The Third Crick Symposium and Talks about TORCs

Last month, the Third Crick Symposium was held in London (UK), with the aim of discussing ways in which basic biological research could progress from “Genetics to molecules to therapies”. In particular, this meeting brought together chemists, biologists and clinicians from what will be the new Francis Crick Institute in London, which is scheduled to open in 2015.

Of note, Professor Charles Swanton (CRUK London Research Institute and UCL Cancer Institute, UK) described four cases of intra-tumour heterogeneity in metastatic renal cell carcinoma (RCC). These findings demonstrated that certain mutations were ubiquitous, shared or unique within specific regions of the metastatic RCC (Gerlinger et al., 2012). For example, using exome sequencing, chromosome aberration analysis and DNA ploidy profiling it was noted that VHL was mutated in all analysed tumour regions within one patient. An activating mutation in mammalian target of rapamycin (mTOR) was also observed in all but one primary tumour region. However, there were distinct SETD2 and KDM5C mutations present within the primary and metastatic regions of the RCC, and these genes have been previously discussed here. This study by Gerlinger et al. highlights that a single biopsy may not represent the mutational load within a tumour, and that certain tumour cell populations may react differently to treatments. Accordingly, therapies which target ubiquitous mutations may prove to be more successful. It would be particularly interesting to see if there is similar intra-tumour heterogeneity within BHD-associated RCCs, as alluded to in this earlier blog post.

The FLCN-associated signalling diagram and the work described above underlines the importance of mTOR complex (mTORC) signalling in the development of RCC. Thus in mid-March, we attended a set of “Talks about TORCs” organised by the Biochemical Society in London. Recent advances in TOR signalling were shared and its role in a variety of processes was introduced. For example, the first talk by Professor Michael Hall (University of Basel, Switzerland) discussed TOR signalling in relation to growth and metabolism. Notably, Professor Hall suggested that mammalian TOR should be renamed mechanistic TOR, as the pathway is not unique to mammals. This was aptly demonstrated by Dr Miguel Navarro (Institute of Parasitology and Biomedicine “López-Neyra”, Spain) with his work on TOR signalling in Trypanosoma brucei (Barquilla et al., 2012). In addition, Professor Thomas Weichhart (Medical University of Vienna, Austria) and Professor Doreen Cantrell (University of Dundee, UK) introduced the role of mTOR signalling in immunity, which is of interest as FLCN/FNIP1 may be associated with B-cell development (as discussed here and here). Professor Linda Partridge (UCL, UK) also introduced the connection between mTOR signalling and ageing. This is especially relevant as recent work has connected FLCN with longevity in C. elegans (Gharbi et al., 2013), which will be discussed in our BHD Research Blog soon. Moreover, Dr Andrew Tee presented his work on ULK1 and mTOR signalling (Dunlop et al., 2011), as well as a poster on BHD. For more information regarding the work of Dr Tee, please look at our lab profile here.

Finally, do visit our Conferences and Events page to keep updated with meetings that are of relevance to BHD syndrome. In particular, the Fifth BHD Symposium and Second HLRCC Symposium will be held in Paris on 28-29th June 2013, and the abstract and earlybird registration deadline is 15th April 2013.

 

  • Barquilla, A., Saldivia, M., Diaz, R., Bart, J., Vidal, I., Calvo, E., Hall, M., & Navarro, M. (2012). Third target of rapamycin complex negatively regulates development of quiescence in Trypanosoma brucei Proceedings of the National Academy of Sciences, 109 (36), 14399-14404 DOI: 10.1073/pnas.1210465109
  • Dunlop EA, Hunt DK, Acosta-Jaquez HA, Fingar DC, & Tee AR (2011). ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding. Autophagy, 7 (7), 737-47 PMID: 21460630
  • 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 DOI: 10.1056/NEJMoa1113205
  • Gharbi, H., Fabretti, F., Bharill, P., Rinschen, M., Brinkkötter, S., Frommolt, P., Burst, V., Schermer, B., Benzing, T., & Müller, R. (2013). Loss of the Birt-Hogg-Dubé gene product Folliculin induces longevity in a hypoxia-inducible factor dependent manner Aging Cell DOI: 10.1111/acel.12081

Familial multiple discoid fibromas and topical rapamycin

In August 2011, a blog post highlighted a study by Starink et al. (2011), which described a condition known as familial multiple discoid fibromas (FMDF). This rare skin disorder is characterised by the early onset of multiple firm papules on the face and ears. Although FMDF shares some characteristics with BHD syndrome, no systemic symptoms or FLCN mutations are observed. A recent study now describes siblings with FMDF which appears to respond to treatment with topical rapamycin (Wee et al., 2013).

