INVESTIGATING THE ROLE OF NESPRIN-4 IN NUCLEAR MECHANICAL REGULATION DURING CONFINED MIGRATION OF BREAST CANCER CELLS

Abstract

Breast cancer metastasis depends on the ability of tumor cells to migrate through physically restrictive environments, where the nucleus acts as a major mechanical barrier. Nuclear mechanics are regulated by the nuclear lamina, chromatin organization, and force transmission through the linker of nucleoskeleton and cytoskeleton (LINC) complex. Nesprin-4, encoded by SYNE4, is a microtubule-associated outer nuclear membrane protein whose role in breast cancer cell mechanics remains poorly defined. This study investigated whether nesprin-4 contributes to nuclear mechanical regulation during confined migration of breast cancer cells. Nesprin-4 expression varies dramatically between different breast cancer cell types and has been shown to correlate negatively with a metastatic phenotype. The study the role of nesprin-4, it was knocked down in luminal MCF7 and T47D cells (which express nesprin-4 endogenously) and overexpressed in triple-negative MDA-MB-231 and MDA-MB-436 cells (which do not express endogenous nesprin-4). Expression changes were confirmed by RT-qPCR, and overexpression was validated at the protein level by western blotting. Confined migration was assessed using transwell assays with 8 μm, 5 μm, and 3 μm pore sizes. Lamin A/C levels were examined by western blotting and immunofluorescence microscopy. SYNE4 modulation produced subtype-dependent effects on migration. In luminal models, SYNE4 knockdown reduced confined migration, with the clearest effects under moderate or high confinement. In triple-negative models, SYNE4 overexpression produced limited or non-significant effects, although MDA-MB-231 cells showed a consistent increase in migration. Total Lamin A/C levels showed no major global change in most models. However, immunofluorescence revealed subtle alterations in Lamin A/C organization at the nuclear periphery, particularly in luminal cells following SYNE4 knockdown. These findings suggest that Nesprin-4 contributes to breast cancer cell behavior in a context-dependent manner and may influence confined migration through effects on nuclear-cytoskeletal coupling and Lamin A/C organization rather than through major disruption of nuclear lamina integrity.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences