Familial Cancer (2013) 12:405–448 DOI 10.1007/s10689-013-9678-z
© Springer Science+Business Media Dordrecht 2013
ABSTRACTS
INVITED LECTURES
L1
NEW INSIGHTS INTO THE BIOLOGY OF CLEAR CELL RENAL CARCINOMA William G. Kaelin, Jr.1,2 1 Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02215, USA 2 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA Biallelic inactivation of the VHL gene is an early and frequent event in clear cell renal carcinoma (ccRCC), which is the most common form of kidney cancer. The VHL protein (pVHL) has many functions including serving as an ubiquitin ligase that targets the alpha subunits of the heterodimeric HIF transcription factor for polyubiquitylation and degradation. Deregulation of HIF, and in particular HIF2, drives pVHL-defective ccRCC and explains the increased VEGF levels and angiogenesis that typify ccRCC. pVHL loss is not, however, sufficient to cause ccRCCother cooperating events are required. Chromosome 3p loss is the most common copy number alteration in ccRCC and is frequently accompanied by 14q loss and 5q gains. 3p contains VHL and several other ccRCC suppressors, including PRBM1, BAP1, and SETD2. 14q contains HIF1_, which acts as a ccRCC suppressor. Our recent studies suggest that the 5q gene SQSTM1, which is involved in autophagy and signaling, promotes ccRCC. Recent sequencing studies have revealed that mTOR pathway genes, such as TSC2 and mTOR, and chromatin regulators, including the abovementioned PBRM1 and SETD2, are mutated in ccRCC. The selection pressure to mutate these proteins might stem from the ability of HIF to inhibit mTOR and to alter chromatin marks by upregulating histone demethylases.
123
406
Abstracts
ABSTRACTS
INVITED LECTURES
L2
TARGETED THERAPY FOR ADVANCED KIDNEY CANCER Bernard Escudier Institut Gustave Roussy, Villejuif, France Treatment of metastatic renal cell carcinoma (mRCC) has dramatically changed in the past 7 years, with the approval of 7 new agents: sorafenib, sunitinib, temsirolimus, bevacizumab plus interferon, everolimus, pazopanib and axitinib. The development of these agents has been encouraged by the demonstration that the VHL-HIF-VEGF pathway was stimulated in RCC, more than in any other cancer. Despite this enrichment of therapies, mRCC remains a lethal disease in the vast majority of patients. Development of new agents continues in many directions: • More active and less toxic drugs, active on the VHL-HIF-VEGF pathway, such as axitinib and tivozanib. Axitinib for example, which is a more potent and more selective VEGF inhibitor, has been shown to be more active that sorafenib in a large randomized phase 3 and recently approved in second line. • New targets are also under investigation, such as cMET, angiopoietin etc… inhibition. Preliminary data are encouraging, and some of these agents are currently in phase III. • Immunotherapy finally has got a new development with promising data with new “targeted immunotherapy”, ipilimumab, anti PD1 antibody and vaccines Interestingly, most of the new targets have been discovered based on findings in genetic disease, primarily VHL disease, but more recently papillary tumors with cMET mutation.
123
Abstracts
407
ABSTRACTS
INVITED LECTURES
L3
DEEP SEQUENCING OF HLRCC-RELATED AND SPORADIC TYPE 2 PAPILLARY RENAL CELL CARCINOMA: THE SEARCH FOR TUMORIGENIC AND PROGRESSION GENES/PATHWAYS Aikseng Ooi1,2, Bin Tean Teh1,2 Van Andel Research Institute, USA 2 National Cancer Center, Singapore
1
Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2) leading to fumarate accumulation. The conventional paradigm posits that the main consequence of fumarate accumulation is HIF-_ stabilization. Paradoxically, FH mutation differs from other HIF-_ stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We and others identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes caused by sustained activation of the stressregulated transcription factor NRF2 (NFE2L2). We demonstrated that over-expression of aldoketo reductase family 1 member B10 (AKR1B10), an AREcontrolled gene, is a biomarker for both hereditary and sporadic PRCC2. More recently, our nextgeneration sequencing studies have identified somatic mutations in NRF2, CUL3, and SIRT1 in sporadic PRCC2, which are responsible for driving the NRF2 activation phenotype. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens demonstrating a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2. We have now completed the studies of the primary and metastatic tissues of a patient with Hereditary Type 2 Papillary RCC (HLRCC) by wholeexome sequencing. Data from the analysis will be presented and discussed.
123
408
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O1
FLCN-DEFICIENCY ABLATES PLITHOTAXIS, ENHANCES CELL-CELL ADHESION, AND LEADS TO CELL-TYPE SPECIFIC DEFECTS IN TGF-BETA SIGNALING Damir Khabibullin1, Doug Medvetz1, Jae Hun Kim2, Robert Steward Jr2, Bomi Gweon2, Erik Zhang1, Andrey Parkhitko3, Venkatesh Hariharan4, Hayden Huang4, Jane Yu1, Jeffrey Fredberg2, Elizabeth Henske1 1 Brigham and Women’s Hospital, Harvard Medical School, Boston MA, USA; 2Harvard School of Public Health, Boston MA, USA; 3Harvard Medical School, Boston MA, USA; 4 Columbia University, New York, NY, USA We recently discovered that FLCN interacts with plakophilin 4 (PKP4, p0071), a component of adherens junctions, and regulates cell adhesion and RhoA signaling in multiple cell types including UOK-257 (Medvetz, Khabibullin…Henske, PLoS One, 2012). In unpublished work, we used traction force microscopy to measure adhesion-dependent coordinated cell migration (plithotaxis), which requires transmission of force across multiple cell-cell junctions. Coordinated cell migration is essential for tissue integrity during development and after injury. UOK2572 cells migrated coordinately (intact plithotaxis) while UOK257 cells showed near complete ablation of plithotaxis. UOK257 cells also migrated 2-fold more slowly than UOK257-2 and exerted 36% weaker traction (cell-substrate) forces (p=0.025). Next, we addressed the impact of FLCN deficiency on lung-derived cells. Consistent with our results in other cell lineages, cell-cell adhesion was increased 3-fold in FLCN-deficient human bronchial epithelial (HBE) cells (p<0.05). Cox4 levels were increased 2.5-fold in FLCN-deficient HBE and small airway epithelial cells (SAEC), suggesting enhanced mitochrondrial number. Striking differences were observed in TGF beta pathway transcription in FLCN-deficient HBE vs. SAEC cells, indicating that some of FLCN’s functions are lineage specific within the lung. Despite this, a TGF beta-dependent cytokine was decreased by 30% in the plasma of Bhd+/- mice (p<0.05), consistent with FLCN’s known role in TGF-beta signaling and suggesting the first candidate plasma biomarker of BHD.
123
Abstracts
409
ABSTRACTS
ORAL COMMUNICATIONS
O2
EXIT FROM PLURIPOTENCY IS GATED BY INTRACELLULAR REDISTRIBUTION OF THE BHLH TRANSCRIPTION FACTOR TFE3 Joerg Betschinger1, Jennifer Nichols1, Sabine Dietmann1, Philip D. Corrin2, Patrick J. Paddison2, Austin Smith1 Wellcome Trust and Medical Research Council Stem Cell Institute, University of Cambridge, United Kingdom; 2Human Biology Division, Fred Hutchinson Cancer Research Centre, Seattle, United States
1
Factors that sustain self-renewal of mouse embryonic stem cells are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale siRNA screen we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ES cell commitment. Tsc2 lies upstream of mammalian target of Rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ES cells to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell intrinsic rheostat for destabilizing ground state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.
123
410
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O3
FOLLICULIN REGULATES LYSOSOME FUNCTION THROUGH MTORC1DEPENDENT PHOSPHORYLATION OF TRANSCRIPTION FACTOR EB Constance S. Petit, Agnès Roczniak-Ferguson, Shawn M. Ferguson Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA While the role of folliculin (FLCN) as a tumor suppressor is well established, the specific molecular mechanisms whereby FLCN exerts this function remain much less well understood. Here we show that FLCN and FLCN-interacting protein 1 (FNIP1) work together to strongly inhibit the nuclear localization of transcription factor EB (TFEB), a master regulator of the expression of genes encoding lysosomal proteins. Building on our previous elucidation of an mTOR complex 1 (mTORC1)-dependent mechanism for the regulation of TFEB (Roczniak-Ferguson et al, Science Signaling, 2012), we have further found that FLCN and FNIP1 act through mTORC1 to promote phosphorylation of TFEB on serine 211 resulting in the interaction of TFEB with 14-3-3 proteins and its retention in the cytoplasm. The increased nuclear abundance of TFEB in cells lacking FLCN has significant consequences on lysosome physiology. Collectively, our results demonstrate that FLCN and FNIP1 form a complex that negatively regulates TFEB and suggest that altered lysosome function could contribute to the pathological consequences of FLCN mutations in Birt-Hogg-Dubé syndrome.
123
Abstracts
411
ABSTRACTS
ORAL COMMUNICATIONS
O4
CONSTITUTIVE AMPK ACTIVATION UPON LOSS OF RENAL TUMOR SUPPRESSOR FOLLICULIN LEADS TO WARBURG EFFECT THROUGH HIF INDUCTION Ming Yan1,2,4, Marie-Claude Gingras1,2,4, Elaine Dunlop3, Anders Bondo Dydensborg1,2, Rachael Preston3, Zahra Jalali1,2, Sylvie Sabourin1,2, Yann Emmanuel Nouët1,2, Brandon Faubert1, Daina Avizonis1, David Mark Davies2, Taren Roughed1, Dmitri Kharitidi1,2, Russell Jones1, Andrew Tee3, Arnim Pause1,2. 1 Goodman Cancer Center, McGill University, Montréal, Québec, H3A 1A3, Canada; 2Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada; 3Institute of Medical Genetics, Cardiff University, Cardiff, Wales, CF14 4XN, UK; 4These authors contributed equally to this work Metabolic reprogramming is an adaptation process of cancer cells characterized by augmented glycolysis, even in the presence of oxygen (Warburg 1956). The Hypoxia Inducible Factor (HIF) has been implicated as the central regulator of the Warburg effect. Normally, upon energy stress, ATP levels are maintained through increased glycolysis and mitochondrial biogenesis, controlled by HIF and the transcriptional coactivator PGC-1, respectively. Both HIF and PGC-1 are induced by AMPK, the master sensor of energy stress. Whereas AMPK directly activates PGC-1 via phosphorylation and transcription, it is unexplored how AMPK activates HIF in normoxia. The renal tumor suppressor Folliculin (FLCN) is a novel AMPK binding protein. Here, we reveal that loss of FLCN activates AMPK in absence of stress, which upregulates PGC-1 leading to enhanced mitochondrial biogenesis and increased reactive oxygen species (ROS) generation. ROS are known HIF activators and we reveal that AMPK/PGC-1 dependent ROS overproduction increases HIF activity as ablation of AMPK or PGC-1 decreases mitochondrial ROS and HIF induction. Strikingly, the antioxidant NAC decreases HIF activity to normal levels. Lastly, we observed that Hif-1_ knockdown in follicular thyroid cancer cells (FTC 133) leads to reduced soft agar colony formation.
