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

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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.