In this study, a 27-year old man and his 22-year old sister presented with multiple skin lesions, but no family history of skin, lung or renal abnormalities. In both cases the lesions developed during childhood/adolescence and involved the face/ears, which helps distinguish FMDF from BHD syndrome. In addition, no systemic manifestations or pathogenic mutations in FLCN were detected. Furthermore, no mutations were identified in TSC1 and TSC2, which also discounts tuberous sclerosis complex (TSC). Much like the lesions described by Starink et al. (2011), hair follicles were seen at the periphery of the papules. However, biopsies also noted an unusual histological feature in some lesions involving a keloidal-like pattern.

Surgical treatment with shave excision, hyfrecation and curettage and cautery was attempted on some lesions with unsatisfactory results. Since rapamycin was shown to improve the appearance of skin lesions in TSC (Haemel et al., 2010; Mutizwa et al., 2011), Wee et al. trialled the application of topical rapamycin at 1 mg/ml on the facial skin of the affected brother. Only a once daily application was tolerated due to local skin irritation, but there was a noticeable improvement in the redness and size of the lesions after 8 weeks of treatment. Further (but less significant) improvement was noted at 16 weeks of treatment, with no detectable absorption of rapamycin into the bloodstream.

The genetic defect which causes FMDF is still under investigation, but the lack of a family history suggests an autosomal recessive pattern of inheritance or a parent with germline mosaicism. Nevertheless, the potential efficacy of rapamycin indicates that dysregulated mTOR signalling may play a role in this disorder (much like TSC and BHD syndrome).

 

  • Haemel AK, O’Brian AL, Teng JM. Topical rapamycin: a novel approach to facial angiofibromas in tuberous sclerosis. Arch Dermatol. 2010 Jul;146(7):715-8. doi: 10.1001/archdermatol.2010.125.
  • Mutizwa MM, Berk DR, Anadkat MJ. Treatment of facial angiofibromas with topical application of oral rapamycin solution (1mgmL(-1) ) in two patients with tuberous sclerosis. Br J Dermatol. 2011 Oct;165(4):922-3. doi: 10.1111/j.1365-2133.2011.10476.x.
  • Starink TM, Houweling AC, van Doorn MB, Leter EM, Jaspars EH, van Moorselaar RJ, Postmus PE, Johannesma PC, van Waesberghe JH, Ploeger MH, Kramer MT, Gille JJ, Waisfisz Q, & Menko FH (2011). Familial multiple discoid fibromas: A look-alike of Birt-Hogg-Dubé syndrome not linked to the FLCN locus. Journal of the American Academy of Dermatology PMID: 21794948
  • Wee, J., Chong, H., Natkunarajah, J., Mortimer, P., & Moosa, Y. (2013). Familial multiple discoid fibromas: unique histological features and therapeutic response to topical rapamycin British Journal of Dermatology DOI: 10.1111/bjd.12315

Video Interview: Dr Frank McCormack – University of Cincinnati & The LAM Foundation, USA

Dr Frank McCormack is a Professor of Medicine and Director of the Division of Pulmonary, Critical Care & Sleep Medicine at the University of Cincinnati. Professor McCormack also leads a research group which investigates pulmonary innate immunity and the mechanisms of pulmonary fibrosis. In addition, Professor McCormack’s clinical interests focus on cystic lung diseases, such as lymphangioleiomyomatosis (LAM).

LAM is caused by mutations in the TSC1 or TSC2 genes and occurs almost exclusively in women, either sporadically or in association with tuberous sclerosis complex (Sato et al., 2002; Henske & McCormack, 2012). LAM and BHD syndrome can present with similar pathophysiology, which has been discussed in a previous blog post, and many findings in the LAM field may be relevant to BHD research.

