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Proc Natl Acad Sci U S A. Nov 3, 2009; 106(44): 18722–18727.
Published online Oct 22, 2009. doi:  10.1073/pnas.0908853106
PMCID: PMC2765925
Medical Sciences

Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2

Abstract

Germline mutations in the BHD/FLCN tumor suppressor gene predispose patients to develop renal tumors in the hamartoma syndrome, Birt-Hogg-Dubé (BHD). BHD encodes folliculin, a protein with unknown function that may interact with the energy- and nutrient-sensing AMPK-mTOR signaling pathways. To clarify BHD function in the mouse, we generated a BHD knockout mouse model. BHD homozygous null (BHDd/d) mice displayed early embryonic lethality at E5.5–E6.5, showing defects in the visceral endoderm. BHD heterozygous knockout (BHDd/+) mice appeared normal at birth but developed kidney cysts and solid tumors as they aged (median kidney-lesion-free survival = 23 months, median tumor-free survival = 25 months). As observed in human BHD kidney tumors, three different histologic types of kidney tumors developed in BHDd/+ mice including oncocytic hybrid, oncocytoma, and clear cell with concomitant loss of heterozygosity (LOH), supporting a tumor suppressor function for BHD in the mouse. The PI3K-AKT pathway was activated in both human BHD renal tumors and kidney tumors in BHDd/+ mice. Interestingly, total AKT protein was elevated in kidney tumors compared to normal kidney tissue, but without increased levels of AKT mRNA, suggesting that AKT may be regulated by folliculin through post translational or post-transcriptional modification. Finally, BHD inactivation led to both mTORC1 and mTORC2 activation in kidney tumors from BHDd/+ mice and human BHD patients. These data support a role for PI3K-AKT pathway activation in kidney tumor formation caused by loss of BHD and suggest that inhibitors of both mTORC1 and mTORC2 may be effective as potential therapeutic agents for BHD-associated kidney cancer.

Keywords: Birt-Hogg-Dubé syndrome, kidney cancer, mouse model, mTOR, tumor suppressor

Birt-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 (13). 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 5′-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 (79). 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.

Results

Role 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 expression was restricted to extraembryonic tissues; however, by E6.5, BHD was expressed in both embryonic and extraembryonic tissues. We saw strong expression in certain tissues including neural ectoderm, headfold, and limb buds, but the signal was relatively weak in the surrounding endoderm and heart.

Next we evaluated BHD homozygous knockout (BHDd/d) embryos from intercrosses of BHD heterozygous knockout (BHDd/+) mice. BHDd/+ mice appeared normal at birth and developed normally, but no BHDd/d mice were born in 75 neonates indicating embryonic lethality (Fig. 1A). No BHDd/d embryos were found at E9.5. BHDd/d embryos were found before E8.5 with lower frequency (9.3%, 15/161) than expected (25%). There were many empty deciduas suggestive of early embryonic death and resorption (21.8%, 26/119 at E5.5 and E6.5). The morphologies of all BHDd/d embryos were abnormal with one exception at E5.5. By gross appearance BHDd/d embryos (Fig. 1 B and D) were thinner or smaller than BHDd/+ (Fig. 1 C and E) or BHD+/+ embryos with occasional bleeding. There were no histological abnormalities in BHD+/+ or BHDd/+ embryos (Fig. 1 F and G). Most BHDd/d embryos lacked an organized cell layer with only a small cell mass (Fig. 1I). We could occasionally see BHDd/d embryos consisting of two types of cells, clear visceral endoderm-like cells and ectoderm cells (Fig. 1H). Immunohistochemical staining with the visceral endoderm (VE) marker DAB2 confirmed that the outer cell layer was VE (Fig. 1 J–M). Interestingly, VE of the BHDd/d embryos showed distorted enlarged cytoplasm and a disorganized structure (Fig. 1 K and M) instead of a cuboidal epithelial layer as seen in the BHDd/+ embryos (Fig. 1 J and L). Also the nuclei of the VE cells were disorientated in BHDd/d embryos (Fig. 1H'), whereas nuclei were aligned along the basal membrane in an orderly fashion in BHD+/+ embryos (Fig. 1F'). BHDd/d embryos also displayed a disorganized ectoderm structure and did not show cavitation or a polarized epithelial cell layer (Fig. 1 H and K).

Fig. 1.
Characterization of BHDd/d mouse embryo phenotype. (A) Embryos were isolated from BHD d/+ intercrosses, observed under a dissection microscope and genotyped by PCR. Numbers in parentheses represent embryos with abnormal appearance, as shown in (B and ...

