How DEFB1 Promotes Lung Adenocarcinoma Growth: Preclinical Potential of a Targeting Antibody
DEFB1 contributes to lung adenocarcinoma growth
A study has identified DEFB1, a gene linked to lung adenocarcinoma proliferation and prognosis, and tested the effects of an antibody that blocks it in cell and animal models. DEFB1 encodes an antimicrobial peptide normally produced by epithelial cells. The researchers investigated its role in tumor growth in lung adenocarcinoma.
To identify genes involved in cancer cell growth, the team performed a genome-wide CRISPR/Cas9 knockout screen. They cross-referenced results from A549 lung adenocarcinoma cells grown in vitro and implanted in mice with a prognostic analysis of 496 lung adenocarcinoma samples in The Cancer Genome Atlas (TCGA). This yielded 15 candidate genes, of which DEFB1 was selected for further study.
A mechanism linking cancer cell proliferation and macrophage changes
Single-cell analysis showed high DEFB1 expression in lung adenocarcinoma cells. Multiplex immunohistochemistry of 118 paired patient surgical specimens also showed higher expression in tumor tissue than in adjacent normal lung tissue. High tumor DEFB1 expression was associated with poorer prognosis. In a progression-free survival analysis based on surgical specimens, the high-expression group had a hazard ratio of 2.52 compared with the low-expression group (p < 0.001). This association in patient data alone does not establish that DEFB1 shortens patients’ survival.
In functional experiments, removing DEFB1 from A549 and PC-9 lung adenocarcinoma cells inhibited proliferation, migration, and invasion. Re-expressing DEFB1 or adding the protein externally restored these suppressed functions.
The researchers also investigated how DEFB1 promotes cancer growth. Co-immunoprecipitation, mass spectrometry, and other analyses showed that DEFB1 interacts with periplakin (PPL). Follow-up functional experiments supported a role for this interaction in epithelial–mesenchymal transition (EMT) and proliferation. EMT is a change associated with cell migration and invasion; these observations should not be interpreted as direct evidence of metastasis in patients.
DEFB1 also bound macrophage migration inhibitory factor (MIF). In co-culture experiments with lung adenocarcinoma cells and THP-1-derived macrophages, DEFB1 promoted a shift toward the M2 phenotype, and the MIF inhibitor ISO-1 attenuated this change. The findings suggest that DEFB1 may affect not only cancer cell growth but also the state of surrounding immune cells.
Effects of the targeting antibody and remaining validation needs
The team generated six monoclonal antibodies that block DEFB1 and selected mAb-5 for further evaluation because it most effectively inhibited cell proliferation. The antibody inhibited cell proliferation and tumor growth in patient-derived organoids and mouse models. The organoids were treated with a control antibody or mAb-5 at 1 μg/mL for seven days. In mice with Kras G12D mutation-induced lung adenocarcinoma engineered to express human DEFB1, tumor growth was delayed and survival was extended. These are preclinical findings, not treatment results in patients.
The safety assessment was limited to four weeks of dosing in healthy male mice humanized for DEFB1. Under those conditions, no clear abnormalities were observed in body weight, blood biochemistry, or major-organ histology, but this does not establish safety in humans. The researchers cited interspecies differences, limited assessment of CD8-positive T cells, insufficient analysis of myeloid-cell function, and the need to improve antibody efficacy as limitations. The available data do not establish a clinical trial timeline or treatment efficacy in patients.
The study design and findings are available in the original Cell Death & Disease paper.
Lung adenocarcinoma, one of the most prevalent malignancies, underscores the urgency of identifying genes linked to its proliferation and prognosis for developing targeted therapies. In this study, we performed genome-wide CRISPR/Cas9 screening both in vitro and in vivo, and subsequently cross-referenced the findings with prognosis-related genes in lung adenocarcinoma. The screening results revealed that DEFB1 promotes lung adenocarcinoma proliferation, while our integrated analysis of single-cell sequencing, multiplex immunohistochemistry, TCGA, and GEO data concurrently demonstrated elevated DEFB1 expression in cancer cells, along with a negative correlation between DEFB1 expression and patient survival. Subsequently, functional studies employing DEFB1 knockout cells, re-expressed DEFB1 cells, and knockout cells supplemented with exogenous DEFB1 revealed that DEFB1 significantly enhances cell proliferation, migration, and invasion. Co-immunoprecipitation combined with mass spectrometry experiments was performed to uncover the mechanism of DEFB1, demonstrating that it interacts with Periplakin (PPL) to induce epithelial-to-mesenchymal transition (EMT) and proliferation, while simultaneously binding to Macrophage Migration Inhibitory Factor (MIF) to enhance M2 macrophage polarization. Furthermore, we developed multiple anti-DEFB1 monoclonal antibodies and found one of them, mAb-5, potently blocked DEFB1's function and inhibited lung adenocarcinoma progression in cell lines, organoids, xenografts, and spontaneous lung cancer models, while maintaining a favorable safety profile. Overall, our study identifies DEFB1 as a novel driver of lung adenocarcinoma, and the anti-DEFB1 monoclonal antibody mAb-5 emerges as a promising therapeutic candidate with significant potential.
This study provides preclinical evidence that an antibody blocking the secreted protein DEFB1 could be a candidate for lung adenocarcinoma drug development. In cells with DEFB1 removed, mAb-5 did not significantly further suppress proliferation; it also blocked the proliferation increase caused by re-expressing DEFB1, supporting target dependence. During development, these results and the patient-derived organoid and mouse data could inform antibody optimization and the design of further efficacy and safety assessments. However, patient selection based on DEFB1 expression, added benefit in combination with existing treatments, and improved survival in people remain unvalidated. The findings should not yet be interpreted as a personalized treatment available to patients.