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1.
J Immunol ; 203(11): 2899-2908, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676673

RESUMO

Folliculin interacting protein 1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with the master metabolic sensor 5' AMP-activated protein kinase (AMPK) and Folliculin, a protein mutated in individuals with Birt-Hogg-Dubé Syndrome. In response to low energy, AMPK stimulates catabolic pathways such as autophagy to enhance energy production while inhibiting anabolic pathways regulated by the mechanistic target of rapamycin complex 1 (mTORC1). We previously found that constitutive disruption of Fnip1 in mice resulted in a lack of peripheral B cells because of a block in B cell development at the pre-B cell stage. Both AMPK and mTORC1 were activated in Fnip1-deficient B cell progenitors. In this study, we found inappropriate mTOR localization at the lysosome under nutrient-depleted conditions. Ex vivo lysine or arginine depletion resulted in increased apoptosis. Genetic inhibition of AMPK, inhibition of mTORC1, or restoration of cell viability with a Bcl-xL transgene failed to rescue B cell development in Fnip1-deficient mice. Fnip1-deficient B cell progenitors exhibited increased nuclear localization of transcription factor binding to IgHM enhancer 3 (TFE3) in developing B cells, which correlated with an increased expression of TFE3-target genes, increased lysosome numbers and function, and increased autophagic flux. These results indicate that Fnip1 modulates autophagy and energy response pathways in part through the regulation of AMPK, mTORC1, and TFE3 in B cell progenitors.

2.
Genes Dev ; 33(23-24): 1641-1656, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31727773

RESUMO

Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein homologous to angiopoietins. Previous studies suggest that tumor cell-derived ANGPTL2 has tumor-promoting function. Here, we conducted mechanistic analysis comparing ANGPTL2 function in cancer progression in a murine syngeneic model of melanoma and a mouse model of translocation renal cell carcinoma (tRCC). ANGPTL2 deficiency in tumor cells slowed tRCC progression, supporting a tumor-promoting role. However, systemic ablation of ANGPTL2 accelerated tRCC progression, supporting a tumor-suppressing role. The syngeneic model also demonstrated a tumor-suppressing role of ANGPTL2 in host tumor microenvironmental cells. Furthermore, the syngeneic model showed that PDGFRα+ fibroblasts in the tumor microenvironment express abundant ANGPTL2 and contribute to tumor suppression. Moreover, host ANGPTL2 facilitates CD8+ T-cell cross-priming and enhances anti-tumor immune responses. Importantly, ANGPTL2 activates dendritic cells through PIR-B-NOTCH signaling and enhances tumor vaccine efficacy. Our study provides strong evidence that ANGPTL2 can function in either tumor promotion or suppression, depending on what cell type it is expressed in.

3.
Mol Cancer Res ; 17(8): 1613-1626, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31043488

RESUMO

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 PRCC-TFE3 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 Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB 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 in vivo 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.

4.
Cancer Sci ; 110(6): 1897-1908, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31006167

RESUMO

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early-stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC-TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)-204-5p levels in urinary exosomes of 40-week-old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA-204-5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR-204-5p-containing exosomes. Notably, we also observed increased miR-204-5p levels in urinary exosomes in 20-week-old renal PRCC-TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40-week-old Tg mice, suggesting that miR-204-5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR-204-5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC-TFE3 fusion gene. These findings suggest that miR-204-5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Cromossomos Humanos X/genética , Neoplasias Renais/genética , MicroRNAs/genética , Translocação Genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Biomarcadores Tumorais/urina , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/urina , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Exossomos/genética , Humanos , Rim/anormalidades , Rim/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/urina , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , MicroRNAs/urina , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo
5.
Histopathology ; 75(2): 254-265, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30908700

