ANTICANCER RESEARCH 32: 4663-4670 (2012) Gene Expression and Protein Array Studies of Folliculin-regulated Pathways ANNE REIMAN1, XIAOHONG LU1, LAURENCE SEABRA1, UNCAAR BOORA1, MICHAEL S. NAHORSKI1, WENBIN WEI2 and EAMONN R. MAHER1,3 1Centre for Rare Diseases and Personalised Medicine and Department of Medical & Molecular Genetics, School of Clinical and Experimental Medicine, University of Birmingham College of Medical and Dental Sciences, Birmingham, U.K.; 2School of Cancer Sciences, University of Birmingham, Birmingham, U.K.; 3West Midlands Regional Genetics Service, Birmingham Women’s Hospital, Birmingham, U.K. Abstract. The familial cancer syndrome Birt-Hogg-Dube microarray data, revealed that EIF2AK2 (PKR) and CASP1 syndrome is characterised by the development of skin were reduced and PLCG2 was increased in folliculin- (fibrofolliculomas) and renal tumours (and lung cysts) and deficient FTC133 cells and in a BHD renal tumour. In view is caused by mutations in the FLCN tumour suppressor gene. of the role of CASP1 in apoptosis we investigated whether Though the FLCN gene product (folliculin) has been linked other apoptosis-related proteins might be regulated by to the regulation of a variety of signalling pathways (e.g. the folliculin and found increased levels of SMAC/Diablo and mTOR, AMPK, TGFbeta and hyoxia-responsive genes) the HtrA2 in folliculin-expressing FTC133 cells. These findings precise function of the folliculin protein is not well-defined. identify novel pathways and targets linked to folliculin In order to identify potential novel pathways linked to tumour suppressor activity. folliculin function we analysed paired isogenic folliculin- deficient and folliculin-expressing cell lines by gene Kidney cancers account for about 2% of all cancers and, expression and protein (Kinexus) arrays. Gene expression worldwide, more than 200,000 new cases are diagnosed each microarray analysis in the folliculin +/- non-renal cancer year (1). When detected early, surgical resection can be line (FTC133), revealed 708 differentially expressed targets curative but though treatment with targeted-antiangiogenic (fold change >2 and p<0.001) with enrichment of genes in therapies have improved survival in RCC, the outcome of the cadherin and Wnt signalling pathways. Comparison of patients with metastatic disease remains poor. Familial forms the differentially expressed genes in the FTC133 datasets and of RCC are infrequent (about 3% of all cases), but the previously reported gene expression data for a folliculin- elucidation of the molecular basis of rare familial forms of deficient renal tumour and the UOK257 renal cell carcinoma kidney cancer has provided seminal insights into the cell line, revealed that RAB27B was dysregulated in all three pathogenesis of the common non-familial forms of RCC. datasets (increased expression in folliculin-deficient cells). Thus, the identification of the gene (VHL) for the rare The Kinexus protein array analysis suggested 73 candidate, syndromic form of inherited RCC, known as von Hippel- differentially expressed, proteins and further investigation by Lindau disease, provided the basis for the discovery that western blot analysis of 5 candidates that were also somatic inactivation of the VHL tumour suppressor gene differentially expressed in the FTC133 gene expression 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 Correspondence to: Prof. E.R. Maher, Department of Medical & response gene pathway (7,8 and references within) and that Molecular Genetics, School of Clinical and Experimental Medicine, HIF-mediated RCC growth may be antagonised by University of Birmingham College of Medical and Dental Sciences, antiangiogenic multi-tyrosine kinase inhibitors, which are now Edgbaston, Birmingham B15 2TT, U.K. Tel: +44 1216272741, Fax: widely used in the treatment of metastatic kidney cancer (9). +44 1216272618, e-mail: [email protected] Birt-Hogg-Dube syndrome is a syndromic form of Key Words: Follicin, gene expression, protein arrays, Birt-Hogg- inherited RCC caused by mutations in the FLCN gene that Dube syndrom, FLCN tumor suppressor gene. encodes the folliculin tumour suppressor protein (10-12). 