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,
Ryan C. Russell,1,
Olga Roche,1,
T. Nadine Burry,1
Jason E. Fish,3
Vinca W. K. Chow,1
William Y. Kim,4
Arthy Saravanan,2
Mindy A. Maynard,1
Michelle L. Gervais,1
Roxana I. Sufan,1
Andrew M. Roberts,1
Leigh A. Wilson,1
Mark Betten,5
Cindy Vandewalle,6
Geert Berx,6
Philip A. Marsden,3,7
Meredith S. Irwin,8,9
Bin T. Teh,5
Michael A. S. Jewett,10 and
Michael Ohh1*
Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada,1 Department of Pathology, University Health Network, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada,2 Department of Medical Biophysics, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada,3 Department of Hematology Oncology, The Lineberger Comprehensive Cancer Center, 102 Mason Farm Road, CB7295, University of North Carolina, Chapel Hill, North Carolina 27599,4 Van Andel Research Institute, 333 Bostwick Avenue Northeast, Grand Rapids, Michigan 49503,5 Department for Molecular Biomedical Research, Molecular and Cellular Oncology, VIB-Ghent University, Technologiepark 927, B-9052 Ghent (Zwijnaarde), Belgium,6 Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada,7 Department of Paediatrics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada,8 Institute of Medical Sciences, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada,9 and Departments of Urology and Surgical Oncology, University Health Network, Princess Margaret Hospital, 610 University Avenue, Toronto, Ontario M5G 2M9, Canada,10
Received 18 May 2006/ Returned for modification 27 June 2006/ Accepted 27 September 2006
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit of hypoxia-inducible factor (HIF) for oxygen-dependent destruction. Although emerging evidence supports the notion that deregulated accumulation of HIF upon the loss of VHL is crucial for the development of clear-cell renal cell carcinoma (CC-RCC), the molecular events downstream of HIF governing renal oncogenesis remain unclear. Here, we show that the expression of a homophilic adhesion molecule, E-cadherin, a major constituent of epithelial cell junctions whose loss is associated with the progression of epithelial cancers, is significantly down-regulated in primary CC-RCC and CC-RCC cell lines devoid of VHL. Reintroduction of wild-type VHL in CC-RCC (VHL–/–) cells markedly reduced the expression of E2 box-dependent E-cadherin-specific transcriptional repressors Snail and SIP1 and concomitantly restored E-cadherin expression. RNA interference-mediated knockdown of HIF in CC-RCC (VHL–/–) cells likewise increased E-cadherin expression, while functional hypoxia or expression of VHL mutants incapable of promoting HIF degradation attenuated E-cadherin expression, correlating with the disengagement of RNA polymerase II from the endogenous E-cadherin promoter/gene. These findings reveal a critical HIF-dependent molecular pathway connecting VHL, an established "gatekeeper" of the renal epithelium, with a major epithelial tumor suppressor, E-cadherin. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The most well-characterized function of VHL is as a substrate-recognition component of the SCF (Skp1/Cdc53/F-box protein)-like E3 ubiquitin ligase complex called ECV (elongins/Cul2/VHL) that selectively ubiquitylates oxygen-dependent prolyl-hydroxylated subunits of hypoxia-inducible factor (HIF) (24, 26, 43, 49). Under hypoxic conditions, HIF is stabilized, recruits p300/CBP, and binds to its constitutively expressed partner HIFß/arylhydrocarbon receptor nuclear translocator (ARNT) (48). The heterodimeric HIF complex binds to a consensus 5'-RCGTG-3' hypoxia-responsive element in enhancers and promoters to trigger the transcription of numerous hypoxia-inducible genes that promote cellular adaptation to hypoxia, enabling cell survival under compromised oxygen availability (48). Concordantly, tumor cells devoid of VHL have enhanced expression of HIF target genes, such as VEGF (vascular endothelial growth factor; angiogenesis), GLUT-1 (glucose transporter 1; anaerobic metabolism), and EPO (erythropoietin; production of oxygen-carrying red blood cells), irrespective of oxygen tension (29). The overexpression of these and other hypoxia-inducible genes likely explains the angiogenic phenotype of VHL-associated tumors and also supports the notion that constitutive stabilization of HIF is causally linked to tumorigenesis. In support, Kaelin and colleagues have shown that forced stable expression of HIF-2 in CC-RCC cells ectopically expressing wild-type VHL overrides the tumor suppressor capacity of VHL and restores the tumorigenic potential of CC-RCC cells in an animal xenograft system (32). Conversely, short hairpin RNA (shRNA)-mediated knock-down of HIF-2 is sufficient to suppress the tumorigenic capacity of RCC cells devoid of VHL (31). Notably, all CC-RCC-causing VHL mutants tested to date have shown a failure in either assembling of ECV complex or binding to HIF (11, 21). However, the critical event(s) downstream of HIF that causes neoplastic transformation of proximal renal tubular epithelial cell (the origin of CC-RCC) is unclear.
