The mammalian TRP cation channel superfamily has 28 family members [29]. While TRP melastatin 3α2 (TRPM3α2), TRP vanilloid 5 (TRPV5), and TRPV6 are highly Ca²⁺-selective, most TRP channels are nonselective [30]. Processes, such as cell apoptosis, proliferation, angiogenesis, invasion, and migration, are under the control of the regulation of the TRP cation channels in intracellular Ca²⁺ concentration (Table 1) [31]. Evidence has shown that TRPV2 mediates the secretion of RANKL via the Ca²⁺-calcineurin-nuclear factor of activated T cells 3 (NFATc3) signaling pathway in multiple myeloma (MM) cells, and RANKL levels are demonstrated in a Ca²⁺ dose-dependent way (Figure 2a) [7]. Furthermore, NFATc3 was found to bind to the promoter of RANKL and induce RANKL expression at the transcriptional level [7]. The RANKL-induced bone remolding contributed to the pathogenesis of MM lesions, but it also provided a likely treatment strategy.

TRPV6 was found to be present at elevated levels in prostate, breast, thyroid, colon, and ovarian carcinomas [48]. Moreover, TRPV6 messenger RNA (mRNA) expression levels were upregulated with the malignant degree of prostate cancer and the highest levels of TRPV6 were detected in prostate cancer with lymphatic metastases and in recurrent lesions [49]. It was found that the TRPV6 channels are translocated to the plasma membrane involving the STIM1/Orai1/TRPC1-mediated ER Ca²⁺ store depletion via the Ca²⁺/Annexin I/S100A11 pathway, leading to increased proliferation and apoptosis resistance (Figure 2b) [50]. Furthermore, prostate cancer cells expressing TRPV6 were directly inoculated into the bone marrow cavity of tibias and promoted the generation of osteoblastic lesions suggesting TRPV6 promotes prostate cancer bone metastasis via numerous osteoblastic lesions [50]. It is well studied that the increased intracellular Ca²⁺ induced by TRPV6 dephosphorylates NFAT to induce cellular proliferation and regulates NF-κB oscillation to resist apoptosis, but the mechanism of the formation of the osteoblastic lesions is unknown [50].

The TRPM7 channels, which play a pivotal role in cell motility, are non-selective channels permeable predominantly to Mg²⁺ and Ca²⁺ [51]. In prostate cancer, an increase in serum Ca²⁺/Mg²⁺ ratio, which is regulated by TRPM7 and facilitates Ca²⁺ entry, leads to an increase in cell proliferation [52]. In a hypoxic environment, the increased TRPM7 results in HIF-1α accumulation. TRPM7-HIF-1α signaling activates downstream Annexin I protein expression mediating EMT, cell migration, and invasion (Figure 2c) [53]. In addition, it is also found that TRPM7 modulates the migration and invasion of breast cancer cells through the Src-MAPK signaling pathway (Figure 2d) [54]. Notably, TRPM7 overexpression promotes neuroblastoma cells to spread to the liver and bone marrow, but the mechanism is unknown [55].

It is well established that many members of the TRP cation channel superfamily play important roles in the mediation of tumor progression. The TRP cation channels also make a contribution to osteoclast differentiation, for example, TRPC1, TRPV4, and TRPV5 are essential for the regulation of osteoclastogenesis [56–58]. In a recent study, osteoporosis and enhanced bone absorption were found in TRPV6 knockout mice [59]. TRPV6 channels decreased the ratios of phosphorylation in the PI3K-AKT pathway which mediates the regulation of osteoclast formation and bone resorption (Figure 2e) [59]. The mechanism of the negative regulation of osteoclast differentiation and fusion and bone absorption by TRPV6 was revealed on a molecular level, and TPRV6 was confirmed to play an important role in bone metabolism [59]. Taken together with the formation of osteoblastic lesions induced by TRPV6, the TRPV6 channels play essential roles in the modulation of tumor progression and osteoclast activation. It makes one wonder, is there a connection between the TRPV6 generated osteoblastic lesions in prostate cancer and the TRPV6 negative regulated osteoclast differentiation? Further studies are required to elucidate the function of the TRP channels on cancer cell bone metastases and the possible link between metastases and the TRP regulation of osteoclast and osteoblast.

SOCE follows the depletion of ER Ca²⁺ storage. Both STIM1 and STIM2 are important for the maintenance of intracellular Ca²⁺ concentration [60]. After reduction of ER intraluminal Ca²⁺, STIM1 is activated and translocated to ER-plasma membrane junctions, where STIM proteins leash and gate Orai1 Ca²⁺ entry channels. STIM2 is more sensitive to changes in ER Ca²⁺ than STIM1, but it is a significantly weaker activator of Orai channels than STIM1.

