Phosphoproteomic Analysis of FLCN Inactivation Highlights Differential Kinase Pathways and Regulatory TFEB Phosphoserines.

In Birt-Hogg-Dubé (BHD) syndrome, germline loss-of-function mutations in the Folliculin (FLCN) gene lead to an increased risk of renal cancer. To address how FLCN inactivation affects cellular kinase signaling pathways, we analyzed comprehensive phosphoproteomic profiles of FLCNPOS and FLCNNEG human renal tubular epithelial cells (RPTEC/TERT1). In total, 15,744 phosphorylated peptides were identified from 4329 phosphorylated proteins. INKA analysis revealed that FLCN loss alters the activity of numerous kinases, including tyrosine kinases EGFR, MET, and the Ephrin receptor subfamily (EPHA2 and EPHB1), as well their downstream targets MAPK1/3. Validation experiments in the BHD renal tumor cell line UOK257 confirmed that FLCN loss contributes to enhanced MAPK1/3 and downstream RPS6K1/3 signaling. The clinically available MAPK inhibitor Ulixertinib showed enhanced toxicity in FLCNNEG cells. Interestingly, FLCN inactivation induced the phosphorylation of PIK3CD (Tyr524) without altering the phosphorylation of canonical Akt1/Akt2/mTOR/EIF4EBP1 phosphosites. Also, we identified that FLCN inactivation resulted in dephosphorylation of TFEB Ser109, Ser114, and Ser122, which may be linked to increased oxidative stress levels in FLCNNEG cells. Together, our study highlights differential phosphorylation of specific kinases and substrates in FLCNNEG renal cells. This provides insight into BHD-associated renal tumorigenesis and may point to several novel candidates for targeted therapies.

FLCN regulates transferrin receptor 1 transport and iron homeostasis.

Birt-Hogg-Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A-TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

Mesenchymal folliculin is required for alveolar development: implications for cystic lung disease in Birt-Hogg-Dubé syndrome.

BACKGROUND: Pulmonary cysts and spontaneous pneumothorax are presented in most patients with Birt-Hogg-Dubé (BHD) syndrome, which is caused by loss of function mutations in the folliculin (FLCN) gene. The pathogenic mechanisms underlying the cystic lung disease in BHD are poorly understood. METHODS: Mesenchymal Flcn was specifically deleted in mice or in cultured lung mesenchymal progenitor cells using a Cre/loxP approach. Dynamic changes in lung structure, cellular and molecular phenotypes and signalling were measured by histology, immunofluorescence staining and immunoblotting. RESULTS: Deletion of Flcn in mesoderm-derived mesenchymal cells results in significant reduction of postnatal alveolar growth and subsequent alveolar destruction, leading to cystic lesions. Cell proliferation and alveolar myofibroblast differentiation are inhibited in the Flcn knockout lungs, and expression of the extracellular matrix proteins Col3a1 and elastin are downregulated. Signalling pathways including mTORC1, AMP-activated protein kinase, ERK1/2 and Wnt-ß-catenin are differentially affected at different developmental stages. All the above changes have statistical significance (p<0.05). CONCLUSIONS: Mesenchymal Flcn is an essential regulator during alveolar development and maintenance, through multiple cellular and molecular mechanisms. The mesenchymal Flcn knockout mouse model provides the first in vivo disease model that may recapitulate the stages of cyst development in human BHD. These findings elucidate the developmental origins and mechanisms of lung disease in BHD.

FLCN-regulated miRNAs suppressed reparative response in cells and pulmonary lesions of Birt-Hogg-Dubé syndrome.

BACKGROUND: Birt-Hogg-Dubé Syndrome (BHDS) characterised by skin fibrofolliculomas, kidney tumour and pulmonary cysts/pneumothorax is caused by folliculin (FLCN) germline mutations. The pathology of both neoplasia and focused tissue loss of BHDS strongly features tissue-specific behaviour of the gene. Isolated cysts/pneumothorax is the most frequent atypical presentation of BHDS and often misdiagnosed as primary spontaneous pneumothorax (PSP). Deferential diagnosis of BHDS with isolated pulmonary presentation (PSP-BHD) from PSP is essential in lifelong surveillance for developing renal cell carcinoma. METHODS: The expression profiles of microRNAs (miRNAs) in cystic lesions of PSP-BHD and PSP were determined via microarray. The selected upregulated miRNAs were further confirmed in the plasma of an expanded cohort of PSP-BHD patients by reverse transcription quantitative PCR (RT-qPCR). Their diagnostic accuracy was evaluated. Moreover, the cellular functions and targeted signalling pathways of FLCN-regulated miRNAs were assessed in various cell lines and in the lesion tissue contexts. RESULTS: Cystic lesions of PSP-BHD and PSP showed different miRNAs profiles with a significant upregulation of miR-424-5p and let-7d-5p in PSP-BHD. The combination of the two effectively predicted BHDS patients. In vitro studies revealed a suppressive effect of FLCN on miR-424-5p and let-7d-5p expressions specifically in lung epithelial cells. The ectopic miRNAs triggered epithelial apoptosis and epithelial transition of mesenchymal cells and suppressed the reparative responses in cells and tissues with FLCN deficiency. CONCLUSION: The upregulation of miR-424-5p and let-7d-5p by FLCN deficiency occurred in epithelial cells and marked the PSP-BHD condition, which contributed to a focused degenerative pathology in the lung of PSP-BHD patients.

