Novel Strategies to Enforce an Epithelial Phenotype in Mesenchymal Cells

Precis: A novel functional assay for E-cadherin expression was used in a genetic screen to identify candidate therapeutic targets to block or reverse EMT as a generalized strategy for treatment of metastatic solid tumors. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.


INTRODUCTION
E-cadherin (CDH1) is a major component of cell-cell junctions in epithelial cells (1). The extracellular domains of E-cadherin connect neighboring cells through Ca 2+ -dependent homotypic interactions, whereas the cytoplasmic domain interacts with components of the adherens junctions, including p120, γ-catenin/plakoglobin and the protooncogene βcatenin (2,3). As an adherens junction component, E-cadherin act as a tumor suppressor not only by contributing to epithelium integrity, thereby preventing metastasis, but also by sequestering β-catenin at the plasma membrane, thereby controlling the mitogenic activity of β-catenin/TCF signaling pathway. E-cadherin expression is tightly regulated at the transcriptional level by repressors (4)(5)(6) and at the post-transcriptional level by phosphorylation, ubiquitination and proteolysis (7,8). Various E-cadherin repressors such as the E-box binding factors Snail, Slug, ZEB1 and ZEB2 and the basic helix-loophelix (bHLH) factor Twist were implicated in E-cadherin regulation and epithelial-tomesenchymal transition during normal development and cancer progression (6).
Recently, microRNAs of the miR-200 family were uncovered as modulators of Ecadherin expression, through regulation of ZEB1 and ZEB2 expression (9,10).
The loss of E-cadherin expression has been extensively documented in cancer metastasis (11,12). Frequently, metastatic tumor cells display decreased E-cadherin expression (13,14) and re-expression of E-cadherin in invasive tumor cell lines reduces their invasive behavior (15,16). Systematic investigations on the regulation of E-cadherin expression have been difficult in the past due to the absence of functional assays amenable to powerful genetic approaches. Here we developed an innovative assay using the E-Research.
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Validation procedure
Cells were transfected by reverse transfection with Dharmafect1 and individual siRNA (D1, D2, D3 and D4, 50 nM final) or a pool of the four silencing reagents (12.5 nM each, 50nM total) and incubated for 72hrs in a 96-well plate format. For real-time PCR analysis, total RNA and first-strand cDNA synthesis was performed using the TaqMan Gene Expression Cells-to-Ct Kit (Applied Biosystems), as recommended by the manufacturer.

DNA constructs
HeLa229 cells were transiently transfected with either wild-type pME18S-FLASH-GFP or the siRNA-resistant form of FLASH. The resistant siRNA form of FLASH was constructed by silent mutagenesis at the siRNA D4-binding sequence site.

Immunofluorescence
For immunofluorescence, cells grown on coverslips were fixed and permeabilized in methanol (at −20°C) for 5 min and stained (at the dilutions shown) for anti-E-cadherin

A functional approach for assessing the level of E-cadherin expression
In a survey for E-cadherin expression in epithelial cells, we found that HT-29 and HeLa229 cells expressed high and low levels of E-cadherin, respectively (Fig. 1A).
Although standard assays such as immuno-fluorescence procedures allowed for the detection of high level of E-cadherin expression in HT-29 cells, similar procedures failed to detect low level of E-cadherin expression in HeLa229 cells (Fig. 1B). To develop a functional assay that reports on differential levels of E-cadherin expression in low  (Fig. 1D, ZEB1 KD 64 % +/-2.7 vs. Mock 14% +/-0.9). We validated these results by using the antibiotic protection assay, a standard approach for measuring the efficiency of L. monocytogenes invasion in mammalian cells (Supplementary Fig. S1). Thus, the combination of the invasive properties of L. monocytogenes, quantitative imaging procedures and the RNAi methodology offers an unprecedented functional approach to investigate the regulatory network that controls E-cadherin expression.

