Proteomic screening identifies the zonula occludens protein ZO-1 as a new partner for ADAM12 in invadopodia-like structures.

Bassil Dekky,Michael Ruff,Vincent Legagneux,Nathalie Théret,Dominique Bonnier

doi:10.18632/oncotarget.25106

Abstract

The epithelial mesenchymal transition (EMT) is a key process for cancer cell invasion and migration. This complex program whereby epithelial tumor cells loose polarity and acquire mesenchymal phenotype is driven by the regulation of cell-cell adhesion and cell-substrate interactions. We recently described the association of ADAM12 with EMT and we now use immunoprecipitation and proteomic approaches to identify interacting partners for ADAM12 during EMT. We identify twenty proteins that are involved in molecular mechanisms associated with adhesion/invasion processes. Integrative network analyses point out the zonula occludens protein ZO-1, as a new potential partner for ADAM12. In silico screening demonstrates that ZO-1 and ADAM12 are coexpressed in breast cancer cell lines sharing EMT signature. We validate the interaction between ZO-1 and ADAM12 in invasive breast cancer cell lines and show that ZO-1 and ADAM12 co-localize in actin- and cortactin-rich structures. Silencing either ADAM12 or ZO-1 inhibits gelatin degradation demonstrating that both proteins are required for matrix degradation. We further show that matrix metalloprotease 14, known to mediate degradation of collagen in invadopodia-like structures interacts with ZO-1. Depletion of PKCε that regulates the recruitment of ADAM12 and ZO-1 to cell membranes induces a decrease in ADAM12 and ZO-1 at invadopodia-like structures and degradation activity. Together our data provide evidence for a new interaction between ADAM12, a mesenchymal marker induced during TGF-β-dependent EMT and ZO-1, a scaffolding protein expressed in tight junctions of epithelial cells, both proteins being redistributed at the invadopodia-like structures of mesenchymal invasive cells to promote PKCε-dependent matrix degradation.

Highlights

The epithelial-mesenchymal transition (EMT) plays a pivotal role during tumor progression and invasion allowing epithelial cells to acquire a migratory phenotype
We recently demonstrated that forced expression of ADAM12L but not ADAM12S in the non-tumorigenic epithelial cell line MCF10A induced EMT [11]
In order to identify new functional partners of ADAM12 during this process, the anti-ADAM12L immunoprecipitates from ADAM12L-overexpressing MCF10A cells were sizeseparated by SDS-PAGE and in-gel digests were analyzed by LC-MS/MS, followed by protein identification through database searching. 253 and 200 proteins were identified in ADAM12L and IgG immunoprecipitates, respectively

Summary

Introduction

The epithelial-mesenchymal transition (EMT) plays a pivotal role during tumor progression and invasion allowing epithelial cells to acquire a migratory phenotype. This complex program occurs through numerous transitional states, some of them being reversible and characterized by cell heterogeneity [1]. SiRNA-based knockdown of ADAM9 in pancreatic cancer cells has been shown to diminish cellular migration, invasion, and to induce the epithelial marker E-cadherin [4] and a role of ADAM17 in promoting EMT has been recently reported in lung adenocarcinoma cells [5] and gastric carcinoma cells [6]. The association of ADAM12 with breast cancer aggressiveness and EMT has been suggested by several lines of evidence that include the ability of ADAM12overexpressing breast cell lines to induce metastasis in vivo [7, 8] and its correlated expression with the presence of metastases in triple-negative breast cancer [9] and with a breast tumor-initiating cell phenotype [10]

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Results

Conclusion