Zinc transporter HKE4 as a new target in antihormone resistance of breast cancer

Background

Oestrogen receptor-positive breast cancers develop resistance to anti-oestrogens by utilising alternative growth factor pathways as observed in our tamoxifen-resistant cell line (TAMR). These include EGFR, IGF1-R and Src signalling as well as increased growth and invasion. Zinc is elevated in breast cancer tissue and has been demonstrated to activate certain growth factor signalling pathways. We have tested the expression level of members of the LIV-1 family of zinc influx transporters and discovered that HKE4 (SLC39A7, ZIP7), previously shown by us capable of increasing the intracellular zinc levels, has increased expression in TAMR. We have therefore investigated whether the development of the more aggressive phenotype observed in our TAMR cells, including activation of these signalling pathways as well as increased growth and invasion, is due to an increase of intracellular zinc and as a direct result of increased expression of HKE4.

Methods

All nine members of the LIV-1 subfamily of ZIP transporters were measured in our model of tamoxifen-resistant breast cancer using Affymetrix arrays. Zinc-induced activation of growth factor signalling pathway components was investigated by western blot and/or fluorescent microscopy. Short-term (15-min) treatments with 20 μM zinc included ionophore, whereas long-term (hours/days) did not. Recombinant LIV-1 family members with a V5 tag were expressed using pcDNA3.1/V5-His-TOPO vector, and siRNA (Dharmacon smartpools with relevant controls) was used to reduce endogenous expression.

Results

HKE4 (SLC39A7), a ZIP transporter from the LIV-1 subfamily, was discovered to be elevated in TAMR cells by Affymetrix analysis and confirmed by PCR and western blot. We have observed that our TAMR cells have a twofold increase in intracellular zinc compared with wild-type cells, using the zinc-specific fluorescent dye Newport Green. Short-term zinc treatment of TAMR cells activates the signalling pathways implicated in antihormone-resistant proliferation and is reduced by both the zinc chelator TPEN and the Src kinase inhibitor SU6556. The same effects are observed after longer term (6 days) zinc treatment with additional increases in cell growth and invasion through Matrigel. Since we have previously demonstrated that HKE4 is capable of increasing intracellular zinc in cells and, more recently, that these TAMR have elevated intracellular zinc levels, we have tested the hypothesis that elevated HKE4 expression is directly responsible for the aggressive phenotype observed in our TAMR cells. Reducing HKE4 levels by siRNA demonstrated a role for this molecule in driving the zinc-induced activation of multiple signalling pathways. In the presence of siRNA for HKE4, the previously observed zinc-induced activation of EGFR, Src, and IGF1-R was eradicated and the EGF-stimulated activation was also decreased. Additionally, we have demonstrated the converse by transfecting recombinant HKE4 into wild-type cells and/or treating them with zinc to observe the activation of these signalling pathways and increases in invasive capability. Interestingly, we have observed a similar role of HKE4 in our model of faslodex-resistant breast cancer.

Conclusion

The presented results propose that HKE4, a member of the LIV-1 subfamily of ZIP transporters, is directly involved in the activation of the aggressive phenotype observed with the development of antihormone resistance, and as such is a potential new target for the prevention of resistance to antihormones in breast cancer progression.