Hypoxia-Inducible Factor-1-Dependent Repression of E-Cadherin in Von Hippel-Lindau Tumor Suppressor–Null Renal Cell Carcinoma Mediated by TCF3, ZFHX1A, and ZFHX1B

Research Article

Hypoxia-Inducible Factor-1-Dependent Repression of E-cadherin in von Hippel-Lindau Tumor Suppressor–Null Renal Cell Carcinoma Mediated by TCF3, ZFHX1A, and ZFHX1B

Balaji Krishnamachary,1 David Zagzag,2 Hideko Nagasawa,1 Karin Rainey,1 Hiroaki Okuyama,1 Jin H. Baek,1 and Gregg L. Semenza1

1Vascular Biology Program, Institute for Cell Engineering; Departments of Pediatrics, Medicine, Oncology, and Radiation Oncology; and McKusick-Nathans Institute for Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland; and 2Microvascular and Molecular Neuro-Oncology Laboratory, Departments of Pathology and Neurosurgery, New York University Cancer Institute of New York University School of Medicine, New York, New York

Abstract the epithelial-mesenchymal transition (10–16). Increased expres- A critical event in the pathogenesis of invasive and metastatic sion of one or more of these repressors has been correlated with cancer is E-cadherin loss of function. Renal clear cell decreased CDH1 mRNA expression in human cancers, but the carcinoma (RCC) is characterized by loss of function of molecular basis for these observations has not been determined. the von Hippel-Lindau tumor suppressor (VHL), which nega- The conventional clear cell type of renal cell carcinoma (RCC) is tively regulates hypoxia-inducible factor-1 (HIF-1). Loss of associated with loss of function of the von Hippel-Lindau tumor suppressor (VHL) protein as a result of VHL gene mutation or E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of hypermethylation (17). A principal function of VHL is to negatively VHL, a dominant-negative HIF-1A mutant, or a short hairpin regulate hypoxia-inducible factor-1 (HIF-1), a transcriptional RNA directed against HIF-1A. In human RCC biopsies, expres- activator of genes that play key roles in angiogenesis, cell sion of E-cadherin and HIF-1A was mutually exclusive. The proliferation/survival, energy metabolism, invasion, and resistance to radiation and chemotherapy (18–23). The constitutively expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, h which repress E-cadherin gene transcription, was increased expressed HIF-1 subunit forms functional heterodimers with a a a a in VHL-null RCC4 cells in a HIF-1–dependent manner. Thus, either HIF-1 or HIF-2 (24, 25). VHL binds to HIF-1 and HIF-2 HIF-1 contributes to the epithelial-mesenchymal transition and targets them for ubiquitination and proteasomal degradation in VHL-null RCC by indirect repression of E-cadherin. (Cancer (26). VHL binding is dependent upon the O2-dependent hydroxylation of specific proline residues in HIF-1a and HIF-2a (27, 28). Res 2006; 66(5): 2725-31) VHL loss of function leading to HIF-1 gain of function is the earliest detectable molecular event in the pathogenesis of RCC (29). Introduction However, the molecular basis for the epithelial-mesenchymal Epithelial cell-cell adhesion in humans and other mammals is transition in RCC has not been determined, and the identification mediated by intercellular