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.
Hereditary renal cell carcinomas (RCCs) require special management so that patients and their families can avoid dialysis and progression to cancer-related death. Birt–Hogg–Dubé syndrome (BHD), also called Hornstein–Knickenberg syndrome, is an inherited disorder characterized by skin fibrofolliculomas, multiple pulmonary cysts, and renal tumors.1, 2 The responsible gene for BHD, folliculin (FLCN), is located at 17p11.2,3 and its protein product, FLCN, forms a complex with interacting partners FNIP1 and FNIP2.4, 5, 6 The principal role of FLCN in human diseases is tumor suppression, and the mechanism involves several pathways, including mammalian target of rapamycin and 5′-AMP-activated protein kinase.4 It is known that a variety of renal tumor subtypes, including clear cell, papillary, chromophobe, and oncocytoma, may develop in BHD patients, although chromophobe RCC and hybrid oncocytic/chromophobe tumors (HOCTs) are predominant.7, 8
Detailed mechanisms of FLCN function in the kidney have been intensively studied using mice with Flcn inactivation in epithelial cells of the kidney,9, 10 or immortalized human kidney epithelial cells treated with FLCN siRNA.11, 12 Only one cell line, UOK257, from a BHD patient’s RCC is currently used for functional studies of FLCN in vitro. UOK257 was established from a high-grade RCC characterized as predominantly clear cell type with aggressive behavior.13 The majority of renal tumors developing in BHD patients are chromophobe RCC and HOCT, and these tumor subtypes tend to be less aggressive in behavior.14, 15, 16 Herein we established an immortalized cell line derived from a chromophobe RCC in a Japanese BHD patient. We characterized the clinicopathologic information, germline mutation, and cytogenetic features of the established cell line.
Materials and methods
Cell Culture Information
A patient with multiple RCCs received genetic counseling and agreed to undergo genetic testing. Written informed consent was obtained from the patient for cytogenetic and clinicopathologic analyses. The study was approved by the Institutional Review Board (IRB) of Yokohama City University. A part of the resected tumor was fixed with 10% buffered formalin and embedded in paraffin. Hematoxylin and eosin (HE) staining was performed for histological diagnosis. The other parts of the tumor were either used for cell culture or snap-frozen and stored in liquid nitrogen until cytogenetic analysis.
The tumor tissue was minced with a sterile scalpel into 1-mm-sized fragments, and treated for 30 min with 1 mg/ml collagenase (Wako Pure Chemical Industries, Osaka, Japan). The cells were rinsed and cultured in RPMI1640 (Life Technologies, Carlsbad, CA, USA) with 10% heat-inactivated fetal bovine serum, and 1% penicillin and streptomycin. One week after plating, adherent epithelial cell overgrowth was observed. These epithelial cells (passage one) were treated with 0.05% trypsin–EDTA and then cultured in 12-well plates at 8 × 104 cells per well. The cells were infected with SV40 large T antigen (SV40gp6)-expressing lentiviral vector pLSVSIN-EF1a (Takara Bio, Shiga, Japan) according to the manufacturers’ instructions. The cells were incubated with the plasmid DNA for 24 h and were then washed, and transferred to 10-cm dishes. When the dishes were subconfluent, the cells were diluted for cloning. After puromycin selection, immortalized SV40gp6-expressing cells was obtained and named BHD-F59RSVT. The expression of SV40gp6 was then confirmed by RT-PCR. BHD-F59RSVT cells were cultured in Renal Epithelial Cell Growth Medium 2 with recombinant human epithelial growth factor (PromoCell GmbH, Heidelberg, Germany), and 1% penicillin and streptomycin. The cells were stepwisely cultured from 96- to 6-well plates, then proliferated in 25-cm2 flasks. Further analysis was performed using one to two passage cells of stably established cells.
