BHD case reports

Patient case studies are an important part of clinical investigations into any disease. Such studies can lead to the identification of associated symptoms, genotype-phenotype correlations and further understanding regarding the onset and progression of symptoms. The only symptoms currently associated with BHD Syndrome are fibrofolliculomaslung cysts, pneumothoraxrenal cysts and renal tumours. However, several other manifestations have been reported in BHD patients and these are listed here and also discussed in more detail here. The association between BHD Syndrome and these symptoms are unknown.

A recent case study presented by Lindor et al. (2012) identified parotid tumours (salivary gland tumours), and subsequently fibrofolliculomas, in a 45 year old woman who was found to have a FLCN mutation. Parotid tumours have been reported previously in BHD individuals, first by Liu et al. (2000), with other reports by Schmidt et al. (2005), Palmirotta et al. (2008) and Maffe et al. (2011). The parotid tumours identified by Lindor et al. are of note as they were found to be oncocytic, a characteristic commonly seen in BHD kidney tumours.

The link between BHD and colorectal cancer has been debated previously in the literature and it is summarised here. A recent paper by Kashiwada et al. (2012) adds further evidence supporting this possible link by identifying a 60 year old woman with BHD who previously had colon carcinoma caused by familial adenomatous polyposis (FAP). Interestingly the woman had both a FLCN mutation and a mutation in the adenomatous polyposis coli (APC) gene, which is known to cause FAP. The authors suggest that the APC mutation could be the ‘driver mutation’ for colon cancer, but that the addition of the FLCN mutation contributed to its development. Further study is needed to investigate this link.

Finally, two separate case reports published this year identified renal angiomyolipomas in BHD patients (Byrne et al., 2012; Tobino and Seyama, 2012). Renal angiomyolipomas are commonly found in patients with Tuberous Sclerosis Complex (TSC), a syndrome with clinical similarities to BHD. It is not surprising that renal angiomyolipomas are seen in both these disorders, as FLCN and TSC1/2 function in the same molecular pathway (as seen in this diagram).

It is important to remember that only the skin, lung and kidney symptoms are currently associated with BHD Syndrome. Further investigation is required to understand the connection, if any, between BHD and other clinical manifestations.

 

  • 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
  • Liu V, Kwan T, & Page EH (2000). Parotid oncocytoma in the Birt-Hogg-Dubé syndrome. Journal of the American Academy of Dermatology, 43 (6), 1120-2 PMID: 11100034
  • Schmidt LS, Nickerson ML, Warren MB, Glenn GM, Toro JR, Merino MJ, Turner ML, Choyke PL, Sharma N, Peterson J, Morrison P, Maher ER, Walther MM, Zbar B, & Linehan WM (2005). Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome. American journal of human genetics, 76 (6), 1023-33 PMID: 15852235
  • Palmirotta R, Donati P, Savonarola A, Cota C, Ferroni P, & Guadagni F (2008). Birt-Hogg-Dubé (BHD) syndrome: report of two novel germline mutations in the folliculin (FLCN) gene. European journal of dermatology : EJD, 18 (4), 382-6 PMID: 18573707
  • Maffé A, Toschi B, Circo G, Giachino D, Giglio S, Rizzo A, Carloni A, Poletti V, Tomassetti S, Ginardi C, Ungari S, & Genuardi M (2011). Constitutional FLCN mutations in patients with suspected Birt-Hogg-Dubé syndrome ascertained for non-cutaneous manifestations. Clinical genetics, 79 (4), 345-54 PMID: 20618353
  • Kashiwada T, Shimizu H, Tamura K, Seyama K, Horie Y, & Mizoo A (2012). Birt-Hogg-Dubé syndrome and familial adenomatous polyposis: an association or a coincidence? Internal medicine (Tokyo, Japan), 51 (13), 1789-92 PMID: 22790147
  • Byrne M, Mallipeddi R, Pichert G, & Whittaker S (2012). Birt-Hogg-Dubé syndrome with a renal angiomyolipoma: further evidence of a relationship between Birt-Hogg-Dubé syndrome and tuberous sclerosis complex. The Australasian journal of dermatology, 53 (2), 151-4 PMID: 22571569
  • Tobino K, & Seyama K (2012). Birt-Hogg-Dubé syndrome with renal angiomyolipoma. Internal medicine (Tokyo, Japan), 51 (10), 1279-80 PMID: 22687807

FLCN phosphorylation and the cell cycle

FLCN phosphorylation was first identified by Baba et al. (2006) when multiple FLCN bands were seen on Western blots. Subsequent studies identified and characterised Serine 62 (S62) and Serine 302 (S302) as phosphorylated residues (Wang et al., 2010; Piao et al., 2009). A third FLCN phosphorylation site, Serine 73 (S73) was identified in a large study by Dephoure et al. (2008). Interestingly, this study found that all three FLCN phosphorylation sites were regulated by the cell cycle.

