Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt–Hogg–Dubé syndrome

Mitsuko Furuya,Hisashi Hasumi,Masaya Baba,Reiko Tanaka,Yasuhiro Iribe,Takahiro Onishi,Yoji Nagashima,Yukio Nakatani,Yasuhiro Isono,Masahiro Yao

doi:10.1038/labinvest.2016.137

Abstract

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.

Highlights

Hereditary renal cell carcinomas (RCCs) require special management so that patients and their families can avoid dialysis and progression to cancer-related death
It is known that a variety of renal tumor subtypes, including clear cell, papillary, chromophobe, and oncocytoma, may develop in Birt–Hogg– Dubé syndrome (BHD) patients, chromophobe RCC and hybrid oncocytic/chromophobe tumors (HOCTs) are predominant.[7,8]
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

Summary

Introduction

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), 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 FNIP2.4–6 The principal role of FLCN in human diseases is tumor suppression, and the mechanism involves several pathways, including mammalian target of rapamycin and 5′-AMPactivated 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, chromophobe RCC and hybrid oncocytic/chromophobe tumors (HOCTs) are predominant.[7,8]. Laboratory Investigation | Volume 97 March 2017 | www.laboratoryinvestigation.org chromophobe RCC and HOCT, and these tumor subtypes tend to be less aggressive in behavior.[14,15,16] 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

Methods

Results

Conclusion