Altered expression of stromal interaction molecule (STIM)-calcium release-activated calcium channel protein (ORAI) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) in cancer: will they become a new battlefield for oncotherapy?

Calcium signals are widespread and rigorously regulate the majority of fundamentally important physiologic processes ranging from cell proliferation to cell apoptosis [1]. The precise and tightly controlled intracellular calcium ion concentration depends on finely tuned modulation by various calcium-transporting processes, including Ca²⁺ channels, pumps, and receptors [2]. These Ca²⁺-transporting molecules strictly regulate the transient or sustained waves, spikes, or oscillations of Ca²⁺ signaling in different cellular compartments and microdomains to maintain a delicate balance between feeding into the cytoplasm and releasing from internal Ca²⁺ stores [3]. Any perturbation and disorder of the delicate Ca²⁺ homeostasis may lead to long-ranging consequences. Therefore, it is not surprising that any derangement of Ca²⁺ channels and/or receptors will contribute to the establishment of many life-threatening diseases, such as cardiopathy [4], heart failure [5], neurodegenerative diseases [6], and cancer [7]. Remarkably, the altered expression or activity of these Ca²⁺ channels or receptors is characterized by the features of specific cancer subtypes, of which the most important is the protein complex consisting of the stromal interaction molecule (STIM), calcium release-activated calcium channel protein (ORAI), and inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs). STIM–ORAI is able to sense and respond to intracellular Ca²⁺ microenvironmental changes that occur during cancer development. This complex primarily mediates the Ca²⁺ influx, with STIM serving as the endoplasmic reticulum (ER) Ca²⁺ sensor and ORAI as the Ca²⁺-selective entry channel. Furthermore, the roles of IP₃Rs are also discussed. There is emerging evidence that IP₃Rs, which regulate the Ca²⁺ flux from the ER into the cytosol and mitochondria, play crucial roles in the apoptotic pathway; additionally, IP₃Rs have been implicated in cellular senescence [3]. The last decade in clinical oncology has been noteworthy because of advances in our understanding of the derangement of Ca²⁺ channels/transporters that are thought to be responsible for the development of cancers [8]. Although enormous explorations have been performed, the molecular mechanisms by which these derangements affect tumorigenesis and tumor progression are far from being fully understood. In this review, we discuss recent progress in understanding the roles of STIM–ORAI and IP₃Rs, with a focus on exploring the mechanism underlying the hijacking of the Ca²⁺-transporting molecules STIM-ORAI and IP₃Rs by malignant cancer cells that leads to tumor onset, growth, and metastasis. The concomitant interaction between STIM–ORAI and IP₃Rs is also discussed. Understanding the molecular basis and pathologic transformations of Ca²⁺-transporting molecules in cancer cells will offer an opportunity for pharmacologic modulation and therapeutic intervention.

External stimulation leads to an increase in cytoplasmic Ca²⁺ from either the entry of extracellular Ca²⁺ across the plasma membrane or the release of Ca²⁺ from internal calcium stores in the ER and sarcoplasmic reticulum (SR) [9]. Both of these functions involve the permeable Ca²⁺ channels that are located on the plasma membrane. Upon the stimulation, extracellular Ca²⁺ can enter the cytoplasm through Ca²⁺ channel transport. Moreover, the plasma membrane is responsible for refilling the internal Ca²⁺ stores when they are depleted. The primary Ca²⁺ entry pathway is store-operated Ca²⁺ entry (SOCE), which includes two key components: a sensitive sensor of calcium store depletion (STIM) and an effective channel that can facilitate calcium entry into the cell (ORAI) [10–12].

STIM, which is predominantly located in the ER, was identified using an RNA interference screen in Drosophila S2 cells; then, two mammalian orthologs (STIM1 and STIM2) were found [13]. Both STIM1 and STIM2 act as sensors of Ca²⁺ store levels in the ER and control calcium refilling by forming connections with ORAI [14]. STIM1 contains an ER luminal N-terminus and a cytosolic C-terminus. The ER luminal portion consists of a canonical Ca²⁺-binding EF-hand (a conventional helix-loop-helix EF motif), a hidden EF hand, and a sterile α-motif (SAM) domain. The cytosolic strand includes three putative coiled-coil (CC1, CC2, and CC3) regions, calcium release-activated calcium (CRAC) modulatory domain (CAD) or a STIM-ORAI-activating region (SOAR), serine or proline-rich segments, and lysine-rich regions [15]. The low Ca²⁺-binding affinity of EF-SAM perfectly matches the detailed alteration of the Ca²⁺ concentration and enables the ER sensor protein to respond to changes in the Ca²⁺ concentration in ER. The Ca²⁺ depletion in the ER leads to the dissociation of Ca²⁺ from the EF hand, thereby destabilizing the entire EF-SAM entity. The CC regions and the serine/proline-rich region promote the oligomerization of STIM, thereby enabling its redistribution into multiple punctae and its localization at ER-plasma membrane junctions. The CC domain has been proven to control the exposure and oligomerization of the SOAR [16]. The structure of STIM2 is similar to that of STIM1; however, STIM1 is widely expressed at both the cell surface and the ER, whereas STIM2 is expressed only in the intracellular space. A growing number of studies have indicated that STIM2 is a potent inhibitor and a feedback regulator of STIM1 via preventing it from forming an aggregate to stabilize basal concentrations of Ca²⁺ in the cytosol and ER [17]. Thus, STIM2 is regarded as a critical regulator of basal Ca²⁺ levels in the human signaling proteome, whereas STIM1 seems to be more involved in the Ca²⁺ entry associated with more pronounced depletion [18, 19].