123
412
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O5
DETECTION AND FUNCTIONAL CHARACTERIZATION OF NEW MUTATIONS IN THE FH GENE Marine Guillaud-Bataille1, Johny Bombled1, Abdel Slama2, Michel Barrois1, Sophie Deveaux3, Patrick Benusiglio3,4, INCa MCUL and HLRCC national group, Stéphane Richard3, Brigitte Bressac-de Paillerets1 1 Service de Génétique, Institut de Cancérologie Gustave Roussy, Villejuif, France; 2Laboratoire de Biochimie, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France; 3Centre Expert National Cancers Rares PREDIR, AP-HP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; 4Consultation d’oncogénétique, Institut de Cancérologie Gustave Roussy, Villejuif, France Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder predisposing to cutaneous and uterine leiomyomas. In 20% of affected families, type 2 papillary renal cell cancer (PRCCII) also occurs, with aggressive course and poor prognosis. HLRCC results from heterozygous germline mutations of the tumor suppressor fumarate hydratase (FH) gene. Since a previously published work at the beginning of 2011 (Gardie et al, J Med Genet, 2011), as part of the French National Cancer Institute inherited predispositions to kidney cancer network, we have screened 84 families for mutation in the FH gene, by sequencing and quantitative PCR. Dosage of the fumarate hydratase enzymatic activity was performed to establish the pathogenicity of the new variants detected. Among the 32 families (38%) with a deleterious mutation, 18 (56%) presented cutaneous and/or uterine leiomyomas alone, 2 (6%) had renal cancers, and 12 (38%) showed both manifestations. We identified 24 different germline FH mutations (2 large deletions, 4 frameshift, 2 nonsense, 5 splice site, 10 missense, and 1 in-frame deletion). Nine of these mutations were novel, and mutations with ambiguous effect were proven to be deleterious by significant reduction of FH enzymatic activity. Five additional new missense variants are under functional characterization. Funded in part by the Institut National du Cancer (INCa)
123
Abstracts
413
ABSTRACTS
ORAL COMMUNICATIONS
O6
IMPROVED DETECTION OF FLCN MUTATIONS IN PATIENTS WITH BHDS Jorge R. Toro1,2, Bethany Friedman3, Sherri Bale3 National Cancer Institute, National Institutes of Health, DHHS, Bethesda, Maryland, USA; Dermatology Department, Veterans Affairs Medical Center, Washington, DC, USA; GeneDx, BioReference Laboratories, Inc., Gaithersburg, MD Maryland, USA. Background: Birt–Hogg–Dubé syndrome (BHDS) (MIM 135150) is an autosomal dominant predisposition to the development of follicular hamartomas (fibrofolliculomas), lung cysts, spontaneous pneumothorax, and kidney neoplasms. Germline mutations in FLCN are associated with the susceptibility for BHDS. To date 153 FLCN germline mutations have been reported in the online Folliculin sequence variation database. Objective: To characterize methods for improved FLCN mutation detection and novel mutations. Methods: Initial screening was conducted with direct bidirectional DNA sequencing of the coding regions and splice sites of exons 4-14 of FLCN. If no mutation was identified by sequencing analysis, large intragenic insertion and deletion mutations were screened by RQ-PCR and targeted arrays comparative genomic hybridization with exon-level resolution. Results: The FLCN mutation detection rate by direct sequencing was 89%. We detected 56 unique novel FLCN germline mutations: 22 deletions, 11 insertions, 13 missense,7 nonsense, 2 splice site and 1 deletion/insertion. To date only eight large unique intragenic mutations have been reported. We identified seven unique large FLCN intragenic deletions: two involving exon 1, one in exon 6 and four encompassing: exons 2-13, exons 6-14, exons 7-8 and exons 10-14. Including this report, to date there are 209 unique FLCN mutations identified: 71 deletions, 32 insertions, 99 substitutions and 7 deletion/insertion. A comprehensive worldwide review of published FLCN mutations and current ongoing efforts to detect novel BHDS causing mutations will be discussed. Conclusion: A systematic approach combining accurate and sensitive methods to detect FLCN mutations provides evidence that most patients with BHDS have mutations in FLCN.
123
414
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O7
BHD: A CLINICAL AND MOLECULAR STUDY OF 177 FRENCH PATIENTS Jérôme Lamoril, Nathalie Clément, Angélique Riffault, Agnès Bourillon, Caroline Kannengiesser, Bernard Grandchamp, Nadem Soufir Département de Génétique Moléculaire, Hôpital Bichat, AP-HP, Paris, France We performed an exhaustive molecular analysis of FLCN (Sanger sequencing, MLPA) in a series of 177 patients. Recruitment was performed via dermatology departments (38%), pneumology departments (46%), and urological departments (16%). FLCN mutations were detected in 59 patients including 23 frameshift, 11 non-sense, 5 splicing, 2 small inframe deletions, 7 missenses and exonic deletions in 11 patients. Four recurrent mutations were observed c.del1376_1407 p.S459del32fsX474 (5 families), c.1285dupC (3 families), c.1300G>A p.Glu434Lys: 3 families, c.1429 C>T, p.R477X : 3 families. Exonic deletions involved exon 1 (4 families), exon 4 (3 families), exons 2, 3, 5, 6, 12 and 14. Fibrofolliculomas were present in 86% of mutated patients (medium age 35 yr, familial in 2/3); pneumothorax in 54% (medium age 34 yr, familial in 2/3). Bullous emphysema was present in 98% of patients who undergo a CT scan. Renal in 20% of mutated families, and bilateral in 3 cases. Colonic cancer was present in 4% of mutated index patients, but also in 10 first degree related and 3 second degree related patients. Thyroid cancer was present in 4% of index cases, and in related in 3 other families. Melanoma was present in 3 families (6%).
123
Abstracts
415
ABSTRACTS
ORAL COMMUNICATIONS
O8
GERMLINE BAP1 MUTATIONS PREDISPOSE TO RENAL CELL CARCINOMAS Tatiana Popova1*, Lucie Hebert1*, Sophie Gad2, Stéphane Richard2, Olivier Caron3, Nadem Soufir5, Brigitte Bressac-de-Paillerets4, Dominique Stoppa-Lyonnet1,6,7, Marc-Henri Stern1,6,4 1 Institut Curie, Inserm U830, Paris 75248, France; 2Génétique Oncologique EPHE, Inserm U753, Institut de Cancérologie Gustave Roussy, Villejuif, 94805, and Centre Expert National Cancers Rares PREDIR, INCa/AP-HP, Hôpital de Bicêtre, Le Kremlin-Bicêtre 94276, France; 3Department of Medicine and 4Service de Génétique and Inserm U946, Institut de Cancérologie Gustave Roussy, Villejuif 94805, France; 5Genetic Department, Bichat Hospital, APHP, Paris 75018, and Inserm U976, Skin Research Center, Saint Louis Hospital, Paris 7 University, Paris 75010, France; 6Institut Curie, Department of Tumor Biology, Paris 75248, France; 7University Paris Descartes 75270, France. *Equal contribution. The genetic cause of some familial non-syndromic renal cell carcinomas (RCC) is unknown. By combining whole exome sequencing and tumor profiling in a RCC-prone family, we identified a germline BAP1 mutation c.277A>G (p.Thr93Ala) as the probable genetic basis of RCC predisposition. This mutation segregated with all four RCC affected relatives. Furthermore, BAP1 was inactivated in RCCs from this family. No BAP1 mutations were identified in 32 familial cases presenting with only RCC. We screened for germline BAP1 deleterious mutations in familial aggregations of cancers within the spectrum of the BAP1-associated tumor predisposition syndrome, including uveal melanoma, malignant pleural mesothelioma and cutaneous melanoma. Among the 11 families identified as carrying germline deleterious BAP1 mutations, 6 families presented with 9 RCC-affected individuals, demonstrating a significantly increased risk for RCC. This strongly argues that RCC belongs to the BAP1 syndrome and that BAP1 is a RCC predisposition gene.
123
416
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O9
FOLLICULIN AND LUNG: IS IT IMPORTANT? Vera P. Krymskaya 1, Elena Goncharova 1, Melane L. Fehrenbach 1, Dmitry Goncharov 1, Seung-Beom Hong1, Masaya Baba2, W. Marston Linehan2, Laura S. Schmidt2,3 1 University of Pennsylvania, Philadelphia, PA, USA; 2Urologic Oncology Branch, CCR, NCI, NIH, Bethesda, MD, 3BSP, SAIC-Frederick, Inc., FNLCR, Frederick, MD, USA Birt-Hogg-Dubé syndrome, caused by germline mutations in the FLCN gene, manifests among others by pulmonary cysts and spontaneous pneumothoraces. How mutational inactivation of FLCN results in lung cyst formation is not established. To study this disorder, doxycyclineinducible conditional knockout animals were generated by crossing FlcnloxP/loxP and CCSP-rtTAtetO-Cre mice for Flcn deletion in proximal lung epithelium or crossing FlcnloxP/loxP and SP-C-rtTAtetOCre mice for loss of Flcn in distal lung epithelium. Pulmonary function tests demonstrated that mice with doxycycline-induced Flcn loss in distal lung epithelium have greater airway resistance, higher compliance and lower tissue elastance than mice with Flcn expression. These differences were not seen in mice with Flcn loss in proximal lung epithelium. The alveolar airspaces of the FlcnloxP/loxP:SP-C-Cre mice on doxycycline were significantly increased (MAAA 5,671 ± 214μm2vs. 3,048 ± 47μm2) compared with Flcn-expressing mice. Lung development was also affected by conditional deletion of Flcn in lung epithelium from gestational day1. Newborn pups from FlcnloxP/loxP:SP-C-Cre mice with induced loss of Flcn were viable and healthy. Morphological analysis, however, demonstrated alveolar airspace enlargement in lungs of pups from doxycycline-induced FlcnloxP/loxP:SP-C-Cre mice (MLI 94 ± 6μm and MAAA 8,256 ± 553μm2) compared to lungs of pups on normal diet (65 ± 3μm and 5110 ± 241μm2, respectively). Collectively, our data demonstrate that loss of Flcn in the distal lung epithelium is important for both structure of the lung tissue and function, and suggests that Flcn may play an important role in lung architecture, development, and function. Funded in part by: NIH/NHLBI (V.P.K.), FNLCR Contract HHSNS261200800001E (L.S.S.).
