Promoter hypermethylation as a mechanism for Lamin A/C silencing in a subset of neuroblastoma cells

Abstract

Nuclear lamins support the nuclear envelope and provide anchorage sites for chromatin. They are involved in DNA synthesis, transcription, and replication. It has previously been reported that the lack of Lamin A/C expression in lymphoma and leukaemia is due to CpG island promoter hypermethylation. Here, we provide evidence that Lamin A/C is silenced via this mechanism in a subset of neuroblastoma cells. Moreover, Lamin A/C expression can be restored with a demethylating agent. Importantly, Lamin A/C reintroduction reduced cell growth kinetics and impaired migration, invasion, and anchorage-independent cell growth. Cytoskeletal restructuring was also induced. In addition, the introduction of lamin Δ50, known as Progerin, caused senescence in these neuroblastoma cells. These cells were stiffer and developed a cytoskeletal structure that differed from that observed upon Lamin A/C introduction. Of relevance, short hairpin RNA Lamin A/C depletion in unmethylated neuroblastoma cells enhanced the aforementioned tumour properties. A cytoskeletal structure similar to that observed in methylated cells was induced. Furthermore, atomic force microscopy revealed that Lamin A/C knockdown decreased cellular stiffness in the lamellar region. Finally, the bioinformatic analysis of a set of methylation arrays of neuroblastoma primary tumours showed that a group of patients (around 3%) gives a methylation signal in some of the CpG sites located within the Lamin A/C promoter region analysed by bisulphite sequencing PCR. These findings highlight the importance of Lamin A/C epigenetic inactivation for a subset of neuroblastomas, leading to enhanced tumour properties and cytoskeletal changes. Additionally, these findings may have treatment implications because tumour cells lacking Lamin A/C exhibit more aggressive behaviour.

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

“…Specifically, is a representative measure of the elastic modulus of the nucleus and is a representative measure of the nuclear stretch modulus. We then find that our measured mean of 0.58 kPa corresponds to = 0.63 kPa and our measured mean of 0.79 kPa corresponds to = 2.5 mN/m, both of which are consistent with the current literature for Young's modulus measurements of the nucleus and stretch modulus of the nuclear membrane respectively (Dahl et al 2004;Dahl et al 2005;Mazumder et al 2008;Schape et al 2009;Liu et al 2014;Neubert et al 2018;Wang et al 2018;Rosso, Liashkovich, and Shahin 2019). Furthermore, we varied the size of our model nucleus and found that and are independent of initial nuclear size ( Figure S6), similar to our results from experiments ( Figure S3).…”

“…Nuclear mechanics are primarily dictated by the nuclear lamina and chromatin, as well as indirectly influenced by the cytoskeleton (Stephens, Banigan, and Marko 2019). The cytoskeleton protects the nucleus both through an actin cap (Khatau et al 2009;Haase et al 2016;Kim et al 2018) and a peri-nuclear cage of the intermediate filament vimentin (Neelam et al 2015;Patteson et al 2019;Rosso, Liashkovich, and Shahin 2019). The nuclear lamina, primarily lamin A/C, has consistently been shown to be a major mechanical constituent of the nucleus through constricted migration, micropipette aspiration, atomic force microscopy, micromanipulation, and other techniques (Dahl et al 2004;Lammerding et al 2004;Dahl et al 2005;Lammerding et al 2006;Lee et al 2007;Pajerowski et al 2007;Schape et al 2009;Swift et al 2013;Hanson et al 2015;Neelam et al 2015;Stephens et al 2017).…”

Lamin A and Prelamin A Counteract Migration of Osteosarcoma Cells

“…Although an increasing number of reports recently implicated lamins in human cancers, their role in OS, the most common primary malignant neoplasia of the skeletal system [39], has been Interestingly, although 72 h mevinolin treatment reduced cell viability in all OS cell lines, the most significant effect of mevinolin was observed in MG63 cells, expressing the highest prelamin A levels (Figure 5a). Moreover, after long-term mevinolin treatment, the highest increase in the percentage of apoptotic cells was observed in MG63 cell culture (Figure 5b).…”

“…Although an increasing number of reports recently implicated lamins in human cancers, their role in OS, the most common primary malignant neoplasia of the skeletal system [39], has been elusive.…”

Lamin Protein Gene Expression in Childhood Acute Lymphoblastic Leukemia

“…20 Neuroblastoma cells with silenced lamin A/C by hypermethylation display more aggressive behavior. 21 Lamin B1 expression is considerably increased in hepatocellular carcinoma and is related to the tumor size, tumor stage and the number of nodules. Increased plasma lamin B1 levels can also be used to predict hepatocellular carcinoma in the early stage.…”