In all, Orai1, -2, and -3 have been identified as plasma membrane Ca²⁺ channels. Although all three proteins are highly homologous to each other, they display notable differences in their features. Orai1 is the most potent to induce Ca²⁺ influx among its homologs, and its depletion significantly inhibits SOCE [61]. SOCE serves a wide set of signaling functions by elevating the cytosolic Ca²⁺ concentration. SOCE has potential roles in cellular proliferation and is inactivated during the division phase (M-phase) of the cell cycle. During the M-phase, STIM1 clustering is inhibited and Orai1 is internalized, thus uncoupling Ca²⁺ store depletion from Orai1 gating [62].

STIM1 and Orai1 are new targets for cancer treatment. Before STIM1’s role in Ca²⁺ influx was suspected, it was implicated that STIM1 could be a tumor suppressor [63]. The role of STIM and Orai in cancer is better studied particularly in the case of breast cancer. Breast cancer cell lines are not homogenous regarding STIM/Orai expression. Orai1 and STIM1 are predominant in the estrogen receptor-negative breast cancer cell lines, but Orai3 and STIM1/2 are the main SOCs in estrogen receptor-positive breast cancer cells [64]. The Orai3-induced Ca²⁺ influx contributed to breast cancer proliferation and survival but not in normal cells, consistent with the down-regulation of Orai3 arresting cell cycle progression and inducing apoptosis in breast cancer cells [65].

Focal adhesions, which are mediated by the interaction of integrin with the extracellular matrix, are relatively stable structures and tend to inhibit cell migration [66, 67]. Cell migration requires a dynamic state of focal adhesion [67]. STIM1 and Orai1 were shown to regulate tumor cell migration partially involving the mediation of the focal adhesion [68]. Increased Ca²⁺ influx might induce tumor cell migration depending on the activation of the focal adhesion kinase (FAK), the Ca²⁺-dependent protease calpain, and other Ca²⁺-sensitive proteins in focal adhesion turnover. SOCE inhibitor SKF96365 inhibited breast cancer cells’ metastasis in mouse models, providing a strong argument that SOCE is vital for breast tumor cell migration and metastasis.

Small conductance Ca²⁺-activated potassium channel protein 3 (SK3), a potassium channel, is a member of the small conductance Ca²⁺-activated potassium channel family [69]. An SK3-Orai1 complex, localized within lipid rafts, was found to be critical for the control of cancer cell migration and osteolytic bone metastases [70]. The SK3-Orai1 complex controls constitutive Ca²⁺ entry and tumor cell migration through store-independent Ca²⁺ signaling. Knocking down of the SK3 channels resulted in a lower metastatic score in breast cancer. Moreover, bone metastases achieved this lower metastatic score, but this reduction was not seen in lung metastases. The formation of the osteolytic lesions increased external Ca²⁺ concentration which amplified Ca²⁺ entry, establishing a vicious circle. Furthermore, the increased intracellular Ca²⁺ upregulated the activity of the Ca²⁺-sensitive protease calpain which could be attributed to bone metastases. Ohmline, a lipid inhibitor of SK3 channels [71], moved the SK3-Orai1 complex outside of lipid rafts and impaired the subsequent SK3-dependent Ca²⁺ entry, tumor cell migration, and bone metastases [70]. Therefore, ohmline could be a promising therapeutic application in preventing and treating breast cancer bone metastases. However, the role of calpain in breast cancer bone metastases needs further study.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates osteoclast differentiation, bone resorption, and bone metastasis via the Orai1 [72]. The expression levels of SGK1 are essentially upregulated during RANKL-induced osteoclastogenesis [72]. It was found that treatment with GSK650394, an SGK1 inhibitor, down-regulated Orai1 levels during osteoclastogenesis and overexpressed Orai1 markedly alleviated the inhibitory effects of GSK650394 on osteoclast differentiation [72]. In addition, SGK1 is functionally relevant for cell migration, which is critically dependent on SOCE [73], and treatment with GSK650394 significantly decreased breast cancer bone metastases in mouse models [72]. These findings present a new perspective on RANKL-induced osteoclastogenesis and breast tumor bone metastases through SGK1-mediated Orai1 overexpression. However, reintroduction of Orai1 did not fully rescue the GSK650394 abolished Ca²⁺ influx [72]. There are possibly other SGK1-mediated Ca²⁺ channels that can be regarded as future therapeutic targets.