As an expert in treating patients with cystic lung disease, Professor McCormack is also the Scientific Director and Chairman of the Scientific Advisory Board of The LAM Foundation. The LAM Foundation was founded in 1995 with the aim to find treatments for, and ultimately cure, LAM through advocacy and research funding. The Foundation has a network of approved LAM Clinics in the United States, which also specialise in the lung symptoms of BHD. Please go to BHDSyndrome.org for more information regarding these BHD Pulmonary Centres.

Furthermore, the LAM Foundation has provided funding and recruitment support for clinical trials for LAM treatments. In particular, the Foundation played a key role in the Multicenter International LAM Efficacy of Sirolimus (MILES) trial, which was co-directed by Professor McCormack. The results of this trial indicate that sirolimus, which is also known as rapamycin, may be useful as a therapy for LAM (McCormack et al., 2011). To learn more about clinical trials in general, and those that are applicable to individuals with BHD syndrome, please click here.

Much like our upcoming BHD symposium, the LAM Foundation also organises an annual LAMposium, which has similar aims to connect patients, families, clinicians and scientists for both information and support. See our Conferences and Events page for more details regarding these and other relevant meetings. The importance of such symposia is aptly demonstrated by Professor McCormack’s comprehensive presentation at the Second BHD Symposium, which was held in Washington DC, USA in 2010. This presentation (which can be accessed here) introduced LAM and the LAM Foundation, as well as discussing potential synergies with BHD.

For more information regarding LAM research, the LAM Foundation and BHD please view our video interview with Professor McCormack, which was filmed at the Fourth BHD Symposium in Cincinnati, USA in 2012. For further reading, the following reference list also highlights the work of Professor McCormack:

 

  • Crouch, E., Nikolaidis, N., McCormack, F., McDonald, B., Allen, K., Rynkiewicz, M., Cafarella, T., White, M., Lewnard, K., Leymarie, N., Zaia, J., Seaton, B., & Hartshorn, K. (2011). Mutagenesis of Surfactant Protein D Informed by Evolution and X-ray Crystallography Enhances Defenses against Influenza A Virus in Vivo Journal of Biological Chemistry, 286 (47), 40681-40692 DOI: 10.1074/jbc.M111.300673
  • Franz, D., Bissler, J., & McCormack, F. (2011). Tuberous Sclerosis Complex: Neurological, Renal and Pulmonary Manifestations Neuropediatrics, 41 (05), 199-208 DOI: 10.1055/s-0030-1269906
  • Henske, E., & McCormack, F. (2012). Lymphangioleiomyomatosis — a wolf in sheep’s clothing Journal of Clinical Investigation, 122 (11), 3807-3816 DOI: 10.1172/JCI58709
  • McCormack, F., Inoue, Y., Moss, J., Singer, L., Strange, C., Nakata, K., Barker, A., Chapman, J., Brantly, M., Stocks, J., Brown, K., Lynch, J., Goldberg, H., Young, L., Kinder, B., Downey, G., Sullivan, E., Colby, T., McKay, R., Cohen, M., Korbee, L., Taveira-DaSilva, A., Lee, H., Krischer, J., & Trapnell, B. (2011). Efficacy and Safety of Sirolimus in Lymphangioleiomyomatosis New England Journal of Medicine, 364 (17), 1595-1606 DOI: 10.1056/NEJMoa1100391
  • Sato T, Seyama K, Fujii H, Maruyama H, Setoguchi Y, Iwakami S, Fukuchi Y, & Hino O (2002). Mutation analysis of the TSC1 and TSC2 genes in Japanese patients with pulmonary lymphangioleiomyomatosis. Journal of human genetics, 47 (1), 20-8 PMID: 11829138
  • Wu, H., Suzuki, T., Carey, B., Trapnell, B., & McCormack, F. (2011). Keratinocyte Growth Factor Augments Pulmonary Innate Immunity through Epithelium-driven, GM-CSF-dependent Paracrine Activation of Alveolar Macrophages Journal of Biological Chemistry, 286 (17), 14932-14940 DOI: 10.1074/jbc.M110.182170
  • Young, L., Gulleman, P., Bridges, J., Weaver, T., Deutsch, G., Blackwell, T., & McCormack, F. (2012). The Alveolar Epithelium Determines Susceptibility to Lung Fibrosis in Hermansky-Pudlak Syndrome American Journal of Respiratory and Critical Care Medicine, 186 (10), 1014-1024 DOI: 10.1164/rccm.201207-1206OC
  • Young, L. (2010). Serum Vascular Endothelial Growth Factor-D Prospectively Distinguishes Lymphangioleiomyomatosis From Other Diseases CHEST Journal, 138 (3) DOI: 10.1378/chest.10-0573