RESUMO

AIMS: Xp11 rearrangement in renal cell carcinoma (RCC) typically involves gene fusion to the gene encoding transcription factor E3 (TFE3), a member of the microphthalmia-associated transcription factor family on chromosome Xp11.2. Dual-colour break-apart fluorescence in-situ hybridisation (FISH) is recommended to confirm histological diagnoses. Recently, RNA-binding motif protein 10 (RBM10), encoded by a gene on chromosome Xp11.3, was identified as a chimeric partner of TFE3; thus, RBM10-TFE3 fusion results from paracentric inversion. RBM10-TFE3 RCC may yield a false-negative result in FISH analysis of TFE3 expression. The aim of the present study was to investigate the clinicopathological features of RBM10-TFE3 RCC. METHODS AND RESULTS: Ten patients with RBM10-TFE3 RCC aged 31-71 years were investigated. Histological analysis, immunostaining, dual-colour break-apart FISH for TFE3, reverse transcription polymerase chain reaction and sequencing analysis were performed. No patient had a history of exposure to chemotherapy. Two of these patients died of RCC, and three were alive but developed metastases. Microscopically, the tumours were composed of a mixed architecture of tubulocystic and papillary patterns with scattered psammoma bodies. The tumours showed strong nuclear immunoreactivity for TFE3. FISH showed consistent closely spaced split signals in the RCCs of four patients, and polysomic signals with occasional closely spaced split signals in the RCCs of six patients. Of the latter six patients, five had renal failure, and four developed tumours in kidneys subjected to haemodialysis. CONCLUSIONS: The present study suggests that the carcinogenesis of RBM10-TFE3 RCC in some, but not all, patients may be associated with chronic kidney disease. The aggressive nature of RBM10-TFE3 RCC should be considered, as five patients experienced metastases.

7.
J Bone Miner Res ; 33(10): 1785-1798, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29893999

RESUMO

Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

8.
Hum Mol Genet ; 2018 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-29767721

RESUMO

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene FLCN is a tumor suppressor for kidney cancer which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, the process by which FLCN-deficiency leads to renal tumorigenesis is unclear. In order to clarify molecular pathogenesis of BHD-associated kidney cancer, we conducted whole-exome sequencing analysis using next-generation sequencing technology as well as metabolite analysis using LC/MS and GC/MS. Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations (CNV) of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. In somatic variant analysis, very few variants were commonly observed in BHD-associated kidney cancer; however, variants in chromatin remodeling genes were frequently observed in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis of BHD-associated kidney cancer revealed metabolic reprogramming towards upregulated redox regulation which may neutralize reactive oxygen species potentially produced from mitochondria with increased respiratory capacity under FLCN-deficiency. BHD-associated kidney cancer displays unique molecular characteristics which are completely different from sporadic kidney cancer, providing mechanistic insight into tumorigenesis under FLCN-deficiency as well as a foundation for development of novel therapeutics for kidney cancer.

9.
BMC Med Genomics ; 11(1): 42, 2018 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720200

RESUMO

BACKGROUND: Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). Nearly 150 pathogenic mutations have been identified in FLCN. The most frequent pattern is a frameshift mutation within a coding exon. In addition, splice-site mutations have been reported, and previous studies have confirmed exon skipping in several cases. However, it is poorly understood whether there are any splice-site mutations that cause translation of intron regions in FLCN. CASE PRESENTATION: A 59-year-old Japanese patient with multiple pulmonary cysts and pneumothorax was hospitalized due to dyspnea. BHD was suspected and genetic testing was performed. The patient exhibited the splice-site mutation of FLCN in the 5' end of intron 9 (c.1062 + 1G > A). Total mRNA was extracted from pulmonary cysts, and RT-PCR assessment and sequence analyses were done. Two distinct bands were generated; one was wild-type and the other was a larger-sized mutant. Sequence analysis of the latter transcript revealed the insertion of 130 base pairs of intron 9 from the beginning of the splice-site between exons 9 and 10. CONCLUSION: To our knowledge, this is the first report of distinct intron insertion using a BHD patient's diseased tissue-derived mRNA. The present case suggests that a splice-site mutation can lead to exon skipping as well as intron reading mRNA. The splicing process may be dependent in part on whether the donor or acceptor site is affected.

