Protein Kinase Cϵ Mediates Polymeric Fibronectin Assembly on the Surface of Blood-borne Rat Breast Cancer Cells to Promote Pulmonary Metastasis

Chia-Sui Chen,Bendicht U. Pauli,Richard Isom,Lynn Huang,Hung-Chi Cheng,Roy A. Levine

doi:10.1074/jbc.m705839200

Chia-Sui Chen, Bendicht U. Pauli + Show 4 more

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https://doi.org/10.1074/jbc.m705839200

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Abstract

Malignant breast cancer cells that have entered the blood circulation from primary mammary fat pad tumors or are grown in end-over-end suspension culture assemble a characteristic, multi-globular polymeric fibronectin (polyFn) coat on their surfaces. Surface polyFn is critical for pulmonary metastasis, presumably by facilitating lung vascular arrest via endothelial dipeptidylpeptidase IV (CD26). Here, we show that cell-surface polyFn assembly is initiated by the state of suspension, is dependent upon the synthesis and secretion of cellular Fn, and is augmented in a dose- and time-dependent manner by plasma Fn. PolyFn assembly is regulated by protein kinase Cepsilon (PKCepsilon), which translocates rapidly and in increasing amounts from the cytosol to the plasma membrane and is phosphorylated. PolyFn assembly is impeded by select inhibitors of this kinase, i.e. bisindolylmaleimide I, Ro-32-0432, Gö6983, and Rottlerin, by the phorbol 12-myristate 13-acetate-mediated and time-dependent loss of PKCepsilon protein and decreased plasma membrane translocation, and more specifically, by stable transfection of lung-metastatic MTF7L breast cancer cells with small interfering RNA-PKCepsilon and dominant-negative PKCepsilon constructs (e.g. RD-PKCepsilon). The inability to assemble a cell surface-associated polyFn coat by knockdown of endogenous Fn or PKCepsilon impedes cancer cells from metastasis to the lungs. The present studies identify a novel regulatory mechanism for polyFn assembly on blood-borne breast cancer cells and depict its effect on pulmonary metastasis.

Highlights

In cancer cell lines selected for enhanced lung colonization (8 –11)
We have firmly established a critical dependence between the ability of the cancer cells to assemble an insoluble, globular polymeric Fn (polyFn)-surface coat and lung colonization in an experimental metastasis model [9, 17], we still do not know whether blood-borne cancer cells use their own cellular Fn or rely on ubiquitous pFn to assemble their polyFn surface coat, how cancer cells regulate the polyFn build-up, and how Fn cell surface deposits are transformed into covalently bonded, insoluble aggregates [9]
To answer some of these questions we examined polyFn genesis in lung-metastatic MTF7L rat breast cancer cells subjected to end-over-end (EoE) suspension culture in serum- or pFn-containing medium, which together induce and augment the build-up of a polyFn surface coat similar to that observed on tumor cells that have entered the blood circulation [9]

Summary

Introduction

In cancer cell lines selected for enhanced lung colonization (8 –11). This pro-metastatic role of Fn is multifaceted, affecting several steps of the metastatic cascade by modulating cell adhesion, motility/invasion, cell cycle progression, and cell survival (for review, see Refs. 12–16). The data presented here show that assembly of the prometastatic, multiglobular polyFn surface coat on MTF7L breast cancer cells depends upon the synthesis and secretion of endogenous, cellular Fn (Fn1: EDAϩEDBϩIIICS120-Fn) and is regulated by membrane-translocation and Ser/Thr phosphorylation of protein kinase C⑀ (PKC⑀).

Results

Conclusion