Blood and lymphatic vessels structurally bear a strong resemblance but never share a lumen, thus maintaining their distinct functions. Although lymphatic vessels initially arise from embryonic veins, ...the molecular mechanism that maintains separation of these two systems has not been elucidated. Here, we show that genetic deficiency of Folliculin, a tumor suppressor, leads to misconnection of blood and lymphatic vessels in mice and humans. Absence of Folliculin results in the appearance of lymphatic-biased venous endothelial cells caused by ectopic expression of Prox1, a master transcription factor for lymphatic specification. Mechanistically, this phenotype is ascribed to nuclear translocation of the basic helix-loop-helix transcription factor Transcription Factor E3 (TFE3), binding to a regulatory element of Prox1, thereby enhancing its venous expression. Overall, these data demonstrate that Folliculin acts as a gatekeeper that maintains separation of blood and lymphatic vessels by limiting the plasticity of committed endothelial cells.
Birt–Hogg–Dubé syndrome is an autosomal dominantly inherited disease that predisposes patients to develop fibrofolliculoma, lung cysts and bilateral multifocal renal tumors, histologically hybrid ...oncocytic/chromophobe tumors, chromophobe renal cell carcinoma, oncocytoma, papillary renal cell carcinoma and clear cell renal cell carcinoma. The predominant forms of Birt–Hogg–Dubé syndrome‐associated renal tumors, hybrid oncocytic/chromophobe tumors and chromophobe renal cell carcinoma are typically less aggressive, and a therapeutic principle for these tumors is a surgical removal with nephron‐sparing. The timing of surgery is the most critical element for postoperative renal function, which is one of the important prognostic factors for Birt–Hogg–Dubé syndrome patients. The folliculin gene (FLCN) that is responsible for Birt–Hogg–Dubé syndrome was isolated as a novel tumor suppressor for kidney cancer. Recent studies using murine models for FLCN, a protein encoded by the FLCN gene, and its two binding partners, folliculin‐interacting protein 1 (FNIP1) and folliculin‐interacting protein 2 (FNIP2), have uncovered important roles for FLCN, FNIP1 and FNIP2 in cell metabolism, which include AMP‐activated protein kinase‐mediated energy sensing, Ppargc1a‐driven mitochondrial oxidative phosphorylation and mTORC1‐dependent cell proliferation. Birt–Hogg–Dubé syndrome is a hereditary hamartoma syndrome, which is triggered by metabolic alterations under a functional loss of FLCN/FNIP1/FNIP2 complex, a critical regulator of kidney cell proliferation rate; a mechanistic insight into the FLCN/FNIP1/FNIP2 pathway could provide us a basis for developing new therapeutics for kidney cancer.
Purpose
Although infliximab (IFX) decreases the risk of blindness due to refractory uveitis in patients with Behçet's disease (BD), there are no standard criteria for IFX switching or withdrawal. To ...evaluate the effect of IFX switching in patients with BD in long-term remission, a prospective, single-arm intervention trial was conducted, switching from IFX to cyclosporine A (CYA).
Study design
A prospective open-label study.
Methods
Eligible patients met the following criteria: administration of IFX without concomitant immunosuppressants for more than 5 years with no episodes of ocular attacks, no retinal vasculitis on fluorescein fundus angiography, negative C-reactive protein in serum, and no extraocular lesions at the time of IFX withdrawal. CYA 5 mg/kg/day was administered from 6 weeks after IFX withdrawal. The primary outcome was the rate of readministration of tumor necrosis factor inhibitors at 1 year after IFX withdrawal.
Results
Three of 45 BD patients treated with IFX for refractory uveitis were included in the study. At 1 year after withdrawal of IFX, no patient had experienced any ocular attacks or needed readministation of IFX. However, extraocular lesions, such as recurrent oral ulcers, folliculitis, and recurrent fevers, occurred in all patients. Liver or renal dysfunction, which may have been caused by CYA, was also observed in all patients.
Conclusions
Although no ocular attacks were observed for at least 1 year after IFX withdrawal, this prospective study indicates that IFX withdrawal should be considered carefully, even for patients in long term remission of ocular and extraocular lesions.
