Loss of tight junction plaque molecules in breast cancer tissues is associated with a poor prognosis in patients with breast cancer

Abstract

The aim of this study was to investigate the expression of Zonula Occludens (ZO) proteins ZO-1, ZO-2 and ZO-3, and MUPP-1 (multi-PDZ domain protein 1), peripheral/plaque proteins that function in maintaining tight junction integrity and in transducing regulatory signalling events in patients with primary breast cancer. Breast cancer primary tumours (n = 114) and background tissues (n = 30) were processed for quantitative-polymerase chain reaction (PCR) analysis, Western blotting and immunostaining. Standardised transcript levels of ZO-1 and MUPP-1 were significantly lower in patients with metastatic disease compared with those remaining disease-free (DF) (median follow-up 72.2 months). Immunohistochemistry confirmed these results, with decreased levels in ZO-1 staining. For both ZO-1 and ZO-3, staining was confined to the intercellular regions in normal tissue, whereas in tumour tissues staining was diffuse and cytosolic. Q-PCR revealed a reduction in the levels of ZO-1 and MUPP-1 in patients with disease recurrence. Prognostic indicators of breast cancer were also inversely correlated with ZO-1 expression. We conclude that low levels of tight junction plaque molecules, such as ZO-1 and MUPP-1, in breast cancer are associated with poor patients prognosis.

Introduction

Tight junctions are directly involved in paracellular sealing and in membrane domain differentiation [1], [2]. The Tight junction is a region where the plasma membrane of adjacent cells forms a series of contacts that appear to completely occlude the extracellular space thereby creating an intercellular barrier and intramembrane diffusion fence [3]. Tight junctions in endothelial cells function as a barrier through which molecules and inflammatory cells can pass and in epithelial cells the tight junction functions in an adhesive manner and can prevent cell dissociation [4], [5]. An important step in the formation of cancer metastases is the interaction with and penetration of the vascular endothelium by dissociated cancer cells. Tight junctions are therefore the first barrier that cancer cells must overcome in order to metastasise. We have previously demonstrated that tight junctions of vascular endothelium function in vivo as a barrier between blood and tissues against metastatic cancer cells [6].

The tight junction structure can be separated into three regions of molecules: (i) transmembrane region includes those molecules that mechanically confer adhesiveness to the cell by (usually) homotypic bonding to the same molecule on adjacent cells. These include Occludin, Claudins (−1 to −26) and junctional adhesion molecules (JAM) −1, −2 and −3. (ii) The plaque or peripheral region of the tight junction includes those molecules that anchor the transmembrane molecules to the tight junction structure and link them to the cell cytoskeleton and signalling pathways; essentially controlling the tight junction structure and function. The Zonula Occludens (ZO) family of MAGUK proteins, including MUPP-1 falls into this category. (iii) The third region is composed of associated molecules, that is, those molecules that often have other roles in the cell, but are known associates of the tight junction, where they function as part of the signalling/control mechanism for the structure (such as AF6, α-catenin and ponsin).

Cell adhesion to adjacent cells and the extracellular matrix is key not only to the organisation of epithelium into a tissue, but also to the regulation of cellular processes such as gene expression, differentiation, motility and growth [7]. Cell adhesion molecules, transmembrane receptors and cytoskeletal factors, all of which are organised into multimolecular complexes, and the activation of signalling pathways, mediate these regulatory functions. Studies suggest that some of the cell adhesion and cytoskeletal proteins may serve an additional and important function, namely, suppression of the malignant phenotype of cells during tumorigenesis [7].

Early studies demonstrated a correlation between the reduction of tight junctions and tumour differentiation and experimental evidence has emerged to place tight junctions on the frontline as the structure that cancer cells must overcome in order to metastasise [8], [9], [10], [11]. Although a considerable body of work exists on tight junctions and their role in a number of diseases, it is only in the last few years that their possible role in tumorigenesis has been studied. To date, most of the work has concentrated on cell lines and, to a limited degree, on colorectal and pancreatic cancers, with only a few studies carried out on breast cancer tissues – which have concentrated on Claudin-1 (SEMP-1), claudin-7 and ZO-1 expression.

It has been shown that Claudin-1 appears to be absent in some breast cancer cell lines, that also have little/no Occludin or ZO-1 [12]. Paraffin-embedded breast cancer samples immunohistochemically-stained for ZO-1 have been examined. It has been found that staining is reduced or lost in 69% of the cancers examined and that ZO-1 staining has a positive correlation with tumour differentiation. Genetic changes in the ZO-1 gene may account for these changes [13]. Recently, it has been demonstrated that loss of claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast [14]. Such findings suggest that the loss of claudin-7 may aid in tumour cell dissemination and augment the cell’s metastatic potential.

Such (albeit) limited evidence, along with work carried out on other types of cancer, indicates that tight junctions may play a key role in breast cancer cell tumorigenesis. In this report, we have examined the role of tight junction molecules in human breast cancer and examined the correlations between levels of plaque proteins, ZO-1, -2, -3 and MUPP-1, and patient prognosis in primary breast cancer tumours.