Research ArticleExperimental Studies

Gene Expression and Protein Array Studies of Folliculin-regulated Pathways

ANNE REIMAN, XIAOHONG LU, LAURENCE SEABRA, UNCAAR BOORA, MICHAEL S. NAHORSKI, WENBIN WEI and EAMONN R. MAHER
Anticancer Research November 2012, 32 (11) 4663-4670;

Abstract

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). 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 and hypoxia-response pathways (23-25). In the knowledge that many tumour suppressors (e.g. pVHL) have multiple functions we analysed matched folliculin-deficient and 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.

Materials and Methods

Cell culture. A matched pair of folliculin-deficient and -expressing cell lines was studied. FTC133 is a metastatic thyroid carcinoma cell line that harbours a somatic FLCN gene mutation (c.1285delC). FTC133 cell line has been described previously (26) but in brief FTC133 cells were obtained from ECACC (Salisbury,UK) and grown in a mixture (1:1) of Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, Steinheim, Germany) and Ham's F-12 (Sigma-Aldrich) and supplemented with 10% FBS (Sigma-Aldrich), 2mM l-glutamine and penicillin/streptomycin. In order to obtain cells stably-expressing FLCN, FTC133 cells were stably transfected with pFLAG-FLCN construct or a construct containing an empty pFLAG-CMV vector (26). Transfected cells were then grown in a selective medium containing 0.5 mg/mL G418.

RNA extraction and gene expression microarray analysis. Total RNA was extracted from exponentially growing FTC133 cells with folliculin-null and -expressing cells (>2×106 cells) using the RNeasy kit (Qiagen, Crawley, UK). RNAs were extracted from triplicate plates of each cell line, labelled and analysed using GeneChip Human Exon 1.0 ST Array (Affymetrix, High Wycombe, UK) following standard Affymetrix protocols. Gene level analysis of the array data was performed using Affymetrix Expression Console with the default settings of “Extended: RMA-Sketch”. Differentially expressed genes between different treatment groups were identified using the limma package (27) with a threshold of p<0.001 and fold change >2. Heatmaps were generated using dChip (http://www.dchip.org/) with the default settings.

Kinexus antibody array. FTC133 folliculin-transfected and folliculin-null cells (>2×106 cells) were washed twice with ice-cold PBS, trypsinized and pelleted by centrifugation and stored at −80°C. The cell pellets were sent to Kinexus Bioinformatics Corporation (Vancouver, Canada) www.kinexus.ca/ for Kinex TM KAM-1.3 chip antibody array. Signal median was subtracted by background median and normalized by its total on each array.

Figure 1.
Figure 1.

Validation of differentially expressed genes in Cadherin and Wnt signaling in FTC133-FLCN-expressing compared to FLCN-null cell line, by real-time qPCR. The experiment was performed in triplicate and the standard deviation was calculated.

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 duplicate onto a nitrocellulose membrane array with apoptotic capture antibodies. Following a 16-h incubation unbound proteins were washed away and a second incubation with streptavidin-HRP linked detection antibodies. Chemiluminescence detection produced a spot corresponding to the amount of bound protein. Peak intensity of the bound protein was calculated and values were obtained on a syngene analysis system.

Immunoblotting. FTC133 cells were lysed in RIPA buffer (Tris pH 7.4, NaCl 150 mM, EDTA 0.5 mM, 1% Triton) containing a protease inhibitor cocktail (Roche Applied Sciences, Indianapolis, IN) and the supernatant removed following centrifugation. The protein concentration of cell lysates was estimated using the DC protein assay kit, according to the manufacturer's instructions (Bio-Rad Laboratories Ltd, Hertfordshire, UK). Equal amounts of total cellular protein were separated on a 7% to 12.5% SDS-PAGE gel and the primary antibodies used were as follows: PLCG2, STK33, β-Actin, Tubulin, EIF2AK2(PKR), SMAC/Diablo and HtrA2 (Abcam), Casp1 and FAK (PTK2) (Cell Signaling Technology).