123
Abstracts
417
ABSTRACTS
ORAL COMMUNICATIONS
O10
FOLLICULIN REGULATES EPITHELIAL-MESENCHYMAL TRANSITION IN AIRWAY EPITHELIUM: IMPLICATIONS FOR CYST FORMATION IN THE BHD LUNG Stephen Land, Claire Scott, Kendra Tosoni, Diane Cassidy, Stuart Booth, Anil Mehta Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK. Pulmonary cysts are a major feature of Birt-Hogg-Dubé (BHD) syndrome but the mechanisms by which folliculin (Flcn) dysfunction drives their formation remain unknown. Airway epithelial turnover is maintained by autocrine vascular endothelial growth factor (VEGF) signalling which induces clearance of aged cells and promotes re-growth. Cells lacking Flcn display slower growth and improper apical-basolateral polarisation suggesting that lung cysts arise from a failure to conserve epithelial phenotype (epithelial-mesenchymal transition, EMT). We tested this hypothesis by examining autocrine VEGF signalling in human bronchial epithelial cells (16HBE14o- or “HBE”) and by correlating Flcn expression with epithelial morphology in primary human nasal epithelial spheroids (HNE). In HBE, Flcn shRNA increased Tissue Inhibitor of Metalloprotease-3 (TIMP-3) mRNA expression and retained VEGF within the extracellular matrix. VEGFR2 autophosphorylation and endocytosis was abolished resulting in muted downstream kinase signalling and growth. In HNE spheroids, Flcn associated with ciliary structures and shared proximity with VEGFR2. In cells undergoing EMT, ciliary collapse was associated with a cystic morphology, loss of epithelial function and suppressed Flcn expression. We conclude that the association of Flcn with autocrine signalling and specialised cell structures points to a central role in epithelial phenotypic conservation and polarity. Supported by The Myrovlytis Trust.
123
418
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O11
FOLLICULIN GENE-ASSOCIATED MULTIPLE CYSTIC LUNG DISEASE Jean-François Cordier1, Camille Taillé2, Benoît Wallaert3, Dominique Valeyre4, the Groupe d’Etudes et de Recherche sur les Maladies “Orphelines” Pulmonaires (GERM“O”P)5 1 National Reference Center and Competence Centers2,3,4 for Rare Pulmonary Diseases in Lyon1, Paris (Bichat) 2, Lille3, Paris (Avicenne)4, France The Birt-Hogg-Dubé syndrome (BHDS) is the rarest and less known of the multiple cystic lung diseases( MCLD) which also comprise especially lymphangioleiomyomatosis and Langerhans cell granulomatosis (“the big three” MCLD). Pneumothorax especially relapsing is a major manifestation occurring in one third of patients with BHDS. MCLD in BHDS is characterized by lung cysts predominating in the lower medial zone of the lung (below the tracheal carina, inner half) and abutting or including the proximal portions of the lower arteries or veins. Lung function is usually relatively preserved. Because they share the common denominator of MCLD, we emphasize the respective distinguishing features of the “big three” in a series of 25 cases of BHDS from specialized centers and network in France. MCLD may be a manifestation of 1) the complete BHDS, 2) the spontaneous familial pneumothorax without overt BHDS, and 3) the only manifestation of FLCN gene mutation.
123
Abstracts
419
ABSTRACTS
ORAL COMMUNICATIONS
O12
BHD VERSUS NON-BHD PATIENTS WITH SPONTANEOUS PNEUMOTHORAX: CHEST CT FINDINGS Maartje J. Binnendijk1, Paul C. Johannesma1, Johan J.P. Gille2, Arjan C. Houweling2, Rinze Reinhard3, Theo M. Starink4, R. Jeroen A. van Moorselaar5, Jan Hein van Waesberghe3, Edward M. Leter2, Quinten Waisfisz2, Fred H. Menko2, Piet E. Postmus1 Departments of 1Respiratory Medicine, 2Clinical Genetics, 3Radiology, 4Dermatology, 5Urology, VU University Medical Center, Amsterdam, The Netherlands Introduction The pulmonary manifestations of Birt-Hogg-Dubé are lung cysts and pneumothorax. The aim of this study is to compare chest CT scans of pneumothorax patients with and without the FLCN mutation typical for BHD syndrome. Materials and methods Chest CT scans of 15 non-BHD (i.e. no FLCN mutation) patients (mean age 44,8) with spontaneous pneumothorax and 16 BHD patients with pneumothorax (mean age 46,2) were studied. All CT scans were scored on presence of cysts, craniocaudal distribution, number, size, and relation to pleura. Results In non-BHD patients, 6 of 15 (40%) had normal findings on CT, while in BHD patients with pneumothorax normal findings were seen in only 1 of 16 patients (6%). If present, the cysts in patients without BHD syndrome were mainly seen in the upper parts of the lung (6/9, 66,7%) and subpleurally (8/9, 89%). In BHD patients the abnormalities dominated the lower parts of the lung (11/15, 70%), and were located subpleurally (33,3%, 5/15), in the parenchyma (13,3%, 2/15), or both (53,3%, 8/15). Conclusion The pattern of abnormalities on chest CT of patients with pneumothorax due to BHD differs from non-BHD pneumothorax patients. A scoring system will be developed and tested on a subsequent group of pneumothorax patients with unknown etiology.
123
420
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O13
BHD PATIENTS WITH AND WITHOUT PNEUMOTHORAX: CHEST CT FINDINGS Maartje J. Binnendijk1, Paul C. Johannesma1, Johan J.P. Gille2, Arjan C. Houweling2, Rinze Reinhard3, Theo M. Starink4, R. Jeroen A. van Moorselaar5, Jan Hein van Waesberghe3, Edward M. Leter2, Quinten Waisfisz2, Fred H. Menko2, Piet E. Postmus1 Departments of 1Respiratory Medicine, 2Clinical Genetics, 3Radiology, 4Dermatology, 5 Urology, VU University Medical Center, Amsterdam, The Netherlands Introduction Birt-Hogg-Dubé syndrome is an autosomal dominant disorder characterized by fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax and renal cancer. The relationship between pulmonary cysts and pneumothorax risk in BHD has not been clarified. The aim is to describe CT characteristics of cysts in patients with BHD syndrome, with and without pneumothorax. Materials and methods Chest CTs of 17 patients with BHD but without pneumothorax (mean age 48,6), and 16 patients with BHD and pneumothorax (mean age 46,2) were studied. The presence of cysts, craniocaudal distribution, number, size, and relation to pleura were noted. Results Among the BHD patients with pneumothorax 15/16 (94%) had cysts, among those without pneumothorax 47% (8/17) had cysts. In BHD patients without pneumothorax, 5/8 (65%) had less than 10 cysts while in BHD patients with pneumothorax more than 10 cysts were seen in 9/15 patients (60%). Cysts in 5/8 (62,2%) in non-pneumothorax patients were located subpleurally and in 3/8 (37,5%) in the parenchyma, in the pneumothorax patients in 5/15 subpleurally (33,3%), 2/15 in the parenchyma (13,3%) and 8/15 in both areas (53,3%). Conclusion Cysts in the lungs of BHD patients are more common in the patients with pneumothorax, both in presence and number. In non-pneumothorax patients is the localization is more subpleurally than pneumothorax patients.
123
Abstracts
421
ABSTRACTS
ORAL COMMUNICATIONS
O14
THORACOSCOPIC SURGERY FOR PREVENTING RECURRENT PNEUMOTHORAX IN BHD SYNDROME: LOWER PLEURAL COVERING TECHNIQUE (LPC) Masatoshi Kurihara, Hideyuki Kataoka, Kuniaki Seyama, Toshio Kumasaka Pneumothorax Research Center & Thoracic surgery division, Nissan Tamagawa Hospital, Japan The study group for pathogenesis and treatments of pneumothorax and cystic lung diseases Introduction Chemical pleurodesis has been generally recommended for diffused cystic lung diseases with recurrent pneumothorax including BHDS. But the results are not always superior. LPC is a newly innovative surgery for preventing recurrent pneumothorax of BHDS. Patients and Methods LPC was thoracoscopically performed in 46 cases of BHDS with recurrent pneumothorax. Multiple lung cysts in BHDS are characteristically distributed over middle and lower lobes. We covered the middle and lower lobes at right lung, and the lower lobe at left lung with absorbable meshes. The regenerated oxidized cellulose (ROC) mesh for covering has the advantage to cause no adhesion to thoracic wall and visceral pleura reinforcement by thickened visceral pleura. Results There is no postoperative pneumothorax in 45 cases of BHDS after LPC. Discussion It is impossible to remove surgically all of lung cysts for BHDS. To remove them also lead to worsening the respiratory function as well. Chemical pleurodesis does not always lead to the good results for recurrent pneumothorax. LPC is surgery of innovative concept to thicken and reinforce visceral pleura by covering pleural surface with ROC mesh. Conclusion LPC for BHDS can prevent recurrent pneumothorax with maintaining respiratory function and no adhesion to thoracic wall.
123
422
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O15
AN UPDATE OF A BIRT-HOGG-DUBÉ SYNDROME (BHD) DATABASE: EVALUATION OF 89 FAMILIES REFERRED FOR SUSPECTED BHD Fred H. Menko, Paul C. Johannesma, Johan J.P. Gille, Arjan C. Houweling, Edward M. Leter, Marianne A. Jonker, Yael Kon, Annelinde Terlou, Theo M. Starink, Elisabeth H. Jaspars, Rinze Reinhard, Jan Hein van Waesberghe, R. Jeroen A. van Moorselaar, Piet E. Postmus, Cora Aalfs, Mirjam M. de Jong, Ernie MHF Bongers, Rogier A. Oldenburg, Theo A. van Os, Karin Y. van Spaendonck-Zwarts, Maurice A.M. van Steensel, Quinten Waisfisz VU University Medical Center, Amsterdam, the Netherlands; Dutch BHD Working Group; Dutch Working Group on Clinical Cancer Genetics Background information In the Netherlands, most families with Birt-Hogg-Dubé syndrome (BHD) have been registered in a central database which on March 1st 2013 numbered 89 families referred for possible BHD. Evaluation of the collected data may clarify the variable clinical expression of BHD, the spectrum of FLCN mutations and differential diagnosis. Aim of the study To evaluate the 89 registered families with respect to clinical diagnosis and molecular background. Results Pneumothorax as indication for referral was relatively frequent in recently ascertained families. Among the 89 families registered 56 had confirmed BHD due to a pathogenic FLCN mutation. The c.610_611delinsTA, p.Ala204X FLCN mutation in exon 6 was identified in ten kindreds and may represent a Dutch founder mutation. Nine families with a common ancestor had familial multiple discoid fibromas. Unusual families include a kindred with skin features compatible with BHD in which the FLCN locus was excluded; this kindred may represent a distinct syndrome. Conclusion The collection of clinical data of BHD families in central registers forms the basis of clinical and molecular evaluation of this syndrome. Future studies will include cancer risk assessment in the families with FLCN mutations and molecular studies of kindreds without an identified FLCN defect.