Syndromic oncocytic tumours: BHD meets Cowden syndrome

Much like BHD, Cowden syndrome (CS) is an autosomal dominant disorder which leads to benign cutaneous lesions and an increased risk of cancer. CS is caused by mutations in PTEN, and its similarities with BHD syndrome have been discussed in a previous blog post. In particular, both BHD and CS patients develop oncocytic tumours, which are epithelial in origin and have an abnormal accumulation of mitochondria. Interestingly, a recent study observed that a parotid and thyroid oncocytoma from a BHD and CS patient respectively were heterozygous for both FLCN and PTEN, which suggests that this double heterozygosity may play a role in syndromic oncocytic tumours (Pradella et al., 2013).

In this study, Pradella et al. noted that a BHD patient with fibrofolliculomas, lung cysts and a history of pneumothorax also had a parotid oncocytoma. Routine peripheral blood sequencing identified a germline heterozygous mutation in FLCN.  To assess whether a complete loss of FLCN caused the development of this parotid oncocytoma, the authors carried out exon sequencing and copy number analysis, but did not detect a loss of the wild-type FLCN allele in the tumour. Mitochondrial DNA (mtDNA) mutations are often discovered in sporadic oncocytomas (Gasparre et al., 2011), however, sequencing of the mtDNA only detected common polymorphisms. The authors also noted that there were no mutations in a number of common nuclear oncogenes, such as PIK3CA, BRAF, CTNNB1, KRAS, HRAS, AKT, KIT, PIK3R1 and ERBB2. The tumour-suppressor genes PTEN and TP53 were then analysed and a somatic deletion of one PTEN allele was observed in the tumour. Array-comparative genomic hybridization confirmed that there was a specific deletion in chromosome 10 which spanned PTEN, with no other major genetic abnormalities detected within the sample.

In light of this finding, the authors returned to a thyroid oncocytoma from a CS patient which had a germline heterozygous PTEN mutation (Pradella et al., 2011), and found the tumour also carried a somatic deletion of FLCN. Molecular karyotyping of the CS oncocytoma revealed a number of chromosomal aberrations, including a deletion in chromosome 10q encompassing PTEN, and a partial loss of chromosome 17p encompassing FLCN. Considering that FNIP1 is located on chromosome 5, it is worth noting that two large duplications in chromosomes 5 and 7 were also observed – as considered in a previous blog post, could this play a part in tumourigenesis?

Finally, no FLCN and/or PTEN mutations were detected in 22 cases of mtDNA-associated sporadic oncocytoma, suggesting that this double heterozygosity may be associated with syndromic oncocytic tumours. As illustrated in our signalling diagram, PTEN and FLCN are known to modulate mTOR signalling. Furthermore, mTORC1 and FLCN are known to affect PGC1a (Cunningham et al., 2007; Klomp et al., 2010; Hasumi et al., 2012), which promotes mitochondrial biogenesis and could provide a mechanism for the mitochondrial hyperplasia seen during oncocytic transformation. However, further research is necessary, for example – could a similar process be taking place within the BHD-associated parotid oncocytomas described here?

