ZEB1 limits adenoviral infectability by transcriptionally repressing the coxsackie virus and adenovirus receptor

Abstract

Background: We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9):2088-95) and TGF-β (Cancer Res. 2006 Feb 1;66(3):1648-57) signaling negatively regulate coxsackie virus and adenovirus receptor (CAR) cell-surface expression and adenovirus uptake. In the case of TGF-β, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT), a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration.

Results: Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic) and MDA-MB-231 (breast) human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET) characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-β-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-β-induced mesenchymal phenotype of PANC-1 cells, TGF-β did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-β may inhibit CAR expression by regulating factor(s) that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal CAR promoter is positively regulated through conserved ETS and CRE elements.

Conclusions: This report provides evidence that inhibition of ZEB1 may improve adenovirus uptake of cancer cells that have undergone EMT and for which ZEB1 is necessary to maintain the mesenchymal phenotype. Targeting of ZEB1 may reverse some aspects of EMT including the down-regulation of CAR.

Figure 1

Structure of the CAR promoter. A. Several CXADR (CAR) upstream fragments were PCR-amplified from human genomic DNA and cloned into a firefly (FF) luciferase vector in endogenous constellation, i.e. without vector sequence between the CAR regulatory region, 5'-UTR and the translational start of the luciferase coding sequence. The resulting 5'-deletion series was transfected into PANC-1, MDA-MB-231 and H460 cells, in combination with pRL-SV40 (Promega) encoding renilla (RL) luciferase. Cells were lysed twenty-four hours post transfection, and promoter activities were measured with the Dual-Luciferase^® Reporter Assay System (Promega). Reporter activities are displayed as fold changes of the FF:RL luciferase RLU (relative light unit) ratios relative to the empty vector. B. Alignment of orthologous CAR promoter sequences and identification of conserved putative ETS and CRE sites [40-42,49]. A region in which mouse CAR transcripts are likely initiated is indicated (TSS) [56]. Human CAR transcripts may start at around 150 bp upstream of the translational start ATG [10]. C. Wild-type (WT), ETS and CRE mutant -291/-1 CAR promoter constructs were transfected into PANC-1 and MDA-MB-231 cells. Cell lysis and measurements of promoter activities were conducted as in A. Error bars represent standard deviations (biological triplicates). p < 0.001 (***), p < 0.01 (**) (Student's t-test: 1-tailed, equal variance).

Figure 2

Effect of TGF-β on EMT markers. MDA-MB-231 and PANC-1 cells were stimulated with TGF-β for four days, then assessed for E-cadherin and vimentin expression/localization by immunofluorescence, or were stained with phalloidin conjugated to Texas Red^® to visualize F-actin. Cell surface expression of E-cadherin is a hallmark of an epithelial phenotype. Vimentin intermediate filaments and F-actin are features of mesenchymal cells.

Figure 3

E2 box-binding repressor expression upon TGF-β stimulation. PANC-1 and MDA-MB-231 cells were treated with recombinant human TGF-β1 for four days. mRNA expression of CXADR (CAR) (A), CDH1 (E-cadherin) (B), ZEB2 (SIP1) (C), ZEB1 (D), SNAI1 (Snail) (E), and SNAI2 (Slug) (F) was measured by TaqMan real-time PCR. Expression data were normalized to H3F3A (H3 histone, family 3A) mRNA levels (arbitrary units). A-F. Error bars represent standard deviations (biological triplicates). p < 0.001 (***), p < 0.01 (**), p < 0.05 (*) (Student's t-test: 1-tailed, equal variance). Absence of * indicates p ≥ 0.05, or decrease of E2 box-binding repressor expression upon TGF-β stimulation.