10.
Pol J Pathol ; 68(4): 284-290, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29517197

RESUMO

The entity of hereditary leiomyomatosis renal cell carcinoma (HLRCC)-associated RCC has been proposed and integrated into the recent International Society of Urologic Pathology (ISUP) of renal tumors. This tumor is characterized by presence of cutaneous and/or uterine leiomyomas and RCC and autosomal dominant hereditary form. Grossly, HLRCC arising in the kidney show the solid tumor with frequent partial cystic area. Microscopically, the tumor typically shows papillary RCC, type 2, with eosinophilic large nucleoli reminiscent of cytomegaloviral inclusion and perinuclear clearing/haloes. Immunohistochemically, tumor cells show the overexpression for 2SC and reduced expression of FH. Germline mutation of fumarate hydratase (FH) gene, the HLRCC responsible gene mapped to chromosome 1q43, has been identified in patients with HLRCC. As the renal cancer in patients with HLRCC generally behave aggressively even in a small size, complete surgical resection and retroperitoneal lymph node resection should be performed promptly when the tumor is discovered. The surveillance of renal tumor in FH gene germline mutation-positive patients should be started from the early age using ultrasound sonography or magnetic resonance imaging.


Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Leiomiomatose/patologia , Síndromes Neoplásicas Hereditárias/patologia , Neoplasias Cutâneas/patologia , Neoplasias Uterinas/patologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biópsia , Carcinoma de Células Renais/química , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , Análise Mutacional de DNA , Diagnóstico Diferencial , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Neoplasias Renais/química , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Leiomiomatose/química , Leiomiomatose/genética , Leiomiomatose/cirurgia , Masculino , Mutação , Síndromes Neoplásicas Hereditárias/genética , Síndromes Neoplásicas Hereditárias/cirurgia , Fenótipo , Valor Preditivo dos Testes , Neoplasias Cutâneas/química , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/cirurgia , Resultado do Tratamento , Neoplasias Uterinas/química , Neoplasias Uterinas/genética , Neoplasias Uterinas/cirurgia
11.
Hum Mol Genet ; 26(2): 354-366, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28007907

RESUMO

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.


Assuntos
Síndrome de Birt-Hogg-Dubé/genética , Doenças Renais Císticas/genética , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Síndrome de Birt-Hogg-Dubé/patologia , Cardiomegalia/genética , Cardiomegalia/patologia , Proliferação de Células/genética , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Mutação em Linhagem Germinativa , Humanos , Rim/patologia , Doenças Renais Císticas/patologia , Neoplasias Renais/patologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto
12.
Lab Invest ; 97(3): 343-351, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27991910

RESUMO

Hereditary renal cell carcinomas (RCCs) are life-threatening disorders not only for the patients but also for their relatives. Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder caused by germline mutations in the folliculin gene (FLCN). The protein product, FLCN, functions as a tumor suppressor, and the affected patients have high risks of developing multiple RCCs. The carcinogenic mechanisms stemming from FLCN dysfunction have been investigated using rodent models and human RCC tissues. However, very limited information has been available about in vitro signaling of human renal cells with genetically mutant FLCN. Herein, we established a new cell line, BHD-F59RSVT, from a BHD patient's chromophobe RCC by transfecting SV40 large T antigen. We investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line. BHD-F59RSVT reflected the patient's FLCN germline mutation, a 3-nt deletion in exon 13 (c.1528_1530delGAG). Neither somatic mutation nor loss of heterozygosity of FLCN was detectable. Chromosome 17p11.2 of the FLCN proximal region demonstrated a trimodal pattern. Genome-wide chromosomal analysis revealed a loss of chromosome 16 and mosaic segmental gains in chromosome 7. BHD-F59RSVT cells were positive when immunostained for cytokeratin 7, supporting their origin from distal convoluted tubules. Western blotting analysis demonstrated severely suppressed FLCN expression at the protein level. The collective findings indicate that the established cell line will be suitable for functional analysis of the typical phenotype of BHD-associated RCC with suppressed FLCN expression.


