The cell cycle is tightly regulated and necessary for development, growth and tissue replacement and repair. The process requires both oncogenes, which drive the cell cycle forward, and tumour suppressor genes, which inhibit the cell cycle and can promote apoptosis in defective cells. Loss of tumour suppressor gene function or over-active oncogene function can lead to uncontrolled cell division, which can ultimately lead to the formation of a tumour. FLCN has been previously linked to cell cycle control and apoptosis, and BHD-associated kidney cancers often show somatic loss of the wild type allele (Vocke et al., 2005), suggesting that FLCN is a tumour suppressor. A study by Laviolette et al. published in PLoS ONE this week, further elucidates FLCN’s role in cell cycle progression and tumour suppression.
To analyse the role of FLCN in cell cycle progression, the authors compared cell cycle profiles, as measured by propidium iodide staining and 5-ethynyl-2’-deoxyuridine uptake following cell synchronisation, between UOK-257 cells completely lacking FLCN, and UOK-257 cells expressing a reconstituted wild type FLCN allele. Cells expressing FLCN showed a significant 2-4 hour delay in progression through S-phase of the cell cycle, which further increased to a 2-6 hour delay in the G2/M phase. This finding was confirmed in MEF cells, indicating that this effect was not a cell-specific phenomenon and that under normal conditions, FLCN slows down cell cycle progression, consistent with a role as a tumour suppressor gene.
The authors used three different constructs carrying FLCN mutations that have been associated with BHD – a missense mutation (K508R), a truncating mutation (c.1408_1418del11), and an in-frame deletion mutant (c.469_471del3) – in order to analyse how these mutations affect cell cycle progression. All three showed cell cycle profiles similar to FLCN-null UOK-257 cells, suggesting that all three mutations had ablated FLCN function significantly. The authors suggest that their cell cycle assay may therefore prove a useful tool to determine the pathogenicity of uncharacterized FLCN variants.
FLCN is known to be differentially phosphorylated throughout the cell cycle at S62, S73 and S302, suggesting that its function may change over time. Laviolette et al. used mass spectrometry analysis and found that S62 and S73 became phosphorylated as the cell cycle progressed. To further investigate the significance of this finding, the authors engineered a phosphomimetic FLCN allele that is constitutively phosphorylated at S62 and S73, and a phosphoinactive allele that is constitutively unphosphorylated at these residues. They found that while both mutants were stably expressed and displayed the same localisation pattern as wild type FLCN, the phosphomimetic mutant was less stable than the wild type FLCN protein, and failed to slow cell cycle progression.
Taken together, these findings suggest that under normal conditions, unphosphorylated FLCN acts to slow down the cell cycle, but as the cell cycle progresses, FLCN becomes phosphorylated and consequently destabilised, removing its inhibitory influence and allowing cells to enter mitosis and to divide at the right time and in a controlled manner. The well known tumour suppressors RB1 and p53 also control cell division and are subject to post-translational modifications that modulate their function during the cell cycle (Chen et al., 1989; Stewart and Pietenpol, 2001), lending further support to the assertion that FLCN is also a tumour suppressor.
FLCN was recently shown to inhibit G1 to S cell cycle progression by inhibiting cyclin D1 expression and by affecting RhoA signalling (Kawai et al., 2013; Medvetz et al., 2012; Nahorski et al., 2012), whereas the authors of this study did not report any G1/early S phase defects. The experiments in these studies were conducted in a number of different cell lines, and under different conditions, which may go some way to explaining the differing observations between studies.
Although FLCN has now been shown to control the cell cycle in several studies, a clear mechanism as to how it exerts its control is still lacking. FLCN is also known to promote apoptosis; a common outcome of tumour suppressor activity when cell cycle control breaks down. Given the intrinsic link between cell cycle disinhibition, escape from apoptosis and tumourigenesis, it is possible that fully characterising FLCN’s role in cell cycle control and apoptosis will shed light on how tumours form in BHD syndrome, and therefore may also suggest an effective therapy.
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www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.