Birt-Hogg-Dubé syndrome (BHDS) is a genetic disorder that increases the risk of developing benign skin cancers, polycystic kidney disease (PKD), lung cysts, and many types of kidney cancers. PKD is characterized by cysts or sacs of fluid or air which is caused by excessive cell proliferation and can lead to renal failure. Understanding the molecular mechanisms leading to BHDS associated diseases is critical to identify and optimize treatment options.
BHDS is caused by disrupted expression of the Bhd gene encoding for the tumor suppressor protein Folliculin. Studies in mice reported that impaired Folliculin expression is associated with PKD progression to kidney failure and kidney cancer. Dysregulation of signaling pathways such as mTOR, a major driver of cell proliferation, and AMPK, a negative regulator of mTOR, are involved but the precise molecular mechanisms remain unclear. Additionally, Fnip1 and Fnip2 (Folliculin interacting proteins 1 and 2) are two proteins interacting with Folliculin but their functions and potential roles in PKD remain unclear.
With the goal to decipher the intricate molecular pathways leading to PKD, Drs. Brian Iritani (a Professor of Comparative Medicine at the UW and FHCRC Basic Sciences Division affiliate) and Ryan Centini (a UW Veterinary Resident), along with collaborators Drs. Hockenbery (Clinical Research Division) and Raftery (Public Health Sciences Division), investigated the role of Fnip1 in PKD. The results of their study were recently published in the journal Plos One.
”We had initially identified Fnip1 in an ENU (N-ethyl-N-nitrosourea) chemical mutagenesis screen in mice where we were trying to identify novel genes involved in immune function”, explained Dr. Iritani. The same Fnip1 null mouse model was used here to focus investigations on Fnip1 functions in kidney disease.
Kidney size and weight were increased along with cyst frequency in the Fnip1-/- mice relative to wild type (WT) mice. These observations were associated with decreased activation of the AMPK pathway, as determined by analysis of AMPK phosphorylation, and increased activation of mTORC1, a major driver of cell growth and proliferation. Increased phosphorylated S6 ribosomal protein, a downstream target of mTORC1 activation, was also found by immunohistochemistry staining in renal tubular epithelial cells lining the cysts. These results suggested that Fnip1 loss of expression could facilitate PKD development through mTORC1 activation, in turn stimulating cell proliferation.
Gene expression analysis by RNAseq and quantitative PCR from cortical kidney tissue samples, indicated that 651 genes were differentially expressed between WT and Fnip1-/- mice. Downregulated genes (n=206) mostly were in the family of membrane ion transporters and lipid metabolism-involved genes while upegulated genes (n=445) were mostly involved in cell adhesion and immune response functions. “Our RNAseq data showed that the major cluster of genes downregulated in Fnip1-/- kidney tissues encoded organic and anion transporters, which likely contribute to fluid imbalance and cyst formation in both PKD and BHDS”, explained Dr. Iritani. Cell metabolism was also different in Fnip1-/- kidney cells as increased metabolic activity, as measured by oxygen consumption and extracellular acidification rate were observed. These observations were supported by RNAseq and metabolomics data suggesting increased mitochondrial metabolism, which is illustrated by changes in expression of genes and proteins involved in Acyl-CoA pathway or fatty acid synthesis among others. In addition, Fnip1 might prevent immune cell infiltration, which could favor tumor growth if the immune cells induce a pro-tumoral and pro-inflammatory state.
According to Dr. Iritani, “in our Fnip1-/- mice, we found increased activation of mTOR in many tissues including B cells, skeletal muscle, heart, kidney, and that loss of Fnip1 potently synergized with TSC1 loss to “superactivate” mTOR. Humans with mutations in Folliculin frequently develop renal cancers which also exhibit hyperactive mTOR. These results suggest that Fnip1 and Folliculin are important in mTOR regulation and human disease, but that the interactions are very complex. The utilization of Fnip1 mice may help decipher the molecular mechanisms governing this interaction”. Indeed, the double deletion of Fnip1 and Tsc1 genes, a suppressor of the mTORC pathway, demonstrated significant synergism between these two genes and acceleration of PKD development. Impressively, mice survival did not exceed 3 to 5 weeks of age due to early PKD development in Fnip1-/- Tsc1-/- mice, while Tsc1-/- mice could survive and average of 24 weeks.
The authors are planning to continue investigating the complex molecular biology mechanisms facilitating PKD development. In addition, “in collaboration with Dr. Jeff Delrow, we made the surprising finding using RNAseq that the major class of upregulated genes in Fnip1 null kidney tissue were immune associated genes. Immunohistochemistry further revealed that macrophages and T lymphocytes accumulated very specifically around the renal cysts where mTOR was also more highly activated. These results suggest that immune cells may contribute to PKD and perhaps renal cancers in BHDS. We are excited to examine this hypothesis”, concluded Dr. Iritani.
Funding for this study was provided by the National Institutes of Health.
Fred Hutch/UW Cancer Consortium faculty members Drs. Brian Iritani, David Hockenbery, Dan Raftery and Jeff Delrow contributed to this research.
Centini R, Tsang M, Iwata T, Park H, Delrow J, Margineantu D, Iritani BM, Gu H, Liggitt HD, Kang J, Kang L, Hockenbery DM, Raftery D, Iritani BM. 2018. Loss of Fnip1 alters kidney developmental transcriptional program and synergizes with TSC1 loss to promote mTORC1 activation and renal cyst formation. PLoS One, 13(6), e0197973.