The actin cytoskeleton-associated protein zyxin acts as a tumor suppressor in Ewing tumor cells

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Abstract

Changes in cell architecture, essentially linked to profound cytoskeleton rearrangements, are common features accompanying cell transformation. Supporting the involvement of the microfilament network in tumor cell behavior, several actin-binding proteins, including zyxin, a potential regulator of actin polymerization, may play a role in oncogenesis. In this work, we investigate the status of zyxin in Ewing tumors, a family of pediatric malignancies of bone and soft tissues, which are mainly associated with a t(11;22) chromosomal translocation encoding the EWS-FLI1 oncoprotein. We observe that EWS-FLI1-transformed murine fibroblasts, as well as human Ewing tumor-derived SK-N-MC cells, exhibit a complete disruption of their actin cytoskeleton, retaining very few stress fibers, focal adhesions and cell-to-cell contacts. We show that within these cells, zyxin is expressed at very low levels and remains diffusely distributed throughout the cytoplasm, instead of concentrating in actin-rich dynamic structures. We demonstrate that zyxin gene transfer into EWS-FLI1-transformed fibroblasts elicits reconstitution of zyxin-rich focal adhesions and intercellular junctions, dramatic reorganization of the actin cytoskeleton, decreased cell motility, inhibition of anchorage-independent growth and impairment of tumor formation in athymic mice. We observe similar phenotypic changes after zyxin gene transfer in SK-N-MC cells, suggesting that zyxin has tumor suppressor activity in Ewing tumor cells.

Introduction

In noncancer cells, adhesion to the extracellular matrix and to neighboring cells plays a central role in the control of cell survival, growth, differentiation and motility [1], [2], [3]. Upon oncogenic transformation, profound changes occur in cell morphology and the organization of the cytoskeleton, in cell motility and in growth factor- or adhesion-dependent cell proliferation (for a review, see Ref. [4]). Disruption of the actin cytoskeleton and a concomitant reduction in the number of focal adhesions are common features accompanying cell transformation induced by various oncogenes. That the actin cytoskeleton plays a fundamental role in oncogenesis is suggested by the association of anchorage-independent growth and tumorigenicity with the rearrangements of the actin filament network observed in transformed cells [5]. Adhesive interactions involve specialized transmembrane receptors that are linked to the cytoskeleton through junctional plaque proteins (for a review, see Ref. [6]). The synthesis of several actin-binding proteins, including α-actinin, vinculin, tropomyosin and profilin, is down-regulated in transformed cells and overexpressing these proteins in tumor cells suppresses the transformed phenotype, which allows them to be considered as tumor suppressors.

Zyxin, a low abundance phosphoprotein, localizes to focal adhesions, to cell–cell contacts, along actin stress fibers and within the lamellipodia of migrating cells [7], [8], [9]. Zyxin directly interacts with α-actinin and Enabled/vasodilator-activated phosphoprotein (Ena/VASP), which connect it to actin filaments within these multicomponent structures. Anchoring of recombinant zyxin to the plasma membrane alters actin stress fiber organization within the cell. Artificial targeting of zyxin to the mitochondrion external membrane induces actin nucleation and polymerization at the surface of the organelle [10], [11]. ActA, a protein encoded by the pathogenic bacterium Listeria monocytogenes, is highly homologous to zyxin and stimulates actin polymerization in vitro [10]. These observations suggest that zyxin, which colocalizes with actin-rich dynamic structures within the cell, may be implicated in the spatial control of actin filament assembly.

Zyxin expression is also deregulated in some cancer cells. It is overexpressed in acute myeloid leukemias [12], in melanomas [13] and upon fiber-induced carcinogenesis in rats [14]. Its synthesis is decreased in human fibroblasts transformed by simian virus 40, in bladder cancer cell lines and in several primary tumors [15], [16]. Together with the actin cytoskeleton rearrangements commonly observed upon cell transformation, these properties of zyxin prompted us to investigate the status of the protein in a model of pediatric cancer, Ewing tumors, which are the second most common malignancies of bone arising in children and young adults. Ewing tumors are mainly associated with a t(11;22)(q24;q12) chromosomal translocation, encoding the chimerical transcription factor EWS-FLI1 [17], [18]. Ectopic expression of EWS-FLI1 is sufficient to transform immortal murine fibroblasts [19], [20], [21], whereas inhibition of EWS-FLI1 expression in Ewing tumor-derived human cell lines, through antisense strategy or RNA interference, impairs anchorage-independent growth and in vivo tumorigenicity of the cells [22], [23], [24]. We show that zyxin levels are very low in EWS-FLI1-transformed NIH 3T3 fibroblasts and in the Ewing tumor-derived human cell line SK-N-MC, which exhibit a disorganized actin cytoskeleton. We demonstrate that zyxin gene transfer into these cells contributes to the suppression of their transformed phenotype, suggesting that zyxin down-regulation may participate in EWS-FLI1-induced oncogenesis.

Section snippets

Cell culture

The NIH 3T3 cell line (3T3) was purchased from ATCC. The EWS-FLI1-transformed NIH 3T3 cell line (3T3-EF) was a kind gift from J. Ghysdael (Orsay, France). These cells are 3T3 fibroblasts that have been stably transduced by the cDNA encoding the type 1 EWS-FLI1 fusion protein inserted downstream of the Mo-MuLV long terminal repeat in the pBabe-puro retroviral vector. All the 3T3-derived cell lines are cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 UI/ml penicillin,

Zyxin expression is down-regulated in EWS-FLI1-transformed 3T3 fibroblasts

In order to investigate the status of zyxin in EWS-FLI1-transformed cells, we used murine 3T3 fibroblasts that have been transduced with the pBabe-puro retroviral vector designed for expression of the EWS-FLI1-encoding cDNA under the control of the CMV promoter. When compared to the parental 3T3 fibroblasts, EWS-FLI1-expressing cells display a more rounded shape, with fewer cytoplasmic extensions and a reduced spreading area. They establish no stable cell-to-cell contacts. Their morphological

Discussion

A number of indirect observations led us to study the contribution of zyxin to the tumoral phenotype in EWS-FLI1-transformed cells: (i) malignant transformation alters cell morphology, cytoskeletal architecture, adhesion and motility [4]; (ii) zyxin associates with specialized actin structures and is implicated in the regulation of cytoskeleton architecture and dynamics [7], [8], [9]; (iii) zyxin expression is deregulated upon transformation of several cell lines [13], [14], [15]. Because the

Acknowledgments

We thank J. Ghysdael for the gift of the 3T3-EF cell line, F.L. Cosset for the gift of the FlyA13 cell line, M.C. Beckerle for the zyxin cDNA and J. Wehland for the 164D4 anti-zyxin antibody. We thank P. Ardouin and co-workers at the Gustave Roussy Institute for assistance in in vivo tumorigenicity experiments. This work was supported by the Centre National de la Recherche Scientifique, the French Ministry of Education and Research and a grant from the Ligue contre le Cancer to V. Amsellem.

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