Spontaneous Kidney Tumor Development in BHDd/+ Mice with Loss of Heterozygosity.

BHDd/+ mice spontaneously developed cysts, complex cysts and solid tumors in their kidneys after the age of 10 months (Fig. 2 A and B). Two of 23 BHD+/+ mice displayed small, isolated simple cysts in their kidneys. The median age of kidney-lesion-free survival for BHDd/+ mice was 23 months (n = 65), compared with an undefined kidney-lesion-free survival for BHD+/+ littermate controls (n = 28, P < 0.0001) (Fig. 2C). No solid kidney tumors developed in BHD+/+ mice (BHDd/+ median tumor-free survival = 25 months, n = 65; BHD+/+ median tumor-free survival = undefined, n = 28; P = 0.0026) (Fig. 2D). The number of kidney lesions in BHDd/+ mice between the ages of 20 and 25 months is shown in Fig. 2E (n = 35; no. cysts per animal, mean = 3.43, SD = 3.13; no. mixed lesions per animal, mean = 0.51, SD = 0.85; no. solid tumors per animal, mean = 0.51, SD = 0.82). Histological examination showed that kidney cysts that develop in BHDd/+ mice were lined by hyperplastic cells with enlarged cytoplasm and nuclei (Fig. 3A). The cysts found in BHD+/+ mice were lined by flat cyst cells characteristic of simple cysts, which were distinct from the hyperplastic cysts that developed in BHDd/+ mice. Complex cysts, defined as cysts with some structure or solid portions in the lumen, were observed. These complex cysts showed papillary protrusions into the lumen (Fig. 3B) as well as regions containing solid tumor (Fig. 3C). Interestingly the solid tumors displayed histologies similar to human kidney tumors that develop in BHD patients, including clear cell (Fig. 3D), oncocytic hybrid consisting of a mixture of chromophobe renal carcinoma and oncocytic cells (Fig. 3E), and oncocytoma (Fig. 3F). Southern blot analysis of tumors that formed in BHDd/+ mice showed a severely reduced wild-type BHD allele signal compared to adjacent normal kidney tissue, supporting LOH at the BHD locus (Fig. 3G). Western blot analysis showed only very weak FLCN expression in the tumors compared with adjacent BHDd/+ kidney or wild-type kidney tissue (Fig. 3H), thus confirming the inactivation of both BHD alleles. Loss of endogenous FLCN expression was also confirmed by immunofluorescent staining using the Duolink in situ proximity ligation assay (PLA), which enables visualization of endogenous FLCN with two different antibodies (Fig. 3 I and J). Importantly all five kidney tumors that were analyzed from BHDd/+ mice showed very low FLCN expression compared to normal mouse kidneys. These results strongly support the premise that kidney tumors, which developed in the BHDd/+ mouse model, were produced as a consequence of loss of FLCN function.

Fig. 2.
Spontaneous kidney tumor development in BHDd/+ mice. BHDd/+ mice and BHD+/+ mice were aged and dissected randomly at different time points when they were moribund or had to be euthanized due to dermatitis. The renal capsule was removed from isolated kidneys ...
Fig. 3.
Histological analysis of BHDd/+ mouse kidney lesions. (A) H&E staining on formalin fixed paraffin embedded kidney samples. Cells lining the cyst show proliferative tubular epithelium unique to BHD cysts. (B) Cyst with papillary projection. (C ...

Activation of the PI3K-AKT-mTOR Signaling Pathway in Kidney Tumors from BHDd/+ Mice and Human BHD Patients.

