WNT7a induces E-cadherin in lung cancer cells.
Journal: 2003/October - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
E-cadherin loss in cancer is associated with de-differentiation, invasion, and metastasis. Drosophila DE-cadherin is regulated by Wnt/beta-catenin signaling, although this has not been demonstrated in mammalian cells. We previously reported that expression of WNT7a, encoded on 3p25, was frequently downregulated in lung cancer, and that loss of E-cadherin or beta-catenin was a poor prognostic feature. Here we show that WNT7a both activates E-cadherin expression via a beta-catenin specific mechanism in lung cancer cells and is involved in a positive feedback loop. Li+, a GSK3 beta inhibitor, led to E-cadherin induction in an inositol-independent manner. Similarly, exposure to mWNT7a specifically induced free beta-catenin and E-cadherin. Among known transcriptional suppressors of E-cadherin, ZEB1 was uniquely correlated with E-cadherin loss in lung cancer cell lines, and its inhibition by RNA interference resulted in E-cadherin induction. Pharmacologic reversal of E-cadherin and WNT7a losses was achieved with Li+, histone deacetylase inhibition, or in some cases only with combined inhibitors. Our findings provide support that E-cadherin induction by WNT/beta-catenin signaling is an evolutionarily conserved pathway operative in lung cancer cells, and that loss of WNT7a expression may be important in lung cancer development or progression by its effects on E-cadherin.
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Proc Natl Acad Sci U S A 100(18): 10429-10434

WNT7a induces E-cadherin in lung cancer cells

+8 authors
Division of Medical Oncology, Departments of Biometrics/Preventive Medicine and Pathology, University of Colorado Health Sciences and Cancer Centers, 4200 East 9th Avenue, Denver, CO 80262; Institut de Biologie Moléculaire et d'Ingénierie Génétique, EA 2224, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers Cédex, France; Laboratoire de Pathologie Cellulaire, Institut National de la Santé et de la Recherche Médicale 9924, Centre Hospitalo-Universitaire Albert Michallon, F-38043 Grenoble, France; Department of Cell and Developmental Biology and Division of Gastroenterology, Department of Genetics, and Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA 19104-6100; Department of Pathology and Center for Reproductive Sciences, Columbia University College of Physicians and Surgeons, New York, NY 10032; and Department of Pathology, Johns Hopkins Medical Center, Baltimore, MD 21231
To whom correspondence should be addressed. E-mail: ude.cshcu@nikbard.yrrah.
Communicated by David M. Prescott, University of Colorado, Boulder, CO, July 3, 2003
Communicated by David M. Prescott, University of Colorado, Boulder, CO, July 3, 2003
Received 2003 Apr 9

Abstract

E-cadherin loss in cancer is associated with de-differentiation, invasion, and metastasis. Drosophila DE-cadherin is regulated by Wnt/β-catenin signaling, although this has not been demonstrated in mammalian cells. We previously reported that expression of WNT7a, encoded on 3p25, was frequently downregulated in lung cancer, and that loss of E-cadherin or β-catenin was a poor prognostic feature. Here we show that WNT7a both activates E-cadherin expression via a β-catenin specific mechanism in lung cancer cells and is involved in a positive feedback loop. Li, a GSK3β inhibitor, led to E-cadherin induction in an inositol-independent manner. Similarly, exposure to mWNT7a specifically induced free β-catenin and E-cadherin. Among known transcriptional suppressors of E-cadherin, ZEB1 was uniquely correlated with E-cadherin loss in lung cancer cell lines, and its inhibition by RNA interference resulted in E-cadherin induction. Pharmacologic reversal of E-cadherin and WNT7a losses was achieved with Li, histone deacetylase inhibition, or in some cases only with combined inhibitors. Our findings provide support that E-cadherin induction by WNT/β-catenin signaling is an evolutionarily conserved pathway operative in lung cancer cells, and that loss of WNT7a expression may be important in lung cancer development or progression by its effects on E-cadherin.

Abstract

Loss of chromosome 3p is one of the earliest and most frequent genetic events in lung cancer (1). Although one predominant 3p tumor suppressor gene has not been identified by mutation analysis, at least five distinct homozygous deletion regions have been described that include several genes with demonstrable relevance to lung cancer development and progression (2-6). Epigenetic silencing appears to be the major mechanism by which the expression of these genes is lost (3, 7).

