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.
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