The cell division cycle involves the replication and separation of cellular material into two daughter cells. The cycle is tightly regulated by a series of kinases, which affect downstream proteins through phosphorylation. During the mitosis phase of the cell cycle, cellular processes undergo several changes, such as the termination of transcription and translation, the condensation of chromosomes and the breakdown of the nuclear envelope. Phosphorylation is instrumental in regulating these processes.

Dephoure et al. used stable isotope labelling, phosphopeptide enrichment and mass spectrometry to identify proteins whose phosphorylation was regulated by the cell cycle. The experiments were performed in HeLa cells arrested in either the G1 or mitotic phase of the cell cycle. The authors identified 14,265 unique phosphorylation sites on 3,682 different proteins. Overall, phosphorylation was upregulated during mitosis – 5,845 sites were phosphorylated only during mitosis, compared to 2,848 sites only phosphorylated during G1 phase. The remaining sites were phosphorylated during both phases of the cell cycle.

Three phosphorylation sites were identified on FLCN: S62, S73 and S302. S62 and S73 were found to be phosphorylated only during mitosis, whereas S302 was phosphorylated only during G1 phase. These results suggest that aspects of FLCN’s function or cellular localisation change during the cell cycle. Indeed, Nahorski et al. (2012) observed FLCN to be localised at cell junctions during interphase (which includes the G1 phase), but at the midbody during cytokinesis (part of the mitotic phase) (discussed in this previous blog post). Perhaps phosphorylation regulates this change in cellular localisation. Phosphorylation of FNIP1 and FNIP2 were not identified in this study, suggesting that these proteins are not phosphorylated during mitosis or the G1 phase.

The authors identified phosphorylation motifs for several kinases and the motif for Cyclin-dependent kinases, [pS/pT]-P, was found in FLCN at sites S62 and S73. However, previous studies have suggested that FLCN is phosphorylated by the AMPK and mTOR signalling pathways, either directly by these kinases or via downstream targets (Baba et al., 2006; Piao et al., 2009; Wang et al., 2010).

The effect of phosphorylation on FLCN’s function is unknown. Phosphorylation can affect many aspects of a protein, including its cellular localisation, conformation, stability, activity and its interactions with other molecules. It would be interesting to look at the phosphorylation status of FLCN during different cellular processes and when bound to an interacting partner, to decipher the function of this post-translational modification.

 

  • Dephoure N, Zhou C, Villén J, Beausoleil SA, Bakalarski CE, Elledge SJ, & Gygi SP (2008). A quantitative atlas of mitotic phosphorylation. Proceedings of the National Academy of Sciences of the United States of America, 105 (31), 10762-7 PMID: 18669648
  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
  • Wang L, Kobayashi T, Piao X, Shiono M, Takagi Y, Mineki R, Taka H, Zhang D, Abe M, Sun G, Hagiwara Y, Okimoto K, Matsumoto I, Kouchi M, & Hino O (2010). Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product. FEBS letters, 584 (1), 39-43 PMID: 19914239
  • Piao X, Kobayashi T, Wang L, Shiono M, Takagi Y, Sun G, Abe M, Hagiwara Y, Zhang D, Okimoto K, Kouchi M, Matsumoto I, & Hino O (2009). Regulation of folliculin (the BHD gene product) phosphorylation by Tsc2-mTOR pathway. Biochemical and biophysical research communications, 389 (1), 16-21 PMID: 19695222
  • Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics PMID: 22965878

Plakophilin-4 is a novel FLCN interacting protein

The identification of the FLCN interacting proteins FNIP1 and FNIP2 led to the discovery that FLCN functions in the AMPK signalling pathway (Baba et al., 2006; Hasumi et al., 2008; Takagi et al., 2008). Nahorski et al. (2012) have now identified Plakophilin-4 (PKP4, also known as p0071) as a novel FLCN interactor, and this has implicated FLCN in the regulation of RhoA signalling.

The FLCN-PKP4 interaction was identified by yeast-2-hybrid analysis and confirmed in vitro using co-immunoprecipitation studies in both Hek293 and ACHN cells. PKP4 is a member of the armadillo superfamily of proteins, which interact with cadherins and have a role in cell-cell contacts. PKP4 is also known to bind the desmosomal protein desmocollin 3a, plakoglobin and desmoplakin, and it has been linked to the regulation of RhoA signalling, cytokinesis and intercellular junction formation. Through its interaction with PKP4, Nahorski et al. suggest that FLCN may also regulate these processes and proceeded to investigate the function of the FLCN-PKP4 complex.

