The effects of dysregulated Ca²⁺ signaling on cell cycle

At early stage of tumor progression, cancer cells normally acquire a vast number of biological alterations that sustain their uncontrolled replicative capacity. Over last few decades, upon the development and technical modernization allowing to probe (Ca²⁺)_i transient oscillation. The functional importance of (Ca²⁺)_i signaling in regulation of cell cycle has been progressively unveiled. As a consequence of the greater than several thousand-fold gradient between (Ca²⁺)_i and extracellular Ca²⁺ [(Ca²⁺)_e] levels [14], the opening of cell surface Ca²⁺ channels leads to an immediate influx of (Ca²⁺)_e across PM. Besides, the transient elevation of (Ca²⁺)_i could be mediated through Ca²⁺ efflux from the internal Ca²⁺ stores such as ER, Golgi complex and the others.

Recently Ca²⁺ signals have emerged to be the hub of controlling G1 phase, G1/S and G2/M phase transitions [15]. In reality, cells are frequently sensitive to depletion of (Ca²⁺)_e in G1, in which Ca²⁺ is critical for the expression of specific genes required for cell division such as FOS, JUN and MYC. Specifically, FOSL1, also known as aka FRA-1, a member of Fos family, is required for Kras-induced lung tumorigenesis in vivo, and promotes human lung adenocarcinoma proliferation and survival [16]. Besides, C-myc functions as a downstream signal of several growth factor receptors such as epidermal growth factor receptor (EGFR), transforming growth factor alpha (TGFα), transforming growth factor beta (TGFβ) receptor, interleukin (IL)-6 receptor, Notch receptor, and Frizzled receptor [17]. Importantly, C-myc also serves as one of the master transcription factors of many target genes that encode for proteins essential for regulation of cell growth and proliferation such as p15, p21, CDK4, CDC25A, E2F1 [18–22].

One of most important pathways regulated by (Ca²⁺)_i signaling towards cell cycle progression is mitogen-activated protein kinase-renin-angiotensin system (MAPK-Ras) pathway. MAPK [rat sarcoma virus (Ras), rapidly accelerated fibrosarcoma (Raf) and mitogen-activated protein kinase (MEK)] pathway keeps a major role in regulating a variety of cellular processes such as proliferation, differentiation and surivial. The abnormal expression of MAPKs is frequently observed in NSCLC [23]. It is best characterized that MAPK pathway is initiated by the external stimuli such as hormones, growth factors (GFs), cytokines and intracellular molecules, following the activation of the RAS upstream receptors including receptor tyrosine kinases (RTKs) and EGFRs. Activation of MAPK-Ras signaling pathway promoting cell cycle progression [24] by retinoblastoma (RB1) phosphorylation, which then triggers upregulation of cyclin D1-induced CDK4 or CDK6, eventually driving G1-to S-phase transition [25–32]. Deregulation of EGFR, also called ErbB-1, was found in a range of 40–89% of NSCLC [33]. Furthermore, (Ca²⁺)_i is also crucial for regulation of several Ca²⁺-dependent cascades such as calciuneurin (CaN) and CaM-kinase. CaN, a Ca²⁺- and camodulin-dependent serine/threonine protein phosphatase, plays a key role in promoting cell cycle progression at G1/S phase transition through cyclin D1 stabilization [34]. Liu et al. [35] identified that CaNAα, an isoform of CaN, which was overexpressed in lung cancer tissues, promoted cell proliferation through accelerating G1-to S-phase transition in SCLC cells in vitro. CaN inhibition by cyclosporin A (CsA) blocked the transcriptional activity of CREB binding protein (CBP) and the nuclear factor of activated T cells (NFAT), leading to alleviate the expression of pro-inflammtory cytokine genes [36, 37]. Noticeably, activation of transcription factors such as CREB and myocyte enhancer factor-2 (MEF-2) was regulated by (Ca²⁺)_i elevation by the (Ca²⁺)_e influx across PM via L-type voltage-gated channels (LTCs) [38]. CsA-triggered CaN inhibition declined CDK2 activity by diminishing the expression of cyclin D1 during G1 [39], cyclin E and cyclin A [40]. Increases in (Ca²⁺)_i concentration result in activation of CaN, which subsequently dephosphorylates NFAT proteins, allowing them to translocate to the nucleus to regulate the expression of the target genes [41].

