H255Y and K508R missense mutations in tumour suppressor folliculin (FLCN) promote kidney cell proliferation

Abstract

Germline H255Y and K508R missense mutations in the folliculin (FLCN) gene have been identified in patients with bilateral multifocal (BMF) kidney tumours and clinical manifestations of Birt-Hogg-Dubé (BHD) syndrome, or with BMF kidney tumours as the only manifestation; however, their impact on FLCN function remains to be determined. In order to determine if FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation leading to pathogenicity, we generated mouse models expressing these mutants using BAC recombineering technology and investigated their ability to rescue the multi-cystic phenotype of Flcn-deficient mouse kidneys. Flcn H255Y mutant transgene expression in kidney-targeted Flcn knockout mice did not rescue the multi-cystic kidney phenotype. However, expression of the Flcn K508R mutant transgene partially, but not completely, abrogated the phenotype. Notably, expression of the Flcn K508R mutant transgene in heterozygous Flcn knockout mice resulted in development of multi-cystic kidneys and cardiac hypertrophy in some mice. These results demonstrate that both FLCN H255Y and K508R missense mutations promote aberrant kidney cell proliferation, but to different degrees. Based on the phenotypes of our preclinical models, the FLCN H255Y mutant protein has lost it tumour suppressive function leading to the clinical manifestations of BHD, whereas the FLCN K508R mutant protein may have a dominant negative effect on the function of wild-type FLCN in regulating kidney cell proliferation and, therefore, act as an oncoprotein. These findings may provide mechanistic insight into the role of FLCN in regulating kidney cell proliferation and facilitate the development of novel therapeutics for FLCN-deficient kidney cancer.

Figure 1.

Imaging and renal pathology of patients with FLCN H255Y and K508R mutations. Case 1: Patient from NCI Family with FLCN H255Y mutation. (A) MRI showing renal tumour in left kidney of patient with FLCN H255Y mutation. (B) Patient underwent a left partial nephrectomy in which 3 hybrid oncocytic tumours were removed. Magnification 200X. Case 2: Bilateral multifocal papillary type 1 RCC patient with FLCN K508R mutation. (C) MRI scan showing multiple renal tumours in right kidney of patient with FLCN K508R mutation. (D) This patient had a right partial nephrectomy in which 22 papillary type 1 renal tumours were removed. Magnification 150X. Case 3: Bilateral multifocal oncocytoma patient with FLCN K508R mutation. (E) MRI scan showing multiple renal tumours in right and left kidneys of patient with FLCN K508R mutation. (F) This patient had a left partial nephrectomy in which 5 renal oncocytomas were removed. Magnification 150X.

Figure 2.

Establishment of a Flcn H255Y mutant transgenic mouse model using BAC recombineering technology. (A) Screening for BAC transgene integration using Southern blotting. The probe detecting the chloramphenicol resistant (CMR) cassette was used for identifying BAC transgenic founder mice. (B) Sequencing result of mouse tail DNA confirming the correct mutant sequence encoding a histidine to tyrosine exchange. (C) Real-time PCR showing Flcn expression levels in kidneys of Flcn^f/+, CDH16-Cre control mice, Flcn ^f/d, CDH16-Cre kidney-targeted knockout mice, and Flcn ^f/d/H255Y, CDH16-Cre kidney-targeted knockout mice expressing the Flcn H255Y mutation. (D) Representative genotyping results for Flcn^f/+, CDH16-Cre mice and Flcn ^f/+/H255Y, CDH16-Cre mice using SNP real-time PCR and mouse tail DNA. N.S., no significance.

Figure 3.

Establishment of a Flcn K508R mutant transgenic mouse model using BAC recombineering technology. (A) Screening for BAC transgene integration using Southern blotting. The probe detecting the chloramphenicol resistant (CMR) cassette was used for identifying BAC transgenic founder mice. (B) Sequencing result of mouse tail DNA confirming the correct mutant sequence encoding a lysine to arginine exchange. (C) Real-time PCR showing Flcn expression levels in kidneys of Flcn^f/+, CDH16-Cre control mice, Flcn ^f/d, CDH16-Cre kidney-targeted knockout mice, and Flcn ^f/d/K508R, CDH16-Cre kidney-targeted knockout mice expressing the Flcn K508R mutation. (D) Representative genotyping results for Flcn^f/+, CDH16-Cre mice and Flcn ^f/+/K508R, CDH16-Cre mice using SNP real-time PCR. N.S., no significance.

Figure 4.

