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Gastroenteropancreatic neuroendocrine tumors (GEPNETs) of the midgut are a heterogeneous group of cancers that typically remain asymptomatic until they metastasize to the liver. However, the mechanism by which these usually indolent neoplasms establish distal metastasis remains unclear. The results presented in this dissertation support the hypothesis that arachidonic acid-induced Ca2+ entry through an Orai3-containing channel enhances GEPNET cell migration and contribute to tumor cell extravasation through the hepatic microvasculature. Using a novel liver slice preparation in combination with ultrastructure analysis, we demonstrated that a subpopulation of BON cells, a well-characterized GEPNET cell line, opportunistically migrated through the discontinuous sinusoidal endothelium and redistributed into the liver parenchyma within 24 hours following their introduction to the sinusoids. In a parallel set of studies, we identified a novel Ca2+ entry pathway through an Orai-containing channel that enhanced the migratory capacity of a subpopulation of these tumor cells. Orai-containing channels are known to underlie Ca2+ entry in response to both store-depletion and activation by arachidonic acid (AA) or its metabolites. In recent years, the store-operated Ca2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. However, the role of AA-induced Ca2+ entry in cancer cell migration has not been adequately assessed. We investigated the involvement of AA-induced Ca2+ entry in migration in BON cells using live cell fluorescence imaging and standard migration assays in combination with pharmacological and gene knockdown methods. We showed that both SOCE and AA-induced Ca2+ entry modes could be selectively activated and inhibited. Administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Moreover, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-activated Ca2+ channel required expression of Orai3 as well as, Orai1. Furthermore, we showed that treatment with AA enhanced migration of BON cells and that this enhancement could be abrogated by selective inhibition of the AA-induced Ca2+ entry, whereas activation of SOCE did not enhance migration. In addition, the AA-induced Ca2+ entry and SOCE appeared to be reciprocally repressive suggesting the possibility that the balance between these two pathways sets the migratory potential of these tumor cells.Taken together, we identified Ca2+ entry through the Orai3-containing channel as a novel signal for BON cell migration that may be exploited to develop therapies to limit recurring GEPNET metastasis.

The Orai3 protein forms a Ca2+ channel with a pharmacological profile distinct from its close relative, the store-operated Ca2+ channel Orai1. Though closely related to Orai1, the function of Orai3 in humans is still unclear. This study attempts to contribute to the body of knowledge by undertaking a pharmacological analysis of Orai3 in insect cells. We describe here the creation of a vector capable of expressing the mammalian Orai3 gene on an insect background. We demonstrate the ability to induce gene expression of Orai3 in these insect cells, and then assess the effectiveness of this system by characterizing the pharmacological properties with the drug 2-aminoethoxydiphenyl borate (2-APB). The results show that the current strategy used to express Orai3 will require additional refinement before the system can be considered generally useful for pharmacological studies, because expression of Orai3 may be affected by native proteins. The interference of expression seems confined to Orai Ca2+ channels, as mammalian STIM1 was also expressed and a response to 2-APB, albeit unexpected, was observed.