Masaya Baba

Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling

Birt–Hogg–Dubé syndrome, a hamartoma disorder characterized by benign tumors of the hair follicle, lung cysts, and renal neoplasia, is caused by germ-line mutations in the BHD(FLCN) gene, which encodes a tumor-suppressor protein, folliculin (FLCN), with unknown function. The tumor-suppressor proteins encoded by genes responsible for several other hamartoma syndromes, LKB1, TSC1/2, and PTEN, have been shown to be involved in the mammalian target of rapamycin (mTOR) signaling pathway. Here, we report the identification of the FLCN-interacting protein, FNIP1, and demonstrate its interaction with 5′ AMP-activated protein kinase (AMPK), a key molecule for energy sensing that negatively regulates mTOR activity. FNIP1 was phosphorylated by AMPK, and its phosphorylation was reduced by AMPK inhibitors, which resulted in reduced FNIP1 expression. AMPK inhibitors also reduced FLCN phosphorylation. Moreover, FLCN phosphorylation was diminished by rapamycin and amino acid starvation and facilitated by FNIP1 overexpression, suggesting that FLCN may be regulated by mTOR and AMPK signaling. Our data suggest that FLCN, mutated in Birt–Hogg–Dubé syndrome, and its interacting partner FNIP1 may be involved in energy and/or nutrient sensing through the AMPK and mTOR signaling pathways.

Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: Relationship to clinicopathological parameters

To delineate more precisely the somatic von Hippel‐Lindau disease (VHL) gene alteration as well as to elucidate its etiologic role in renal tumorigenesis, we examined a total of 240 sporadic renal cell carcinomas (RCCs) for somatic VHL gene alterations by DNA‐SSCP followed by sequencing, methylation‐specific PCR assay, microsatellite LOH study, and Southern blot analysis. Intragenic mutation of the VHL gene was found exclusively in clear‐cell or variant‐type RCCs at a frequency of 51% (104/202). Hypermethylation of the VHL promoter region was detected in an additional 11 clear‐cell RCCs. Microsatellite analysis demonstrated that LOH of the VHL locus was found in 140/155 (90%) informative clear‐cell RCCs. The VHL gene therefore seems to be inactivated in a two‐hit manner by intragenic mutation or hypermethylation plus allelic loss in clear‐cell RCC. Genomic rearrangement of the VHL gene detected by Southern analysis was not found (0/216 cases); this is in contrast to germ lines in which Southern aberrations consisted of 7–19% of the mutations. Clinicopathologic data demonstrated that VHL mutation/LOH did not vary according to tumor progression in clear‐cell RCC, including tumor diameter, stage, grading, distant metastasis, and lymph node metastasis. Interestingly, VHL mutation was significantly less frequent in RCCs occurring in younger (≦ 55 years) than that in older (≧ 56 years) patients. These data suggested that the inactivation of the VHL tumor‐suppressor gene is a specific genetic change in clear‐cell RCC, and that it may occur at an early or first step in the clear‐cell tumorigenic pathway rather than as a late event.

Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2

Germline mutations in the BHD/FLCN tumor suppressor gene predispose 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 (BHDd/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 BHDd/+ 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 BHDd/+ 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 BHDd/+ 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.

Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma

The gene expression profiles of 33 renal cell carcinomas (RCCs) and nine normal kidney samples were examined using high‐density oligonucleotide microarrays in an attempt to identify biomolecular markers for the diagnosis of tumour subtypes and also for prediction of prognosis. Hierarchical clustering demonstrated that clear‐cell RCC, chromophobe RCC, and normal kidney tissue showed distinctive gene expression profiles. The mean expression levels of 149 of 12 500 genes were more than three times higher in clear‐cell RCC than in chromophobe RCC and normal kidney tissue. Among the genes whose expression was upregulated in clear‐cell RCC, adipose differentiation‐related protein (ADFP) and nicotinamide N‐methyltransferase (NNMT) were selected for further analysis. Consistent with the results of the microarray, increased levels of ADFP and NNMT mRNA were found more frequently in clear‐cell RCCs than in other non‐clear‐cell tumour subtypes using real‐time quantitative PCR. Immunohistochemistry for ADFP showed strong and unique tumour cell staining patterns in the majority of clear‐cell RCCs. More importantly, patients bearing tumours with higher AFDP mRNA levels showed significantly better survival in both univariate and multivariate analyses. ADFP is a lipid storage droplet‐associated protein and its transcription is considered to be regulated by the von Hippel–Lindau/hypoxia‐inducible factor pathway. It is known that clear‐cell RCC contains abundant lipids and cholesterols. Thus it is likely that sustained upregulation of ADFP following VHL inactivation is involved in the morphological appearance of clear‐cell RCC. Moreover ADFP expression status may provide useful prognostic information as a biomolecular marker in patients with clear‐cell RCC. Copyright

Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through hypoxia-inducible factor.

Loss of the von Hippel–Lindau gene (VHL) expression ca-uses deregulation of contact inhibition of cell growth, which might be one of the bases of the tumor suppressor function of VHL. Here we show that this function of the VHL gene product (pVHL) depends on cell autonomous events. To identify the target gene of pVHL, which is directly involved in the contact inhibition, we compared the gene expression profile between VHL-deficient renal carcinoma 786-O cells and those infected with an adenovirus vector encoding VHL. In addition to known pVHL-regulated genes, such as vascular endothelial growth factor and carbonic anhydrase, we found cyclinD1 as a new target of pVHL at a high cell density. In VHL-expressing cells (VHL (+) cells), the cyclinD1 mRNA expression level diminishes at a high cell density, while it remains at a relatively high level in VHL-deficient cells (VHL (−) cells). The cyclinD1 expression level was also abnormally high in VHL (−) cells at a high cell density. Consequently, the phosporylation level of the retinoblastoma (Rb) protein remained high in these cells, whereas there was no phosporylated Rb in VHL (+) cells under the contact inhibition. The abnormal expression of cyclinD1 at a high cell density was observed even in VHL (+) cells under the hypoxic state. Moreover, ectopic expression of a HIF mutant resistant to pVHL-mediated proteolysis causes the abnormal cyclinD1 expression in VHL (+) cells. Taken together, these observations indicate that VHL is required for the downregulation of cyclinD1 at a high cell density through HIF.

Inactivation of the FLCN tumor suppressor gene induces TFE3 transcriptional activity by increasing its nuclear localization.

Background Germline mutations in a tumor suppressor gene FLCN lead to development of fibrofolliculomas, lung cysts and renal cell carcinoma (RCC) in Birt-Hogg-Dubé syndrome. TFE3 is a member of the MiTF/TFE transcription factor family and Xp11.2 translocations found in sporadic RCC involving TFE3 result in gene fusions and overexpression of chimeric fusion proteins that retain the C-terminal DNA binding domain of TFE3. We found that GPNMB expression, which is regulated by MiTF, was greatly elevated in renal cancer cells harboring either TFE3 translocations or FLCN inactivation. Since TFE3 is implicated in RCC, we hypothesized that elevated GPNMB expression was due to increased TFE3 activity resulting from the inactivation of FLCN. Methodology/Principal Findings TFE3 knockdown reduced GPNMB expression in renal cancer cells harboring either TFE3 translocations or FLCN inactivation. Moreover, FLCN knockdown induced GPNMB expression in FLCN-restored renal cancer cells. Conversely, wildtype FLCN suppressed GPNMB expression in FLCN-null cells. FLCN inactivation was correlated with increased TFE3 transcriptional activity accompanied by its nuclear localization as revealed by elevated GPNMB mRNA and protein expression, and predominantly nuclear immunostaining of TFE3 in renal cancer cells, mouse embryo fibroblast cells, mouse kidneys and mouse and human renal tumors. Nuclear localization of TFE3 was associated with TFE3 post-translational modifications including decreased phosphorylation. Conclusions/Significance Increased TFE3 activity is a downstream event induced by FLCN inactivation and is likely to be important for renal tumor development. This study provides an important novel mechanism for induction of TFE3 activity in addition to TFE3 overexpression resulting from Xp11.2 translocations, suggesting that TFE3 may be more broadly involved in tumorigenesis.

PTEN/MMAC1/TEP1 mutations in human primary renal-cell carcinomas and renal carcinoma cell lines.

