The Ca2+ 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 Ca2+ levels via direct transport of Ca2+, some Ca2+ pumps and channels are able to contribute to tumorigenesis via mechanisms that are independent of their ability to transport Ca2+ or effect global Ca2+ homeostasis in the cytoplasm. Here, we review some of the most recent studies that present evidence of altered Ca2+ channel or pump expression in tumorigenesis and discuss the importance and complexity of localized Ca2+ signaling in events critical for tumor formation.
- local Ca2+, ORAI
the versatility and complexity of the Ca2+ signal is widely established, with Ca2+ ions involved, either directly or indirectly, in almost every aspect of cellular processes (5, 49). Comprising an extensive range of signaling components, including channels, pumps, and exchangers, the Ca2+ signaling system is important in the regulation of processes relevant to tumorigenesis, including cell proliferation and apoptosis (5, 49). Research has increasingly focused on the identification of Ca2+ transporting proteins, with altered expression in cancer, with several publications reviewing this area (49, 63). The traditional dogma has been that Ca2+ pumps and channels contribute to tumorigenesis through alterations in global cytosolic free Ca2+ concentration. However, very recent discoveries challenge this traditional emphasis and highlight the importance of localized Ca2+ signals from intracellular compartments and subcellular microdomains. These studies also suggest that some Ca2+ pumps and channels can promote tumor progression via unique mechanisms, which are independent of Ca2+ transporting function. This review aims to provide an insight into the most recent developments and findings relating to aberrant expression of Ca2+ transporters in various cancer types and also address current controversial and unexplored areas in the field.
Ca2+ Pumps and Channels in Cancer: Recent Progress and Issues
Over the past four years, there has been a significant increase in published work reporting on Ca2+ channels and pumps that are associated with cancer. There has also been a significant increase in the types of cancers that are associated with alterations in the expression of specific Ca2+ transporters. A summary of important Ca2+ transport proteins that are reported to have altered expression or activity in some of the most common cancers is shown in Table 1.
An association between the plasma membrane Ca2+-ATPase (PMCA), a p-type ATPase responsible for the extrusion of Ca2+ ions from the cytosol into the extracellular domain, and cancer was established in 1997. SV40 transformed human skin and lung fibroblasts were shown to have reduced levels of PMCA protein (65). Since that time, further work has been published in the area of PMCAs and cancer. An upregulation of the PMCA2 isoform has been shown to occur in some breast cancer cell lines (40), and PMCA also has a potential role in regulating the proliferation of MCF-7 breast cancer cells (41). More recently, work by other laboratories has extended the study of PMCA2 expression to human clinical samples and defined a possible role of PMCA in the acquisition of apoptotic resistance (77). When PMCA2 is overexpressed in T47D breast cancer cells, alterations are seen in the magnitude and recovery of Ca2+ transients by an increased capacity to extrude Ca2+ from the cytoplasm, significantly reducing apoptosis in response to a Ca2+ ionophore (77). PMCA2 overexpression may thus contribute to cancer progression via the acquisition of an apoptotic resistant phenotype. Further linking PMCA2 and tumor characteristics, VanHouton et al. (77) also found an association between PMCA2 levels and tumor grade, nodal metastases, and poor clinical outcome.
As suggested by the early work of Reisner et al. (65) in SV40 transformed cells, PMCA can also contribute to tumorigenesis via reduced expression. PMCA4 mRNA levels are downregulated as an early event in human colon tumorigenesis (2). Correspondingly, when HT-29 colon cancer cells are differentiated with either sodium butyrate or by culturing postconfluence, they have an isoform-specific increase in PMCA4 expression (1, 2). Compromised Ca2+ efflux may provide colon cancer cells with a growth advantage through the promotion of proliferative pathways; this remodeling in PMCA expression does not occur to a degree that would sensitize colon cancer cells to apoptotic stimuli (2).