junctional complexes consisting of tight of proteins regulated by VHL in addition to HIF-1 has raised the junctions, adherens junctions, and desmosomes. E-cadherin, which possibility that key steps in RCC pathogenesis are independent of is the principal component of adherens junctions and desmosomes HIF-1 (17). In this study, we investigated whether dysregulated HIF- in epithelial cells, mediates adhesion by homophilic interactions 1 activity contributes to down-regulation of E-cadherin expression, between cells (1). A defining step in the pathogenesis of carcinomas loss of cell-cell adhesion, and the epithelial-mesenchymal transi- is the epithelial-mesenchymal transition, during which E-cadherin– tion in RCC. mediated cell-cell adhesion is lost, and cells acquire invasive and metastatic properties (2, 3). In addition to its direct effects on Materials and Methods cell-cell adhesion, E-cadherin loss of function in cancer cells also Construction of enhanced green fluorescent protein and HIF-1ADN activates signal transduction pathways that promote proliferation, retroviral shuttle vectors. Enhanced green fluorescent protein (EGFP) invasion, and metastasis (1). sequences were amplified using pEGFP-N (Clontech, Palo Alto, CA) as E-cadherin loss of function occurs via a variety of molecular template and inserted into retroviral vector pQCXIH (Clontech). The open mechanisms in different human cancers (4), including mutations reading frame from pCEP-4-HIF-1aDN (30) was amplified by PCR and in the CDH1 gene that encodes E-cadherin (5, 6), increased inserted into retroviral vector pQCXIN (Clontech). E-cadherin degradation (7), and decreased CDH1 mRNA expression Retrovirus production and generation of stable cell lines. 293-T cells associated with CDH1 hypermethylation indicating transcriptional were seeded at 7 Â 106 per 10-cm dish. Retroviral vector DNA (12 Ag) silencing (8, 9). The repressors SLUG, SNAIL, TCF3 (also known as encoding EGFP or HIF-1aDN was cotransfected with plasmids encoding E12/E47), ZFHX1A (also known as yEF1 or ZEB1), and ZFHX1B gag/pol (6 Ag) and VSVG (1.5 Ag) using LipofectAMINE 2000. Viral (also known as SIP1 or ZEB2) have been shown to bind to the supernatant was collected 48 hours after transfection and added to RCC4 cells in the presence of polybrene (8 Ag/mL; Sigma, St. Louis, MO). The proximal promoter of CDH1, repress its transcription, and induce procedure was repeated at 60 and 72 hours after transfection. Hygromycin (400 Ag/mL) or G418 (800 Ag/mL) was added to the culture media, starting at 96 hours after transfection of pQCXIH-EGFP or pQCXIN-HIF-1aDN, Requests for reprints: Gregg L. Semenza, Johns Hopkins University School of respectively. Pooled clones of resistant cells were cultured in DMEM Medicine, Broadway Research Building, 733 North Broadway, Suite 671, Baltimore, MD (Mediatech, Herndon, VA) with 10% fetal bovine serum (FBS; Gemini, 21205. Fax: 443-287-5618; E-mail: [email protected]. I2006 American Association for Cancer Research. Calabasas, CA) and 1% penicillin/streptomycin starting 24 hours before all doi:10.1158/0008-5472.CAN-05-3719 studies. www.aacrjournals.org 2725 Cancer Res 2006; 66: (5). March 1, 2006