Total AKT protein levels, AKT1, and AKT2 as well as total AKT, were dramatically elevated in kidney tumors compared with normal kidney tissue (Fig. 4A). Levels of AKT phosphorylation were also dramatically elevated at both the PDK1 phosphorylation site (Thr308) and the mTORC2 phosphorylation site (Ser473) (15) (Fig. 4 A and B). AKT mRNA levels were measured by qRT-PCR in the same samples (Fig. 4C). Contrary to the Western blot results, mRNA levels of AKT1 and AKT2 were not significantly different between tumors and normal kidneys. Increased phosphorylation of downstream effectors of AKT signaling would support AKT activation in kidney tumors that developed in BHDd/+ mice. Indeed, p-GSK3, p-FOXO1, and p-FOXO3a were elevated in tumors compared with normal kidneys (Fig. 4 D–F). We also saw Cyclin D1 elevation, which might be a consequence of GSK3 phosphorylation (Fig. 4D). mTOR phosphorylation on serine 2448, which reflects mTORC1 activation (16), was higher in tumors than in normal kidneys. Furthermore, mTOR phosphorylation on serine 2481, indicating mTORC2 activation (17), was also higher in tumors compared with normal kidney tissue (Fig. 5A). Interestingly Rictor was more highly expressed in tumors from BHDd/+ mice, which may lead to mTORC2 activation and result in higher AKT phosphorylation on serine 473. Levels of phospho(p)-S6 kinase (Thr421/Ser424) and phospho S6 ribosomal protein (Ser240/244), readouts of mTORC1 activation, were higher in tumors than in normal kidney tissue, although total protein levels of S6K and S6R were also elevated (Fig. 5A). Consistent with the Western blot results, we were able to see stronger p-mTOR (Ser2448) and p-S6R (Ser240/244) staining in cells lining the cysts and in tumors from BHDd/+ mice when compared to adjacent normal kidney (Fig. 5 B–G).

Fig. 4.
Elevated total AKT and phosphorylated AKT protein in kidney tumors arising in BHDd/+ mice. (A) Western blotting was performed on the protein lysates isolated from normal kidneys and kidney tumors. Both total AKT and phospho-AKT expressions were up-regulated ...
Fig. 5.
mTOR pathway activation in kidney tumors from BHDd/+ mice. (A) Western blotting was performed on the protein lysates isolated from normal kidneys and kidney tumors. Both mTOR phosphorylation sites (Ser2448 and Ser2481) were more phosphorylated in tumors ...

Finally we evaluated these protein levels in human BHD tumors and normal kidneys. Western blotting and immunofluorescent staining showed higher levels of p-AKT(Ser473) in kidney tumors compared to normal kidney controls (Fig. 6 A, E, and F). We quantified p-AKT(Ser473), p-S6K(Thr421/Ser424), and p-S6R(Ser240/244) using a Meso Scale Discovery multiplex microtiter plate assay, a quantitative assay system using a combination of electrochemiluminescence detection and patterned arrays (Fig. 6 B–D). The protein levels of p-AKT (Ser473) were more than 30 times higher in human BHD tumors than in normal kidneys. Levels of p-S6K (Thr421/Ser424) protein and p-S6R protein were also higher in the BHD kidney tumors (Fig. 6 C and D). We compared the genotype and phenotype of the BHD patients whose kidney tumors were evaluated in this study (Fig. 6G). The kidney tumors were surgical specimens from three different female patients with different types of germline BHD mutations: missense, splicing defect and frameshift. All of the patients with different BHD mutations showed the classic triad of BHD phenotypic features- fibrofolliculomas, pulmonary cysts and renal tumors (Fig. S3). Importantly, the molecular phenotype was similar among the tumors derived from these three different types of BHD alterations, underscoring the importance of PI3K-AKT-mTOR activation for kidney tumorigenesis in BHD patients.

Fig. 6.
Activation of the PI3K-AKT-mTOR signaling pathway in kidney tumors from human BHD patients. (A) Western blotting was performed on protein lysates of normal kidneys and kidney tumors from BHD patients, showing elevation of p-AKT, total AKT1, and total ...

Discussion

The early embryonic lethality of BHD homozygous knockout mice supports an essential role for BHD in mouse development. Embryonic ectoderm-like cells of BHDd/d embryos did not form the proamniotic cavity or bilayered ectoderm structure. The visceral endoderm (VE) cell layer was disorganized with misaligned nuclei suggesting loss of polarity. Interestingly, VE cells displayed swollen cytoplasm with enlarged vacuoles. In addition to nutrient uptake and transport, the anterior VE plays an active role in formation of the proamniotic cavity during the postimplantation period (18). The existence of swollen vacuoles may suggest a defect in phagocytosis, trafficking, or digestion. The loss of polarity of VE cells resulting from BHD inactivation may compromise their function. LKB1, working upstream of AMPK, regulates cell polarity (19) and loss of LKB1 or AMPK function is associated with a defect in cell polarity. The BHD encoded protein, folliculin (FLCN), interacts with FNIP1/2, which associate with AMPK. Loss of BHD may lead to a defect in cell polarity by altering LKB1/AMPK signaling resulting in embryonic lethality of BHDd/d mice.