The ORAI gene encodes a family consisting of three proteins (ORAI1, ORAI2, and ORAI3). Each of these proteins consists of 4 TM-spanning segments and 3 cytosolic strands, which include the N-terminus, the second loop con-necting TM2 and TM3, and the C-terminus [20]. The ORAI1 C-terminus forms the cytosolic extension, and the C-terminal putative CC domain is important for binding to the SOAR/CAD domain of STIM1. By binding to the intracellular C-terminus, STIM1 recruits ORAI to the puncta in the plasma membrane.

The primary intracellular hysiologic functions of STIM and ORAI are straightforward (Fig. 1a). STIM detects the decrease in Ca²⁺ stores in the ER and moves within the ER to ER-plasma membrane junctions. Then, STIM recruits ORAI to the ER-plasma membrane junctions, where the two proteins form a close contact. The formation of the active STIM–ORAI complex at the conformational gate of the SOCE channel allows Ca²⁺ entry [21].

The IP₃R is the most ubiquitous intracellular Ca²⁺ channel, and its isoforms (IP₃R1, IP₃R2, and IP₃R3) have been identified in vertebrates [22]. The majority of cell types express more than one isoform but have a predominant one. The three IP₃R isoforms have distinct but overlapping expression patterns. IP₃R1 is expressed in neuronal cells, IP₃R2 is expressed in liver and muscle cells, and IP₃R3 is expressed in most cultured cell types [23]. The general domain structure of IP₃Rs (which exist as tetramers) has been determined; IP₃Rs contain a binding site for IP₃ in the N-terminal region, the channel domain, and the determinants for tetramer formation in the C-terminus [24, 25].

IP₃Rs predominantly reside in the ER. IP₃ is produced by phospholipase C and binds to IP₃Rs to induce calcium release from the ER upon cell activation by endogenous or exogenous hormones, growth factors, or neurotransmitters. Notably, IP₃-induced Ca²⁺ release is typically regulated by the Ca²⁺ concentration in the cytosol and ER. Furthermore, the activities of IP₃Rs are biphasically regulated by cytosolic Ca²⁺. The concentration–response relationship is a typical bell-shaped curve, indicating that the IP₃-mediated Ca²⁺ release is potentiated at a low Ca²⁺ concentration and inhibited at a higher concentration [26]. The Ca²⁺ storage in the lumen of the ER also regulates IP₃Rs, which can prevent excessive ER depletion at low levels of store filling [27]. IP₃R-mediated Ca²⁺ elevation regulates fundamental cellular functions, such as fertilization, cell cycle entry, cell division, metabolism, and transcription [28]. An important function of IP₃Rs is to decide the cell fate by controlling the mitochondrial Ca²⁺ elevation and mitochondrial metabolism. Cell survival or apoptosis is encoded in the frequency and amplitude of Ca²⁺ oscillations mediated by IP₃Rs and decoded by different Ca²⁺-sensitive kinases or phosphatases that in turn regulate the target proteins. When IP₃Rs transport appropriate amounts of Ca²⁺ from the ER to the mitochondria, they catalyze the conversation of pyruvate to acetyl-coenzyme A (CoA) to produce adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide 2′-phosphate (NADPH). The insufficient transport of Ca²⁺ to the mitochondria induces cellular autophagy. Conversely, when activated IP₃Rs excessively transport Ca²⁺ from the ER to the mitochondria, the mitochondrial Ca²⁺ is overloaded, which induces a dissipation of the mitochondrial potential, the opening of the permeability transition pore, and the release of pro-apoptotic factors such as cytochrome c [29]; this process ultimately triggers cell apoptosis. Therefore, IP₃Rs play pivotal roles in the apoptotic process via controlling the cellular response to apoptotic signals and conferring oncogenic features to the cell [30].

STIM and ORAI have been found to be abundantly expressed in human cancer tissues and multiple tumor cell lines. Abnormal spatial and temporal changes in these two proteins have been found to be involved in many aspects of tumorigenesis, including cancer cell proliferation, migration, and apoptosis resistance.

