or BAY-1816032 (IC50 approximately 29 nM), which offer superior selectivity, for future preclinical work. Validation was also restricted to a single cell line, so generalizability across endometrial carcinoma subtypes, and any relevance in vivo, remains untested; the authors call for studies using additional cell lines, patient-derived xenografts, organoid cultures, and CRISPR-based knockout systems.

The translational message is deliberately cautious. BUB1 emerges from this work as a clinically correlated prognostic marker and a biologically plausible target whose inhibition disrupts proliferation, survival under anchorage-independent conditions, and migration in endometrial cancer cells in vitro. Before any bedside application, the observed associations must be converted into demonstrated causal mechanisms, more selective inhibitors must be tested in representative models, and questions of drug delivery, toxicity, and acquired resistance must be resolved. For now, the study adds BUB1 to the growing list of mitotic checkpoint kinases under investigation as vulnerabilities in gynecological malignancy.

The epidemiological backdrop of the study underscores why new molecular targets are being sought. UCEC is described as the most prevalent gynecologic malignancy, with recent global estimates of approximately 67,000 to 68,000 new cases and more than 13,000 deaths annually. Incidence rose by roughly 4–5% per year in earlier decades, and mortality continues to increase by about 1.5% annually, trends attributed in part to obesity, metabolic disorders, aging populations, and lifestyle-associated risk factors. The disease also shows marked racial and ethnic disparities: Black women experience disproportionately higher mortality and are more frequently diagnosed with aggressive non-endometrioid subtypes compared with White women, despite similar incidence rates and significantly lower five-year survival, reported at approximately 63% versus 84%. Socioeconomic inequality, delayed diagnosis, differences in tumor biology, and limited access to targeted therapies are cited as contributing factors.

Clinically, abnormal uterine bleeding remains the most common presenting symptom, particularly in postmenopausal women, while advanced disease may manifest with pelvic pain, weight loss, lymphatic dissemination, and distant metastasis. Established risk factors include obesity, diabetes mellitus, hypertension, prolonged estrogen exposure, polycystic ovarian syndrome, nulliparity, and hereditary syndromes such as Lynch syndrome. Histologically, UCEC spans a spectrum from well-differentiated endometrioid tumors to aggressive serous, clear cell, and carcinosarcoma forms, and this biological diversity contributes to therapeutic resistance and progression. Molecular classification studies have identified diverse mutational signatures involving PTEN, PIK3CA, TP53, KRAS, ARID1A, POLE, and mismatch repair deficiency pathways, with PI3K-AKT pathway dysregulation among the most frequent alterations in endometrial carcinoma.

The biological rationale for examining BUB1 rests on its canonical role in cell division. The BUB1 gene encodes a conserved serine/threonine kinase that functions as a core component of the spindle assembly checkpoint, regulating chromosome alignment, kinetochore signaling, and mitotic fidelity. Through interactions with BUB3, MAD1, MAD2, and BUBR1, the protein helps ensure accurate chromosome segregation and prevents aneuploidy. Dysregulation of BUB1 promotes chromosomal instability, a hallmark of tumorigenesis associated with uncontrolled proliferation, genomic heterogeneity, and therapeutic resistance. Beyond this mitotic role, prior work in other cancer types, including breast, colorectal, and ovarian cancers, has linked abnormal BUB1 expression to tumor progression, poor clinical outcomes, and therapy resistance, and BUB1 overexpression or hyperactivation has been associated with enhanced PI3K-Akt signaling, increased Akt phosphorylation, and possible ERK1/2 activation, with reciprocal feedback that can stabilize BUB1 expression.

Anoikis resistance, the process the study interrogates, is the ability of cancer cells to evade apoptosis triggered by detachment from the extracellular matrix. It enables tumor cells to endure during circulation, promoting dissemination and colonization of distant organs, and is orchestrated by a network of anoikis-related genes regulating cell survival, extracellular matrix signaling, apoptosis, and metastatic spread. Established contributors include BCL2, MCL1, ITGA5, ITGB1, FAK/PTK2, SRC, EGFR, AKT1, PIK3CA, TWIST1, SNAI1, ZEB1, and MUC16, which act through epithelial–mesenchymal transition, focal adhesion signaling, PI3K-AKT activation, and cytoskeletal remodeling. The authors note that the specific regulators of anoikis resistance in UCEC had remained largely undefined, and that the contribution of mitotic checkpoint regulators such as BUB1 to this process had not previously been investigated in this cancer.

Methodological details add context to the findings. Transcriptomic data were normalized and analyzed with the limma package in R, using differential expression thresholds of absolute log2 fold change greater than 2 and adjusted p below 0.05, with multiple comparisons controlled by the Benjamini–Hochberg false discovery rate. Enrichment analyses employed Gene Ontology, Gene Set Enrichment Analysis, and Kyoto Encyclopedia of Genes and Genomes tools, while the protein interaction network was built in STRING version 11.5 and visualized in Cytoscape, with co-expression cross-validated through TIMER 2.0. Genomic alterations were profiled using the Gene Set Cancer Analysis database, and immune deconvolution quantified macrophages, neutrophils, CD4-positive T cells, and dendritic cells across the three algorithms at a significance threshold of p below 0.05.

The choice of inhibitor also reflects practical constraints. In vitro kinase assays show that 2OH-BNPP1 achieves low micromolar potency against BUB1, with an IC50 of approximately 0.60 µM comparable to BAY-320 at 0.56 µM, but it exhibits broader kinase inhibition with potential off-target activity against PDGFRβ, CSF1R, and VEGFR2/3 at sub-micromolar concentrations. BAY-1816032 demonstrates intracellular BUB1 inhibition with an IC50 of approximately 29 nM and strong selectivity, though its availability and validation in UCEC contexts remain limited. The authors accordingly position 2OH-BNPP1 as a proof-of-concept tool compound and suggest that future preclinical evaluation could combine BUB1 inhibition, preferably with more selective next-generation inhibitors, with DNA-damaging agents, taxanes, or immune checkpoint blockade, while also addressing drug delivery, toxicity, and acquired resistance through compensatory pathway activation or BUB1 kinase domain mutations.

Subject of Research: The role of the BUB1 kinase in endometrial carcinoma progression and its inhibition by 2OH-BNPP1 in vitro

Article Title: Pharmacological inhibition of BUB1 suppresses UCEC progression by modulating the PI3K-AKT pathway and reversing anoikis resistance and immune evasion: a multi-omics and experimental study

Article References: Ali, W., Al Mamun, M. A., He, Y., Muhammad, N., Ahmad, S., Umar, M., & Kulsoom (2026). Pharmacological inhibition of BUB1 suppresses UCEC progression by modulating the PI3K-AKT pathway and reversing anoikis resistance and immune evasion: a multi-omics and experimental study. Future Journal of Pharmaceutical Sciences, 12(1), Article 85. https://doi.org/10.1186/s43094-026-01017-3

Image Credits: AI Generated

DOI: 10.1186/s43094-026-01017-3

Keywords: BUB1, endometrial carcinoma, UCEC, PI3K-AKT pathway, anoikis resistance, 2OH-BNPP1, spindle assembly checkpoint, immune infiltration, TCGA, prognostic biomarker, kinase inhibitor, HEC-1-B