Proper regulation of cell-cell adhesion is vital during cell growth, differentiation, and tissue development. E-cadherins, homophilic adhesion molecules, and their associated catenins are the major constituents of cell junctions in polarized epithelial cells (46). Increased expression of E-cadherin is associated with the differentiation of mesenchymal cells into tubular epithelial cells of the adult nephron. Conversely, loss of cell-cell adhesion is frequently associated with tumor progression and metastasis (46). In support of this paradigm, the loss of E-cadherin is associated with the progression of numerous carcinoma types, and forced expression of E-cadherin suppresses tumor development and invasion in various in vitro and in vivo tumor model systems, establishing E-cadherin as a critical tumor suppressor of the epithelium (46). Recently, Krishnamachary et al. and Esteban et al. independently showed that the loss of VHL in CC-RCC cells results in the loss of E-cadherin expression in an HIF-dependent manner (16, 34). Krishnamachary et al. argue that the regulation of E-cadherin expression is exclusively HIF-1-dependent, while Esteban et al. demonstrated dependency on both HIF-1 and HIF-2 (16, 34). Furthermore, Krishnamachary et al. showed that the activation of the HIF-1 pathway upon the loss of VHL transactivates E-cadherin transcriptional repressors TCF3 (also known as E12/E47), ZFHX1A (
EF1 or ZEB1), and SIP1 (Smad-interacting protein-1; also known as ZEB-2 or ZFHX1B), which correlated with the down-regulation of E-cadherin transcription (34).
Here, we examined over 100 primary CC-RCC samples via Affymetrix oligonucleotide arrays and show that the expression of E-cadherin transcripts is significantly down-regulated in CC-RCC. In addition, the analysis of 56 CC-RCC samples on tissue microarrays and immunohistochemistry on CC-RCC nephrectomy specimens from 13 patients revealed a strong positive correlation between the expression of VHL and E-cadherin. Small interfering RNA (siRNA)-mediated knockdown of endogenous VHL or functional hypoxia resulted in dramatic attenuation of E-cadherin expression. Importantly, reintroduction of wild-type VHL, but not a CC-RCC-causing VHL mutant incapable of promoting HIF degradation, in CC-RCC (VHL–/– HIF-1–/–) cells fully restored E-cadherin expression, in part, via HIF-dependent regulation of E2 box-dependent transcriptional repressors Snail and SIP1 but not Slug, TCF3, or ZFHX1A, and subsequent engagement of RNA polymerase (Pol) II on endogenous E-cadherin promoter/gene. These findings provide a compelling molecular mechanism governing the transcription of E-cadherin by the VHL-HIF pathway and strengthen the potential involvement of E-cadherin in the development of CC-RCC.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(P531A) (786-VHL+HIF-2) or empty control (786-VHL+MOCK) via retrovirus were previously described (32) and were generously provided by William G. Kaelin. 786-O subclones stably expressing pRetroSUPER-empty or pRetroSUPER-HIF2 shRNA were previously described (31).
Antibodies.
Monoclonal anti-HA antibody (12CA5) was obtained from Roche Molecular Biochemicals. Monoclonal anti-VHL antibody (IG32) was as previously described (30). Anti-ß-catenin, anti-lamin A/C, and anti--tubulin antibodies were obtained from Santa Cruz (Santa Cruz, CA), Abcam (Cambridge, MA), and Sigma-Aldrich (Oakville, Ontario, Canada), respectively. Anti-E-cadherin antibody was obtained from BD Transduction Labs (Mississauga, Canada). Anti-HIF-2 antibody was obtained from Novus Biologicals Inc. (Littleton, CO).
Plasmids. Mammalian expression plasmid pRc-CMV-HA-VHL(WT) was described previously (39). Expression plasmid encoding Snail was generously provided by Paul Hamel. E-cadherin core promoter (–308/+21)-luciferase reporter plasmids (E-cad prom-luc wild type [WT] or mutE2, which contains inactivating mutations in both E2 boxes) and expression plasmids encoding SIP1 were previously described (13). Plasmids containing shRNA against E-cadherin were generated using the Invitrogen pENTR/U6 system. Inserts containing hairpin DNA were generated using Invitrogen's Block iT siRNA designer, and insert cassettes were bought polyacrylamide gel electrophoresis (PAGE) purified from Invitrogen using the pENTR recommended protocol. The four generated shRNAs were chosen based on their predicted ability of silencing and their placement within the E-cadherin transcript. Each shRNA has 21 mer of complementary sequences starting at positions 1184, 1660, 1973, and 2128 of the E-cadherin gene (accession number NM004360). A CGAA loop is included in all plasmids. The nontargeting or scrambled shRNA was created in a similar manner with a sequence of 5'-GGAGCACGGTATTCGGGTCTACTAA-3', which bears no sequence similarity with the E-cadherin transcript.