For western blotting of FLCN, rabbit monoclonal antibody D14G9 (Cell Signaling, Danvers, MA, USA) was used. Mouse monoclonal antibody against β-actin was purchased from Sigma (St Louis, MO, USA). For cytostaining and histopathologic studies, we used rabbit polyclonal antibodies against FLCN (ab93196, Abcam, Cambridge, UK) and smooth muscle actin (SMA; ab5694, Abcam). We also utilized mouse monoclonal antibodies against cytokeratin 7 (CK7; Dako, Glostrup, Denmark), CD82 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mitochondria (Clone 113-1, BioGenex, Fremont, CA, USA), CD10 (Dako), RCC (Dako), and cytokeratin AE1/AE3 (Dako).
BHD-F59RSVT cells were seeded on chamber slides, and 24 h later, the slides were washed with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100. After washing three times with PBS and blocking with 5% blocking reagent for 30 min, cells were labeled with primary antibodies for 90 min, and then incubated with Alexa Fluor 488 goat anti-mouse IgG or anti-rabbit IgG (Life Technologies) for 30 min at room temperature. Working dilutions were 1:50 for FLCN and mitochondria and 1:100 for other antibodies. UOK257-2 was used for FLCN-positive control. This cell line restores wild-type FLCN.11
Four-μm-thick paraffin sections of the original tumor and BHD-F59RSVT cell pellets were subjected to immunohistochemistry. After deparaffinization and rehydration, sections were autoclaved at 121 °C for 15 min, after which they were treated with the diluted antibodies at 4 °C overnight. Immunohistochemical staining was done using the EnVision+ kit (Dako), followed by 3, 3′-diaminobenzidne staining for visualization. Working dilutions were 1:50 for FLCN and CK7, and 1:100 for other antibodies.16
DNA from peripheral blood leukocytes was obtained using the LabPass Blood Mini kit (Cosmo GENETECH, Seoul, Korea). DNA from renal tissue was obtained using a QIAamp DNA Mini kit (Qiagen, Hidden, Germany) according to the manufacturers’ instructions.
Direct Sequencing and PCR
Exons 1–14 of the FLCN gene were amplified by PCR using the primers described previously.3 In addition, the copy number of each exon was assessed using quantitative PCR to detect large rearrangements.17 PCR conditions were described in our previous study.18 After purification, DNA was labeled with a Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Cleveland, OH, USA) and DNA sequencing was done using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems).
The promoter status of FLCN was examined by a methylation-specific PCR approach using a CpGenome Turbo Bisulfate Modification kit (Merck Millipore, Darmstadt, Germany). DNA was treated with sodium bisulfate according to the manufacturers’ instructions. The specific primers and PCR conditions were described previously.19 The PCR product was treated with RsaI for 2 h at 37 °C. The restriction enzyme digestion product was then visualized in 2% agarose gel.
Lysates (12.5 μg protein) were electrophoresed on SDS-PAGE and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG (Santa Cruz Biotechnology) were used as the secondary antibodies. Bands were detected using an enhanced chemiluminescence system, according to the Hybond ECL protocol (GE Healthcare, Buckinghamshire, UK).
RNA Isolation and RT-PCR
Total RNAs were isolated from cells using TRIzol Reagents and reverse transcription was done using SuperScript III according to the manufacturer’s protocols (Life Technologies). The primers used for PCR were as follows: SV40gp6, (F) 5′-IndexTermGGCTACTGCTGACTCTCAACATTCT-3′ and (R) 5′-IndexTermAGCAGAGCCTGTAGAA CCAAACATT-3′; GAPDH (F) 5′-IndexTermAAGGTGAAGGTCGGAGTCAAC-3′ and (R) 5′-IndexTermCATGAGTCCTTCCACGATACC-3′. The expression levels of FLCN mRNA were investigated according to the method previously described.11
CytoScan HD Array and Copy-Number Variation Analysis
A CytoScan HD Array (Affymetrix, Santa Clara, CA, USA) was used to analyze genomic alterations according to the manufacturer’s protocol. In brief, 250 ng of genomic DNA from BHD-F59RSVT was digested with the restriction enzyme Nsp I and then ligated to an adapter, followed by PCR amplification. The data were analyzed using Affymetrix Chromosome Analysis Suite (ChAS) version 3 to detect copy-number variation (CNV) and loss of heterozygosity (LOH). Definitions of CNV and LOH were presented in our previous study.20 The allelic patterns were interpreted as follows. A normal log2 ratio with trimodal allele peaks indicated biparental disomy (normal). A normal log2 ratio with bimodal allele peaks indicated uniparental disomy (UPD). An increased log2 ratio with tetramodal allele peaks indicated trisomy. A decreased log2 ratio with bimodal allele peaks indicated monosomy. A decreased log2 ratio with tetramodal allele peaks indicated mosaic loss.