Abstract
Immune checkpoint inhibitors (ICIs) activate anti-tumor activity by inhibiting immune checkpoint molecules that suppress inflammatory T-cell activity. However, ICIs can initiate excessive ...immune responses, thereby causing immune-related adverse events (irAEs). ICI-associated uveitis (ICIU) is an irAE that affects the eyes. Although Vogt–Koyanagi–Harada disease (VKH)-like uveitis is a common form of ICIU, it is unclear which factors determine the ICIU form. We retrospectively reviewed the medical records of nine ICIU cases treated with ICIs for malignancies. We also performed HLA typing in seven cases to investigate the association between HLA and disease type. Fisher's exact test was used for the statistical analysis. Five of the ICIU cases were VKH-like ICIUs, and four were non-VKH-like ICIUs. No association was found between mean age, sex, primary disease, ICI, time to onset, and disease type. Four patients with VKH-like uveitis underwent HLA genotyping and were all positive for
HLA-DRB1*04:05
. All 3 patients with non-VKH-like uveitis were negative for
HLA-DRB1*04:05
. Statistical analysis showed that
HLA-DRB1*04:05
was significantly associated with developing VKH-like ICIU (
P
= 0.029). In ICIU,
HLA-DRB1*04:05
was associated with the pathogenesis of VKH-like uveitis, suggesting that ICI-associated VKH-like uveitis has a similar pathogenesis to VKH.
Significance The role of FLCN as a tumor suppressor in kidney cancer has been well documented, whereas the functional roles of folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1 and FNIP2) in ...kidney are unknown. In this study, we demonstrate that double inactivation of Fnip1 and Fnip2 leads to enlarged polycystic kidneys or kidney cancer, which mimics the phenotypes seen in Flcn -deficient kidneys and underscores the significance of Fnip1 and Fnip2 in kidney tumor suppression. Moreover, we found that Fnip1/Fnip2 mRNA ratios differ among organs, which may reflect tissue-specific roles for each Fnip . Our findings define Fnip1 and Fnip2 as critical components of the Flcn complex that are essential for its tumor suppressive function and will aid in the development of novel therapeutics for kidney cancer.
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.
Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The ...Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix-loop-helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the
transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that
(Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that
is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical
model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. IMPLICATIONS: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.
Germline mutations in the BHD/FLCN tumor suppressor gene pre-dispose patients to develop renal tumors in the hamartoma syndrome, Birt-Hogg-Dubé (BHD). BHD encodes folliculin, a protein with unknown ...function that may interact with the energy- and nutrient-sensing AMPK-mTOR signaling pathways. To clarify BHD function in the mouse, we generated a BHD knockout mouse model. BHD homozygous null $(BHD^{d/d})$ mice displayed early embryonic lethality at E5.5-E6.5, showing defects in the visceral endoderm. BHD heterozygous knockout $(BHDd^{/+})$ mice appeared normal at birth but developed kidney cysts and solid tumors as they aged (median kidney-lesion-free survival = 23 months, median tumor-free survival = 25 months). As observed in human BHD kidney tumors, three different histologic types of kidney tumors developed in $BHD^{d/+}$ mice including oncocytic hybrid, oncocytoma, and clear cell with concomitant loss of heterozygosity (LOH), supporting a tumor suppressor function for BHD in the mouse. The PI3K-AKT pathway was activated in both human BHD renal tumors and kidney tumors in $BHD^{d/+}$ mice. Interestingly, total AKT protein was elevated in kidney tumors compared to normal kidney tissue, but without increased levels of AKT mRNA, suggesting that AKT may be regulated by folliculin through post translational or post-transcriptional modification. Finally, BHD inactivation led to both mTORC1 and mTORC2 activation in kidney tumors from $BHD^{d/+}$ mice and human BHD patients. These data support a role for PI3K-AKT pathway activation in kidney tumor formation caused by loss of BHD and suggest that inhibitors of both mTORC1 and mTORC2 may be effective as potential therapeutic agents for BHD-associated kidney cancer.
