Prelude therapeutics: Transglutaminase type 2 inhibitors target Kv10.1 channels in triple negative MDA-MB-231 breast cancer cells

Type 2 Transglutaminase (TG2) in addition to being endowed with transmidating activity by which it regulates several extra- and intracellular functions, can also act as membrane G protein. In this context, it has been reported that TG2 is involved in the regulation of potassium current in several models, which however express different types of potassium channels. In this study we have analyzed the intracellular effects of TG2 using specific inhibitors, which could affect not only the catalytic activity, but also the GTPase function, modifying conformational states. We applied this approach using patch-clamp technique coupled to the administration of the specific inibitor NC9 in breast cancer MDA-MB-231 single-cell, because they are rappresentative of triple negative phenotype, a tumour still difficult to treat. We applied a current-voltage protocol ranging from -90 to +70 mV (20 mV step) to control cells (n=14) and NC9 treated cells (n=6). The TG2 inhibitor led to significant decrease in the membrane current. The treatment of MDA-MB-231 cells with AA9, another TG2 inhibitor, was more effective than NC9 decreasing by 90.1±3.3% the +70 mV current-voltage (two way ANOVA, P<0.001). Since the main ion involved in the genesis of the membrane current is the K+ ion, we attempted to identify the possible K+-channel target of TG2. The literature suggests that the most abundant K+-channel in cancer cells is Kv10.1. In order to demonstrate that it is present in MDA-MB-231 cells, we repeated the patch clamp experiments by treating the cells with its specific inhibitor 4-amino-pyridine. This treatment led to reduction of the membrane current equal to 88.4±3.5% comparable to that obtained with AA9. These results suggest an involvement of TG2 in the regulation of the Kv10.1 channels, and this effect is of particular interest because this type of channel, generally expressed in excitable cells, is of exclusive use in MDA-MB-231 cells, in which other potassium channels result mutated or altered. This aspect makes it an important therapeutic target for targeted therapy. The consequences of this alteration in the membrane current remain to be investigated, since it is associated in non-neuronal models with secretion processes, as in the case of pancreatic beta cells, in which TG2 appears to direct the traffic of vesicles for insulin secretion. The analysis of the secretome of these tumor cells will contribute to define other players involved and orchestrated by TG2.