Identification of novel regulators of E-cadherin expression
To identify novel regulators of E-cadherin expression, we used the functional approach presented in Fig. 1 and screened the Dharmacon siRNA library covering the Druggable Human Genome (ThermoFisher). This library harbors pools of four independent siRNA duplexes for a given gene, targeting a total of 7,000 genes. We identified 52 pools whose transfection led to increased infection levels that reproducibly deviated from the mean by at least 2 Standard Deviation (SD) units in three independent experiments. The main challenge in RNAi studies is associated with the unintended silencing of genes displaying limited sequence homology with the targeted gene, a phenomenon referred to as the "offtarget effect" (24). We have previously shown that validation of RNAi screens requires the use of independent silencing reagents in order to establish a functional relationship between the silencing of the targeted gene and the observed phenotype (25). To validate the identified candidate genes, we re-tested individually the four siRNA duplexes that constituted the pools used in the primary screen. We identified 78 duplexes that conferred an increase in L. monocytogenes invasion. These duplexes corresponded to 4 pools displaying the phenotype for three or four of the four siRNA duplexes, 19 pools displaying the phenotype for two of the duplexes tested and 26 pools displaying the phenotype for only one of the four duplexes (Supplementary Table 1 Table 2).

Transcriptional and post-transcriptional regulators of E-cadherin expression
We further tested whether the identified regulators controlled E-cadherin expression at the transcriptional or post-transcriptional level. To this end, we tested whether increased Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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and total protein levels ( Supplementary Fig. S2B, WCL, D1-D4). We obtained similar results for ZEB1, HDAC1, Caspase 7 (CASP7) and polycomb group (PcG) protein PCGF1 ( Supplementary Fig. S3). We also performed surface biotinylation experiments in order to quantify the level of E-cadherin at the plasma membrane. Biotinylated proteins were collected by streptavidin beads pull-down, and subsequent E-cadherin blotting showed an increase in surface E-cadherin in FLASH-depleted cells ( Supplementary Fig.   S2B, IP:streptavidin). Altogether these results not only confirmed the previously reported role of ZEB1 and HDAC1 in E-cadherin regulation (5,6,9,26,27), but also uncovered previously unappreciated roles for FLASH, PCGF1 and CASP7 as transcriptional regulators of E-cadherin expression.
As opposed to the situation observed for the depletion of the transcriptional regulators, depletion of FBXL5 did not alter significantly the levels of E-cadherin mRNA

Depletion of the newly identified E-cadherin regulators affects the anchorageindependent growth of HeLa229 cells
We next studied the impact of silencing the expression of the identified candidate genes on the ability of HeLa229 cells to proliferate independently of both external and internal regulatory cues in a soft agar assay. We silenced the expression of the identified candidate with two independent siRNA duplexes (Fig. 3A). We found that the increase in of E-cadherin expression in cells depleted for CASP8AP2 (FLASH), FBXL5, HDAC1, PCGF1, Caspase 7, Caveolin 2, MMP14 and ZEB1 resulted in a significant suppression of anchorage-independent cell growth in soft agar (Fig. 3B). These results suggest that the increase in E-cadherin expression resulting from the silencing of the identified candidate genes reduces the proliferating capacity of Hela229 cells and support the notion that these newly identified E-cadherin regulators constitute potential targets for therapeutic intervention.

Full validation of FLASH as a regulator of E-cadherin expression
We next conducted a full validation of the specific involvement of FLASH in E-cadherin expression. To this end, we first confirmed that siRNA treatment with the four duplexes targeting FLASH expression led to FLASH depletion at the mRNA as well as the protein level (Fig. 4A). We also ruled out a potential off-target activity for siRNA duplex D4 by taking advantage of the C911 mismatch approach in which bases 9 through 11 of the siRNA duplexes were replaced by their complement (28). We found that transfection of