Immunofluorescence microscopic analysis, using MCF-7 cells, and bimolecular fluorescence complementation (BiFC) analysis, using HeLa cells, was used to investigate the intracellular localisation of FLCN and PKP4. As the localisation of PKP4 changes during mitosis, different stages of the cell cycle were studied. During interphase, FLCN and PKP4 were shown to co-localise most strongly at cell junctions, with a more dispersed co-localisation throughout the cytoplasm. The authors note that the FLCN signal at cell junctions is more consistent with a transient role, rather than that of a structural component. During cytokinesis, FLCN and PKP4 co-localise at the midbody.

PKP4 regulates RhoA signalling through a direct interaction with RhoA and Ect2, a RhoA GEF. Ect2 has been associated with renal cell carcinoma, as described in this previous blog post. To investigate FLCN’s effect on RhoA signalling, paired isogenic cell lines were used which demonstrated that FLCN deficiency is associated with increased expression of RhoA. Moreover, FLCN deficiency also resulted in increased activity of RhoA, as measured by the levels of GTP-bound RhoA present in the cells.

PKP4 loss is known to cause cytokinesis defects, multinucleation and a dysregulation of RhoA signalling. The authors demonstrate that FLCN loss also leads to an increase in multinucleated cells, suggesting that FLCN is required for cytokinesis. Furthermore, a wound healing assay showed that FLCN deficient cells migrated faster than cells which contained FLCN. Interestingly, inhibiting the downstream RhoA signalling pathway, using the ROCK inhibitor Y-27632, ameliorated this migratory phenotype. Finally, the effect of FLCN on cell junction formation was investigated and it was seen that knockdown of FLCN delayed tight junction formation and reduced the amount of Claudin-1 and E-Cadherin, components of tight junctions and adherin junctions respectively.

The exact function of the FLCN-PKP4 complex remains unknown, however this study has suggested a new role for FLCN in cytokinesis, cell junction formation and the regulation of RhoA signalling. RhoA is often overexpressed in cancers and it has been linked to metastasis. Perhaps the inhibition of this signalling pathway could be a potential therapy to treat metastatic renal tumours associated with BHD syndrome.

 

  • Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (Plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics PMID: 22965878
  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
  • Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, & Schmidt LS (2008). Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene, 415 (1-2), 60-7 PMID: 18403135
  • Takagi Y, Kobayashi T, Shiono M, Wang L, Piao X, Sun G, Zhang D, Abe M, Hagiwara Y, Takahashi K, & Hino O (2008). Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein. Oncogene, 27 (40), 5339-47 PMID: 18663353

Video interview: Dr Laura Schmidt – National Cancer Institute, NIH, USA

This week we would like to introduce you to the work of Dr Laura Schmidt, a staff scientist in the Urologic Oncology Branch at the National Institutes of Health in Bethesda, USA.

Dr Schmidt was instrumental in the identification of the inherited kidney cancer genes FLCN (Nickerson et al., 2002) and MET (Schmidt et al., 1997), of which mutations cause BHD Syndrome and hereditary papillary renal cell carcinoma (HPRCC) respectively. Dr Schmidt was also involved in the identification of mutations in the VHL gene (Gnarra et al., 1994), which causes von Hippel-Lindau disease, and the FH gene (Toro et al., 2003), which leads to the development of hereditary leiomyomatosis and renal cell carcinoma (HLRCC).

Dr Schmidt’s work on BHD Syndrome focuses on elucidating the function of FLCN through several lines of investigation. One of these involved the identification of the Folliculin-interacting partners, FNIP1 and FNIP2 (Baba et al., 2006; Hasumi et al., 2008), which were also found to bind AMPK. It is through this association that it is now known that FLCN plays a role in cellular energy homeostasis.

Other methods used by Dr Schmidt to determine the function of FLCN include studying the phenotype of animal models that have knockout of FLCN, as well as genotype-phenotype correlation studies in patient groups. The latter is highlighted in a recent publication where large intragenic deletions and duplications were found in the FLCN gene (Benhammou et al., 2011). Although no genotype-phenotype correlation has been identified to date, this type of study could one day help to understand the different symptoms of BHD and their differential onset.

To find out more about Dr Schmidt and her work at the NIH, watch our video interview (with its accompanying transcript and audio-only files). Video interviews are also available for other members of the Urologic Oncology Branch, including Dr W. Marston Linehan, Dr Masaya Baba and Lindsay Middelton.