Transient receptor potential (TRP) channels are arguably the most comprehensively studied class of Ca2+ signaling proteins in the context of cancer (43, 71). Recent studies such as those linking TRPM8 and TRPV6 channels with prostate cancer (6, 22) have provided new depth into the potential significance of TRP channel expression in cancer. Although the overexpression of TRPV6 in prostate cancer has been established, current research aims to investigate the mechanisms by which TRPV6 overexpression contributes to prostate carcinogenesis. TRPV6-mediated Ca2+ influx stimulates activation of the Ca2+-dependent transcription factor nuclear factor of activated T-cells (NFAT) to modulate proliferation and promote resistance to apoptosis in prostate cancer cells (42). Another channel implicated in prostate cancer development is a member of the melastatin subfamily of TRP channels, TRPM8, which is overexpressed in early-stage prostate cancer (90). However, as the disease progresses to a metastatic, androgen-independent stage, TRPM8 expression is downregulated (35, 64). New research has identified a natural agonist for TRPM8 in the prostate. Gkika et al. (28) show that prostate-specific antigen (PSA), a well-known prostate cancer marker, promotes TRPM8 activity via the bradykinin/protein kinase C signaling pathway. Using cell surface biotinylation, they showed that the increased TRPM8 current was due to an increase in functional TRPM8 channels localized to the plasma membrane. Migration assays show that PSA reduces cell motility (28).
Further research into the molecular mechanisms underlying prostate cancer progression shows that TRPV2 has a role. TRPV2 mRNA levels are approximately 12 times greater in patients with late-stage metastatic prostate cancer compared with samples from localized primary solid tumors, suggesting TRPV2 as a potential prognostic marker for this late-stage of disease (48). Overexpression appears to bestow elevated cytosolic Ca2+ levels to the cell, which enhances their ability to migrate and invade into adjacent tissue, through direct regulation of key invasion proteases such as the matrix metalloproteinases MMP2 and MMP9 (48). Interestingly, another recent study reported a progressive decline in TRPV2 levels corresponding to increasing histological grade in human glioma tissue. Small interfering RNA (siRNA) silencing of TRPV2 resulted in increased cell proliferation and resistance to apoptosis via a pathway dependent on extracellular signal-regulated kinase activation (51). Given the association of TRPV2 with prostate cancer and glioma, further studies of TRPV2 in a variety of cancer models are warranted.
Recent work has also extended the association between TRP channels and cancer to other TRP classes and other cancers as exemplified by the marked overexpression of TRPC3 protein in human ovarian cancer samples (89). TRPC3 knockdown using siRNA leads to a decrease in cell proliferation via the induction of G2/M phase cell cycle arrest, suggesting a functional consequence of the channel's overexpression (89). In support of canonical TRP channels playing an essential role in cell cycle progression, a similar involvement of TRPC6 was recently reported in human glioma cells, where inhibition of TRPC6 activity or expression attenuated intracellular Ca2+ influx, also arresting cells at the G2/M phase and suppressing cell growth (13).
The most rapid acceleration of knowledge in recent years in the field is related to the study of ORAI ion channels in cancer. This is in part due to the identification of STIM proteins and ORAI channels as components of store-operated Ca2+ entry in 2005 and 2006, respectively (45, 66, 80). Store-operated Ca2+ entry is the predominant manner of Ca2+ influx in nonexcitable cells (79). The most widely studied proteins of these classes are STIM1, which functions as an endoplasmic reticulum (ER) Ca2+ sensor, and ORAI1, which is an essential component of the channel pore (61, 79). Studies on the role of store-operated Ca2+ entry in tumor metastasis were limited until Yang et al. (88) provided evidence for the role of STIM1 and ORAI1 in the migration of MDA-MB-231 breast cancer cells. The study was extended into an animal model, using immunodeficient NOD/SCID mice to confirm a reduction of breast tumor metastasis with STIM1 and ORAI1 knockdown (88).
