The transition element zinc, which has recently been identified as an intracellular second messenger, has been implicated in various signaling pathways, including those leading to cell proliferation. Zinc channels of the ZIP (ZRT1- and IRT1-like protein) family [also known as solute carrier family 39A (SLC39A)] transiently increase the cytosolic free zinc (Zn(2+)) concentration in response to extracellular signals. We show that phosphorylation of evolutionarily conserved residues in endoplasmic reticulum zinc channel ZIP7 is associated with the gated release of Zn(2+) from intracellular stores, leading to activation of tyrosine kinases and the phosphorylation of AKT and extracellular signal-regulated kinases 1 and 2. Through pharmacological manipulation, proximity ligation assay, and mutagenesis, we identified protein kinase CK2 as the kinase responsible for ZIP7 activation. Together, the present results show that transition element channels in eukaryotes can be activated posttranslationally by phosphorylation, as part of a cell signaling cascade. Our study links the regulated release of zinc from intracellular stores to phosphorylation of kinases involved in proliferative responses and cell migration, suggesting a functional role for ZIP7 and zinc signals in these events. The connection with proliferation and migration, as well as the activation of ZIP7 by CK2, a kinase that is antiapoptotic and promotes cell division, suggests that ZIP7 may provide a target for anticancer drug development.

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Antiestrogens such as tamoxifen are the mainstay of treatment for estrogen receptor-positive breast cancer. However, their effectiveness is limited by the development of endocrine resistance, allowing tumor regrowth and progression. Importantly, in vitro MCF7 cell models of acquired tamoxifen resistance (TamR cells) display an aggressive, invasive phenotype in which activation of epithelial growth factor receptor/IGF-I receptor/Src signaling plays a critical role. In this study, we report that TamR cells have increased levels of zinc and zinc transporter, ZIP7 [solute carrier family 39 (zinc transporter) member 7, also known as SLC39A7], resulting in an enhanced response to exogenous zinc, which is manifested as a greatly increased growth factor receptor activation, leading to increased growth and invasion. Removal of ZIP7, using small interfering RNA, destroys this activation of epithelial growth factor receptor/IGF-I receptor/Src signaling by reducing intracellular zinc levels. Similarly, it also blocks the activation of HER2, -3, and -4. These data suggest that intracellular zinc levels may be a critical factor in determining growth factor responses and that the targeting of zinc transporters may have novel therapeutic implications. We show that ZIP7 is a critical component in the redistribution of zinc from intracellular stores to the cytoplasm and, as such, is essential for the zinc-induced inhibition of phosphatases, which leads to activation of growth factor receptors. Removal of ZIP7 therefore offers a means through which zinc-induced activation of growth factor receptors may be effectively suppressed and provides a mechanism of targeting multiple growth factor pathways, increasing tumor kill, and preventing further development of resistance in breast cancer.

De novo and acquired resistance to the anti-tumour drug gefitinib (ZD1839; Iressa), a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been reported. We have determined whether signalling through the IGF-I receptor (IGF-1R) pathway plays a role in the gefitinib-acquired resistance phenotype. Continuous exposure of EGFR-positive MCF-7-derived tamoxifen resistant breast cancer cells (TAM-R) to 1 mM gefitinib resulted in a sustained growth inhibition (90%) for 4 months before the surviving cells resumed proliferation. A stable gefitinibresistant subline (TAM/TKI-R) was established after a further 2 months and this showed no detectable basal phosphorylated EGFR activity. Compared with the parental TAM-R cells, the TAM/ TKI-R cells demonstrated (a) elevated levels of activated IGF-1R, AKT and protein kinase C (PKC)d, (b) an increased sensitivity to growth inhibition by the IGF-1R TKI AG1024 and (c) an increased migratory capacity that was reduced by AG1024 treatment. Similarly, the EGFR-positive androgen-independent human prostate cancer cell line DU145 was also continuously challenged with 1 mM gefitinib and, although substantial growth inhibition (60%) was seen initially, a gefitinibresistant variant (DU145/TKI-R) developed after 3 months. Like their breast cancer counterparts, the DU145/TKI-R cells showed increases in the levels of components of the IGF-1R signalling pathway and an elevated sensitivity to growth inhibition by AG1024 compared with the parent DU145 cell line. Additionally, DU145/TKI-R cell migration was also decreased by this inhibitor. We have therefore concluded that in breast and prostate cancer cells acquired resistance to gefitinib is associated with increased signalling via the IGF-1R pathway, which also plays a role in the invasive capacity of the gefitinib-resistant phenotype.