When huffing and puffing Ca²⁺ goes global, breast cancer cells are unmoved

Ca²⁺ signaling mediates numerous cell functions, including cell proliferation and migration, and as such plays a dominant role in many aspects of cancer (1). The canonical receptor-evoked Ca²⁺ signal is initiated by activation of PLC that hydrolyzes PI(4,5)P₂ to IP₃ and diacylglycerol. IP₃ opens IP₃ receptors (IP₃Rs), releasing Ca²⁺ stored in the ER to increase cytoplasmic Ca²⁺ ([Ca²⁺]_i) rapidly. Ca²⁺ release from the ER causes unfolding and homo or hetero dimerization of the ER Ca²⁺ sensors STIM1 and STIM2. STIM1 recruits and activates primarily the Ca²⁺ influx channel Orai1. Ca²⁺ influx sustains the intracellular Ca²⁺ signal and provides the Ca²⁺ needed to replenish the ER Ca²⁺ store. In addition to activating Orai1, STIM proteins tether the ER and PM to form and stabilize junctions between them. In fact, the ER/PM junctions are where Ca²⁺ signaling complexes are formed and the Ca²⁺ signal is initiated and regulated (2, 3). The function of all pathways generating the receptor-evoked Ca²⁺ signal, including STIM1 and STIM2, are involved in the classical and emerging hallmarks of cancers (1).

Physiologically, most Ca²⁺ signals are in the form of repetitive short Ca²⁺ increases called Ca²⁺ puffs that are confined to cellular domains and which only infrequently globalize to cause a large and sustained increase in [Ca²⁺]_i. Ca²⁺ puffs involve repetitive transient activation of IP₃Rs to transiently release Ca²⁺ from the ER; the frequency of Ca²⁺ puffs is determined by the strength of cell stimulation, and thus the level of IP₃. Confined Ca²⁺ puffs are common in polarized cells like exocrine, neuronal, and muscle cells, as well as at the leading edge of migrating cells (4, 5). However, the mechanism of transition from Ca²⁺ puffs to global Ca²⁺ signals is not well understood, nor is the role of each form of the Ca²⁺ signal patterns in cell proliferation, migration, and tumorigenesis. A study in this issue by Militsin et al. (6) provides fresh insight on these two imperative questions.

STIM1 and STIM2 play a unique role in Ca²⁺ signaling by mediating the communication between the cell surface (PM) and cell interior (ER) and by dynamically regulating cell signaling hubs at the ER/PM junctions (7, 8). Militsin et al. examined the role of STIM1 and STIM2 in the MDA-MB-231 breast cancer cell line by generating STIM1^−/− (S1KO), STIM2^−/− (S2KO), and STIM1^−/−+STIM2^−/− double knockout (S1/S2DKO) cancer cells. Measuring in vitro cell proliferation and colony formation and in vivo tumor growth revealed that both STIMs are required for these activities. However, a surprising observation was made when comparing the effects of S1KO, S2KO, and S1/2DKO on cell adhesion, cell migration, and focal adhesion. While S1KO and S2KO cells showed reduced activity, S1/2DKO cells showed normal cell adhesion, migration, and focal adhesion. Interestingly, cell migration and focal adhesion indicate formation of polarized cell domains, which are controlled by dynamic ER/PM junction gradients (9), in which the STIM isoforms have critical roles (7, 8).

These findings not only reveal the participation of STIM isoforms in breast cancer initiation, migration, and invasion but also raise the question of why removing the two STIM isoforms restored, rather than eliminated, their involvement in these activities. Both pharmacological inhibitors and genetic modifications demonstrated that the unexpected results in S1/2DKO cells were due to altered IP₃R function (6). Moreover, analysis of the pattern of Ca²⁺ signals evoked by IP₃R activation in cells expressing the various STIM combinations revealed a novel mechanism of STIM-mediated IP₃R gating. Additionally, the Ca²⁺ signal pattern had a novel and remarkable role in cell migration and cancer invasion (6). As illustrated in Fig. 1, controlled IP₃R activation in WT breast cancer cell lines resulted in periodic Ca²⁺ puffs that never globalized. By contrast, similar IP₃R activation in S1KO and S2KO resulted in Ca²⁺ puffs that rapidly globalized to generate a diffuse Ca²⁺ pattern. Notably, IP₃R activation in S1/2DKO resulted in periodic Ca²⁺ puffs that never globalized. Overall, the findings by Militsin et al. provide strong support that STIM1 and STIM2 facilitate the opening of IP₃Rs, in addition to their well-established role in opening Orai channels. By opening IP₃R, STIMs set the Ca²⁺ signaling pattern to either Ca²⁺ puffs or a diffuse global increase in [Ca²⁺]_i. In cancer, the Ca²⁺ puff pattern supports cell migration and invasion, while global [Ca²⁺]_i induction restricts cell migration.

The findings by Militsin et al. also raise the key questions of how STIMs regulate IP₃Rs and why Ca²⁺ signaling in S1/2DKO cells does not globalize. The authors suggest STIMs indirectly regulate IP₃R gating since the two proteins could not be co-immunoprecipitated in MDA-MB-231 cell lysates. However, co-immunoprecipitation of native STIM1-IP₃Rs and FRET between the expressed proteins as part of a signaling complex has been reported in several studies (for example, [3]). Therefore, modification of IP₃R function by the N terminus of STIM1 and STIM2, which has been described in colorectal cancer (10), may partly account for STIM-mediated IP₃R gating. An additional mechanism can be IP₃R sensitization by PI(4,5)P₂, which facilitates Ca²⁺ release (11). STIMs assemble Ca²⁺ signaling complexes at PI(4,5)P₂-rich domains that may be necessary for PI(4,5)P₂-mediated sensitization of IP₃Rs and globalization of the Ca²⁺ signal. Finally, the most vital mechanism may be the control of ER Ca²⁺ content by STIMs. ER Ca²⁺ content controls STIM clustering (7) and IP₃R activity (12), whereas STIM deletion reduces ER Ca²⁺ content (6). Reduced ER Ca²⁺ would, therefore, be expected to cluster STIM1 and STIM2 homodimers at the ER/PM junctions to facilitate and enhance IP₃R-mediated Ca²⁺ release and globalize the Ca²⁺ signal (Fig. 1, middle).

Altered IP₃R clustering may account for the puzzling observations in S1/2DKO cells. IP₃-mediated Ca²⁺ release increases with increased IP₃R clustering (4). IP₃R clustering takes place at the ER/PM junctions (3), and STIM proteins have a major role in ER/PM junction formation and stabilization (7, 8). In S1/2DKO cells, the complete absence of STIM proteins reduces ER/PM junction formation, thereby preventing IP₃R clustering. This mechanism may underlie the failure of Ca²⁺ puffs to globalize in complete STIM deficiency (Fig. 1, right), although other mechanisms may also contribute. Considering the critical role of STIM in regulation and shaping Ca²⁺ signal patterns in cancer and the observation that STIM expression changes during tumorigenesis (10), it is important and clinically relevant to further explore the mechanisms by which STIMs gate IP₃Rs to shape Ca²⁺ signals.