Immunoprecipitation and immunoblotting. Immunoprecipitation and Western blotting were performed as described previously (50). In brief, cells were lysed in EBC buffer (50 mM Tris [pH 8.0], 120 mM NaCl, 0.5% NP-40) supplemented with a cocktail of protease and phosphatase inhibitors (Roche, Laval, Canada). Immunoprecipitates immobilized on protein A-Sepharose beads (Amersham Biosciences, Piscataway, NJ) were washed five times with NETN buffer (20 mM Tris [pH 8.0], 120 mM NaCl, 1 mM ETDA, 0.5% NP-40), eluted by boiling in sodium dodecyl sulfate (SDS)-containing sample buffer, and size-fractionated by SDS-PAGE. Resolved proteins were then electrotransferred onto polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA), immunoblotted with the various antibodies, and visualized by chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Hypoxia treatment of cells. Cells were maintained at 1% O2 for the times indicated in the legend to Fig. 6 in a ThermoForma (Marietta, OH) hypoxia chamber (5% CO2, 10% H2, 85% N2). Cell lysates were prepared in the chamber in hypoxic environment prior to further experimentation.
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TMAs. Tissue microarrays (TMAs) consisting of quadruplicate representative 1.0-mm cores from 56 CC-RCCs with various Fuhrman grades (grades 1 to 4) and stages (pT1-pT4) were used to analyze the correlation between E-cadherin and VHL protein expression patterns. Five-micrometer sections from the CC-RCC TMAs were stained with anti-E-cadherin and anti-VHL antibodies as described above in "Immunohistochemical staining." The slides were then scanned using a ScanScope (Aperio Technologies, Vista, CA) and scored by two observers. In order to be considered for evaluation, at least half of the TMA core was required to be present on the slide with at least 50% of the tissue present being tumor cells. Cores were scored as being either positive or negative. Only tumors with three or more cores in agreement were used for further analysis. Based on these criteria, 70% (39 out of 56) of the tumors on the TMA were suitable for the correlation analysis between E-cadherin and VHL.
Migration assay. Cells were seeded on uncoated six-well plates, and scraping the cells with a sterile pipette tip generated the wounds. Cells were washed once and maintained in warm DMEM containing 0.5% fetal bovine serum (FBS) for the duration of the migration assay. A Nikon Eclipse TE200 inverted microscope equipped with a heated stage and a Hamamatsu digital camera (model C4742-95) was used to capture the images at the time points indicated in the legend to Fig. 3. Each wound measurement was taken in triplicate, and the experiment was repeated three times. Percent wound closure was determined by the measurement of the surface area of the wound at various time points relative to the starting wound surface area using the AxioVision AC program (version 4.2.0.0).
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Subcellular fractionation. Cells were resuspended in buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol) supplemented with protease and phosphatase inhibitors (Roche, Laval, Canada) and 1 mM dithiothreitol (DTT) and subsequently lysed with 0.1% Triton X-100. Samples were incubated for 7 min on ice and centrifuged. While the supernatant was recovered (cytoplasmic fraction), the pellet was washed with buffer A and resuspended in buffer B (0.2 mM EGTA [pH 8], 3 mM EDTA [pH 8]) supplemented with protease and phosphatase inhibitors (Roche, Laval, Canada) and 1 mM DTT. After a 30-min incubation period, samples were centrifuged, and the resulting supernatant was isolated (nuclear fraction).
Dual-luciferase assay. U2OS osteosarcoma cells grown on six-well plates were transfected with a total of 2.5 µg of expression plasmids using Fugene 6 (Roche). E-cad prom-luc WT or mutE2 (0.9 µg per transfection) was used to measure E-cadherin core promoter-mediated transcription, and 0.1 µg of the Renilla luciferase plasmid, pRL-SV40 (Promega), was used as a transfection control. An empty pcDNA3.1 plasmid (Invitrogen) was used to maintain a constant final amount of transfected DNA. Cells were lysed 48 h after transfection, and luciferase assays were performed using the Dual-Luciferase Reporter Assay system (Promega), and relative light units (RLUs) were measured using a Lumat LB9507 luminometer (Berthold Technologies). Firefly luciferase RLUs were normalized against Renilla luciferase RLUs and standardized to the result of transfection with only the E-cad prom-luc WT, which was arbitrarily set to 1.0. Experiments and transfections were performed in triplicate with one representative experiment presented. Error bars represent standard deviations.
Microarray analysis. We have established a large gene expression profiling database of renal tumors, some of which have previously been published (18, 63). For this study, we selected a total of 105 renal tumors of clear-cell type and 12 normal kidney tissue samples. The Affymetrix HGU133 Plus 2.0 GeneChip oligonucleotide arrays were used for all 117 cases. The HGU133 Plus 2.0 arrays contain 54,675 probe sets, representing approximately 47,000 transcripts and variants. The manufacturer's recommended protocol (1) was followed for expression profiling. Briefly, for oligonucleotide expression profiling, 5 to 20 µg of total RNA was used to prepare antisense biotinylated RNA. A subset of cases was spiked with external poly(A) RNA positive controls (Affymetrix, Santa Clara, CA). Synthesis of cDNA was performed with the use of T7-oligo(dT) primer. In vitro transcription was performed using a Bioarray Transcript Labeling Kit (Enzo, New York). The biotinylated cRNA was subsequently fragmented, and 15 µg was hybridized to each array at 45°C for 16 h. Scanning was performed in a GeneChip 3000 scanner. Quality assessment was performed in a GeneChip Operating System 1.4 (Affymetrix) using global scaling to a target signal of 500. Quality assessment was also performed using denaturing gel electrophoresis. Median background was 73, the median scaling factor was 3.06, and the median GADPH (glyceraldehyde-3-phosphate dehydrogenase) 3'/5' ratio was 1.03, indicative of a high overall array and RNA quality.
