The importance of targeting signalling mechanisms of the SLC39A family of zinc transporters to inhibit endocrine resistant breast cancer

Aim: Zinc is a key secondary messenger that can regulate multiple signalling pathways within cancer cells, thus its levels need to be strictly controlled. The Zrt, Irt-like protein (ZIP, SLC39A) family of zinc transporters increase cytosolic zinc from either extracellular or intracellular stores. This study examines the relevance of zinc transporters ZIP7 and ZIP6 as therapeutic targets in tamoxifen resistant (TAMR) breast cancer. Methods: A series of in vitro assays, including immunohistochemistry, immunofluorescence, flow cytometry, and western blotting were used to evaluate levels and activity of ZIP7 and ZIP6 in models of TAMR and sensitive (MCF-7) breast cancer. Analyses of these transporters in the clinical setting were performed using publicly available online resources: Gene Expression Profiling Interactive Analysis (GEPIA)2 and Kaplan-Meier Plotter (KmPlot). Results: Both total and activated levels of ZIP7 were significantly elevated in TAMR cells versus responsive MCF-7 cells. This was accompanied by an associated increase in free cytoplasmic zinc leading to amplification of downstream signals. Consistent with our proposed model, activated ZIP6 levels correlated with mitotic cells, which could be efficiently inhibited through use of our anti-ZIP6 monoclonal antibody. Mitotic inhibition translated to impaired proliferation in both models, with TAMR cells displaying increased sensitivity. Analysis of matched tumour and normal breast samples from patients revealed significant increases in both ZIP7 and ZIP6 in tumours, as well as family member ZIP4. Kaplan-Meier analysis revealed that high ZIP7 levels correlated with decreased overall and relapse-free survival (RFS) of patients, including patient groups who had received systemic endocrine therapy or tamoxifen only. In contrast, high ZIP6 levels were significantly linked to improved overall and RFS in all patients, as well as RFS in patients that received systemic endocrine therapy. Conclusions: TAMR cells displayed increased activity of both ZIP7 and ZIP6 transporters compared to anti-hormone responsive cells, suggesting their potential as novel therapeutic targets following development of resistant disease

Biological effects of fulvestrant on estrogen receptor positive human breast cancer: Short, medium and long-term effects based on sequential biopsies.

We report the first study of the biological effect of fulvestrant on ER positive clinical breast cancer using sequential biopsies through to progression. Thirty-two locally/systemically advanced breast cancers treated with first-line fulvestrant (250 mg/month) were biopsied at therapy initiation, 6 weeks, 6 months and progression and immunohistochemically-analyzed for Ki67, ER, EGFR and HER2 expression/signaling activity. This series showed good fulvestrant responses (duration of response [DoR] = 25.8 months; clinical benefit = 81%). Ki67 fell (p < 0.001) in 79% of tumours by 6 months and lower Ki67 at all preprogression time-points predicted for longer DoR. ER and PR significantly decreased in all tumours by 6 months (p < 0.001), with some declines in ER (serine 118) phosphorylation and Bcl-2 (p = 0.007). There were modest HER2 increases (p = 0.034, 29% tumours) and loss of any detectable EGFR phosphorylation (p = 0.024, 50% tumours) and MAP kinase (ERK1/2) phosphorylation (p = 0.019, 65% tumours) by 6 months. While ER remained low, there was some recovery of Ki67, Bcl-2 and (weakly) EGFR/MAPK activity in 45–67% patients at progression. Fulvestrant's anti-proliferative impact is related to DoR, but while commonly downregulating ER and indicators of its signaling and depleting EGFR/MAPK signaling in some patients, additional elements must determine response duration. Residual ER at fulvestrant relapse explains reported sensitivity to further endocrine therapies. Occasional modest treatment-induced HER2 and weakly detectable EGFR/HER2/MAPK signaling at relapse suggests targeting of such activity might have value alongside fulvestrant in some patients. However, unknown pathways must drive relapse in most. Ki67 has biomarker potential to predict fulvestrant outcome and as a quantitative measure of response

ELF5 drives lung metastasis in luminal breast cancer through recruitment of Gr1+ CD11b+ myeloid-derived suppressor cells

During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis–free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer

Insulin-like growth factor-1 receptor signalling and acquired resistance to gefitinib (ZD1839; IRESSATM) in DU145 human prostate cancer cells

Introduction: Although gefitinib (ZD1839; IRESSATM), a specific EGF-R tyrosine kinase inhibitor (TKI), has been shown to possess anti-tumour activity in a range of cancer types including androgen refractory prostate cancer, de novo and acquired resistance to the inhibitor has been reported clinically (Ranson, The Oncologist 2001; 58 (suppl 2A):114-22). In this study, we examined the involvement of the IGF1-R in the acquisition of resistance to gefitinib in the DU145 model of prostate cancer. Methods: The DU145 human prostate cancer cell line was continuously exposed to 1mM gefitinib. Alterations in signalling pathways were assessed using immunocytochemical, Western blotting and/or RTPCR techniques. Matrigel invasion assays were performed and cell proliferation was assessed by evaluating anchorage dependent growth. Cell sensitivity to the specific IGF1-R TKI, AG1024, (1-10mg/ml) was also measured. Results: Continuous exposure of the DU145 cells to 1mM gefitinib resulted in an approximate 60% initial growth inhibition after 10 days treatment. The surviving cells however, showed slow but steady increases in growth over the following 2 months, reaching a stable growth rate which far exceeded that of the parental cell line after a total of 3 months. This gefitinib-resistant subline designated DU145/TKI-R, demonstrated reduced EGF-R expression and negligible basal phosphorylated EGF-R activity. Compared with the parental DU145 cells, the DU145/TKI-R cells showed i) an increased production of IGFII mRNA, ii) elevated levels of both total and phosphorylated IGF1-R and PKCd, and iii) a marked increased sensitivity to growth inhibition by the IGF1-R inhibitor AG1024. Significantly, while 1mM gefitinib initially reduced the invasion of the DU145 cells through matrigel by 40%, the DU145/TKI-R cells overcame this inhibition. Furthermore, the migratory activity of the DU145/TKI-R cells was substantially reduced (45%) by treatment with AG1024 (10mM). Conclusion: In prostate cancer cells, acquired resistance to gefitinib is associated with increased signalling through the IGF1-R, which also plays a role in the invasive capacity of the gefitinib-resistant phenotype. Thus, therapeutic strategies that target the IGF1-R may increase the efficacy and duration of response to gefitinib