Recent knowledge of NFATc4 in oncogenesis and cancer prognosis

Epigenetics is the study of heritable changes in gene expression without alteration of the underlying DNA sequence that are mediated by epigenetic factors like methylation of DNA, modification of histone and non-coding RNA regulation [29]. It is reported that NFATc4 protein expression can be suppressed by several microRNA: miR-34a-5p [30], miR-29a-3p [31], miR-133a [32], miR-182 [14], miR-145-5p [33], miR-7134-5p [34], and miR-140 [11]. However, DNA and histone modification haven’t been explored yet.

At the resting state, a balance between the tonic activity of calcineurin phosphatase and the protein kinase is required to maintain cytosolic localization of NFATc4 [35]. Upon an increase of intracellular calcium, activated calcineurin dephosphorylates NFATc4 and promotes its nuclear translocation. Transcription termination promotes rephosphorylation of NFATc4, then NFATc4 can be translocated to the cytosol. Except phosphorylation and dephosphorylation, NFATc4 can also be subjected to ADP-ribosylation, ubiquitination, and deacetylation (Table 3). Specifically, ADP-ribosylation at NFATc4 DNA binding domain could augment the transcriptional activity of NFATc4 [36] whereas ubiquitination of NFATc4 promoted NFATc4 degradation and thus suppressed its transcriptional activity [37]. Also, it has been shown that the deacetylation of NFATc4 by SIRT6 could suppress NFATc4 dephosphorylation [38].

Besides post-translational modification, NFATc4 transcriptional activity is also modulated by the interaction proteins (Table 4).

Lung cancer is the leading cause of cancer induced death worldwide. The past two decades of research have yielded encouraging results about the disease mechanism and therapy of lung cancer. Nonetheless, the need of new molecular targets remain to be intensively elucidated to prompt the development of diagnosis and new therapies. Chen’s research revealed that the NFATc4 positive rate is 28.3% in tumor tissues and 1.3% in adjacent normal tissues from 159 non-small cell lung cancer patients [12]. They found a positive correlation between the expressions of NFATc4 and COX-2 which were both overexpressed in non-small cell lung carcinoma. In addition, NFATc4 transcriptionally regulated COX-2 expression and prevented human bronchial epithelial cells BEAS-2B from apoptosis caused by arsenite and vanadium treatment [55, 56]. Another research reported that knockdown of NFATc4 expression resulted in a radioprotective effect on A549 lung cancer cell line [57]. The survival analysis of GSE31210 dataset from GEO database indicates that high NFATc4 level tends to a favorable prognosis (Fig. 4A).

Although the research of NFATc4 in lung carcinoma is a bit less, NFATc4 has presented relatively high expression in lung cancer and involved in radiotherapy and the prognosis.

Accumulated evidences have indicated that NFATc4 plays essential roles in regulating the proliferation, invasion and migration of breast cancer cells. It has been shown that the expression of NFATc4 can be detected in breast cancer cells and a subset of breast cancer patients. NFATc4 is overexpressed in breast cancer with 65% positive in cancerous tissue and 15% positive in noncancerous tissue. Notably, it was found that NFATc4 not only interacted with the ligand-independent transactivation domain of ER (ERα and ERβ) but also increased its binding to the estrogen-responsive elements resulting in the up-regulation of pS2 and cathepsin D expression. Meanwhile, knockdown of NFATc4 reduced the cell growth rate in ZR-75-1 cells which showed high NFATc4 expression level [48].

Tumor invasion and metastasis may lead to cancer progression and poor outcomes. NFATc4 also shows potential regulation of these aspects in breast cancer cells. Interestingly, NFATc4 expression is restricted to ERα positive cell lines at both transcriptional and translational levels. Remarkably, NFATc4 inhibited cell invasion and migration through regulating actin redistribution. Furthermore, research identified that NFATc4/ERα decreased cell migration capacity through transcriptionally repression of LCN2 expression [13]. Meanwhile, overexpression of ERα promoted the nuclear translocation of NFATc4. NFATc4/ERα and NFATc4/ERβ inhibited the transcriptional activity of IL-2 in breast cancer cells, especially ERα. Phosphorylation of ERα at different sites affected the regulation of ERα on NFATc4 transcriptional activity [47]. Additionally, NFATc4 is highly co-expressed with androgen receptor (AR) which is expressed in 88% ER-positive breast tumors [58].

Extracellular vesicles (EVs) are kinds of two-layer secreted vesicles which play pivotal roles in mediating cell signal transduction and pathological process. The tumor-derived EVs are thought as the promising therapeutic approaches through mediating intercellular communication in the tumor microenvironment. EVs produced by breast cancer cells expressing NFATc4 inhibited cell invasion effectively among different types of cancer cell lines (triple negative breast cancer, invasive melanoma, glioblastoma, and pancreatic cancer). Intra-tumor EVs injection presented a significant inhibition of the tumor volume and metastasis in tumor-bearing mice. To further explain EVs function, de Camargo et al. demonstrated EVs from the NFATc4-expressing cells decreased the cell proliferation rate and induced cell apoptosis through macrophages recruitment in the 2D cell culture system [59]. In addition, NFATc4 down-regulated during (E)-4-chloro-2-((3-ethoxy-2-hydroxybenzylidene) amino) phenol (ACES) induced apoptosis through p38-β/SAPK in MCF-7 breast cancer cells, indicating that NFATc4 could be a latent cancer therapeutic target [60].