Statistical analyses were performed in the statistical environment R 2.2, utilizing packages from the Bioconductor project. The MAS 5 algorithm was used to perform preprocessing of the CEL files, including background adjustment, quartile normalization, and summarization. The means and the standard errors for E-cadherin gene expressions were calculated for each of the group of samples. A two-tailed Student's t test was used to determine statistically significant differences between various groups.
siRNA-mediated VHL knockdown. siGENOME SMARTpool targeted to VHL was used (Dharmacon, Austin, TX). A nontargeting scrambled siRNA duplex was used as a negative control (5'-CCAUUCCGAUCCUGAUCCG-3'). HEK293A (VHL+/+) cells grown on six-well tissue culture plates were transfected with scrambled and VHL siRNA at a final concentration of 200 nM. Briefly, 8 µl of Oligofectamine (Invitrogen) was incubated with 48 µl of Opti-MEM I (Gibco/Invitrogen) for 8 min. The Oligofectamine mixture was added to the siRNA diluted in 175 µl of Opti-MEM I and incubated for 20 min before adding the resulting mixture to 800 µl of Opti-MEM I into the wells. After 3 h, 300 µl of DMEM containing 30% heat-inactivated FBS (Sigma) was added to the plates. RNA was extracted 48 h after transfection using an RNeasy kit (QIAGEN, Mississauga, Ontario, Canada) treated with RNA-free DNase (Ambion, Texas), and first-strand cDNA synthesis was performed.
Quantitative real-time PCR. For first-strand cDNA synthesis, 1 µl of oligo(dT)23 primer (Sigma) was incubated with 5 µg of RNA and distilled H2O (total reaction volume of 20 µl) for 10 min at 70°C in a thermal cycler (MJ Research, Boston, MA). The mixture was cooled to 4°C, at which time 4 µl of 5x first-strand reaction buffer, 2 µl of 0.1 M DTT, 1 µl of a 10 mM concentration of each deoxynucleoside triphosphate, and 1 µl of Superscript II reverse transcriptase (Invitrogen) were added. cDNA synthesis was performed for 1.5 h at 42°C, followed by 15 min at 70°C in the thermal cycler. Human genomic DNA standards (human genomic DNA was obtained from Roche, Mannheim, Germany) or cDNA equivalent to 20 ng of total RNA were added to the quantitative PCR (qPCR) reaction mixture in a final volume of 10 µl containing 1x PCR buffer (without MgCl2), 3 mM MgCl2, 0.25 units of Platinum Taq DNA polymerase, a 0.2 mM concentration of each deoxynucleoside triphosphate, 0.3 µl of SYBR Green I, 0.2 µl of ROX reference dye, and a 0.5 µM concentration of each primer (Invitrogen). Amplification conditions were as follows: 95°C (3 min), 40 cycles of 95°C (10 s), 65°C (15 s), 72°C (20 s), and 95°C (15 s). qPCR was performed using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Gene-specific oligonucleotide primers designed using Primer Express (Applied Biosystems) were as follows: Snail primer set (5'-TTCAACTGCAAATACTGCAACAAG-3' and 5'-CGTGTGGCTTCGGATGTG-3'), SIP1 primer set (5'-CCACACTTCGCGGCTTCTT-3' and 5'-CGATCTGCGAAGTCTTGTTTGT-3'), E-cadherin primer set (5'-GTCATCCAACGGGAATGCA-3' and 5'-TGATCGGTTACCGTGATCAAAA-3'), GLUT-1 primer set (5'-CACCACCTCACTCCTGT-TACTT-3' and 5'-CAAGCATTTCAAAACCATGTTTCTA-3'), VEGF primer set (5'-CTCTCTCCCTCATCGGTGACA-3' and 5'-GGAGGGCAGAGCTGAGTGTTAG-3'), and U1AsnRNP1 primer set (5'-CAACGACAGCCGAGACATGTA-3' and 5'-AGCCTCCATCAAATACCCATTC-3'). SYBR Green I fluoresces during each cycle of the qPCR by an amount proportional to the quantity of amplified cDNA (the amplicon) present at that time. The point at which the fluorescent signal is statistically significant above background is defined as the cycle threshold (CT). Expression levels of the various transcripts were determined by taking the average CT value for each cDNA sample performed in triplicate and measured against a standard plot of CT values from amplification of serially diluted human genomic DNA standards. Since the CT value is inversely proportional to the log of the initial copy number, the copy number of an experimental mRNA can be obtained from linear regression of the standard curve. A measure of the relative difference in copy number was determined for each mRNA. Values were normalized to expression of U1AsnRNP1 mRNA, expressed relative to scrambled siRNA samples (arbitrarily set to 1.0), and represented as the mean value of three independent experiments performed in triplicate ± standard deviations.