Orai3 channels, frequently overexpressed in NSCLC, mediate Ca²⁺ entry via store-operated Ca²⁺ entry (SOCE) and promote cell cycle progression via Atk pathway [42]. Orai3 silencing downregulated MAPK kinase pathway via diminishing the phosphorylation form of ERK1/2, and expression of C-myc, which triggered cell cycle arrest in G1 phase in breast cancer [43]. On the contrary, overexpression of Ca²⁺ release-activated Ca²⁺ channel protein 1 (Orai1), also known as CRACM1, triggered reduction of store-operated Ca²⁺ influx and attenuation of EGF-mediated proliferative signaling and driving cell cycle arrest in A549 lung cancer cells [44]. Furthermore, antigen-stimulated opening of Ca²⁺ release activated Ca²⁺ (CRAC) channel, a highly Ca²⁺-selective store-operated channel, enables the refilling of ER Ca²⁺ stores and maintain the persistency of Ca²⁺ oscillations which are first identified essential for T cell proliferation and cytokine production [45]. Conformational change and redistribution of stromal interaction molecule 1 (STIM1), the ER Ca²⁺ sensor, and Orai1, a key subunit of CRAC channel pore are required for activation of CRAC channel, which, in turn, triggers Ca²⁺ release from ER lumen into cytosol through activating IP₃R and/or (Ca²⁺)_e influx [46, 47]. Also, the depolarization-induced opening of VGCC Ca_v1.2 is directly suppressed by STIM1, causing a sustained internalization of VGCC Ca_v1.2 [48]. Heretofore, Wang, et al. [49] identified that STIM1 was significantly overexpressed in lung cancer tissues as compared to that of non-neoplastic lung tissues; furthermore, Ge, et al. [50] revealed that STIM1 knockdown induced cell cycle arrest at G2/M and S phases through alleviating expression of CDK1 and 2 in A549 and SK-MES-1 cells, and abolishing tumorigenesis and growth of lung cancer cells in nude mice xenograft. Upon reaching to cytosol, Ca²⁺ often forms complexes with the molecular components of “(Ca²⁺)_i signaling molecular toolkit” specific for each cell type given. Among such direct effectors essential for (Ca²⁺)_i signaling are CaM and Ca²⁺/CaM-dependent protein kinases II (CaMKII), protein phosphatase 2B (PP2B) and protein kinase C (PKC), modulating the transcriptional activity of various transcription factors for a large number of genes required for cell cycle progression [51]. Using 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine) (KN-62), a specific CaMKII antagonist, Williams, et al. [52] observed that blockade of CaMKII activity inhibited the exponentially proliferative capacity of SCLC cells through ameliorating cell cycle arrest at S phase. Altogether, the Ca²⁺ channels/pumps/exchangers and Ca²⁺-handling proteins identified affect cell cycle progression in lung cancer cells (Table 1).

The effects of dysregulated Ca²⁺ signaling on apoptosis

Apoptosis, a programmed cell death (PCD), is important for removal of mutated or transformed cells from the body essential for embryogenesis, development and tissue homeostasis of multicellular organisms. Principally, apoptosis comprises two core pathways: (1) the extrinsic pathway and (2) the intrinsic pathway, which are sequentially referred to as death receptor (DR)-mediated pathway and mitochondrial pathway [53]. Though stimulated in different manners, these pathways commonly converge into the same destination. Both the intrinsic and extrinsic apoptotic mechanisms lead to the activation of caspase 8, an initiator of a series of the apoptotic events through activating caspase 3, 6 and 7, subsequently resulting in cell collapse, chromatin condensation, breakdown of nuclear DNA, formation of apoptotic bodies and recognition of apoptotic cells by phagocytic cells [54].

