Birt-Hogg-Dubé syndrome is caused by mutations in the FLCN gene. The FLCN protein acts as a tumour suppressor and BHD patients have a high risk of developing renal cell carcinoma (RCC). The mechanisms of tumour formation in BHD have been investigated using mouse models and human RCC tissues. However, in vitro signalling studies of human renal cells with mutant FLCN are still scarce. In a recent study, Furuya et al. (2016) established a new cell line from a BHD patient’s chromophobe RCC. The authors investigated FLCN mutations, chromosome profiles, and cytopathologic characteristics of the cell line to confirm its suitability for functional analysis of the typical phenotype of BHD-associated RCC with impaired FLCN.
The only cell line from a BHD patient’s RCC available and used for functional studies of FLCN in vitro is UOK257. UOK257 was established from a high-grade RCC characterized as predominantly clear cell type with aggressive behaviour. However, most renal tumours developing in BHD patients are the less aggressive subtypes chromophobe RCC and hybrid oncocytic/chromophobe tumours (HOCT). Therefore, the authors established an immortalized cell line derived from a chromophobe RCC in a Japanese BHD patient.
The patient was a 52-year-old Japanese man with repeated episodes of pneumothorax, skin papules, multiple pulmonary cysts, bilateral renal tumours and a family history of pneumothorax. Genetic analysis revealed a pathogenic FLCN mutation – a 3-bp deletion in exon 13 (c.1528_30delGAG) confirming the diagnosis of BHD Syndrome. The patient underwent nephrectomy, and multiple tumours were resected. All tumours were histologically classified as chromophobe RCCs or HOCTs.
The immortalized cell line named BHD-F59RSVT was established from one of the resected chromophobe RCCs by transfecting SV40 large T antigen. BHD-F59RSVT had granular eosinophilic cytoplasm and demonstrated positive immunostaining for mitochondria. These morphologic characteristics were similar to those of the original RCC. The cells exhibited the same mutation pattern as that in the germ line. The cell line, as well as the original tumour tissue, had neither a second hit nor promotor methylation of FLCN. However, western blot analysis revealed that FLCN expression at the protein level was suppressed. It is apparent that a second hit of FLCN may not be essential for FLCN suppression. FLCN mRNA levels were also suppressed in the cell line and original tumour compared with those in sporadic RCC.
Chromosomal analysis of BHD-F59RSVT revealed a loss of chromosome 16. Other chromosomes demonstrated balanced chromosomal copy numbers. The pattern was distinctively different from that of sporadic chromophobe RCC. Deletion of 16p was detected in one of the primary culture cells suggesting that a subset of primary culture cells had segmental loss of chromosome 16p during proliferation, leading to an allelic loss in the immortalization process. This chromosomal alteration during the immortalization process was not seen in previously reported BHD-associated RCCs (Iribe et al., 2016; Klomp et al., 2010). More than one RCC-associated gene is on chromosome 16, including TSC2 and PKD1. As FLCN and TSC2 can both be negative regulators of mTOR signalling, TSC2 in this cell line might be exposed to continuous stress of accelerated mTOR signalling for cell proliferation and protein synthesis.
There is the possibility of transfection-associated effects. SV40 large T antigen can inactivate several tumour suppressor genes including p53, pRB, and SEN6. Even though chromosome segments containing these genes showed no pathologic event the authors do not exclude the possibility of small intragenic mutations and/or functional alterations of these genes. However, in preliminary experiments using nude mice, inoculated BHD-F59RSVT failed to form a tumour mass in 8 weeks. Although longer surveillance is needed to characterize BHD-F59RSVT in vivo, this cell line may reflect a slow-developing and less aggressive nature of the patient’s tumour.
BHD-F59RSVT may be a suitable model to study molecular pathways in the presence of impaired FLCN. BHD-F59RSVT was established from an RCC with a typical chromophobe phenotype reflecting the nature of RCCs occurring in most BHD patients. The unique features of BHD-F59RSVT may help clarifying the distinct tumour formation mechanism in BHD syndrome.
- Furuya, M., Hasumi, H., Baba, M., Tanaka, R., Iribe, Y., Onishi, T., Nagashima, Y., Nakatani, Y., Isono, Y., & Yao, M. (2016). Establishment and characterization of BHD-F59RSVT, an immortalized cell line derived from a renal cell carcinoma in a patient with Birt–Hogg–Dubé syndrome Laboratory Investigation DOI: 10.1038/labinvest.2016.137