ChIP. Chromatin immunoprecipitation (ChIP) was performed as published previously using the Upstate ChIP assay kit (17). Five micrograms of anti-RNA polymerase II (N-20) antibody (Santa Cruz) was added to sheared, formaldehyde cross-linked chromatin preparations from 1 x 106 cells, and immunoprecipitation was performed overnight at 4°C. A control immunoprecipitation without the addition of antibody was also performed in parallel. An 18-µl aliquot (of a total of 1,800 µl) of chromatin was removed prior to immunoprecipitation to serve as an input control. The cross-links were reversed by addition of 2 µl of 5 M NaCl, and the sample was diluted 1 in 10 before real-time PCR was performed. Immune complexes were collected with protein A-agarose beads, and, after extensive washing, immune complexes were released, formaldehyde cross-links were reversed, and DNA was purified by phenol-chloroform extraction. Following ethanol precipitation, DNA was resuspended in 30 µl of water. Real-time PCR was performed on 2 µl of anti-Pol II immunoprecipitated DNA, 2 µl of no-antibody control, and 2 µl of the diluted input sample. Real-time PCR was performed in triplicate using SYBR green chemistry. Copies of the target gene were determined using genomic DNA as a standard curve (where 1 ng of genomic DNA is equivalent to 300 copies of a single copy gene). Immunoprecipitated DNA (IP DNA) was determined by subtracting the number of copies of the no-antibody control from the anti-Pol II immunoprecipitated DNA and dividing by the number of copies in the diluted input sample. Primers were designed to amplify the human E-cadherin promoter (forward, 5'-CCACGCACCCCCTCTCAGT-3'; reverse, 5'-GAGCGGGCTGGAGTCTGAAC-3'), human E-cadherin exon 10 (forward, 5'-CCGTGGATGTGCTGGATGTGA-3'; reverse, 5'-TGGGCAGTGTAGGATGTGATTTC-3'), and the human cyclophilin A promoter (forward, 5'-CCTCATGTGTCGTCCCCATCA-3'; reverse, 5'-CGCCCGTTTTATACCACGTTCG-3').
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To further validate the positive correlation between VHL and E-cadherin expression, TMAs consisting of 56 CC-RCC cores in quadruplicate were generated. Thirty-nine of the CC-RCC samples met the quality standard criteria (see Materials and Methods) and were analyzed for E-cadherin and VHL protein expression patterns. While only 33% (5/15) of the tumors that stained negative for VHL (15/39) stained positive for E-cadherin, the majority (67% or 16/24) of tumors that stained positive for VHL (24/39) also stained positive for E-cadherin (Fig. 1C). However, a positive stain for VHL does not formally indicate the presence of a wild-type VHL, as, for example, a subtle point mutation will likely produce a positive staining signal. Thus, additional mutational analysis will be required to generate a more precise E-cadherin:VHL correlation index.
Knockdown of endogenous VHL results in a dramatic attenuation of E-cadherin expression.
Reconstitution of 786-O (VHL–/– HIF-1–/–) or RCC4 (VHL–/–) renal carcinoma cells with HA-VHL dramatically restored the expression of E-cadherin protein and mRNA, as measured by Western blotting and quantitative real-time PCR, respectively (Fig. 2A and B). In addition, siRNA-mediated knockdown of endogenous VHL in HEK293A embryonic kidney cells resulted in marked down-regulation of E-cadherin expression (Fig. 2C). Microarray (Fig. 1A) and real-time PCR data strongly suggest that E-cadherin regulation by VHL is at the pretranslational level. The cytoplasmic domain of E-cadherin is in a complex with ß-catenin, implicating a potential "outside-in" signaling where a loss of E-cadherin would release ß-catenin to associate with the leukocyte enhancer factor /T-cell factor to regulate the transcription of cell cycle-related genes (e.g., cyclin D1) or invasion-related genes (e.g., metalloproteinase matrilysin and fibronectin) (46). Interestingly, an increased level of cyclin D1 has been observed in RCC cells devoid of VHL at high cell density (4), and cells expressing tumor-causing VHL mutants fail to assemble proper extracellular fibronectin matrices (50, 62). However, both the overall expression and subcellular localization of ß-catenin remained unaffected by VHL (Fig. 2A and see Fig. S1 in the supplemental material), suggesting that ß-catenin-mediated transcription is likely not involved in potential outside-in signaling via the loss of E-cadherin in the context of CC-RCC.
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VHL regulates E-cadherin expression via an HIF-dependent mechanism.
