Chandler Paige, Kochupurakkal Bose S, Alam Samina, Richardson Andrea L, Soybel David I, Kelleher Shannon L
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.
Mol Cancer. 2016 Jan 5;15:2. doi: 10.1186/s12943-015-0486-y.
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.
与正常乳腺上皮相比，锌（Zn）在乳腺肿瘤和恶性细胞系中高度蓄积。导致锌蓄积的机制以及锌失调的后果目前尚不清楚。
利用微阵列评估人类乳腺肿瘤和乳腺癌细胞系中锌转运体和金属硫蛋白（MTs）表达的差异。采用实时PCR和免疫印迹法分析代表性的腔面（T47D）、基底样（MDA-MB-231）和非恶性（MCF10A）细胞系中锌转运体的表达情况。通过X射线荧光成像评估人类肿瘤中锌的分布。使用FluoZin-3成像、定量分析和原子吸收光谱法测量细胞系中锌的分布和含量。分别使用博伊登小室、MTT法和流式细胞术检测MDA-MB-231细胞中锌转运体2（ZnT2）过表达对侵袭、增殖和细胞周期的功能影响。
对人类乳腺肿瘤和乳腺癌细胞系的基因表达谱分析确定了锌转运网络中的亚型特异性失调。乳腺肿瘤组织的X射线荧光成像显示，腔面型乳腺肿瘤边缘锌高度蓄积，而基底样肿瘤中锌分布更均匀。与MCF10A细胞相比，T47D和MDA-MB-231细胞均高度蓄积锌，但T47D细胞蓄积的锌是MDA-MB-231细胞的2.5倍。FluoZin-3成像表明，锌在T47D细胞中被隔离到众多大囊泡中，但在MDA-MB-231细胞中保留在细胞质中，且存在于较少且较大的无定形亚细胞区室中。锌定位的差异反映了锌转运体ZnT2的相对丰度；T47D细胞过表达ZnT2，而MDA-MB-231细胞由于蛋白酶体降解不表达ZnT2蛋白。为了确定MDA-MB-231细胞中缺乏ZnT2的功能相关性，对细胞进行转染以表达ZnT2。ZnT2过表达导致锌囊泡化、细胞周期改变、凋亡增强以及增殖和侵袭减少。
对恶性乳腺肿瘤和细胞系中锌转运网络的全面分析表明，锌管理的不同亚型特异性失调可能是乳腺癌诸如分级、侵袭性、转移潜能和对治疗反应等表型特征的基础。