Expression of BAC transgene carrying Flcn H255Y mutant allele does not rescue kidney-targeted Flcn knockout mouse phenotype. (A) Histology of Flcn-deficient kidney with Flcn H255Y mutant expression (Flcn ^f/d/H255Y, CDH16-Cre) at 3 weeks of age compared to Flcn-deficient kidney (Flcn ^f/d, CDH16-Cre) and control kidney (Flcn^f/+, CDH16-Cre). Multi-cystic kidneys are shown with hyperplastic kidney cells protruding into the cystic lumen in both Flcn ^f/d/H255Y, CDH16-Cre mice and Flcn ^f/d, CDH16-Cre mice. (B) Kidney to body weight ratio of kidney-targeted Flcn knockout mice with Flcn H255Y mutant expression (Flcn ^f/d/H255Y, CDH16-Cre) at 3 weeks of age compared to kidney-targeted Flcn knockout mice (Flcn ^f/d, CDH16-Cre) and control mice (Flcn^f/+, CDH16-Cre). No significant difference was seen between Flcn ^f/d/H255Y, CDH16-Cre and Flcn ^f/d, CDH16-Cre mice. (C) Kaplan-Meier survival plot of kidney-targeted Flcn knockout mice with Flcn H255Y mutant expression compared to kidney-targeted Flcn knockout and control mice; n= 8 mice for each genotype. No significant difference was seen between Flcn ^f/d/H255Y, CDH16-Cre and Flcn ^f/d, CDH16-Cre mice. (D) Western blot analysis of mTORC1 downstream effectors in Flcn-deficient kidneys with Flcn H255Y mutant expression, Flcn-deficient kidneys and control kidneys. N.S., no significance.

Figure 5.

Expression of BAC transgene carrying Flcn K508R mutant allele partially, but not completely rescues the kidney-targeted Flcn knockout mouse phenotype. (A) Histology of Flcn-deficient kidney with Flcn K508R mutant expression (Flcn ^f/d/K508R, CDH16-Cre) at 3 weeks of age compared to Flcn-deficient kidney (Flcn ^f/d, CDH16-Cre) and control kidney (Flcn^f/+, CDH16-Cre). Fewer cysts develop in Flcn ^f/d/K508R, CDH16-Cre mice compared to Flcn ^f/d, CDH16-Cre mice with no protruding hyperplastic cells. (B) Kidney to body weight ratio of kidney-targeted Flcn knockout mice with Flcn K508R mutant expression (Flcn ^f/d/K508R, CDH16-Cre) at 3 weeks of age showed significantly decreased % kidney/body weight relative to kidney-targeted Flcn knockout mice (Flcn ^f/d, CDH16-Cre). (C) Kaplan-Meier survival plot of kidney-targeted Flcn knockout mice with Flcn K508R mutant expression demonstrates 3-fold increase in survival (61 vs 20 days) compared to kidney-targeted Flcn knockout mice; n= 8 mice for each genotype. (D) Western blot analysis of mTORC1 downstream effectors in Flcn-deficient kidneys with Flcn K508R mutant expression, Flcn-deficient kidneys and control kidneys at 3 weeks of age showing suppression of the mTOR pathway activation in the Flcn ^f/d/K508R, CDH16-Cre mice compared with Flcn ^f/d, CDH16-Cre mice. N.S., no significance.

Figure 6.

Expression of BAC transgene carrying Flcn K508R mutant alleles in heterozygous Flcn knockout mice drives a late onset Flcn-deficient phenotype. (A) Histology of polycystic kidneys that developed in a portion of heterozygous Flcn knockout mice with Flcn K508R mutant expression (Flcn ^d/+/K508R) at 3 weeks and 9 months of age, respectively. Insert shows hyperplastic cells protruding into the cystic lumen.(B) PCR-based genotyping of DNA from macrodissected FFPE Flcn ^d/+/K508R cystic kidney tissue generated PCR products of the correct size for the Flcn deleted allele and for the Flcn wild-type allele and/or Flcn K508R transgene (both are the same size). DNA from macrodissected FFPE Flcn ^d/+kidneys was genotyped and included as a control along with PCR-based genotyping of mouse tail DNA for comparison. +, wild-type allele; d, delete allele; Kid, kidney tissue. (C) Sequence verification of both wild-type Flcn and Flcn K508R transgene in PCR product from (B) amplified from DNA extracted from macrodissected Flcn ^d/+/K508R cystic kidneys. +, wild-type allele. (D) Gpnmb expression (read-out of Flcn deficiency) measured by qRT-PCR was statistically significantly higher in Flcn ^d/+/K508R kidneys compared to Flcn ^d/+mouse kidneys. Statistical significance measured by two-way ANOVA. Two representative mouse kidneys for each genotype are shown. P <0.05 for significance. (E) Histology of cardiac hypertrophy that developed in a portion of heterozygous Flcn knockout mice with Flcn K508R mutant expression at 9 months of age. (F) Muscle diameter analysis of (E).