RNA isolation and quantitative real-time reverse-transcription PCR. mouse (goat anti-mouse, Santa Cruz Biotechnology, Santa Cruz, CA) or Total RNA was isolated from cells using QIAshredder and RNeasy Mini Kits rabbit (donkey anti-rabbit, Amersham, Arlington Heights, IL) immunoglob- (Qiagen, Chatsworth, CA). cDNA was prepared using the iScript cDNA ulin was used for detection. h-Actin was detected using a goat polyclonal Synthesis kit (Bio-Rad, Richmond, CA). cDNA samples were diluted 1:15, antibody against h-actin and HRP-conjugated donkey anti-goat secondary and real-time PCR was performed using IQ SYBR Green Supermix and antibody (Santa Cruz Biotechnology). Immunoblots were developed using specific primers in the iCycler Real-time PCR detection system (Bio-Rad). enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ). Primers were designed using Beacon Designer 2.1 software (sequences Transepithelial resistance assays. In a modified Boyden chamber (BD available on request). Annealing temperature was optimized for each primer Biosciences), 1 Â 104 cells resuspended in 2.5 mL of media were added to pair. The expression of each target mRNA relative to 18S rRNA (R) was the upper chamber, and 3.5 mL of media were added to the lower chamber, ÀD(DCt) calculated based on the threshold cycle (Ct)asR =2 , where DCt = Ct, in triplicate. Resistance was measured using an epithelial voltohmmeter target À Ct, 18s and D(DCt)=DCt, RCC4 À DCt, RCC4-VHL or DCt, RCC4-GFP À DCt, (EVOM, World Precision Instruments, Sarasota, FL). A blank with no cells RCC4-DN or DCt, GFP-EV À DCt, GFP-shr2265. was maintained, and the average resistance from the blank was subtracted Immunoblot assays. Cells were lysed in radioimmunoprecipitation from the sample. Resistance (R) per unit area was calculated as (Rsample À 2 assay buffer supplemented with protease inhibitors; 60-Ag aliquots of Rblank) Â pd /4, where d = diameter (in cm) of the chamber. protein were resolved by 7% PAGE and transferred to a nitrocellulose Immunocytochemistry. Cells (5 Â 103 per well) were seeded into an membrane. A monoclonal antibody (mAb) directed against the extracellular eight-well chamber slide precoated with poly-D-lysine. Cells were fixed with domain (clone HECD1, Zymed Laboratories, South San Francisco, CA; 4% paraformaldehyde for 10 minutes and washed with 0.5% bovine serum 1:3,000 dilution) or intracellular domain (clone 36, BD Biosciences, San Jose, albumin (BSA) in PBS. Blocking was carried out at room temperature for 30 CA; 1:3,000 dilution) of E-cadherin was used. Immunoblot assays of HIF-1a minutes using normal goat serum. Upon permeabilization with 0.1% Triton (mAb H1a67) and HIF-2a (rabbit polyclonal antibody, NB-100-122, Novus X-100 in 0.5% BSA/PBS, cells were incubated with primary antibody against Biologicals, Littleton, CO) were performed using 100-Ag aliquots of lysate. E-cadherin (clone 36, BD Biosciences) overnight. Cells were incubated with Horseradish peroxidase (HRP)–conjugated secondary antibody against phycoerythrin-conjugated secondary antibody (BD Biosciences) at 1:500