 

  • Cunningham JT, Rodgers JT, Arlow DH, Vazquez F, Mootha VK, & Puigserver P (2007). mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex. Nature, 450 (7170), 736-40 PMID: 18046414
  • Gasparre G, Romeo G, Rugolo M, & Porcelli AM (2011). Learning from oncocytic tumors: Why choose inefficient mitochondria? Biochimica et biophysica acta, 1807 (6), 633-42 PMID: 20732299
  • Hasumi H, Baba M, Hasumi Y, Huang Y, Oh H, Hughes RM, Klein ME, Takikita S, Nagashima K, Schmidt LS, & Linehan WM (2012). Regulation of mitochondrial oxidative metabolism by tumor suppressor FLCN. Journal of the National Cancer Institute, 104 (22), 1750-64 PMID: 23150719
  • 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
  • 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 PMID: 23386036
  • Pradella LM, Zuntini R, Magini P, Ceccarelli C, Neri I, Cerasoli S, Graziano C, Gasparre G, & Turchetti D (2011). Two distinct thyroid tumours in a patient with Cowden syndrome carrying both a 10q23 and a mitochondrial DNA germline deletion. Journal of medical genetics, 48 (11), 779-82 PMID: 21926107

The Fifth BHD Symposium and Second HLRCC Symposium 2013

We are happy to announce that registration for the Fifth BHD and Second HLRCC Symposium is now open, with discounted rates available till 31st March 2013. The Symposium will be hosted by Professor Stéphane Richard at the École du Louvre in Paris, France on 28-29th June 2013. Please do visit the dedicated Symposium website for more information regarding accommodation and registration options.

The rationale behind the Symposia was recently discussed by Colledge & Solly (2012), and one of the main aims of the meeting is to update the community with the latest research in Birt-Hogg-Dubé syndrome (BHD) and Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). For oral/poster presentations, the deadline for abstract submission is 29th March 2013 and further details can be found here.

In addition, we are delighted that our keynote speakers will be Professor William G. Kaelin Jr. from the Harvard Medical School and Dana-Farber Cancer Institute in Boston, USA, Dr Bernard Escudier from Institute Gustave-Roussy in Villejuif, France and Professor Bin Tean Teh from the National Cancer Centre Singapore and Duke-National University of Singapore Graduate Medical School. Each has a particular interest in the molecular basis of renal cell carcinoma and its treatment (with more information available upon clicking their names above).

There will also be a Patient and Family session on 29th June organised by genetic counsellors Lindsay Middelton (National Cancer Institute, National Institutes of Health, Bethesda, USA) and Sophie Deveaux (Le Centre predispositions aux tumeurs du rein, Hôpital de Bicêtre, Le Kremlin-Bicètre, France). This will be a great opportunity for families to meet each other and experts in the field, in order to ask questions and share thoughts.

Additionally, a conference dinner has been planned for everyone on 28th June, so we hope to see you in Paris! In the meantime, do check back regularly with BHDSyndrome.org for more updates and other conferences of interest.

 

  • Colledge VL, & Solly J (2012). The rare disease challenge and how to promote a productive rare disease community: case study of Birt-Hogg-Dubé symposia. Orphanet journal of rare diseases, 7 PMID: 22950632

 

BHD and metalloproteinase activity

In 2011, a study by Tobino et al. discussed the differences between BHD syndrome and the cystic lung disease lymphangioleiomyomatosis (LAM), as described here. The importance of these differences has been underlined by a recent case report in which a BHD patient was initially diagnosed and treated for LAM (Pimenta et al., 2012). Interestingly, this report indicates that matrix metalloproteinases (MMPs) may also be involved in the development of BHD lung cysts, as they are in LAM.

In this report, a 44-year old female presented with difficulty breathing after exertion and a spontaneous pneumothorax. CT scans showed bilateral cystic lesions, a biopsy and pleurodesis was performed, and the patient was diagnosed with LAM. Matrix degradation by MMPs is thought to play a role in cyst development in LAM (McCormack, 2008), and inhibition of MMPs with doxycycline has been used as a treatment for this condition (Moses et al., 2006). After 6 months of doxycycline therapy, Pimenta et al. noted an improvement in the patient’s lung function, as well as a decrease in the serum and urinary levels of MMP-9.

However, upon follow-up the authors found papules on the patient’s neck and upper thorax, as well as a family history of cystic lung disease and pneumothorax. Histological analysis of the lung biopsy showed an absence of markers for LAM, as well as cyst walls formed by collapsed alveoli or thickened pleural tissue, as has been observed in BHD lung samples by Furuya et al. (2012). Consequently, a diagnosis of BHD syndrome was established.