We previously reported that the expression of WNT7a, encoded at 3p25, was absent or markedly reduced in most lung cancer cell lines and direct tumors (8). We also identified a homozygous deletion of β-catenin, encoded at 3p21, in the mesothelioma cell line, NCI-H28 (8). This deletion was independently confirmed and shown to be confined to the β-catenin gene (9). Signaling through the canonical WNT pathway inhibits phosphorylation of β-catenin by GSK3β thereby preventing its proteasome-mediated destruction (10). In turn, β-catenin binds and activates TCF/LEF transcription factors (11). More recently, β-catenin has been shown to activate transcription factors other than TCF/LEF, including the retinoic acid and vitamin D receptors, which promote differentiation (12, 13), and the androgen receptor (14, 15). Each appears capable of competing with TCF/LEF for β-catenin binding. Thus, WNT/β-catenin signaling may have very different consequences depending on the cellular context.

β-catenin has a second role linking E-cadherin to the actin cytoskeleton (10). Loss of E-cadherin induces an epithelialmesenchymal transition with increased tumorigenicity (16). We used a lung tumor microarray to report that loss of E-cadherin or β-catenin had a severe effect on patient survival (17). In a multivariate analysis, E-cadherin loss remained significant. Other than methylation, mechanisms regulating E-cadherin expression in lung cancer have not been thoroughly investigated. Although in Drosophila the E-cadherin homologue is known to be a Wnt target gene (18), this has not been observed in colon cancer cells where many of the known WNT targets have been identified. E-cadherin can be downregulated in epithelial cells by transcription factors that bind E-box elements in its promoter. Four such factors have been identified: Snail, Slug, ZEB, and E12/E47 (19-23). At least for Snail and ZEB, repression involves CtBP binding, which in turn recruits histone deacetylases (HDACs) leading to chromatin inaccessibility (24). Which factors predominate in lung cancer is unknown.

We were intrigued by the loss in lung tumors of two 3p-encoded WNT pathway genes and wondered whether WNT signaling could affect E-cadherin expression. Although Wnt7a up-regulates β-catenin in some contexts (25-27), during limb development Wnt7a signals through a non-β-catenin pathway (28). Our results demonstrate that apparent physiologic levels of WNT7a positively regulate E-cadherin expression in lung cancer cells via β-catenin. Moreover, downregulation of E-cadherin was uniquely correlated with ZEB1 expression and could be reversed by RNA interference against ZEB1. Pharmacologic reversal of E-cadherin and WNT7a losses could be achieved with the use of inhibitors of GSK3β, HDACs, or in some cases only with combined treatment. These findings have potential implications for lung cancer treatment and provide support for the hypothesis that E-cadherin regulation by WNT/β-catenin signaling is evolutionarily conserved.

Real-time RT-PCR analysis of E-cadherin, WNT7a, and WNT3a mRNAs in H661 cells at 6, 12, and 18 h after activated (S37A) β-catenin adenovirus, low-dose (41 nM) TSA, or treatment with both. The Ct values were normalized to GAPDH and 18S RNA with identical results. Fold induction over untreated control cells was calculated by using the estimate that every PCR cycle difference, during exponential amplification, represents a 2-fold change in mRNA levels.

Normalized real-time quantitative RT-PCR data were generated from 10 NSCLC and 3 mesothelioma lines for the indicated genes (see Methods). Relative E-cadherin protein expression levels were determined by Western blot and densitometric scanning. Correlations were evaluated by using the Spearman statistic (upper number) and significance (P, lower number). Significant correlations are shown in bold.

H661 cells were mock-transfected, transfected with the indicated siRNA as described in the text, or treated with siRNA and either TSA or Li. The Ct values were normalized to GAPDH, and the fold induction was calculated as in Table 1.

Acknowledgments

We thank Drs. C. Korch and P. Bunn for their comments, J. Jacobsen for tissue culture, and the University of Colorado Cancer Center DNA Sequencing Core (supported by National Institutes of Health Grant CA46934). This work was supported by Lung Cancer Specialized Program of Research Excellence Grant CA58187 and Early Detection Research Network Grant CA85070. J.R. and S.K. were supported by Association pour la Recherche sur le Cancer and Ligue Nationale Contre le Cancer, comités de la Vienne et de la Charente.

Acknowledgments

Notes

Abbreviations: NSCLC, non-small-cell lung cancer; TSA, trichostatin A; Ct values, cycle threshold values; IMPase, inositol monophosphatase; HDAC, histone deacetylase; siRNA, small interfering RNA.

Notes
Abbreviations: NSCLC, non-small-cell lung cancer; TSA, trichostatin A; Ct values, cycle threshold values; IMPase, inositol monophosphatase; HDAC, histone deacetylase; siRNA, small interfering RNA.

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