Felicity M. Davis

Calcium channels and pumps in cancer: changes and consequences

Increases in intracellular free Ca2+ play a major role in many cellular processes. The deregulation of Ca2+ signaling is a feature of a variety of diseases, and modulators of Ca2+ signaling are used to treat conditions as diverse as hypertension to pain. The Ca2+ signal also plays a role in processes important in cancer, such as proliferation and migration. Many studies in cancer have identified alterations in the expression of proteins involved in the movement of Ca2+ across the plasma membrane and subcellular organelles. In some cases, these Ca2+ channels or pumps are potential therapeutic targets for specific cancer subtypes or correlate with prognosis.

Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent

Signals from the tumor microenvironment trigger cancer cells to adopt an invasive phenotype through epithelial–mesenchymal transition (EMT). Relatively little is known regarding key signal transduction pathways that serve as cytosolic bridges between cell surface receptors and nuclear transcription factors to induce EMT. A better understanding of these early EMT events may identify potential targets for the control of metastasis. One rapid intracellular signaling pathway that has not yet been explored during EMT induction is calcium. Here we show that stimuli used to induce EMT produce a transient increase in cytosolic calcium levels in human breast cancer cells. Attenuation of the calcium signal by intracellular calcium chelation significantly reduced epidermal growth factor (EGF)- and hypoxia-induced EMT. Intracellular calcium chelation also inhibited EGF-induced activation of signal transducer and activator of transcription 3 (STAT3), while preserving other signal transduction pathways such as Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. To identify calcium-permeable channels that may regulate EMT induction in breast cancer cells, we performed a targeted siRNA-based screen. We found that transient receptor potential-melastatin-like 7 (TRPM7) channel expression regulated EGF-induced STAT3 phosphorylation and expression of the EMT marker vimentin. Although intracellular calcium chelation almost completely blocked the induction of many EMT markers, including vimentin, Twist and N-cadherin, the effect of TRPM7 silencing was specific for vimentin protein expression and STAT3 phosphorylation. These results indicate that TRPM7 is a partial regulator of EMT in breast cancer cells, and that other calcium-permeable ion channels are also involved in calcium-dependent EMT induction. In summary, this work establishes an important role for the intracellular calcium signal in the induction of EMT in human breast cancer cells. Manipulation of calcium-signaling pathways controlling EMT induction in cancer cells may therefore be an important therapeutic strategy for preventing metastases.

Targeting EMT in cancer: opportunities for pharmacological intervention.

The spread of cancer cells to distant organs represents a major clinical challenge in the treatment of cancer. Epithelial-mesenchymal transition (EMT) has emerged as a key regulator of metastasis in some cancers by conferring an invasive phenotype. As well as facilitating metastasis, EMT is thought to generate cancer stem cells and contribute to therapy resistance. Therefore, the EMT pathway is of great therapeutic interest in the treatment of cancer and could be targeted either to prevent tumor dissemination in patients at high risk of developing metastatic lesions or to eradicate existing metastatic cancer cells in patients with more advanced disease. In this review, we discuss approaches for the design of EMT-based therapies in cancer, summarize evidence for some of the proposed EMT targets, and review the potential advantages and pitfalls of each approach.

ORAI1-Mediated Calcium Influx in Lactation and in Breast Cancer

The entry of calcium into the mammary epithelial cell from the maternal plasma (i.e., calcium influx mechanisms) during lactation is poorly understood. As alterations in calcium channels and pumps are a key feature of some cancers, including breast cancer, understanding these calcium influx pathways may have significance beyond mammary biology. We show that the store-operated calcium influx protein, Orai1, is increased during lactation whereas the Orai1 activator Stim1, but not Stim2, is downregulated. Stim2 siRNA reduced basal calcium levels in a lactation model. Our results suggest that calcium influx is remodeled in mammary epithelial cells during lactation, with calcium influx increased through Orai1, activated by Stim2. Breast cancer cell lines had increased levels of ORAI1. ORAI1 siRNA in breast cancer cells reduced store-operated calcium entry and remodeled the calcium influx associated with invasive stimuli. Analysis of microarray data from 295 breast cancers showed that the transcriptional breast cancer subtype with the poorest prognosis (basal) was associated with an altered relationship between the ORAI1 regulators STIM1 and STIM2, and that women with breast cancers with STIM1high/STIM2low tumors had a significantly poorer prognosis. Our studies show that during lactation there is a remodeling in the nature of calcium influx and that alteration in the ORAI1 influx pathway may be a feature of some breast cancers, particularly those with the poorest prognosis. Our studies suggest that this pathway may be a novel therapeutic target for breast cancer treatment in these women. Mol Cancer Ther; 10(3); 448–60. ©2011 AACR.