 

  • Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML, Duray P, Merino M, Choyke P, Pavlovich CP, Sharma N, Walther M, Munroe D, Hill R, Maher E, Greenberg C, Lerman MI, Linehan WM, Zbar B, & Schmidt LS (2002). Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome. Cancer cell, 2 (2), 157-64 PMID: 12204536
  • Schmidt L, Duh FM, Chen F, Kishida T, Glenn G, Choyke P, Scherer SW, Zhuang Z, Lubensky I, Dean M, Allikmets R, Chidambaram A, Bergerheim UR, Feltis JT, Casadevall C, Zamarron A, Bernues M, Richard S, Lips CJ, Walther MM, Tsui LC, Geil L, Orcutt ML, Stackhouse T, Lipan J, Slife L, Brauch H, Decker J, Niehans G, Hughson MD, Moch H, Storkel S, Lerman MI, Linehan WM, & Zbar B (1997). Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nature genetics, 16 (1), 68-73 PMID: 9140397
  • Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H, Latif F, Liu S, Chen F, & Duh FM (1994). Mutations of the VHL tumour suppressor gene in renal carcinoma. Nature genetics, 7 (1), 85-90 PMID: 7915601
  • Toro JR, Nickerson ML, Wei MH, Warren MB, Glenn GM, Turner ML, Stewart L, Duray P, Tourre O, Sharma N, Choyke P, Stratton P, Merino M, Walther MM, Linehan WM, Schmidt LS, & Zbar B (2003). Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America. American journal of human genetics, 73 (1), 95-106 PMID: 12772087
  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
  • Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, & Schmidt LS (2008). Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene, 415 (1-2), 60-7 PMID: 18403135
  • Benhammou JN, Vocke CD, Santani A, Schmidt LS, Baba M, Seyama K, Wu X, Korolevich S, Nathanson KL, Stolle CA, & Linehan WM (2011). Identification of intragenic deletions and duplication in the FLCN gene in Birt-Hogg-Dubé syndrome. Genes, chromosomes & cancer, 50 (6), 466-77 PMID: 21412933

Rabs, GEFs and DENNs

Last week’s blog described the crystal structure of the C-terminal domain of FLCN, which was published in a recent paper by Nookala et al. (2012). FLCN was found to have a similar structure to that of DENN domain proteins, which function as Rab guanine nucleotide exchange factors (GEFs). As such, Nookala et al. proposed a novel function for FLCN as a Rab GEF. Here, we introduce Rab GTPases, GEFs and DENN domains and discuss the future research which may be used to confirm FLCN’s function.

Rabs are the largest family of small GTPases, with around 70 identified in humans. GTPases cycle between an active GTP-bound state and an inactive GDP-bound state, regulating many processes within the cell. Rab GTPases specifically regulate membrane trafficking. Molecules move around the cell within vesicles, which bud from one membrane before fusing with an organelle or membrane where the cargo is released. Rabs ensure that the vesicle, and hence its cargo, is delivered to the correct destination.

Rabs are activated by GEFs which facilitate the release of GDP, allowing GTP, which is present at much higher levels in the cell than GDP, to bind to the Rab. Several DENN GEFs have been shown to facilitate this exchange through a direct interaction with their associated Rab. GEFs are localised to specific areas in the cell, ensuring that Rabs are only activated at the correct location. GEFs can be classified by their distinct protein domains; however GEFs for most Rabs are yet to be identified.

DENN domain proteins are a class of Rab GEFs. Differentially expressed in normal cells and neoplasia (DENN) domain proteins were first identified due to their variable mRNA expression levels in tissues and cell lines (Chow and Lee, 1996). Prior to the identification of FLCN as a DENN protein, 18 other human DENN proteins had been identified. The DENN domain is poorly characterised but conserved throughout evolution. It consists of three regions: the upstream (u-DENN), core (c-DENN) and downstream (d-DENN) regions, which are separated by linkers of various lengths.

The first structure of a DENN domain was determined by Wu et al. (2011), who described the structure of DENN1B-S bound to Rab35. The c-DENN and d-DENN regions of DENN1B-S overlay almost exactly with the C-terminal domain of FLCN. The u-DENN region of DENN1B-S was found to contain a longin domain, a domain which is found in several proteins involved in membrane trafficking. Structure prediction programs suggest that the N-terminal of FLCN also contains a longin domain (Nookala et al., 2012), but it would be interesting to study this further as it would give support to FLCN’s proposed role in membrane trafficking.

The dysregulation of DENN proteins has been associated with diseases such as asthma, Alzheimer’s and several cancers. Additionally, DENND2B functions as a tumour suppressor. It is possible that mutations in FLCN cause a dysregulation of membrane trafficking which leads to the symptoms of BHD syndrome. It would be interesting to study vesicle transport in FLCN-null cells, as well as other processes that are regulated by small GTPases.