We next asked whether the regulation of E-cadherin expression by VHL is mediated through the activity of HIF. RCC4-VHL and 786-VHL cells were maintained under normoxic (21% oxygen) (Fig. 4A) or hypoxic (1%) (see Fig. S2 in the supplemental material) conditions for 16 h and analyzed by Western blotting. The expression of E-cadherin was dramatically reduced under hypoxia while preserving the expression status of VHL (Fig. 4A). The effect of hypoxic treatment was confirmed by the increase in HIF-2 expression (Fig. 4A). Although this result suggests that hypoxia-induced stabilization of HIF results in repression of E-cadherin expression, it is formally possible that VHL, independent of HIF, regulates the expression of E-cadherin in an oxygen-dependent manner. Therefore, we examined various VHL mutants that have retained or lost the ability to regulate HIF. Certain non-CC-RCC-associated VHL mutants have been shown to retain the ability to regulate HIF activity (11, 21, 62). For example, The L188V mutation allows proper oxygen-dependent degradation of HIF and is associated with a subclass of VHL disease (type 2C), which is clinically characterized by the exclusive development of pheochromocytoma (21). In contrast, invariably all CC-RCC-associated VHL mutations tested to date, such as C162F, result in a complete loss of the ability to mediate the destruction of HIF via the ubiquitin-proteasome pathway (11, 21). 786-O cells ectopically expressing VHL(C162F) showed negligible expression of E-cadherin, while those expressing VHL(L188V) showed higher detectable levels of E-cadherin, albeit at a lower level than observed in cells expressing VHL(WT) (Fig. 4B). In addition, 786-O (VHL–/– HIF-1–/–) cells stably expressing wild-type VHL (786-VHL) infected with retroviruses that express functional and stable HIF-2(P531A) (escapes VHL recognition) demonstrated a reduced level of E-cadherin relative to 786-VHL cells infected with "empty" retrovirus (Fig. 4C, compare lanes 1 and 2). Notably, the level of E-cadherin was inversely proportional to the level of HIF-2 (Fig. 4C). Conversely, 786-O subclones infected with retroviruses that express HIF-2-specific shRNA demonstrated a markedly increased level of E-cadherin relative to 786-O cells infected with empty retrovirus (Fig. 4D). In addition, the activity of the exogenous E-cadherin promoter-driven luciferase reporter was much higher in 786-O cells reconstituted with wild-type VHL (786-WT; low HIF activity) than in 786-MOCK (high HIF activity) cells (Fig. 4E). Taken together, these results strongly suggest that HIF negatively regulates E-cadherin expression, at least in part, at the level of transcription.
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Quantitative real-time PCR analysis showed a significant attenuation of both Snail and SIP1 in CC-RCC 786-O cells restored with VHL (Fig. 5A). However, negligible changes in the expression were noted for the other known E-cadherin transcriptional repressors Slug, TCF3, and ZFHX1A (data not shown). As expected, restoration of VHL reduced the expression of HIF target genes, VEGF and GLUT-1, and increased E-cadherin expression (Fig. 5A; compare with results shown in Fig. 2). These results suggest the possibility that VHL may increase the expression of E-cadherin by down-regulating the transcriptional repressors Snail and SIP1. In a complementary experiment, we tested the ability of VHL in the transactivation of E-cadherin promoter-driven luciferase reporter (Fig. 5B). As expected, E-cadherin promoter containing both E2 boxes had lower basal transcriptional activity relative to E-cadherin promoter with point mutations in the E2 boxes that abrogate Snail/SIP1 binding (Fig. 5B). Importantly, the addition of VHL markedly increased the wild-type E-cadherin promoter-driven luciferase transcription, but had insignificant effect on the E2 mutant E-cadherin promoter (Fig. 5B). As predicted, the increase in VHL-mediated transactivity of wild-type E-cadherin promoter-luciferase was dampened by the addition of SIP1 or/and Snail in a dosage-dependent manner (Fig. 5C). Coexpression analysis indicated that Snail or SIP1 had negligible effect on the steady-state level of VHL (see Fig. S3 in the supplemental material; also data not shown). These results demonstrate that the E2 boxes are functionally important in upregulating E-cadherin transcription by VHL, in part, via the down-regulation of SIP1 and/or Snail. However, neither SIP1 nor Snail individually or in combination achieved a complete inhibition of E-cadherin promoter-driven reporter activity. This suggests that there exist other yet-to-be-defined VHL/HIF-mediated E-cadherin-specific transcriptional repressors or that full repression requires the concerted actions of multiple repressors and involves, in addition to the E2 boxes, other elements within the E-cadherin promoter.
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To further establish a functional role of VHL/HIF in E-cadherin gene transcription, 786-O cells stably expressing wild-type VHL were exposed to hypoxia. Assessment of Pol II binding to genomic regions corresponding to coding regions of E-cadherin (exon 10) revealed that hypoxia decreased E-cadherin transcription (Fig. 6B). A similar decrease in Pol II binding was demonstrated at the promoter of E-cadherin (data not shown). As expected, hypoxia elicited a time-dependent increase in VEGF mRNA expression (Fig. 6B). Taken together, these results suggest that VHL activity, specifically E3 ligase function to negatively regulate HIF, is required for the transcription of the E-cadherin gene. Conversely, cellular hypoxia or loss of HIF-associated function of VHL results in the activation of HIF and disengagement of Pol II from the E-cadherin promoter, resulting in the down-regulation of E-cadherin transcription. However, it is not formally known whether HIF-mediated engagement of Pol II on E-cadherin promoter is strictly SIP1/Snail-dependent.
