Elsevier

European Journal of Cancer

Volume 44, Issue 12, August 2008, Pages 1770-1779
European Journal of Cancer

Overexpression of CEACAM6 promotes migration and invasion of oestrogen-deprived breast cancer cells

https://doi.org/10.1016/j.ejca.2008.05.016Get rights and content

Abstract

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast and lung and is associated with tumourigenesis, tumour cell adhesion, invasion and metastasis. In this study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERα)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation, and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6–15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced phosphorylated Akt and c-Src expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen-deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.

Introduction

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is a glycosylphosphatidylinositol-anchored cell surface protein that functions as a homotypic intercellular adhesion molecule.1 It is overexpressed in a number of human malignancies including pancreatic cancer, gastrointestinal cancer and breast cancers2, 3, and increased levels of CEACAM6 are inversely correlated to the differentiation state of cancer cells. Previous studies have shown that CEACAM6 is overexpressed in pancreatic adenocarcinoma cells, and its overexpression is associated with greater in vivo metastatic ability and increased invasiveness and migration.4, 5 More recently, Poola and co-workers6 reported that the expression of CEACAM6 in atypical ductal hyperplasia was associated with the development of invasive breast cancer (IBC). Currently, however, the role of CEACAM6 overexpression in breast cancer migration and invasion is not known.

Invasion and metastasis are the hallmarks of cancer malignancy, and they are the primary cause of patient mortality during breast cancer progression.7 Invasion refers to the ability of cancer cells to penetrate through the membranes that separate them from healthy tissues and blood vessels, and metastasis refers to the spreading of cancer cells to other parts of the body.8 In order for a transformed cell to metastasize, it must first lose adhesion, penetrate and invade the surrounding extracellular matrix (ECM), enter the vascular system and adhere to distant organs.8 These processes require extensive alterations in gene expression profiles, including the down-regulation of genes involved in cell anchorage and the up-regulation of genes involved in cell motility and matrix degradation.7, 9, 10

Aromatase inhibitors (AIs) are anti-oestrogen agents that suppress oestrogen production in peripheral tissues and breast tumours by inhibiting or inactivating aromatase, the enzyme which catalyses the conversion of androgens to oestrogens in post-menopausal women.11 Several randomized trials12, 13, 14, 15 have shown that third generation AIs are superior to adjuvant tamoxifen in terms of improved disease-free survival and less side-effects. Unfortunately, one of the consequences of prolonged oestrogen deprivation/suppression is the development of drug resistance.16, 17 Previous studies have shown that acquisition of tamoxifen resistance in breast cancer cells is associated with a significant increase in motility and invasion18, 19 along with increased CEACAM6 expression20; however, it is unknown whether acquired resistance to oestrogen deprivation affects tumour cell migration and invasion and whether CEACAM6 plays a role in this process.

In this study, we investigated the role of CEACAM6 in cellular migration and invasion of breast cancer cells that have acquired resistance to oestrogen deprivation. We found that CEACAM6 was significantly overexpressed in oestrogen-deprived MCF-7:5C and MCF-7:2A breast cancer cells and that these cells were markedly more migratory and invasive than parental MCF-7 cells. Suppression of CEACAM6 expression by small interfering RNA (siRNA) completely reversed the invasive phenotype of MCF-7:5C and MCF-7:2A cells. E-cadherin and β-catenin were also significantly reduced in these cells. The mechanism of action of CEACAM6 appears to involve, in part, the c-Src and Akt signalling pathways.

Section snippets

Reagents

17 Beta-oestradiol was purchased from Sigma Chemical Co. (St Louis, MO); PP2 was purchased from EMD Biosciences Inc. (La Jolla, CA); LY294002 was purchased from Promega (Madison, WI); fulvestrant was obtained as a generous gift from AstraZeneca (Macclesfield, United Kingdom); Affymetrix Human Genome U133 Plus 2.0 Arrays were purchased from Affymetrix (Santa Clara, CA); foetal bovine serum (FBS), cell culture medium and other reagents were purchased from Invitrogen (Carlsbad, CA).

Cell lines and culture conditions

Wild-type MCF-7

Characterisation of long-term oestrogen-deprived breast cancer cells

The growth of oestrogen-deprived MCF-7:5C and MCF-7:2A cells is compared to parental MCF-7 cells in Fig. 1A. Both MCF-7:5C and MCF-7:2A cells grew robustly in the absence of oestrogen whereas MCF-7 cells grew minimally without oestrogen. The doubling times were 2.7, 3.4, and 6 d for MCF-7:5C, MCF-7:2A and MCF-7 cells, respectively. We also examined cell morphology changes associated with resistance to long term oestrogen deprivation using phase-contrast microscopy. Fig. 1B shows that MCF-7 cells

Discussion

Despite advances in detection and treatment of metastatic breast cancer, mortality from this disease remains high because current therapies are limited by the emergence of therapy-resistant cancer cells. In this study, we showed that oestrogen deprivation significantly increased the motility and invasiveness of two ERα-positive human breast cancer cell lines that have acquired resistance to oestrogen deprivation, and that these cells overexpressed the invasive gene CEACAM6. Furthermore,

Conflict of interest statement

None declared.

Acknowledgments

We thank Dr. Chris Wambi (Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA) for his valuable comments and critical review of this manuscript. This work was supported by the NIH Career Development Grant 1K01CA120051-01A2; the American Cancer Society Grant IRG-9202714; the Department of Defense Breast Program under award number BC050277 Center of Excellence; Fox Chase Cancer Center Core Grant NIH P30 CA006927; Weg Fund of Fox Chase Cancer Center; and the Hollenbach

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