Subsequent to this work was the finding of McAndrew et al. (47) that ORAI1 levels are significantly elevated (up to 21-fold) in some breast cancer cell lines, relative to normal 184A1 breast cells, and that ORAI1 siRNA-mediated inhibition in MCF-7 and MDA-MB-231 cells attenuates store-operated Ca2+ entry and viable cell number. ORAI1 activity may be augmented in some breast cancers via mechanisms distinct from an increase in ORAI1 expression level. An analysis of microarray data from 295 clinical samples of breast cancers showed that tumors defined transcriptionally as basal-like were characterized by high STIM1 and low STIM2 mRNA expression (47). This increase in STIM1 relative to STIM2 was associated with a poorer patient prognosis (47). This study also showed that STIM1 siRNA-mediated inhibition had more pronounced effects on store-operated Ca2+ entry than inhibition of STIM2 (47). This work suggests that basal breast cancer cells may have augmented store-operated Ca2+ entry.
The association between store-operated Ca2+ entry and cancer is now being extended to the less well-studied ORAI channels such as ORAI3. Faouzi et al. (19) report that ORAI3 mRNA is overexpressed in the estrogen receptor-positive breast cancer cell lines MCF-7 and T47D compared with the nontumorigenic MCF-10A breast cell line. ORAI3 mRNA is also increased in some human breast tumors compared with normal breast samples (19). Downregulation of ORAI3 inhibits cancer cell proliferation, contributes to cell cycle arrest at the G1 phase, and increases apoptotic cell death (19). ORAI3 silencing in the nontumorigenic MCF-10A cells did not change proliferation (19), suggesting that the ORAI3 signaling pathway is a potential therapeutic target in some human breast cancers. Work by Motiani et al. (50) has linked ORAI3 to the store-operated Ca2+ entry pathway used in estrogen receptor-positive breast cancer cells and report elevated ORAI3 protein in some estrogen receptor-positive cell lines. Collectively, these results highlight an interesting and diverse role for the store-operated Ca2+ pathway components in breast tumorigenesis and attest to the value of further research in this area.
There are limited studies on the role of the principal components of store-operated Ca2+ entry in cancers originating from areas outside of the breast. However, interest in other cancer types is an emerging area as reflected in recent work linking ORAI1 to prostate cancer (23). ORAI1 is downregulated during the transition to an androgen-independent phenotype in prostate cancer cells. ORAI1 inhibition in LNCaP cells results in resistance to apoptosis mediated by the sarco-endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (23). Furthermore, downregulation of ORAI1 in LNCaP cells protects the cells from chemotherapy-induced apoptosis, using cisplatin and oxaliplatin, two clinically used agents (23). Hence, advanced prostate cancers may acquire resistance to treatment in part due to ORAI1 downregulation. These results reflect the importance of the ORAI/STIM proteins and the store-operated Ca2+ signaling pathway in the pathogenesis of cancers and will provoke further research in this field.
Ca2+ Transporters and Tumorigenesis: New Mechanisms and New Complexities
The majority of studies in the context of Ca2+ transport in cancer have of course focused on exploring the relationship between how alterations in the expression of a Ca2+ channel or pump mediate their effects via the direct movement of Ca2+ ions through the pore of the channel or pump. However, recent studies in cell lines of both tumorigenic and nontumorigenic origin offer alternative mechanisms. Tumorigenic processes, in some cases, may be promoted via unique mechanisms that are independent of the Ca2+ transporting function of the protein or effects on global cytosolic free Ca2+.
The contribution of the secretory pathway Ca2+ ATPases (SPCAs) in the context of cancer was largely unexplored until recent work by Feng et al. (20) on SPCA2. SPCA2 is a Golgi-localized p-type Ca2+-ATPase first characterized in 2005 (78, 86). SPCA2 mRNA levels are upregulated in luminal-like breast cancer cell lines (20). The consequence of SPCA2 upregulation is an increase in cell proliferation and tumorigenicity (as assessed by growth on soft agar). Detailed examination into the mechanisms responsible for the effect revealed the ability of SPCA2 to localize to the plasma membrane and elicit constitutive Ca2+ influx through direct interaction of its NH2-terminus with ORAI1 (20). The separation of the ORAI1 activation function from the ion transporting ability of SPCA2 was demonstrated by the ability of ion transport-deficient mutants to activate Ca2+ influx, NFAT signaling, and proliferation. This study provides an example of how a Ca2+ pump can augment cancer processes via a mechanism distinct from its primary Ca2+ transport function. Physiologically, such a mechanism may be important in processes requiring sustained Ca2+ secretion such as lactation, where SPCA2 expression is increased (18).
