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 the present 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, knockdown of CEACAM6 expression completely inhibited the invasiveness of MCF-7:5C and MCF-7:2A cells and caused a reduction in phosphorylated c-Src and pAkt expression. A significant reduction in E-cadherin and β-catenin was also observed in MCF-7:5C and MCF-7:2A cells compared to parental MCF-7 cells. To our knowledge, this study is the first to demonstrate a critical role for CEACAM6 in migration and invasion of breast cancer cells that have acquired resistance to oestrogen deprivation.
Previous studies have reported that overexpression of CEACAM6 in pancreatic adenocarcinoma cells is associated with enhanced cellular invasiveness and increased metastatic potential in vivo
and that this effect is completely attenuated by suppression of CEACAM6 expression (4
). Recently, Scott and coworkers (20
) reported that CEACAM6 was upregulated by 20-fold in tamoxifen-resistant MCF-7 cells compared to tamoxifen-sensitive cells and that hormone sensitivity could be partially restored in the tamoxifen-resistant cells by siRNA silencing of CEACAM6. This in vitro
data was substantiated in clinical breast cancer where it was demonstrated that CEACAM6 was overexpressed in primary breast tumours that subsequently relapsed following adjuvant tamoxifen and in a multivariate analysis, only CEACAM6 remained a significant predictor of recurrence (31
). These findings are consistent with our present study which shows that CEACAM6 is significantly upregulated in oestrogen deprived breast cancer cells that have acquired resistance to oestrogen suppression and knockdown of CEACAM6 expression reverses the invasive phenotype of these cells. The fact that CEACAM6 is identified independently in two model systems using endocrine agents with distinct modes of action suggests that it may play an important role in endocrine resistance. Currently, the mechanism by which CEACAM6 facilitates invasion is not fully understood. However, there is evidence that CEACAM6, along with other GPI-anchored proteins, are capable of modulating the activity of intracellular tyrosine kinases such as c-Src (32
). In particular, studies by Duxbury and coworkers (30
) showed that c-Src activity was increased in CEACAM6-overexpressing BxPC3 human pancreatic cancer cells and decreased following suppression of CEACAM6 expression and that inhibition of c-Src activity significantly suppressed CEACAM6-mediated cellular invasiveness. We found that phosphorylated c-Src was significantly elevated in MCF-7:5C and MCF-7:2A cells and that suppression of CEACAM6 expression reduced its level in these cells. Pharmacological blockade of c-Src using the Src tyrosine kinase inhibitor pyrazolopyrimidine (PP2) also inhibited the invasiveness of MCF-7:5C and MCF-7:2A cells. In addition, we found markedly elevated levels of phosphorylated Aktser473
in MCF-7:5C and MCF-7:2A cells which were dramatically reduced following CEACAM6 suppression. Akt is a serine/threonine protein kinase that mediates cell survival, proliferation (35
), tumour cell migration and invasion and metastasis (37
) and previous studies have shown that c-Src activates the PI3K/Akt signaling pathway (38
). Thus, it is possible that activation of both c-Src and Akt might play a role in mediating CEACAM6-induced migration and invasion.
The epithelial-to-mesenchymal transition (EMT) plays a key role in metastasis and is characterized by the conversion of epithelial cancer cells to a more motile phenotype that facilitates invasion. A critical molecular feature of EMT is the downregulation of E-cadherin (39
), a cell adhesion molecule present in the plasma membrane of most normal epithelial cells. E-cadherin acts de facto
as a tumour suppressor inhibiting invasion and metastasis and is frequently repressed or degraded during transformation. In our study, E-cadherin and β-catenin were significantly decreased whereas N-cadherin was markedly increased in invasive MCF-7:5C and MCF-7:2A cells compared to noninvasive MCF-7 cells. In addition, our cell morphology studies showed EMT-like changes in MCF-7:5C and MCF-7:2A cells compared to MCF-7 cells. A variety of signal transduction pathways impinge on the regulation of E-cadherin levels or subcellular distribution. In particular, Akt/PKB has been shown to repress transcription of the E-cadherin gene which leads to conversion of epithelial cells into invasive mesenchymal cells (40
). We have found that MCF-7:5C and MCF-7:2A cells both overexpress phosphorylated Akt and gene ontology analysis of expression data obtained for MCF-7:5C and MCF-7:2A cells reveal that the P13K/Akt signalling pathway is significantly (p = 0.002) altered compared to parental MCF-7 cells.
In conclusion, we have identified CEACAM6 as a critical gene in the regulation of migration and invasion of breast cancer cells that have acquired resistance to oestrogen deprivation. Since aromatase inhibitors are now considered the standard of care for the hormonal treatment of early breast cancer in postmenopausal women, this finding has important clinical implications for these patients because it suggests that extended use of aromatase inhibitors may potentially lead to the development of metastatic disease. CEACAM6 can thus serve as a powerful predictor of future recurrence and may also represent a promising new therapeutic target for breast cancer.