Figure 1. VHL and HIF-1 regulate E-cadherin expression in RCC4 cells. A, immunoblot analysis of whole-cell lysates prepared from VHL-null RCC4 cells and from a subclone transfected with an expression vector encoding VHL (RCC4-VHL) using antibodies that recognize the intracellular (E-cadICD ) or extracellular (E-cadECD ) domain of E-cadherin or other proteins. B, quantitative real-time reverse transcription-PCR analysis of E-cadherin and VEGF mRNA levels in RCC4 and RCC4-VHL cells. The levels of E-cadherin and VEGF mRNA in RCC4-VHL cells were quantified relative to the levels in RCC4 cells. C, immunoblot analysis of cell lysates prepared from RCC4 subclones that express GFP (RCC4-GFP) or a dominant-negative form of HIF-1a (RCC4-DN). D, quantitative real-time reverse transcription-PCR analysis of E-cadherin and VEGF mRNA levels in RCC4-DN cells relative to RCC4-GFP cells.

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Figure 2. Immunocytochemical analysis of E-cadherin expression in RCC4 subclones. RCC4 cells and subclones expressing VHL (RCC4-VHL), GFP (RCC4-GFP), or a dominant-negative form of HIF-1a (RCC4-DN) were analyzed by indirect immunofluorescence using an anti–E-cadherin antibody. Nuclei were counterstained with 4V,6-diamidino-2-phenylindole (DAPI). Bar, 200 Am. dilution for 1 hour at room temperature. Before mounting the slide, 4V,6- E-cadherin antibody was diluted 1:100 in PBS, applied, and incubated diamidino-2-phenylindole (Molecular Probes, Eugene, OR) was added to 300 overnight at room temperature. Primary antibody was detected using nmol/L. Phycoerythrin-conjugated IgG2A (R&D Systems, Inc., Minneapolis, biotinylated goat anti-mouse antibody followed by streptavidin-HRP MN) was used as an isotype-matched control. The sections were photo- conjugate and visualized with 3,3-diaminobenzidene enhanced with copper graphed under fluorescent microscopy (Olympus BX51 mounted with sulfate. Slides were washed in distilled water, counterstained with Olympus DP70 camera). hematoxylin, dehydrated, and mounted. Retroviral expression of short hairpin RNA in RCC4 cells. Short Statistical analysis. Student’s t test and ANOVA were performed using hairpin RNA (shRNA) specific for HIF-1a was identified using the Excel (Microsoft, Redmond, WA). manufacturer’s design tool (OligoEngine, Seattle, WA). Oligonucleotides meeting the standards was annealed and cloned into BglII- and HindIII- digested pSUPER-Retro-neo-GFP (OligoEngine). In this vector, shRNA and Results and Discussion GFP mRNA expression are driven by H1 and phosphoglycerate kinase Expression of E-cadherin in RCC4 cells is positively promoter sequences, respectively. Retrovirus was generated by cotransfect- regulated by VHL. To investigate the effect of VHL loss of ing 293T cells with the shRNA vector and a second vector encoding gag/pol function on E-cadherin expression in clear cell renal carcinoma, and VSVG using LipofectAMINE 2000 (Invitrogen, San Diego, CA). The we performed immunoblot assays on VHL-null RCC4 cells and a supernatant was collected 48 hours after transfection, centrifuged, and subclone transfected with an expression vector encoding VHL directly used for infection of 1 Â 105 RCC4 cells per well in a six-well plate, a using 2 mL of viral supernatant and polybrene at a final concentration of (RCC4-VHL). RCC4 cells expressed high levels of HIF-1 and HIF- 8 Ag/mL. Three rounds of retroviral infection were performed at 24-hour 2a protein under nonhypoxic conditions, as previously described intervals. A pool of cells was selected by treatment with G418 at a (32), showing loss of physiologic regulation in the absence of VHL, concentration of 800 Ag/mL. Cells were maintained in DMEM containing which was corrected by restoration of VHL expression (Fig. 1A). In 10% FBS, 1% penicillin/streptomycin, and 600 Ag/mL G418. contrast, levels of the HIF-1h subunit and h-actin were unaffected Immunohistochemistry. Formalin-fixed, 6-Am-thick sections were by the presence or absence of VHL. E-cadherin protein was not stained for HIF-1a using Catalyzed Signal Amplification (DAKO, Carpin- detected in lysates of VHL-null RCC4 cells using antibodies teria, CA) and mAb H1a67 (1 mg/mL, Novus Biologicals; 1:10,000 dilution) directed against either the extracellular domain, which is the site as previously described (31). Nuclei were counterstained with hematoxylin. of homophilic interactions, or the intracellular domain, which As a negative control, nonimmune serum was used in lieu of the primary interacts with catenins that link E-cadherin to the cytoskeleton. antibody. Immunohistochemistry for E-cadherin was performed using mouse anti-human E-cadherin clone 4A2C7 (Invitrogen). Epitope retrieval Upon restoration of VHL expression, increased levels of E-cadherin was performed in 10 mmol/L citrate buffer (pH 6) for 20 minutes in a 1,200- protein were detected with both antibodies (Fig. 1A). Immunocy- W microwave oven at 90% power. Antibody incubation and detection were tochemistry confirmed the low levels of E-cadherin protein in carried out on a NEXes instrument (Ventana Medical Systems, Tucson, AZ). RCC4 cells and the markedly increased E-cadherin levels in RCC4- Endogenous peroxidase activity was blocked with hydrogen peroxide. VHL cells (Fig. 2). www.aacrjournals.org 2727 Cancer Res 2006; 66: (5). March 1, 2006

Figure 3. Transepithelial resistance in RCC4 subclones. A, RCC4 and RCC4-VHL cells were cultured for 6 days, and transepithelial resistance was measured daily. Points, mean for three independent cultures; bars, SE. Differences between paired samples were statistically significant (P < 0.0001, ANOVA). B, to analyze cell proliferation, 2 Â 104 cells per well were inoculated in six-well dishes on day 0, and the number of viable cells were determined on days 1 to 3 by trypan blue exclusion. C, transepithelial resistance in RCC4-GFP and RCC4-DN cells was determined as described above. The differences between subclones were statistically significant (P < 0.0001, ANOVA). D, proliferation of RCC4-GFP and RCC4-DN cells was analyzed as described above.

