Nuclear Factor of Activated T Cells in Cancer Development and Treatment

Cancer Letters 361 (2015) 174–184

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Cancer Letters

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Mini-review Nuclear factor of activated T cells in cancer development and treatment Jiawei Shou a, Jing Jing b, Jiansheng Xie c, Liangkun You a, Zhao Jing a, Junlin Yao a, Weidong Han a,c,*, Hongming Pan a,c a Department of Medical Oncology, Institute of Clinical Science, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China b Department of Medical Oncology, Tongde Hospital of Zhejiang Province, Hangzhou, Zhejiang, China c Laboratory of Cancer Biology, Institute of Clinical Science, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China

ARTICLE INFO ABSTRACT

Article history: Since nuclear factor of activated T cells (NFAT) was ﬁrst identiﬁed as a transcription factor in T cells, various Received 7 January 2015 NFAT isoforms have been discovered and investigated. Accumulating studies have suggested that NFATs Received in revised form 4 March 2015 are involved in many aspects of cancer, including carcinogenesis, cancer cell proliferation, metastasis, Accepted 4 March 2015 drug resistance and tumor microenvironment. Different NFAT isoforms have distinct functions in different cancers. The exact function of NFAT in cancer or the tumor microenvironment is context dependent. Keywords: In this review, we summarize our current knowledge of NFAT regulation and function in cancer devel- NFAT opment and treatment. NFATs have emerged as a potential target for cancer prevention and therapy. Calcineurin Tumor microenvironment © 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY- Metastasis NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Drug resistance

Introduction NFAT therapeutics. Insights into NFAT functions will help to elucidate the NFAT signaling axis mechanism and develop cancer therapies. Nuclear factor of activated T cells (NFAT) was first identified as a transcription factor nearly three decades ago. NFAT was original- The NFAT family and regulation ly found in nuclear extracts from activated T cells. Shaw et al. previously reported that NFAT binds to the interleukin-2 (IL-2) pro- There are five NFAT family members, known as NFAT1 (NFATc2/ moter to activate T cells [1]. NFAT was not well studied until the NFATp), NFAT2 (NFATc1/NFATc), NFAT3 (NFATc4), NFAT4 (NFATc3/ development of immunosuppressants such as cyclosporine A (CsA) NFATx) and NFAT5 (tonicity enhancer binding protein) [4,12].NFAT and tacrolimus (FK506), which block calcineurin activity and inhibit proteins contain an amino-terminal transactivation domain (TAD) NFAT nuclear translocation and consequent target gene transcrip- and a carboxy-terminal domain. NFAT1-4 has two additional con- tion [2]. It was later found that NFAT was also expressed in other served domains, the Rel-homology region (RHR) and NFAT homology cells and tissues, including muscle, bone, neurons, viscera and skin region (NHR). RHR is similar to the DNA-binding domain of the Rel [3–6]. Since the first research demonstrating the relationship family (also known as nuclear factor-κB family), and NHR is a reg- between NFAT and cancer cells was published [7], more evidence ulatory domain with the calcineurin-binding site. Thus, the Ca2+/ has emerged to reveal the roles of NFAT in cancer development and calcineurin pathway regulates NFAT1-4. In contrast, NFAT5 can’t be treatment. activated by calcineurin, as it lacks an NHR [13,14] (Fig. 1A). NFAT plays crucial role in regulating cancer initiation and pro- In resting cells, the NHR is heavily phosphorylated at serine resi- gression, such as proliferation, invasion, migration and angiogenesis dues within several conserved serine-rich sequence motifs (SRR-1, [8–10]. In this review, we focus on the connection between NFATs SPxx repeat, and SRR-2), which can be dephosphorylated by several and three hallmarks of cancer: drug resistance, metastasis and the types of stimulation [15]. When dephosphorylated, the nuclear lo- tumor microenvironment [11]. In addition, we also review the func- calization sequence (NLS) within NFAT is exposed, resulting in the tion of NFAT in carcinogenesis, cell proliferation, and the potential protein’s nuclear translocation. In the nucleus, NFAT cooperates with other transcription factors, such as activator protein 1 (AP1, Fos- Jun complexes), GATA4, MEF families and forkhead box P3 (FOXP3), to initiate target gene transcription [16–18]. NHR rephosphorylation * Corresponding author. Tel.: +86 571 86006926; fax: +86 571 86044817. leads to nuclear export signal (NES) exposure and NLS masking, E-mail address: [email protected] (W. Han). leading to NFAT translocation from the nucleus to the cytoplasm http://dx.doi.org/10.1016/j.canlet.2015.03.005 0304-3835/© 2015 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/). J. Shou et al./Cancer Letters 361 (2015) 174–184 175

Fig. 1. General structure of NFAT and calcineurin. (A) NFAT proteins consist of the Rel-homology region (RHR), the NFAT homology region (NHR) and a C-terminal domain, while NFAT5 doesn’t possess the NHR. The NHR contains an N-terminal transactivation domain (TAD), the calcineurin binding site, the nuclear localization sequence (NLS) and the nuclear export signal (NES). It also harbors several serine-rich motifs including SRR1, SRR2, SP1, SP2 and SP3. The RHR comprises the DNA binding domain and the recognition points with other transcriptional binding partners such as Fos and Jun. (B) The calcineurin protein has two subunits calcineurin A (CnA) and calcineurin B (CnB). CnA contains a phosphatase catalytic domain and a regulatory domain, the latter owns a CnB-binding domain, a calmodulin-binding domain and an auto-inhibitory domain (AID). CnB has a main latch region with four Ca2+-binding EF-hand motifs, which can be recognized by immunosuppressive complexes and competitively binds to CnA.