For more information and references on Rabs and DENN domain proteins, please see the reviews by Yoshimura et al. (2010) and Marat et al. (2011).

 

  • Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montano 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 Biol DOI: 10.1098/rsob.120071
  • Wu X, Bradley MJ, Cai Y, Kümmel D, De La Cruz EM, Barr FA, & Reinisch KM (2011). Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. Proceedings of the National Academy of Sciences of the United States of America, 108 (46), 18672-7 PMID: 22065758
  • Chow VT, & Lee SS (1996). DENN, a novel human gene differentially expressed in normal and neoplastic cells. DNA sequence : the journal of DNA sequencing and mapping, 6 (5), 263-73 PMID: 8988362
  • Marat AL, Dokainish H, & McPherson PS (2011). DENN domain proteins: regulators of Rab GTPases. The Journal of biological chemistry, 286 (16), 13791-800 PMID: 21330364
  • Yoshimura S, Gerondopoulos A, Linford A, Rigden DJ, & Barr FA (2010). Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors. The Journal of cell biology, 191 (2), 367-81 PMID: 20937701

The crystal structure of FLCN suggests a novel function as a Rab GEF

BHD Syndrome was first described in 1977 and although the FLCN gene was discovered in 2002 by Nickerson et al., the function of the protein has remained unknown. The majority of FLCN mutations found in BHD patients result in truncation of the protein and a loss of the C-terminal domain, suggesting an important role for this region. An interesting paper by Nookala et al. (2012) now presents the crystal structure of the C-terminal domain of FLCN, which gives a novel insight into the function of the protein.

Using X-ray crystallography, Nookala et al. determined the structure of the C-terminal domain of FLCN (residues 341-579) to 2 Å (PDB ID: 3V42). The domain is composed of a core β-sheet with helices packed on one side, followed by an all helical region. The authors noted that this fold was remarkably similar to that of DENN1B-S, with both proteins having the same strand order and orientation. In fact, overlaying the structures gave an r.m.s.d. of 2.8 Å over 170 residues, despite there being only an 11% sequence identity.

Differentially expressed in normal cells and neoplasia (DENN) proteins are Rab guanine nucleotide exchange factors (GEFs), which activate Rab GTPases by promoting GDP-GTP exchange. More information on Rabs and DENN domain proteins will be provided over subsequent blog posts. The crystal structure of DENN1B-S was recently solved, bound to its cognate Rab GTPase (Wu et al., 2011).

Since FLCN appears to possess a DENN-like fold, the author next tested whether FLCN has GEF activity towards small GTPases. Using biochemical analysis, the C-terminal domain of FLCN was shown to have GEF activity, in vitro, towards Rab35. This activity was also confirmed with the full length FLCN protein. Rab35 is involved in early endocytic trafficking, recycling events and cytokinesis, and these results now suggest that FLCN may also function in these pathways. Nookala et al. add, however, that FLCN GEF activity towards other small GTPases cannot yet be excluded.

The DENN proteins have three discrete domains, namely the upstream (u-DENN), core (c-DENN) and downstream (d-DENN) regions. The FLCN C-terminal domain maps to the c-DENN and d-DENN regions and, using secondary structure prediction programmes, the FLCN N-terminal domain appears to contain a fold similar to that of the u-DENN domain. Interestingly, FLCN also contains a Zinc-finger domain at the N-terminal of the protein, which is not found in other DENN proteins.

As evidenced from this paper, knowing the structure of a protein can go a long way to helping determine its function. Nookala et al. have proposed a novel function for FLCN as a Rab GEF and this will now open up many new avenues of research. It would be interesting to study endocytic transport in BHD patients, as a dysregulation of this pathway, caused by loss of the FLCN GEF activity, may contribute to the symptoms of BHD. Knowing the structure of FLCN will also assist in the development of drugs and therapies to treat BHD. This new discovery is an exciting step forward and it will no doubt play a big role in shaping the future direction of BHD research.

 

  • Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montano 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 Biol DOI: 10.1098/rsob.120071
  • Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML, Duray P, Merino M, Choyke P, Pavlovich CP, Sharma N, Walther M, Munroe D, Hill R, Maher E, Greenberg C, Lerman MI, Linehan WM, Zbar B, & Schmidt LS (2002). Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome. Cancer cell, 2 (2), 157-64 PMID: 12204536
  • Wu X, Bradley MJ, Cai Y, Kümmel D, De La Cruz EM, Barr FA, & Reinisch KM (2011). Insights regarding guanine nucleotide exchange from the structure of a DENN-domain protein complexed with its Rab GTPase substrate. Proceedings of the National Academy of Sciences of the United States of America, 108 (46), 18672-7 PMID: 22065758

FH-deficiency leads to increased AMPK activity and protection from apoptosis

Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) is a kidney cancer syndrome caused by mutation of Fumarate Hydratase (FH), a TCA cycle enzyme which catalyses the conversion of fumarate to malate. The accumulation of fumarate in FH-deficient cells results in the stabilisation of HIFα subunits, but it is unknown if this drives tumourigenesis.

