Fnip1 and Fnip2 play critical roles in kidney tumour suppression in cooperation with Flcn in mice

Folliculin (FLCN), through its association with renal tumours in BHD and knockout mouse models, is now recognised as a tumour suppressor. The loss of Flcn in kidney cells is linked to pro-tumourigenic changes in AMPK and mTOR activity, increased expression of PPARGC1A (PGC1α) and increased mitochondrial biogenesis (Baba et al., 2008, Hasumi et al., 2012). Two folliculin interacting proteins, FNIP1 and FNIP2, are known to facilitate FLCN function. A report from Hasumi et al., 2015 aimed to clarify the function of Fnip1 and Fnip2 and identifies them as having a critical role in kidney tumour suppression.

It has previously been shown that the loss of Fnip1 and the loss of Flcn in specific tissues resulted in the same phenotypes; both disrupt B-cell development (Baba et al., 2012, Park et al., 2012, Park et al., 2014) and alter skeletal muscle physiology and metabolism (Hasumi et al., 2012, Reyes et al., 2015). With both proteins the same downstream pathways show aberrant behaviour suggesting a connected functional role for Flcn and Fnip1. Knockdown of Flcn in mouse kidneys results in an enlarged, polycystic pathology, but Hasumi et al report that knockdown of either Fnip1 or Fnip2 in kidneys does not produce a phenotype, and that constitutive knockdown of Fnip2 has no effect on any tissue.

The 74% similarity in human FNIP1 and FNIP2 sequences, in addition to both binding to the C-terminal of FLCN and also AMPK, suggests some functional redundancy. Constitutive Fnip1-/-, Fnip2-/- double knockdown, like Flcn-/- (Hasumi et al., 2009), was shown to be embryonic lethal indicating an essential role for Fnips in development. However single constitutive knockdown of either Fnip1 or Fnip2 does not affect embryonic development, indicative of functional redundancy. In addition FNIP1 or FNIP2 expression is sufficient to reduce PPARGC1A expression and slow cell proliferation in Fnip1/Fnip2-null mouse embryonic fibroblasts.

To explain why Fnip1-/- but not Fnip2-/- mice show a phenotype, Hasumi et al. compared Fnip1 and Fnip2 mRNA levels and identified higher Fnip1 expression in bone marrow, cardiac and skeletal muscle samples. The reported levels of Fnip1 and Fnip2 in bone marrow, whilst statistically significant, are not drastically dissimilar which could indicate a tissue-specific function of Flcn-Fnip1. In the kidneys Fnip1 and Fnip2 levels are similar and, as demonstrated by a lack of a renal phenotype, the loss of one Fnip can be tolerated. The variation in expression ratio seen in different tissues could indicate tissue specific functions for each Fnip, an area that requires further investigation.

The relative expression levels of FNIP1 and FNIP2 in human tissues are different to mice – FNIP1 shows higher expression in skeletal muscle but not cardiac muscle, expression of both FNIPs is similar in bone marrow, and the kidneys show greater expression of FNIP2 (Hasumi et al., 2008). This should be considered in future studies especially if FNIP1 and FNIP2 are found to have additional functions in specific tissues.

Targeted double knockout of both Fnip1 and Fnip2 in the kidneys results in the production of large, polycystic kidneys associated with the same molecular changes and reduced survival as Flcn loss. Additional knockdown of Flcn in Fnip1/Fnip2-deficient kidneys did not increase the size or change the histology of the kidneys. This indicates a shared pathway and further supports the hypothesis of a shared tumour suppressing function between Flcn and Fnip1/2 that can be disrupted by loss of either component.

The heterozygous Flcn+/- mouse is a more accurate model of BHD in humans, as it shows kidney tumour formation at 24 months associated with a second hit mutation and complete loss of Flcn function (Hasumi et al., 2009). Due to the functional redundancy in the Fnips in the kidney, demonstrated by the lack of renal phenotypes in Fnip1 or Fnip2 single knockout mice, a comparative model would have only one functional Fnip allele. Homozygous constitutive knockdown of Fnip1 vastly reduces lifespan due to immunodeficiency (Park et al., 2012), but Fnip2-/- mice show no phenotype. Therefore Hasumi et al., (2015) produced a constitutive Fnip1+/-, Fnip2-/- knockdown mouse model to assess the role of Fnips in tumour suppression.