123
Abstracts
423
ABSTRACTS
ORAL COMMUNICATIONS
O16
FUMARATE PROMOTES ONGOING PSEUDOHYPOXIA THROUGH TBK1MEDIATED PHOSPHORYLATION OF RELA (P65) Karthigayan Shanmugasundaram1, EunHee Shim2, Karen Block1, Sunil Sudarshan2 Department of Medicine, University of Texas Health Sciences Center at San Antonio, USA; 2 Department of Urology, University of Alabama at Birmingham, USA
1
Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an inherited kidney cancer resulting from inactivating mutations of fumarate hydratase (FH). Tumor formation may result from the biochemical consequences due to FH loss, which leads to aberrant accumulation of the transcription factor hypoxia inducible factor (HIF), a phenomenon referred to as pseudohypoxia. To date, HIF-1_ accumulation in HLRCC tumors is thought to result from inhibition of prolyl hydroxylases (PHDs). PHDs normally target HIF for degradation mediated by the E3 ubiquitin ligase pVHL (von Hippel-Lindau). Here, we demonstrate that fumarate promotes HIF-1_ mRNA and protein accumulation in a VHL-independent manner. Cell models of FH loss demonstrate activation of RelA (p65), a component of the NFgB pathway. NF-gB signaling has previously been shown to promote HIF_ subunit transcription. Here we demonstrate that fumarate promotes p65 phosphorylation through the upstream kinase TBK1 (Tank Binding Kinase 1). Correspondingly, chromatin immunoprecipitation studies demonstrate that fumarate promotes p65 accumulation at the HIF-1_ promoter. Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1_ accumulation in cellular models of FH loss. Collectively, our data demonstrate a novel signaling node activated in HLRCC and could identify new therapeutic targets for treating FH deficient tumors.
123
424
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O17
HLRCC: INSIGHTS FROM MOUSE MODELS TO MAN Julie Adam1, Ming Yang1, Christina Bauerschmidt1, Tomoyoshi Soga2,3, Patrick J. Pollard1,4 Cancer Biology and Metabolism Group, 4Oxford-Keio Metabolomics Consortium, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, UK; 2Institute for Advanced Biosciences and 3Oxford-Keio Metabolomics Consortium, Keio University, 246-2 Mizukami, Tsuruoka, Yamagata 997-0052, Japan 1
Fumarate hydratase (FH) catalyses the hydration of fumarate to malate with roles in both mitochondria (Krebs cycle) and cytoplasm (metabolising fumarate generated in arginine synthesis and the purine nucleotide cycle). Using murine models we have shown that loss of FH activity, leading to accumulation of the oncometabolite fumarate, has multiple independent cellular consequences. These include the non-enzymatic modification via succination of cysteine residues in multiple proteins, including Kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase 2, resulting in their disrupted function. Also, we have demonstrated in vitro that in Fh1-null cells fumarate competitively inhibits 2-oxoglutarate-dependent dioxygenases, particularly hypoxia-inducible factor prolyl hydroxylases (HIF-PHDs), leading to HIF-stabilisation and activation of HIF-dependent oncogenic pathways. Re-expression of cytosolic FH ameliorates constitutive activation of both hypoxia and antioxidant response pathways, despite a persistent defect in oxidative metabolism. Previously we determined in a mouse model of Fh1-deficiency that hyperplastic renal cyst formation is HIF- and PHDindependent and have now demonstrated that re-expression of cytosolic FH is sufficient to rescue this phenotype and associated urea cycle metabolic defects. Our ongoing studies utilising high-resolution, mass spectrometry-based metabolomic analyses have emphasised the importance of cytosolic FH and extra-mitochondrial metabolic pathways in HLRCC.
123
Abstracts
425
ABSTRACTS
ORAL COMMUNICATIONS
O18
DIFFERENTIAL EXPRESSION OF CD44/DPYS IN HEREDITARY AND SPORADIC PAPILLARY RENAL CELL CARCINOMA TYPE 2 (PRCC2) DPYS AS THE POTENTIAL DIAGNOSTIC BIOMARKER Victoria Perrier-Trudova1,2,3, Aikseng Ooi2, Sophie Gad1, David Petillo2, Vincent Molinié4, Puay-Hoon Tan5, Aye Aye Thike5, Min-Han Tan6, Betty Gardie1, Stéphane Richard1,7, Bin Tean-Teh2,3 1 Laboratoire de Génétique Oncologique EPHE, INSERM U753, Faculté de Médecine Paris-Sud, Le KremlinBicêtre et Institut de cancérologie Gustave Roussy, Villejuif, France; 2Laboratory of Cancer Genetics, Van Andel Research Institute, Grand Rapids, MI 49503, USA; 3National Cancer Center Singapore-VARI Translational Lab, Singhealth Research Facilities & Duke-NUS Graduate Medical School, Singapore; 4 Service de Pathologie, Groupe Hospitalier Saint-Joseph, Paris, France; 5Department of Pathology, Singapore General Hospital, Singapore, Singapore; 6National Cancer Center Singapore, Singapore; 7 Centre Expert National Cancers Rares PREDIR, AP-HP, Hôpital Bicêtre, Le Kremlin-Bicêtre, France Papillary renal cell carcinoma type 2 (PRCC2) is known to be very aggressive type of tumor without efficient therapy. Hereditary form of PRCC2 is caused by Fumarate Hydratase (FH) gene mutation that accompanied Hereditary Leiomyomatosis and Renal Cell Carcinoma (HLRCC) disorder. In sporadic form of PRCC2 somatic mutation of FH gene has not been reported, however recent studies have shown somatic alterations of NRF2, CUL3 and SIRT1 genes. Both forms of tumors have the similar histopathological characteristics with bad survival prognosis. In this study we performed comparative genetic and molecular analysis carried out on renal tumors issued from PRCC2 patients. We performed microarray gene expression profiling on 5 hereditary, 19 sporadic cases and 8 normal renal tissues. Afterward we realized immunohistochemical staining of DPYS and CD44 in independent series of tumors and normal renal tissues. Our results show DPYS staining in 1/12 (8.3%) in hereditary cases and in 27/27 (100%) in sporadic PRCC2. CD44 overexpression in 12/12 (100%) in HLRCC cases and in 11/32 (34,5%) in sporadic cases. Here we propose to use DPYS as potential diagnostic biomarker and CD44 as the new protein potentially responsible for carcinogenesis in HLRCC. Eventually DPYS/ CD44 could be investigated as novel therapeutic targets in PRCC2. Singapore HLRCC Group: Bernice Wong Huimin, Song Ling Poon, Ee Yan Siew, Swe Swe Myint, Weng Khong, Choon Kiat Ong, Huang Da Chuan, Waraporn Chan-on, Chutima Subimerb, Heng Hong Lee, Anna Gan, Vikneswari Rajasegaran French HLRCC Group: Brigitte Bressac-de Paillerets, Johny Bombled, Bernard Escudier, Virginie Verkarre. Funded by the Fondation Institut Gustave Roussy.
123
426
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O19
THE ROLE OF MITOCHONDRIAL DNA MUTATIONS IN SYNDROMIC RENAL ONCOCYTOMAS Martin Lang, Cathy D. Vocke, Maria J. Merino, Laura S. Schmidt, W. Marston Linehan Urologic Oncology Branch and Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892; Basic Science Program, SAIC-Frederick, Inc., Frederick National Lab, Frederick, MD 21702 Oncocytomas are mostly benign tumors of epithelial origin, characterized by dense accumulation of defective mitochondria. A correlation of the oncocytic phenotype with the occurrence of disruptive mitochondrial DNA (mtDNA) mutations has been shown in sporadic oncocytomas of different organs, including renal oncocytomas (RO). However, the role mtDNA mutations play in renal tumors of Birt-Hogg-Dubé (BHD) patients and other RO with an apparent genetic component has not been investigated to date. In order to address this question we sequenced the entire mtDNA in seven cases of RO and 23 hybrid and chromophobe renal cell carcinomas from BHD patients, 24 samples of apparently sporadic bilateral and multifocal (BMF) RO as well as 9 oncocytomas from affected members of a family with recurrent RO (FRO). Our results show that BMF oncocytomas harbor disruptive mtDNA mutations affecting complex I of the respiratory chain and are related to renal oncocytosis. Also FRO samples present with mtDNA mutations, indicating a role in mtDNA quality control for the – yet unknown – gene responsible for this syndrome. However, BHD oncocytomas do not show disruptive mtDNA mutations, nor are mtDNA mutations found in other types of renal tumors of BHD patients. These results strengthen the role of FLCN in mitochondrial biogenesis. Funded in part by FIRC “Fellowship for abroad - 2011” to ML and by FNLCR Contract HHSN261200800001E to LSS.
123
Abstracts
427
ABSTRACTS
ORAL COMMUNICATIONS
O20
A FNIP1 BINDING PROTEIN IDENTIFIED BY YEAST TWO- SCREENING LINKS THE FLCN/FNIP1 COMPLEX WITH MEMBRANE TRAFFICKING Masaya Baba1, Hisashi Hasumi1, Yukiko Hasumi1, Ying Huang1, Robert M. Hughes1, Laura S. Schmidt1,2, W. Marston Linehan1 1 Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA; 2Basic Science Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, USA FNIP1 was cloned as a novel FLCN-interacting protein with no characterized functional domains. Endogenous FNIP1/FLCN interactions were confirmed and FNIP1 expression was demonstrated to alter FLCN phosphorylation status. FNIP1 knockout mice present with B-cell developmental defects identical to hematopoietic-targeted FLCN conditional knockout mice. Although these physical, physiological and genetic interactions between FLCN and FNIP1 emphasize the importance of FNIP1 to FLCN function, the precise molecular details are unknown. Based on protein analysis of size-exclusion column fractions FLCN and FNIP1 are components of a high molecular weight protein complex, suggesting the existence of additional FNIP1 or FLCN binding roteins. To elucidate FNIP1 function for further clarification of FLCN function, we conducted yeast two-hybrid screenings to search for FNIP1 binding proteins. Positive clones were isolated and verified for binding to FNIP1 by transient expression in mammalian cells, immunoprecipitation and western blotting. Among the positive clones, FNIP1 binding to a protein involved in membrane trafficking was confirmed. Moreover this protein was shown to bind to FLCN and AMPK through FNIP1. Transiently expressed FLCN colocalized with this FNIP1 binding protein in mammalian cells. These data suggest that the FLCN/FNIP1 complex plays a role in membrane trafficking. We will discuss the physiological meaning of these molecular interactions. Funded in part by FNLCR Contract HHSNS261200800001E.