A recent paper by Bardella et al. (2012) has found that FH-deficient cells are protected from apoptosis. When FH was knocked down by shRNA in human renal cells (both RPTEC and HK-2 cells), the amount of apoptosis was reduced when treated with stimuli inducing cell death (compared to FH-positive cells). The authors confirmed that HIF-1α and HIF-2α accumulate in FH-null cells, but by knockdown of both HIF subunits, it was found that the protection from apoptosis was independent of HIF-1α and HIF-2α.

The authors next considered whether kinases were involved in the protection from apoptosis. ERK1/2, MEK1 and AMPK were all more active in FH-deficient cells than FH-positive cells. When FH-deficient cells were treated with apoptotic stimuli, only AMPK was further activated. This activation of AMPK was shown to be HIF independent. Functional and knock down assays confirmed that AMPK activation protects cells from apoptosis. The increase in AMPK activation was found to be due to the accumulation of fumarate, with the authors suggesting that the increased fumarate could activate G-protein coupled receptors, which may lead to AMPK activation.

The mechanism by which AMPK activation leads to reduced apoptosis was investigated. The BCL2 family of apoptotic proteins was studied and it was seen that the expression and phosphorylation of these proteins were affected by FH suppression. In particular, the pro-apoptotic protein BAD was more phosphorylated in FH-deficient cells. Treatment with an AMPK activator confirmed that AMPK was directly responsible for the increase in phosphorylation, and therefore inhibition, of BAD.

Perhaps avoiding apoptosis could contribute directly to tumourigenesis in FH-null cells. The results in this paper suggest an oncogenic role for AMPK, which is independent of HIF. This correlates with results from Adam et al. (2011) who found that Fh1-associated cyst formation in mice is Hif independent (see this previous blog). The results in this recent paper do, however, differ from those presented by Tong et al. (2011, discussed here), who found that AMPK levels were reduced in FH-deficient cells. Bardella et al. suggest that more stringent conditions used in the Tong paper might lead to the selection of more aggressive cancer cells, which likely have AMPK suppression.

A connection between FLCN and apoptosis has been proposed in papers by Cash et al. (2011, discussed here) and Lim et al. (2012, discussed here). It is also known that FLCN has a role to play in the activation of AMPK during MNU-induced apoptosis (Lim et al., 2012). Understanding more about how AMPK can promote or inhibit tumourigenesis may further help in unravelling BHD syndrome and perhaps in the development of therapies for both BHD and HLRCC.

 

  • Bardella C, Olivero M, Lorenzato A, Geuna M, Adam J, O’Flaherty L, Rustin P, Tomlinson I, Pollard PJ, & Di Renzo MF (2012). Cells Lacking the Fumarase Tumor Suppressor Are Protected from Apoptosis through a Hypoxia-Inducible Factor-Independent, AMPK-Dependent Mechanism. Molecular and cellular biology, 32 (15), 3081-94 PMID: 22645311
  • Adam J, Hatipoglu E, O’Flaherty L, Ternette N, Sahgal N, Lockstone H, Baban D, Nye E, Stamp GW, Wolhuter K, Stevens M, Fischer R, Carmeliet P, Maxwell PH, Pugh CW, Frizzell N, Soga T, Kessler BM, El-Bahrawy M, Ratcliffe PJ, & Pollard PJ (2011). Renal cyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway: roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer cell, 20 (4), 524-37 PMID: 22014577
  • Tong WH, Sourbier C, Kovtunovych G, Jeong SY, Vira M, Ghosh M, Romero VV, Sougrat R, Vaulont S, Viollet B, Kim YS, Lee S, Trepel J, Srinivasan R, Bratslavsky G, Yang Y, Linehan WM, & Rouault TA (2011). The glycolytic shift in fumarate-hydratase-deficient kidney cancer lowers AMPK levels, increases anabolic propensities and lowers cellular iron levels. Cancer cell, 20 (3), 315-27 PMID: 21907923
  • Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46 PMID: 21258407
  • Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2012). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair, 11 (3), 259-66 PMID: 22209521

Fourth BHD Symposium Abstracts

The abstracts from the Fourth BHD Symposium have now been published online in the journal Familial Cancer. The Symposium was held in March 2012 in Cincinnati, USA, and hosted over 90 BHD researchers, clinicians and patients. The latest findings and on-going research in the field of BHD were presented over the two days. The abstracts are a great overview of the Symposium, so do take a look, especially if you were unable to attend the meeting.