Notably, pulmonary symptoms worsened following an interruption of the doxycycline treatment due to gastric intolerance. As doxycycline was producing a clinical effect, the distribution of MMP-9 in the lung biopsy was assessed by immunohistochemistry. Using this technique, a large number of MMP-9-positive cells, which were mostly macrophages and neutrophils, were observed in the cyst wall when compared to normal adjacent lung tissue.

It is important to note that the authors did not report the results of a genetic test, which is the only way to conclusively confirm the presence of a FLCN mutation and a diagnosis of BHD syndrome. However, the potential role of FLCN in maintaining lung matrix integrity has been discussed in previous BHD studies (Kalhan et al., 2007; Fröhlich et al., 2008). Furthermore, MMPs are known to be involved in cell migration and angiogenesis (Jackson 2002; Elkington & Friedland, 2006), which are also linked to FLCN (as seen in our recently updated signalling diagram). Therefore, this study highlights an association between MMP activity and BHD syndrome, which could be used as a potential therapeutic target.

 

  • Elkington PT, & Friedland JS (2006). Matrix metalloproteinases in destructive pulmonary pathology. Thorax, 61 (3), 259-66 PMID: 16227332
  • Fröhlich BA, Zeitz C, Mátyás G, Alkadhi H, Tuor C, Berger W, & Russi EW (2008). Novel mutations in the folliculin gene associated with spontaneous pneumothorax. The European respiratory journal : official journal of the European Society for Clinical Respiratory Physiology, 32 (5), 1316-20 PMID: 18579543
  • Furuya M, Tanaka R, Koga S, Yatabe Y, Gotoda H, Takagi S, Hsu YH, Fujii T, Okada A, Kuroda N, Moritani S, Mizuno H, Nagashima Y, Nagahama K, Hiroshima K, Yoshino I, Nomura F, Aoki I, & Nakatani Y (2012). Pulmonary cysts of Birt-Hogg-Dubé syndrome: a clinicopathologic and immunohistochemical study of 9 families. The American journal of surgical pathology, 36 (4), 589-600 PMID: 22441547
  • Jackson C (2002). Matrix metalloproteinases and angiogenesis. Current opinion in nephrology and hypertension, 11 (3), 295-9 PMID: 11981259
  • Kalhan R, Yeldandi AV, & Jain M (2007). A 48-year-old woman with skin lesions, renal masses, and spontaneous pneumothorax. Chest, 131 (2), 624-7 PMID: 17296671
  • McCormack FX (2008). Lymphangioleiomyomatosis: a clinical update. Chest, 133 (2), 507-16 PMID: 18252917
  • Moses MA, Harper J, & Folkman J (2006). Doxycycline treatment for lymphangioleiomyomatosis with urinary monitoring for MMPs. The New England journal of medicine, 354 (24), 2621-2 PMID: 16775248
  • Pimenta SP, Baldi BG, Nascimento EC, Mauad T, Kairalla RA, & Carvalho CR (2012). Birt-Hogg-Dubé syndrome: metalloproteinase activity and response to doxycycline. Clinics (Sao Paulo, Brazil), 67 (12), 1501-4 PMID: 23295609
  • Tobino K, Hirai T, Johkoh T, Kurihara M, Fujimoto K, Tomiyama N, Mishima M, Takahashi K, & Seyama K (2012). Differentiation between Birt-Hogg-Dubé syndrome and lymphangioleiomyomatosis: quantitative analysis of pulmonary cysts on computed tomography of the chest in 66 females. European journal of radiology, 81 (6), 1340-6 PMID: 21550193

Signalling diagram update

As has been noted in our blog review of 2012, a great deal of research in the BHD field has been published in the past year. Thus, it is only appropriate to reflect these changes in an updated version of our interactive signalling diagram.

The FLCN-associated signalling diagram retains many of the features from previous versions, such as the clickable links which give further information about the various factors and signalling pathways, as well as clickable references which take you to the relevant PubMed entry or directly to the article for those that are open access. In addition, these references can be found in our regularly updated BHD Literature Database, and those that are freely available are also found in our BHD Article Library.

