Elevation of Cytoplasmic Calcium Suppresses Microtentacle Formation and Function in Breast Tumor Cells

SIMPLE SUMMARY: Calcium is a versatile and ubiquitous signaling molecule that long-term dysregulation can increase the spread of cancer to various parts of the body but that short-term effects are understudied. Disseminated cancer cells in circulation have distinct extensions or protrusions, called microtentacles, that enhance their ability to attach to surfaces or other cells. In this study, we show rapidly increasing cellular calcium with the compounds of Ionomycin and Thapsigargin decreases the microtentacle frequency and clustering functions on cancer cells in a detached and suspended environment. Acute calcium-induced signaling events promoted changes to actin contraction and rearrangement responsible for suppressing microtentacles. The results from this study support clinical trial data from Thapsigargin derivatives, suggesting Ca(2+) modulating therapies can potentially be used to promote cellular shape and structure changes in free-floating tumor cells to reduce metastasis. ABSTRACT: Cytoskeletal remodeling in circulating tumor cells (CTCs) facilitates metastatic spread. Previous oncology studies examine sustained aberrant calcium (Ca(2+)) signaling and cytoskeletal remodeling scrutinizing long-term phenotypes such as tumorigenesis and metastasis. The significance of acute Ca(2+) signaling in tumor cells that occur within seconds to minutes is overlooked. This study investigates rapid cytoplasmic Ca(2+) elevation in suspended cells on actin and tubulin cytoskeletal rearrangements and the metastatic microtentacle (McTN) phenotype. The compounds Ionomycin and Thapsigargin acutely increase cytoplasmic Ca(2+), suppressing McTNs in the metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-436. Functional decreases in McTN-mediated reattachment and cell clustering during the first 24 h of treatment are not attributed to cytotoxicity. Rapid cytoplasmic Ca(2+) elevation was correlated to Ca(2+)-induced actin cortex contraction and rearrangement via myosin light chain 2 and cofilin activity, while the inhibition of actin polymerization with Latrunculin A reversed Ca(2+)-mediated McTN suppression. Preclinical and phase 1 and 2 clinical trial data have established Thapsigargin derivatives as cytotoxic anticancer agents. The results from this study suggest an alternative molecular mechanism by which these compounds act, and proof-of-principle Ca(2+)-modulating compounds can rapidly induce morphological changes in free-floating tumor cells to reduce metastatic phenotypes.