Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.

Neuronal Calcium Sensor 1 is up-regulated in response to stress to promote cell survival and motility in cancer cells.

Changes in intracellular calcium (Ca2+ ) signaling can modulate cellular machinery required for cancer progression. Neuronal calcium sensor 1 (NCS1) is a ubiquitously expressed Ca2+ -binding protein that promotes tumor aggressiveness by enhancing cell survival and metastasis. However, the underlying mechanism by which NCS1 contributes to increased tumor aggressiveness has yet to be identified. In this study, we aimed to determine (a) whether NCS1 expression changes in response to external stimuli, (b) the importance of NCS1 for cell survival and migration, and (c) the cellular mechanism(s) through which NSC1 modulates these outcomes. We found that NCS1 abundance increases under conditions of stress, most prominently after stimulation with the pro-inflammatory cytokine tumor necrosis factor α, in a manner dependent on nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). We found that NFκB signaling is activated in human breast cancer tissue, which was accompanied by an increase in NCS1 mRNA expression. Further exploration into the relevance of NCS1 in breast cancer progression showed that knockout of NCS1 (NCS1 KO) caused decreased cell survival and motility, increased baseline intracellular Ca2+ levels, and decreased inositol 1,4,5-trisphosphate-mediated Ca2+ responses. Protein kinase B (Akt) activity was decreased in NCS1 KO cells, which could be rescued by buffering intracellular Ca2+ . Conversely, Akt activity was increased in cells overexpressing NCS1 (NCS1 OE). We therefore conclude that NCS1 acts as cellular stress response protein up-regulated by stress-induced NFκB signaling and that NCS1 influences cell survival and motility through effects on Ca2+ signaling and Akt pathway activation.