Figure 4

E2 box-dependent repression of the CAR promoter and binding of ZEB1 to CAR promoter oligonucleotides and chromatin. A. PANC-1 cells were transfected with CAR promoter/firefly (FF) luciferase constructs (-291/-1) in combination with pRL-SV40 (Promega) encoding renilla (RL) luciferase, an inducible Myc-ZEB1 expression construct, and a "Tet-OFF" plasmid allowing induction of ZEB1 by absence (-Dox), and repression by addition (+Dox) of doxycycline to the culture medium. Cells were lysed forty-eight hours post transfection, and promoter activities were measured with the Dual-Luciferase^® Reporter Assay System (Promega). Reporter activities are displayed as fold changes of the FF:RL luciferase RLU ratios relative to the empty vector. Error bars represent standard deviations (biological duplicates). B. In PANC-1 cells ectopically expressed Myc-tagged ZEB1 was precipitated with streptavidin-agarose resin and biotinlyated E-cadherin or CAR promoter oligonucleotides, and then subjected to immunoblotting and detection with an anti-Myc tag antibody. C. Mutations at the E2 boxes in the constructs transfected in A, and in the oligonucleotides used to precipitate ZEB1 (B). E-cadherin promoter mutations are described [39]. D. ChIP assay conducted with PANC-1 cells transiently transfeced with Myc-ZEB1 and stimulated with TGF-β. Myc-ZEB1 was precipitated with an anti-Myc Tag antibody. Co-precipitated DNA was amplified with CAR promoter-specific primers flanking E2 boxes 1 and 2. Shown are SYBR Green I real-time PCR data, normalized to input DNA (prior to precipitation with anti-Myc Tag antibody or control IgG). E. SYBR Green real-time PCR demonstrating overexpression of the total ZEB1 levels in the experiment shown in D. Abbreviations: TCL, total cell lysate; Dox, doxycyline; WT, wild-type; Bx 1, E2 box 1; Bx 2, E2 box 2; Bx 1+2, E2 boxes 1+2; Bx1+3, E2 boxes 1+3 ([39]). p < 0.001 (***), p < 0.01 (**), p < 0.05 (*), n.s.: not significant (Student's t-test: 1-tailed, equal variance).

Figure 5

ZEB1 promotes EMT. A-C. Immunoblots. PANC-1 cells were pre-treated with TGF-β1 for two days and then transfected twice (day 0 and day 2) with ZEB1 siRNAs in the continued presence of TGF-β1. Four days after the initial transfection, cells were harvested. A. By up-regulating epithelial proteins such as E-cadherin and CAR, knockdown of ZEB1 antagonizes TGF-β-induced EMT in PANC-1 cells. Similarly, silencing of ZEB1 expression in MDA-MB-231 cells up-regulates E-cadherin and CAR, and down-regulates the mesenchymal marker fibronectin. B. PANC-1 cells were treated with TGF-β1, and harvested at the indicated time-points for analysis of the total protein fractions. C. PANC-1 cells were treated with TGF-β1 and subjected to cell fractionation. Abbreviations: C, TGF-β1 solvent control [4 mM HCl/0.1% (v/w) BSA]; UT, untransfected; Ctrl #1, siControl ON-TARGETplus Non-targeting siRNA #1 (Dharmacon); Ctrl #2, firefly luciferase-targeting siRNA; ZEB1 siRNA #1/#2, ZEB1-targeting siRNAs. Ctrl #2 and ZEB1 siRNA sequences are provided in Additional file 1 (Table S3). Chinese Hamster Ovary cells stably expressing human CAR (CHO+), or vector (CHO-) [9]. Loading controls are shown as β-actin, β-tubulin, GAPDH and PARP signals, with GAPDH as a cytoplasmic, and PARP as a nuclear marker.

Figure 6

ZEB1 as a constitutive repressor of the CAR promoter in PANC-1 cells. Our data supports a model in which ZEB1 constitutively binds to E2 box 1 (Fig. 4B) in the human CAR promoter thereby controlling the basal levels of CAR. TGF-β may further repress the promoter via E2 box 2 and its adjacent Smad-binding element (SBE) by a mechanism likely involving Snail-Smad3/4 [37]. Knockdown of ZEB1 increases CAR expression despite the presence of Snail-Smad3/4. Factors binding to the ETS-CRE region (Fig. 1C), here speculatively indicated as ETS or CREB, strongly induce CAR expression ("+++"), while ZEB1 and Snail-Smad3/4 negatively ("-") regulate the CAR promoter. Net CAR expression indicates overall promoter activity. See text for details.

Figure 7

Up-regulation of cell-surface CAR levels upon ZEB1 knockdown coincides with increased adenoviral infectability. A and B. Cell-surface CAR levels following ZEB1 knockdown were measured by flow cytometry on PANC-1 cells treated with TGF-β1 (A), or on MDA-MB-231 cells (B). Error bars represent standard deviations (biological duplicates). C and D. Ad-GFP uptake following ZEB1 knockdown was determined by flow cytometry measuring GFP levels (C), or by real-time PCR for virus copy number (D). Error bars represent standard deviations. A-D. CAR and GFP signals are expressed as geometric means of the fluorescence signal intensities normalized to the untreated controls (A-C), virus copy numbers as relative adenovirus fiber levels (D). Abbreviations: UT, untransfected; Ctrl #1, siControl ON-TARGETplus Non-targeting siRNA #1 (Dharmacon); Ctrl #2, firefly luciferase-targeting siRNA; ZEB1 siRNA #1/#2, ZEB1-targeting siRNAs. Ctrl #2 and ZEB1 siRNA sequences are provided in Additional file 1 (Table S3). p < 0.001 (***), p < 0.01 (**) (Student's t-test, 1-tailed, equal variance).