Spectral Karyotyping Analysis
Spectral karyotyping (SKY) analysis was performed to characterize aberrant chromosome using the 24-color SKY probe system (Applied Spectral Imaging (ASI), Carlsbad, CA, USA). The chromosome preparations were hybridized with the SKY probe mixture for 48 h at 37 °C. The multicolor hybridization was visualized by Spectra Cube (ASI) and images were analyzed using SKYVIEW software (ASI). Five cell lines were analyzed. Chromosomal abnormalities were described according to an International System for Human Cytogenetic Nomenclature (ISCN) 2013. If two or more cells contained an identical structural aberration, the aberrations were considered recurring, and if three or more cells demonstrated an identical chromosomal loss, the loss was considered clonal.
Clinicopathologic Manifestations and Germline FLCN Mutation
A 52-year-old Japanese man with repeated episodes of pneumothorax was admitted to our hospital for resection of multiple renal tumors. He had experienced skin papules in adolescence, and papules developed in the arms, legs, head, neck, and trunk. His mother and a cousin had episodes of pneumothorax. Although his mother, an aunt, and a grandmother also reported subtle skin papules, none consulted dermatologists (Figure 1a). His mother underwent colon cancer surgery at the age of 72, but no neoplasm was detected in the kidney. Computed tomography of the proband revealed multiple pulmonary cysts and bilateral renal tumors (Figure 1b). His medical record, together with a family history of pneumothorax, alerted us to the possibility of BHD. Genetic counseling was performed, and informed consent was obtained from the patient for FLCN genetic testing after approval of IRB. A 3-bp deletion in exon 13 (c.1528_30delGAG) was detected (Figure 1c). The mutation was reported by Toro et al as pathogenic, predicting amino-acid change p.Glu510del.2 The patient was diagnosed with BHD. His mother and siblings were not willing the genetic testing.
He underwent asynchronous bilateral nephron-sparing surgeries, and multiple tumors were resected (seven from the left and four from the right). All tumors exhibited oncocytic cytoplasm with perinuclear halo, and they were histologically diagnosed as HOCTs (n=3) and chromophobe RCCs (n=8). A few biopsied skin papules were histologically diagnosed as fibrofolliculomas. The patient recovered without complication, and receives periodic medical check-up.
Cytology and FLCN Status of BHD-F59RSVT
An immortalized cell line BHD-F59RSVT was established from one of the resected chromophobe RCCs. The tumor was obtained from the patient’s right kidney, a 12 × 14-mm-sized nodule with central bleeding. The tumor cells had granular or oncocytic cytoplasms, and round to raisinoid nuclei with perinuclear halos. Tumor cells were positively immunostained for CK7 and CD82, but they were negative for CD10 and RCC marker (Figures 2a–e). These immunostaining patterns indicated that the tumors were derived from distal tubules or collecting ducts.
