Zinc signaling and metallothioneins sub/isoform expression in breast cancer: Implications for prognostic and therapeutic purposes

这项提议是DFG-捷克科学基金会(GACR)合作计划中的一个联合研究项目的德国部分。它将与捷克共和国布尔诺理工大学化学和生物化学系主任Vojtech Adam教授合作进行。我们的两个小组在研究锌及其主要的细胞内结合蛋白金属硫蛋白(MT)的生物学作用方面有着长期的经验。MT与锌的结合受蛋白质不同亚型的表达和翻译后修饰的控制，包括金属结合的半胱氨酸硫醇的氧化和聚合。锌是人体必需的微量元素，由300多种酶和更多的其他蛋白质组成，如转录因子。此外，游离锌作为第二信使，控制哺乳动物细胞的激活、增殖和存活。因此，锌/MT系统可能在癌细胞生长中起着至关重要的作用。本研究旨在利用代表不同乳腺癌亚型的细胞系作为体外模型，阐明锌和MT在不同类型乳腺癌(BCA)中的作用。我们将调查游离锌和总锌的水平和在细胞内的分布，以及不同的MT物种。基于这些数据，我们将研究锌依赖的信号转导在BCA细胞增殖和存活中的作用，特别是在刺激与BCA相关的受体，即表皮生长因子受体、孕激素受体和雌激素受体时。我们将进一步阐明常用的细胞抑制剂(顺铂、阿霉素、依托泊苷)和锌/MT系统之间潜在的相互作用。这包括细胞抑制剂对锌水平、MT和锌依赖信号的影响，以及锌/MT状态对BCA细胞生长、细胞周期分布和活性的影响。在最后的部分中，我们将在一个小鼠模型中可视化锌在组织/肿瘤切片中的分布(动物实验将由Brno的伙伴小组进行)，以将体外发现的结果与体内环境中生长的细胞进行比较。总之，这些结果将确定锌/MT在选定的BCA亚型中的重要性，以便为改善预后和治疗应用奠定基础。最终目的将是利用锌和MT的分布和形态对BCA亚型进行分类，并将锌相关信号作为调控BCA细胞生长和存活的潜在起点。

This proposal constitutes the German part of a joint research project within the DFG- Czech Science Foundation (GACR) cooperation program. It will be performed in collaboration with Prof. Vojtech Adam, head of Department of Chemistry and Biochemistry at Brno University of Technology, Czech Republic. Our two groups have longstanding experience investigating the biological roles of zinc and its main intracellular binding protein, metallothionein (MT). Zinc binding by MT is controlled by expression of different isoforms of the protein, and post-translational modifications including oxidation of metal-binding cysteine thiols and polymerization. The essential trace element zinc is a constituent of over 300 enzymes and an even higher number of other proteins, such as transcription factors. Moreover, free zinc acts as a second messenger, controlling activation, proliferation, and survival of mammalian cells. The zinc/MT system may therefore have a crucial role in cancer cell growth. The present project aims to elucidate the role of zinc and MT in different forms of breast cancer (BCa), using cell lines representing different breast cancer subtypes as in vitro models. We will investigate the levels and intracellular distribution of free and total zinc, as well as the different MT species. Based on these data, we will examine the role of zinc-dependent signal transduction for the proliferation and survival of BCa cells, in particular in response to stimulation of receptors relevant for BCa, namely receptors for Epidermal Growth Factor, Progesterone, and Estrogen. We will further elucidate potential reciprocal interactions between commonly used cytostatics (cisplatin, doxorubicin, etoposide) and the zinc/MT system. This includes the impact of cytostatics on zinc levels, MT, and zinc-dependent signaling, as well as the impact of zinc/MT status on cytostatic effects on growth, cell cycle distribution, and viability of BCa cells. In the final part we will visualize the zinc distribution in tissue/tumor sections in a murine model (animal experiments will be performed by the partner group in Brno) to compare the in vitro findings to cells grown in an in vivo environment. Together, these results shall identify the importance of zinc/MT within selected BCa subtypes, in order to lay the groundwork for improved prognostic and therapeutic applications. The ultimate aims will be utilizing zinc and MT distribution and speciation for classification of BCa subtypes, and zinc-related signaling as a potential starting point for modulating BCa cell growth and survival.