Although FLCN has been shown to function in several biological pathways, such as mTOR and AMPK signalling (as illustrated in our signalling diagram), its precise function is currently unknown. In order to identify novel FLCN targets, Reiman et al. used gene expression and protein arrays to analyse isogenic FTC133 FLCN-positive and FLCN-null cell lines.
Gene expression microarray analysis found 708 genes to be differentially expressed between the two cell lines. Pathway analysis found a preponderance of differentially expressed Wnt and cadherin signalling genes, with 23 and 18 genes dysregulated respectively. The role of Wnt signalling in renal cell carcinoma (RCC) was discussed in a recent blog post and these results suggest that the pathway may play a role in BHD-associated kidney tumourigenesis. FLCN has also been previously linked to the cadherin signalling pathway via its role in cell-cell adhesion and RhoA signalling (Medvetz et al., 2012, Nahorski et al., 2012), also discussed in previous blog posts here and here.
Two microarray data sets investigating the role of FLCN have been published previously. Firstly, Hong et al. (2010) compared gene expression in isogenic FLCN-positive and FLCN-null UOK257 cells, while Klomp et al. (2010) investigated gene expression in six BHD renal tumours compared to sporadic renal tumours. Comparison of all data sets showed that one gene was differentially expressed in all three: Rab27b, a small GTPase which enhances breast cancer growth and invasiveness (Hendrix et al., 2010). This finding is particularly interesting in light of the recent report by Nookala et al. (2012) that FLCN has guanine nucleotide exchange factor (GEF) activity towards Rab35. Together, these findings suggest a role for FLCN in the regulation of Rabs and potentially membrane trafficking.
Reiman et al. also performed protein array analysis and compared this data with the gene expression data in order to prioritise gene validation. Eight genes were differentially expressed in both experiments, five of which had suitable antibodies for validation by Western blotting. The dysregulation of three proteins was confirmed in both FTC133 and primary tumour cells from a BHD patient: EIF2AK2 and CASP1 were shown to be upregulated by FLCN, whereas PLCG2 is downregulated. EIF2AK2 is a protein kinase reported to have tumour suppressor activity (Koromilas et al., 1992), and the dysregulation of the transmembrane signalling enzyme PLCG2 has been observed in Wilms tumour (Maschietto et al., 2008), a paediatric kidney cancer. Therefore, both could plausibly promote BHD-associated renal oncogenesis.
CASP1 activity induces cell death, and given that FLCN has been previously linked to apoptosis (Cash et al., 2010, Lim et al., 2012), the authors investigated whether other apoptosis genes were dysregulated in the FTC133 cell lines. Using an apoptotic protein array, they found that FLCN upregulates Diablo and HtrA2. Both genes are part of the mitochondrial apoptotic pathway (Martinez-Ruiz et al., 2008), and decreased Diablo expression has been linked to poor prognosis in RCC (Mizutani et al., 2005).
Taken together, these results identify several pathways and targets of FLCN, several of which have been previously implicated in BHD-associated or other renal cancers, thereby shedding further light on FLCN’s role in renal tumorigenesis and suggesting further avenues of research.
- Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46 PMID: 21258407
- Hendrix A, Braems G, Bracke M, Seabra M, Gahl W, De Wever O, & Westbroek W (2010). The secretory small GTPase Rab27B as a marker for breast cancer progression. Oncotarget, 1 (4), 304-8 PMID: 21304180
- Hong SB, Oh H, Valera VA, Stull J, Ngo DT, Baba M, Merino MJ, Linehan WM, & Schmidt LS (2010). Tumor suppressor FLCN inhibits tumorigenesis of a FLCN-null renal cancer cell line and regulates expression of key molecules in TGF-beta signaling. Molecular cancer, 9 PMID: 20573232
- Klomp JA, Petillo D, Niemi NM, Dykema KJ, Chen J, Yang XJ, Sääf A, Zickert P, Aly M, Bergerheim U, Nordenskjöld M, Gad S, Giraud S, Denoux Y, Yonneau L, Méjean A, Vasiliu V, Richard S, MacKeigan JP, Teh BT, & Furge KA (2010). Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression. BMC medical genomics, 3 PMID: 21162720
- Koromilas AE, Roy S, Barber GN, Katze MG, & Sonenberg N (1992). Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science (New York, N.Y.), 257 (5077), 1685-9 PMID: 1382315
- Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2012). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair, 11 (3), 259-66 PMID: 22209521
- Martinez-Ruiz G, Maldonado V, Ceballos-Cancino G, Grajeda JP, & Melendez-Zajgla J (2008). Role of Smac/DIABLO in cancer progression. Journal of experimental & clinical cancer research : CR, 27 PMID: 18822137
- Maschietto M, de Camargo B, Brentani H, Grundy P, Sredni ST, Torres C, Mota LD, Cunha IW, Patrão DF, Costa CM, Soares FA, Brentani RR, & Carraro DM (2008). Molecular profiling of isolated histological components of wilms tumor implicates a common role for the Wnt signaling pathway in kidney and tumor development. Oncology, 75 (1-2), 81-91 PMID: 18784435
- Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H, & Henske EP (2012). Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion. PloS one, 7 (11) PMID: 23139756
- Mizutani Y, Nakanishi H, Yamamoto K, Li YN, Matsubara H, Mikami K, Okihara K, Kawauchi A, Bonavida B, & Miki T (2005). Downregulation of Smac/DIABLO expression in renal cell carcinoma and its prognostic significance. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 23 (3), 448-54 PMID: 15572731
- Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics PMID: 22965878
- Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, & Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open biology, 2 (8) PMID: 22977732
- Reiman A, Lu X, Seabra L, Boora U, Nahorski MS, Wei W, & Maher ER (2012). Gene Expression and Protein Array Studies of Folliculin-regulated Pathways. Anticancer research, 32 (11), 4663-70 PMID: 23155228
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.