Real-time qRT-PCR. Total RNA was extracted from FTC-133 cells (>5×106 cells) using the RNeasy kit (Qiagen Inc, Valencia, CA) and converted into cDNA using Superscript III reverse transcriptase. TaqMan Universal PCR Master Mix and the validated probes for the genes of interest were purchased from Applied Biosystems. The PCR conditions were denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for15 sec and annealing and extension at 60°C for 1 min. Each gene was analysed in triplicate and normalized to GAPDH mRNA levels.

Results

Gene expression profiles of FLCN null and FLCN restored FTC133. mRNA extracted from paired isogenic FTC-133 cell lines stably transfected with Flag-FLCN or an empty Flag vector was hybridised to Affymetrix Human Exon 1.0 ST Arrays. 458 and 250 genes were >2-fold up- and down-regulated respectively in the folliculin expressing FTC-133 cell line, compared to folliculin null cell line (supplementary Table I; supplementary tables can be obtained from E.R.Maher{at}bham.ac.uk). We validated the results of the microarray expression analysis by real time PCR analysis for selected differentially expressed genes (FZD1, CDH11, BCDHB5 and BCDHB8). Each of the genes was confirmed to be down-regulated in a folliculin expressing FTC133 cell line (Figure 1).

View this table:
Table I.

Pathways de-regulated in FTC133-FLCN-null versus FLCN-restored cell line.

View this table:
Table II.

Genes de-regulated in Cadherin and Wnt pathways in FTC133-FLCN-null versus FLCN-restored cell line.

Pathway analysis of folliculin-dependent differentially expressed genes. Pathway analysis of the 708 differentially expressed genes (fold change >2 and p<0.001) in folliculin-expressing and null FTC-133 cells, using David bioinformatics program (28, 29) showed abnormal regulation of the known oncogenic pathways including cadherin and the Wnt pathway (Table I and Figure 1). There was a considerable overlap between these two pathways with 17 common genes (Table II). Thus 18 genes were de-regulated in the cadherin signalling pathway (17 down-regulated and 1 up-regulated by folliculin) whilst 23 genes belonging to Wnt signalling were de-regulated (21 down-regulated and 2 up-regulated by folliculin) (Table II). PCDHB5 and PCDHB8 showed the greatest down-regulation (20- and 14-fold respectively).

Previously Hong et al. (23) reported gene expression microarray data for folliculin-expressing and null UOK257 renal cancer cell lines and Klomp et al. (30) described gene expression microarray data for 6 kidney tumours from patients with BHD using Affymetrix Human Genome U133 Plus 2.0 Arrays. Comparison of the differentially expressed genes in the paired FTC133 cell lines and those described by Hong et al. (23) and Klomp et al. (30) identified just one gene that demonstrated altered expression in all three datasets (Figure 2). Thus RAB27B 2 (Rab27B, member of RAS oncogene family) was down-regulated by folliculin (Figure 2). However, a combined analysis of microarray data for FTC133 and 6 BHD-associated kidney tumours revealed 7 genes (TSPAN7, PCDH18, LPCAT2, CD36, RAB27B, KIT and NCKAP5), down-regulated and 36 genes up-regulated by folliculin in both datasets (Figure 3).

Figure 2.
Figure 2.

Genes down- and up-regulated by FLCN in three datasets. FTC mut up or down=genes up-or down-regulated in FTC133 FLCN-null cell line. Tumour up or down=genes up- or down-regulated in BHD tumours (30); UOK257 up or down=genes up-or down-regulated in the UOK257 FLCN-null renal cancer cell line (23).

Down-regulation of the expression of Rab27B and LPCAT in folliculin-expressing FTC133 cell line was confirmed by real-time qRT-PCR (Figure 4).