The VGCCs transport intracellular Ca²⁺ cations into intracellular Ca²⁺ transients initiating numerous physiological activities [26]. VGCCs are composed of three different subfamilies, the CaV1 (L-type) Ca²⁺ channel family, the CaV2 Ca²⁺ channel family, and the CaV3 (T-type) Ca²⁺ channel family, and are specified to ten members, CaV1.1, CaV1.2, CaV1.3, CaV1.4, CaV2.1, CaV2.2, CaV2.3, CaV3.1, CaV3.2, and CaV3.3 [74]. The CaV1 subfamily and the CaV2 subfamily are primarily responsible for the initiation of contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells, and initiation of synaptic transmission at fast synapses [26]. However, pieces of evidence have suggested that CaV3 mediates cellular processes including tumorigenesis and cancer progression by regulating intracellular Ca²⁺ levels [75].

T-type VGCCs expression levels are upregulated in many cancers and, thus, CaV3 channels are regarded as promising therapeutic targets. CaV3.1 isoform is a tumor-suppressor candidate and is reported to promote apoptosis and prevent tumor proliferation in breast cancer cells [76]. The CaV3.2 channels were not involved in the proliferation of MCF-7 breast cancer cells [76]. However, CaV3.1 was aberrantly upregulated and indicated a positive role in the regulation of proliferation in prostate cancer [77]. CaV3.1 together with CaV3.2 isoforms were found to increase gradually from normal skin to common nevi, dysplastic nevi, and melanoma samples with differences in distribution. Notably, metastatic melanoma showed the highest CaV3.2 expression levels which significantly differed from all other groups [78]. These results suggest that CaV3.1 and CaV3.2 channels may contribute to tumorigenesis and metastases. Therefore, further studies are required to identify the role of T-type VGCCs in cancers and bone metastasis, which is significant for the regulation of Ca²⁺ homeostasis.

Connexin 43 [Cx43, encoded by gap junction protein alpha 1 (GJA1)] belongs to the connexin family which is the major constituent of gap junctions, widely connects osteocytes and osteoblasts in bone, and directs Ca²⁺ flow [79, 80]. Prostate cancer and breast cancer bone metastasis showed the highest levels of Cx43 expression among all sites of metastases, suggesting that bone colonization requires Ca²⁺ flows from osteoblasts to cancer cells via the Cx43-based gap junctions [12]. Importantly, arsenic trioxide (As₂O₃) can inhibit Ca²⁺ signaling through downregulation of Cx43 and affecting Ca²⁺ influx, making it a promising therapeutic agent for clinical practice (Figure 3) [12].

Ca²⁺/CaM-dependent protein kinase kinase α (CaMKKα) and β are the upstream kinases in the CaMK signaling cascade [84]. CaMKKα and β are activated through Ca²⁺/CaM binding and intramolecular phosphorylation. Activated CaMKKs phosphorylate and activate CaMKI and CaMKIV, adenosine monophosphate (AMP)-activated protein kinase (AMPK), or AKT (PKB). These kinases then phosphorylate downstream proteins, such as cyclic AMP (cAMP) response element-binding protein (CREB), activating transcription factor-1 (ATF-1), CAAT-enhancer-binding protein (C/EBP), and serum response factor (SRF) [85]. Notably, different from CaMKKα, which is solely dependent on Ca²⁺/CaM for activity, CaMKKβ also can be activated in the absence of Ca²⁺/CaM. Glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) regulate CaMKKβ activities through phosphorylation [86]. Unlike CaMKI and CaMKIV, CaMKII requires Ca²⁺/CaM complexes for activation, independent of CaMKKs [87].

CaMKKβ was identified as a downstream target protein of androgen receptor that has been known as prostate cancer bone metastasis enhancer (Table 2). Androgen-dependent regulation of CaMKKβ allowed tumor cells to migrate toward a more nutrient-rich environment, such as bone marrow, by activating and phosphorylating AMPK [88, 89]. Furthermore, CaMKKβ is a critical regulator of bone remodeling and macrophage function, creating a favorable microenvironment for colonizing and tumor growth of prostate cancer cells [90]. CaMKKβ stimulates osteoclast differentiation via CaMKKβ-CaMKIV-phosphorylated cAMP response element binding (pCREB) signaling cascade its downstream target, the NFATc1, the primary mediator during osteoclastogenesis [91]. However, in osteoblast, the CaMKKβ-CaMKIV pathway suppresses type I adenylate cyclase-cAMP regulated activities of protein kinase A (PKA), resulting in inhibited osteoblast differentiation [91]. Among immune cells, CaMKKβ was found to be restrictedly expressed in cells of the monocytic/macrophage lineage [92]. CaMKKβ ablation impaired macrophages’ ability such as cytokine secretion, and morphological changes, and CaMKKβ knockdown mice showed resistance to irritants that lead to systemic inflammation. Above all, dysregulation of CaMKKβ remodels bone into a favorable environment for tumor cells. Knockdown of CaMKKβ inhibits tumor growth, resists macrophage-induced inflammation, and improves the bone microenvironment. Further studies are still needed to investigate the molecular mechanisms of how CaMKKβ mediates prostate cancer cells’ metastatic abilities.