Kidney tumor development in BHDd/+ mice mimics the kidney tumor phenotype found in humans with BHD. Previously we reported a kidney-targeted conditional BHD knockout mouse model, which produced enlarged highly cystic kidneys displaying profoundly increased cell proliferation and hyperplastic morphologic changes. However, the animals died of renal failure at 21 days of life and therefore did not live long enough to develop kidney tumors. Furthermore, additional genetic and/or epigenetic events may be required for tumor formation. BHDd/+ mice developed hyperplastic kidney cysts, complex cysts and solid tumors at different frequencies and with different latency periods, providing evidence to support a multistep process in BHD-associated kidney carcinogenesis. Loss of both copies of BHD in all analyzed tumors arising in BHDd/+ mice supports BHD inactivation as the initiating step for kidney tumorigenesis in BHD.

Although other naturally-occurring animal models for BHD have been described (20, 21), they may harbor additional genetic changes that could confound studies of the functional consequences of BHD inactivation. The genetically engineered mouse model in this report provides a “clean” system with which to pursue FLCN functional studies. Hartman et al. (22) has described another BHD heterozygously targeted mouse model in which a gene trap cassette inserted in intron 8 encodes a FLCN-βgeo fusion protein. These investigators reported a lower incidence of kidney tumors than in our BHDd/+ model, (three tumors in 31 BHD+/− mice and 0 tumors in 15 wild-type mice), which may possibly be due to a shorter observation period (17 vs. 30 months). Reduced phospho-S6R (Ser235/236) immunostaining of paraffin-embedded tumors led these investigators to conclude that mTOR activity was suppressed in kidney tumors that developed in Bhd+/− mice. The inconsistencies between the results of Hartman et al. and our results may be due to differences in gene targeting strategy. In our model, mRNA transcribed from the BHDd allele creates a frameshift resulting in a premature termination codon at the beginning of exon 8 and will be degraded by nonsense mediated decay (NMD). In fact, no truncated forms of FLCN protein were detected by Western blotting in our BHDd/+ mouse kidneys. However, although Hartman et al. did not evaluate the presence of the FLCN-βgeo fusion protein, successful selection of targeted embryonic stem cells by G418 screening would necessitate the expression of a FLCN-βgeo fusion protein that retains the N-terminal half of FLCN in the embryonic stem cells and, presumably, also in the Bhd+/− mouse tumors. Since Hartman et al. did not confirm LOH of BHD, it is not clear if the kidney tumors that developed in the Bhd+/− mice were caused by homozygous inactivation of BHD or by another molecular mechanism. If the FLCN-βgeo fusion protein has partial function it could down-regulate mTOR and explain the discrepancy between the mTOR activation seen in tumors that developed in our BHDd/+ mice and the reduced mTOR activity reported by Hartman in tumors that developed in the gene trap mouse model for BHD. One additional difference between the two studies was the method of tissue preservation for immunostaining, which may affect the antigenicity of certain proteins. In this report freshly frozen tissues in OCT compound were analyzed, whereas paraffin embedded tissues were used in the Hartman's study.

We found PI3K-AKT-mTOR pathway activation in both kidney tumors from BHDd/+ mice and human BHD tumors, consistent with the kidney-targeted BHD knockout kidney results, supporting a role for PI3K-AKT-mTOR pathway in both BHD null kidney tumorigenesis and in the development of hyperplastic kidney cysts. Interestingly, total AKT protein levels were elevated in those tumors without changes in AKT mRNA levels. Therefore FLCN may be involved in regulation of total AKT protein levels through post-translational or post transcriptional modification. It is possible that elevated total AKT could result in higher AKT activation as indicated by elevated AKT phosphorylation (Thr308/Ser473). AKT downstream target molecules were also highly phosphorylated in tumors from BHDd/+ mice, supporting the possibility that elevated total AKT may be driving the activation of the AKT pathway. Downstream of AKT, we found mTORC1 was activated in tumors from BHDd/+ mice. The high levels of p-AKT (Ser473) suggest mTORC2 activation. As expected, we saw elevated levels of p-mTOR on Ser2481, a readout of mTORC2 activity (17), in the mouse kidney tumors. Additionally we observed elevated Rictor expression in tumors from BHDd/+ mice. mTORC2 activity is regulated by Rictor expression level (23), and Facchinetti et al. reported that mTORC2 phosphorylated AKT on the turn motif and stabilized AKT (24). Taken together, the high expression level of Rictor and subsequent activation of mTORC2 may be the primary mechanism by which AKT activation occurs in BHD null tumors. Previously we found that FLCN phosphorylation was partially blocked by rapamycin (7), suggesting that FLCN function may be regulated by mTOR. This may support a hypothesis whereby FLCN is on a negative feedback loop suppressing PI3K-AKT-mTOR signaling (Fig. S4).