Basic research highlights include a new FLCN-interacting protein, independently identified by both Dr Douglas Medvetz at Brigham and Women’s Hospital and Michael Nahorski at the University of Birmingham, UK. Sander Basten, a PhD student in the lab of Professor Rachel Giles at the University Medical Center Utrecht, also presented the latest data on the PTC124 drug, which was described in last week’s blog.

There were also several clinical presentations, including an account of the long-term surveillance of 73 FLCN-mutation carriers given by Dr Paul Johannesma from the VU Medical Center in Amsterdam. Dr Shunsuke Koga, from Chiba University, Japan, described the lung phenotypes seen in 11 BHD families – work which has been discussed in a previous blog post.

Further highlights of the researchers sessions and patient day can be found in previous blog posts (here and here respectively). The Fifth BHD Symposium will be held in Europe in spring 2013 – watch out for further details on BHDSyndrome.org.

 

  • The Fourth BHD Symposium Abstracts (2012). The Fourth Birt-Hogg-Dubé Symposium, Cincinnati, USA, 28(th)-30 (th) March, 2012. Familial cancer PMID: 22752245

FNIP1 deletion leads to increased apoptosis in pre-B cells

The work of Dr Masaya Baba, at the National Cancer Institute, was highlighted in last week’s blog. Dr Baba was part of the team who identified Folliculin-interacting protein 1 (FNIP1) and a recent paper by this group now suggests a function for the protein (Baba et al., 2012). This new paper ties in well with the study by Park et al. (2012) which was discussed in a previous blog post. Both papers independently identified a similar function for FNIP1 in B cell development.

Baba and authors generated Fnip1 knockout mice and upon inspection of the mice, found them to have a reduced spleen size as compared to heterozygous or wild type controls. After further investigation, the thymocyte numbers and peripheral T cells were found to be normal, but there was seen to be a reduced number of pre-B cells and an accumulation of pro-B cells. These results suggest there is a developmental block at the pro-B cell to pre-B cell stage.

The FLCN-FNIP1 complex is involved in mTOR signalling (Baba et al., 2006) and it was considered whether defects in this pathway were causing the B cell developmental arrest. Unlike Flcn-/- mice, Fnip1-/- mice had no cysts in their kidneys, suggesting the mTOR pathway was unaffected. The phosphorylation of mTOR and its downstream target, ribosomal protein S6, were unchanged between wild type and mutant mice, further suggesting that mTOR signalling is not affected by Fnip1 deletion. Finally, treatment with rapamycin did not bypass the B cell developmental arrest, confirming that the block in B cell development is independent of mTOR signalling.

Subsequent experiments investigated whether the lack of pre-B cells resulted from increased cell death. Fnip1-/- pre-B cells had enhanced caspase activity relative to wild type cells, and also an increase in the number of dead cells as seen by DAPI staining. This suggests that Fnip1 is required for B cell survival and that deletion of Fnip1 leads to apoptosis of precursor B cells. This was confirmed by introducing an anti-apoptotic Bcl2 transgene into Fnip1-/- cells, which bypassed the B cell block and rescued the B cell population.

This study also investigated the effect of Flcn knockout on B cell development. Interestingly, Flcn-/- mice had the same B cell phenotype as the Fnip1-/- mice, suggesting that both Flcn and Fnip1 are required for B cell development. BHD syndrome and FLCN mutations have not previously been associated with B cell defects; however patients with BHD have monoallelic FLCN mutations and so perhaps loss of the second FLCN allele in B cells is required for the developmental defect (as is thought to be the case for the development of RCC in the kidney).

Although the studies by Park et al. and Baba et al. both show B cell developmental defects caused by Fnip1 deletion, the proposed reason for these defects differs. Park et al. suggest Fnip1 deletion causes energy stress which leads to B cell developmental arrest, whereas Baba et al. suggest Fnip1 deletion causes increased apoptosis of pre-B cells. Given that FLCN and FNIP2 have been implicated in MNU-induced apoptosis (Lim et al., 2012 – discussed in this blog post), it would be interesting to investigate this link with apoptosis further. Future work investigating if FNIP2 also has a role in B cell development will be interesting and will shed more light on the FLCN-FNIP complex.

