We show that alcohol increases the invasive ability of breast cancer cells in a dose-dependent manner. This suggests that alcohol may increase the ability of the cancer to metastasize. In fact, both animal and epidemiological findings suggest that alcohol increase the metastatic ability of breast cancers . Vaeth et al. showed that frequent alcohol drinkers were 1.45-times more likely to be diagnosed with later stage breast cancer than infrequent drinkers . Additionally, animal studies suggest that alcohol consumption increases the incidence of lung metastasis . Thus, it is critical to understand the mechanism by which alcohol promotes the invasive ability of breast cancer cells in order to develop prevention and treatment options for cancer metastasis. Our data suggest that alcohol increases the invasive ability of breast cancer cells via the Nm23 metastasis suppressor gene. More importantly, we show that the invasive ability associated with alcohol can be blocked by regulating Nm23 levels.
The expression of integrins (e.g., ITGA5) in cancer cells is essential as they allow the cells to attach to the endothelium found within the blood vessels of organs such as the lungs (a secondary site for tumor metastasis) . Thus, the levels of integrins such as ITGA5 determine how aggressively the cancer cells may spread to secondary tissues. Our data shows that alcohol exposure increases the expression of the fibronectin receptor subunit ITGA5 in T47D breast cancer cells. Furthermore, overexpression of Nm23 can block the effects of alcohol on ITGA5 expression. Additionally, results show that suppression of Nm23 by siRNA increases the expression of ITGA5 in the cancer cells, thus, indicating that Nm23 regulates ITGA5 expression. Furthermore, we show that down-regulation of ITGA5 is sufficient to block the effects of alcohol on the invasion of T47D cells. Further investigation with other breast cancer cell lines will be necessary before conclusive statements can be made regarding the involvement of the Nm23-ITGA5 pathway in alcohol-induced breast cancer cell invasiveness. Nevertheless, our results indicate that alcohol decreases the expression of Nm23, thereby allowing ITGA5 to be expressed, which in turn allows T47D breast cancer cells to obtain a more invasive phenotype.
Further investigation is also necessary to better understand how alcohol regulates Nm23 expression and how Nm23 regulates ITGA5 expression. It is well accepted that alcohol may promote breast cancer development via the estrogen signaling pathway . As breast cancer cells are able to produce estrogen in vitro, the binding of estrogen to the estrogen receptor α (ERα) may activate downstream PI3K/Akt and MAPK/ERK pathways to promote cell migration [29, 30]. In a recent study, it was reported that estrogen negatively regulates Nm23 expression in vitro. Thus, the modulation of Nm23 expression shown in this study as a result of alcohol exposure may be mediated by estrogen levels. As a NDP kinase, Nm23 may modify cytoskeleton organization and protein trafficking, possibility through ITGA5, to promote cell migration and adhesion to the extracellular matrix (ECM). Previous studies have shown that Nm23 decreases activity of Rac1, a specific nucleotide exchange factor, through binding of Tiam1 [32, 33]. Reduction of Rac1 activation induces the activity of RhoA, a component in the ITGA5-mediated cellular adhesion and migration signalling pathway [34, 33]. Indeed, estrogen has been found to activate RhoA and this activity is necessary for cytoskeletal remodelling and for the enhancement of breast cancer cell migration and invasion . Thus, down-regulation of Nm23 by alcohol may promote RhoA activation through estrogen regulation to favor ITGA5-mediated breast cancer progression.
The ECM and adhesion molecules play a critical role in the invasive phenotype of cancer cells . For example, the binding of integrins to ECM proteins stimulates the phosphorylation of focal adhesion kinase (FAK); this activated FAK can activate signaling pathways such as PI3K, MAPK, and ERK . These pathways have been shown to regulate cell adhesion, motility, invasion, and metastasis . Integrins are heterodimer cell surface receptors composed of α and β subunits. The integrin α5 subunit (ITGA5) dimerizes exclusively with the β1 integrin (ITGB1) to form the classic fibronectin receptor (α5/β1 or ITGA5B1) . The interaction of α5/β1 with fibronectin (FN) plays an important role in the adhesion of cancer cells to the extracellular matrix . Moreover, previous studies have shown that interaction of α5/β1 with FN promotes activation of the ERK and PI3K signaling pathways, which in turn stimulates cells to invade and produce MMPs (e.g., MMP-1 MMP-9) to facilitate invasion . In our studies, we show that the integrin α5 subunit expression is necessary for alcohol to increase the invasive ability of T47D breast cancer cells. It is possible that alcohol stimulates signaling pathways such as ERK and PI3K, via α5/β1, which then increases the invasive phenotype of T47D breast cancer cells. Consequently, activated integrins may facilitate the movement and metastasis of breast cancer cells. In future studies, we will determine if alcohol affects signaling pathways such as FAK, ERK, and PI3K via ITGA5 and elucidate the role of estrogen in alcohol-mediated down-regulation of Nm23.