Growing attention has been paid to the special role of IP₃Rs in tumorigenesis and tumor metastasis. Over the last 20 years, IP₃Rs have been regarded as key regulators that control cell death and survival in a variety of cellular systems. Interfering with the amount of IP₃R-mediated Ca²⁺ transport from the ER to the mitochondria determines the susceptibility of cells to apoptotic stimulation. Because IP₃Rs can promote senescence and/or apoptosis, the available evidence indicates that down-regulating IP₃Rs or dampening their activities can decrease cellular sensitivity to apoptotic signaling, finally resulting in the survival of cells with oncogenic features. An in vitro study showed that knockdown of IP₃R1 prevented apoptosis in bladder cancer cells and rendered them resistant to chemotherapeutics [58]. Conversely, overexpression of IP₃Rs might increase the sensitivity of cancer cells to cisplatin [59]. As the molecular bridge between the ER and mitochondria, IP₃Rs are also hijacked by different proto-oncogenes to give rise to cells with oncogenic features [60], such as Akt/protein kinase B (PKB) [61], Bcl-2 family members [62, 63], Bax inhibitor-1 (BI-1) [64, 65], and K-ras-induced actin-interacting protein (KRAP) [66]. Recently, it has become clear that Bcl-2 directly targets the central modulation domain of IP₃Rs through its tetrahydrobiopterin (BH4) domain to inhibit their functions [61]. The spatiotemporal interaction of BH4 and IP₃Rs hindered mitochondrial Ca²⁺ accumulation by abrogating Ca²⁺ transport from the ER to the outer mitochondrial membrane (Fig. 1d), therefore, the IP₃R-BH4 complex counteracted the pressure of pro-apoptotic proteins to protect tumor cells [67]. The Bcl-2 family was also shown to enhance basal Ca²⁺ leakage through sensitization of IP₃Rs to basal IP₃ levels lower than the Ca²⁺ concentration in the ER [68, 69]. The low levels of Ca²⁺ in the ER destroyed the mitochondrial Ca²⁺ overload and decreased the susceptibility of the cells to apoptosis. Importantly, a peptide tool that was designed to disrupt the IP₃R-BH4 complex could effectively induce an intracellular Ca²⁺ overload and provoke cell death in diffuse large B cell lymphoma (DLBCL) cells [70]. However, Kang et al. [71] reported that the invasion and migration of tumor cells were suppressed by caffeine, which is a well-known inhibitor of IP₃Rs. The expression levels of IP₃R3 in colon cells were directly related to tumor aggressiveness [72]. These results suggest that the regulatory mechanisms of IP₃Rs may vary in different types of cancer, and many mechanisms are not fully understood.

The binding of IP₃ to IP₃Rs releases intracellular Ca²⁺, leading to a reduction in the Ca²⁺ concentration in the lumen of ER, which in turn activates the STIM sensor to allow extracellular Ca²⁺ to refill the empty ER Ca²⁺ stores across the ORAI in the plasma membrane. In rapidly growing cancers, IP₃Rs are blocked by a variety of anti-apoptosis proteins, resulting in Ca²⁺ overload in the ER. The harsh microenvironment perturbs the STIM-ORAI functions and induces the accumulation of misfolded proteins in the ER. This triggers an adaptation program referred to as “ER stress.” Chronic ER stress kills normal cells but can contribute to tumor cell dormancy, thereby permitting survival in the stressed environment until more favorable conditions are encountered. Overexpression of STIM could reverse ER stress, implying that Ca²⁺ overload restrains the STIM functions in cancer cells [73]. Oncogenic KRAS mutations could reduce the Ca²⁺ store content in the ER via promoting IP₃R1 overexpression to suppress agonist-induced Ca²⁺ release and mitochondrial Ca²⁺ accumulation in cancer cells [74]. Nevertheless, the relationships between STIM–ORAI and IP₃Rs are not completely understood, and further investigations are needed to elucidate the mechanisms by which cancer cells control the functions of STIM-ORAI and IP₃Rs.

Multiple roles of STIM–ORAI and IP₃Rs in several types of human cancer have made them attractive drug targets for tumor therapy. Inhibiting ORAI1 by pharmacologic antagonists in cultured epithelial cells derived from ESCC patients impeded ESCC cell proliferation, invasion, and migration [75]. Importantly, the growth of ESCC in vivo was significantly suppressed when ORAI1-mediated SOCE was knocked down by siRNA or blocked by pharmacologic inhibitors in xenografted nude mice. SKF-96365 and 2-APB, which are inhibitors of store-operated Ca²⁺ entry, inhibited the growth and metastasis of tumor cells after 1 week of treatment [76, 77]. No increase in metastasis was observed in mouse cancer models [78], even 2 weeks after withdrawal of SKF-96365. Similar phenomena have been found in cervical and esophageal cancer mouse models.

To date, “proof of principle” studies of the Ca²⁺ signal channels have shown that STIM-ORAI and IP₃Rs either do not differ or are overexpressed in tumor tissues compared to those in normal tissues. However, the roles of STIM-ORAI and IP₃Rs may be over- or underestimated depending on the use of the pharmacologic inhibitors or siRNA-mediated gene knockdown approaches in cancer cells. Moreover, only a relatively limited amount of information concerning STIM-ORAI and IP₃Rs is available to date due to their complicated and comprehensive functions in tumor cells. Despite a wealth of data describing their functions, the elucidation of their roles in cancer is still at the beginning stages. How STIM-ORAI and IP₃Rs affect carcinogenesis in vivo, the relationship between these proteins and Ca²⁺ oscillations in cancer cells, and whether the participation of these proteins in the cancer procedure is a general mechanism need to be investigated.