During carcinogenesis, cancer cells acquired specific mechanisms of protection against apoptosis [55]. Among these, Ca²⁺ has been emerged as an important element exploiting its specialized effects towards regulation of apoptosis [56]. Under specific conditions at the initial step of mitochondria-induced apoptosis, overload of mitochondrial Ca²⁺, a vital sensitizer of the mitochondrial permeability transition (MPT) triggers mitochondrial swelling, perturbation of the mitochondrial outer membrane [57]. MPT pore-triggered release of the pro-apoptotic factors such as cytochrome c, apoptosis inducing factor (AIF), procaspase-9, Smac/DIABLO, and endonuclease G into cytosol causes a massive activation of proteases (caspases) and phospholipases [58–61]. To resist to apoptosis, cancer cells typically acquire the highly protective mechanisms against abolishment of mitochondria-triggered Ca²⁺ signals.

ER-derived Ca²⁺ signals, critical for regulating the apoptosis-related events, are also engaged in the mitochondria-induced apoptosis via the mitochondria-associated membranes (MAMs) juxtaposed between ER and mitochondria [62, 63]. Ca²⁺ storage in the ER is accomplished by the action of SERCA and of the intraluminal ER Ca²⁺-binding proteins such as BiP, calreticulin and calnexin whereas the release of Ca²⁺ from the ER is virtually mediated by IP₃Rs. The role of MAMs for regulation of (Ca²⁺)_i homeostasis is mediated by IP₃R3, RyR and SERCA [63]. Upon ER-derived Ca²⁺ signal-induced mitochondrial remodeling, B-cell lymphoma-2 (Bcl-2) proteins the first anti-apoptotic proteins identified regulate apoptosis via regulating Ca²⁺ transfer between ER and mitochondria [64]. These proteins are functionally categorized into the anti-apoptotic group (Bcl-2, BCL-X_L, and Mcl-1) and the pro-apoptotic group (Bax, Bak, Bim, Bid, etc.). Anti-apoptotic Bcl-2 proteins regulates apoptosis by modulating the ER-mitochondrial Ca²⁺ transfer via the MAMs [65] while overexpression of pro-apoptotic Bcl-2 decreased both ER-Ca²⁺ release either by the direct control of IP₃R3-mediated pore opening or by lowering the Ca²⁺ content of the ER, which weakens Ca²⁺-triggered MPT, and thus enables cancer cell to resist to apoptosis [66]. Abnormal upregulation of pro-apoptotic Bcl-2 is frequently observed in various types of cancer cells such as gastric, colon, breast and lung cancer [67–70]. Indeed, alleviation of ER-Ca²⁺ levels and signals has been observed in pro-apoptotic Bax and Bak-knockout murine embryonic fibroblasts (MEFs) [71]. Strikingly, enhanced ER Ca²⁺ levels by ectopic expression of SERCA2 rescue their sensitivity to death stimuli, suggesting the functional necessity of Bcl-2 proteins in regulating the ER-mitochondrial Ca²⁺ gateway and cell death in MEFs [71]. Bcl-2 mutant has also been reported to reduce ER Ca²⁺ by inhibition of SERCA2 as a consequence of a reduction of SOCE [72, 73]. Bergner, et al. [74] reported that a reduction of Ca²⁺ content correlated with a decreased expression of SERCA2 pumping Ca²⁺ into the ER, an increased expression of IP₃R releasing Ca²⁺ from the ER in various types of lung cancer cell lines. In contrast, the detailed mechanisms underlying Bcl-2-mediated regulation of SOCE remains controversial. Depletion of Ca²⁺ in the ER causes translocation of the SOC channel activator, STIM1, to the PM [75]. Thereafter, binding of STIM1 to Orai1 and/or transient receptor potential channel 1 (TRPC1) forces them to open for allowing Ca²⁺ entry across the PM [75].

