Gene Expression and Protein Array Studies of Folliculin-regulated Pathways

The familial cancer syndrome Birt-Hogg-Dube syndrome is characterised by the development of skin (fibrofolliculomas) and renal tumours (and lung cysts) and is caused by mutations in the FLCN tumour suppressor gene. Though the FLCN gene product (folliculin) has been linked to the regulation of a variety of signalling pathways (e.g. the mTOR, AMPK, TGFbeta and hyoxia-responsive genes) the precise function of the folliculin protein is not well-defined. In order to identify potential novel pathways linked to folliculin function we analysed paired isogenic folliculin- deficient and folliculin-expressing cell lines by gene expression and protein (Kinexus) arrays. Gene expression microarray analysis in the folliculin +/- non-renal cancer line (FTC133), revealed 708 differentially expressed targets (fold change >2 and p<0.001) with enrichment of genes in the cadherin and Wnt signalling pathways. Comparison of the differentially expressed genes in the FTC133 datasets and previously reported gene expression data for a folliculin- deficient renal tumour and the UOK257 renal cell carcinoma cell line, revealed that RAB27B was dysregulated in all three datasets (increased expression in folliculin-deficient cells). The Kinexus protein array analysis suggested 73 candidate, differentially expressed, proteins and further investigation by western blot analysis of 5 candidates that were also differentially expressed in the FTC133 gene expression microarray data, revealed that EIF2AK2 (PKR) and CASP1 were reduced and PLCG2 was increased in folliculin- deficient FTC133 cells and in a BHD renal tumour. In view of the role of CASP1 in apoptosis we investigated whether other apoptosis-related proteins might be regulated by folliculin and found increased levels of SMAC/Diablo and HtrA2 in folliculin-expressing FTC133 cells. These findings identify novel pathways and targets linked to folliculin tumour suppressor activity. Kidney cancers account for about 2% of all cancers and, worldwide, more than 200,000 new cases are diagnosed each year (1). When detected early, surgical resection can be curative but though treatment with targeted-antiangiogenic therapies have improved survival in RCC, the outcome of patients with metastatic disease remains poor. Familial forms of RCC are infrequent (about 3% of all cases), but the elucidation of the molecular basis of rare familial forms of kidney cancer has provided seminal insights into the pathogenesis of the common non-familial forms of RCC. Thus, the identification of the gene (VHL) for the rare syndromic form of inherited RCC, known as von Hippel- Lindau disease, provided the basis for the discovery that somatic inactivation of the VHL tumour suppressor gene occurred in most sporadic clear-cell RCC (2-6). Furthermore, subsequent investigations demonstrated that VHL inactivation leads to stabilisation of hypoxia-inducible factor (HIF)-1 and HIF-2 transcription factors and activation of the hypoxic response gene pathway (7,8 and references within) and that HIF-mediated RCC growth may be antagonised by antiangiogenic multi-tyrosine kinase inhibitors, which are now widely used in the treatment of metastatic kidney cancer (9). Birt-Hogg-Dube syndrome is a syndromic form of inherited RCC caused by mutations in the FLCN gene that encodes the folliculin tumour suppressor protein (10-12).