Figure 6. BK_Ca-DEC expression contributes to the metabolic remodeling and growth of murine and human BCCs and is present in primary tumor samples.

(A) Schematic representation of the fate of glucose in glycolysis. The tricarboxylic acid (TCA) cycle or lactate secretion via monocarboxylate transporters (MCT) can be inhibited, either using NaN₃ or BAY-8002. GLUT: Glucose transporter, HKs: Hexokinases. (B) Representative cytosolic lactate concentration ([LAC]_cyto) of a MMTV-PyMT WT cell over-time in response to administration or removal of NaN₃ and BAY-8002 at time points indicated. Dashed lines indicate slopes taken for assessment of the ‘Warburg index’. (C – G) Average Warburg indices ± SEM of MMTV-PyMT WT (C, F, left), MMTV-PyMT BK-KO (C, F, right), MDA-MB-453 cells (D, G) and MCF-7 cells (E) calculated from the experiments as shown in (B), either under control conditions, in the presence of paxilline or iberiotoxin (C, D), upon expression of BK_Ca^RFP or BK_Ca-DEC^RFP (E), or upon cell treatment with a scrambled siRNA (siScrbl), or siRNA against a common BK_Ca sequence targeting all known splice variants (siBK), or a siRNA specifically designed to knockdown BK_Ca-DEC (siDEC) (F, G). (H, I) Normalized MTT absorbance over-time of MMTV-PyMT WT (H, left) and BK-KO cells (H, right), and MDA-MB-453 cells (I), either under control conditions, or in the presence of paxilline or iberiotoxin. (J), Representative images and corresponding statistics of colony formation assays using MMTV-PyMT WT or BK-KO cells in the presence or absence of O₂. (K – N) mRNA expression of BK_Ca and BK_Ca-DEC as performed by Nanostring analysis of 551 BC patient samples. (K) Log2 expression counts of BK_Ca and BK_Ca-DEC. The threshold for positive expression level was set to log2 = 5.5 (dashed line). (L) Log2 expression counts of BK_Ca-DEC blotted over the log2 expression counts of BK_Ca. 10 of the 551 patient samples showed expression of BK_Ca-DEC above the threshold of log2 = 5.5 (dashed line), whereas 541 patient samples were BK_Ca-DEC negative. (M) Correlation of the log2 expression counts of BK_Ca-DEC positive samples with the log2 expression counts of BK_Ca in the primary human BC material. (N) Summarizing scheme of BK_Ca in cancer cell homeostasis. N (independent experiments) / n (cells analyzed) = (C): 4/26 WT ctrl, 6/28 WT +PAX, 4/39 WT +IBTX, 4/17 BK-KO ctrl, 5/18 BK-KO +PAX, 4/27 BK-KO +IBTX, (D): 7/29 ctrl, 5/13+PAX, (E): 5/27 ctrl, 5/20+BK_Ca^RFP, 7/26 BK_Ca-DEC^RFP, (F): 5/22 WT siScrbl, 5/28 WT siBK, 4/26 WT siDEC, 5/24 BK-KO siScrbl, 5/24 BK-KO siBK, 5/29 BK-KO siDEC, (G): 5/21 siScrbl, 5/22 siBK, 5/19 siDEC, (H – J): 4 for all. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (C, E, F, G, I), One-Way ANOVA test followed by Tukey’s MC test (H) or Mann-Whitney test (D). †p≤0.01, ‡p≤0.001 compared to respective WT condition, Unpaired t-test ctrl, (C, J) or Mann-Whitney test (+IBTX, C, F).

Figure 6—figure supplement 1. Effects of siRNA treatment on expression of BK_Ca and BK_Ca-DEC, and physiologic consequences of BK_Ca inhibition in BCCs.

(A) Schematic representation of siRNAs used for subsequent silencing experiments. Either an siRNA targeting all known BK_Ca isoforms, referred to as siBK (orange), or an siRNA specifically designed against the DEC exon (green), referred to as siDEC (violet), were used. (B, C) Relative mRNA expression levels ± SEM of BK_Ca and BK_Ca-DEC in MMTV-PyMT WT (B) and MDA-MB-453 cells (C), as analyzed by qPCR. Cells were either treated with a scrambled siRNA as a control (siScrbl), siRNA against all known BK_Ca isoforms (siBK) or siRNA specifically targeting BK_Ca-DEC (siDEC), respectively. N = 4 siBK MMTV-PyMT WT, 3 for all others. (D–F) Representative images (D) and corresponding statistics (E, F) of MMTV-PyMT WT cells (D, upper line and E, left panel), MMTV-PyMT BK-KO cells (D, middle line and E, right panel) and MDA-MB-453 cells (D, lower line and F) immune-stained for the proliferation marker KI-67 (Alexa488, green), and Hoechst-33342 (blue) for visualizing all nuclei. Experiments were either performed under control conditions (ctrl, left column), or in the presence of 5 µM paxilline (+PAX, middle column) or iberiotoxin (+IBTX, right column). *p≤0.05, Kruskal-Wallis test followed by Dunn’s MC test. #p≤0.05 compared to respective WT condition, Mann-Whitney test. N = 5 for WT ctrl, BK-KO +PAX and MDA-MB-453+IBTX, N = 6 for all others. (G) Normalized colony sizes of colony formation assays performed using MMTV-PyMT WT and BK-KO cells. Cells were cultivated for 7 days either in the presence or absence of O₂. *p≤0.05, Mann-Whitney test. N = 4 independent experiments for all conditions.