The BHD-F59RSVT cells were plump or oval in shape. HE and cytostaining showed eosinophilic granular cytoplasm, supporting their oncocytic nature. Cytostaining analysis demonstrated that the cells expressed CK7, mitochondrial antigen, and cytokeratin AE1/AE3, but were negative for SMA, supporting the interpretation that these oncocytic epithelial cells were derived from distal tubules (Figures 2d–g). The cells were scarcely immunostained for FLCN (Figure 2h). Sequence analysis of exons 1–14 and donor/acceptor sites of introns was performed to clarify somatic second hit mutations of FLCN. BHD-F59RSVT demonstrated neither small intragenic mutations nor LOH of FLCN. The cells exhibited the same mutation pattern (c.1528_30delGAG) as that in the germ line (Supplementary Figure S1). Quantitative PCR analysis of exons 1–14 argued against potential deletion/duplication in the FLCN gene (data not shown). Further cytogenetic analysis of FLCN proximal regions in chromosome 17p11.2 demonstrated normal allele peaks and a trimodal pattern (Figure 2i). Methylation was not detectable in FLCN (data not shown); however, western blot analysis revealed that FLCN expression at the protein level was severely suppressed in BHD-F59RSVT as well as in the original RCC tissue (Figure 2j). Quantitative RT-PCR demonstrated suppressed FLCN mRNA in BHD-F59RSVT and original tumor compared with those in sporadic RCCs. Significant difference was not observed between BHD-F59RSVT and normal kidneys in which FLCN was preserved at the protein level (Supplementary Figure S2).
Chromosomal Analysis and CNV
Chromosomal analysis of BHD-F59RSVT revealed a loss of chromosome 16 and slight mosaic gains of chromosome 7 (Figure 3a). Other chromosomes demonstrated balanced chromosomal copy numbers. The pattern was distinctively different from that of sporadic chromophobe RCC characterized by the losses of chromosomes 1, 2, 6, 10, 13, 17, and 21.21
CNV analysis demonstrated numerous segmental gains, losses, and LOHs distributed over all chromosomes (Figure 3b; Supplementary Figure S3). Mosaic losses were consistently detected throughout in chromosome 16, and mosaic gains were detected in chromosome 7. There were 66 copy-number gains (2861 kbp in sum) and 9 mosaic gains (96 059 kbp in sum), and 294 copy-number losses (93 952 kbp in sum. It was not possible to distinguish between losses and mosaic losses because of low-level mosaic detection). Each chromosome had at least one LOH lesion. There were 105 LOHs (305 306 kbp in sum). In chromosome 16, LOH lesions showed either monosomy or chromosomal loss. In other chromosomes, almost all regions were copy-neutral LOH, that is, UPD. In SKY analysis, allelic loss of chromosome 16 was detected in three of five cells (Figure 3c), which was considered to be clonal according to ISCN. Other abnormalities were as follows: allelic loss of chromosome 22 was in one cell, and a translocation involving chromosome 7 in 2 of 5 cells.
We also examined chromosomal state of the original tumor for comparison. The tumor demonstrated balanced copy numbers including chromosome 16 (Supplementary Figure S4A). The LOH and gain/loss patterns were almost same to those in BHD-F59RSVT (Supplementary Figure S4B). In the original tumor, regional LOHs of chromosome 7 and 16 were almost identical to those in BHD-F59RSVT, but mosaic gains/losses were not detected. SKY analysis using the primary culture cells of the original tumor was performed. One of five cells demonstrated deletion of the short arm of chromosome 16 (Supplementary Figure S4C). Collectively, it was suggested that BHD-F59RSVT lost 16p during clonal selection, leading to an allelic loss of chromosome 16 in the immortalization process.
We established a new cell line that carries a mutant FLCN gene. It originated from a chromophobe RCC in a patient with BHD. The cell line, as well as the original tumor tissue, had neither a second hit nor promotor methylation of FLCN; however, its protein product (FLCN) was severely suppressed. It is assumed that some unknown mechanisms suppressed FLCN production both in vivo and in vitro. It is apparent that a second hit of FLCN may not be essential for FLCN suppression. BHD-F59RSVT had granular eosinophilic cytoplasm and demonstrated positive immunostaining for mitochondria. These morphologic characteristics were similar to those of the original RCC. BHD-F59RSVT may be a suitable model for investigating molecular pathways in the presence of impaired FLCN.