0250-7005/2012 $2.00+.40 4663 ANTICANCER RESEARCH 32: 4663-4670 (2012) Individuals with germline FLCN mutations are also predisposed to develop skin (fibrofolliculomas) and colorectal tumours and lung cysts (13-15). The precise function of the folliculin tumour suppressor protein is not well-defined although two, folliculin interacting proteins (FNIP1 and FNIP2) were linked to the mTOR and AMPK signalling pathways (16-18) and mice with kidney-targeted homozygous inactivation of Flcn developed renal tumours and cysts that demonstrated mTOR activation (19-20). Interestingly, though, mTOR inhibitors have shown promise as treatments for metastatic RCC (21), some studies have suggested that the effect of folliculin on mTOR signaling may not be straightforward and can be context-dependent (22). Recently, folliculin has been reported to regulate TGF-beta signaling Figure 1. Validation of differentially expressed genes in Cadherin and and hypoxia-response pathways (23-25). In the knowledge Wnt signaling in FTC133-FLCN-expressing compared to FLCN-null cell that many tumour suppressors (e.g. pVHL) have multiple line, by real-time qPCR. The experiment was performed in triplicate and functions we analysed matched folliculin-deficient and the standard deviation was calculated. folliculin-expressing cell lines by gene expression and protein arrays, in order to determine which signaling pathways might be regulated by the folliculin tumour suppressor protein. Apoptotic antibody arrays. Analysis of samples on arrays was as per manufacturer’s recommendation (R&D systems Abingdon, UK.). In short, 250ugs total cellular proteins were spotted in Materials and Methods duplicate onto a nitrocellulose membrane array with apoptotic capture antibodies. Following a 16-h incubation unbound proteins Cell culture. A matched pair of folliculin-deficient and -expressing were washed away and a second incubation with streptavidin-HRP cell lines was studied. FTC133 is a metastatic thyroid carcinoma linked detection antibodies. Chemiluminescence detection produced cell line that harbours a somatic FLCN gene mutation (c.1285delC). a spot corresponding to the amount of bound protein. Peak intensity FTC133 cell line has been described previously (26) but in brief of the bound protein was calculated and values were obtained on a FTC133 cells were obtained from ECACC (Salisbury,UK) and syngene analysis system. grown in a mixture (1:1) of Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich, Steinheim, Germany) and Ham’s F-12 Immunoblotting. FTC133 cells were lysed in RIPA buffer (Tris pH (Sigma-Aldrich) and supplemented with 10% FBS (Sigma-Aldrich), 7.4, NaCl 150 mM, EDTA 0.5 mM, 1% Triton) containing a 2mM l-glutamine and penicillin/streptomycin. In order to obtain protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, cells stably-expressing FLCN, FTC133 cells were stably transfected IN) and the supernatant removed following centrifugation. The with pFLAG-FLCN construct or a construct containing an empty protein concentration of cell lysates was estimated using the DC pFLAG-CMV vector (26). Transfected cells were then grown in a protein assay kit, according to the manufacturer’s instructions (Bio- selective medium containing 0.5 mg/mL G418. Rad Laboratories Ltd, Hertfordshire, UK). Equal amounts of total cellular protein were separated on a 7% to 12.5% SDS-PAGE gel RNA extraction and gene expression microarray analysis. Total RNA and the primary antibodies used were as follows: PLCG2, STK33, was extracted from exponentially growing FTC133 cells with folliculin- β-Actin, Tubulin, EIF2AK2(PKR), SMAC/Diablo and HtrA2 null and -expressing cells (>2×106 cells) using the RNeasy kit (Qiagen, (Abcam), Casp1 and FAK (PTK2) (Cell Signaling Technology). Crawley, UK). RNAs were extracted from triplicate plates of each cell line, labelled and analysed using GeneChip Human Exon 1.0 ST Array Real-time qRT-PCR. Total RNA was extracted from FTC-133 cells (Affymetrix, High Wycombe, UK) following standard Affymetrix (>5×106 cells) using the RNeasy kit (Qiagen Inc, Valencia, CA) and protocols. Gene level analysis of the array data was performed using converted into cDNA using Superscript III reverse transcriptase. Affymetrix Expression Console with the default settings of “Extended: TaqMan Universal PCR Master Mix and the validated probes for the RMA-Sketch”. Differentially expressed genes between different genes of interest were purchased from Applied Biosystems. The treatment groups were identified using the limma package (27) with a PCR conditions were denaturation at 95˚C for 10 min followed by threshold of p<0.001 and fold change >2. Heatmaps were generated 40 cycles of denaturation at 95˚C for15 sec and annealing and using dChip (http://www.dchip.org/) with the default settings. extension at 60˚C for 1 min. Each gene was analysed in triplicate and normalized to GAPDH mRNA levels. Kinexus antibody array. FTC133 folliculin-transfected and 6 folliculin-null cells (>2×10 cells) were washed twice with ice-cold Results PBS, trypsinized and pelleted by centrifugation and stored at –80˚C. The cell pellets were sent to Kinexus Bioinformatics Corporation