Zinc (Zn) hyper-accumulates in breast tumors and malignant cell lines compared to normal mammary epithelium. The mechanisms responsible for Zn accumulation and the consequence of Zn dysregulation are poorly understood.
Microarrays were performed to assess differences in the expression of Zn transporters and metallothioneins (MTs) in human breast tumors and breast cancer cell lines. Real-time PCR and immunoblotting were employed to profile Zn transporter expression in representative luminal (T47D), basal (MDA-MB-231), and non-malignant (MCF10A) cell lines. Zn distribution in human tumors was assessed by X-ray fluorescence imaging. Zn distribution and content in cell lines was measured using FluoZin-3 imaging, and quantification and atomic absorption spectroscopy. Functional consequences of ZnT2 over-expression in MDA-MB-231 cells including invasion, proliferation, and cell cycle were measured using Boyden chambers, MTT assays, and flow cytometry, respectively.
Gene expression profiling of human breast tumors and breast cancer cell lines identified subtype-specific dysregulation in the Zn transporting network. X-ray fluorescence imaging of breast tumor tissues revealed Zn hyper-accumulation at the margins of Luminal breast tumors while Zn was more evenly distributed within Basal tumors. While both T47D and MDA-MB-231 cells hyper-accumulated Zn relative to MCF10A cells, T47D cells accumulated 2.5-fold more Zn compared to MDA-MB-231 cells. FluoZin-3 imaging indicated that Zn was sequestered into numerous large vesicles in T47D cells, but was retained in the cytoplasm and found in fewer and larger, amorphous sub-cellular compartments in MDA-MB-231 cells. The differences in Zn localization mirrored the relative abundance of the Zn transporter ZnT2; T47D cells over-expressed ZnT2, whereas MDA-MB-231 cells did not express ZnT2 protein due to proteasomal degradation. To determine the functional relevance of the lack of ZnT2 in MDA-MB-231cells, cells were transfected to express ZnT2. ZnT2 over-expression led to Zn vesicularization, shifts in cell cycle, enhanced apoptosis, and reduced proliferation and invasion.
This comprehensive analysis of the Zn transporting network in malignant breast tumors and cell lines illustrates that distinct subtype-specific dysregulation of Zn management may underlie phenotypic characteristics of breast cancers such as grade, invasiveness, metastatic potential, and response to therapy.
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The online version of this article (doi:10.1186/s12943-015-0486-y) contains supplementary material, which is available to authorized users.
Keywords: Breast cancer, Basal/Luminal, Metallothionein, Zinc, Zinc importer, Zinc transporter
Breast cancer is a heterogeneous disease at the molecular, histopathological, and clinical level. Through gene expression profiling, four subtypes based on expression of estrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor 2 (HER2) are recognized including: Luminal A (ER+/PR+/HER2−), Luminal B (ER+/PR+/HER2+), Basal (ER−/PR−/HER2−) and HER2- enriched (ER−/PR−/HER2+). These subtypes differ in incidence , aggressiveness, and response to therapy [2, 3]. Recently, it has been reported that breast tumors accumulate zinc (Zn) to levels well above those observed in normal tissue . The degree of Zn accumulation is associated with cancer progression  and malignancy . However, the mechanisms responsible for Zn accumulation, and the relationship between Zn accumulation and breast cancer subtype are not understood.
A multitude of cellular processes are regulated by Zn including transcription, cell signaling, proliferation, invasion, apoptosis, and autophagy . Cellular Zn metabolism is tightly regulated by a “Zn transporting network” which consists of 24 Zn transporting proteins that transport Zn into discrete sub-cellular compartments. The ZnT family of Zn transporters (SLC30A1-10 gene family) contains 10 members (ZnT1-10)  that export Zn from the cytoplasm, either directly across the cell membrane or into intracellular compartments. The ZIP family of Zn transporters (SLC39A1-14 gene family) contains 14 members (ZIP1-14)  and facilitates Zn import into the cytoplasm, either from across the cell membrane or from within a sub-cellular compartment. Cellular Zn management is also regulated by metallothioneins (MTs) , which are Zn binding proteins that buffer cytoplasmic Zn. ZnT2-mediated Zn accumulation into vesicles and MT-binding are the two primary mechanisms through which cells protect themselves from Zn toxicity, and both are positively regulated by Zn exposure through the activation of four metal responsive elements (MREs) in their promoters [11, 12].
Over-expression of several Zn transporters (ZIP6, ZIP7, ZIP10, and ZnT2) [13–19] is associated with Zn hyper-accumulation in breast tumors and several breast cancer cell lines. ZIP6 over-expression has been noted in ER+ subtypes  and is associated with less aggressive tumors . Similarly, ZnT2 over-expression accumulates Zn in vesicles which protects ER+ T47D cells from Zn toxicity . In contrast, ZIP10 is over-expressed in highly invasive, basal-like cell lines (MDA-MB-231 and MDA-MB-435S cells) and potentiates invasion . Similarly, ZIP7 over-expression in tamoxifen-resistant MCF7 cells is associated with enhanced motility . In addition to Zn transporters, MT over-expression is documented in ~88 % of invasive ductal carcinoma tissue biopsies , and is generally associated with poor prognosis  and high histological grade . However, reports of Zn transporter dysregulation are sporadic and a comprehensive analysis of Zn management in specific breast cancer subtypes has not been reported.
We reasoned that the molecular portrait of the Zn transporting network may be very different between malignant subtypes, and perhaps even a driver of their phenotypic behaviors. Herein, we used targeted genomic, proteomic, and Zn profiling in breast tumors and malignant cell lines that have characteristic features of Luminal (low-invasive, ER+/PR+/HER2−; T47D cells) and Basal (highly invasive, ER−/PR−/HER2−; MDA-MB-231 cells) subtypes. We observed subtype-specific differences in Zn management between Luminal and Basal breast tumors, and in cell culture models of luminal and basal-like breast cancer cells. Importantly, we found that Zn sequestration in vesicles through expression of ZnT2 profoundly reduced the proliferative and invasive phenotype of MDA-MB-231 cells, indicating that Zn dysregulation is subtype-specific, which may inform the development of novel diagnostic or therapeutic strategies.
The distribution of Zn accumulation in breast tumors differs between Luminal and Basal tumors
We first utilized X-ray fluorescence microscopy to determine the spatial distribution of Zn in Luminal and Basal tumors and adjacent normal tissue (Fig. 1). Spatial analysis revealed differences in Zn content and localization within the malignant regions. In Luminal breast tumors, Zn primarily accumulated around the tumor periphery. In Basal breast tumors, Zn was more evenly distributed throughout the malignant tissue. When compared with the distribution of calcium (Ca), some differences were noted such that Zn overlapped closely with Ca in Basal tumors, but this was less consistent in Luminal tumors.