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

Abstract

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.

Keywords: Birt–Hogg–Dubé syndrome; FNIP1; FNIP2; folliculin; kidney tumor.

Fig. 1.

Neither kidney-targeted Fnip1 nor Fnip2 knockout mice show a kidney phenotype. (A) Conditional Fnip1 knockout mice were crossbred with CDH16-Cre transgenic mice. Inactivation of Fnip1 mRNA was confirmed by real-time PCR. n = 6 each at 3 wk of age. Mean ± SD. Student t test (Left). The size of the Fnip1-deficient kidney was not significantly different from that of the control kidney. n = 11 each at 3 wk of age. Mean ± SD. Student t test (Middle). Representative H&E staining of 3-wk old control and Fnip1-deficient kidneys did not show differences except for infrequent tiny cysts (Right). (B) Fnip2 gene-targeting vector was constructed by recombineering methodology using homologous recombination. A neomycin resistance (Neo r) cassette flanked by Frt (bar) and loxP (triangle) sequences was inserted into intron 11 for positive selection, and the thymidine kinase gene was included for negative selection. A second loxP sequence was inserted into intron 13. Correctly targeted embryonic stem cells were identified by Southern blot analysis and injected into blastocysts to produce chimeras. Backcrossing to C57BL/6 mice produced heterozygous F1 offspring with germline transmission of the Fnip2 floxed (f)-Neo allele. The Neo cassette flanked by Frt sites was excised in vivo by crossing with mice expressing the Flp recombinase transgene under the β-actin promoter. To produce the Fnip2 deleted (d) allele, Fnip2 f/+ mice were crossed with mice expressing the Cre recombinase transgene under the ubiquitous β-actin promoter. Deletion of exon 12 and 13 resulted in a frameshift and premature termination codon in exon 14, which was predicted to cause mRNA degradation by the nonsense-mediated decay mRNA surveillance system. (C) The targeted embryonic stem cells were screened by Southern blotting of BamH1- and EcoRV-digested DNA, using two different external probes located outside the targeting sequence, as shown in B. (D) PCR-based genotyping was performed using DNA extracted from mouse tails for routine monitoring of inheritance in offspring. Locations of PCR primers are indicated by arrows. (E) Kidney-specific inactivation of Fnip2 was achieved by crossing with CDH16-Cre transgenic mice. Inactivation of Fnip2 mRNA was confirmed by real-time PCR. n = 6 each at 3 wk of age. Mean ± SD. Student t test (Left). Size of Fnip2-deficient kidney was not significantly different from that of control kidney. n = 6 each at 3 wk of age. Mean ± SD. Student t test (Middle). Representative H&E staining of 3-wk old control and Fnip2-deficient kidneys did not show any difference in histology (Right).

Fig. 2.

Fnip1 and Fnip2 expression differs from organ to organ. (A) Muscle-targeted Fnip1 knockout mice show red-colored muscle relative to control muscle (6 wk of age). (B) Western blotting shows increased mitochondrial biogenesis and decreased pAMPK in Fnip1-deficient muscle. α-tubulin served as a loading control. n = 3 each at 6 wk of age. (C) Fnip1-deficient heart is enlarged relative to control heart (8 wk of age). (Scale bar: 5 mm.) (D) Histology of Fnip1-deficient heart shows enlarged cardiac fibers (8 wk of age). (Scale bars: 3 mm and 60 μm.) (E) Muscle diameter of Fnip1-deficient hearts was increased. n = 20 each at 8 wk of age. P < 0.0001, Student t test. (F) Western blotting of Fnip1-deficient hearts showed decreased pAMPK and activated mTORC1 signaling molecules. Gapdh served as a loading control. n = 3 each at 6 wk of age. (G) Western blotting shows the restoration of either FNIP1 or FNIP2 in Fnip1/Fnip2 null MEFs (DKO). α-tubulin served as a loading control. (H) Restoration of either FNIP1 or FNIP2 suppressed Ppargc1a expression in Fnip1/Fnip2 null MEFs (DKO). n = 3 each. Mean ± SD. P < 0.001 for DKO+FNIP1, P < 0.0001 for DKO+FNIP2; Student t test. (I) Restoration of either FNIP1 or FNIP2 suppressed ATP levels in Fnip1/Fnip2 null MEFs (DKO). n = 6 each. Mean ± SD. P < 0.0001. Student t test. (J) ddPCR showed Fnip1 expression was significantly higher than Fnip2 expression in bone marrow, heart, and quadriceps of C57BL/6 mice but was not significantly higher than Fnip2 expression in kidney of C57BL/6 mice. n = 6 each at 6 wk of age. Mean ± SD. P < 0.001 for bone marrow, heart, quadriceps. N.S., not significant. Student t test.

Fig. 3.