123
428
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O21
FOLLICULIN AND FNIP ARE EVOLUTIONARY CONSERVED REGULATORS OF AMPK FUNCTION Arnim Pause1,2, Yann Nouët1,2,7, Zhara Jalali1,2,7, Elite Possik1,2,7, Ming Yan1,2, Marie-Claude Gingras1,2, Laëtitia Chotard1,2, Fanny Dupuy1, Elea Prezel1,2, Russell Jones1, Richard Roy4, Christian Rocheleau3, David Hall6 1 Goodman Cancer Research Center, McGill University, Montréal, Québec, H3A 1A3, Canada. 2 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. 3 Department of Medicine, McGill University, Montreal, Quebec, H3A 2B2, Canada. 4 Department of Biology, McGill University, Montréal, Québec, H3A 1B1, Canada. 6 Department of Neuroscience, Albert Einstein College of Medicine, NY10461, New York, USA. 7 These authors contributed equally to this work. AMPK plays a key role in energy sensing and metabolism. Energy depletion activates AMPK, leading to restored cellular energy levels by inhibiting anabolic energy consuming pathways and stimulating catabolic energy producing pathways. Here, we identified the BHD gene product FLCN as well as its binding partner FNIP as negative regulators of AMPK function using C. elegans and mammalian cells. Our results show that loss of FLCN/FNIP interaction with AMPK results in constitutive AMPK activation. Strikingly, we show that this constitutive AMPK activation leads to heightened ATP levels in worms and cells, which is dependent on increased autophagy. These results demonstrate that in the absence FLCN or FNIP, AMPK can no longer sense energy levels in the cell since AMPK could not be further activated by an AMP mimetic or energy deprivation treatments. Interestingly, upon metabolic stress or energy deprivation, FLCN and FNIP dissociate from AMPK leading to its activation by a kinase supporting the notion that FLCN/FNIP are components of the AMPK regulatory system. In conclusion, we identify FLCN and FNIP as new players regulating AMPK-function in response to metabolic stress, which involves AMPK mediated upregulation of aerobic glycolysis via HIF1alpha, mitochondrial biogenesis via PGC-1 and autophagy via ULK1.
123
Abstracts
429
ABSTRACTS
ORAL COMMUNICATIONS
O22
FLCN IS INVOLVED IN AUTOPHAGY REGULATION Elaine A. Dunlop1, Sara Seifan1, Ewelina Rozycka2, Ross Tomaino3, Barry Coull4, Ravi Nookala5, Tijs Classens4, James T. Murray2, John Blenis3, Maurice A.M. van Steensel4, Simon Wilkinson6, Andrew R. Tee1 1 Institute of Cancer and Genetics, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK; 2Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK: 3 Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; 4 Department of Dermatology, Maastricht University Medical Center, Maastricht, The Netherlands; 5 Department of Biochemistry, University of Cambridge , Sanger Building, 80 Tennis Court Road, Cambridge CB2 1GA, UK; 6Edinburgh Cancer Research UK Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Birt-Hogg-Dubé (BHD) syndrome is a rare genetic disorder and the BHD gene product, folliculin (FLCN), is considered a tumour suppressor. Mutations in the BHD gene predispose BHD patients to hair follicle tumours, pneumothorax and renal cancer. Of importance, we now have direct evidence that FLCN plays a role in the regulation of autophagy. Autophagy helps maintain cellular homeostasis through removal of damaged or non-essential organelles and macromolecules. FLCN appears to function at the level of autophagy initiation and we have uncovered that phosphorylation is important during this process which also involves specific members of the Rab family of small G proteins. Additionally, the interaction of FLCN with its binding partner, FNIP2, appears to be important for the direct association of FLCN with the autophagy machinery. As dysregulated autophagy is implicated in tumour development, this novel link between FLCN and the autophagy machinery could explain the enhanced cancer progression seen in BHD patients.
123
430
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O23
DELETION OF FLCN CAUSES CARDIAC HYPERTROPHY THROUGH AMPKMTOR AXIS DEREGULATION IN MICE Yukiko Hasumi1, Masaya Baba1, Hisashi Hasumi1, Ying Huang1, Antoine Smith2, Hyoungbin Oh1, Mara E. Klein1, Robert M. Hughes1, Kunio Nagashima3, Laura S. Schmidt1,4, Robert S. Adelstein2, W. Marston Linehan1 1 Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892; 2Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, 20892; 3Image Analysis Laboratory, Advanced Technology Program, and 4Basic Research Program, SAICFrederick, Inc., Frederick National Lab, Frederick, Maryland 21702 Non-physiologic cardiac hypertrophy is an irreversible disease leading to fatal heart failure. Cardiac hypertrophy occurs in response to various growth signals, of which the AMPK-mTOR signaling pathway is well recognized. Here, we show that loss of tumor suppressor FLCN causes fatal dilated cardiomyopathy with deregulated energy homeostasis in mice. mTORC1 was upregulated in hearts of mice with cardiac-targeted conditional FLCN inactivation. Rapamycin treatment significantly reduced heart size and ameliorated cardiac function. PhosphoAMPKa(T172) levels were reduced in FLCN-deficient hearts relative to littermate controls, although they were nonresponsive to metformin treatment. ATP levels were elevated in FLCNdeficient hearts and FLCN-null MEFs leading to the hypothesis that excess energy resulting from FLCN inactivation attenuated AMPK activity. Cardiac-targeted genetic inactivation of PPARGC1A, a key molecule for energy homeostasis, in mice with FLCN-deficient hearts significantly reduced heart size and prolonged survival. PPARGC1A inactivation restored AMPK activity and suppressed mTORC1 in FLCN-deficient hearts. However, rapamycin treatment did not affect heart size of cardiac-targeted FLCN/PPARGC1A double-knockout mice suggesting that PPARGC1A is critical for FLCN-deficient cardiac hypertrophy through deregulation of the AMPKmTOR-axis. These data support an important role for FLCN in cardiac function and may provide novel insight into the mechanism of cardiac hypertrophy development. Funded in part by FNLCR Contract HHSNS261200800001E.
123
Abstracts
431
ABSTRACTS
ORAL COMMUNICATIONS
O24
FOLLICULIN IN ORGANISMAL LONGEVITY AND CELLULAR STRESS RESISTANCE Hakam Gharbi1,2, Francesca Fabretti1, Puneet Bharill1, Markus Rinschen1, Sibylle Brinkkötter1, Peter Frommolt2, Volker Burst1, Bernhard Schermer1,2,3, Thomas Benzing1,2,3, Roman-Ulrich Müller1,2,3 1 Department 2 of Internal Medicine and Center for Molecular Medicine, University of Cologne, Cologne, Germany; 2Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany; 3Systems Biology of Aging Cologne, University of Cologne, Cologne, Germany Birt-Hogg-Dubé (BHD) is an autosomal dominant multi-tumor syndrome. FLCN, the protein encoded by the BHD gene, appears to be involved in a densely entangled signaling network comprising AMPK, HIF, TGF-, JAK-STAT and mTOR signaling pathways. To better understand the role of FLCN, we chose the model organism Caenorhabditis elegans. We can now show that loss of the C. elegans ortholog flcn-1 plays a role in lifespan regulation. Just like pVHL (von Hippel-Lindau protein), FLCN is a renal tumor suppressor protein which exerts its effect on nematode lifespan and stress resistance in a manner involving the hypoxiainducible factor (HIF). flcn-1 mutants live longer and show increased thermotolerance as compared to wild type. While loss of hif-1 abrogates this lifespan extension, HIF-1 stabilization through vhl-1 deficiency does not further increase it. Moreover, these effects are independent from insulin-like signaling: daf-16 (the ortholog of human FOXO3) deficiency does neither influence lifespan extension nor heat resistance mediated by flcn-1 and knock-down of daf-2 (insulin receptor-like gene) expression had no effect on the lifespan extension mediated by FLCN. Using the nematode to examine flcn-1 holds the promise to help greatly getting a better understanding of the molecular basis underlying the BHD syndrome due to the unique features of this amazing model organism.
123
432
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O25
ALTERATIONS IN METABOLIC FLUX UNDER FLCN DEFICIENCY Hisashi Hasumi1, Masaya Baba1, Yukiko Hasumi1, Ying Huang1, Hyoungbin Oh1, Robert M. Hughes1, Mara E. Klein1, Shoichi Takikita4, Kunio Nagashima3, Oksana Gavrilova5, Samuel W. Cushman6, Laura S. Schmidt1,2, W. Marston Linehan1 1 Urologic Oncology Branch, CCR, NCI, NIH, Bethesda, MD USA; 2Basic Science Program and 3Electron Microscopy Lab, Advanced Technology Program, SAIC-Frederick, Inc., Frederick National Lab, Frederick, MD USA; 4Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, MD USA; 5Mouse Metabolic Core Facility and 6Diabetes, Endocrinology and Obesity Branch, NIDDK, National Institutes of Health, Bethesda, MD USA Birt-Hogg-Dubé (BHD) syndrome is an inherited kidney cancer syndrome, which predisposes patients to renal tumors, fibrofolliculomas, and lung cysts. The causative gene, FLCN, is a tumor suppressor that encodes folliculin (FLCN), a novel protein with unknown function. To investigate a role for FLCN in cell metabolism, we conducted metabolic assays using muscletargeted FLCN conditional knockout mice. Muscle-targeted FLCN knockout mice displayed red-colored muscle with increased mitochondrial biogenesis. FLCN-deficient muscle also demonstrated increases in metabolites related to fatty acid oxidation, cofactors for the electron transport chain, and respiratory capacity. Interestingly, glucose clamp tests revealed that insulin sensitivity was increased in muscle-targeted FLCN knockout mice. Glucose uptake and biogenesis of glucose transporters were increased in FLCN-deficient muscle. However, 3-phosphoglycerate was dramatically decreased whereas fructose 1,6-bisphosphate was significantly increased in FLCNdeficient muscle. Metabolites of glycogenesis and pentose phosphate pathway were increased as well suggesting that increased ATP production in FLCNdeficient muscle is attributed to increased mitochondrial biogenesis under FLCN deficiency and that increased glucose uptake drives the production of glycogen and co-enzymes involved in redox reactions. These data support an important role for FLCN in metabolism and may lead to novel management and targeted therapeutic approaches for patients with BHD-associated kidney cancer. Funded by FNLCR Contract HHSNS261200800001E.