[19]. This reversible phosphorylation controls NFAT nuclear and cy- catalytic subunit A (CnA) and a regulatory subunit B (CnB). The CnA toplasmic shuttling to switch its transcriptional activity. contains a phosphatase catalytic domain, a CnB-binding domain, a NFAT is primarily activated by store-operated calcium entry via calmodulin-binding domain and a carboxy-terminal auto-inhibitory calcium release-activated calcium (CRAC) channels. The CRAC chan- domain (AID) [26,27]. Under resting condition, the AID interacts with nels include the CRAC channel protein 1 (ORAI1) and stromal the calmodulin-binding domain to inhibit its phosphatase activity. interaction molecule 1 (STIM1) in non-excitable cells [20–23]. Phos- In the activated state, increased cytoplasmic Ca2+ concentration assists pholipase Cγ (PLCγ) activation initiates Ca2+ mobilization. Ligand calmodulin in displacing the AID and promotes CnB binding to CnA binding to cell surface receptors, such as immunoreceptors, recep- by activating Ca2+-binding EF-hand motifs in the CnB. These events tor tyrosine kinases and G-protein-coupled receptor (GPCR), induces ultimately lead to calcineurin activation. The activated calcineurin PLC activation and subsequent phosphatidylinositol-4, 5-bisphosphate dephosphorylates multiple phospho-residues in NFAT NHR, leading