The complexity of ORAI channel regulation is further highlighted by the ability of STIM2 to activate ORAI1 in a store-independent manner (30). STIM2 has now been reported to exist in distinct functional forms: the widely investigated STIM2 and another population, referred to as pre-STIM2, that is able to escape ER targeting (30). Immunolocalization revealed that cytosolic pre-STIM2 localizes to the plasma membrane, where it interacts with ORAI1, increasing basal Ca2+ concentration in a manner that is independent of ER Ca2+ store depletion (30). The increase in basal Ca2+ concentration by pre-STIM2 results in the induction of NFAT and nuclear factor-κB (NF-κB)-mediated gene transcription (30). Further to this, a third population, which is actually a fragment of the STIM2 signal peptide, upon its release from the ER membrane into the cytoplasm, becomes a regulator of gene transcription in a Ca2+ independent fashion (30). Together, these findings support the repositioning of paralogs in protein families into subcellular compartments as an emerging method for the functional diversification of replicated genes (30). Given the role of SPCA2 regulation of ORAI1 in breast cancer, assessment of pre-STIM2 in cancers, including those aside from the breast would now seem a priority. Figure 1 is a schematic representation that summarizes regulators of ORAI1 channels and activation associated with tumorigenesis as described in this review.
SPCA2, which as discussed above, activates ORAI1, is not the only example of a Ca2+ transporter with dual functionality. In neuronal cells, the voltage-gated channel CaV1.2 can produce a COOH-terminal fragment of CaV1.2 known as Ca2+ channel-associated transcriptional regulator (CCAT) (29). CCAT translocates into the nucleus, binds to nuclear proteins such as p54(nrb)/NonO, and regulates the expression of genes critical for neuronal signaling (29). The potential significance of such mechanisms in cancer is still to be explored.
Another recent study by Wang et al. (83) provides further insight into mechanisms by which Ca2+ channels may be regulated, suggesting a crucial link between receptor-induced Ca2+ store depletion and the control of voltage-activated signals. This group showed that STIM1 activation by store depletion not only leads to the activation of ORAI channels but also attenuates the activity of CaV1.2 (83). Although the action of STIM1 on CaV1.2 is independent of ORAI channels, its effects on both of these Ca2+ channels are spatially and functionally connected. ORAI channels trap STIM1 in puncta and cause STIM1 to become closely associated with CaV1.2 channels (83). One could postulate that cancer cells could have deregulation of these complex channel regulation mechanisms.
The arachidonic acid pathway has been implicated as a key inflammatory pathway involved in the cellular signaling contributing to prostate carcinogenesis (56, 76). Arachidonic acid is an inhibitor of TRPM8, a channel overexpressed in androgen-dependent prostate cancer (75, 90). However, the physiological pathways and surface receptors mediating the effect of arachidonic acid on cancer progression remain largely uninvestigated. Bavencoffe et al. (4) have recently reported on the role of arachidonic acid release in the complex regulation of TRPM8 through the M3-type muscarinic acetylecholine receptor-coupled signaling cascade. Their results showed that the application of a nonselective muscarinic agonist suppressed the activity of TRPM8 channels expressed in HEK-293 cells via stimulation of cytosolic phospholipase A2 (cPLA2) and subsequent generation of arachidonic acid. Further research into other surface receptors as regulators of ion channel activity in cancer cells is now required.
Localized Ca2+ Signaling
Changes in global cytosolic Ca2+ homeostasis through alterations in Ca2+ transporter expression or activity has been the focus of researchers characterizing changes in Ca2+ signaling in cancer cells (49). Spatially restricted Ca2+ signaling within specific cellular compartments or discrete cytosolic domains provides an additional layer of complexity in the regulation of cellular processes important in tumorigenesis; however, this has not yet been extensively assessed in the context of cancer.
