Small Molecule-facilitated Degradation of ANO1 Protein: A NEW TARGETING APPROACH FOR ANTICANCER THERAPEUTICS

ANO1, a calcium-activated chloride channel, is highly expressed and amplified in human cancers and is a critical survival factor in these cancers. The ANO1 inhibitor CaCC(inh)-A01 decreases proliferation of ANO1-amplified cell lines; however, the mechanism of action remains elusive. We explored the mechanism behind the inhibitory effect of CaCC(inh)-A01 on cell proliferation using a combined experimental and in silico approach. We show that inhibition of ANO1 function is not sufficient to diminish proliferation of ANO1-dependent cancer cells. We report that CaCC(inh)-A01 reduces ANO1 protein levels by facilitating endoplasmic reticulum-associated, proteasomal turnover of ANO1. Washout of CaCC(inh)-A01 rescued ANO1 protein levels and resumed cell proliferation. Proliferation of newly derived CaCC(inh)-A01-resistant cell pools was not affected by CaCC(inh)-A01 as compared with the parental cells. Consistently, CaCC(inh)-A01 failed to reduce ANO1 protein levels in these cells, whereas ANO1 currents were still inhibited by CaCC(inh)-A01, indicating that CaCC(inh)-A01 inhibits cell proliferation by reducing ANO1 protein levels. Furthermore, we employed in silico methods to elucidate novel biological functions of ANO1 inhibitors. Specifically, we derived a pharmacophore model to describe inhibitors capable of promoting ANO1 degradation and report new inhibitors of ANO1-dependent cell proliferation. In summary, our data demonstrate that inhibition of the channel activity of ANO1 is not sufficient to inhibit ANO1-dependent cell proliferation, indicating that the role of ANO1 in cancer only partially depends on its function as a channel. Our results provide an impetus for gaining a deeper understanding of ANO1 modulation in cells and introduce a new targeting approach for antitumor therapy in ANO1-amplified cancers.