To determine whether the dysregulation of E-cadherin protein expression of E-cadherin mRNA relative to RCC4-GFP cells reflected changes in steady-state mRNA levels, quantitative real- (Fig. 1D). Increased E-cadherin protein expression in RCC4-DN cells time reverse transcription-PCR was performed. A 27-fold decrease relative to RCC-GFP cells was also shown by immunocytochemistry in E-cadherin mRNA expression in RCC4 cells relative to RCC4- (Fig. 2). Thus, in RCC4 cells, the loss of E-cadherin expression that VHL cells was shown (Fig. 1B). In contrast, vascular endothelial is associated with VHL loss of function is mediated by HIF-1. growth factor (VEGF) mRNA expression was increased 5.5-fold in Evidence of epithelial-mesenchymal transformation of RCC4 cells compared with RCC4-VHL cells, which is consistent VHL-null RCC4 cells. To investigate the functional consequences with previous reports (32). Thus, VEGF expression is induced, associated with the presence or absence of E-cadherin expression whereas E-cadherin expression is repressed in RCC4 cells with in RCC4 cells, we analyzed transepithelial resistance as a measure VHL loss of function. of cell-cell adhesion in RCC4 and RCC4-VHL cells over 6 days Expression of E-cadherin in RCC4 cells is negatively in culture. VHL loss of function was associated with a highly regulated by HIF-1. To determine whether the dysregulated significant reduction in transepithelial resistance at all time points expression of HIF-1a and HIF-2a in RCC4 cells contributed to (Fig. 3A). Comparison of RCC4-GFP and RCC4-DN cells revealed the loss of E-cadherin expression, the RCC4-DN subclone was that expression of the dominant-negative form of HIF-1a, which established by stable transfection of RCC4 cells with a retroviral was associated with a derepression of E-cadherin expression, also expression vector encoding a dominant-negative form of HIF-1a resulted in an increase in transepithelial resistance at all time that competes with endogenous HIF-1a and HIF-2a for binding to points relative to RCC4-GFP cells (Fig. 3C). The rate of RCC4 and HIF-1h, resulting in the formation of HIF-1aDN:HIF-1h hetero- RCC4-GFP cell proliferation was increased compared with dimers that cannot bind to DNA or activate transcription (30). RCC4-VHL and RCC4-DN, respectively (Fig. 3B and D). Thus, the In addition, the dominant-negative form of HIF-1a unexpectedly transepithelial resistance measurements, which were not corrected inhibited HIF-2a protein expression (Fig. 1C). RCC4-DN cells for cell number, understate the differences between subclones. showed increased expression of E-cadherin protein relative to From these results, we conclude that VHL loss of function in RCC4 RCC4-GFP cells, which were stably transfected with a retroviral cells is associated with a HIF-1–dependent loss of transepithelial expression vector encoding GFP. resistance, which is consistent with a loss of adherens junctions Loss of HIF-1 activity was associated with 4.2-fold decreased due to reduced E-cadherin expression. VEGF mRNA expression in RCC4-DN cells compared with RCC- HIF-1A is specifically required for E-cadherin repression in GFP cells (Fig. 1D). RCC4-DN cells showed 9.8-fold increased RCC4 cells. HIF-1aDN competes with both HIF-1a and HIF-2a for