The Fnip1+/-, Fnip2-/- mice, like the Flcn+/- mice (Hasumi et al., 2009), develop renal tumours by 24 months old – presumably associated with the loss of the remaining functional Fnip1 allele but the authors did not confirm this. As seen in BHD patients (Pavlovich et al., 2002) the tumours showed varied histology, increased mTOR activity and PPARGC1A expression. These similarities in tumour biology further support the conclusion that tumourigenesis associated with a loss of Flcn or both Fnip1 and Fnip2 occurs via the same pathways.

In conclusion, in mice, a loss of total Fnip function mimics loss of Flcn function in kidneys and indicates that Fnips also play an important role in tumour suppression. It may be that the loss of interaction between FLCN and a FNIP is responsible for the downstream events that result in tumourigenesis. Further work is needed to elucidate the generic and tissue-specific key targets of the FLCN-FNIP1 and FLCN-FNIP2 complexes that could be targeted as the basis of new cancer treatments.

  • Baba M, Furihata M, Hong SB, Tessarollo L, Haines DC, Southon E, Patel V, Igarashi P, Alvord WG, Leighty R, Yao M, Bernardo M, Ileva L, Choyke P, Warren MB, Zbar B, Linehan WM, Schmidt LS. 2008. Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys. Journal of the National Cancer Institute, Jan 16;100(2):140-54. PubMed PMID: 18182616
  • Baba M, Keller JR, Sun HW, Resch W, Kuchen S, Suh HC, Hasumi H, Hasumi Y, Kieffer-Kwon KR, Gonzalez CG, Hughes RM, Klein ME, Oh HF, Bible P, Southon E, Tessarollo L, Schmidt LS, Linehan WM, & Casellas R. 2012. The folliculin-FNIP1 pathway deleted in human Birt-Hogg-Dubé syndrome is required for murine B-cell development. Blood, 120 (6), 1254-61 PMID: 22709692
  • Hasumi H, Baba M, Hong SB, Hasumi Y, Huang Y, Yao M, Valera VA, Linehan WM, Schmidt LS. 2008. Identification and characterization of a novel folliculin-interacting protein FNIP2. Gene, May 31;415(1-2):60-7. PubMed PMID: 18403135
  • Hasumi Y, Baba M, Ajima R, Hasumi H, Valera VA, Klein ME, Haines DC, Merino MJ, Hong SB, Yamaguchi TP, Schmidt LS, Linehan WM. 2009. Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2. Proc Natl Acad Sci U S A, Nov 3;106(44):18722-7. PubMed PMID: 19850877.
  • Hasumi H, Baba M, Hasumi Y, Huang Y, Oh H, Hughes RM, Klein ME, Takikita S, Nagashima K, Schmidt LS, Linehan WM. 2012. Regulation of mitochondrial oxidative metabolism by tumor suppressor FLCN. Journal of the National Cancer Institute, Nov 21;104(22):1750-64. PubMed PMID: 23150719
  • Hasumi H, Baba M, Hasumi Y, Lang M, Huang Y, Oh HF, Matsuo M, Merino MJ, Yao M, Ito Y, Furuya M, Iribe Y, Kodama T, Southon E, Tessarollo L, Nagashima K, Haines DC, Linehan WM, & Schmidt LS (2015). Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn. Proceedings of the National Academy of Sciences of the United States of America PMID: 25775561
  • Park H, Staehling K, Tsang M, Appleby MW, Brunkow ME, Margineantu D, Hockenbery DM, Habib T, Liggitt HD, Carlson G, & Iritani BM. 2012. Disruption of Fnip1 reveals a metabolic checkpoint controlling B lymphocyte development. Immunity, 36 (5), 769-81 PMID: 22608497
  • Park H, Tsang M, Iritani BM, & Bevan MJ. 2014. Metabolic regulator Fnip1 is crucial for iNKT lymphocyte development. Proceedings of the National Academy of Sciences of the United States of America, 111 (19), 7066-71 PMID: 24785297
  • Reyes NL, Banks GB, Tsang M, Margineantu D, Gu H, Djukovic D, Chan J, Torres M, Liggitt HD, Hirenallur-S DK, Hockenbery DM, Raftery D, & Iritani BM. 2015. Fnip1 regulates skeletal muscle fiber type specification, fatigue resistance, and susceptibility to muscular dystrophy. Proceedings of the National Academy of Sciences of the United States of America, 112 (2), 424-9 PMID: 25548157
  • Pavlovich CP, Walther MM, Eyler RA, Hewitt SM, Zbar B, Linehan WM, Merino MJ. Renal tumors in the Birt-Hogg-Dubé syndrome. American Journal of Surgical Pathology. 2002 Dec;26(12):1542-52. PubMed PMID: 12459621


Leave a Reply