Von Hippel-Lindau (VHL) syndrome is characterised by the development of tumours in multiple organ systems, including the kidney, and is caused by mutations in the VHL gene. As well as causing tumour development in the context of VHL syndrome, VHL mutations are a common and early event in sporadic cases of clear cell renal cell carcinoma (ccRCC), with somatic VHL mutations seen in 80% of ccRCCs (Gerlinger et al., 2012). A paper published this week in PLoS ONE by Bastola et al. reports that the VHL gene’s tumour suppressor function is mediated by the BHD gene, FLCN. This work was partially funded by the Myrovlytis Trust/ BHD Foundation.
As VHL mutation is a common cause of ccRCC, Bastola et al. reconstituted VHL expression in the VHL-null 786-O cell line (786-O VHL(+)), and used microarray mRNA expression analysis to screen for genes whose expression was affected by VHL. There was a significant enrichment in genes positively regulated by VHL on the Smith-Magenis Syndrome locus on chromosome 17; a region which includes the FLCN gene. RT-PCR confirmed this finding, showing that FLCN expression was increased 1.5-2 fold in both 786-O and A498 cells with reconstituted FLCN expression. The effect on FLCN protein expression was greater, with a 3-4 fold increase observed in VHL positive cells, suggesting that VHL may additionally regulate FLCN expression through a post-transcriptional mechanism. Indeed, this group has previously shown that VHL regulates miR-204 expression (Mikhaylova et al., 2012); thus VHL may regulate a miRNA that controls the translation of the FLCN protein. Furthermore, FLCN protein expression was found to be decreased in ccRCC tumours carrying VHL mutations, while no difference in FLCN expression was observed in ccRCC with wildtype VHL, indicating that a loss of FLCN function may be a causative factor in VHL-associated tumorigenesis.
To determine whether FLCN contributed to VHL’s tumour suppressor activity, the authors knocked down FLCN expression in 786-O VHL(+) cells using RNAi, and injected these cells into the kidney capsules of nude mice. The incidence and size of tumours was larger in FLCN knockdown cells compared with FLCN wildtype 786-O VHL(+) xenografts. Additionally, the FLCN-depleted resultant tumours had a highly malignant and dedifferentiated phenotype, indicating that FLCN loss contributes to tumorigenesis in these mice.
As FLCN is known to regulate mTOR signalling, Bastola et al. assessed mTORC1 signalling in FLCN knock down 768-O VHL(+) cells and in tumours from the xenograft experiments. Both in vitro and in vivo experiments showed that mTORC1 signalling was reduced in the FLCN knockdown cells, suggesting that dysregulated mTORC1 signalling was not responsible for the increased tumour growth seen in these cells. However, FLCN’s effect on mTOR signalling has been reported to be context dependent (Hudon et al., 2010), and the authors of this study did not rule out that in other contexts and cell types, dysregulated mTOR signalling may be responsible for FLCN-associated pathogenesis.
This group had previously shown that VHL loss leads to tumorigenesis through the induction of autophagy (Mikhaylova et al., 2012). Autophagy describes the process of recycling old proteins within the cell, thus allowing cells to survive in the absence of nutrients. Thus erroneous activation of autophagy may allow inappropriate cell survival, as has been observed in tuberous sclerosis complex cells (Parkhitko et al., 2011). Autophagy is activated by LC3B and inhibited by LC3C. Mikhaylova et al. found that VHL inhibits LC3B and activates LC3C by respectively activating miR204 expression and inhibiting HIF accumulation, overall inhibiting autophagy. FLCN has previously been shown to prevent HIF1 accumulation (Preston et al., 2010), so Batola et al. investigated whether FLCN regulates LC3B or LC3C activity: they observed an accumulation of LC3B and a decrease of LC3C protein, indicating that FLCN controls LC3B and LC3C – and therefore also autophagy induction – similarly to VHL.