 

  • Baba M, Keller JR, Sun HW, Resch W, Kuchen S, Suh HC, Hasumi H, Hasumi Y, Kieffer-Kwon KR, Gonzalez CG, Hughes RM, Klein ME, Oh HF, Bible P, Southon E, Tessarollo L, Schmidt LS, Linehan WM, & Casellas R (2012). The Folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dube syndrome is required for mouse B cell development. Blood PMID: 22709692
  • Park H, Staehling K, Tsang M, Appleby MW, Brunkow ME, Margineantu D, Hockenbery DM, Habib T, Liggitt HD, Carlson G, & Iritani BM (2012). Disruption of fnip1 reveals a metabolic checkpoint controlling B lymphocyte development. Immunity, 36 (5), 769-81 PMID: 22608497
  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
  • Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2012). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair, 11 (3), 259-66 PMID: 22209521

A role for FNIP1 in B cell development

Folliculin-interacting protein 1 (FNIP1) was identified in 2006 by Baba et al. as a protein which interacts with the C-terminus of FLCN. Although the function of FNIP1 is unknown, the protein was also found to interact with and be phosphorylated by AMPK, an enzyme involved in cellular energy homeostasis. A recent paper by Park et al. (2012) has now identified a role for FNIP1 in B lymphocyte development.

The authors used an ethylnitrosourea (ENU) mutagenesis screen to identify genes involved in immune cell development. One pedigree of mice, which had an absence of B lymphocytes, was found to have a deletion in the Fnip1 gene. The Fnip1-/- mice had several additional phenotypes, including alterations in skeletal muscle, increased liver glycogen content and hypertrophic cardiomyopathy. In wild-type mice, Fnip1 was found to be highly expressed in many tissues and at an equal level throughout B-cell development.

Loss of Fnip1 appeared to halt B-cell development at the pre-B cell stage. This was investigated further and the authors found the block to occur at the transition from large pre-B cell to small pre-B cell. Fnip1-/- pre-B cells showed no defects in cell division; therefore only maturation of the B cell was inhibited. B cell development was rescued by retroviral expression of Fnip1, confirming Fnip1 is directly responsible for the observed phenotype.

To identify the cause of this inhibition of B cell development, gene expression levels were compared between Fnip1-/- and wild-type mice using cDNA microarray technology. Compared to the wild-type, over 500 genes were differentially expressed in Fnip1-/- mice. The majority of these genes were involved in cell metabolism and mitochondrial biogenesis, and in particular, there was an increase in the expression of Pgc1α and Pgc1β (which are regulators of fatty acid oxidation). These mice also had increased numbers of mitochondria and an increase in phospho-S6 ribosomal protein (S6R), a downstream target of mTORC1, suggesting an increase in mTOR-mediated metabolism.

In addition, the effect of Fnip1 loss on AMPK signalling in pre-B cells was investigated and it was seen that the phosphorylation of the mTORC1 component Raptor by AMPK was unaffected. However, where AMPK activation in wild-type pre-B cells inhibited the phosphorylation of S6R, suggesting a decrease in mTOR function, in Fnip1-/- pre-B cells, activation of AMPK failed to inhibit this phosphorylation. These results suggest that Fnip1 is not required for the activation of AMPK, but it is important for AMPK to inhibit mTOR.

The authors suggest that Fnip1 loss results in a nutrient and energy deficit and that the role of Fnip1 in B cells could be to help maintain metabolic homeostasis. When Fnip1 is lost, homeostasis is dysregulated and B-cell development is arrested in response to nutrient stress. It would be interesting to investigate the role of FNIP1 in BHD Syndrome and the function of its interaction with FLCN. Does FNIP1 also have a role to play in the regulation of metabolism in BHD Syndrome? It is known that metabolism and mitochondrial biogenesis are dysregulated in BHD (Preston et al., 2011; Klomp et al., 2010). Perhaps dysregulation of FNIP1, through loss of FLCN, could contribute to these cellular effects and the observed symptoms of BHD.

 

  • Park H, Staehling K, Tsang M, Appleby MW, Brunkow ME, Margineantu D, Hockenbery DM, Habib T, Liggitt HD, Carlson G, & Iritani BM (2012). Disruption of fnip1 reveals a metabolic checkpoint controlling B lymphocyte development. Immunity, 36 (5), 769-81 PMID: 22608497
  • Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF 3rd, Hartley JL, Furihata M, Oishi S, Zhen W, Burke TR Jr, Linehan WM, Schmidt LS, & Zbar B (2006). Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America, 103 (42), 15552-7 PMID: 17028174
  • Klomp JA, Petillo D, Niemi NM, Dykema KJ, Chen J, Yang XJ, Sääf A, Zickert P, Aly M, Bergerheim U, Nordenskjöld M, Gad S, Giraud S, Denoux Y, Yonneau L, Méjean A, Vasiliu V, Richard S, MacKeigan JP, Teh BT, & Furge KA (2010). Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression. BMC medical genomics, 3 PMID: 21162720
  • 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