BHD-F59RSVT was established from an RCC with a typical chromophobe phenotype. Thus, this cell line is expected to reflect the nature of RCCs occurring in the majority of BHD patients. As Klomp et al and our group described previously, BHD-associated oncocytic RCCs have different chromosomal characteristics from those in sporadic chromophobe RCCs.20, 22, 23 BHD-F59RSVT exhibited balanced state of chromosomes 2, 6, 10, 13, 17, and 21. Frequent losses of these chromosomes are observed in sporadic chromophobe RCCs.21, 24 The unique features of BHD-F59RSVT may contribute to clarification of the distinct carcinogenic mechanism in which impaired FLCN is involved. On the other hand, BHD-F59RSVT showed a loss of chromosome 16. This chromosomal alteration during the immortalization process was not seen in previously reported BHD-associated RCCs.20, 22 More than one RCC-associated gene is on chromosome 16, including TSC2 and PKD1. Deletion of 16p was detected in one of the primary culture cells. It was likely that a subset of primary culture cells had segmental loss of chromosome 16p during proliferation, leading to an allelic loss in the immortalization process. As FLCN and TSC2 are both negative regulators of mTOR signaling, TSC2 in this cell line might be exposed to continuous stress of accelerated mTOR signaling for cell proliferation and protein synthesis.
There is also a concern about miscellaneous effects associated with transfection. SV40 large T antigen potentially inactivates several tumor suppressor genes including p53, pRB, and SEN6. Although chromosome segments containing these genes showed no pathologic event in BHD-F59RSVT, we cannot deny the possibility of small intragenic mutations and/or functional alterations of these factors. At least, we did not detect highly malignant transformation possibly related to SV40 large T antigen. In our preliminary in vivo experiment using nude mice, inoculated BHD-F59RSVT failed to form a tumor mass in 8 weeks (data not shown). Although longer surveillance is needed to characterize BHD-F59RSVT in vivo, this cell line may reflect less aggressive nature of the patient’s tumor that developed slowly without destructive infiltration.
The role of FLCN in the kidney cells is incompletely understood. The kidney-specific Flcn knockout mice develop polycystic kidneys and die due to renal failure.9 Cystic change of the kidney is also often observed in patients with BHD.18 Therefore, FLCN in renal epithelium appears to be involved not only in carcinogenesis but also in some metabolism-associated morphologic alterations. BHD-F59RSVT has the nature of distal tubules of the kidney, enriched with mitochondria, and impaired expression of FLCN. We hope that BHD-F59RSVT with heterozygous FLCN mutation will contribute to the studies of renal pathology, including renal carcinogenesis, polycystic kidney, and metabolic disorders in which FLCN is possibly involved.
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We thank Ms Hiromi Soeda, Mr Masato Kawashima, and members of the pathology laboratories in Yokohama City University Hospital for excellent assistance. This work was supported by JSPS KAKENHI grant number 26460422 (MF), 26460456 (Yo.N.), 15K08374 (Yu.N.), 15604475 (MB), 15551829 (MB), and 15K10600 (MY), the Mitsubishi Foundation 26329 (MF), Yokohama Foundation for Advancement of Medical Science (MF), Yokohama Academic Foundation (MF), Project Mirai Cancer Research Grants (MF), and Project for Development of Innovative Research on Cancer Therapeutics (P-DIRECT), Ministry of Education, Culture, Sports, Science and Technology of Japan (MY).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Laboratory Investigation website
The authors established a new renal cell carcinoma cell line, BHD-F59RSVT, from a typical chromophobe renal cell carcinoma that developed in a patient with Birt-Hogg-Dubé Syndrome. Detailed cytogenetic and microscopic features of BHD-F59RSVT and its original tumor are described in this paper. BHD-F59RSVT has a heterozygous FLCN mutation, and the expression of its protein product, the tumor suppressor folliculin, is markedly suppressed.
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Furuya, M., Hasumi, H., Baba, M. et al. Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt–Hogg–Dubé syndrome. Lab Invest 97, 343–351 (2017). https://doi.org/10.1038/labinvest.2016.137