A study recently published in the Journal of Clinical Investigation draws together a number of earlier observations of how FLCN functions: that FLCN binds AMPK via the FNIPs, that loss of FLCN leads to HIF hyperactivity and metabolic transformation, and that loss of FLCN leads to increased PGC1a expression and consequent mitochondrial activity. This study was performed in collaboration by two labs – those of Professor Arnim Pause at McGill University in Canada, and Dr Andy Tee at Cardiff University in Wales – and was funded in part by the Myrovlytis Trust.
By comparing wildtype mouse embryonic fibroblasts (MEFs), Flcn-null MEFs, and FLCN-null MEFs in which FLCN had been reinstated, Yan et al. elucidate the mechanism through which loss of FLCN leads to metabolic transformation.
Yan et al. show that loss of FLCN leads to hyperactivity of AMPK, as also reported by Possik et al and described in last week’s blog. A non-phosphorylateable S62A FLCN mutant was unable to bind AMPK and also led to AMPK dysregulation. This shows that FLCN inhibits AMPK by directly binding to it, and preventing AMPK’s catalytic site from being activated by phosphorylation.
Increased AMPK activation lead to increased PGC1α expression and a consequent increase in the number of mitochondria per cell. Although the number of mitochondria was only increased 1.2-fold in this experiment, gradual accumulation of mitochondria over time following FLCN loss may explain why BHD patients are predisposed to developing oncocytic tumours (Lindor et al., 2012, Pradella et al., 2013, Raymond et al., 2013).
Increased numbers of mitochondria led to an increased amount of mitochondrial respiration and reactive oxygen species (ROS) production. This, in turn led to increased expression of HIF1a and its target genes, and subsequent metabolic transformation, particularly increased glycolysis and ATP production, despite cells being in normoxic conditions. This is a metabolic transformation known as the Warburg effect, which is common in many cancers and increases tumorigenic potential.
Immunohistochemical analysis of a BHD tumour showed increased expression of mitochondrial markers, increased HIF1a nuclear expression and increased expression of HIF targets. Therefore this pathway is likely to contribute to the development of kidney cancer in BHD patients.
Although not directly analysed in this study, a recent study from Professor Pause’s group showed that FLCN loss led to hyperactive AMPK signalling, which led to increased ATP production and stress resistance due to increased autophagy in FLCN-null MEFs. Whether activation of HIF signalling and autophagy to promote metabolic transformation and stress resistance are part of a single larger pathway, or two separate tumorigenic pathways activated by loss of FLCN is not currently known. However, the HIF1a target BNIP3 – whose expression is increased in the absence of FLCN (this study and Preston et al., 2011) – is known to stimulate autophagy (Bellot et al., 2009), as does ROS (Zhang et al., 2013), suggesting that the two processes might constitute a single tumorigenic signalling cascade.
Interestingly, FLCN has been shown to activate AMPK function to promote cell survival in Type II Alveolar Cells and to activate mTOR signalling at the lysosome, which functions antagonistically to AMPK signalling. Considered as a whole, it seems likely that FLCN function varies, not only in different cell types, but also within the same cell in response to environmental cues which modify its localisation and binding partners. This lends further weight to the hypothesis that FLCN may act as a molecular switch to control cellular metabolism in response to environmental changes.
- Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouysségur J, & Mazure NM (2009). Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Molecular and cellular biology, 29 (10), 2570-81 PMID: 19273585
- Lindor NM, Kasperbauer J, Lewis JE, & Pittelkow M (2012). Birt-Hogg-Dube syndrome presenting as multiple oncocytic parotid tumors. Hereditary cancer in clinical practice, 10 (1) PMID: 23050938
- Possik E, Jalali Z, Nouët Y, Yan M, Gingras MC, Schmeisser K, Panaite L, Dupuy F, Kharitidi D, Chotard L, Jones RG, Hall DH, & Pause A (2014). Folliculin regulates ampk-dependent autophagy and metabolic stress survival. PLoS genetics, 10 (4) PMID: 24763318
- Pradella LM, Lang M, Kurelac I, Mariani E, Guerra F, Zuntini R, Tallini G, MacKay A, Reis-Filho JS, Seri M, Turchetti D, & Gasparre G (2013). Where Birt-Hogg-Dubé meets Cowden syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours. European journal of human genetics : EJHG, 21 (10), 1169-72 PMID: 23386036
- Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, Harper KT, Brinkhuizen T, Menko FH, Davies DM, Land SC, Pause A, Baar K, van Steensel MA, & Tee AR (2011). Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene, 30 (10), 1159-73 PMID: 21057536
- Raymond VM, Long JM, Everett JN, Caoili EM, Gruber SB, Stoffel EM, Giordano TJ, Hammer GD, & Else T (2014). An oncocytic adrenal tumour in a patient with Birt-Hogg-Dubé syndrome. Clinical endocrinology, 80 (6), 925-7 PMID: 23848572
- Yan M, Gingras MC, Dunlop EA, Nouët Y, Dupuy F, Jalali Z, Possik E, Coull BJ, Kharitidi D, Dydensborg AB, Faubert B, Kamps M, Sabourin S, Preston RS, Davies DM, Roughead T, Chotard L, van Steensel MA, Jones R, Tee AR, & Pause A (2014). The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation. The Journal of clinical investigation PMID: 24762438
- Zhang J, Kim J, Alexander A, Cai S, Tripathi DN, Dere R, Tee AR, Tait-Mulder J, Di Nardo A, Han JM, Kwiatkowski E, Dunlop EA, Dodd KM, Folkerth RD, Faust PL, Kastan MB, Sahin M, & Walker CL (2013). A tuberous sclerosis complex signalling node at the peroxisome regulates mTORC1 and autophagy in response to ROS. Nature cell biology, 15 (10), 1186-96 PMID: 23955302
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.