Based on these findings, it would be of interest to determine if autophagy plays any role in the development of BHD symptoms, as this may identify novel therapeutic targets for BHD. Chloriquine – an autophagy inhibitor – is currently being tested in combination with Rapamycin as a therapy for the cystic lung disease LAM. Thus, if inappropriate autophagy induction is a causative factor of BHD pathogenesis, it is possible that chloroquine may prove a viable therapy.
FLCN’s effect of LC3B expression seems to be independent of miR-204 expression, meaning that whether FLCN’s control on LC3B and LC3C is downstream or independent of VHL tumour suppressor activity is unclear. Interestingly, the LC3 family of proteins are required for membrane formation of autophagosomes, and the acquisition of cargo. FLCN, and its interacting proteins FNIP1 and FNIP2, are predicted to play a role in membrane trafficking (Nookala et al., 2012; Zhang et al., 2012), suggesting that it may be through this pathway that it interacts with LC3B and LC3C, perhaps in concert with FNIP1 and/or FNIP2. However, given the strong correlation between VHL and FLCN expression, it seems likely that FLCN is at least partially responsible for VHL’s tumour suppressor activity.
This study represents an important milestone in BHD and FLCN research: it links FLCN function with that of VHL, which is a well-characterised tumour suppressor and a common cause of both sporadic and syndromic kidney cancers, thereby suggesting that FLCN is also an important factor in the development of kidney cancer, in addition to its role in BHD syndrome. We have previously written about the idea that rare diseases should be considered as fundamental diseases, and this study is proof of concept of how studying the causative genes of two rare syndromes – FLCN and VHL – can be relevant to, and hopefully inform the development of a therapy for, both rare and common diseases.
- Bastola P, Stratton Y, Kellner E, Mikhaylova O, Yi Y, Sartor MA, Medvedovic M, Biesiada J, Meller J, & Czyzyk-Krzeska MF (2013). Folliculin Contributes to VHL Tumor Suppressing Activity in Renal Cancer through Regulation of Autophagy. PloS one, 8 (7) PMID: 23922894
Gerlinger, M., Rowan, A., Horswell, S., Larkin, J., Endesfelder, D., Gronroos, E., Martinez, P., Matthews, N., Stewart, A., Tarpey, P., Varela, I., Phillimore, B., Begum, S., McDonald, N., Butler, A., Jones, D., Raine, K., Latimer, C., Santos, C.,
Nohadani, M., Eklund, A., Spencer-Dene, B., Clark, G., Pickering, L., Stamp, G., Gore, M., Szallasi, Z., Downward, J., Futreal, P., & Swanton, C. (2012). Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing New England Journal of Medicine, 366 (10), 883-892 PMID: 22397650
Hudon V, Sabourin S, Dydensborg AB, Kottis V, Ghazi A, Paquet M, Crosby K, Pomerleau V, Uetani N, Pause A (2010). Renal tumour suppressor function of the Birt-Hogg-Dubé syndrome gene product folliculin. Journal of Medical Genetics, 47 (3), 182-9 PMID: 19843504
Mikhaylova O, Stratton Y, Hall D, Kellner E, Ehmer B, Drew AF, Gallo CA, Plas DR, Biesiada J, Meller J, & Czyzyk-Krzeska MF (2012). VHL-regulated MiR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal clear cell carcinoma. Cancer cell, 21 (4), 532-46 PMID: 22516261
Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open biology, 2 (8), 120071 PMID: 22977732
Parkhitko A, Myachina F, Morrison TA, Hindi KM, Auricchio N, Karbowniczek M, Wu JJ, Finkel T, Kwiatkowski DJ, Yu JJ, & Henske EP (2011). Tumorigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)-dependent. Proceedings of the National Academy of Sciences of the United States of America, 108 (30), 12455-60 PMID: 21746920
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
Zhang D, Iyer LM, He F, & Aravind L (2012). Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease. Frontiers in genetics, 3 PMID: 23248642
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.