Extensive allelotyping studies have implicated several tumor‐suppressor loci on chromosomes 3p, 5q, 6q, 8p, 9pq, 10q, 11q, 14q, 17p, 18q and 19p in human kidney tumorigenesis. The PTEN (also called MMAC1 and TEP1) gene, a candidate tumor suppressor located at chromosome 10q23.3, is mutated in a variety of sporadic malignancies as well as in patients with Cowden disease. To investigate the potential role of the PTEN gene in renal tumorigenesis, we searched for abnormalities of the gene in 68 primary renal‐cell carcinomas (RCCs) as well as in 17 renal carcinoma–derived cell lines, using DNA‐SSCP, sequencing and microsatellite analysis. Five of 68 (7.5%) primary RCCs exhibited intragenic mutations (3 missense, 1 deletion and 1 splice‐site), and 1 of 17 (5.9%) cell lines had an insertion mutation. Loss of heterozygosity of the PTEN gene occurred in 25% of primary RCCs, including the 3 cases with intragenic mutation and the 1 PTEN‐mutated cell line. Clinical and histopathological examinations revealed that 4 of the 5 primary tumors with PTEN mutation were high‐grade, advanced clear‐cell RCCs with distant metastases or renal vein tumor invasions, resulting in poor prognostic courses. The other was a low‐stage papillary/chromophilic RCC. Our data suggest that PTEN mutation is observed in a subset of RCCs and that, especially in clear‐cell RCCs, it occurs as a late‐stage event and may contribute to the invasive and/or metastatic tumor phenotype.

Inactivation of von Hippel-Lindau Gene Induces Constitutive Phosphorylation of MET Protein in Clear Cell Renal Carcinoma

It is well known that inactivation of von Hippel-Lindau (VHL) gene predisposes for human clear cell renal carcinoma (CCRC). However, details about critical roles of VHL inactivation during tumorigenesis are still unknown. MET protein is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), which regulates cell growth, cell morphology, and cell motility. We showed that MET protein overexpressed in CCRC cells was phosphorylated without HGF/SF. This constitutive phosphorylation of MET protein in CCRC cells was inhibited by the rescue of exogenous wild-type VHL gene without a decrease in expression level of MET protein. Interestingly, wild-type VHL gene suppressed the phosphorylation of MET protein only under high cell density conditions. Additionally, MET protein activated by the inactivation of VHL gene modified cell adherence, including N-cadherin and beta-catenin. When activation of MET protein in CCRC cells was inhibited by the MET inhibitor K252a, the growth of CCRC cells in vitro and the tumorigenesis induced by CCRC cells in nude mice were suppressed. From these results, we concluded that inactivation of VHL gene induced constitutive phosphorylation of MET protein and modified intercellular adherence structure to trigger the cell growth released from contact inhibition, finally resulting in tumorigenesis. This is one of the mechanisms of CCRC oncogenesis, and MET protein has potential as a molecular target for novel CCRC therapies.

Regulation of Mitochondrial Oxidative Metabolism by Tumor Suppressor FLCN

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.

Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth

In spite of the general recognition of von Hippel-Lindau (VHL) as a tumor suppressor gene, the physiological and pathological importance of VHL protein in cell growth regulation and tumorigenesis remains unclear. Here we show that in normal human renal proximal tubule epithelial cells (RPTEC), the steady-state amount of VHL protein is strictly regulated by cell density. The cellular VHL content is more than 100-fold higher in dense cultures than in sparse cultures. The increase in VHL protein at high cell density was also observed for NIH3T3 fibroblasts, suggesting the generality of the phenomenon. The growth rates of renal cell carcinoma cells lacking an intact VHL gene and their derivatives with wild-type or mutant VHL expression vector do not differ significantly when they are growing in log-phase. Importantly, however, there is a difference when they reach confluency: cells lacking wild-type VHL grew continuously, while cells expressing exogenous VHL protein showed relatively limited cell growth. Using an ecdysone-inducible VHL expressing cell line, we also show that the growth inhibition at high cell density can be released by attenuating the VHL expression. Taken together, we propose that VHL protein functions as a growth suppressor at high cell density, and this might be the basis of the tumor suppressor function of VHL.