(PIP2) hydrolysis into diacylglycerol (DAG) and inositol trisphosphate to NFAT cytoplasmic–nuclear trafficking. Thus, calcineurin plays a 2+ (IP3). IP3 binds to the Ca -permeable IP3 receptor (IP3R) on the en- key role in NFAT dephosphorylation [10,28]. doplasmic reticulum (ER) membrane and stimulates Ca2+ release from In addition to promoting nuclear translocation, calcineurin keeps the stores into the cytosol. Subsequently, STIM1 senses the de- NFAT dephosphorylated and constitutively activated in the nucleus. crease in ER Ca2+, which then forms oligomers and moves to the ER– Therefore, calcineurin inhibition could be an effective way to promote plasma membrane junction, where it gates the plasma membrane NFAT nuclear export and cytoplasmic retention. CsA and FK506 calcium channel ORAI1. This STIM1–ORAI1 interaction induces a con- inhibit calcineurin by binding to its CnB domain. Both compounds formational change in ORAI1, resulting in a persistent rise in are widely used as immunosuppressants in the clinic, mainly in organ intracellular Ca2+ concentration. In addition to the STIM–ORAI system, transplantation and dermatology. In addition, there are several en- there are other Ca2+ channels, including voltage-gated Ca2+ and tran- dogenous calcineurin inhibitors such as Down’s syndrome candidate sient receptor potential ion channels, which are also involved in Ca2+ region 1 (DSCR1), calcineurin-binding protein 1 (CABIN1) and influx across the plasma membrane into the cytoplasm [24,25]. The A-kinase anchor protein 79 (AKAP79) [29–31]. Interestingly, DSCR1 increased cytoplasmic Ca2+ leads to activation of the Ca2+ sensor is upregulated in patients with Down’s syndrome, and these pa- calmodulin (CaM) and CaM further activates the phosphatase tients are less predisposed to malignancy [32]. It was later found calcineurin [4,26]. As shown in Fig. 1B, calcineurin consists of a that DSCR1 suppresses tumor growth by inhibiting the calcineurin 176 J. Shou et al./Cancer Letters 361 (2015) 174–184 pathway [33]. Additionally, blocking targets upstream of the Ca2+/ skin cancer [53,54]. Constitutive NFAT2 activation induces skin and calcineurin/NFAT pathway by inhibiting STIM1/ORAI1 or other Ca2+ ovary cancers in transgenic mice, suggesting that NFAT2 has dual- channels could be an alternative approach in calcineurin inhibition. characterized effects [49]. Similarly, CsA or FK506 pretreatment In addition to calcineurin, several serine/threonine protein kinases impairs ultraviolet B-induced apoptosis of human keratinocytes [55], that regulate NFAT have been identified. These kinases are gener- while CsA and FK506 enhanced ultraviolet A-induced human fi- ally classified into two classes: export and maintenance kinases. broblast cell death [56]. These inconsistencies may be because Export kinases rephosphorylate NFAT in the nucleus to induce its different cell types express distinct primary NFAT isoforms. Human relocation to the cytoplasm, while maintenance kinases keep NFAT keratinocytes may express NFAT1 as its major isoform while fibro- fully phosphorylated and keep them in the cytoplasm. Glycogen syn- blasts primarily express NFAT2. thase kinase 3β (GSK3β) phosphorylates the SP2 motif of NFAT1 and NFATs also regulate mammalian cell proliferation. Constitutive the SP2/3 of NFAT2 [34]. NFAT phosphorylation by protein kinase A NFAT2 activation not only induces cell transformation but also pro- (PKA) or the dual specificity tyrosine phosphorylation-regulated motes colony formation in fibroblasts [57]. Similarly, NFAT2 induces kinases (DYRKs) is a prerequisite for GSK3β phosphorylation [35]. c-Myc transcription in pancreatic cancer cells and c-Myc interacts PI3K/Akt signaling can phosphorylate and inhibits GSK3β, and it is with the NFAT complexes, leading to cyclin D upregulation and one of the most highly affected pathways in cancer cells [36].However, anchorage-independent growth [58]. Additionally, sequential in- it has also been reported that GSK3β protects NFAT1 from E3-ubiquitin duction of NFAT2 and c-Myc facilitates TGF-β-promoted cell growth ligase MDM2-mediated proteolysis [37,38]. Mitogen-activated protein in various cancer cells [59]. NFAT1 suppresses cyclin dependent kinases (MAPKs), which are excessively activated in human carci- kinase 4 (CDK4) and cyclin A2 expression to control lymphocyte cell noma, promote NFAT cytoplasmic translocation. P38 MAPKs cycle and proliferation [60,61]. In NFAT1 and NFAT4 deficient mice, selectively phosphorylate Ser168 and Ser170 in NFAT3’s NHR, and immune cells are resistant to activation-induced cell death, Jun N-terminal kinase (JNK) phosphorylates the calcineurin- hyperactivation and defective Fas ligand production, highlighting targeting domain of NFAT2 and NFAT4 [39,40]. There are also their tumor suppressor activities [62,63]. However, NFAT1 has been regulators that have both maintenance and export kinase func- shown to induce MDM2 transcription and inactivate p53 in breast tions, such as DYRKs and casein kinase 1 (CK1). The DYRK family cancer cells [64]. Baumgart et al. previously report that NFAT1 ex- was first discovered in a genome-wide small interference RNA screen pression correlates with advanced stage pancreatic cancer by in Drosophila melanogaster [35], and it is composed of two family silencing the tumor suppressor gene p15 [65]. Taken together, these members: DYRK1 and DYRK2. DYRK1 works as an NFAT export kinase, data suggest that NFAT1 is a tumor suppressor in untransformed while DYRK2 functions as a maintenance kinase. Both can directly cells, but may act as an oncogene in cancer cells. phosphorylate NFAT1’s SP3 motif. CK1 functions as a maintenance Previous studies also revealed that NFAT2 plays a critical role in kinase by phosphorylating the SRR1 motif and as an export kinase the switch from stem cell dormancy to proliferation. As the down- by docking at a conserved motif near NFAT1’s N-terminus [41]. stream of bone morphogenetic protein 4, NFAT2 inhibits CDK4 to Interestingly, there is another type of NFAT kinase, which can maintain skin stem cell dormancy [6]. Another study demon- phosphorylate NFAT and enhance their transcriptional activity. strated that constitutively activated NFAT2 leads to significant Cot/Tpl2 kinase and the PKC pathway promote NFAT1 nuclear trans- expansion of CD34+ cells and initiates skin squamous cell-like car- location by phosphorylating Ser53 and Ser56 of the N-terminal TAD. cinoma [66]. NFAT2 may be involved in acquiring stem cell-like CsA does not inhibit this transactivation, which indicates that NFAT1 properties and self-renewal capacity, including tumor cell epithe- can be activated independent of calcineurin [42,43]. Another recent lial to mesenchymal transition (EMT) [67] (Table 1). study has revealed that human vaccinia-related kinase 2 (VRK2) has similar functions. VRK2 phosphorylates Ser32 in the N-terminal TAD NFATs in tumor metastasis followed by calcineurin activation [44]. In addition to NFAT kinases, caspase 3 has been shown to cleav- Metastasis is the leading cause of human cancer death. Even in age two potential sites in the NFAT1 TAD [45]. Poly-ADP-ribose patients with decades of disease-free survival, metastatic tumors polymerase 1 (PARP1) promotes NFAT stability and transcrip- sometimes recur. It is well known that angiogenesis and tional activity in T cells [46,47]. Post-translational modifications, lymphangiogenesis are necessary for cancer metastasis. Cancer cells including ubiquitination and sumoylation, also modulate NFAT, which become aggressive and motile through EMT, and invade the blood makes the NFAT family’s regulatory network more complex [38,48] or lymphatic vessels to circulate [68]. It is estimated that 0.01% of (Fig. 2). cancer cells in circulation will survive and initiate new tumor growth in distant organs [69]. Although the metastatic process is clear, the NFATs in tumorigenesis and proliferation molecular mechanism is still unclear. Emerging evidence has suggested that NFATs play important roles in the metastasis process. It is well known that carcinogen-induced genetic or epigenetic aberration leads to tumorigenesis. NFAT is a ubiquitous transcrip- NFATs in tumor invasion and migration tion factor in vertebrates, and its dysregulation induces the expression of various target genes involved in cancer develop- Invasion and migration acquisition is the first step of cancer me- ment. However, NFAT isoforms have distinct and diverse functions tastasis, and it is always combined with EMT [70]. Jauliac et al. first in the tumorigenic process. Constitutive NFAT2 activation inhibits reported that NFAT1 expression promotes breast cancer cell inva- fibroblast cell differentiation and induces malignant transforma- sion and migration, and wild-type NFAT5 expression only promotes tion. In contrast, constitutively active NFAT1 induces cell cycle arrest migration. Meanwhile, NFAT1 and NFAT5 overexpressions have been and apoptosis and inhibits H-rasV12-induced transformation [49]. found in invasive primary breast cancer cells, and this NFAT1/5 NFAT1 deficient mice have an increased susceptibility to chemical overexpression is accompanied with increased α6β4 integrin ex- carcinogen-induced tumorigenesis. Other carcinogens, including pression [71]. The α6β4 integrin is anchored to hemidesmosomes nickel compounds and asbestos, have been reported to increase NFAT and correlates with the actin cytoskeleton in cancer cells. NFATs are activity by diverse mechanisms [50–52]. These results suggest that downstream of α6β4 integrin signaling, as α6β4 integrin regu- NFAT1 is a tumor suppressor and NFAT2 is an oncogene. However, lates NFAT5. Another aberrant pathway in breast cancer is the PI3K/ there are also some contradictory reports. In organ-transplant pa- Akt pathway [36]. Although PI3K/Akt activity leads to nuclear NFAT tients, long-term treatment of CsA or FK506 increases the risk of retention and increased NFAT activity by inhibiting GSK3β, breast J. Shou et al./Cancer Letters 361 (2015) 174–184 177

