LCN2 loss reduces invasion and alters stress pathways in TNBC cells

Disrupting lipocalin-2 (LCN2) in MDA-MB-231 triple-negative breast cancer cells reduced migration, invasion and adhesion to uncoated surfaces in a laboratory study. Researchers used CRISPR/Cas9 editing to isolate single-cell-derived LCN2 knockout clones and combined RNA sequencing, protein analysis, microscopy and functional assays to characterize the consequences. The deficient cells largely retained the elongated, mesenchymal phenotype of the parental line, while their gene-expression profile shifted toward inflammatory, translational and stress-related programs. Transient re-expression of LCN2 partially restored migration and invasion, supporting a functional role for the protein in tumor-cell movement and extracellular matrix interactions.

The study used an established human cell-line model rather than patients or animals. MDA-MB-231 cells were originally derived from a pleural effusion of a woman with metastatic breast adenocarcinoma and are widely used to investigate triple-negative breast cancer, a subtype lacking estrogen receptors, progesterone receptors and HER2 expression. Before editing, the investigators checked the parental and control cell lines by short tandem repeat profiling. Two guide RNAs and a donor construct were then used to create LCN2-deficient cells, from which individual clones were isolated. Western blotting and reverse-transcription quantitative PCR confirmed that selected clones lacked detectable functional LCN2 under basal conditions and after cytokine stimulation. Multicolor fluorescence in situ hybridization showed that knockout clone 15 retained the complex, aneuploid architecture of MDA-MB-231 without evidence of additional large-scale rearrangements after editing, although it represented a more homogeneous subpopulation than the heterogeneous parental culture.

Light and fluorescence microscopy showed that the knockout cells generally remained elongated, adherent and disorganized in growth, resembling their parental counterparts. A subset of deficient cells, however, developed a markedly enlarged morphology. These cells contained prominent F-actin stress fibers and, in some cases, multiple or enlarged nuclei. Transmission electron microscopy showed broadly comparable nuclei, mitochondria, endoplasmic reticulum and other organelles in parental and knockout cells, with somewhat more lysosomes in clone 15. The authors described that observation cautiously as potentially consistent with increased lysosomal activity or cellular turnover. Protein and RNA measurements showed continued expression of core mesenchymal markers such as vimentin, fibronectin 1 and alpha-smooth muscle actin. E-cadherin was weak in parental cells and undetectable in the knockout clones, while the epithelial-to-mesenchymal transition gene set did not display a coordinated shift. The results therefore did not show that LCN2 loss drove the cells toward a clearly epithelial phenotype.

RNA sequencing compared parental MDA-MB-231 cells with knockout clone 15 using three independent biological replicates per group. Principal component analysis separated the knockout replicates from the parental samples; the first two components accounted for 78.77% and 9.44% of the total variance, respectively, or 88.21% together. Pathway enrichment indicated that genes increased in clone 15 were associated with chemokine response, cytokine-mediated signaling, ribosome biogenesis, rRNA processing, translation and canonical NF-kappaB-related functions. Genes decreased in the knockout were associated with mitochondrial organization, metabolic pathways, extracellular matrix organization and protein trafficking. The investigators explicitly treated this broad sequencing result as clone-specific until it could be confirmed in additional independent knockout clones. The focused analysis also showed that key mesenchymal genes VIM and CDH2 remained largely unchanged, while FN1 and ITGA5 increased. Although CDH1, KRT8 and KRT18 decreased, EPCAM, CLDN1 and OCLN were unchanged or increased, providing no consistent epithelial signature.

Selected inflammatory changes were assessed with independent methods. RT-qPCR confirmed higher expression of NFKBIA, CASP1, CXCL1 and IL1B in knockout clone 15, and Western blotting detected increased basal abundance of the NF-kappaB regulators IκBα and IκBζ. Both parental and knockout cells remained responsive to interleukin-1 beta and tumor necrosis factor alpha, including induction of IκBζ. Phosphorylation of NF-kappaB p65 was lower in the knockout under several stimulation conditions, while total p65 abundance did not change. Because the protein analysis occurred 24 hours after cytokine exposure, it cannot establish whether the pathway’s rapid, transient early activation was affected; the observations instead suggest a change in the sustained response. RNA sequencing also detected marked upregulation of CXCL8, but its protein abundance did not mirror the transcriptional result, illustrating why transcript-level changes require protein validation.