Assuntos
Síndrome de Birt-Hogg-Dubé/genética , Carcinoma de Células Renais/genética , Mutação em Linhagem Germinativa , Neoplasias Renais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Síndrome de Birt-Hogg-Dubé/complicações , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/patologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Análise Mutacional de DNA/métodos , Saúde da Família , Feminino , Humanos , Neoplasias Renais/complicações , Neoplasias Renais/patologia , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Linhagem , Proteínas Proto-Oncogênicas/metabolismo , Cariotipagem Espectral/métodos , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo
13.
Genes Dev ; 30(9): 1034-46, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27151976

RESUMO

The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat.


Assuntos
Tecido Adiposo Bege/metabolismo , Metabolismo Energético/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Estrogênicos/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Quinases Ativadas por AMP/genética , Animais , Temperatura Baixa , Ativação Enzimática/genética , Regulação da Expressão Gênica/genética , Camundongos , Camundongos Knockout , Obesidade/enzimologia , Obesidade/genética , Oxirredução , Proteínas Proto-Oncogênicas/metabolismo , Receptores Estrogênicos/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo
14.
Clin Genet ; 90(5): 403-412, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27220747

RESUMO

Birt-Hogg-Dubé syndrome (BHD) is a rare genetic disorder characterized by fibrofolliculomas, pulmonary cysts and renal cell carcinomas (RCCs). The affected individuals inherit germline mutations in the folliculin gene (FLCN). We investigated the mutation spectrum and clinicopathologic findings of 312 patients from 120 different families (119 Japanese and 1 Taiwanese). A total of 31 different FLCN sequence variants were identified. The majority were c.1285dupC (n = 34), c.1533_1536delGATG (n = 25), and c.1347_1353dupCCACCCT (n = 19). Almost all patients presented with pulmonary cysts. The incidence of RCCs in FLCN mutation carriers over the age of 40 was 34.8% (40/115). Fifty-five RCC lesions were surgically resected; most were either chromophobe RCC (n = 24; 43.6%) or hybrid oncocytic/chromophobe tumors (19; 34.5%). Seventy-six of 156 FLCN mutation carriers (120 probands and 36 sibs, 48.7%) had skin papules; however, cutaneous manifestations were so subtle that only one patient voluntarily consulted dermatologists. Japanese Asian BHD families have three FLCN mutational hotspots. Recurrent episodes of pneumothoraces are the major symptoms suggestive of a BHD diagnosis in our cohort. Characteristic features of lung and kidney lesions may be more informative than fibrofolliculomas as diagnostic criteria for BHD in the Japanese Asian population.


Assuntos
Síndrome de Birt-Hogg-Dubé/genética , Carcinoma de Células Renais/genética , Epidemiologia Molecular , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Síndrome de Birt-Hogg-Dubé/epidemiologia , Síndrome de Birt-Hogg-Dubé/fisiopatologia , Carcinoma de Células Renais/epidemiologia , Carcinoma de Células Renais/fisiopatologia , Cistos/fisiopatologia , Feminino , Mutação em Linhagem Germinativa , Humanos , Japão/epidemiologia , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , Pele/fisiopatologia
15.
Stem Cells ; 34(4): 1068-82, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27095138