Protein array expression profiles of FLCN-null and FLCN-restored FTC-133 lines. The Kinex™ KAM-1.2 microarray-platform was utilised to identify differentially expressed proteins in paired isogenic FTC-133 folliculin-null and FLCN-restored cell lines.

The Kinex™ protein array interrogates 800 proteins (approximately 500 antibodies assess protein expression and 300 phosphoantibodies detect for phosphorylation-specific proteins). 73 targets were differentially expressed in FTC-133 folliculin null and expressing cell lines (fold change >1.5) (supplementary Table II; supplementary tables can be obtained from E.R.Maher{at}bham.ac.uk).

Pathway analysis of antibody array data using David bioinformatics program (28) revealed de-regulation of proteins participating in apoptosis and MAPK signaling pathways in FTC-133 folliculin-null compared to folliculin-restored cell line. However, as previous studies have suggested that the protein array analysis can lead to false-positive findings, we compared the results of the antibody array and gene expression microarray in the paired FTC-133 cell lines in order to prioritise the validation of differentially expressed proteins. Eight candidate folliculin targets were differentially expressed in both experimental systems (PTK2 (FAK), MAP3K1, EIF2AK2 (PKR), PLCG2, SGK3, SLK, STK33 and Casp1) (see Table III). Western blot analysis of the cell lines was undertaken for 5 targets, for which suitable antibodies were available and revealed that, compared to folliculin-null cells, folliculin-expressing cells expressed increased EIF2AK2 (PKR) and Casp1 but lower levels of PLCG2 and STK33 (no significant difference in expression of FAK could be detected) (Figure 5). We then performed western blot analysis of renal tumour removed from a patient with BHD (with a germline FLCN missense mutation Arg239Cys and loss of the wild-type allele in the tumour) for EIF2AK2 (PKR), Casp1, PLCG2 and STK33. This demonstrated reduced levels of PKR and Casp1 and increased levels of PLCG2 in the tumour tissue, compared to the matched normal tissue (i.e. similar to the folliculin-null/expressing cell lines) but STK33 levels (that were reduced in folliculin-expressing cell lines) were reduced/absent in BHD-associated kidney tumour (compared to matched normal kidney tissue) (Figure 5).

Figure 3.
Figure 3.

Genes down- and up-regulated by FLCN in combined FTC133 and BHD tumour datasets. FTC mut up or down=genes up-or down-regulated in FTC133-FLCN-null cell line. Tumour up or down=genes up- or down-regulated in BHD tumours (30).

Figure 4.
Figure 4.

Validation of expression levels of LPCAT2 and Rab27B in folliculin-expressing and folliculin-null FTC133 cell lines by real-time qPCR. The experiment was performed in triplicate and the standard deviation was calculated.

View this table:
Table III.

Combined analysis of the Kinexus antibody array and gene expression array in FTC133-FLCN compared to FTC133-FLCN-null cell lines.

Figure 5.
Figure 5.

Validation of differentially expressed proteins. A) Expression levels of STK33, PKR, PLCG2, FAK and Casp1 in FTC33 folliculin-null cell line and FTC133 folliculin-expressing (FTC133-FLCN) cell line. B) Expression levels of the same proteins in BHDS-associated renal tumour compared to normal kidney. Equal amounts of protein were separated on a 7% to 12.5% SDS-PAGE gel and probed for PLCG2, STK33, EIF2AK2(PKR), Casp1 and PTK2(FAK). β-Actin or Tubulin was used as a loading control. Blots were exposed to photographic film and scanned using a flat bed scanner. Tum=BHDS associated tumour tissue. Norm=matched normal kidney tissue.

In view of the role of CASP1 in apoptosis we investigated FTC-133-null and -expressing cell lines for the expression of other proteins involved in apoptosis using apoptotic protein arrays (R & D Systems Abingdon, UK). Two different FLCN-expressing FTC-133 cell lines displayed increased levels of SMAC/Diablo, HtrA2 compared to FTC-133 folliculin-null cell line (Figure 6) supporting the role of folliculin in apoptosis.