Loss of LCN2 was associated with altered expression of genes involved in iron handling, antioxidant responses and ferroptosis-related regulation. The knockout clone showed increased TFRC, FTH1 and FTL, while SLC7A11, a transporter associated with cellular cysteine availability, was also upregulated. GPX4 and ACSL4 transcripts decreased, whereas SLC40A1 did not change significantly. The antioxidant-response pattern was similarly mixed: NQO1, GCLM, KEAP1 and mitochondrial SOD2 increased, while SOD1 decreased. These findings are gene-expression associations, not direct measurements of intracellular iron, lipid peroxidation, reactive oxygen species or ferroptotic death. The direction of the individual changes does not support a simple conclusion that the cells became either more or less susceptible to ferroptosis. Previous work has implicated LCN2 in protection against ferroptosis in MDA-MB-231 cells, but the present experiment did not repeat those functional tests and instead provides a molecular profile for further investigation.

The clearest measured effects appeared in motility. In Transwell assays, knockout clones 7 and 15 migrated less effectively than parental MDA-MB-231 cells, while the non-migratory MCF-7 breast cancer cell line served as a control. The same clones showed reduced invasion through Geltrex-coated membranes. These differences were not explained by faster or slower proliferation: ATP-based measurements at 24 and 48 hours found no significant change, and the proliferation markers MKI67, TOP2A and CCNA2 were not significantly altered. Transient re-expression of human LCN2 increased migration relative to GFP-transfected knockout controls and partially restored invasion. This rescue supports the association between LCN2 loss and the functional phenotype, although it was incomplete and used transient overexpression, which does not reproduce every aspect of endogenous LCN2 expression and regulation.

Adhesion depended strongly on the material beneath the cells. LCN2-deficient clones attached less well to uncoated culture vessels than parental cells, yet their adhesion to collagen type I was comparable to the parental level and their attachment to Geltrex was enhanced. Transcripts and proteins for the extracellular matrix receptors ITGA2 and ITGA6 increased in the knockout cells. This pattern may represent an adaptive response to altered interactions with the extracellular environment, but receptor abundance alone does not establish integrin activity, adhesive strength or the turnover of focal adhesions. The study did not directly test whether alpha2 or alpha6 integrins caused the migration, invasion or adhesion changes. It also found that the enriched ribosome-biogenesis and translation signatures did not correspond to increased proliferation, demonstrating that transcriptional pathway labels do not by themselves reveal the resulting cellular behavior.

Overall, the experiments support LCN2 as a regulator of morphology, motility and cell–matrix interactions in MDA-MB-231 cells. Its absence altered inflammatory, metabolic, iron-related and extracellular matrix gene programs, enlarged a subset of cells and weakened migration, invasion and adhesion to uncoated surfaces, without eliminating the line’s mesenchymal identity or measurably changing proliferation. The use of authenticated cells, verified knockouts, multiple functional assays and a partial rescue gives the findings internal consistency, but the broad RNA-sequencing analysis centered on one clone. Other limitations include the lack of direct ferroptosis measurements, incomplete agreement between RNA and protein abundance, absence of a canonical senescence-marker panel, and no use of three-dimensional cultures or in vivo metastasis models. The results therefore identify LCN2-dependent pathways in one experimental cell model rather than demonstrating a clinical effect or a general mechanism across triple-negative breast cancers.

Subject of Research: Experimental characterization of LCN2 loss in MDA-MB-231 triple-negative breast cancer cells

Article Title: Comprehensive characterization of the cellular and molecular consequences of LCN2 loss in MDA-MB-231 triple-negative breast cancer cells

Article References: Keller, D. T., Kankel, S., Liehr, T., Hardt, K. S., Buhl, E. M., Weiskirchen, R., & Schröder-Lange, S. K. (2026). Comprehensive characterization of the cellular and molecular consequences of LCN2 loss in MDA-MB-231 triple-negative breast cancer cells. Frontiers in Cell and Developmental Biology, 14, Article 1909534. https://doi.org/10.3389/fcell.2026.1909534

Image Credits: AI Generated

DOI: 10.3389/fcell.2026.1909534

Keywords: LCN2, triple-negative breast cancer, MDA-MB-231, CRISPR knockout, cell migration, tumor invasion, extracellular matrix, RNA sequencing, ferroptosis, NF-kappaB signaling, Comprehensive, characterization