Oncogenic K-RAS, which degenerates ER Ca²⁺ dynamics [76], and Akt, which not only phosphorylates and inactivates several pro-apoptotic Bcl-2 such as Bad, Bax and hexokinase-2, but more importantly diminishes mitochondrial Ca²⁺ overload via alleviating IP₃R opening, also contribute towards inhibition of the intrinsic apoptosis inhibition [77, 78]. Concomitantly, downregulation of protein phosphatase and tensin homolog (PTEN) in NSCLC tumors [79] antagonized F-box and leucine rich repeat protein 2 (FBXL2)-induced ubiquitination of IP₃R3, thereby stabilizing IP₃R3 in ER [80]. Besides, EGFRs with its aberrant expression and constitutive activation in in NSCLC [81] stimulate three of the most well-characterized signaling branches such as Ras-MAPK, phosphoinositol 3 kinase (PI3K)-protein kinase B (PKB)/Akt and phospho lipase C (PLC)-PKC pathways, enhancing ER-mitochondria Ca²⁺ transfer, thereby abolishing mitochondria-induced apoptosis. Also, loss of promyelocytic leukemia protein (PML) isoform IV, a suppressor of transcriptional activity of EGFR, for instance, on cyclin D1 gene promoter in lung cancer cells [82] also contributed to the EGFR-mediated mitochondria-induced apoptosis and cell cycle arrest [81].

The effects of dysregulated Ca²⁺ signaling on metastatic lung cancer

Metastasis, a term used to describe the spread of cancer cells from the primary tumor to surrounding tissues and to distant organs, is the major cause of morbidity and mortality in cancer patients. Among all solid tumors, SCLC is one of the most aggressive malignancy associated with a majority of patients diagnosed with metastatic disease [83]. Metastatic SCLC cells easily dissociate from lungs to disseminate throughout bloodstream and/or lymph system to anatomically distant organs such as lymph nodes, brain, liver and bone [83].

Progress of metastatic cascades primarily begins with the loss of cell-extracellular microenvironment [extracellular matrix (ECM)] as well as cell-cell attachment. Cells are connected to the ECM at focal adhesion points by structural complexes linking membrane spanning integrins to the cytoskeleton. Therefore, migratory capacity of cancer cells are principally assessed by the rate of focal adhesion assembly and disassembly. Noticeably, Ca²⁺ pulses promote the association of focal adhesion kinase (FAK), which regulates focal adhesion turnover, with the focal adhesion complex (FAC). More detailed, Ca²⁺ pulses strengthen the FAK at the specific sites where it is phosphorylated in a CaMKII-dependent manner. The movement of migrating cells is initialized by the extension of the protrusive front edge, which is known as lamellipodia. For cell protrusion, actin polymerization in lamellipodia and filopodia is required [84]. The attachment of lamellipodia to the substratum and contraction of the rear edge enable cells to move towards the lamellipodia. Establishment of a gradient difference of (Ca²⁺)_i levels, which was lower in the front, and higher in the rear of the migrating, polarized cells, caused rear retraction, focal adhesion (at the rear) and protrusion (at the front) [85, 86]. Following protrusion, the cell front starts to retract and locally adhere to ECM in lamella [87], which plays a pivotal in actomyosin contractility and F-actin disassembly in a treadmill-like manner [88]. Furthermore, actin and myosin, two of the important structural constituents, are regulated indirectly by Ca²⁺ signaling via the activation of the cyclic element-mediated Ca²⁺-dependent kinases, named calpain [89], and regulation of Rac1, RhoA, Cdc42, protein kinase A (PKA) [90, 91], and local Ca²⁺ signals between lamellipodia and lamella [92]. For retraction of the rear edge, Ca²⁺ signaling play a vital role in maintaining contractivity and stabilizing the directional movement via modulating the Ca²⁺ influx through L-type Ca²⁺ channels [93]. Ca²⁺-dependent MLC kinase (MLCK)-mediated phosphorylation of myosin light-chain (MLC) triggers myosin II-induced actomycin contractility [94], promoting the retraction and adhesion more efficiently [95, 96].