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. HIF-1–Dependent Repression of E-cadherin dimerization with HIF-1h. HIF-1 heterodimers containing HIF-1a GFP-shr2265 cells compared with RCC4-GFP-EV cells (Fig. 4D). or HIF-2a have an overlapping but distinct set of transcriptional Thus, interference with HIF-1a expression in RCC4 cells is targets (32, 33). To determine whether HIF-1a was specifically sufficient for derepression of E-cadherin and increased cell-cell required for the loss of E-cadherin expression in VHL-null RCC4 adhesion. cells, we established by retroviral infection stable RCC4 subclones Mutually exclusive expression of HIF-1A and E-cadherin in expressing GFP alone (RCC4-GFP-EV) or GFP and a shRNA (RCC4- human RCC biopsies. To investigate whether the relationship GFP-shr2265), which modestly reduced the levels of HIF-1a protein between VHL, HIF-1a, and E-cadherin that we have shown in RCC4 (Fig. 4A) and mRNA (Fig. 4B) and the levels of VEGF mRNA. cells is clinically relevant, we analyzed E-cadherin expression by Despite the modest level of inhibition, E-cadherin protein and immunohistochemistry. In sections of normal kidney, E-cadherin mRNA levels were increased significantly in RCC4-GFP-shr2265 was specifically detected within the distal convoluted tubules compared with RCC4-GFP-EV cells. Immunocytochemistry but not in proximal tubules (Fig. 5, top). Next, 13 RCC biopsies were revealed that compared with RCC4-GFP-EV cells, E-cadherin analyzed. Eleven of these biopsies were classic clear cell renal expression in RCC4-GFP-shr2265 cells was markedly increased carcinomas, in which HIF-1a was strongly expressed in the nuclei (Fig. 4C). The increase in E-cadherin expression was associated of all tumor cells, and E-cadherin expression was not detected with a significant increase in the transepithelial resistance of RCC4- (Fig. 5, middle). These data are difficult to interpret because it is

Figure 4. Effect of HIF-1a loss of function in RCC4 cells. A, immunoblot analysis of RCC4 subclones stably transfected with a retrovirus encoding GFP alone (GFP-EV) or GFP and a shRNA that interferes with HIF-1a mRNA expression (GFP-shr2265). B, the levels of HIF-1a, VEGF, and E-cadherin mRNAs in GFP-shr2265 compared with GFP-EV cells was determined by quantitative real-time reverse transcription-PCR. C, immunocytochemical analysis of E-cadherin expression in GFP-EV and GFP-shr2265 subclones of RCC4 cells. Indirect immunofluorescence was performed using an anti–E-cadherin antibody, and nuclei were stained with 4V,6-diamidino- 2-phenylindole (DAPI). D, transepithelial resistance in GFP-EV and GFP-shr2265 subclones of RCC4 cells. Differences between subclones were statistically significant (P < 0.0001, ANOVA).

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not possible to determine whether the tumors arose from proximal or distal tubules. The most informative cases were two RCCs with mixed clear cell and granular morphology, both of which manifested the following pattern of HIF-1a and E-cadherin expression. In the areas of clear cell morphology, nuclear HIF-1a overexpression was detected, and E-cadherin expression was absent (Fig. 5, bottom). In contrast, the contiguous areas of granular morphology lacked detectable HIF-1a expression and manifested strong E-cadherin expression. Thus, within individual human RCC biopsies, expression of HIF-1a and E-cadherin was mutually exclusive. HIF-1–mediated induction of repressors of CDH1 gene transcription in RCC4 cells. To investigate the mechanism by which increased HIF-1 activity in VHL-null RCC4 cells led to reduced E-cadherin mRNA and protein expression, we determined the levels of mRNAs encoding known transcriptional repressors of the CDH1 gene, which encodes E-cadherin. Analysis of mRNA expression by quantitative RT-PCR revealed increased levels of TCF3 (E12/E47), ZFHX1A (yEF1/ZEB1), and ZFHX1B (SIP1/ZEB2) mRNAs in RCC4 compared with RCC4-VHL cells (Fig. 6A, top) and in RCC4-GFP compared with RCC4-DN cells (Fig. 6A, bottom). Among these three repressors, ZFHX1B showed the greatest loss of expression in response to VHL or HIF-1aDN and was the only repressor whose expression was decreased in response to shr2265 (data not shown), which may reflect the more modest reduction in HIF-1 transcriptional activity achieved in RCC4-GFP-shr2265 cells Figure 5. Immunohistochemical analysis of E-cadherin and HIF-1a protein compared with RCC4-VHL or RCC4-DN cells. expression in human RCC biopsies. Sections of normal kidney (top) and RCC Taken together, our data support a model in which dysregulated (middle and bottom) were stained with a mAb against E-cadherin (Ecad)or HIF-1a. In normal kidney, E-cadherin was expressed in distal convoluted tubules HIF-1 activity resulting from VHL loss of function in RCC4 cells (arrowheads) but not in proximal tubules (arrows). Control, primary antibody induces the expression of multiple known repressors of CDH1 gene was omitted. In serial sections from a RCC (middle), HIF-1a expression is transcription, leading to a dramatic loss of E-cadherin mRNA and detected in tumor cell nuclei, whereas E-cadherin expression is not detected. In serial sections from a RCC (bottom), HIF-1a expression is detected in the protein expression and decreased cell-cell adhesion (Fig. 6B). nuclei of tumor cells with clear cell morphology (arrows) located in the upper left Whereas E-cadherin mRNA is repressed in RCC4 cells in a HIF-1– quadrant of the field, whereas E-cadherin expression is detected within tumor cells with granular morphology (arrows) located in the lower right quadrant that dependent manner, exposure of RCC4-VHL cells to hypoxia do not express detectable levels of HIF-1a. paradoxically represses TCF3, ZFX1A, and ZFX1B mRNA and