One major difference with the figure is that it highlights the factors and pathways that appear to be directly linked to FLCN, while related pathways which provide context for these associations are faded. Furthermore, there are now two pop-up boxes that can be opened and repositioned independently. The first shows the C-terminal structure of FLCN which was determined by Nookala et al. (2012) (as previously described here). It also illustrates the FLCN-associated interactome, which outlines the potential interacting partners of FLCN and their downstream effects. The second pop-up box notes the post-translational modifications that have been detected in FLCN and its interactors, such as phosphorylation and ubiquitination (which have been discussed in the blog here and here respectively).

An update of the “What is BHD?” text in the For Researchers section is coming soon too. In the meantime, if you have any comments or suggestions regarding the diagram or any other aspect of the website, please do not hesitate to email us at contact@BHDSyndrome.org or fill in our feedback form.

 

  • 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) PMID: 22977732

Research update: FLCN-FNIP2-AMPK and MNU-induced apoptosis

Previous studies in mouse cell lines have suggested that FLCN, FNIP2 and AMPK are involved in N-methyl-N-nitrosourea (MNU)-induced apoptosis (Lim et al., 2011 – described here). Using a human cell line, further work from the same group has now shown that FNIP2 appears to be stabilised by MNU treatment, and that this stability is modulated by FLCN and AMPK (Sano et al., 2012).

Sano et al. used a HeLa MR cell line which lacks the enzyme MGMT that repairs MNU-induced DNA lesions, and confirmed that FNIP2 is involved in the induction of apoptosis following MNU exposure (using flow cytometry with miRNA-knockdown). Western blot analysis went on to show that FNIP2 protein levels gradually increased after MNU treatment, while those of FLCN and AMPKα remained unchanged. However, qRT-PCR showed no change in the levels of FNIP2 mRNA after MNU treatment, indicating that the observed increase in FNIP2 protein levels was not due to transcriptional effects. Using HeLa MR cells which expressed FLAG-tagged FNIP2, it could be seen that the proteosome inhibitor MG132 led to a significant increase in FNIP2 protein levels, while inhibiting protein synthesis with cyclohexamide led to a significant decrease. In both cases, the levels of FLCN and AMPKα remained constant. Consequently, the authors suggested that FNIP2 may be regulated at the post-transcriptional level following MNU exposure.

As FNIP2 is known to interact with FLCN and AMPK (Hasumi et al., 2008; Takagi et al., 2008; Lim et al., 2011), the authors investigated whether these factors affected the levels of FNIP2. siRNA knockdown of FLCN in HeLa MR cells led to a decrease in FNIP2 protein levels, and treatment with MNU elicited no significant changes. In contrast, siRNA knockdown of AMPKα led to an increase in FNIP2 levels, which increased further after MNU treatment. Together these results suggest that FLCN and AMPK may have opposing effects on the stability of FNIP2. Moreover, the inhibition of AMPK kinase activity with compound C led to an increase in the levels of FNIP2, but no increase was observed after MNU exposure. This result suggests that the kinase activity of AMPK is also linked to the stability of FNIP2.

Finally, immunoprecipitation of FLAG-tagged FNIP2 from HeLa MR cells treated with MNU over 72 hours showed that FLCN interacted with FNIP2 throughout this period. However, it could be seen that the interaction between FNIP2 and AMPKα gradually decreased over the same timescale. Although the exact mechanistic details appear to be quite complex, it is clear that FLCN, FNIP2 and AMPK are associated with MNU-induced apoptosis in vitro.

 

  • 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
  • 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
  • Sano S, Sakagami R, Sekiguchi M, & Hidaka M (2012). Stabilization of MAPO1 by specific binding with folliculin and AMP-activated protein kinase in O(6)-methylguanine-induced apoptosis. Biochemical and biophysical research communications PMID: 23201403
  • 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

Research involving Rab35 and Rab27B

As discussed in a previous blog post, the Rab family of small GTPases are known to be involved in membrane trafficking.  Two members of this family have now been associated with FLCN: Rab27B, using microarray studies (Hong et al., 2010; Klomp et al., 2010; Reiman et al., 2012), reviewed here, and Rab35, in an in vitro guanine nucleotide exchange assay (Nookala et al., 2012), reviewed here. However, the exact function of these Rabs is yet to be defined and further research is now shedding light on this issue.