It is well-characterized that “local Ca²⁺ pulses” in the front of migrating cells are released from ER via IP₃-induced activation of IP₃Rs [97], which is generated through activation of RTK-PLC-dependent signaling pathways. It is therefore proposed that ER-derived Ca²⁺ release by the axis of RTK-PLC-IP₃R would be major source of Ca²⁺ pulses in the front of migrating cells. Indeed, EGFR, also known as HER1, belonging to the ErbB family of structurally related RTKs that comprises four isoforms: ErbB2 (HER2), ErbB3 (HER3) and ErbB4/HER4 [98], is overexpressed and constitutively activated in 62% of NSCLC cases [99]. It is clear that EGFR is the key activator of the ERK/MAPK, Akt-PI3K, and PLCγ-PKC signaling cascades [100]; furthermore, Tsai, et al. [101] reported RTK and PLC were enriched at the leading edge of migrating cells, in correlation with intensity of local Ca²⁺ pulses in the cell front.

In addition to RTK, G-protein coupled receptors (GPCRs) on local Ca²⁺ pulses in the cell front via activating PLC, which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP₂) to release IP₃, which binds to IP₃R to trigger transient Ca²⁺ release [102, 103]. In a meanwhile, depletion of ER luminal Ca²⁺ sensitizes SOC channels located at PM to infux (Ca²⁺)_e across PM [104]. Chantôme, et al. [105] once reported that the interaction of Orai1 with SK3 channel, a potassium channel belonging to the small conductance Ca²⁺-activated potassium (KCa) channel family, regulated the constitutive (Ca²⁺)_e entry through Orai1 localization within the lipid raft, which affected the migratory ability of breast cancer cells. Disruption of interaction between SK3 and Orai1 from lipid rafts weakened SK3-mediated Ca²⁺ entry, migration and bone metastasis [105]. Moreover, STIM/Orai-mediated SOCE is also essential for elevation of (Ca²⁺)_i level. STIM1, the ER Ca²⁺ sensor, and Orai3, constituent a native SOC entry essential for NSCLC progression [42, 50]. Increasing evidence implied that STIM1 assisted the turnover of cell matrix adhesion complexes, thereby enhancing cell migration by maintaining local Ca²⁺ pulses in the front of migrating cells [106]. In migrating cells, local Ca²⁺ pulses near its leading edge cause depletion of Ca²⁺ in the front ER, resulting in activation of STIM1 at the cell front [101]. More specifically, STIM1 was remarkably translocated to the ER-PM junction in cell front rather than cell rear during cell migration, thereby promoting maintenance of cell polarity and motility [101].

In addition, inhibition of the activity of Ca²⁺ permeable channels, PMCA and NCX, by the specific blockers, which are vanadate (V⁵⁺) and KB-R7943, respectively, led to a decrease in migratory capacity of MDCK-F cells, suggesting these channels were of significant importance for cell migration [107]. Furthermore, inactivation of ER membrane-located SERCA, which is responsible for pumping (Ca²⁺)_i into the ER lumen, triggering a leak of the ER lumical Ca²⁺ into cytosol [101]. The high (Ca²⁺)_i concentration caused MLCK saturation and myosin contractility [101]. Indeed, Atousa Arbabian [108] once reported that dysfunctional SERCA diminishes the ER luminal Ca²⁺, thereby disabling further Ca²⁺ signaling through IP₃Rs, suggesting a physiological importance of SERCA on lung cancer progression, invasion and metastasis.