RESUMO

Folliculin (FLCN) is an autosomal dominant tumor suppressor gene that modulates diverse signaling pathways required for growth, proliferation, metabolism, survival, motility, and adhesion. FLCN is an essential protein required for murine embryonic development, embryonic stem cell (ESC) commitment, and Drosophila germline stem cell maintenance, suggesting that Flcn may be required for adult stem cell homeostasis. Conditional inactivation of Flcn in adult hematopoietic stem/progenitor cells (HSPCs) drives hematopoietic stem cells (HSC) into proliferative exhaustion resulting in the rapid depletion of HSPC, loss of all hematopoietic cell lineages, acute bone marrow (BM) failure, and mortality after 40 days. HSC that lack Flcn fail to reconstitute the hematopoietic compartment in recipient mice, demonstrating a cell-autonomous requirement for Flcn in HSC maintenance. BM cells showed increased phosphorylation of Akt and mTorc1, and extramedullary hematopoiesis was significantly reduced by treating mice with rapamycin in vivo, suggesting that the mTorc1 pathway was activated by loss of Flcn expression in hematopoietic cells in vivo. Tfe3 was activated and preferentially localized to the nucleus of Flcn knockout (KO) HSPCs. Tfe3 overexpression in HSPCs impaired long-term hematopoietic reconstitution in vivo, recapitulating the Flcn KO phenotype, and supporting the notion that abnormal activation of Tfe3 contributes to the Flcn KO phenotype. Flcn KO mice develop an acute histiocytic hyperplasia in multiple organs, suggesting a novel function for Flcn in macrophage development. Thus, Flcn is intrinsically required to maintain adult HSC quiescence and homeostasis, and Flcn loss leads to BM failure and mortality in mice.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Diferenciação Celular/genética , Estrona/genética , Células-Tronco Hematopoéticas/patologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células da Medula Óssea/patologia , Linhagem da Célula/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout
16.
Int J Urol ; 23(3): 204-10, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26608100

RESUMO

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.


Assuntos
Síndrome de Birt-Hogg-Dubé , Neoplasias Renais , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Animais , Síndrome de Birt-Hogg-Dubé/genética , Síndrome de Birt-Hogg-Dubé/patologia , Síndrome de Birt-Hogg-Dubé/terapia , Proteínas de Transporte/metabolismo , Técnicas de Inativação de Genes , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Neoplasias Renais/terapia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Nefrectomia , Proteínas Proto-Oncogênicas/metabolismo , Ratos , Sirolimo/administração & dosagem , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo
17.
Proc Natl Acad Sci U S A ; 112(13): E1624-31, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25775561

RESUMO

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.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Alelos , Animais , Proteínas Reguladoras de Apoptose/genética , Síndrome de Birt-Hogg-Dubé/genética , Proteínas de Transporte/genética , Modelos Animais de Doenças , Feminino , Rim/patologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Doenças Renais Policísticas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética
18.
Hum Mol Genet ; 23(21): 5706-19, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24908670

RESUMO

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-ß-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.


Assuntos
Cardiomegalia/genética , Cardiomegalia/metabolismo , Estrona/genética , Inativação Gênica , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Cardiomegalia/complicações , Cardiomegalia/tratamento farmacológico , Cardiomegalia/patologia , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Transgênicos , Renovação Mitocondrial , Tamanho do Órgão/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosforilação , Transdução de Sinais , Sirolimo/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Função Ventricular/efeitos dos fármacos
19.
Cell Rep ; 7(2): 412-23, 2014 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-24726356

RESUMO

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.


Assuntos
Apoptose , Síndrome de Birt-Hogg-Dubé/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Alvéolos Pulmonares/metabolismo , Mucosa Respiratória/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Síndrome de Birt-Hogg-Dubé/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Deleção de Genes , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Alvéolos Pulmonares/patologia , Ratos , Mucosa Respiratória/patologia , Proteínas Supressoras de Tumor/genética
20.
J Natl Cancer Inst ; 104(22): 1750-64, 2012 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-23150719

RESUMO

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.


Assuntos
Transformação Celular Neoplásica/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Músculos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Síndrome de Birt-Hogg-Dubé/genética , Western Blotting , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Mitocôndrias/metabolismo , Renovação Mitocondrial , Músculos/patologia , Oxirredução , Consumo de Oxigênio , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosforilação , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase em Tempo Real , Transativadores/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética
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