Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype characterised by the absence of targetable hormone receptors and increased metastatic rates. As nuclear softening strongly contributes to TNBC’s enhanced metastatic capacity, increasing the nuclear stiffness of TNBC cells may present a promising therapeutic avenue. Previous evidence has demonstrated the ability of Sirtuin 2 (SIRT2) inhibition to induce cytoskeletal reorganisation, a key factor in regulating nuclear mechanics. Thus, our study aimed to investigate the effect of SIRT2 inhibition on the nuclear mechanics and migratory behaviour of TNBC cells. To achieve this, SIRT2 was pharmacologically inhibited in MDA-MB-231 cells using AGK2, a SIRT2-specific inhibitor. Although SIRT2 inhibition had no effect on LINC complex composition, the AGK2-treated MDA-MB-231 cells displayed more prominent perinuclear organisations of acetylated α-tubulin, vimentin, and F-actin. Additionally, the nuclei of the AGK2-treated MDA-MB-231 cells exhibited greater resistance to collapse under osmotic shock. Scratch-wound assays also revealed that SIRT2 inhibition led to polarity defects in the MDA-MB-231 cells, while in vitro space-restrictive invasion assays highlighted their reduced migratory capacity upon AGK2 treatment. Taken together, our findings suggest that SIRT2 inhibition promotes a perinuclear cytoskeletal organisation in MDA-MB-231 cells, which enhances their nuclear rigidity and impedes their invasion through confined spaces in vitro.

Eukaryotic cells tether the nucleoskeleton to the cytoskeleton via a conserved molecular bridge, called the LINC complex. The core of the LINC complex comprises SUN-domain and KASH-domain proteins that directly associate within the nuclear envelope lumen. Intra- and inter-chain disulphide bonds, along with KASH-domain protein interactions, both contribute to the tertiary and quaternary structure of vertebrate SUN-domain proteins. The significance of these bonds and the role of PDIs (protein disulphide isomerases) in LINC complex biology remains unclear. Reducing and non-reducing SDS-PAGE analyses revealed a prevalence of SUN2 homodimers in non-tumorigenic breast epithelia MCF10A cells, but not in the invasive triple-negative breast cancer MDA-MB-231 cell line. Furthermore, super-resolution microscopy revealed SUN2 staining alterations in MCF10A, but not in MDA-MB-231 nuclei, upon reducing agent exposure. While PDIA1 levels were similar in both cell lines, pharmacological inhibition of PDI activity in MDA-MB-231 cells led to SUN-domain protein down-regulation, as well as Nesprin-2 displacement from the nucleus. This inhibition also caused changes in perinuclear cytoskeletal architecture and lamin downregulation, and increased the invasiveness of PDI-inhibited MDA-MB-231 cells in space-restrictive in vitro environments, compared to untreated cells. These results emphasise the key roles of PDIs in regulating LINC complex biology, cellular architecture, biomechanics, and invasion.

SYNE2 mutations have been associated with skeletal and cardiac muscle diseases, including Emery-Dreifuss muscular dystrophy (EDMD). Here, we present a 70-year-old male patient with muscle pain and elevated serum creatine kinase levels in whom whole-exome sequencing revealed a novel heterozygous SYNE2 splice site mutation (NM_182914.3:c.15306+2T>G). This mutation is likely to result in the loss of the donor splice site in intron 82. While a diagnostic muscle biopsy showed unspecific myopathological findings, immunofluorescence analyses of skeletal muscle and dermal cells derived from the patient showed nuclear shape alterations when compared to control cells. In addition, a significantly reduced nesprin-2 giant protein localisation to the nuclear envelope was observed in patient-derived dermal fibroblasts. Our findings imply that the novel heterozygous SYNE2 mutation results in a monoallelic splicing defect of nesprin-2, thereby leading to a rare cause of myalgia and hyperCKemia.