E-cadherin expression is cell density dependent.
E-cadherin expression in CC-RCC cells is also cell density dependent as measured by Western blotting and quantitative real-time PCR (see Fig. S4A and B in the supplemental material). Interestingly, the expression of VHL is strictly regulated by cell density where the steady-state amount of VHL in human renal proximal tubule epithelial cells was shown to increase more than 100-fold in dense cultures relative to sparse cultures (3). In addition, other components of the VHL E3 ligase complex showed a similar cell density-dependent regulation (44). Importantly, HIF-2 level was elevated in sparsely growing cells with low levels of VHL and significantly reduced or undetectable in confluent cells containing abundant VHL (44). Moreover, the ability of VHL to shuttle between the nucleus and the cytoplasm is also regulated by cell density (37), which in turn may influence the ability of VHL to regulate HIF activity (20). Thus, cell density-dependent expression of E-cadherin may be due to a corresponding cell density-dependent regulation of VHL stability/function.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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via the ubiquitin-proteasome pathway (29). The loss of VHL or VHL mutations associated with the development of CC-RCC invariably results in the accumulation/hyperactivation of HIF due to a failure in VHL's ability to either bind or ubiquitylate HIF. Here, we propose that HIF—stabilized by hypoxia in the presence of wild-type VHL or upon mutation/loss of VHL—activates the transcriptional repressors SIP1 and Snail (likely via the HIF-engagement of the hypoxia-responsive element 5'-GCGTG-3' found in the Snail promoter at position –86 to –82; SIP1 promoter/enhancer has not been defined), preventing Pol II engagement on the E-cadherin promoter and resulting in the down-regulation of E-cadherin expression (Fig. 7). There is, however, an additional pathway to consider (described below).
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In the current work, we demonstrated that the loss of VHL leads to the dramatic down-regulation of E-cadherin in CC-RCC. VHL-dependent transactivation of E-cadherin was dependent on the conserved E2 boxes known to recruit transcriptional repressors Snail and SIP1 to the promoter of E-cadherin. Reintroduction of VHL in CC-RCC cells devoid of VHL showed a reduction in the expression of both Snail and SIP1, thereby explaining, at least in part, the resulting restoration of E-cadherin expression. Transcriptional repressors principally block transcription by inhibiting the engagement of Pol II to the promoter. In support, Snail has been shown to repress E-cadherin expression through the binding of histone deacetylase, promoting repressive changes in chromatin structure and thereby impairing the recruitment of Pol II and transcription (52). Consistent with this view, wild-type VHL enhanced the recruitment of Pol II to the E-cadherin promoter/gene, whereas hypoxia or a tumor-causing VHL mutant with a failure in targeting HIF for ubiquitin-mediated destruction dramatically decreased the association of Pol II with the E-cadherin gene. Thus, VHL or functional hypoxia directly affects Pol II engagement on E-cadherin DNA likely via HIF-dependent regulation of E-cadherin-specific transcriptional repressors, revealing a previously unrecognized regulation of a major epithelial tumor suppressor E-cadherin.
During the preparation of the present report, Krishnamachary et al. and Esteban et al. showed that the loss of VHL in CC-RCC cells results in the loss of E-cadherin expression in a HIF-dependent manner (16, 34). Krishnamachary et al. argue that the regulation of E-cadherin expression is exclusively HIF-1 dependent, as 786-O (HIF-1 –/–) cells failed to express E-cadherin after VHL rescue (16, 34). However, our data clearly demonstrate that HIF-2, in the absence of HIF-1, can restore E-cadherin expression upon reintroduction of VHL in 786-O cells. Although the reason for this discrepancy is unclear, the relative contribution of HIF-1 versus HIF-2 in the development of CC-RCC is an emerging area of research. For example, the introduction of a HIF-1 mutant that escapes VHL recognition into CC-RCC cells reconstituted with wild-type VHL does not produce a tumorigenic phenotype in SCID mice (41). However, the treatment of these VHL-restored CC-RCC cells with an HIF-1-oxygen-dependent degradation domain peptide that can block VHL binding to HIF substrates restored the tumorigenic phenotype (41). This finding suggests that although HIF-1 is dispensable, other HIF subunits (or possibly other ECV substrates) are associated with the tumor suppressor function of VHL. In support of this notion, Kondo et al. demonstrated that, unlike HIF-1, the nondegradable HIF-2 was able to restore the tumor phenotype in CC-RCC cells expressing wild-type VHL (32), suggesting that HIF-2 is the relevant oncogenic player in the development of CC-RCC. Furthermore, Rankin et al. used the PEPCK (phosphoenolpyruvate carboxykinase) promoter to generate transgenic mice in which Cre-recombinase is expressed in the renal proximal tubules and hepatocytes (54). Conditional inactivation of VHL in PEPCK-Cre mouse resulted in glomerular and tubular renal cysts, increased serum erythropoietin levels, and polycythemia (54). The inactivation of ARNT, but not HIF-1, rescued the conditional VHL knockout mouse from developing renal cysts (53, 54), further supporting the notion that another partner of ARNT such as HIF-2, but not HIF-1, plays a critical role in the transformation of renal proximal tubules.