Ca2+ gradients, such as those maintained by the ER, mitochondria, nucleus, and the Golgi help confer versatility to the Ca2+ signal, enabling the control of many cellular processes within a single cell (5). An example of the deregulation of Ca2+ stores that may occur in cancer is seen in the regulation of ER Ca2+ homeostasis by the anti-apoptotic protein BCL2. Ca2+ transfer from the ER to the mitochondria, if sufficient in magnitude, leads to the activation of cell death pathways (59). Many studies have now established a role for BCL2 in the inhibition of Ca2+ transfer between these often closely apposed organelles (9, 33, 59). A reduction in the luminal ER free Ca2+ level ([Ca2+]ER), inhibition of Ca2+ release from the ER, and inhibition of Ca2+ accumulation by mitochondria are all possible mechanisms for the anti-apoptotic effects of BCL2 (14, 33) and are a further link between Ca2+ signaling and processes important in cancer. A recent study by Giorgi et al. (27) identified a role for the promyelocytic leukemia (Pml) protein in ER-mitochondrial Ca2+ transfer. Pml is present at ER and ER-mitochondrial contact sites. Studies comparing Pml+/+ and Pml−/− mouse embryonic fibroblasts showed that ER-localized Pml is associated with a higher [Ca2+]ER and a larger Ca2+ transient in response to apoptotic stimuli. Loss of functional Pml on the ER [as reported in some cancer cells (27)], results in an impairment of Ca2+ transfer between the ER and mitochondria and the evasion of Ca2+-mediated cellular apoptosis (27). Hence, therapeutic modulation of targets that regulate [Ca2+]ER and/or ER-mitochondrial Ca2+ transfer may be able to augment apoptosis in cancer cells without disrupting global Ca2+ homeostasis.
Nuclear Ca2+ signaling has been the subject of debate since the first reports of differential Ca2+ signaling in the nucleus (26). The notion of a nuclear-cytoplasmic Ca2+ gradient has obvious implications for the regulation of gene transcription during tumorigenesis. One possible mechanism for a differential Ca2+ concentration between the cytosol and the nucleus may be buffering of Ca2+ by perinuclear mitochondria (26). Careful studies in cancer cells are required before the potential impact of this phenomenon on aberrant gene transcription in cancer cells and tumor progression can be fully understood.
Posttranslational protein modification and the trafficking of secreted proteins is the responsibility of the Golgi, which maintains a relatively high luminal Ca2+ concentration compared with the cytosol (60). This Ca2+ gradient appears to be predominately mediated via SPCAs. Recent studies assessing the potential significance of SPCA1 upregulation in the basal breast cancer subtype has shown that the silencing of SPCA1 inhibits the processing of the insulin-like growth factor receptor-1 (31). This change occurs without an impact on general cytosolic free Ca2+ homeostasis (e.g., changes in [Ca2+]CYT recovery after stimulation). Hence, some Ca2+ transporters may contribute to tumor progression through alterations in Golgi luminal Ca2+. Rather than altering cytosolic Ca2+ dynamics, SPCA1 may contribute to cancer pathways by altering the activity of Ca2+-regulated enzymes that are responsible for the posttranslational modification of proteins important in cell proliferation, apoptosis, and differentiation (31).
Spatial Heterogeneity in Cytosolic Ca2+ Signaling
The importance of localized Ca2+ signaling during excitation-transcription coupling is increasingly investigated in neurons and during the phenotypic conversion of vascular smooth muscle cells (46, 81). Ma et al. (46) demonstrated a role for local Ca2+/calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) in decoding Ca2+ signaling via L-type voltage-gated channels in sympathetic neurons. The ability of CaM to act as both a local and global Ca2+ sensor, via its COOH- and NH2-terminal lobes, respectively, highlights the importance of this protein in integrating the spatiality of the Ca2+ signal (73). CaM and CaMKII are deregulated in several cancers including thyroid and prostate cancers (67, 68) and the ability of localized Ca2+ signaling to regulate these proteins highlights the need for this aspect of their regulation to be further explored in tumorigenesis. Localized Ca2+ events, defined as Ca2+ “flickers,” are also an important regulator of migration in human embryonic lung fibroblasts exposed to a growth-factor gradient (84) and are another example of a pathway that requires further assessment in cancer cells.
