Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn

Folliculin (FLCN)-interacting proteins 1 and 2 (FNIP1, FNIP2) are homologous binding partners of FLCN, a tumor suppressor for kidney cancer. Recent studies have revealed potential functions for Flcn in kidney; however, kidney-specific functions for Fnip1 and Fnip2 are unknown. Here we demonstrate that Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. We observed no detectable phenotype in Fnip2 knockout mice, whereas Fnip1 deficiency produced phenotypes similar to those seen in Flcn-deficient mice in multiple organs, but not in kidneys. We found that absolute Fnip2 mRNA copy number was low relative to Fnip1 in organs that showed phenotypes under Fnip1 deficiency but was comparable to Fnip1 mRNA copy number in mouse kidney. Strikingly, kidney-targeted Fnip1/Fnip2 double inactivation produced enlarged polycystic kidneys, as was previously reported in Flcn-deficient kidneys. Kidney-specific Flcn inactivation did not further augment kidney size or cystic histology of Fnip1/Fnip2 double-deficient kidneys, suggesting pathways dysregulated in Flcn-deficient kidneys and Fnip1/Fnip2 double-deficient kidneys are convergent. Heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed kidney cancer at 24 mo of age, analogous to the heterozygous Flcn knockout mouse model, further supporting the concept that Fnip1 and Fnip2 are essential for the tumor-suppressive function of Flcn and that kidney tumorigenesis in human Birt–Hogg–Dubé syndrome may be triggered by loss of interactions among Flcn, Fnip1, and Fnip2. Our findings uncover important roles for Fnip1 and Fnip2 in kidney tumor suppression and may provide molecular targets for the development of novel therapeutics for kidney cancer.Germline alteration of the folliculin (FLCN) gene, a novel tumor suppressor, is responsible for Birt–Hogg–Dubé (BHD) syndrome, an inherited kidney cancer syndrome characterized by cutaneous fibrofolliculomas, pulmonary cysts, and an increased risk for the development of kidney cancer (1–4). Genetic studies using Flcn knockout mice have defined important roles for Flcn in metabolism. Kidney-targeted Flcn knockout mice developed enlarged polycystic kidneys with elevated mechanistic target of rapamycin complex 1 (mTORC1) activity (5) and increased mitochondrial biogenesis through up-regulated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Ppargc1a) (6). Muscle-targeted Flcn knockout mice displayed both red-colored muscle with increased mitochondrial biogenesis caused by elevated Ppargc1, and cardiac hypertrophy with up-regulated mTORC1, which were ameliorated by Ppargc1a inactivation, suggesting that Flcn might serve as a critical link connecting mTORC1 with Ppargc1a-driven mitochondrial biogenesis (5–7). Importantly, mice heterozygous for Flcn inactivation develop renal tumors at 24 mo of age with increased mTORC1/2 activity and Ppargc1a expression, which mimics the human BHD tumor phenotype (6, 8–10), suggesting potential therapeutic targets in metabolic pathways for BHD-associated kidney cancer.The first FLCN interacting protein FNIP1 was identified through protein–protein interaction studies of the FLCN protein (11). FNIP1 binds to the C terminus of FLCN and to AMP-activated protein kinase (AMPK) (11), a critical molecule for energy sensing, further underscoring a central role for the FLCN/FNIP1 pathway in cellular metabolism. A second folliculin-interacting protein FNIP2 was discovered through bioinformatics searches for sequences similar to FNIP1 (12, 13). Similar to FNIP1, FNIP2 was found to bind to the C terminus of FLCN and to AMPK (12), suggesting a potential functional redundancy with FNIP1. Recent studies with Fnip1 knockout mouse models have demonstrated that Fnip1 is required for B-cell development (14, 15). Interestingly, a Flcn knockout mouse model using the tamoxifen-inducible ER (mutated form of the ligand-binding domain of the estrogen receptor)-Cre system also displayed defects in B-cell development (14), suggesting Fnip1 knockout mice might develop phenotypes similar to those that develop as a consequence of Flcn deficiency.Furthermore, FLCN has been shown to have a variety of functions that might potentially link AMPK, mTOR, and Ppargc1a with other important pathways. Crystallographic studies have shown that the C terminus of FLCN may be distantly related to Differentially Expressed in Normal Cells and Neoplasia (DENN) domain proteins and may possess guanine nucleotide exchange factor activity toward RAB35 (16). FLCN modulates TFE3 localization (17), which may play an important role in the exit of stem cells from pluripotency (18), and interacts with other signaling pathways including the von Hippel-Lindau–hypoxia inducible factor–vascular endothelial growth factor axis (19–21), the TGF-beta pathway (22, 23), Rho A signaling (24, 25), cell cycle regulation (26, 27), Rag-mediated amino acid sensing (28, 29), and autophagy (30, 31). These findings underscore FLCN as an important molecule, inactivation of which affects multiple pathways.To clarify the function of FLCN-interacting proteins Fnip1 and Fnip2, we inactivated Fnip1 and/or Fnip2 in mouse kidneys, muscle, and heart and investigated the effect on the mTOR pathway and mitochondrial metabolism. The absolute mRNA copy number of Fnip1 and Fnip2 was measured using droplet digital PCR (ddPCR) technology. To evaluate functional synergy of Fnip1 and Fnip2 with Flcn, we also inactivated Flcn, Fnip1, and Fnip2 simultaneously in mouse kidneys. Finally, we searched for latent tumor development in Fnip1 and Fnip2 knockout mice.