Functional analysis of BK-type oncogenic K+ channels with nanobody-based probes in 2D and 3D HER2-positive human breast cancer cells and mouse models

使用基于纳米抗体的探针在 2D 和 3D HER2 阳性人乳腺癌细胞和小鼠模型中对 BK 型致癌 K 通道进行功能分析

• 批准号: 523982968
• 负责人: Professor Dr. Robert Lukowski
• 金额: --
• 依托单位: Fachrichtung Pharmakologie, Toxikologie und Klinische Pharmazie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/523982968?language=en
• 关键词: Functional; analysis; BK; type; oncogenic

Extracellular potassium ions (K+ex) in the tumor microenvironment (TME) and the activity K+ channels have been shown to impact initiation and progression of solid cancer. In this respect the calcium ion (Ca2+)-and voltage activated K+ channel of big conductance (BK), seems to be of utmost importance. Overexpression of individual subunits of the BK channel has been associated with higher malignancy and poorer prognosis of human breast cancer (BC), while we recently showed that the absence of BK attenuates BC onset and development in relevant preclinical mouse models. Based on a positive correlation between BK status and HER2+ tumors, we hypothesize that modulation of BK activity and the resulting K+ex changes play an important role in regulating malignant BC cell behaviors. However, the precise role of BK, particularly (i) its subunit distribution and interaction in BC cells, (ii) modulatory roles in raising local or global K+ex pools within the TME, and (iii) the functional interplay with HER2-depdendent signaling pathways, remains poorly understood. To address these questions, we will develop novel nanobodies (Nbs) against cancer-associated subunits of BK for advanced optical analyses of BK and its subunits in 2D and 3D BC cell models. In addition, these tools will be combined with our recently engineered Nb-fused FRET-based K+ biosensors specifically targeting human HER2. To investigate the putative effect of BK in modulating functionally relevant changes of K+ex, BK-positive and BK-negative 2D and 3D human HER2+ BC models will be monitored with Nb-based FRET imaging probes and the obtained K+ signals will be correlated with multiple hallmarks of BC malignancy. In parallel, molecular and proteomic analysis of the effects of HER2 signaling on BK and vice versa will provide information on the mechanistic link between these oncogenic factors and their contribution to K+ex in BC. To investigate the relevance of this interplay in vivo, we will generate, pharmacologically manipulate, and intravitally image spontaneously arising and xenografted BK-proficient and BK-deficient human HER2+ BC mouse models. We anticipate that the Nb-based probes developed in this project, in combination with disease-relevant cellular and in vivo models, will guide new opportunities to elucidate the role of K+ channels in cancer development and as potential targets for novel therapeutic approaches in HER2+-related malignancies.

肿瘤微环境 (TME) 中的细胞外钾离子 (K+ex) 和活性 K+ 通道已被证明会影响实体癌的发生和进展。在这方面，钙离子 (Ca2+) 和电压激活的大电导 K+ 通道 (BK) 似乎至关重要。 BK 通道单个亚基的过度表达与人类乳腺癌 (BC) 的恶性程度较高和预后较差有关，而我们最近表明，在相关临床前小鼠模型中，BK 的缺失会减弱 BC 的发病和发展。基于 BK 状态与 HER2+ 肿瘤之间的正相关性，我们假设 BK 活性的调节以及由此产生的 K+ex 变化在调节恶性 BC 细胞行为中发挥重要作用。然而，BK 的确切作用，特别是 (i) 它的亚基分布和在 BC 细胞中的相互作用，(ii) 在 TME 内提高局部或全局 K+ex 库的调节作用，以及 (iii) 与 HER2 依赖的信号通路的功能相互作用，仍然知之甚少。为了解决这些问题，我们将开发针对 BK 癌症相关亚基的新型纳米抗体 (Nbs)，以便在 2D 和 3D BC 细胞模型中对 BK 及其亚基进行高级光学分析。此外，这些工具将与我们最近设计的基于 Nb 融合 FRET 的 K+ 生物传感器相结合，专门针对人类 HER2。为了研究 BK 在调节 K+ex 功能相关变化中的假定效果，将使用基于 Nb 的 FRET 成像探针监测 BK 阳性和 BK 阴性 2D 和 3D 人类 HER2+ BC 模型，并将获得的 K+ 信号与 BC 恶性肿瘤的多个标志相关。同时，对 HER2 信号传导对 BK 的影响进行分子和蛋白质组学分析，反之亦然，将提供有关这些致癌因素及其对 BC 中 K+ex 的贡献之间的机制联系的信息。为了研究这种体内相互作用的相关性，我们将生成、药理学操作和活体成像自发产生和异种移植的 BK 丰富和 BK 缺陷的人类 HER2+ BC 小鼠模型。我们预计，该项目中开发的基于 Nb 的探针，与疾病相关的细胞和体内模型相结合，将为阐明 K+ 通道在癌症发展中的作用提供新的机会，并作为 HER2+ 相关恶性肿瘤新治疗方法的潜在靶点。