Fig. 2. The NFAT pathway and regulation. Immunoreceptors, receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCR) activate phospholipase Cγ (PLCγ), 2+ which hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 binds to Ca -permeable IP3 receptor (IP3R) on the endoplasmic reticulum (ER) and stimulates Ca2+ release into the cytosol. With the loss of calcium, stromal interaction molecule (STIM) reforms into oligomers and interacts with calcium release-activated calcium channel protein (ORAI), which then induces a sustained influx of extracellular Ca2+. Transient receptor potential (TRP) Ca2+ channels are other ways for Ca2+ entry. Then the calcium sensor protein calmodulin (CaM) is activated by Ca2+ and further activates calcineurin, which dephosphorylates NFAT, leading NFAT cytoplasmic–nuclear trafficking. In the nucleus, NFAT forms dimers or cooperates with other transcription partners to initiate target gene transcription. Some transcription partners can be activated by RAS/RAF/MEK/ERK signaling. NFAT is also regulated by various NFAT kinases. Glycogen synthase kinase 3β (GSK 3β), dual specificity tyrosine phosphorylation-regulated kinases (DYRKs), casein kinase 1 (CK1), mitogen-activated protein kinase (p38 MAPK) and Jun N-terminal kinase (JNK) rephosphorylate and inactivate NFAT, while human vaccinia-related kinase 2 (VRK2) and Cot kinase cooperating with protein kinase C (PKC) promote NFAT nuclear translocation. NFAT could be cleaved by caspase 3 and ubiquitinated by MDM2 which is activated via the PI3K/AKT pathway, while poly-ADP-ribose polymerase 1 (PARP1) stabilizes and activates NFAT. There are also several endogenous calcineurin inhibitors, including Down syndrome candidate region 1 (DSCR1), calcineurin-binding protein 1 (CABIN1), and A-kinase anchor protein 79 (AKAP79), as well as pharmacological calcineurin inhibitors: cyclosporine A (CsA) and tacrolimus (FK506), which bind to cyclophilin and FK506 binding protein (FKPB) respectively.

Table 1 NFATs in tumorigenesis and proliferation.

NFAT isoforms Cell types Potential mechanism Reference

NFAT1 3T3 cell Inducing cell cycle arrest and apoptosis, inhibiting H-rasV12-induced transformation and [49] reducing the risk in carcinogen-induced tumorigenesis. T cell Suppressing CDK4 and cyclin A2 and inducing FasL and AICD. [60–62] Breast cancer Inducing MDM2 transcription and inhibiting p53. [64] Pancreatic cancer Silencing tumor suppressor gene p15 (INK4b). [65] NFAT2 3T3, Skin and ovary cells Inhibiting cell differentiation, promoting colony formation, inducing malignant transformation [49,57] and CD34+ stem cell expansion. Colon cancer and pancreatic cancer Inducing expression of c-Myc and cyclin D, and facilitating TGF-β-promoted cell growth. [58,59] Skin stem cell Maintaining dormancy of stem cells by inhibiting CDK4. [6] NFAT3 Null NFAT4 T cell Decreasing lymphoproliferation by inhibiting NFAT2-induced IL-4 expression and increasing AICD. [62,63] 178 J. Shou et al./Cancer Letters 361 (2015) 174–184

Fig. 3. NFATs in tumor invasion and migration. The most important acquired property for tumor invasion and migration is the epithelial to mesenchymal transition (EMT). NFATs are proved to be related to this section as their transcriptional targets are involved. The α6β4 integrin on tumor cells activates NFATs, which transcript multiple target genes such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), autotaxin and matrix metalloproteinases (MMPs). COX-2 synthesizes PGE2, while autotaxin catalyzes lysophosphatidic acid (LPA). Both PGE2 and PLA bind to PGE2 receptor (EP) and LPA receptor (LPAR), respectively, to promote tumor invasion and migration. MMPs promote the proteolysis of extracellular matrix (ECM), and facilitate tumor cells migrating through the ECM. NFAT3 cooperates with ERα to reduce migration through inhibiting lipocalin 2 (LCN2) expression in breast cancer cell.

