Mechanisms Leading to Increased Growth and Survival Signalling in Breast Cancer Cells Zinc Transporters and Endocrine Resistance

Recent studies within our group have begun to define a previously unidentified role for zinc in contributing to ER dependent and independent forms of endocrine resistance through its capacity to sustain the activity of growth factor signalling. Zinc is a metal ion which is involved in the regulation of many cellular processes, including proliferation and survival, and increased zinc levels have been shown to inactivate several phosphatases involved in the dephosphorylation and inactivation of EGFR, HER2, IGF-1R and c-src (Haase and Maret, 2005). Exposure of our tamoxifen resistant cells to zinc, therefore, serves to activate signalling from these growth factor receptors and raise the intracellular activity of MAPK and AKT to promote tumour cell growth and motility (Taylor et al., 2008). Significantly, we have shown that acquired tamoxifen resistance in vitro is associated with increased basal zinc levels and increased expression of a key zinc transporter, ZIP7 (HKE4/SLC39A7), which facilitates the release of zinc from its intracellular stores. Silencing of this gene through the use of siRNA to ZIP7, reduces zinc levels, blocks growth factor responses and inhibits cell growth, providing evidence for a central importance of zinc in resistance (Taylor et al., 2008). Importantly, the expression of a further family member located on tumour cell membranes, LIV-1 (Taylor et al., 2007), has been previously shown by our group to predict breast cancer spread to the regional lymph nodes and may link zinc transport to other features of tumour progression noted in our endocrine resistant cells. Hypo- and Hyper-Methylation of DNA in Endocrine Resistance

In general terms, the hyper-methylation/hypo-methylation of gene promoters can have a profound effect on gene transcription, leading, at its extremes, to gene silencing or increased gene expression respectively. In anti-oestrogen resistant MCF-7 cells, a recent study by Fan et al. (2006) has suggested that a hypo-methylated state predominates and results in the increased expression and activation of multiple growth regulatory pathways, including EGFR/HER2 and related proteins, PKA signalling elements, cytokines and cytokine receptors, Wnt/p-catenin, Notch signalling elements and IFN signalling components. Characteristically, however, considerable differences between the induced gene expression profiles of SERMs and SERDs exist, emphasising once again the individual nature and diverse mechanisms of actions of endocrine agents which target the same receptor.

Interestingly, although DNA hyper-methylation was not as evident as hypo-methylation in the study of Fan et al. (2006), it is of some potential importance in endocrine resistance since it is an established mechanism for inactivating tumour suppressor and pro-apoptotic genes which would subsequently allow growth factor associated growth signalling to occur more efficiently. Certainly, several investigations have described the capacity of anti-oestrogenic drugs to promote gene inactivation through the hyper-methylation of CpG islands in oestrogen regulated genes (Jensen et al., 1999; Leu et al., 2004). In our own studies (see Chapter 4), we have shown that exposure of MCF-7 cells to either tamoxifen or fulvestrant for prolonged periods (> 2 years) effectively silences a substantial cohort of oestrogen regulated growth tumour suppressor genes which when re-expressed by treatment of the cells with 5-AZA are predominantly growth inhibitory in the presence of oestradiol. Evidently, the remodelling of chromatin structure by anti-hormonal drugs, through the hypo- and hyper-methylation of DNA, appears to provide complimentary signals to promote resistant growth, acting to stimulate positive growth regulatory signals, while inhibiting negative ones. Epithelial Mesenchymal Transition (EMT) in Endocrine Resistance

Importantly in our models, endocrine resistance is frequently hall-marked by a partial epithelial mesenchymal transition (EMT) which characteristically involves a reprogramming of cells towards a less differentiated, more invasive, phenotype. Studies from our own group have highlighted the breadth of induced genes which may contribute to this altered phenotype (Gee et al., 2006). Among these are CD44, notably encompassing the CD44v3 isoform (see Chapter 8), which acts as a co-receptor for erbB family members (Yu et al., 2002; Ghatak et al., 2005) and c-Met, the pro-invasive tyrosine kinase receptor target for HGF/scatter factor (Orian-Rousseau et al., 2002). Since, in the case of long-term fulvestrant resistance, such poorly differentiated cells are largely unresponsive to EGFR/HER2 blockade and express only modest levels of these growth factor receptors, their aggressive phe-notype must rely on an entirely different cohort of signalling pathways from those identified in earlier forms of resistance (see Chapter 4). Loss of Tumour Suppressors in Endocrine Resistance

A recent enhanced retroviral mutagen study has revealed that a disruptive insertion into the allele coding for the p27 cyclin-dependent kinase (CDK) inhibitor created oestrogen-independent and anti-oestrogen resistant breast cancer cells that still contained functional ER. Several notable changes were observed to the signalling pathways of the cells, including increased CDK2 activity, hyper-phosphorylation of AIB1 which enhanced its co-activator activity on the transcription of E2F1 and growth factor binding protein 2-associated binder 2 (Gab2) and Akt activity were increased following E2F1 over-activation (Yuan et al., 2007). Similarly, loss of the retinoblastoma tumour suppressor (RB) protein, a common aberration in breast cancer (Dublin et al., 1998) also leads to increased CDC2 activity (Varma et al., 2007), increased E2F-regulated gene expression (Bosco et al., 2007), cell cycle progression and an endocrine resistant phenotype in vitro (Bosco et al., 2007, Varma et al., 2007), as does loss of the cell cycle inhibitor p21 (Cariou et al., 2000). Evidently, loss of cell cycle inhibitors allow passage of anti-hormone treated cells through the cell cycle, together with enhancing aspects of pathways positively regulated by growth factor signalling.

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