Discussion

We investigated paired folliculin-expressing and -deficient cancer cell lines using gene expression microarrays and Kinex™ antibody arrays to detect differentially expressed transcripts and proteins. Previously gene expression analysis of folliculin-null and -replete UOK257 RCC cells was reported by Hong et al. (23) and of 6 BHD renal tumours (in comparison to normal renal tissue) by Klomp et al. (30), but the gene expression microarrays we utilised interrogated a larger number of transcripts and we report on the first analysis of non-renal folliculin-deficient cells. We found that folliculin regulates the cadherin and Wnt pathways. Both of these pathways have been implicated in a wide diversity of human cancer types and de-regulation of cadherin signalling in the folliculin-null UOK257 cell line was also reported by Hong et al. (23). Specific microarrays detected changes in the cadherin and Wnt signal pathways that were confirmed independently, thus, folliculin suppressed the expression of CDH11, PCDHB5, PCDHB8 and FZD1. Though CDH11 (cadherin 11) has been reported to have tumour suppressor properties (31, 32), CDH11 has also been reported to be overexpressed in a metastatic prostate cell line and to increase invasiveness (33, 34). RAB27B was found to be de-regulated in both folliculin-deficient renal and non-renal carcinoma cell lines and also in BHD renal tumours. Previously, RAB27B has been linked to oncogenesis in non-BHD-related tumours and reported to enhance breast cancer growth and invasiveness (35).

The Kinexus protein array has not been previously used to analyse folliculin-deficient cell lines. In order to prioritise the large number of potential “hits” we prioritised those targets that were also dysregulated in the gene microarray dataset. Though this strategy might overlook proteins that were dysregulated by post-transcriptional mechanisms, even among those targets that were apparently dysregulated at both transcript and protein level, some targets could not be validated by western blotting. Previous studies have shown that the Kinexus antibody array is more sensitive than western blotting and so a proportion of false positive results are expected (36). Three proteins (EIF2AK2 and Casp1 and PLCG2) were also confirmed to be dysregulated in a BHD renal tumour. EIF2AK2/PKR has been reported to have tumor suppressor activity (37-39) and de-regulation of PLCG2, has been noted in Wilms tumour (40). Caspase 1 (CASP1) expresssion was reduced in folliculin-deficient cancer cell lines and renal tumour. CASP1 is a member of the cysteine-aspartic acid protease family that induces cell apoptosis when activated. The observation that CASP1 levels were reduced by folliculin deficiency suggested that folliculin might promote apoptosis. Moreover, increased levels of Smac/Diablo and HtrA2/OMI were detected in the presence of folliculin. Smac (Second mitochondria-derived activator of caspase) protein, also known as Direct Inhibitor of Apoptosis-Binding protein with LOw pI (DIABLO) and high temperature requirement protein A2 HtrA2/OMI participate in the mitochondrial apoptotic pathway where these proteins are released from the mitochondrial inter-membrane space in response to various stimuli (41). Smac/DIABLO has been shown to be down-regulated in RCC and the absence of Smac/DIABLO expression was predictive of worse prognosis (42). Recently Cash et al. (24) also linked folliculin to apoptosis, by reporting that folliculin deficiency resulted in resistance to apoptosis and reduced levels of the pro-apoptotic protein Bim. Hence our findings further contribute to the elucidation of the mechanisms of folliculin tumour suppressor activity.

Figure 6.
Figure 6.

Expression levels of SMAC/Diablo and HtrA2/OMI in FTC133 folliculin-null and -expressing cell lines.

Acknowledgements

We thank the Myrovlytis Trust for financial support and John Arrand (Birmingham Cancer Research UK Cancer Center) for performing the gene expression microarray analysis.

  • Received August 6, 2012.
  • Accepted September 3, 2012.