Loss of FLCN inhibits canonical WNT signaling via TFE3.

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt-Hogg-Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/ß inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/ß inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression-two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/ß inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/ß inhibition. Finally, we found that a constitutively active ß-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.

The Birt-Hogg-Dubé (BHD) syndrome related protein FLCN has recently been implicated in the vesicular trafficking processes by interacting with several Rab family GTPases. In the previous studies, we have shown that FLCN could inhibit the binding of overexpressed PAT1, which is a membrane-bound amino acid transporter, to the lysosome in human embryonic kidney 293 cells. This tends to stabilize the lysosomal amino acid pool that is a critical signal to activate the mTORC1 signaling pathway. However, the mechanisms of FLCN during this process remain unexplored. Here we report that FLCN can bind through its C-terminal DENN-like domain to the recycling transport regulator, Rab11A. Suppression of either Rab11A or FLCN facilitated the localization of the overexpressed PAT1 to the lysosome and inhibited its targeting on the plasma membrane. As a consequence, the mTORC1 was down-regulated. The in vitro GEF activity assay does not support FLCN modifies the Rab11A activity directly. Instead, we found FLCN promoted the loading of PAT1 on Rab11A. Our data uncover a function of FLCN in the Rab11A-mediated recycling pathway and might provide new clues to understand BHD.This article has an associated First Person interview with the first author of the paper.

A Case of Birt-Hogg-Dubé (BHD) Syndrome Harboring a Novel Folliculin (FLCN) Gene Mutation.

BACKGROUND Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder clinically characterized by pulmonary cysts, spontaneous pneumothorax, renal cell cancer, and skin fibrofolliculomas. The disorder is caused by germline mutations in the FLCN gene. CASE REPORT A 56-year-old female was admitted to our hospital with a diagnosis of bilateral spontaneous pneumothorax. A computed tomography (CT) scan of the chest revealed bilateral multiple bullae predominantly located in the subpleural and mediastinal areas in the bilateral upper and lower lobes. Although she was cured by thoracic cavity drainage, she underwent resection of bilateral lung bullae because she had a prior history of right pneumothorax at 37- and 45-years of age. She had no signs of renal tumor but had fibrofolliculoma in her face and a family history of pneumothorax, we therefore suspected BHD syndrome. DNA sequence analyses determined that there was a two base pair deletion in exon 4 of the FLCN gene, confirming the diagnosis of BHD syndrome. CONCLUSIONS Here we report a case of BHD syndrome with a previously unreported FLCN mutation.

FLCN, a novel autophagy component, interacts with GABARAP and is regulated by ULK1 phosphorylation.

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.

Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development.

Folliculin-interacting protein 1 (Fnip1) is an adaptor protein that physically interacts with AMPK, an energy-sensing kinase that stimulates mitochondrial biogenesis and autophagy in response to low ATP, while turning off energy consumption mediated by mammalian target of rapamycin. Previous studies with Fnip1-null mice revealed that Fnip1 is essential for pre-B-cell development. Here we report a critical role of Fnip1 in invariant natural killer T (iNKT) cell development. Thymic iNKT development in Fnip1(-/-) mice was arrested at stage 2 (NK1.1(-)CD44(+)) but development of CD4, CD8, γδ T-cell, and NK cell lineages proceeded normally. Enforced expression of a Vα14Jα18 iNKT TCR transgene or loss of the proapoptotic protein Bim did not rescue iNKT cell maturation in Fnip1(-/-) mice. Whereas most known essential transcription factors for iNKT cell development were represented normally, Fnip1(-/-) iNKT cells failed to down-regulate Promyelocytic leukemia zinc finger compared with their WT counterparts. Moreover, Fnip1(-/-) iNKT cells contained hyperactive mTOR and reduced mitochondrial number despite lower ATP levels, resulting in increased sensitivity to apoptosis. These results indicate that Fnip1 is vital for iNKT cell development by maintaining metabolic homeostasis in response to metabolic stress.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation.

