The transcription factor Fra-2 promotes mammary tumour progression by changing the adhesive properties of breast cancer cells

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Abstract

The transcription factor Fra-2 (Fos-related antigen-2) has been implicated in invasion of breast cancer cells, but there is only sparse information about its role in clinical tumours. In the present study, we analysed Fra-2 mRNA expression in a cohort of 167 patients, and found significant correlations between high Fra-2 expression and nodal involvement or reduced disease-free survival. To get more information about the underlying mechanisms, we generated stably transfected MDA-MB231 breast cancer cells with increased Fra-2 expression. Compared with the controls, these clones did not differ in proliferation and motility, but had higher invasive potential. By global gene expression analysis and subsequent validation of selected genes, we identified a number of proteins involved in cell–cell or cell–matrix interactions that were up- or down-regulated in Fra-2 overexpressing cells, e.g. connexin 43, ICAM-1, L1-CAM, integrin beta 2, integrin beta 4, and integrin alpha 6. The association of Fra-2 overexpression and high ICAM-1 or L1-CAM levels could also be demonstrated in our clinical cohort of mammary tumours. In both MDA-MB231 and MCF7 cells, we found an increased attachment of Fra-2 transfectants to components of the extracellular matrix. In addition, we could show a striking increase in the number of rolling cells in flow-through assays using E-selectin coated capillaries, which might indicate a higher capacity of extravasation. In conclusion, our data obtained on breast cancer cell lines and clinical tissue samples suggest that overexpression of Fra-2 promotes breast cancer progression and metastasis by deregulation of genes involved in cell–cell and cell–ECM contacts.

Introduction

Activating protein-1 (AP-1) is a transcription factor complex composed of members of the Jun, Fos, and activating transcription factor (ATF) family that bind as hetero or homodimers to the regulatory sequences of various target genes. Inhibition of AP-1 activity results in blocked proliferation, migration, invasion and experimental metastasis of tumour cells in various systems.1

The transcription factor Fra-2 (Fos-related antigen 2) was identified as a serum-inducible gene with homologies to c-Fos, FosB and Fra-1, but with significantly lower transforming activity compared with c-Fos.2, 3 Like the other members of this Fos family, Fra-2 forms heterodimers with Jun family members. In contrast to c-Fos and FosB, but similar to Fra-1, Fra-2 proteins lack a C-terminal transactivating domain and do not stimulate artificial AP-1-responsive promoters in vitro.4, 5 Animal models have shown that Fra-2 is required for proper bone and cartilage development, and Fra-2 −/− pups exhibit growth retardation and die within one week after birth.6 On the other hand, ectopic expression of Fra-2 in transgenic mice results in extensive fibrosis, predominantly in the lung, leading to premature mortality.7 Investigations on human clinical tissues indicate that Fra-2 might play an important role in the progression of various human tumour types in vivo.8 Fra-2 overexpression was found in salivary gland tumours9, colorectal cancer10 and adult T-cell leukaemia11, and in anaplastic large cell lymphomas. In the latter case, Fra-2 is amongst the genes which are affected by the characteristic t2;5 translocation leading to enhanced Fra-2 expression in the tumour cells.12

In breast cancer cell lines and tumour tissues, Fra-2 is detectable in variable amounts and phosphorylation states.13 In a Western blot study with 75 breast cancer samples, we found a significant association of strong Fra-2 expression with a high frequency of recurrence.14 In experimental models, Fra-2 expression is associated with an increased invasive potential and motility, whereas proliferation was hardly influenced at all.14, 15 In mouse mammary adenocarcinoma cell lines (CSMLO) with different metastatic potential, strong Fra-1 and Fra-2 expression was present in the metastatic cells, whereas c-Fos and FosB were undetectable.16 In transfection experiments with these cell lines, overexpression of Fra-2 resulted in activation of osteopontin, thrombospondin and CD44 which are all known to be involved in metastasis.17

In order to further investigate the role of Fra-2 in breast cancer progression, Fra-2 mRNA expression was analysed in a cohort of 164 mammary carcinomas with long-term follow-up data. In addition, we performed stable transfection leading to Fra-2 overexpression in the ER-negative MDA-MB231 cells. Amongst the genes which are differentially regulated in these cells, we identified a number of genes involved in cell–cell or cell–ECM interaction which suggested that the effect of Fra-2 is at least partly due to its influence on cell adhesion, especially during extravasation from the bloodstream. This hypothesis was further confirmed by ECM adhesion assays and analysis of attachment to E-selectin-coated surfaces in a laminar flow system. The correlation of Fra-2 expression with two relevant Fra-2 target genes, the cell adhesion molecules ICAM-1 and L1-CAM, was further analysed in our cohort of clinical breast cancer samples.

Section snippets

Patients

For array-based Fra-2 mRNA detection, samples from 167 patients (mean age 56.2 years; range 29–85 years) were analysed. All patients were treated for breast cancer at the University Medical Center Hamburg-Eppendorf, Germany, Department of Gynecology, between the years 1991 and 2002. Patient selection was based upon availability of tumour tissue. All patients gave written informed consent to access their tissues and review their medical records in accordance with the principles of the

Fra-2 expression in clinical tumour tissues

Fra-2 (FOSL2) mRNA expression in 167 breast cancer samples was evaluated by microarray analysis. Since the Affymetrix genchips harboured three probesets for FOSL2 (205409_at, 218880_at and 218881_s_at), we first compared the expression data of these sets and found highly significant correlations between all of them (p < 0.001; not shown). Since similar correlations with established prognostic markers were found for all probesets, we chose one of them (218881_s_at) for further analysis. Expression

Discussion

This study was undertaken to examine the role of Fra-2 expression in malignant progression of breast cancer in an experimental system and clinical tumours. Similar to our prior western blot study with a smaller cohort14, the present analysis of RNA expression data of 167 breast cancer samples has shown that high Fra-2 expression is associated with nodal involvement and early relapse, suggesting that Fra-2 might regulate target genes which are involved in the metastatic cascade in breast cancer.

Conflict of interest statement

Ralph M. Wirtz is employed at Siemens Healthcare Diagnostics Products GmbH, Cologne, Germany. For the other authors, there are no conflicting interests.

Acknowledgements

We gratefully acknowledge the excellent technical assistance of Katrin Beck and Sylke Krenkel and we thank the department directors Fritz Jänicke and Thomas Löning for generous support. Ulrich Richter is supported by a scholarship from the Werner Otto Foundation, which is gratefully acknowledged. This work was supported by the Deutsche Krebshilfe, Grant No. 106110.

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