References

  1. ↵
    1. Ferlay J,
    2. Autier P,
    3. Boniol M,
    4. Heanue M,
    5. Colombet M,
    6. Boyle P
    : Estimates of the cancer incidence and mortality in Europe in 2006. Ann Oncol 18(3): 581-592, 2007.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Latif F,
    2. Tory K,
    3. Gnarra J,
    4. Yao M,
    5. Duh FM,
    6. Orcutt ML,
    7. Stackhouse T,
    8. Kuzmin I,
    9. Modi W,
    10. Geil L,
    11. et al
    : Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 260(5112): 1317-1320, 1993.
    OpenUrlAbstract/FREE Full Text
    1. Foster K,
    2. Crossey PA,
    3. Cairns P,
    4. Hetherington JW,
    5. Richards FM,
    6. Jones MH,
    7. Bentley E,
    8. Affara NA,
    9. Ferguson-Smith MA,
    10. Maher ER
    : Molecular genetic investigation of sporadic renal cell carcinoma: analysis of allele loss on chromosomes 3p, 5q, 11p, 17 and 22. Br J Cancer 69(2): 230-234, 1994.
    OpenUrlPubMed
    1. Herman JG,
    2. Latif F,
    3. Weng Y,
    4. Lerman MI,
    5. Zbar B,
    6. Liu S,
    7. Samid D,
    8. Duan DS,
    9. Gnarra JR,
    10. Linehan WM,
    11. et al
    : Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci USA 91(21): 9700-9704, 1994.
    OpenUrlAbstract/FREE Full Text
    1. Clifford SC,
    2. Prowse AH,
    3. Affara NA,
    4. Buys CH,
    5. Maher ER
    : Inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene and allelic losses at chromosome arm 3p in primary renal cell carcinoma: a VHL-independent pathway in clear cell renal tumourigenesis. Genes Chromosomes Cancer 22(3): 200-209, 1998.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Banks RE,
    2. Tirukonda P,
    3. Taylor C,
    4. Hornigold N,
    5. Astuti D,
    6. Cohen D,
    7. Maher ER,
    8. Stanley AJ,
    9. Harnden P,
    10. Joyce A,
    11. Knowles M,
    12. Selby PJ
    : Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer Cancer Res 66(4): 2000-2011, 2006.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Maxwell PH,
    2. Wiesener MS,
    3. Chang GW,
    4. Clifford SC,
    5. Vaux EC,
    6. Cockman ME,
    7. Wykoff CC,
    8. Pugh CW,
    9. Maher ER,
    10. Ratcliffe PJ
    : The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399(6733): 271-275, 1999.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Kaelin WG Jr..
    : The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer 8(11): 865-873, 2008.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Chowdhury S,
    2. Larkin JM,
    3. Gore ME
    : Recent advances in the treatment of renal cell carcinoma and the role of targeted therapies. Eur J Cancer 44(15): 2152-2161, 2008.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Nickerson ML,
    2. Warren MB,
    3. Toro JR,
    4. Matrosova V,
    5. Glenn G,
    6. Turner ML,
    7. Duray P,
    8. Merino M,
    9. Choyke P,
    10. Pavlovich C P,
    11. Sharma N,
    12. Walther M,
    13. Munroe D,
    14. Hill R,
    15. Maher E,
    16. Greenberg C,
    17. Lerman MI,
    18. Linehan WM,
    19. Zbar B,
    20. Schmidt LS
    : Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dube syndrome. Cancer Cell 2: 157-164, 2002.
    OpenUrlCrossRefPubMed
    1. Vocke CD,
    2. Yang Y,
    3. Pavlovich CP,
    4. Schmidt LS,
    5. Nickerson ML,
    6. Torres-Cabala CA,
    7. Merino MJ,
    8. Walther MM,
    9. Zbar B,
    10. Linehan WM
    : High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dube-associated renal tumors. J Natl Cancer Inst 97: 931-935, 2005.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Lim DHK,