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Folliculin controls lung alveolar enlargement and epithelial cell survival through E-cadherin, LKB1, and AMPK.

Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD) syndrome, which is caused by mutations of the tumor suppressor gene folliculin (FLCN). The underlying mechanism of the lung manifestations in BHD is unclear. We show that BHD lungs exhibit increased alveolar epithelial cell apoptosis and that Flcn deletion in mouse lung epithelium leads to cell apoptosis, alveolar enlargement, and an impairment of both epithelial barrier and overall lung function. We find that Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Correspondingly, Flcn-null cell survival is rescued by the AMPK activator AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcn(f/f):SP-C-Cre mice. Our data suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival, attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.

Folliculin regulates cyclin D1 expression through cis-acting elements in the 3' untranslated region of cyclin D1 mRNA.

Birt-Hogg-Dubé syndrome (BHDS) is an autosomal dominantly inherited disease characterized by spontaneous pneumothorax, hair folliculomas and renal tumors. The responsible gene, BHD, is a tumor suppressor and encodes folliculin. Folliculin is an evolutionarily conserved protein (~67 kDa) with no apparent functional motif and its role has not yet been fully elucidated. In this study, we found that knockdown of BHD increased the levels of cyclin D1 in HeLa cells. A reporter assay with the cyclin D1 gene (CCND1) promoter region indicated that this increase was not caused by activation of transcription through known cis-acting elements. We examined the possibility of post-transcriptional mechanism using reporter constructs containing fragments of the cyclin D1 3' untranslated region (3'UTR). Transfection of control cells with a construct carrying a medial 1.3 kb 3'UTR fragment resulted in a significant reduction in luciferase activity. This effect was largely prevented by knockdown of BHD. Our results suggest that the post-transcriptional regulation of the CCND1 expression by BHD may be associated with microRNA(s) or RNA binding protein(s) that bind to the 3'UTR.

Unique mutation, accelerated mTOR signaling and angiogenesis in the pulmonary cysts of Birt-Hogg-Dubé syndrome.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, renal tumors and pulmonary cysts with repeated pneumothorax. This disorder is caused by mutations in the gene that encodes folliculin (FLCN). FLCN is known to be involved in the signaling of mammalian target of rapamycin (mTOR). We investigated the lung of a BHD patient who presented with a unique mutation. A 33-year-old woman visited our hospital due to repeated pneumothorax. Histopathologic study of the resected lung demonstrated multiple epithelial cysts. An increase of blood vessels was observed in the vicinity of subpleural cysts. Genomic DNA analysis revealed heterozygous mutation at the 3' end of intron 5 of the FLCN gene. Total mRNA and protein were extracted from the resected lung tissue. RT-PCR and sequence analysis demonstrated the production of exon 6-skipped FLCN mRNA. In Western blotting, the band intensities of phospho-mTOR, phospho-S6, phospho-Akt, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were increased in the BHD lung compared with normal lungs. Histopathologic analysis demonstrated strong immunostainings of mTOR signaling molecules in cyst-lining cells. Collective data indicates that dysregulation of mTOR signaling facilitates S6-mediated protein synthesis and HIF-1α-mediated angiogenesis, which may contribute to the development of pulmonary cysts in this disorder.

The Birt-Hogg-Dubé tumor suppressor Folliculin negatively regulates ribosomal RNA synthesis.

Birt-Hogg-Dubé syndrome (BHD) is a human cancer disorder caused by mutations in the tumor suppressor gene Folliculin (FLCN) with unknown biological functions. Here, we show that the Drosophila homolog of FLCN, dFLCN (a.k.a. dBHD) localizes to the nucleolus and physically interacts with the 19S proteasomal ATPase, Rpt4, a nucleolar resident and known regulator of rRNA transcription. Downregulation of dFLCN resulted in an increase in nucleolar volume and upregulation of rRNA synthesis, whereas dFLCN overexpression reduced rRNA transcription and counteracted the effects of Rpt4 on rRNA production by preventing the association of Rpt4 with the rDNA locus. We further show that human FLCN exhibited evolutionarily conserved function and that Rpt4 knockdown inhibits the growth of FLCN-deficient human renal cancer cells in mouse xenografts. Our study suggests that FLCN functions as a tumor suppressor by negatively regulating rRNA synthesis.

