Nuclear envelope protein lamin A can define breast cancer cell phenotype.

Introduction: : Breast cancer is the most common malignancy among women in the developed world, with metastasis accounting for over 90% of the related deaths. Therapies for metastatic breast cancer remain only partially effective due to the disease's heterogeneity, therapy resistance, and immune evasion, which can be driven by epithelial-mesenchymal transition (EMT). Notably, recent studies found that the most aggressive metastatic subpopulations reside in a partial EMT (pEMT) state.

The nuclear envelope protein lamin A, encoded by the LMNA gene, regulates nuclear stiffness and integrity, chromatin organization, and gene expression; tumor cells with reduced lamin A levels are associated with more advanced disease and increased metastatic potential, but the reasons behind this effect are unclear. Reduced lamin A expression contributes to nuclear morphology abnormalities, a hallmark of aggressive cancers, and enhances the ability of cells to migrate through tight spaces, like the tumor microenvironment. However, these physical effects alone cannot explain the correlation between decreased lamin A levels and poor prognosis, as our previous studies identified numerous proteins and cellular functions altered by reduced lamin A levels.

Motivated to better understand the correlation between lamin A expression and poor patient prognosis in breast cancer, we investigated the function of lamin A in defining breast cancer cell epithelial-mesenchymal phenotypes. In our preliminary work, we uncovered a novel link between lamin A expression and EMT phenotype regulation, where reduced lamin A levels promote the emergence of the pEMT phenotype, providing a potential mechanistic link between reduced lamin A expression and breast cancer progression.

Materials and

Methods: : To understand the relationship between lamin A expression and EMT, we utilized MCF7 breast cancer cells engineered to undergo EMT via doxycycline (dox)-induction and quantified relative lamin A expression before, during, and after EMT induction using immunofluorescence imaging and western blots. To evaluate the epithelial and mesenchymal properties of the cells during the transition and their relationship to changes in lamin A expression, we measured the expression of cell surface and stem-like markers CD24 and CD44—both previously implicated in EMT progression—using flow cytometry. A CD44high/CD24low profile is associated with a mesenchymal phenotype.

To identify the causal relationship between lamin A and the pEMT phenotype in vivo, we analyzed mammary glands from mice with partial or complete deletion of the Lmna gene. Specifically, we removed the developing mammary glands from 4-week old mice and quantified the epithelial (E-Cadherin) and mesenchymal (slug) protein expression using immunofluorescence image quantification.

Finally, to assess the relevance of our findings in breast tumor progression, we crossed mice with reduced Lmna expression with the MMTV-PyMT model for spontaneous mammary tumor formation, resulting in mice which spontaneously formed mammary tumors with wild-type or reduced levels of lamin A. After 20 weeks, we removed the primary tumors and quantified the epithelial (E-Cadherin) protein expression using immunofluorescence imaging and histology grade using hematoxylin and eosin staining.

Results, Conclusions, and Discussions:: Using human cell lines and mouse models, we identified that reduced—but not absent—lamin A levels are associated with a pEMT phenotype, suggesting a potential mechanism which links reduced lamin A expression to breast cancer aggressiveness.

In MCF7 cells, we observed that lamin A expression, low in epithelial cells, increased substantially during EMT (Fig. 1A), suggesting a potential involvement of lamin A in the EMT process. The most pronounced increase occurred on day three of EMT (Fig. 1B), which coincides with dual expression of the pEMT markers CD24 and CD44 (Fig. 1C), revealing a novel correlation between lamin A expression and the pEMT phenotype.
Moreover, we identified that mammary glands from mice with reduced lamin A levels (Lmna+/–) had elevated epithelial and mesenchymal protein expression compared to wild-type (Lmna+/+) glands (Fig. 2), indicating that mammary epithelial cells with reduced lamin A expression have a pEMT phenotype. In contrast, mammary glands with complete lamin A depletion (Lmna–/–) expressed only E-Cadherin, indicating that no EMT had occurred. Together, these findings suggest that reduced lamin A levels promote a pEMT phenotype, whereas complete lamin A depletion impairs or prevents EMT completely.

Finally, we found that tumors with reduced lamin A expression simultaneously exhibited higher histological grades, indicative of more advanced disease, and increased epithelial expression, typically associated with pre-EMT states, compared to wild-type controls (Fig. 3). Although high histology grade and strong epithelial protein expression are usually considered contradictory, their coexistence in our data supports the hypothesis that reduced lamin A expression promotes a pEMT phenotype.

Overall, this work identified that reduced lamin A levels promote a pEMT phenotype, establishing a link between lamin A and EMT and offering a potential mechanistic explanation for the poorly understood correlation between lamin A and poor patient outcomes. Unlike previous studies, which have primarily focused on lamin A’s role in individual steps of the metastatic cascade, we holistically investigated how reduced lamin A levels broadly influence cancer cell phenotype. Future studies based on this work could inform the development of novel therapeutic strategies aimed at inhibiting metastasis by targeting pEMT-related mechanisms.

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