The Birt-Hogg-Dube disease occurs as a result of germline mutations in the human Folliculin gene (FLCN), and is characterized by clinical features including fibrofolliculomas, lung cysts and multifocal renal neoplasia. Clinical and genetic evidence suggest that FLCN acts as a tumor suppressor gene. The human cell line UOK257, derived from the renal cell carcinoma of a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into UOK257 cells delays cell cycle progression, due to a slower progression through the late S and G2/M-phases. Similarly, Flcn –/– mouse embryonic fibroblasts progress more rapidly through the cell cycle than wild type controls (Flcn flox/flox). The reintroduction of tumor-associated FLCN mutants (FLCN ΔF157, FLCN 1–469 or FLCN K508R) fails to delay cell cycle progression in UOK257 cells. Additionally, FLCN phosphorylation (on Serines 62 and 73) fluctuates throughout the cell cycle and peaks during the G2/M phase in cells treated with nocodazole. In keeping with this observation, the reintroduction of a FLCN phosphomimetic mutant into the UOK257 cell line results in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that the tumor suppression function of FLCN may be linked to its impact on the cell cycle and that FLCN phosphorylation is important for this activity. Additionally, these observations describe a novel in vitro assay for testing the functional significance of FLCN mutations and/or genetic polymorphisms.

The Birt-Hogg-Dube (BHD) autosomal dominantly inherited syndrome has clinical features that include the development of skin fibrofolliculomas, lung cysts, and renal neoplasia. Renal tumors occur in approximately one third of individuals with BHD. The gene responsible for BHD syndrome is Folliculin (FLCN), which encodes a 68kD phosphoprotein (FLCN). One allele of the FLCN gene is mutated in the germline of >90% of BHD patients and somatic mutations or loss of the wild type FLCN allele are observed in the renal tumors of patients with BHD, suggesting that FLCN acts as a tumor suppressor protein. The human renal cell carcinoma cell line UOK257, derived from a patient with a germline mutation in the FLCN gene, harbors a truncated version of the FLCN protein. Reconstitution of the wild type FLCN protein into the FLCN-deficient UOK257 cells decreased their proliferation rate. Here we tested whether this difference in cell proliferation rate may be partly attributed to the effect of FLCN on cell cycle progression. We synchronized FLCN-deficient UOK257 cells and their isogenic, wild type or mutant FLCN counterparts, by double thymidine block and followed cell cycle progression after release. UOK257 cells expressing wild type FLCN exhibited a slower progression through the late S and G2/M-phases of the cell cycle compared to the vector only controls (FLCN-deficient). The reintroduction of tumor-associated FLCN mutants failed to delay cell cycle progression in UOK257 cells, indicating that the effect of wild type FLCN on cell cycle progression may contribute to its tumor suppressor function. FLCN phosphorylation on serines S62 and S73 fluctuates throughout the cell cycle and peaks in cells arrested at the G2/M boundary following treatment with nocodazole. In keeping with this observation, the reintroduction of a FLCN S62/73 phosphomimetic mutant into the UOK257 cell line resulted in faster progression through the cell cycle compared to those expressing the wild type FLCN protein. These findings suggest that orderly changes in FLCN phosphorylation during the cell cycle are linked to and may be required for its effect on cell cycle progression and possibly its activity as a tumor suppressor protein. We expect that these studies will provide insight into the role of FLCN in kidney tumorigenesis and will identify putative new targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2171. doi:1538-7445.AM2012-2171

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