123
Abstracts
433
ABSTRACTS
ORAL COMMUNICATIONS
O26
PERMANENT GENETIC CORRECTION OF THE PHENOTYPE OF BHD CELLS USING EPISOMALLY MAINTAINED DNA VECTORS Suet Ping Wong, Richard P. Harbottle Section of Molecular Medicine, Imperial College London, United Kingdom, SW7 2AZ The simple, stable and efficient application of episomal DNA vectors to genetically modify dividing cells without the risk of integration-mediated genotoxicity provides a valuable tool in cell biology research. Here, we demonstrate the utility of Scaffold/Matrix Attachment Region (S/MAR) DNA plasmid vectors to rapidly and simply generate novel genetically modified cell lines. In this study we utilize these vectors to model the restoration of a functional wild-type copy of the gene implicated in the renal cancer Birt-Hogg-Dube (BHD) in a cell-line (UOK257) derived from a BHD patient. Persistent genetic correction of UOK257 cells with an S/MAR-FLCN plasmid (UOK257-FS) restores FLCN expression and normalises downstream TGF` signalling. The corrected cells also show a reduced growth rate in vitro and suppressed xenograft tumour development in vivo, compared to the original cell line. This study demonstrates the suitability of episomally maintained S/MAR DNA vectors to successfully model the persistent functional expression of a therapeutic gene in a cancer cell line. It should also establish this class of vectors as effective tools for investigating signalling components differentially regulated in different cancers and to aid the identification of novel cancer markers for diagnosis and therapy.
123
434
Abstracts
ABSTRACTS
ORAL COMMUNICATIONS
O27
EFFICACY OF AZD8055, A DUAL MTORC1/MTORC2 KINASE INHIBITOR, IN THE NIHON RAT TUMOR MODEL OF BHD Laura S. Schmidt1,5, Lilia V. Ileva2, Joseph D. Kalen2, Diana C. Haines3, Daniel L. Logsdon4, Craig L. Driver4, Amy M. James4, Yukiko Hasumi5, Masaya Baba5, Sylvie Guichard6, Ramaprasad Srinivasan5, W. Marston Linehan5 1 Basic Science Program, 2Small Animal Imaging Program, 3Pathology/Histology Laboratory and 4 Laboratory of Animal Sciences Program, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD USA; 5Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD USA; 6AstraZeneca, Oncology Innovative Medicines, Waltham, MA USA Patients with Birt-Hogg-Dubé syndrome caused by germline mutations in the FLCN gene have an increased risk for developing bilateral multifocal kidney tumors requiring life-long screening for early diagnosis and effective management of their disease. Molecular targeted therapeutics for BHD-associated renal tumors have not been administered in the clinical setting because response data in preclinical in vivo models are lacking. Our previous studies in genetically engineered Flcn-deficient mouse models demonstrated that FLCN inactivation leads to dysregulation of the mTOR pathway with partial reversal of the kidney phenotype by rapamycin treatment. In this study we evaluated the efficacy of a novel agent targeting the mTOR pathway in the Nihon rat tumor model that more closely mimics the human BHD kidney phenotype. This model harbors a germline frameshift mutation in the rat Flcn gene leading to kidney tumors that are measurable on imaging by 6 months of age. Thirty-nine week-old rats were orally administered AZD8055, a dual mTORC1/mTORC2 kinase inhibitor, at 7.5 mg/ kg/b.i.d. or vehicle, and monitored at 3-week intervals by in vivo magnetic resonance imaging with Magnevist. After 15 weeks of treatment, there were no significant differences in tumor size or growth between control and treatment arms. Future plans include screening secondgeneration mTOR inhibitors in this preclinical tumor model of BHD. Funded in part by FNLCR Contract HHSNS261200800001E.
123
Abstracts
435
ABSTRACT
Saturday, June 29, 2013 Conference room: MICHEL-ANGE AMPHITHEATER 3:20 pm
LATE BREAKING COMMUNICATION BIRT-HOGG-DUBÉ SYNDROME IS A CILIOPATHY Monique N. H. Luijten1,+, Sander G. Basten2,3,+, Tijs Claessens1,5,+, Marigje Vernooij1, Claire L. Scott4, Renske Janssen1, Jennifer A. Easton1, Miriam A. F. Kamps1, Maaike Vreeburg6, Jos L. V. Broers7, Michel van Geel1, Fred H. Menko8, Richard P. Harbottle9, Ravi K. Nookala10, Andrew R. Tee5, Stephen C. Land4, Rachel Giles2, Barry J. Coull1,*, Maurice A. M. van Steensel1,6 1 Department of Dermatology and GROW School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands 2 Department of Nephrology and Hypertension University Medical Centre Utrecht, Heidelberglaan 100 F03.233, 3584CX Utrecht, The Netherlands 3 Department of Medical Oncology, University Medical Centre Utrecht, Heidelberglaan 100 F02.126 3584CX Utrecht, The Netherlands 4 Division of Cardiovascular and Diabetes Medicine, Medical Research Institute Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY Scotland, UK 5 Institute of Medical Genetics Cancer, Genetics Building, Cardiff University, Heath Park Way, Cardiff CF14 4XN UK 6 Department of Molecular Cell Biology GROW School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands 7 Department of Clinical Genetics Maastricht University Medical Centre Maastricht The Netherlands 8 Department of Clinical Genetics VU Medical Centre Amsterdam, The Netherlands 9 Gene Therapy Research Group Molecular Medicine Imperial College London Sir Alexander Fleming Building Imperial College Road South Kensington London SW7 2AZ UK 10 Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts, and benign skin tumours. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumour suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicates that FLCN localises to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarised growth of kidney cells and deregulates canonical WNT signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.
123
436
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C1
PHENOTYPIC AND GENOTYPIC DESCRIPTION OF 10 BHD FAMILIES FROM 2 DISTINCT PREDIR REGIONAL CENTERS. Caroline Abadie1, Catherine Dugast1, Brivael Gery1, Carole Corsini2, Sylvie Odent1, Sophie Giraud3, Stéphane Richard4,5, Isabelle Coupier2 1 Unité de génétique Clinique, Centre hospitalier Univeristaire Rennes, Rennes, France; 2Service de génétique médicale, Oncogénétique, Centre hospitalier Universitaire Montpellier, Montpellier, France; 3 Laboratoire de génétique moléculaire, hôpital Edouard Herriot, hospices civils de Lyon, université Claude-Bernard, Lyon, France; 4Réseau National et Centre Expert National Cancers Rares PREDIR, Service d’Urologie, AP-HP, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France; 5Laboratoire de Génétique Oncologique EPHE, INSERM U753, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre et Institut de cancérologie Gustave Roussy, Villejuif, France Association of benign cutaneous, lung lesions and increased kidney cancer risk characterize the Birt-Hogg-Dubé (BHD) syndrome, whose early diagnosis is challenging. In France, the PREDIR center encompasses 25 regional centers aiming an accurate and effective management of patients with renal tumour predispositions like BHD syndrome. We report phenotypic and genotypic description of 10 BHD families coming from 2 geographical distinct regional PREDIR centers, Rennes (West) and Montpellier (South of France). Folliculin gene analysis was performed for 23 patients including 13 for relatives with 7 already symptomatic. Mean age for symptomatic cases was similar for index cases (50) than for relatives (51). Typical fibrofolliculomas were seen in 17 patients (100% of symptomatic cases), spontaneous pneumothorax in 7 patients and no kidney cancer case but cysts found in 6 patients. Interestingly, we report one case of parotid oncocytoma, thyroid benign lesions and colon polyps for 8 and 5 patients respectively. Folliculin gene analysis revealed 8 distinct deleterious mutations including 2 recurrent in each centers, the hotspot c.1285dupC (South) and the splice mutation c.1300G>A (West). Our findings are consistent with literature despite bias of dermatologic recruitment in both PREDIR centers but maintain the debate of thyroid and colonic associated lesions
123
Abstracts
437
ABSTRACTS
POSTER COMMUNICATIONS
C2
RADIOFREQUENCY ABLATION: AN ALTERNATIVE TREATMENT TO SURGERY IN BIRT-HOGG-DUBÉ SYNDROME Mohamed Karji Almarzooqi1, Stéphane Richard2,3, Dominique Joly3,4,5, Arnaud Méjean3,5,6, Olivier Hélénon1,6, Jean Michel Corréas1,3,6 1 Department of Adult Radiology, Necker University Hospital, Paris; 2Génétique Oncologique EPHE, Faculté de Médecine Paris-Sud, Le Kremlin Bicêtre and INSERM U753, Villejuif, France; 3 Centre Expert National Cancers Rares PREDIR, AP-HP/INCa, Le Kremlin Bicêtre; 4Department of Adult Nephrology, Necker University Hospital, Paris; 5Paris-Descartes University, Paris; 6 Department of Urology, Necker University Hospital 1 European Hospital Georges Pompidou, Paris, FRANCE Birt-Hogg-Dubé (BHD) syndrome is an autosomal, dominantly inherited disease including skin lesions, pulmonary cysts, pneumothorax and kidney lesions (including oncocytoma, chromophobe RCC and hybrid tumors). They require specific management in order to limit renal function degradation. Treatment options rely on partial nephrectomy or minimally conservation therapy. Radiofrequency ablation (RFA) was used successfully in 3 BHD patients and 7 tumors, with diameter ranging from 18 to 45 mm, from April 2007 to June 2012. Procedural success and adverse events following RFA were assessed for each procedure. All procedures were conducted under local anesthesia and conscious sedation. The primary success rate was 100% despite the size of the largest lesions, probably because of their limited degree of vascularity as assessed by dynamic MRI. No death, peri-renal hemorrhage or bowel perforation was reported. After one procedure the patient developed transient acute renal failure due to subcapsular hematoma and contrast-induced nephropathy following low-osmolality iodinated contrast media administration. In all other procedures, the post procedural renal function assessed by the GFR calculated using the MDRD formula was significantly different to the preprocedural dosage. RFA is an efficient and safe alternative to surgery, allowing conservative management of renal tumors.