cancer cells with activated PI3K/Akt signaling show decreased mo- its overexpression increases the invasive potential of tumor cells tility and invasion. Further investigation explains that Akt promotes through MMP-7 and MMP-9 upregulation [82]. Additional study has NFAT1 ubiquitination exclusively by the E3 ubiquitin ligase MDM2 claimed that NFAT2 activation promotes metastasis of mouse bone [38]. Liu et al. and Chen et al. also find that NFAT1 is overexpressed tumor cells via MMP-2 upregulation [83]. in human lung cancer cells and NFAT1 knockdown inhibits cell in- Interestingly, NFAT3 has an anti-metastatic function in estro- vasion and migration [72,73]. gen receptor α positive (ERα+) breast cancer cells [84].NFAT3 Mechanistically, NFATs are transcription factors for invasion and cooperates with ERα to prevent cancer cell migration, and it inhib- migration-associated genes, such as cyclooxygenase-2 (COX-2) and its lipocalin 2 expression to reduce cancer cell invasion. Conversely, autotaxin. NFAT1 induces COX-2 expression, which catalyzes pros- NFAT3 downregulation leads to actin reorganization, which corre- taglandin synthesis, including prostaglandin E2 (PGE2), in breast lates with increased migration and invasion. Taken together, different epithelial cells [74]. Knockdown or chemical inhibition of COX-2 NFAT isoforms have diverse functions, making it more difficult and limits breast cancer cell invasion. In contrast, COX-2 or PGE2 complicated to target NFAT in anti-cancer therapies (Fig. 3). overexpression enhances cell invasion [74]. Another NFAT target gene is autotaxin, which converts lysophosphatidylcholine into NFATs in tumor angiogenesis and lymphangiogenesis lysophosphatidic acid (LPA), and LPA acts as both mitogen and motogen in breast cancer. Autotaxin is overexpressed in an α6β4/ The most common pathway for cancer metastasis is the human NFAT-dependent way and facilitates breast cancer cell metastasis vessel system, which includes blood and lymphatic vessels. In ad- in vitro and in vivo [75]. dition to providing nutrients and oxygen for tumor proliferation and Furthermore, NFAT1 can initiate glypican-6 (GPC-6) transcrip- survival, tumor neovascularization provides a way for tumor me- tion, which activates JNK and p38 MAPK through Wnt5A signaling tastasis. Neovascular primitive structure, malformation, blood flow and increases breast cancer cell invasive migration [76]. However, disturbance and hyperpermeability all contribute to metastasis. In as mentioned above, p38 MAPK also functions as an NFAT1 export vertebrates, calcineurin/NFAT signaling is essential for cardiovas- kinase. This may provide a feedback and self-regulation of the NFAT- cular system formation [85,86]. Thus, it is reasonable that NFATs may MAPK loop. NFAT1 hyperactivity may not be completely inhibited be involved in angiogenesis and lymphangiogenesis, which are es- by NFAT kinases, and this ultimately leads to the pro-metastasis phe- sential for cancer dissemination. notype. Foldynova-Trantirkova et al. reported that CK1ε mutants Several soluble factors have been identified to correlate with activate the Wnt/Rac1/JNK and NFAT pathways, resulting in de- tumor angiogenesis, including vascular endothelial growth factor creased breast cancer cell adhesion and increased migration [77]. A (VEGFA), acidic/basic fibroblast growth factor (a-FGF/b-FGF) and Matrix metalloproteinases (MMPs) are well-known basement mem- secreted frizzle-related protein 2 (SFRP2) [11,87,88]. VEGFA activates brane proteolytic enzymes that are activated during tumor invasion PLCγ by binding to the VEGF receptor (VEGFR), while FGF23 binds [78], and they are induced by NFATs in myocytes, glomerular to FGF receptor 1c (FGFR1c). Both lead to calcineurin/NFAT signal- mesangial cells and astrocytes [79–81]. One recent study has found ing activation [89]. SFRP2 increases NFAT4 nuclear translocation that NFAT1 is overexpressed in glioblastoma multiforme cells, and through the noncanonical Wnt/Ca2+/NFAT pathway [87]. After they J. Shou et al./Cancer Letters 361 (2015) 174–184 179 localize to the nucleus, NFATs enhance transcription of some target glioma cell survival [98]. In large B-cell lymphoma (LBCL), NFAT2 genes involved in angiogenesis, such as COX-2, tissue factor (TF) and is constitutively active and interacts with NF-κB to upregulate their caspase-8 inhibitor cellular Fas-associated death domain-like downstream target gene CD154 and B-lymphoma stimulator (BLyS), interleukin 1β-converting enzyme inhibitory protein (c-FLIP). COX-2 which can maintain lymphoma cell survival. NFAT2 knockdown by plays a pro-angiogenic role by enhancing PGE2 synthesis. COX-2 in- siRNA induces LBCL apoptosis, suggesting that NFAT2 constitutive hibitor or CsA can inhibit angiogenesis in vitro and in vivo.However, activation plays a pivotal role in maintaining LBCL survival [99]. In- CsA fails to prevent b-FGF-induced angiogenesis, suggesting that terestingly, NFAT3 acts as a tumor suppressor. It is required in NFAT activation by b-FGF does not require the canonical pathway doxorubicin-mediated apoptosis in human glioma cells, as the NFAT3 [90]. Later, c-FLIP was found to be an angiogenesis enhancer as down- target gene tumor necrosis factor α (TNF-α) is required to initiate stream of b-FGF/NFAT1 signaling. Meanwhile, pigment epithelium- caspase-dependent apoptosis [100]. NFAT family members can also derived factor (PEDF) acts as an inhibitor of FGF-activated NFAT1 induce caspase-independent apoptosis [101,102]. by activating JNKs, leading to decrease c-FLIP expression and inhi- As discussed above, NFATs have potential pro-survival func- bition of angiogenesis [91]. TF is another crucial angiogenesis initiator tions, as tumor cells may develop drug resistance via NFAT pathway induced by the VEGFA/NFAT pathway [92]. activation. ABT-737 induces cancer cell apoptosis by binding to anti- More powerful evidence for NFAT involvement in tumor angio- apoptotic Bcl-2 family member proteins. But Bcl-2-A1 is upregulated genesis lies in animal models and human pathophysiology. The by TCR/calcineurin/NFAT pathway in activated T lymphocytes or ABT- endogenous