    2. Rehal PK,
    3. Nahorski MS,
    4. Macdonald F,
    5. Claessens T,
    6. vanGeel M,
    7. Gijezen L,
    8. Gille JJ,
    9. Giraud S,
    10. Richard S,
    11. van Steensel M,
    12. Menko FH,
    13. Maher ER
    : A new locus-specific database (LSDB) for mutationsin the folliculin (FLCN) gene. Hum Mutat 31: E1043-1051, 2010.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Menko FH,
    2. van Steensel MA,
    3. Giraud S,
    4. Friis-Hansen L,
    5. Richard S,
    6. Ungari S,
    7. Nordenskjöld M,
    8. Hansen TV,
    9. Solly J,
    10. Maher ER,
    11. European BHD Consortium
    : Birt-Hogg-Dubé syndrome: diagnosis and management.Lancet Oncol 10(12): 1199-1206, 2009.
    OpenUrlCrossRefPubMed
    1. Toro JR,
    2. Wei MH,
    3. Glenn GM,
    4. Weinreich M,
    5. Toure O,
    6. Vocke C,
    7. Turner M,
    8. Choyke P,
    9. Merino MJ,
    10. Pinto PA,
    11. Steinberg SM,
    12. Schmidt LS,
    13. Linehan WM
    : BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dube syndrome: a new series of 50 families and a review of published reports. J Med Genet 45: 321-331, 2008.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Nahorski MS,
    2. Lim DH,
    3. Martin L,
    4. Gille JJ,
    5. McKay K,
    6. Rehal PK,
    7. Ploeger HM,
    8. van Steensel M,
    9. Tomlinson IP,
    10. Latif F,
    11. Menko FH,
    12. Maher ER
    : Investigation of the Birt-Hogg-Dube tumour suppressor gene (FLCN) in familial and sporadic colorectal cancer. J Med Genet 47(6): 385-390, 2010.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Baba M,
    2. Hong SB,
    3. Sharma N,
    4. Warren MB,
    5. Nickerson ML,
    6. Iwamatsu A,
    7. Esposito D,
    8. Gillette WK,
    9. Hopkins RF 3rd.,
    10. Hartley JL,
    11. Furihata M,
    12. Oishi S,
    13. Zhen W,
    14. Burke TR Jr..,
    15. Linehan WM,
    16. Schmidt LS,
    17. Zbar B
    : Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proceedings of the National Academy of Sciences of the United States of America 103(42): 15552-15557, 2006.
    OpenUrlAbstract/FREE Full Text
    1. Hasumi H,
    2. Baba M,
    3. Hong SB,
    4. Hasumi Y,
    5. Huang Y,
    6. Yao M,
    7. Valera VA,
    8. Linehan WM,
    9. Schmidt LS
    : Identification and characterization of a novel folliculin interacting protein FNIP2. Gene 415(1-2): 60-67, 2008.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Takagi Y,
    2. Kobayashi T,
    3. Shiono M,
    4. Wang L,
    5. Piao X,
    6. Sun G,
    7. Zhang D,
    8. Abe M,
    9. Hagiwara Y,
    10. Takahashi K,
    11. Hino O
    : Interaction of folliculin (Birt-Hogg-Dube gene product) with a novel Fnip1-like (FnipL/Fnip2) protein. Oncogene 27(40): 5339-5347, 2008.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Baba M,
    2. Furihata M,
    3. Hong SB,
    4. Tessarollo L,
    5. Haines DC,
    6. Southon E,
    7. Patel V,
    8. Igarashi P,
    9. Alvord WG,
    10. Leighty R,
    11. Yao M,
    12. Bernardo M,
    13. Ileva L,
    14. Choyke P,
    15. Warren MB,
    16. Zbar B,
    17. Linehan WM,
    18. Schmidt LS
    : 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 100: 140-154, 2008.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Chen J,
    2. Futami K,
    3. Petillo D,
    4. Peng J,
    5. Wang P,
    6. Knol J,
    7. Li Y,
    8. Khoo SK,
    9. Huang D,
    10. Qian CN,