Kidney-targeted Fnip1/Fnip2 double-knockout mice develop enlarged polycystic kidneys. (A) Fnip1 and Fnip2 alleles were deleted specifically in kidney using CDH16-Cre transgenic mice. Double inactivation of Fnip1 and Fnip2 targeted to the kidney resulted in enlarged kidneys relative to the controls (3 wk of age). (B) T2 weighted images (T2WI) of MRI show multiple round-shaped structures in Fnip1/Fnip2 double-knockout kidneys at 3 wk of age (Left). Striations of medulla and renal pelvis are seen (Right). (C) H&E staining shows enlarged polycystic kidneys in 3-wk-old kidney-specific Fnip1/Fnip2 double-knockout mice. (Scale bars: 500 μm.) (D) H&E staining reveals detailed histology of kidneys from 3-wk-old kidney-targeted Fnip1/Fnip2 double-knockout mice displaying hyperplastic cells protruding into the lumen (arrow) within the medulla (Upper). Normal glomeruli (G) and proximal renal tubules (P) were observed in the cortex (Lower). (Scale bars: 50 μm and 20 μm.) (E) Kidney-specific Fnip1/Fnip2 double-knockout mice show an increased kidney/body weight ratio. Homozygous Fnip1/heterozygous Fnip2 double-knockout mice show a slightly increased kidney/body weight ratio as a result of occasionally observed tiny cysts. Mean ± SD. Two-sided Student t test. Three weeks of age. (F) Survival curve of kidney-specific Fnip1/Fnip2 double-knockout mice. Proportion surviving ± SD. Log rank test. n = 14 each at 3 wk of age.

Fig. 4.

Fnip1/Fnip2 double-deficient kidneys are identical to Flcn-deficient kidneys. (A) Western blotting shows increased Ppargc1 and phospho-proteins of the AKT-mTOR pathway in Fnip1/Fnip2 double-deficient kidney. Increased phospho-Ulk1 at Ser757, one of the readouts of mTORC1 activity, correlates with the accumulated SQSTM1/p62, normally degraded by autophagy. Mouse Fnip2 protein is not shown because of technical difficulty developing a unique Fnip2 antibody that does not cross-react with mouse Fnip1. Gapdh served as a loading control. n = 3 at 3 wk of age. (B) Immunofluorescence shows increased staining of p-mTOR (S2448) and pS6R (S240/244) in Fnip1/Fnip2 double-deficient kidney relative to control kidney. Nuclei were stained with DAPI (Blue). Representative of three mice at 3 wk of age. (C) Respiratory capacity of isolated mitochondria is increased in Fnip1/Fnip2 double-deficient kidney relative to control kidney. State 3 respiration of complex I and complex II, and complex IV-dependent respiration, were measured by Seahorse XF96 analyzer. Mean ± SD. n = 4 at 3 wk of age. Student t test (two-sided). (D) Electron microscope images show increased mitochondrial mass in Fnip1/Fnip2 double-deficient kidney compared with control kidney. Arrows indicate mitochondria. (Scale bars: 10 μm and 500 nm.) Percentage of mitochondrial area per cell was quantified for the indicated genotypes. Thirteen cells were evaluated for each genotype. Mean ± SD. Student t test (two-sided). Three weeks of age. (E) The kidney/body weight ratios of 3-wk-old mice with Flcn-deficient, Fnip1/Fnip2 double-deficient, and Flcn/Fnip1/Fnip2 triple-deficient kidneys show no significant differences. Mean ± SD, n = 8 each. Student t test (two-sided). (F) The histologies of kidneys from 3-wk-old Flcn-deficient, Fnip1/Fnip2 double-deficient, and Flcn/Fnip1/Fnip2 triple-deficient mice show no differences. Arrows indicate hyperplastic cells protruding into the cyst lumens that were observed in all of the genotypes. (Scale bars: 1 mm, 50 μm, and 20 μm.)

Fig. 5.

Fnip1/Fnip2 double-knockout mice develop kidney cancer. (A) Double knockdown of FNIP1 and FNIP2 in human kidney cancer cell line caused increased cell proliferation. UOK257 FLCN-null kidney cancer cell line was reconstituted with wild-type FLCN in a doxycycline-inducible manner (Upper Left). FNIP1 and FNIP2 expression was knocked down with siRNA (Lower Left). Cell proliferation was evaluated using MTT assay at days 1, 3, and 5 (Right). CT, Control siRNA; F1, FNIP1 siRNA; F2, FNIP2 siRNA; F1/2, FNIP1 siRNA + FNIP2 siRNA. Mean ± SD. Student t-test (two-sided). n = 5 each. N.S., no significance. (B) Kidney tumor-free survival demonstrates that heterozygous Fnip1/homozygous Fnip2 double-knockout mice developed renal tumors at a median age of 796 d. Neither heterozygous Fnip1 knockout mice nor homozygous Fnip2 knockout mice developed kidney cancer. Kidney tumor-free survival ± SD. Log rank test. n = 25, 15, and 42 for Fnip1^d/+, Fnip2^d/d, and Fnip1^d/+/Fnip2^d/d, respectively. (C) H&E staining shows kidney tumor development in heterozygous Fnip1/homozygous Fnip2 double-knockout mice. Tumor developed from cyst wall (Tumor1 of Mouse1, 692 d old) or within the kidney without prominent infiltration (Tumor2 of Mouse1). Cells lining the cyst walls (Cyst1 of Mouse2) were occasionally piled up (Cyst2 of Mouse2, 670 d old). The most frequent histology was the hybrid oncocytic tumor (Tumor1–Tumor3 of Mouse2). Papillary projections from the cyst wall were occasionally observed (Mouse3, 699 d old). (Scale bars: 1 mm, 100 μm, and 20 μm.) (D) Western blotting shows increased Ppargc1a and phospho-proteins of the AKT-mTOR pathway in kidney tumors from Fnip1/Fnip2 double-knockout mice. Gapdh served as a loading control. n = 7 each, 628–699 d old. (E) Immunostaining of kidney tumors from Fnip1/Fnip2 double-knockout mice demonstrate increased phospho-mTOR and pS6R (downstream readout of mTOR) compared with adjacent normal kidney.