calcineurin suppressor DSCR1 and maintenance kinase 737-resistant chronic lymphocytic leukemia. Bcl-2-A1 is a Bcl-2 DYRK1A are upregulated in Down’s syndrome patients. Baek et al. family member with an anti-apoptotic function. Therefore, block- previously report that Down’s syndrome could suppress tumor ing Bcl-2-A1-dependent anti-apoptotic signaling via CsA is critical growth. Using transgenic mice, they found that DSCR1 and DYRK1A to prevent ABT-737 resistance [103]. block angiogenesis by inhibiting the VEGFA/calcineurin/NFAT sig- Imatinib is the first targeted drug that greatly improves the naling pathway [33]. The congenital disease infantile hemangioma outcome of Philadelphia chromosome positive (Ph+) chronic myeloid is also related to VEGFR/NFAT signaling dysregulation [93].Inthe leukemia (CML) patients by inhibiting Bcr–Abl [104]. Unfortu- region of disorganized vasculature, VEGFR1 is downregulated in an nately, these cancers often acquire imatinib resistance and become NFAT-dependent manner. VEGFR1 downregulation leads to VEGF- more aggressive. A recent study revealed that the Wnt/Ca2+/NFAT dependent VEGFR2 activation. A subset of these patients harbor pathway is involved in imatinib resistance. Wnt5A-bound Frizzled mutations in VEGFR2 or integrin-like receptor TEM8, which forms (FZD) binds to GPCR and activates phospholipase C, which acti- a complex with β1 integrin, inhibits integrin/NFAT signaling and vates NFAT2. The NFAT2 target gene IL-4 promotes CML cell survival reduces VEGFR1 expression. Conversely, wild-type VEGFR2 or TEM8 upon Bcr–Abl Inhibition. Wnt/Ca2+/NFAT signaling inhibition sen- overexpression restores the normal phenotype [93]. sitizes Ph+ leukemia cells to Bcr–Abl inhibitors, including imatinib Lymphatic metastasis is another route for tumor dissemina- and the second-generation inhibitor dasatinib in vitro and in vivo tion. Solid tumors utilize pre-existing lymph vessels or generate new [105]. Griesmann et al. reported that the Wnt/Ca2+/NFAT pathway lymphatic vasculature to reach distant organs. Chen et al. previ- also mediates pancreatic cancer cell resistance to drug-induced apop- ously reported that NFAT expression is significantly higher in lung tosis. Wnt5A knockdown leads to a remarkable increase in cancer than in adjacent normal lung tissues. Moreover, NFAT1 ex- gemcitabine-induced apoptosis [106]. As a downstream Wnt5A pression was significantly higher in patients with lymph node target, NFAT1 functions in an anti-apoptosis mechanism. metastasis than without lymph node metastasis. Therefore, posi- Another successful targeted drug is the first-generation of epi- tive NFAT1 expression indicates a poor prognosis in lung cancer [73]. dermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), VEGFC and platelet-derived growth factor BB (PDGF-BB) have been which greatly helps in the treatment of lung cancer patients with extensively studied in lymphangiogenesis [94,95]. VEGFC and PDGF- EGFR sensitive mutations [107]. Gefitinib and erlotinib inhibit the BB co-expression is also related to lymphangiogenesis in lung cancer EGFR signaling cascade by reversibly binding to EGFR’s adenosine [96]. The results above suggest that both the VEGFC/NFAT1 and PDGF/ triphosphate binding sites. However, EGFR-TKIs have similar limi- NFAT1 pathways are involved in tumor lymphangiogenesis. NFAT2 tations as Bcr–Abl inhibitors. Nearly all patients acquired resistance also plays a critical role in lymphatic-valve morphogenesis and pat- to TKIs via diverse mechanisms, such as a secondary T790M mu- terning [97]. As downstream of VEGFA/VEGFR2 signaling pathway, tation, c-Met amplification or PI3K mutation. The second- it interacts with prospero homeobox 1 (PROX1) or FOXC2 to promote generation TKI afatinib, which irreversibly binds to EGFR still could lymphangiogenic gene transcription, including FGFR3, VEGFR3 and not overcome the effect of the T790M mutation. Recently, signal podoplanin (Fig. 4). transducer and activator of transcription 3 (STAT3) activation has As discussed above, NFATs can facilitate tumor angiogenesis been shown to be involved in TKI resistance [108,109].STAT3 and lymphangiogenesis. Consequently, NFATs can promote tumor induces Wnt5A activation, which can stimulate the noncanonical progression and metastasis. To date, the anti-VEGF antibody and Wnt/Ca2+/NFAT pathway, as discussed above. Moreover, NFAT2 con- VEGFR tyrosine kinase inhibitors (TKIs) have been used to inhibit stitutive activation induces autocrine of growth factors, which can tumor angiogenesis and lymphangiogenesis, but the result is activate STAT3 in turn [110]. Interestingly, nuclear NFAT2 can co- not so satisfactory. Due to NFAT’s crucial role in angiogenesis and operate with STAT3 to control IL-6, IL-4 and other cytokine lymphangiogenesis, further investigation is required to explore expressions [111]. This feedback loop promotes cancer cell surviv- new effective methods in targeting NFAT and its down or up- al under stress, such as anti-cancer drug treatment. Our preliminary stream genes. study also shows that CsA greatly enhances gefitinib-induced apoptosis in both EGFR sensitive and resistant cell lines through STAT3 NFATs in tumor survival and drug resistance inhibition (unpublished data). The BRAF inhibitor vemurafenib and MEK inhibitor trametinib provide clinically significant improve- Accumulating evidence indicates that the NFAT family is in- ments in melanoma patients [112,113]. Recent reports have volved in the development of drug resistance to chemotherapeutics demonstrated that NFAT inhibition augments vemurafenib and or targeted agents. It has been reported that the dysregulation of trametinib anti-cancer effects [114,115]. Spreafico et al. have found NFAT signaling confers malignant cells with pro-survival potential that combination treatment with CsA reverses resistance to the under stress conditions. CsA induces apoptosis in rat glioma cells, MEK inhibitor selumetinib in a patient-derived tumor xenograft which indicates that the calcineurin/NFAT pathway can promote model of colorectal cancer [116] (Table 2). 180 J. Shou et al./Cancer Letters 361 (2015) 174–184