123
438
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C3
CUTANEOUS AND RENAL PRESENTATION OF PTEN HAMARTOMA TUMOR SYNDROME Blandine Bel1, Mathilde Funes de la Vega2, Eric Mourey3, Laurence Faivre4, Michel Longy5, Pierre Vabres1 Dept of Dermatology, CHU Dijon, France; 2Dept of Anatomopathology, CHU Dijon, France; 3 Dept of Urology, CHU Dijon, France; 4Dept of Genetics, CHU Dijon, France; 5Dept of Genetics, Institut Bergonié, Bordeaux, France
1
Introduction The most common neoplasms associated with PTEN hamartoma tumor syndrome (PHTS) are breast, thyroid and endometrial carcinomas. Increased risk of renal cancer has also been suggested. Observation A 16 year-old patient had multiple arteriovenous malformations (AVMs) with macrocrania and penile freckling. No other signs of Cowden / Bannayan syndrome were present. Chromophobe renal cell carcinoma (RCC, Fuhrman grade II) was serendipitously diagnosed on MRI imaging of AVMs. He underwent total nephrectomy. A de novo germ line PTEN mutation (c.808delA; p.Met270Cys fsX6) was found. Immunohistochemistry of the renal tumor showed complete loss of PTEN expression. Discussion A recent study found a history of nine RCCs among 219 PTEN mutation positive patients (ageadjusted standardized incidence ratio = 31.7; median age 49). Six were papillary and two were chromophobe RCCs. PTEN expression was abolished in five papillary RCCs, and one chromophobe RCC exhibited patchy labelling. Here we found complete loss of PTEN expression in a chromophobe RCC. Hence, PTEN could be a major gene involved in RCC genesis. Conclusion Early-onset chromophobe RCC may be a diagnostic clue to PHTS. This observation also raises the issue of RCC screening and surveillance in patients with PTEN mutations.
123
Abstracts
439
ABSTRACTS
POSTER COMMUNICATIONS
C4
METASTATIC RENAL CELL CARCINOMA IN BIRT-HOGG-DUBÉ PATIENTS Patrick Benusiglio1, Sophie Deveaux1, Arnaud Méjean1, Jean-Michel Corréas1, Bernard Escudier1,2, Stanislas Ropert3, Ayhan Ulusakarya4, Sophie Ferlicot1, Sophie Giraud1,5, Stéphane Richard1 1 Centre Expert National Cancers Rares PREDIR, AP-HP/INCa, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; 2Département de Médecine Oncologique, Institut Gustave Roussy, Villejuif, France; 3 Service d’Oncologie Médicale, Centre Hospitalier Universitaire Cochin, Paris, France; 4 Service d’Oncologie Générale, Centre Hospitalier Universitaire Paul Brousse, Villejuif, France; 5 Laboratoire de Génétique, Hôpital Edouard Herriot, Lyon, France Metastatic renal cell carcinoma (RCC) is rare in patients with Birt-Hogg-Dubé syndrome (BHD). In the two largest series of BHD families, the investigators reported that out of 18 and 14 carriers with RCC, zero and five had metastatic disease, respectively. We studied retrospectively all French BHD patients with a history of RCC, and report herein those with metastatic disease. We explored the French Hereditary RCC database. It contains detailed information for all individuals with a FLCN mutation. Out of 15 BHD patients with RCC, three had metastatic disease. Patient 1 had unclassified RCC age 35, he had undergone successful nephrectomy at diagnosis. He presented age 58 with an isolated lung metastasis that was surgically removed. Patient 2 had right nephrectomy age 56 for a chromophobe RCC. Multiple involved retroperitoneal lymph-nodes had to be left in place. Patient 3 was diagnosed age 42 with oncocytoma-chromophobe bilateral RCC and liver metastases. We have not heard from patient 1 since metastasectomy, while patients 2 and 3 are clinically well seven years after metastatic disease was diagnosed, and the retroperitoneal and liver metastases are radiologically stable. In conclusion, the prognosis associated with metastatic RCC in BHD seems favorable as metatastic disease can remain stable for years. Funded in part by the Ligue Nationale contre le Cancer (Comités du Cher et de l’Indre).
123
440
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C5
A CLINICOPATHOLOGIC STUDY OF 72 INDIVIDUALS IN 33 ASIAN BHD FAMILIES Mitsuko Furuya1, Reiko Tanaka3, Masahiro Yao2, Yoji Nagashima1, Shunsuke Koga4, Yukio Nakatani5 Department of Pathology1 and Urology2, Yokohama City University, Yokohama Japan. 3 Medical Mycology Research Center, Chiba University, Chiba, Japan. 4,5Department of Neurology4 and Diagnostic Pathology5, Chiba University Graduate School of Medicine, Chiba, Japan
1,2
We investigated clinicopathologic features of 72 individuals from 33 Asian Birt-Hogg-Dubé syndrome (BHD) families. Genetic testing revealed 10 different mutations including 6 unique patterns. Histopathologic analysis of the lung specimens of BHD patients who received thoracic surgery (n=12) elucidated the complexity of pulmonary cysts in morphology, clinical course and genetic condition. The cells making up the pulmonary cysts stained positive for phospho-mTOR, phospho-S6, HIF-1, and VEGF, suggesting deranged mTOR and angiogenic signaling. Not only patients with large cysts but also with small-sized cysts undetectable by thoracic CT caused repeated pneumothorax. Among 41 patients who received genetic testing, 15 (36.6%) had renal cell carcinomas (RCC). 8 of 15 (53.3%) RCC cases belonged to the families with cytosine duplication (c.1285dupC) in the C8 tract of exon 11. Among 12 individuals who had c.1285dupC mutation confirmed by genetic testing, 7 had RCC (58.3%). On the other hand, none of 9 individuals who belonged to the families with c.1347_1353dupCCACCCT in exon 12 had RCC. The results indicate possible propensity of developing RCC in the patients with specific mutation patterns. 2 patients with RCC had lung metastasis, and the histology of both cases was papillary RCC.
123
Abstracts
441
ABSTRACTS
POSTER COMMUNICATIONS
C6
FOLLICULIN IS AN EVOLUTIONARY CONSERVED REGULATOR OF AMPK FUNCTION MEDIATING METABOLIC STRESS SURVIVAL TROUGH AUTOPHAGY Zahra Jalali1,2,7, Yann Nouët1,2,7,Elite Possik1,2,7, Ming Yan1,2, Marie-Claude Gingras1,2 Laëtitia Chotard1,2, Fanny Dupuy1, Elea Prezel1,2, Russell Jones1, Richard Roy4, Christian Rocheleau3, David Hall6, Arnim Pause1,2 5 1 Goodman Cancer Research Center, McGill University, Montréal, Québec, H3A 1A3, Canada. 2 Department of Biochemistry, McGill University, Montréal, Québec, H3G 1Y6, Canada. 3 Department of Medicine, Division of Experimental Medicine, McGill University, Montreal, Quebec, H3A 2B2, Canada; 4Department of Biology, McGill University, Montréal, Québec, H3A 1B1, Canada. 6 Department of Neuroscience, Albert Einstein College of Medicine, NY10461, New York, USA. 7These authors contributed equally to this work AMK is highly conserved enzyme complex that plays a key role in energy sensing and metabolism. Energy depletion activates AMPK, leading to restored cellular energy levels by inhibiting anabolic energy consuming pathways and stimulating catabolic energy producing pathways. AMPK was recently shown to directly regulate autophagy, a catabolic mechanism characterized by degradation of cellular components leading to generation of ATP and resistance to metabolic stress. Here, we identified the BHD gene product FLCN as a negative regulator of AMPK function using C. elegans and mammalian cells. Our results show that loss of FLCN interaction with AMPK results in its constitutive activation. We consequently tested whether FLCN loss of could withstand various energy stresses. We observed a significant AMPKdependent resistance to all metabolic stresses upon loss of FLCN. FLCN deletion enhanced AMPKdependent autophagy and ablation of autophagy genes abrogated resistance to metabolic stresses in both systems. Strikingly, we show that FLCN interacts with AMPK thereby competitively preventing complex formation between AMPK and the autophagy initiating kinase ULK1 leading to autophagy inhibition. Upon metabolic stress, FLCN dissociates from AMPK leading to an increased formation of the AMPK/ULK1 complex, which in turn initiated autophagy. Importantly, we demonstrate that increased autophagy in FLCN knockout animals and cells is sufficient to sustain energy (ATP) availability leading to blocked apoptosis and enhanced survival. In conclusion, we identify FLCN as a new player regulating AMPK-ULK1 interaction and autophagy in response to metabolic stress to sustain ATP levels and trigger resistance to energy stress.
123
442
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C7
PREVALENCE OF BIRT-HOGG-DUBÉ SYNDROME AMONG PATIENTS WITH SPONTANEOUS PNEUMOTHORAX: PRELIMINARY RESULTS Paul C. Johannesma1, Maartje J. Binnendijk1, Rinze Reinhard2, Theo M. Starink3, Yaël Kon3, Annelinde Terlou3, R. Jeroen A. van Moorselaar4, Jan-Hein van Waesberghe2, Arjan C. Houweling5, Edward M. Leter5, Quinten Waisfisz5, Johan J.P. Gille5, Fred H. Menko5, Pieter E. Postmus1 Departments of 1Respiratory Medicine, 2Radiology, 3Dermatology, 4Urology, 5Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands Introduction Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant condition characterized by variable expression of skin fibrofolliculomas, pneumothorax and renal cancer. The prevalence of BHD among patients who present with spontaneous pneumothorax (SP) has not been fully clarified. Aim of the study To assess the prevalence of BHD among unselected patients with a history of spontaneous pneumothorax of unknown cause. Material and Methods We invited 380 patients, who were treated for SP between 1990 and 2005 to participate in this study aimed at diagnosing possible BHD. 93 patients returned a questionnaire and 24 patients participated in the study. Preliminary results Among the 24 patients 11 had a positive family history for pneumothorax. A pathogenic FLCN mutation was found in one of 13 cases with non-familial SP (male, age 26) and in one case with familial pneumothorax (female, age 20). Both cases had multiple basal long cysts, the patient with familial SP had undiagnosed fibrofolliculomas. No renal tumours or renal cysts were found in either patient. Conclusion FLCN mutations were found in both a familial and an apparently sporadic case of SP. Due to the low response rate we will extend our studies to 60 patients diagnosed with SP since 2005. These studies will show whether FLCN mutation analysis may be indicated not only in familial SP but also in non-familial cases.