FLASH regulates ZEB1 expression
As previously shown (5,6,9) and as illustrated in this study ( Fig. 2H and Supplementary   Fig. S2), the transcriptional repressor ZEB1 acts as negative regulator of E-cadherin expression. We tested whether the depletion of FLASH, PCGF1 and CASP7 may lead to an increase in E-cadherin transcription by modulating ZEB1 expression. We found that FLASH depletion, but not CASP7 or PCGF depletion, led to a decrease in ZEB1 protein expression (Fig. 5A, FLASH KD). We next determined whether FLASH depletion affected ZEB1 expression at the transcriptional or post-transcriptional level. We used 4 different siRNA duplexes to silence FLASH in HeLa229 cells and quantitative real-time PCR revealed that ZEB1 mRNA levels were not affected in the FLASH-depleted cells We next carried out co-immuno-precipitation experiments with cytosolic and nuclear extracts of HeLa229 cells to determine whether FLASH and ZEB1 may form a complex.
As expected, ZEB1 and FLASH were largely identified in the nuclear fraction of HeLa229 cells (Fig. 5E, Lysate). Using an anti-FLASH antibody, we showed coimmuno-precipitation of FLASH and ZEB1 with extracts from mock-treated cells, but not with extracts from FLASH-depleted cells (Fig 5E, IP, Mock vs. FLASH KD). As expected, the converse experiments using an anti-ZEB1 antibody also showed coimmuno-precipitation of ZEB1 and FLASH (Fig. 5F) chromatin immuno-precipitation assay with an anti-ZEB1 antibody, as previously described (20,29). Quantitative PCR of the E-cadherin promoter region revealed that FLASH depletion led to a 2.5 decrease in the occupancy of the E-cadherin promoter by ZEB1 (Fig. 5G). Altogether, these results are in agreement with the notion that FLASH modulates E-cadherin expression by regulating ZEB1 protein levels, potentially through formation of a complex, which impacts the occupancy of the E-cadherin promoter and therefore the level of E-cadherin expression.  Table 3). We also compared the gene expression profiles of mock-treated and FLASH-depleted cells. As expected, we found that FLASH depletion increased the expression of the ZEB1-regulated genes, including CDH1 and EPCAM ( Fig. 6A and Supplementary Table 3). In addition, we found that FLASH-depletion led to a > 4-fold increase in the transcription of more than these microarray data by real time PCR (Fig. 6D). These results not only establish the role of FLASH in the regulation of ZEB1-dependent genes, but also uncover a role for FLASH in the negative regulation of ZEB1-independent genes previously associated with tumor suppression in mesenchymal cells.

siRNA duplexes that mimic microRNAs
In addition to the pools that passed our validation procedures for gene specificity, we ZNF281_D1 may regulate E-cadherin expression by mimicking the activity of a yet uncovered human homolog of C. elegans miR-70. We refer to those siRNA duplexes that potentially mimic the activity of endogenous microRNAs as miR-mimics.

Effects of miR-mimics on the epithelial phenotype
We explored the mechanisms supporting the activity of miR-mimics PON3_D2 and ZNF281_D1. Transfection with miR-mimics led to a strong increase in L. monocytogenes infection (Fig. 7A, Infection, D1) paralleled by high levels of E-cadherin mRNA (Fig.   7B, D1), total E-cadherin protein (Fig. 7C, WCL, D1) and an increased availability of Ecadherin protein at the plasma membrane (Fig. 7C, IP:streptavidin, D1). Strong Ecadherin expression was still detected 21 days after a single transfection with 50nM siRNA (Supplementary Fig. S6B) and the lowest effective dose tested that induced Ecadherin expression was 5nM ( Supplementary Fig. S6C). Increased E-cadherin expression ( Supplementary Fig. S7A) was paralleled by the decreased expression of its transcriptional repressors, including ZEB1, ZEB2 and SNAIL ( Supplementary Fig. S7B,   S7C and S7D). To examine the ability of ZNF281_D1 to enforce an epithelial phenotype, we also examined occludin expression, a critical component of tight-junctions involved in the establishment and maintenance of cell polarity (31). In mock-treated Hela229 cells, occludin expression was not detectable (Fig. 7D, Mock). However, occludin expression was restored in miR-mimic-transfected cells (Fig. 7D, PON3_D2 and ZNF281_D1). In addition to the transcriptional up-regulation of epithelial markers, such as E-cadherin ( Supplementary Fig. S7A) and occludin ( Supplementary Fig. S7E), we also observed repression of mesenchymal markers, such as vimentin (Supplementary Fig. S7F). Finally, inverse correlation between CAV2 and E-cadherin expression was found in invasive breast cancer (34). Our results indicate that CAV2 depletion leads to an accumulation of E-cadherin at the plasma membrane, suggesting that CAV2 regulates E-cadherin endocytosis.

Transcriptional regulation of E-cadherin expression
We identified CASP7 and PCGF1 as regulators whose depletion led to an increase in Ecadherin transcription. Given its cytoplasmic localization, it is likely that CASP7 plays an indirect role in E-cadherin transcription. Interestingly, a recent report revealed that CASP3-induced cleavage of δ-catenin generates a fragment that localizes to the nucleus and binds to a zinc finger transcriptional factor (ZIFCAT), potentially modulating its function (35). We speculate that CASP7 might exhibit a similar role and process unknown factor(s) that modulate E-cadherin transcription in the nucleus. In addition to CASP7, we identified the Polycomb repressive complex (PRC) component PCGF1 as a regulator of E-cadherin transcription. PRC components are divided into two subcomplexes (PRC1 and PRC2) which play an important role in embryonic development and carcinogenesis through gene silencing (36,37). A role for PRC2 in E-cadherin repression has been formerly described (38

