Loss of FLCN leads to constitutive AMPK activation, increased autophagy and stress resistance in C. elegans

As discussed on this blog previously, dysregulated autophagy seems to be a common feature of many hereditary kidney cancers. A study from Professor Arnim Pause’s laboratory at McGill University in Canada has shown that the loss of FLCN leads to constitutive activation of AMPK, which stimulates autophagy and stress resistance in both C. elegans and mammalian cells.

Using a C. elegans model of BHD, Possik et al. (2014) show that worms lacking a functional Flcn allele were more resistant to oxidative stress. Deletion of aak2 or par4, the C. elegans homologues of AMPK and its activator LKB1 respectively, reversed this phenotype. This indicates that the deletion of flcn leads to hyperactivity of AMPK, and is responsible for increased stress tolerance in these animals.

Electron microscopy, fluorescence microscopy and western blotting revealed elevated autophagic flux in flcn-null animals, leading to an increase in intracellular ATP compared with wild-type cells. This increased energy store protected flcn-null cells from apoptotic cell death, allowing flcn-null worms to survive oxidative stresses better than their wild-type counterparts.

Similar results were observed in FLCN-null mouse embryonic fibroblasts (MEFs), indicating that this pathway is evolutionarily conserved and that FLCN inhibits AMPK – thus inhibiting autophagy and stimulating apoptosis – in mammals. Furthermore, inhibiting autophagy using siRNAs directed to the ATG7 gene in FTC-133 tumour cells, which lack FLCN, led to a reduction of colonies forming in a soft agar assay, indicating that increased autophagy increases the tumorigenic potential of FLCN-null cells.

A C. elegans BHD model published last year showed increased longevity and stress resistance, which was dependent on hif-signalling. Possik et al. only observed increased longevity in their flcn-null worms when high concentrations of the drug FUDR – which is commonly used in C. elegans ageing studies to prevent eggs hatching – was added to growth medium. They therefore attribute this difference to the different culture conditions used in the two studies.

Possik et al.’s data correspond well with several studies that have been published in the last year, indicating that FLCN inhibits autophagy through multiple mechanisms: DBHD-null Drosophila show increased levels of autophagy; FLCN inhibits the activity of the pro-autophagy transcription factors TFE3 and TFEB, via FLCN’s interaction with mTOR; FLCN activity reduces the ratio of LC3B protein to LC3C, thus inhibiting autophagy; and increased autophagy is observed in FLCN-null cells and led to increased apoptosis  following chemotherapy or radiotherapy.

Goncharova et al. reported that the AMPK activator AICAR reversed some of lung symptoms in a mouse model of BHD, and suggested that AICAR might prove to be a good preventative drug to inhibit the development of lung cysts. Possik et al. show that AMPK was maximally activated in FLCN-null MEFs, meaning that AICAR treatment did not exacerbate the pathology of these cells. However, if in BHD patients AMPK is over, but not fully, activated, it is possible that AICAR could actually speed the development of kidney tumours by further activating AMPK, thus highlighting the potential difficulty of developing a systemic treatment for BHD.

 

  • Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, Li H, Gonzales L, Baba M, Linehan WM, Gow AJ, Margulies S, Guttentag S, Schmidt LS, & Krymskaya VP (2014). Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK. Cell reports, 7 (2), 412-23 PMID: 24726356
  • 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 Genet, 10 (4):e1004273. PMID: 24763318

www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.

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