123
Abstracts
443
ABSTRACTS
POSTER COMMUNICATIONS
C8
EXOME SEQUENCING AND SNP ARRAY ANALYSIS OF HLRCC SYNDROMIC UTERINE LEIOMYOMAS Kati Kämpjärvi, Miika Mehine, Pia Vahteristo, Lauri A. Aaltonen Department of Medical Genetics, Genome-Scale Biology Research Program, University of Helsinki, Helsinki, Finland Women with Hereditary Leiomyomatosis and Renal Cell Cancer syndrome (HLRCC) have an 8-to 9-fold increased risk of developing uterine leiomyomas (ULMs). These lesions are benign smooth muscle tumors of the uterus which may cause various symptoms including severe pain, excessive bleeding, infertility, and pregnancy complications. They are the most common indication for hysterectomy. We recently identified somatic mutations in mediator complex subunit 12 (MED12) in as many as 70% of sporadic ULMs. Traditionally, ULMs have been considered rather stable, although some recurrent genomic aberrations affecting mainly chromosomes 7q, 12q15, and 6p21 have been reported. To systematically characterize the genomic landscape of FH (fumarate hydratase) deficient ULMs, we analyzed altogether 10 ULMs from two Finnish HLRCC-family members and one sporadic ULM sample with two somatic changes in FH as well as their corresponding myometrial samples utilizing exome sequencing and SNP arrays. These efforts did not reveal any recurrent somatic variations or copy number alterations in addition to the changes affecting the FH locus at 1q42. FH inactivation damages the tricarboxylic acid cycle and causes a severe metabolic stress for the cells, which may be sufficient to induce formation of these benign uterine lesions
123
444
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C9
INTERPRETATION OF UNCLASSIFIED VARIANTS IN THE FUMARATE HYDRATASE-(FH) GENE BY USING IMMUNOHISTOCHEMISTRY FOR 2-SUCCINYL CYSTEINE (2SC) C. Marleen Kets1, Christina A. Hulsbergen-van der Kaa2, Maran J. Olderode-Berends3, Fred H. Menko4, Arjen R. Mensenkamp1 1 Department of Human Genetics, Radboud University Nijmegen Medical Centre, the Netherlands; 2 Department of Pathology, Radboud University Nijmegen Medical Centre, the Netherlands; 3 Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands; 4Department of Clinical Genetics, VU University Medical Center, Amsterdam, the Netherlands. Carriers of germline FH mutations are at risk of developing multiple leiomyomas and renal cancer as part of the HLRCC (hereditary leiomyomatosis and renal cell cancer) syndrome. Missense mutations in the FH gene are not always clearly pathogenic. In those cases more information on the effect of the mutation at the cellular level is helpful. In HLRCC intracellular fumarate accumulates to high levels reacting with cysteine sulphydryl groups to form the stable protein S-(2-succinyl) cysteine (2SC). Using immunohistochemistry for 2SC for instance in a leiomyoma, presence of 2SC-modified proteins can be shown, indicating fumarate hydratase deficiency. At Radboud University Nijmegen Medical Centre we analyzed cutaneous and uterine leiomyomas and a renal cell carcinoma of four patients with unclassified variants and four patients with a truncating mutation in the FH-gene by 2SC-immunohistochemistry. In all cases accumulation of 2SC-modified proteins was shown, in line with pathogenicity of the unclassified variants. As expected no 2SC proteins were expressed in the control tissues nor in four tumours from patients in which no mutation was found. Conclusion: Accumulation of 2SC in tumours of patients with unclassified variants the FH gene detected by immunohistochemistry strongly supports the pathogenicity of these variants in a clinical diagnostic setting.
123
Abstracts
445
ABSTRACTS
POSTER COMMUNICATIONS
C10
MALIGNANT FIBROUS HISTIOCYTOMA OCCURRING IN A PREVIOUSLY UNDIAGNOSED BIRT-HOGG-DUBÉ PATIENT: A CASE-REPORT Nicolas Poulalhon, Luc Thomas Department of dermatology, Lyon 1 University, Centre Hospitalier Lyon Sud, 69495 Pierre-Bénite cedex, France Malignant fibrous histiocytoma (MFH), also called undifferentiated pleomorphic sarcoma (World Health Organization, 2002), is a malignant neoplasm arising most often in soft tissue or bone. We describe the case of a 52-year old male who presented with a 1-cm, fast-growing hyperkeratotic nodule on his left leg, documented histologically as a MFH developed in the dermis (FNCLCC grade: 2). His medical history included left pleural talcage due to bilateral relapsing spontaneous pneumothorax. On physical examination, we noticed multiple whitish papules located on his nose, cheeks and supra-clavicular area. Whole-body computed tomography was unremarkable except for multiples lung cysts. Thus, our patient was treated by surgery only (complementary excision with 2-cm lateral margins). Besides, pathological examination of a representative papule of the trunk concluded to fibrofolliculoma. Therefore, Birt-Hogg-Dubé syndrome (BHD) was diagnosed. A heterozygous germinal mutation in FLCN gene was found in exon 14 (c.1566del; p.Lys523fs). To our knowledge, the onset of MFH in BHD patients has not been described before; however, melanoma and other primary cutaneous sarcomas (dermatofibrosarcoma protuberans, leiomyosarcoma) have been reported in this setting. Therefore, it would seem advisable to include skin examination not only in the initial management, but also in the follow-up of BHD patients.
123
446
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C11
EXPLORING FLCN-HIF1ı CROSS-REGULATION: IMMUNOHISTOCHEMICAL AND MOLECULAR STUDIES Laura Maria Pradella1, Claudia Ligorio 2, Giovanni Tallini 2, Marco Seri 1, Daniela Turchetti 1, Giuseppe Gasparre1 1 University of Bologna, Department of Medical and Surgical Sciences, Unit of Medical Genetics, Bologna, Italy; 2University of Bologna, Bellaria Hospital, Section of Anatomic Pathology “M. Malpighi”, Bologna, Italy. The BHD gene, FLCN, is a tumor suppressor gene that does not undergo LOH in all tumors1. We previously showed that in two patients with BHD and Cowden syndrome, which displays large similarities to BHD, lack of FLCN LOH may be mirrored by somatic inactivation of one PTEN allele2. These findings occur within the context of a peculiar cancer phenotype, namely oncocytoma, where mitochondrial biogenesis is aberrantly activated, concordantly with the prominent role of FLCN (and PTEN) on PGC1_, the master regulator of mitochondrial proliferation. Sporadic oncocytomas generally lack HIF1_ stabilization3, whose activity is not compatible with a mitochondrial biogenesis increase. We reasoned that FLCN-HIF_ crossregulation, as previously suggested, may be important in oncocytic tumors and provide insight into the different cancer phenotype that may develop according to whether loss of the wild-type FLCN allele occurs. We hence optimized immunohistochemistry for FLCN and HIF1_. Preliminary results on sporadic and syndromic oncocytoma and controls show that lack of HIF1_ correlates with lack of FLCN, even in FLCN+/+ or FLCN+/- samples, suggesting post-transcriptional mechanisms may phenocopy a second FLCN somatic hit. Molecular studies in an oncocytic cell models preliminarily support the hypothesis that HIF1_ stabilization may positively regulate FLCN expression. 1
Schmidt LS et al., Fam Cancer 2012. Pradella LM et al., EJHG 2013. 3 Porcelli AM et al., HMG 2010. 2
123
Abstracts
447
ABSTRACTS
POSTER COMMUNICATIONS
C12
PULMONARY ARTERIAL HYPERTENSION IN A PATIENT WITH BIRT-HOGGDUBÉ SYNDROME AND RENAL INSUFFICIENCY Laurent Savale1, Elise Artaud-Macari1, Barbara Girerd1, David Montani1, Marc Humbert1, Gérard Simonneau1, Olivier Sitbon1, Sophie Giraud2,3, Stéphane Richard2 1 AP-HP, Service de Pneumologie, DHU Thorax Innovation, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; 2 Centre Expert National Cancers Rares PREDIR, AP-HP/INCa, Hôpital Bicêtre, Le Kremlin-Bicêtre, France; 3 Laboratoire de Génétique, Hôpital Edouard Herriot, Lyon, France Birt-Hogg-Dubbé (BHD) syndrome is an autosomal dominant disorder characterized by mutations in the gene encoding folliculin (FLCN). FLCN is known to be involved in the signalling of mammalian target of rapamycin (mTOR) which may play a role in the pathogenesis of pulmonary hypertension. We report a case of pulmonary hypertension (PAH) occurring in a 54 year-old female patient with BHD syndrome, in a context of chronic renal insufficiency. The main clinical presentations of BHD syndrome were multiple cutaneous lesions, two episodes of pneumothorax and multifocal renal tumours that required bilateral nephrectomy. She subsequently underwent two failed kidney transplantation attempts in 2008 and 2010. She was referred to the French Reference Center for Severe Pulmonary Hypertension in 2011 with a one year history of progressive dyspnea. Right heart catheterization confirmed precapillary pulmonary hypertension (PAH). Chronic renal insufficiency is known to be a risk factor for PAH. However, the role of FLCN in cell function and its complex cross talk with mTOR suggests that the association of PAH and BHD syndrome may be relevant. The patient received the oral dual endothelin receptor antagonist in association with a phophodiesterase 5 inhibitor. In this case report, we hypothesize that FLCN mutations may be predisposing factor for the development of PAH, with chronic renal insufficiency and dialysis as a potential trigger.
123
448
Abstracts
ABSTRACTS
POSTER COMMUNICATIONS
C13
FLCN IS INVOLVED IN AUTOPHAGY REGULATION Elaine A. Dunlop1, Sara Seifan1, Ewelina Rozycka2, Ross Tomaino3, Barry Coull4, Ravi Nookala5, Tijs Classens4, James T. Murray2, John Blenis3, Maurice A.M. van Steensel4, Simon Wilkinson6, Andrew R. Tee1 1 Institute of Cancer and Genetics, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK; 2 Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK: 3Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA; 4 Department of Dermatology, Maastricht University Medical Center, Maastricht, The Netherlands; 5 Department of Biochemistry , University of Cambridge , Sanger Building, 80 Tennis Court Road, Cambridge CB2 1GA, UK; 6Edinburgh Cancer Research UK Centre, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Birt-Hogg-Dubé (BHD) syndrome is a rare genetic disorder and the BHD gene product, folliculin (FLCN), is considered a tumour suppressor. Mutations in the BHD gene predispose BHD patients to hair follicle tumours, pneumothorax and renal cancer. Of importance, we now have direct evidence that FLCN plays a role in the regulation of autophagy. Autophagy helps maintain cellular homeostasis through removal of damaged or non-essential organelles and macromolecules. FLCN appears to function at the level of autophagy initiation and we have uncovered that phosphorylation is important during this process which also involves specific members of the Rab family of small G proteins. Additionally, the interaction of FLCN with its binding partner, FNIP2, appears to be important for the direct association of FLCN with the autophagy machinery. As dysregulated autophagy is implicated in tumour development, this novel link between FLCN and the autophagy machinery could explain the enhanced cancer progression seen in BHD patients.
123