    11. Zhao P,
    12. Dykema K,
    13. Zhang R,
    14. Cao B,
    15. Yang XJ,
    16. Furge K,
    17. Williams BO,
    18. Teh BT
    : Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia. PloS one 3: e3581, 2008.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Molina AM,
    2. Motzer RJ
    : Current algorithms and prognostic factors in the treatment of metastatic renal cell carcinoma. Clin Genitourin Cancer 6(Suppl 1): S7-13, 2008.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Hartman TR,
    2. Nicolas E,
    3. Klein-Szanto A,
    4. Al-Saleem T,
    5. Cash TP,
    6. Simon MC,
    7. Henske EP
    : The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis Oncogene 28(13): 1594-1604, 2009.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Hong SB,
    2. Oh H,
    3. Valera VA,
    4. Stull J,
    5. Ngo DT,
    6. Baba M,
    7. Merino MJ,
    8. Linehan W,
    9. Schmidt LS
    : Tumor suppressor FLCN inhibits tumorigenesis of a FLCN null renal cancer cell line and regulates expression of key molecules in TGF-beta signaling. Mol Cancer 9: 160, 2010.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Cash TP,
    2. Gruber JJ,
    3. Hartman TR,
    4. Henske EP,
    5. Simon MC
    : Loss of Birt-Hogg-Dube tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene 30(22): 2534-2546, 2011.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Preston RS,
    2. Philp A,
    3. Claessens T,
    4. Gijezen L,
    5. Dydensborg AB,
    6. Dunlop EA,
    7. Harper T,
    8. Brinkhuizen T,
    9. Menko FH,
    10. Davies DM,
    11. Land SC,
    12. Pause A,
    13. Baar K,
    14. van Steensel MA,
    15. Tee AR
    : Absence of the Birt-Hogg-Dubé gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene 30(10): 1159-1173, 2011.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Lu X,
    2. Wei W,
    3. Fenton J,
    4. Nahorski MS,
    5. Rabai E,
    6. Reiman A,
    7. Seabra L,
    8. Nagy Z,
    9. Latif F,
    10. Maher ER
    : Therapeutic targeting the loss of the birt-hogg-dube suppressor gene. Mol Cancer Ther 10: 80-89, 2011.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Smyth GK
    : Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology, Volume 3, Article 3.
  28. ↵
    1. Huang DW,
    2. Sherman BT,
    3. Lempicki RA
    : Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc 4(1): 44-57, 2009.
    OpenUrlCrossRef
  29. ↵
    1. Huang DW,
    2. Sherman BT,
    3. Lempicki RA
    : Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37(1): 1-13, 2009.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Klomp JA,
    2. Petillo D,
    3. Niemi NM,
    4. Dykema KJ,
    5. Chen J,
    6. Yang XJ,
    7. Sääf A,
    8. Zickert P,
    9. Aly M,
    10. Bergerheim U,
    11. Nordenskjöld M,
    12. Gad S,
    13. Giraud S,
    14. Denoux Y,
    15. Yonneau L,
    16. Méjean A,
    17. Vasiliu V,
    18. Richard S,
    19. MacKeigan JP,
    20. Teh BT,
    21. Furge KA
    : Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression. BMC Med Genomics 3: 59, 2010.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Marchong MN,
    2. Yurkowski C,
    3. Ma C,
    4. Spencer C,
    5. Pajovic S,
    6. Gallie B
    : Cdh11 acts as a tumor suppressor in a murine retinoblastoma model by facilitating tumor cell death. PLoS Genet 6(4): e1000923, 2010.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Delic S,
    2. Lottmann N,
    3. Jetschke K,
    4. Reifenberger G,
