Birt-Hogg-Dubè can be thought of as a ciliopathy. Ciliopathies are a set of rare diseases resulting from defects in cilia; membrane-protruding organelles that provide either propulsion (motile cilia) or a sensory function (primary cilia) in mammalian cells. Like BHD, cystic lesions in the kidneys are a major manifestation of ciliopathies such as polycystic kidney disease and Bardet-Biedl syndrome, which result from mutations in the ciliary proteins that regulate cilia formation and retraction, a process known as ciliogenesis. In 2013 Luijten et al., reported that FLCN localizes to the primary cilium and influences various parameters of ciliogenesis. Furthermore, knock-down of FLCN in polarized epithelial cells of the kidney interferes with canonical Wnt signaling, an important cilia-associated pathway, and distorts the ability of these cells to organize into 3D spheroid cultures.
In 2016, Zhong et al. published an interesting study that further examined the role of FLCN in cilia, this time in the context of flow stress and mTOR signaling. Primary cilia of kidney cells experience flow stress when fluid passes over the cell surface causing the cilia to bend, and this is transduced into a cellular response by the suppression of mTORC1 signaling in an AMPK-dependent manner (Boehlke et al., 2013). Excitingly, the paper by Zhong et al., implicates a role for FLCN in this process, thereby providing another mechanism by which mTOR signaling could be dysregulated in BHD.
Zhong et al. first established that an interaction between FLCN with KIF3A (a subunit of the cilia motor protein Kinesin-2) occurred in a cilia-dependent manner and was absent in cell populations lacking cilia, and therefore, all subsequent experiments were conduct on ciliated cells. This is consistent with the findings of Dodding et al., 2011, which predicted that FLCN houses a WD motif that enables it to bind Kinesisn-1, a cytoplasmic motor protein that is associated with intracellular transport. Next the study sought to confirm the observation that flow stress on ciliated cells causes a suppression of mTOR signaling by measuring the levels of P-S6, a product of mTOR phosphorylation. In normal cells, flow stress caused a down-regulation of P-S6, which is consistent with a reduction of mTOR activity, however, in FLCN knockout (UOK-257) and knock-down cells (siRNA) this P-S6 down-regulation failed to occur in response to the stress. The investigators also looked at the P-AMPK signaling that is upstream of the pathway and that acts as a repressor of mTOR signaling. In normal cells, the flow stress increased the expression of P-AMPK (which in turn decreases mTOR activity) but this increase was less apparent in the FLCN deficient knockdown cells. This behavior was also mirrored by P-TSC2, a signaling molecule down stream of P-AMPK (see diagram). Taken together, this is strong evidence that FLCN is required to down-regulate mTORC1 in flow stress signals, the very pathway strongly implicated in FLCN tumor suppressor role (Tsun et al., 2013).
Next, using another series of intricate co-immunoprecipitation experiments, the authors demonstrated that FLCN recruits the protein LKB1 to activate AMPK (see diagram below) This is consistent with the findings of Goncharova et al. 2014, who report that FLCN controls AMPK activity via LKB1 for cell survival processes in the lung alveoli. Like previous experiments, this process was cilia dependent and only occurred in response to fluid stress. When FLCN was knocked down the interaction of LKB1 and AMPK was significantly reduced. These experiments were nicely complemented with imaging and FLCN knockdown experiments that demonstrated that FLCN was required for the localisation of both p-AMPK and LKB-1 to the basal bodies, the microtubule structure present at the base of the cilia.
In summary, Zhong et al., have identified a novel role for FLCN in the mechanosensing function of cilia when exposed to fluid stress. A reduction in FLCN levels interferes with the fluid stress–induced suppression of mTORC1 and causes prolonged activation of this pathway. Notably, hyperexcitation of mTOR pathways is well documented in cancer biology and this study is consistent with of a wealth of recent literature that implicates a role for cilia within cancer and tumorigenesis. Unfortunately, the paper did not look to rescue the effects of FLCN knockdown/knockout on fluid stress signaling, which would have given them a good opportunity to test the effects of FLCN mutations on the process. Nevertheless, the study opens some interesting follow-up opportunities, including understanding if the process of mechanosensing involved in BHD cyst formation and tumourigenesis .
Image from Zhong et al., 2016.