Birt-Hogg-Dubé: tumour suppressor function and signalling dynamics central to folliculin.

The cellular function of folliculin (FLCN) is a mystery that still needs to be solved. It is known that mutation of FLCN can predispose Birt-Hogg-Dubé (BHD) patient's to renal cell carcinoma , renal and lung cysts, as well as skin fibrofolliculomas. FLCN has been classed as a tumour suppressor, but it is probable that cystic and the skin manifestations do not occur as a consequence of FLCN loss of heterozygosity. Discovery that FLCN is a direct substrate of AMP dependent protein kinase (AMPK) placed FLCN on the cell signalling map, downstream of AMPK. This breakthrough suggested that FLCN might be involved in cell energy homeostasis. Over these more recent years, BHD research has become much more complicated and interesting from a cell signalling perspective. Folliculin has been linked to numerous cell pathways that are known to cause cancer, involving cell growth, metabolism, cell adhesion, cell motility, cytokinesis, and cell survival. The collective evidence implies that FLCN may have a broader housekeeping role in the cell. Of particular importance, FLCN was recently been reported to have guanine exchange factor activity towards the small G protein Rab35 and implicates FLCN in vesicular trafficking and/or membrane sorting. This newer discovery will undoubtedly help in the continued challenge of solving the signalling puzzle that shrouds FLCN function.

Birt-Hogg-Dube syndrome: clinicopathological features of the lung.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant inherited disorder characterised by fibrofolliculomas, renal tumours, pulmonary cysts and pneumothorax. The pulmonary cysts and repeated episodes of pneumothorax are the clinical hallmarks for discovering families affected by the syndrome. This disorder is caused by mutations in the gene coding for folliculin (FLCN). FLCN forms a complex with FLCN-interacting protein 1 (FNIP1) and FNIP2 (also known as FNIPL), and the complex cross-talks with signalling molecules such as 5'-AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR). Heterozygous Flcn knockout mice and rats with Flcn gene mutations develop renal cysts, adenomas and/or carcinomas. These findings suggest that FLCN functions as a tumour suppressor that inhibits renal carcinogenesis. However, the mechanisms of the formation of pulmonary cysts and pneumothorax associated with heterozygous mutations in FLCN are poorly understood. Resected lung specimens from patients with BHD are often misdiagnosed by pathologists as non-specific blebs or bullae or emphysema, and patients with BHD who have pulmonary cysts and repeated pneumothorax frequently do not receive appropriate medical investigations. This review discusses the clinical and pathological features of lungs of patients with BHD, focusing on the diagnostic pathology and possible mechanisms of cyst formation.

Gene expression and protein array studies of folliculin-regulated pathways.

The familial cancer syndrome Birt-Hogg-Dube syndrome is characterised by the development of skin (fibrofolliculomas) and renal tumours (and lung cysts) and is caused by mutations in the FLCN tumour suppressor gene. Though the FLCN gene product (folliculin) has been linked to the regulation of a variety of signalling pathways (e.g. the mTOR, AMPK, TGFbeta and hyoxia-responsive genes) the precise function of the folliculin protein is not well-defined. In order to identify potential novel pathways linked to folliculin function we analysed paired isogenic folliculin-deficient and folliculin-expressing cell lines by gene expression and protein (Kinexus) arrays. Gene expression microarray analysis in the folliculin +/- non-renal cancer line (FTC133), revealed 708 differentially expressed targets (fold change >2 and p<0.001) with enrichment of genes in the cadherin and Wnt signalling pathways. Comparison of the differentially expressed genes in the FTC133 datasets and previously reported gene expression data for a folliculin-deficient renal tumour and the UOK257 renal cell carcinoma cell line, revealed that RAB27B was dysregulated in all three datasets (increased expression in folliculin-deficient cells). The Kinexus protein array analysis suggested 73 candidate, differentially expressed, proteins and further investigation by western blot analysis of 5 candidates that were also differentially expressed in the FTC133 gene expression microarray data, revealed that EIF2AK2 (PKR) and CASP1 were reduced and PLCG2 was increased in folliculin-deficient FTC133 cells and in a BHD renal tumour. In view of the role of CASP1 in apoptosis we investigated whether other apoptosis-related proteins might be regulated by folliculin and found increased levels of SMAC/Diablo and HtrA2 in folliculin-expressing FTC133 cells. These findings identify novel pathways and targets linked to folliculin tumour suppressor activity.