    5. Riemenschneider MJ
    : Identification and functional validation of CDH11, PCSK6 and SH3GL3 as novel glioma invasion-associated candidate genes. Neuropathol Appl Neurobiol Jul 4, 2011.
  33. ↵
    1. Chu K,
    2. Cheng CJ,
    3. Ye X,
    4. Lee YC,
    5. Zurita AJ,
    6. Chen DT,
    7. Yu-Lee LY,
    8. Zhang S,
    9. Yeh ET,
    10. Hu MC,
    11. Logothetis CJ,
    12. Lin SH
    : Cadherin-11 promotes the metastasis of prostate cancer cells to bone. Mol Cancer Res 6(8): 1259-1267, 2008.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Huang CF,
    2. Lira C,
    3. Chu K,
    4. Bilen MA,
    5. Lee YC,
    6. Ye X,
    7. Kim SM,
    8. Ortiz A,
    9. Wu FL,
    10. Logothetis CJ,
    11. Yu-Lee LY,
    12. Lin SH
    : Cadherin-11 increases migration and invasion of prostate cancer cells and enhances their interaction with osteoblasts. Cancer Res 70(11): 4580-4589, 2010.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Hendrix A,
    2. Braems G,
    3. Bracke M,
    4. Seabra M,
    5. Gahl W,
    6. De Wever O,
    7. Westbroek W
    : The secretory small GTPase Rab27B as a marker for breast cancer progression. Oncotarget 1(4): 304-308, 2010.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Pelech S,
    2. Jelinkova L,
    3. Susor A,
    4. Zhang H,
    5. Shi X,
    6. Pavlok A,
    7. Kubelka M,
    8. Kovarova H
    : Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation. J Proteome Res 7(7): 2860-2871, 2008.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Koromilas AE,
    2. Roy S,
    3. Barber GN,
    4. et al
    : Malignant transformation by a mutant of the IFN-inducible dsRNA-dependent protein kinase. Science 257: 1685-1689, 1992.
    OpenUrlAbstract/FREE Full Text
    1. Meurs EF,
    2. Galabru J,
    3. Barber GN,
    4. Katze MG,
    5. Hovanessian AG
    : Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc. Natl Acad. Sci USA 90(1): 232-236, 1993.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Donze O,
    2. Jagus R,
    3. Koromilas AE,
    4. Hershey JW,
    5. Sonenberg N
    : Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J 14(15): 3828-3834, 1995.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Maschietto M,
    2. de Camargo B,
    3. Brentani H,
    4. Grundy P,
    5. Sredni ST,
    6. Torres C,
    7. Mota LD,
    8. Cunha IW,
    9. Patrão DF,
    10. Costa CM,
    11. Soares FA,
    12. Brentani RR,
    13. Carraro DM
    : 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, 2008.
    OpenUrlPubMed
  41. ↵
    1. Martinez-Ruiz G,
    2. Maldonado V,
    3. Ceballos-Cancino,
    4. Reyes Grajeda JP,
    5. Melendez-Zajgla J
    : Role of Smac/DIABLO in cancer progression J Exp Clin Cancer Res 27(1): 48, 2008.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Mizutani Y,
    2. Nakanishi H,
    3. Yamamoto K,
    4. Li YN,
    5. Matsubara H,
    6. Mikami K,
    7. Okihara K,
    8. Kawauchi A,
    9. Bonavida B,
    10. Miki T
    : Down-regulation of Smac/DIABLO expression in renal cell carcinoma and its prognostic significance. J Clin Oncol 23(3): 448-454, 2005.
    OpenUrlAbstract/FREE Full Text
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Gene Expression and Protein Array Studies of Folliculin-regulated Pathways
ANNE REIMAN, XIAOHONG LU, LAURENCE SEABRA, UNCAAR BOORA, MICHAEL S. NAHORSKI, WENBIN WEI, EAMONN R. MAHER
Anticancer Research Nov 2012, 32 (11) 4663-4670;
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