A role for FLASH in ZEB1-regulated E-cadherin transcription
In addition to CASP7 and PCGF1, we identified FLASH as a transcriptional regulator of E-cadherin expression. The role of FLASH in transcriptional regulation has been formerly described and recent studies support its emerging role as a co-factor in specific transcription processes (41,42). For instance, FLASH was found to act as a co-factor of c-Myb and enhance the expression of c-Myb-dependent genes (43). As our results suggest that FLASH interacts with ZEB1 in the nucleus and FLASH depletion decreases the E-cadherin promoter occupancy by ZEB1, we speculate that FLASH may be a component of the ZEB1-containg repressor complex. Alternatively and since FLASH depletion impacted the protein level of ZEB1, we cannot exclude the possibility that FLASH regulates the stability or the nuclear localization of ZEB1 through unknown mechanisms.

A role for FLASH in the regulation of the mesenchymal phenotype
To further understand the role of ZEB1 and FLASH in the regulation of gene expression in mesenchymal cells, we conducted gene expression profile analyses. In addition to Ecadherin, our results revealed novel genes involved in tumorigenesis whose expression is findings are consistent with data showing a negative correlation between EPCAM and ZEB1 in several cancer cell lines (47,48). The roles of JAM-A, DIRAS3 and MARVELD3 as potential tumor suppressors and promoters of epithelial phenotype have been previously described (49-51). To the best of our knowledge, this is the first report on the negative correlation between ZEB1 and JAM-A, DIRAS3 and MARVELD3.
These results reveal that in addition to E-cadherin, ZEB1 regulates the expression of genes whose products may contribute to the epithelial phenotype.
In agreement with our findings showing that FLASH regulates ZEB1 expression, our gene expression profile analyses showed that FLASH regulates the expression of ZEB1dependent genes. In addition, we uncovered that FLASH regulates the expression of numerous ZEB1-independent genes. These genes not only include histone genes, as previously reported (30, 42), but also numerous genes whose products may contribute to the epithelial phenotype, including DHRS2, DKK1 and TFPI-2. Previous studies showed that mitochondrial Hep27 (DHRS2) is a c-Myb target gene that inhibits Mdm2 and stabilizes p53 (52). Also, a role for DKK1 as a tumor suppressor by Wnt-signaling inhibition was documented (53). Tissue factor pathway inhibitor-2 (TFPI-2) is a matrixassociated serine protease inhibitor, which has been previously described as a tumor suppressor gene in several types of cancer (54, 55). Our results therefore support the notion that FLASH represses the expression of numerous genes that support the epithelial phenotype and therefore is a critical determinant of the mesenchymal phenotype.

Novel strategies for enforcing an epithelial phenotype
Research.
on January 2, 2021. © 2014 American Association for Cancer cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on May 20, 2014; DOI: 10.1158/0008-5472.CAN-  Our genetic investigation led to the identification of several novel regulators of Ecadherin expression that may constitute potential targets in the context of therapeutic intervention aiming at preventing the epithelial-to-mesenchymal transition. In particular, our results revealed that FLASH is a major enforcer of the mesenchymal phenotype whose inhibition effectively restores the expression of numerous genes that support the epithelial phenotype. Similarly, our results revealed that siRNA duplexes such as miRmimics, not only restored very high levels of E-cadherin expression, but also enforced a mesenchymal-to-epithelial transition in mesenchymal cells. In addition to miR-mimic-70 (ZNF281_D1) and miR-mimic-200 (PON3_D2), our genetic screen uncovered other siRNA duplexes displaying very potent properties with respect to E-cadherin expression (Supplementary Table 1). Although, the seed-region of these siRNA did not match the seed-region of any known human miRNAs, these siRNA duplexes may mimic the activity of yet undiscovered, endogenous microRNAs. We note, however, that we cannot exclude the possibility that these siRNA molecules may exert their effects on E-cadherin expression independently of the siRNA and miRNA pathways, through uncovered mechanism(s). Also, we acknowledge that the targets we identified may be cell-specific and various cancer cell lines with low expression of E-cadherin may require depletion of other regulators in order to restore high levels of E-cadherin. Nonetheless, these molecules should be considered as a novel class of compounds that display remarkably potent activities on E-cadherin expression and the enforcement of the epithelial phenotype.   performed for E-cadherin promoter using SYBR-green and Ct values were normalized to β-globin.