We show here that the depletion of E-cadherin promotes the invasive potential of VHL-reconstituted CC-RCC cells, which is in keeping with the canonical function of E-cadherin. Krishnamachary et al. recently provided evidence that loss of VHL promotes epithelial-mesenchymal transformation, which is consistent with a loss of adherens junction due to reduced E-cadherin expression (34). There are, however, other notable VHL-dependent events bearing on CC-RCC progression to consider. CXCR4 is a chemokine receptor that aids in the metastasis of tumor cells to organs abundant in CXCR4-specific ligand, stromal cell-derived factor-1 (SDF-1). Staller et al. showed that the expression of chemokine receptor CXCR4 increases upon the loss of VHL, suggesting a potential mechanism of CC-RCC metastasis (61). In addition, Zagzag et al. recently demonstrated that CC-RCC and hemangioblastoma cells devoid of VHL overexpress not only CXCR4 but also its ligand SDF-1 (66). These findings suggest that loss-of-function of VHL can establish an autocrine signaling pathway providing selective survival advantage and increased tendency for metastasis. The impact of the individual events (i.e., VHL-mediated E-cadherin versus CXCR4/SDF-1 regulation) in CC-RCC progression is not yet established, but nevertheless remains an important question to address.
In addition, VHL binds directly to fibronectin and promotes proper assembly of fibronectin extracellular matrix (ECM) (21, 50). All disease-associated VHL mutants examined to date exhibit decreased or undetectable binding to fibronectin (21, 50), underscoring the importance of VHL-mediated fibronectin assembly in the pathogenesis of VHL disease. In fact, alterations in the fibronectin component of the ECM have been correlated with cellular transformation (22). Conversely, multimeric forms of fibronectin or the overproduction of fibronectin receptors, such as 5ß1 integrin, have been shown to promote differentiation and suppress the proliferative and metastatic potentials of transformed cells in various model systems (22, 51, 56). Lieubeau-Teillet et al. demonstrated that CC-RCC cells lacking VHL grow as tightly packed amorphous spheroids in a three-dimensional growth assay, indicative of an undifferentiated phenotype (38). In contrast, CC-RCC cells reconstituted with VHL form loose aggregates, which, on microscopic and ultrastructural examination, exhibit evidence of epithelial differentiation, such as trabecular and tubular structures (38). Davidowitz et al. demonstrated that VHL expressing CC-RCC cells grown on ECM differentiate into organized epithelial monolayers, whereas VHL-deficient cells are branched and disorganized and fail to arrest under high cell density (14). Koochekpour et al. showed that CC-RCC cells lacking functional VHL produce increased levels of matrix metalloproteinases and attenuated levels of tissue inhibitors of metalloproteinases and demonstrate increased invasiveness on growth factor-reduced matrigel (an artificial ECM) in response to hepatocyte growth factor/scatter factor (33). The lack of functional VHL has also been associated with overproduction of carbonic anhydrases 9 and 12, which are involved in the acidification of the microenvironment, favoring the growth and invasive properties of tumor cells (25, 42, 59). Thus, in addition to regulating E-cadherin expression, VHL is seemingly required for multiple additional events governing epithelial transformation from cellular differentiation to invasiveness.
There are other examples of cancer-causing mutations that often increase the expression of HIF, which provides a mechanistic explanation for hypervascular tumors including CC-RCC that develop in the absence of VHL mutations. Mutations in the TSC2 tumor suppressor gene increase the level of HIF via the mammalian target of rapamycin-dependent and -independent mechanisms that may involve chromatin remodeling (9). Loss of PTEN, which has been observed in the brain tumor glioblastoma multiforme, results in increased HIF-1 levels via the activation of the Akt/protein kinase B signaling cascade (68). The increased expression of HER2 receptor tyrosine kinase in breast cancer and the loss of p53 in various tumors enhance HIF-1-dependent transcription, often correlating with tumor aggressiveness (36, 55). Although these examples support the notion that there are multiple important regulators of HIF to ultimately promote oncogenic transformation, whether non-VHL-associated HIF activation likewise results in the down-regulation of E-cadherin via the activation of SIP1/Snail family of transcriptional repressors is an important question that remains to be resolved.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published ahead of print on 23 October 2006.
Supplemental material for this article may be found at http://mcb.asm.org/.
A.J.E., R.C.R., and O.R. contributed equally to this work.
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-tubulin antibody. E-cadherin signal intensities were quantified using a Kodak Image Station 2000R densitometer and normalized against the corresponding 