Work by Charrasse et al. (2012) showed that Rab35 co-localises and interacts with N-cadherin and M-cadherin at cell-cell contacts, using both immunofluorescence and immunoprecipitation experiments in C2C12 and HeLa cells. shRNA studies in C2C12 cells then went on to show that Rab35 is necessary for N- and M-cadherin localisation at cell-cell contacts, and that it is involved in adherens junction formation. This is of particular interest as studies have suggested a role for FLCN in cadherin signalling (Hong et al., 2010; Reiman et al., 2012) and cell-cell adhesion (Nahorski et al., 2012; Medvetz et al., 2012). Consequently, could FLCN-loss impact on these cellular processes through dysregulated Rab35?

Additionally, a clinical study by McGrath et al. (2012) suggested that a mutation in the Rab27B effector protein EXPH5 plays a role in the development of an inherited skin disorder. Closer analysis of the skin lesions by electron microscopy showed a disruption of keratinocyte adhesion in the lower epidermis, as well as aggregated keratin filaments and a perinuclear accumulation of vesicles within the cells. A similar alteration in the keratin filament network and keratinocyte adhesion was noted after shRNA knockdown of EXPH5 in normal human keratinocytes. Notably, Medvetz et al. observed that keratin levels were regulated by FLCN and PKP4. Thus, could Rab27B and EXPH5 also be involved in this process?

It is encouraging that studies have identified both Rab27B and Rab35 as potential targets of FLCN, and that similar themes appear to be emerging from additional studies. However, Rabs are known to be involved with a number of different organelles and processes (Galvez et al., 2012), so it must be noted that current knowledge does not exclude the possibility that FLCN may interact with other Rabs or even an alternative family of small GTPases.

 

  • Charrasse S, Comunale F, De Rossi S, Echard A, & Gauthier-Rouvière C (2012). Rab35 regulates cadherin-mediated adherens junction formation and myoblast fusion. Molecular biology of the cell PMID: 23197472
  • Galvez T, Gilleron J, Zerial M, & O’Sullivan GA (2012). SnapShot: Mammalian Rab proteins in endocytic trafficking. Cell, 151 (1), 234-23400 PMID: 23021225
  • Hong SB, Oh H, Valera VA, Stull J, Ngo DT, Baba M, Merino MJ, Linehan WM, & Schmidt LS (2010). Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-beta signaling. Molecular cancer, 9 PMID: 20573232
  • 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
  • McGrath JA, Stone KL, Begum R, Simpson MA, Dopping-Hepenstal PJ, Liu L, McMillan JR, South AP, Pourreyron C, McLean WH, Martinez AE, Mellerio JE, & Parsons M (2012). Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility. American journal of human genetics, 91 (6), 1115-21 PMID: 23176819
  • Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H, & Henske EP (2012). Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion. PloS one, 7 (11) PMID: 23139756
  • 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, 21 (24), 5268-79 PMID: 22965878
  • 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) PMID: 22977732
  • Reiman A, Lu X, Seabra L, Boora U, Nahorski MS, Wei W, & Maher ER (2012). Gene Expression and Protein Array Studies of Folliculin-regulated Pathways. Anticancer research, 32 (11), 4663-70 PMID: 23155228

The Fifth BHD Symposium – 28-29th June 2013

It is our pleasure to announce the Fifth BHD Symposium which will be hosted by Professor Stéphane Richard at the École du Louvre in Paris, France on 28-29th June 2013.

As with previous Symposia, the aim is to promote a supportive international community for both BHD researchers and patients. In fact, Colledge & Solly (2012) recently discussed the BHD Symposia and how they can be used as a model for other rare disease meetings.

Details from the previous BHD Symposia can be found on our Conferences and Events page. In particular, the Fourth BHD Symposium was the largest meeting to date, which provided an excellent environment for discussions and collaborations. Further highlights from the scientific and patient sessions of this meeting can be found in our blog here and here respectively.

In the meantime, please do check BHDSyndrome.org for more information about the Fifth BHD Symposium as it becomes available.

 

  • Colledge VL, & Solly J (2012). The rare disease challenge and how to promote a productive rare disease community: case study of Birt-Hogg-Dubé symposia. Orphanet journal of rare diseases, 7 PMID: 22950632