Fig. 4. NFATs in tumor angiogenesis and lymphangiogenesis. Neovascular and neolymphatic are two main approaches for tumor cells to detach from initial location and spread to distant organs. Vascular endothelial growth factor (VEGF), ﬁbroblast growth factor (FGF), secreted frizzle-related protein 2 (SFRP2) and platelet-derived growth factor BB (PDGF-BB) are secreted into ECM by various cells, including tumor cells, ﬁbroblasts, endothelial cells and precursors. On the neovascular side, VEGFA and FGF bind to each receptor, while SFRP2 cooperates with Wnt to activate non-canonical Wnt/Ca2+/NFAT pathway. Both lead to the expression of angiogenesis factors, including COX-2, PGE2, tissue factor (TF) and cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein (c-FLIP). Pigment epithelium-derived factor (PEDF) inhibits NFAT through JNK signaling, and indirectly attenuates the angiogenesis induced by c-FLIP. On the neolymphatic side, VEGFC, FGF and PDGF-BB promote NFAT nuclear translocation, and NFAT interacts with prospero homeobox protein 1 (PROX1) or FOXC2 to transcript neolymphatic-related genes, such as FGFR, VEGFR and podoplanin.

Table 2 NFATs in drug resistance.

Compounds NFAT isoforms Potential mechanism Tumor types Reference

Doxorubicin NFAT3 Caspase-dependent apoptosis initiated by NFAT3-activated TNF-α. Glioma [100] Gemcitabine NFAT1 Wnt5A/NFAT1 pathway inhibition increases apoptosis. Pancreatic cancer [106] Dasatinib and Imatinib NFAT1 IL-4 activated by NFAT1 leads to treatment failure. CML [105] ABT-737 NFAT Bcl-2-A1 induces drug resistance by calcineurin/NFAT pathway CML [103] Geﬁtinib and Erlotinib NFAT2 STAT3 is activated by NFAT2 in various approaches. Lung cancer [110,111] PD0325901 and vemurafenib NFAT1 Enhanced apoptosis by inhibiting calcineurin/NFAT1 pathway Melanoma [114,115] Selumetinib NFAT1 Increased apoptosis via Inhibiting Wnt/Ca2+/NFAT1 signaling. Colorectal cancer [116]

