Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.
ABSTRACT: Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanism by which FLCN prevents kidney cancer remains unknown. Here, we show that disrupting FLCN in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.
Project description:Germline inactivating mutations in Folliculin (FLCN) cause Birt–Hogg–Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing to kidney tumors. FLCN is a conserved, essential gene that has been linked to diverse cellular processes but the mechanisms by which FLCN prevents kidney cancer remain unknown Here we show that FLCN loss activates E-box target genes in human renal tubular epithelial cells (RPTEC/TERT1), including RRAGD, yet without modifying mTORC1 activity. Surprisingly, inactivation of FLCN or its binding partners FNIP1/FNIP2 activates interferon response genes but independently of interferon. Mechanistically, FLCN loss promotes recruitment of STAT2 to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence the immune response. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint novel prognostic biomarkers.
Project description:Birt-Hogg-Dube' syndrome characterized by increased risk for renal neoplasia is caused by germline mutations in the BHD/FLCN gene encoding a novel tumor suppressor protein, folliculin(FLCN), which interacts with FNIP1 and 5'-AMP-activated protein kinase(AMPK). Here we report the identification and characterization of a novel FNIP1 homolog FNIP2 that also interacts with FLCN and AMPK. C-terminally-deleted FLCN mutants, similar to those produced by naturally-occurring germline mutations in BHD patients, were unable to bind FNIP2. These data taken together with our previous results that demonstrated FNIP1 binding to the C-terminus of FLCN suggest that FLCN tumor suppressor function may be facilitated by interactions with both FNIP1 and FNIP2 through its C-terminus. Furthermore, we demonstrate that FNIP1 and FNIP2 are able to form homo- or heteromeric multimers suggesting that they may function independently or cooperatively with FLCN. Differential expression of FNIP1 and FNIP2 transcripts in some normal tissues may indicate tissue specificity for these homologs. Interestingly FNIP1 and FNIP2 were oppositely expressed in human clear cell renal cell carcinoma (RCC), and coordinately expressed in chromophobe RCC and oncocytoma, suggesting their differential function in different histologic variants of RCC.
Project description:Folliculin-interacting protein 1 and 2 (FNIP1 and FNIP2) play critical roles in preventing renal malignancy through their association with the tumor suppressor FLCN. Mutations in FLCN are associated with Birt-Hogg-Dubé (BHD) syndrome, a rare disorder with increased risk of renal cancer. Recent studies indicated that FNIP1/FNIP2 double knockout mice display enlarged polycystic kidneys and renal carcinoma, which phenocopies FLCN knockout mice, suggesting that these two proteins function together to suppress renal cancer. However, the molecular mechanism functionally linking FNIP1/FNIP2 and FLCN remains largely elusive. Here, we demonstrated that FNIP2 protein is unstable and subjected to proteasome-dependent degradation via ?-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and subsequent lysosomal association of mTOR, which in turn promotes the proliferation of renal cancer cells. These results indicate that SCF?-TRCP negatively regulates the FLCN complex by promoting FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer.
Project description:Birt-Hogg-Dubé (BHD) syndrome is an inherited renal cancer syndrome in which affected individuals are at risk of developing benign cutaneous fibrofolliculomas, bilateral pulmonary cysts and spontaneous pneumothoraces, and kidney tumours. Bilateral multifocal renal tumours that develop in BHD syndrome are most frequently hybrid oncocytic tumours and chromophobe renal carcinoma, but can present with other histologies. Germline mutations in the FLCN gene on chromosome 17 are responsible for BHD syndrome--BHD-associated renal tumours display inactivation of the wild-type FLCN allele by somatic mutation or chromosomal loss, confirming that FLCN is a tumour suppressor gene that fits the classic two-hit model. FLCN interacts with two novel proteins, FNIP1 and FNIP2, and with AMPK, a negative regulator of mTOR. Studies with FLCN-deficient cell and animal models support a role for FLCN in modulating the AKT-mTOR pathway. Emerging evidence links FLCN with a number of other molecular pathways and cellular processes important for cell homeostasis that are frequently deregulated in cancer, including regulation of TFE3 and/or TFEB transcriptional activity, amino-acid-dependent mTOR activation through Rag GTPases, TGF? signalling, PGC1?-driven mitochondrial biogenesis, and autophagy. Currently, surgical intervention is the only therapy available for BHD-associated renal tumours, but improved understanding of the FLCN pathway will hopefully lead to the development of effective forms of targeted systemic therapy for this disease.