Furthermore, increased cytolytic Ca²⁺ levels induced by Cx43 activate CaMKII mediating tumor cells’ bone colonization [12]. Nuclear Ca²⁺ signaling induces the CaMKII-dependent MeCP2 phosphorylation on serine 421 of MeCP2 and releases MeCP2 from silenced promotors in many cellular contexts [101, 102]. Decreased levels of MeCP2 enriched TFs, NFAT, and MEF2 which are associated with the promotion of EMT, migration, angiogenesis, and invasion [103, 104], in bone metastases. Moreover, evidence has suggested that CaMKII may be co-regulatory with the Notch signaling pathway which plays a critical role in the development of osteometric properties by prostate cancer bone metastatic cells [105].

It has been established that CaMKII is involved in the differentiation of both osteoblasts and osteoclasts. A collagen-binding motif derived from osteopontin induces an influx of extracellular Ca²⁺ via Ca²⁺ channels and promotes osteoblastic differentiation via Ca²⁺/CaMKII/extracellular signal-regulated kinase (ERK)/activating protein-1 (AP-1) signaling pathway [106]. More importantly, increased osteoclastic resorption and subsequent bone loss are common features of bone metastases. Once osteoclasts are stimulated, activated CaM complexes combine with CaMKII to regulate the expression of NFATc1 and tartrate-resistant acid phosphatase (TRAP/ACP5), an osteoclast marker, leading to macrophage differentiation into osteoclasts [107]. Zoledronic acid, a bisphosphonate, significantly decreases the Ca²⁺ levels, inhibits the expression of CaM and CaMKII, and prevents osteoclasts differentiation, providing effective therapy for patients with skeletal involvement from advanced cancers [107]. Furthermore, CaMKII induces c-fos gene expression and subsequent AP-1 activation, which can, in turn, drive NFAT2 expression and is involved in osteoclast differentiation and bone remodeling [108]. CaMKII also mediates leukemia inhibitory factor (LIF)-induced phosphorylation of serine-782 in the glycoprotein 130 (gp130) tail, which leads to internalization and downregulation of the gp130 receptor on the cell surface, suggesting that CaMKII may promote osteoclastogenesis by inhibiting the gp130 receptor signaling cascade [108]. Zoledronic acid has proven to be efficient to treat bone metastases targeting osteoclastogenesis, but zoledronic acid has nonnegligible side effects and limited application [107]. The molecular mechanisms of Ca²⁺ signaling leading to osteoclastogenesis may provide more specific targets to the treatment regimen for bone metastases, and needs further studies.

Calcineurin is a conserved Ca²⁺-CaM-dependent serine-threonine phosphatase that controls signaling pathways relevant to the migration, invasiveness, and metastatic potency of cancer cells. Increased cytolytic Ca²⁺ levels activate calcineurin mediating tumor cell bone colonization [12]. Calcineurin showed a similar effect as CaMKII to increase NFAT and MEF2 expression levels and inhibition of calcineurin also impedes bone colonization [12]. Calcineurin dephosphorylates resident NFAT proteins in the cytoplasm and triggers NFAT nuclear accumulation and activation [109]. RANK activation evokes Ca²⁺ oscillation by Ca²⁺ released from the ER and SOCE promotes CRC bone metastases through the calcineurin/NFATc1/ACP5 axis [110]. In addition, calcineurin/NFATc1 signaling promotes breast cancer metastasis to bone and brain and upregulates IGFI [111]. Regulator of calcineurin 1 isoform 4 (RCAN1.4) was found to reduce calcineurin activity and block nuclear translocation of NFATc1 [112]. Hence, RCAN1.4 is competent to reduce proliferation, migration, and metastases [112]. Moreover, RCAN1.4 was identified as a super suppressor of breast cancer and a potential therapeutic target for late-stage breast cancer patients with bone and brain lesions by ablation of calcineurin/NFATc1 signaling [111].

Furthermore, calcineurin Aα (CnAα), an isoform of calcineurin, is significantly overexpressed in small cell lung cancer (SCLC) tissues with bone metastasis in contrast to tumor cells where bone metastasis was absent [113]. CnAα is located in nuclear SCLC cells with bone metastases, but in non-metastatic tumors, CnAα is mainly located in the cytosol [113]. Downregulation of CnAα by lentiviral vector-mediated RNA interference (RNAi) reduced cell migration and invasion, and inhibited adhesion to the bone matrix, hampering metastasis development of SCLC with no change in the apoptosis rate of tumor cells [114].