NFATs in the tumor microenvironment Therefore, it is necessary to treat both tumor cells and the tumor microenvironment. The molecular mechanism of the interaction between Since Stephen Paget proposed the famous ‘seed and soil’ hypoth- tumor cells and its microenvironment remains elusive. Recently, esis, the tumor microenvironment has been a focus in cancer research several studies have demonstrated that NFATs exert important func- [117]. It is now well recognized that the tumor microenvironment tions in the tumor microenvironment, which significantly affect cancer is involved in every stage of cancer, including tumorigenesis, cancer initiation and development. proliferation, metastasis and drug resistance [69]. The tumor mi- Numerous cytokines are involved in tumorigenesis, angiogen- croenvironment is a complex system composed of endothelial cells, esis and metastasis. NFAT signaling activation in tumor cells results fibroblasts, pericytes, muscle cells, immune cells and their precur- in inflammatory chemokine production, which can recruit inflam- sors. It is an integral and essential component of cancer [118]. matory cells, including mast cells, neutrophils and monocytes, to J. Shou et al./Cancer Letters 361 (2015) 174–184 181 the tumor [119]. Thus a feedforward loop forms between inflam- microenvironment. In conclusion, NFAT2 activation possesses both mation, the immune response and cancer. CXCL12 and CCL21 are cell autonomous and cell non-autonomous mechanisms for estab- chemokines that are upregulated in advanced breast cancer, and lishing the tumor microenvironment in situ [66]. Studies have also these chemokines act as a chemotactic factor to promote breast been performed examining the microenvironment of the pre- cancer cell invasion [120,121]. Likewise, macrophages infiltrate into metastatic site or niche. Loss of DSCR1, an endogenous calcineurin the tumor microenvironment and secrete epidermal growth factor inhibitor, leads to excessive calcineurin/NFAT/angiopoietin-2 (ANG-2) (EGF) and colony-stimulating factor 1 (CSF1) to facilitate tumor pro- pathway activation in lung endothelium and the establishment of liferation and migration [122]. NFATs can induce CSF1 in both lung metastasis [123]. Increased VEGF level in the lung microen- immune cells and tumor cells, indicating a dual effect of NFATs in vironment triggers calcineurin/NFAT/ANG-2 signaling activation and tumor progression. promotes angiogenesis in the pre-metastatic niche. Although there Recently, there are some direct pieces of evidence indicating was no difference in the numbers of lung metastases in NFAT1 knock- that NFAT signaling establishes the tumorigenic microenviron- out mice, the tumors are smaller in size [124]. Loss of NFAT1 reduces ment in vivo. In inducible constitutively active NFAT2 transgenic transforming growth factor β (TGF-β) expression and recruits more mice, spontaneous cancers were found in the skin and ovary. In- mononuclear cells. triguingly, both cells expressing constitutively active NFAT2 and In addition, NFAT1 has been reported to promote tumor- neighboring wild-type cells upregulated c-Myc and STAT3 in skin induced T-cell anergy [125]. As the main antitumor immune response and ovary tumors. This phenomenon suggests that NFAT2 consti- character, T cells are attenuated by the tumor-associated immuno- tutive activation affects the microenvironment and further affects suppressive microenvironment. Normally, NFAT activation the neighboring cells. Meanwhile, several cytokines and cytokine results in T cell excitation. In B16 melanoma or TC-1 lung cancer receptors are upregulated and contribute to the inflammatory bearing animals, NFAT1 deficient mice harbor more effective

Fig. 5. NFATs in tumor microenvironment. The tumor microenvironment is a complex system which is involved in all aspects discussed above, including tumor migration, angiogenesis and drug resistance. Here we briefly focus on inflammatory cells and other neighboring cells. NFAT facilitates cytokines secreted into the tumor microenvironment, which induce inflammatory cells infiltrating in the tumor microenvironment. Macrophages produce epidermal growth factor (EGF) and colony-stimulating factor 1 (CSF1) for tumor proliferation and migration. T cells are induced into an anergic condition due to decreased interleukin 2 (IL-2) and interferon-γ (IFN-γ) by NFAT1. NFAT promotes the expression of signal transducer and activator of transcription 3 (STAT3), c-Myc, MMPs and transforming growth factor-β (TGF-β) in neighboring cells at primary tumor location, while in pre-metastatic niche, VEGF activates the calcineurin/NFAT signaling and leads to the high level of angiopoietin-2 (ANG-2), which induces the neovascularization for the circulating cancer cell (CTC) settling down. 182 J. Shou et al./Cancer Letters 361 (2015) 174–184 tumor-infiltrating CD4+ T cells, which can inhibit tumor growth. unknown. Perhaps more likely, kinases or NFAT binding partners These NFAT1−/− CD4+ T cells produce more IL-2 and interferon-γ that regulate NFATs may be dysfunctional in cancer. Therefore, ad- (IFN-γ) than wild-type mice, and are resistant to tumor-induced dressing the issues discussed here will be pivotal in understanding anergy. Interestingly, NFAT1 depletion does not affect T-cell acti- NFAT function in cancer and developing novel drugs against the NFAT vation, likely due to a compensatory effect by NFAT2 (Fig. 5). pathway.

Targeting the NFAT pathway for cancer therapy Acknowledgments

As discussed above, the two classical calcineurin inhibitors CsA This work is supported by the National Natural Science Foun- and FK506 are widely used as immunosuppressant in organ recipi- dation of China grant number 81272593 (www.nsfc.gov.cn)toH. ents. CsA binds to the immunophilin protein cyclophilin, while FK506 Pan, and the National Natural Science Foundation of China grant binds to FK binding protein (FKBP). Both drug–immunophilin com- number 81372621 (www.nsfc.gov.cn) to W. Han. plexes directly interact with calcineurin and inhibit its phosphatase activity, leading to NFAT inactivation [2,126]. The peptide called VIVIT prevents the interaction between calcineurin and NFATs to block Conflict of interest NFAT activation [127]. VIVIT has more potent anticancer function in vitro and in vivo [128]. However, the chemical inhibitors and The authors declare no conflict of interest. peptide target proteins upstream of NFATs, which will lead to broad NFAT inhibition. 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