Figure 6. VHL and HIF-1 regulate TCF3, ZFHX1A, and ZFHX1B mRNA expression in RCC4 subclones. A, the expression of TCF3, ZFHX1A, and ZFHX1B mRNAs in RCC4-VHL relative to RCC4 cells (top) or RCC4-DN relative to RCC4-GFP cells (bottom) was determined by quantitative real-time reverse transcription-PCR. B, molecular basis for loss of cell-cell adhesion in VHL-null RCC4 cells. HIF-1 induces increased expression of the mRNAs encoding ZFHX1A, ZFHX1B, and TCF3, which are transcriptional repressors of the CDH1 gene encoding E-cadherin, which is the principal determinant of epithelial cell-cell adhesion.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. HIF-1–Dependent Repression of E-cadherin induces E-cadherin mRNA expression in a HIF-1–independent may not also occur in some cases of RCC. In support of manner (data not shown), thus indicating that there is considerable this hypothesis, the VHL-null RCC line 786-0 does not express complexity underlying the regulated expression of E-cadherin and E-cadherin after VHL rescue (data not shown). Further studies are its repressors. required to determine the extent to which this mechanism is The demonstration of a reciprocal relationship between HIF-1a responsible for loss of E-cadherin expression in other human and E-cadherin expression in human RCC biopsies suggests cancers. that the mechanism deduced from molecular analysis of RCC4 subclones may have clinical relevance. VHL loss of function Acknowledgments leading to HIF-1 gain of function is the earliest detectable molecular event in the pathogenesis of RCC (29), but the key HIF- Received 10/13/2005; revised 12/14/2005; accepted 12/29/2005. Grant support: NIH/USPHS grant P50-CA103175. 1–mediated changes in gene expression that induce RCC The costs of publication of this article were defrayed in part by the payment of page transformation were previously undetermined. Our data implicate charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. HIF-1–mediated loss of E-cadherin expression as a critical event We thank Linzhao Cheng (Johns Hopkins University, Baltimore, MD) for providing underlying the epithelial-to-mesenchymal transition in RCC. retroviral packaging vectors and advice on viral production: Luis Chiriboga (New York However, as described in the Introduction, multiple mechanisms University, New York, NY) for technical assistance, Garry Cutting (Johns Hopkins University) for use of his voltohmmeter, Karen Padgett (Novus Biologicals, Littleton, resulting in E-cadherin loss of function have been reported in CO) for providing anti-HIF-2a antibodies, and Celeste Simon (University of other cancers, and there is no reason to suppose that these Pennsylvania, Philadelphia, PA) for providing RCC4 and RCC4-VHL cells.

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www.aacrjournals.org 2731 Cancer Res 2006; 66: (5). March 1, 2006

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. Hypoxia-Inducible Factor-1-Dependent Repression of E-cadherin in von Hippel-Lindau Tumor Suppressor −Null Renal Cell Carcinoma Mediated by TCF3, ZFHX1A, and ZFHX1B

Balaji Krishnamachary, David Zagzag, Hideko Nagasawa, et al.

Cancer Res 2006;66:2725-2731.

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