Birt-Hogg-Dubé syndrome ͉ kidney cancer ͉ mouse model ͉ mTOR ͉ tumor suppressor B irt-Hogg-Dubé (BHD) syndrome is an inherited kidney cancer syndrome which predisposes patients to develop hair follicle tumors, lung cysts, spontaneous pneumothorax, and an increased risk of renal neoplasia (1-3). We previously identified germline mutations in the BHD (FLCN) gene in patients with BHD (4). About one-third of BHD patients develop bilateral multifocal renal tumors that are most frequently chromophobe renal tumors and renal oncocytic hybrid tumors with features of chromophobe renal carcinoma and renal oncocytoma (5). Somatic mutations in the wild-type copy of BHD and loss of heterozygosity at chromosome 17p11.2 have been identified in human BHD tumors, indicating that BHD is a classical tumor suppressor gene (6). The BHD protein folliculin (FLCN) is a 64-kDa protein with no known functional domains (4). We reported two FLCN binding proteins FNIP1 and FNIP2, which interact with 5Ј-AMP-activated protein kinase (AMPK), an important energy sensor in cells that negatively regulates mammalian target of rapamycin (mTOR), the master switch for cell growth and proliferation (7-9). These findings suggest that FLCN may play a role in cellular energy and nutrient sensing through interactions with the AMPK-mTOR signaling pathway. Mutations in several other tumor suppressor genes, including LKB1 (10), PTEN (11), and TSC1/2 (12), have been shown to lead to dysregulation of PI3K-AKT-mTOR signaling and to the development of other hamartoma syndromes. We and others previously reported the generation of a conditionally targeted BHD allele and kidney-directed BHD inactivation in the mouse using the cadherin16 (KSP)-Cre transgene (13,14). Although BHD homozygous deletion in kidney epithelial cells was sufficient to cause uncontrolled cell proliferation and hyperplastic cell transformation, the kidney-targeted BHD-knockout mice lived only approximately 3 weeks and did not produce kidney tumors. A BHD heterozygous knockout mouse model that develops tumors with age will more accurately reflect tumor development in the human BHD patient and may be a better model for understanding how BHD inactivation leads to tumor initiation and progression. Here we report the analysis of an embryonic lethal phenotype that occurs in a BHD homozygous knockout mouse model and characterize and compare the kidney tumors that develop in a BHD heterozygous knockout mouse model with human BHD kidney tumors. ResultsRole of BHD during Early Embryogenesis. We have analyzed mouse BHD mRNA expression levels by qRT-PCR in wild-type embryos and adult tissues (Fig. S1). We detected consistent BHD mRNA expression from E8.5 to E12.5 with 4-fold elevation at E19 and high expression in adult heart, pancreas, and prostate with moderate expression in adult brain, kidney, liver, and lung. BHD mRNA expression was further analyzed during early embryogenesis by whole mount in situ hybridization (Fig. S2). BHD mRNA was expressed consistently throughout embryogenesis. At E5.5, BHD expr...

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