title: Subtype-specific accumulation of intracellular zinc pools is associated with the malignant phenotype in breast cancer.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The Interdisciplinary Graduate Program in Physiology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA.|The Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA.
authors.forename: Paige
authors.initials: P
authors.lastname: Chandler
authors.affiliation: Dana Farber Cancer Institute, Boston, MA, 02115, USA.|Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.
authors.forename: Bose S
authors.initials: BS
authors.lastname: Kochupurakkal
authors.affiliation: The Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA.|The Department of Surgery, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA.
authors.forename: Samina
authors.initials: S
authors.lastname: Alam
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, 02115, USA.
authors.forename: Andrea L
authors.initials: AL
authors.lastname: Richardson
authors.affiliation: The Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA.|The Department of Surgery, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA.
authors.forename: David I
authors.initials: DI
authors.lastname: Soybel
authors.affiliation: The Interdisciplinary Graduate Program in Physiology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA. slk39@psu.edu.|The Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA. slk39@psu.edu.|The Department of Pharmacology, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA. slk39@psu.edu.|The Department of Surgery, Penn State Hershey College of Medicine, Hershey, PA, 17033, USA. slk39@psu.edu.
authors.forename: Shannon L
authors.initials: SL
authors.lastname: Kelleher
mesh_terms: D017209:Apoptosis; D001943:Breast Neoplasms / Q000145:classification* / Q000235:genetics / Q000473:pathology*; D027682:Cation Transport Proteins / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D046988:Proteasome Endopeptidase Complex; D015032:Zinc