Project description:Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt-Hogg-Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.
Project description:Folliculin (FLCN) is a tumor-suppressor protein mutated in the Birt-Hogg-Dubé (BHD) syndrome, which associates with two paralogous proteins, folliculin-interacting protein (FNIP)1 and FNIP2, forming a complex that interacts with the AMP-activated protein kinase (AMPK). Although it is clear that this complex influences AMPK and other metabolic regulators, reports of its effects have been inconsistent. To address this issue, we created a recessive loss-of-function variant of Fnip1 Homozygous FNIP1 deficiency resulted in profound B-cell deficiency, partially restored by overexpression of the antiapoptotic protein BCL2, whereas heterozygous deficiency caused a loss of marginal zone B cells. FNIP1-deficient mice developed cardiomyopathy characterized by left ventricular hypertrophy and glycogen accumulation, with close parallels to mice and humans bearing gain-of-function mutations in the ?2 subunit of AMPK. Concordantly, ?2-specific AMPK activity was elevated in neonatal FNIP1-deficient myocardium, whereas AMPK-dependent unc-51-like autophagy activating kinase 1 (ULK1) phosphorylation and autophagy were increased in FNIP1-deficient B-cell progenitors. These data support a role for FNIP1 as a negative regulator of AMPK.
Project description:Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (?E510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.
Project description:Germline mutations in the novel tumor suppressor gene FLCN are responsible for the autosomal dominant inherited disorder Birt-Hogg-Dubé (BHD) syndrome that predisposes to fibrofolliculomas, lung cysts and spontaneous pneumothorax, and an increased risk for developing kidney tumors. Although the encoded protein, folliculin (FLCN), has no sequence homology to known functional domains, x-ray crystallographic studies have shown that the C-terminus of FLCN has structural similarity to DENN (differentially expressed in normal cells and neoplasia) domain proteins that act as guanine nucleotide exchange factors (GEFs) for small Rab GTPases. FLCN forms a complex with folliculin interacting proteins 1 and 2 (FNIP1, FNIP2) and with 5' AMP-activated protein kinase (AMPK). This review summarizes FLCN functional studies which support a role for FLCN in diverse metabolic pathways and cellular processes that include modulation of the mTOR pathway, regulation of PGC1? and mitochondrial biogenesis, cell-cell adhesion and RhoA signaling, control of TFE3/TFEB transcriptional activity, amino acid-dependent activation of mTORC1 on lysosomes through Rag GTPases, and regulation of autophagy. Ongoing research efforts are focused on clarifying the primary FLCN-associated pathway(s) that drives the development of fibrofolliculomas, lung cysts and kidney tumors in BHD patients carrying germline FLCN mutations.
Project description: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.
Project description:Birt-Hogg-Dube (BHD) syndrome is an autosomal dominant disorder characterized by hamartomas of skin follicles, cystic lung disease, and renal neoplasia. Affected individuals carry heterozygous mutations in Folliculin (FLCN), a tumor suppressor gene that becomes biallelically inactivated in kidney tumors by second-hit mutations. Similar to other factors implicated in kidney malignancies, Folliculin has been shown to modulate activation of mammalian target of rapamycin (mTOR). However, its precise in vivo function is largely unknown because germline deletion of Flcn results in early embryonic lethality in animal models. We here describe mice deficient in the newly characterized Folliculin-Interacting Protein 1 (Fnip1). In contrast to Flcn, Fnip1-/- mice develop normally, are not susceptible to kidney neoplasia, but display a striking pro-B cell block that is independent of mTOR activity. We show that this developmental arrest results at least in part from impaired V(D)J recombination and caspase-induced cell death, and that pre-recombined V(D)J and Bcl2 transgenes reconstitute pre-B and mature B cell populations respectively. We also demonstrate that conditional deletion of Flcn recapitulates the pro-B cell arrest of Fnip1-/- mice. Our studies thus demonstrate that the Flcn-Fnip complex deregulated in BHD syndrome is absolutely required for B cell differentiation and that it functions both through mTOR dependent and independent pathways. RNASeq data for two pro-B cell subsets (fraction B and CC') isolated from wt and Fnip1-/- mice