Each kidney tumor from human BHD patients that was analyzed showed PI3K-AKT-mTOR activation, regardless of type of BHD mutation. Most frameshift or splicing defect mutations found in BHD patients are predicted to produce aberrant mRNAs that would be degraded by nonsense mediated decay. Two different missense mutations have been reported to date; however, the physiological significance of these mutations has yet to be determined. Tumor 1 with the H255Y missense mutation (Fig. 6 A and G) showed the same molecular phenotype as the tumors from patients with germline frameshift or splicing mutations, suggesting loss of function of this mutant FLCN protein. Our data are consistent with a common consequence of BHD inactivation in mouse and man, regardless of BHD mutation type, and support activation of AKT signaling as an important mechanism driving kidney tumorigenesis in BHD syndrome. Rapamycin does not inhibit mTORC2 effectively, which may explain its partial effect on kidney-targeted BHD knockout mice (13). Taken together, data generated from our two BHD mouse models suggest that mTORC2 as well as mTORC1 inhibition may be needed for the development of an effective form of therapy for patients with BHD-associated kidney cancer.

Materials and Methods

Development of BHD Knockout Mouse Model.

The BHD heterozygous knockout mice were generated as previously described (13). Details of the targeting strategy are described in the SI Methods (Fig. S5). All mice which were used in these experiments were housed in the National Cancer Institute (NCI)-Frederick animal facility according to the NCI-Frederick Animal Care and Use Committee guidelines.

PCR-Based BHD Genotyping.

Mouse genomic DNA was isolated from tails (weaned neonates), yolk sacs (E8.5 or later), and whole embryos (E7.5 or earlier). Primers and details are in the SI Methods.

Quantitative Real Time-PCR.

The qRT-PCR for BHD, AKT1, and AKT2 was performed as described in the SI Methods.

Whole Mount in Situ Hybridization.

The embryos were collected from wild-type C57BL/6 mice intercrossed at different stages of gestation and processed for whole mount in situ hybridization as previously described (25). Negative staining was confirmed using a sense probe with wild-type embryos. Probe information is in the SI Methods.

Histological and Immunohistochemical Analysis.

Embryo sections were stained with hematoxylin and eosin (H&E) or with DAB2 antibody (BD Bioscience) for immunohistochemical evaluation performed as previously described (26). The slides were read by at least three persons, including two pathologists (M.J.M. and D.C.H.).

Western Blotting and Antibodies.

Immunoblotting was performed as described in the SI Methods.

Tissue Genotyping by Southern Blotting.

Nonradioactive Southern blotting was performed with DIG OMNI System for PCR Probes according to the manufacturer's protocol (Roche). Probe information is in the SI Methods.

Endogenous FLCN Detection by Duolink System.

Duolink in situ PLA was performed per manufacturer's instruction (OLink Biosciences). For further information, see the SI Methods.

Human Sample Preparation and MSD Analysis.

Renal tumors were obtained from BHD patients surgically treated at the Urologic Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD with patient permission under a National Institutes of Health Institutional Review Board (IRB)-approved protocol #97-C-0147. All patients signed informed consent. MSD (Meso Scale Discovery) 96-well multispot AKT signaling pathway (phospho-AKT [Ser-473]/total GSK3β/phospho-S6K [Thr-421/Ser-424]), and phospho-S6R (Ser-240/244) assays were carried out according to the manufacturer's protocol. For further information, see the SI Methods.

Immunofluorescence Imaging of the AKT-mTOR Pathway.

Immunostaining for p-AKT, p-mTOR, and p-S6R was performed on frozen sections as described in the SI Methods.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Louise Cromwell for excellent technical support with the mouse studies, Serguei Kozlov for helpful discussions, Yelena Golubeva for her technical support regarding laser microdissection (LCM), Jaime Rodriguez-Canales and Jeffrey Hanson for their technical expertise regarding LCM procedures, and Kristin K. Biris for her technical support regarding in situ hybridization. This research was supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute (NCI), Center for Cancer Research; federal funds from the NCI, NIH, under Contract HHSN261200800001E.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0908853106/DCSupplemental.

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