Non-Stimulated, Agonist-Stimulated and Store-Operated Ca2+ Influx in MDA-MB-468 Breast Cancer Cells and the Effect of EGF-Induced EMT on Calcium Entry

In addition to their well-defined roles in replenishing depleted endoplasmic reticulum (ER) Ca2+ reserves, molecular components of the store-operated Ca2+ entry pathway regulate breast cancer metastasis. A process implicated in cancer metastasis that describes the conversion to a more invasive phenotype is epithelial-mesenchymal transition (EMT). In this study we show that EGF-induced EMT in MDA-MB-468 breast cancer cells is associated with a reduction in agonist-stimulated and store-operated Ca2+ influx, and that MDA-MB-468 cells prior to EMT induction have a high level of non-stimulated Ca2+ influx. The potential roles for specific Ca2+ channels in these pathways were assessed by siRNA-mediated silencing of ORAI1 and transient receptor potential canonical type 1 (TRPC1) channels in MDA-MB-468 breast cancer cells. Non-stimulated, agonist-stimulated and store-operated Ca2+ influx were significantly inhibited with ORAI1 silencing. TRPC1 knockdown attenuated non-stimulated Ca2+ influx in a manner dependent on Ca2+ influx via ORAI1. TRPC1 silencing was also associated with reduced ERK1/2 phosphorylation and changes in the rate of Ca2+ release from the ER associated with the inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (time to peak [Ca2+]CYT = 188.7±34.6 s (TRPC1 siRNA) versus 124.0±9.5 s (non-targeting siRNA); P<0.05). These studies indicate that EMT in MDA-MB-468 breast cancer cells is associated with a pronounced remodeling of Ca2+ influx, which may be due to altered ORAI1 and/or TRPC1 channel function. Our findings also suggest that TRPC1 channels in MDA-MB-468 cells contribute to ORAI1-mediated Ca2+ influx in non-stimulated cells.

Ion channels and transporters in cancer. 4. Remodeling of Ca(2+) signaling in tumorigenesis: role of Ca(2+) transport.

The Ca(2+) signal has major roles in cellular processes important in tumorigenesis, including migration, invasion, proliferation, and apoptotic sensitivity. New evidence has revealed that, aside from altered expression and effects on global cytosolic free Ca(2+) levels via direct transport of Ca(2+), some Ca(2+) pumps and channels are able to contribute to tumorigenesis via mechanisms that are independent of their ability to transport Ca(2+) or effect global Ca(2+) homeostasis in the cytoplasm. Here, we review some of the most recent studies that present evidence of altered Ca(2+) channel or pump expression in tumorigenesis and discuss the importance and complexity of localized Ca(2+) signaling in events critical for tumor formation.

Essential role of Orai1 store-operated calcium channels in lactation

Significance All mammals, from platypuses to humans, produce relatively immature offspring that are wholly dependent on their mother’s milk for their postnatal growth and development. However, the dynamic signaling and molecular mechanisms responsible for the transport of key constituents (e.g., calcium) into milk and for alveolar unit contraction and milk ejection are not fully understood. Using genetically modified mouse models, we demonstrate that the store-operated Ca2+ channel Orai1 delivers over 50% of the calcium ions present in milk. We also reveal an unanticipated role of Orai1 as a master regulator of oxytocin-mediated alveolar unit contractility, milk ejection, and pup survival. These results provide a unique mechanistic insight into the fundamentally mammalian process of lactation. The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca2+ channel subunit, Orai1, is required for both optimal Ca2+ transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca2+ in mammary myoepithelial cells followed by slow, irregular Ca2+ oscillations. These oscillations, and not the initial Ca2+ transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca2+ is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca2+ oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Remodeling of purinergic receptor-mediated Ca2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells.

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPRTETRA) and observed significant changes in the potency of ATP (EC50 0.175 µM (−EGF) versus 1.731 µM (+EGF), P<0.05), and the nature of the ATP-induced Ca2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca2+ homeostasis. To determine whether changes in ATP-mediated Ca2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

Altered purinergic receptor‐Ca2+ signaling associated with hypoxia‐induced epithelial‐mesenchymal transition in breast cancer cells

Hypoxia is a feature of the microenvironment of many cancers and can trigger epithelial‐mesenchymal transition (EMT), a process by which cells acquire a more invasive phenotype with enriched survival. A remodeling of adenosine 5′‐triphosphate (ATP)‐induced Ca2+ signaling via purinergic receptors is associated with epidermal growth factor (EGF)‐induced EMT in MDA‐MB‐468 breast cancer cells. Here, we assessed ATP‐mediated Ca2+ signaling in a model of hypoxia‐induced EMT in MDA‐MB‐468 cells. Like EGF, hypoxia treatment (1% O2) was also associated with a significant reduction in the sensitivity of MDA‐MB‐468 cells to ATP (EC50 of 0.5 μM for normoxic cells versus EC50 of 5.8 μM for hypoxic cells). Assessment of mRNA levels of a panel of P2X and P2Y purinergic receptors following hypoxia revealed a change in levels of a suite of purinergic receptors. P2X4, P2X5, P2X7, P2Y1 and P2Y11 mRNAs decreased with hypoxia, whereas P2Y6 mRNA increased. Up‐regulation of P2Y6 was a common feature of both growth factor‐ and hypoxia‐induced models of EMT. P2Y6 levels were also significantly increased in basal‐like breast tumors compared to other subtypes and breast cancer patients with higher P2Y6 levels showed reduced overall survival rates. P2Y6 siRNA‐mediated silencing and the P2Y6 pharmacological inhibitor MRS2578 reduced hypoxia‐induced vimentin protein expression in MDA‐MB‐468 cells. P2Y6 inhibition also reduced the migration of mesenchymal‐like MDA‐MB‐231 breast cancer cells. The up‐regulation of P2Y6 appears to be a common feature of the mesenchymal phenotype of breast cancer cells and inhibition of this receptor may represent a novel therapeutic target in breast cancer metastasis.

The functions of store-operated calcium channels

Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca2+ stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.