Calcium channel remodeling in colon cancer cellsimplications for channel inactivation and reversal by polyamine synthesis inhibition

Resumen

Colorectal cancer is one of the most prevalent and deadly forms of cancer, particularly in Spain. Ongoing clinical trials are aimed at preventing colorectal cancer, especially in those patients at high risk. Combination of difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, with sulindac, a non-steroidal anti-inflammatory drug (NSAID) is yielding promising results but the mechanisms are not well known. Our group has proposed that intracellular Ca2+ homeostasis is remodeled in colorectal cancer. Particularly, colorectal cancer cells displayed changes in store-operated channels that make them more sustained and contribute to cancer hallmarks. Accordingly, in this work we asked whether SOC remodeling influences SOC inactivation and whether cancer chemopreventive compounds may prevent colorectal cancer acting on SOC remodeling and inactivation. To address these issues we measure store-operated Ca2+ entry (SOCE) and store-operated currents in normal colonic and colorectal cancer cells using calcium imaging and patch-clamp electrophysiology. Expression of molecular players was tested by qRT-PCR, western blotting and immunofluorescence. Formation of channel complexes was tested using co-immunoprecipitation. Apoptosis resistance and cell proliferation were tested by flow cytometry and cell counting was estimated using a cell counter. Finally, ROS production was measured using fluorescence imaging. The results show that cancer cells over express ornithinedecarboxylase, the limiting step in polyamine synthesis. In addition, ODC inhibition by DFMO reverses Ca2+ remodeling and cancer cell hallmarks. Specifically, polyamine depletion induced by DFMO downregulates TRPC1 and STIM1, decreases channel complex between these molecular players and reverses SOC remodeling in cancer cells. In addition, the results show that the mitochondrial Na+/Ca2+ exchanger (NCLX) is overexpressed in colon cancer cells and contribute together with enhanced Na+ influx by TRPC1 channels to clear mitochondrial Ca2+ loads, thus preventing ROS production and ROS-induced inactivation of SOCE. In summary, our results suggest that SOC remodeling in cancer prevent SOC inactivation leading to sustained Ca2+ signaling. In addition, they provide evidence that colorectal cancer chemoprevention by DFMO, particularly when combined with sulindac, reverses SOC remodeling in colorectal cancer leading to channel inactivation.