Regulation of gene transcription via NFAT is a defining feature of local store-operated Ca2+ entry currents (21), which as discussed previously in this review, is a Ca2+ influx pathway increasingly linked to cancer. The NFAT family of transcription factors regulates many cancer-related processes including cellular differentiation, migration, and angiogenesis. Kar et al. (36) recently demonstrated that thapsigargin-induced store-operated Ca2+ entry induces NFAT1 nuclear translocation. Through a series of elegant experiments, this pathway was shown to be dependent on the local Ca2+ concentration near the Ca2+ channel pore. EGTA-AM, a cytosolic Ca2+ chelator with a slow Ca2+ buffering on-rate, significantly reduced the [Ca2+]CYT response; however, EGTA-AM failed to block NFAT translocation to the nucleus, indicative of a dependence on localized Ca2+ signaling. Overexpression and activation of TRPC3, which produces a similar Ca2+ transient to store-operated Ca2+ entry, did not induce NFAT translocation (36). Collectively, these results implicate a mechanism whereby the spatial Ca2+ concentration gradient in the cytosolic microdomain adjacent to Ca2+ channels, such as ORAI1, are important in gene transcription. The recently identified associations with ORAI channels and some cancers provide new impetus to explore this phenomenon in cancer cells.
The link between Ca2+ transporters and cancer may be through specific plasma membrane domains such as plasmalemmal lipid rafts and caveolae (54). Caveolae are membranous invaginations that serve as signaling complexes via the clustering of related signaling proteins into discrete microdomains (55). Upon ER Ca2+ store depletion, the fraction of the Ca2+ channel TRPC1 and the ORAI1 channel activator STIM1 localized to the lipid raft domain increases (53). Several studies have demonstrated an interaction between ORAI1 and caveolin-1 (54), and when combined with studies showing that pharmacological disruption of lipid rafts impairs store-operated Ca2+ entry (53), provide compelling evidence for the importance of plasma membrane microdomains in this type of Ca2+ influx. This relationship has significant implications in cancer, as the caveolae coat protein caveolin-1 is linked to processes important in cancer metastasis (87) and is enriched in clinical breast cancer samples associated with a poor prognosis (16).
One important implication of localized Ca2+ signaling may be a divergence between studies investigating the expression of Ca2+ transporters in cancer and those assessing function. Proteins with a relatively low endogenous expression may still wield significant responses in cells due to regions of privileged Ca2+ signaling in the vicinity of downstream targets, and thus should not be disregarded as having a role in tumorigenesis (Fig. 2). Conversely, models founded on heterologous expression of proteins that signal via localized Ca2+ may be misleading. Forced expression of these proteins may skew the ratio between the protein of interest and its natural downstream signaling partners. Although these studies may provide valuable information for research, they should not be interpreted in isolation, rather in conjunction with studies assessing the functional role of these proteins in physiology and pathophysiology.
In summary, this review has focused on very recent discoveries of changes in the expression and/or activity of important Ca2+ transporters in cancer cells, such as the overexpression of TRPC3 in ovarian cancer and ORAI-mediated Ca2+ influx in some breast cancer cells. We have also described unusual mechanisms by which some Ca2+ transporters contribute to tumorigenesis in a manner distinct from their primary transporting function or global cytosolic Ca2+ levels. Over the next five years, we will see current advanced approaches to the study of Ca2+ signaling being used to define the mechanisms by which Ca2+ transporters contribute to tumorigenesis.
This work was funded by the National Health and Medical Research Council of Australia (NHMRC) (569645 and 631347) and an Australian Postgraduate Award scholarship to J. M. Lee and an NHMRC Biomedical Postgraduate Research Scholarship to F. M. Davis.
S. J. Roberts-Thomson and G. R Monteith have a patent covering use of ORAI1 as a novel target.
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