Birt-Hogg-Dubé (BHD) syndrome is a hereditary hamartoma syndrome that predisposes patients to develop hair follicle tumors, lung cysts, and kidney cancer. Genetic studies of BHD patients have ...uncovered the causative gene, FLCN, but its function is incompletely understood.
Mice with conditional alleles of FLCN and/or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), a transcriptional coactivator that regulates mitochondrial biogenesis, were crossbred with mice harboring either muscle creatine kinase (CKM) -Cre or myogenin (MYOG) -Cre transgenes to knock out FLCN and/or PPARGC1A in muscle, or cadherin 16 (CDH16)- Cre transgenes to knock out FLCN and/or PPARGC1A in kidney. Real-time polymerase chain reaction, immunoblotting, electron microscopy, and metabolic profiling assay were performed to evaluate mitochondrial biogenesis and function in muscle. Immunoblotting, electron microscopy, and histological analysis were used to investigate expression and the pathological role of PPARGC1A in FLCN-deficient kidney. Real-time polymerase chain reaction, oxygen consumption measurement, and flow cytometry were carried out using a FLCN-null kidney cancer cell line. All statistical analyses were two-sided.
Muscle-targeted FLCN knockout mice underwent a pronounced metabolic shift toward oxidative phosphorylation, including increased mitochondrial biogenesis (FLCN ( f/f ) vs FLCN ( f/f ) /CKM-Cre: % mitochondrial area mean = 7.8% vs 17.8%; difference = 10.0%; 95% confidence interval = 5.7% to 14.3%; P < .001), and the observed increase in mitochondrial biogenesis was PPARGC1A dependent. Reconstitution of FLCN-null kidney cancer cells with wild-type FLCN suppressed mitochondrial metabolism and PPARGC1A expression. Kidney-targeted PPARGC1A inactivation partially rescued the enlarged kidney phenotype and abrogated the hyperplastic cells observed in the FLCN-deficient kidney.
FLCN deficiency and subsequent increased PPARGC1A expression result in increased mitochondrial function and oxidative metabolism as the source of cellular energy, which may give FLCN-null kidney cells a growth advantage and drive hyperplastic transformation.
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.
Cardiac hypertrophy, an adaptive process that responds to increased wall stress, is characterized by the enlargement of cardiomyocytes and structural remodeling. It is stimulated by various growth ...signals, of which the mTORC1 pathway is a well-recognized source. Here, we show that loss of Flcn, a novel AMPK-mTOR interacting molecule, causes severe cardiac hypertrophy with deregulated energy homeostasis leading to dilated cardiomyopathy in mice. We found that mTORC1 activity was upregulated in Flcn-deficient hearts, and that rapamycin treatment significantly reduced heart mass and ameliorated cardiac dysfunction. Phospho-AMP-activated protein kinase (AMPK)-alpha (T172) was reduced in Flcn-deficient hearts and nonresponsive to various stimulations including metformin and AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). ATP levels were elevated and mitochondrial function was increased in Flcn-deficient hearts, suggesting that excess energy resulting from up-regulated mitochondrial metabolism under Flcn deficiency might attenuate AMPK activation. Expression of Ppargc1a, a central molecule for mitochondrial metabolism, was increased in Flcn-deficient hearts and indeed, inactivation of Ppargc1a in Flcn-deficient hearts significantly reduced heart mass and prolonged survival. Ppargc1a inactivation restored phospho-AMPK-alpha levels and suppressed mTORC1 activity in Flcn-deficient hearts, suggesting that up-regulated Ppargc1a confers increased mitochondrial metabolism and excess energy, leading to inactivation of AMPK and activation of mTORC1. Rapamycin treatment did not affect the heart size of Flcn/Ppargc1a doubly inactivated hearts, further supporting the idea that Ppargc1a is the critical element leading to deregulation of the AMPK-mTOR-axis and resulting in cardiac hypertrophy under Flcn deficiency. These data support an important role for Flcn in cardiac homeostasis in the murine model.
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