Based on survival analysis, NFATc4 is an unfavorable prognostic factor in breast cancer (Fig. 4B). Thus, these observations collectively highlighted the critical role of NFATc4 in breast cancer development. On the one hand, NFATc4 could interact with ER and transcriptionally regulate downstream genes, and promote the cell growth. On the another hand, NFATc4 presenting high expression at mRNA and protein levels in ERα-positive cells repressed LCN2 and led to inhibition of invasion and migration capacities of breast cancer cells. NFATc4 could also play an important role in tumor immune microenvironment through EVs from NFATc4-expressing cells.

Ovarian cancer ranks the fifth cancer-induced death among women. Patients diagnosed with ovarian cancer are treated with complete resection surgery and neoadjuvant or adjuvant chemotherapy. Recently, it is considered that many patients were overtreated especially those at early stage. It is important to develop the molecular signature to indicate more suitable individual therapeutic strategy. Accumulating studies identified that NFATc4 is involved in the ovarian cancer, too. NFATc4 is overexpressed in ovarian cell lines 3D spheroids comparing with adherent cells [16]. In ovarian quiescent cancer stem-like cells (CSCs), NFATc4 is also overexpressed [15]. NFATc4 directly bound to the CXCR4 promoter at the DNA sequence of -114 to + 59 relative to the transcriptional start site and correspondingly increased CXCR4 expression. When the HeyA8 ovarian cell line 3D spheroids were treated with Cyclosporine A (a usual NFAT inhibitor), the cells dissociated from the 3D spheroids. CXCR4 also presented as a key driver in tumor progression in HeyA8 xenograft model. Moreover, phospho-ERK participated in modulating CXCR4 expression through inhibiting NFAT transactivation [16]. Interestingly, NFATc4 is enriched in ovarian quiescent CSCs and inhibited the cell growth through arresting cells at G0 phase. It was found that NFATc4-involved quiescence could play a role in cisplatin resistance. Cisplatin treatment activated NFATc4 and promoted NFATc4 translocation into the nucleus in multiple ovarian cell lines. Remarkably, MYC overexpression may partially inhibit NFATc4-regulated quiescence, which indicated a potential therapy for cisplatin resistance patients [15]. Besides, NFATc4 acted as a risk factor in the survival of ovarian cancer analyzed from the GSE26193 dataset (Fig. 4C).

As a transcriptional factor, activated NFATc4 took part in ovarian cancer progression in various regulatory ways. NFATc4 could promote tumor formation through CXCR4 expression up-regulation whereas NFATc4 inhibited cell proliferation by down-regulating MYC during cisplatin resistance-associated quiescence.

Skin-derived malignancies include squamous cell carcinoma, basal cell carcinoma, and malignant melanoma. These malignancies partially related to harmful factors exposure. NFATc4, a stress response transcriptional factor, was found overexpressing in many skin cancer cell lines comparing with HaCaT cells. Consistently, NFATc4 showed high protein level in various tissues of skin malignancies. The ectopic expression and activation of NFATc4 may markedly facilitate the progress of skin cancer. Overexpressing NFATc4 increased the cell proliferation, colony formation, migration, invasion, as well as tumor volume [19], while knockdown of NFATc4 by siRNA or inhibiting NFATc4 activity by tacrolimus (FK506) or ascomycin (FK520) suppressed skin cancer cell migration and invasion [18]. Further study elucidated that CDK3 phosphorylated NFATc4 at Ser259 and enhanced NFATc4 effect on skin cancer, and EGF treatment could promote CDK3/NFATc4-mediated cell transformation and proliferation. Of note, both CDK3 and NFATc4 are highly expressed among various types of skin tumor tissues compared with normal tissues [19]. Survival curve drawn with TCGA-SKCM indicates a poor outcome in patients with high NFATc4 level.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignances for its dramatically low 5-year survival rate. A recent study showed that NFATc4 is positively expressed in cell nucleus of human chronic pancreatitis and it is highly induced during pancreatic acinar-to-ductal metaplasia. NFATc4 could play a pivotal role in caerulein-induced pancreatic cancer initiation through NFATc4 transcriptional overexpression of SOX9 [20].

Since the blood-brain barrier and the crucial function of brain, malignant brain tumors remain a therapeutic challenge, in spite of surgical and medical advancement. NFATc4 was required and activated in doxorubicin-mediated cell death as well as doxorubicin-induced inhibition of  cell migration and invasion in glioblastoma cell lines [22]. In Schwannoma, NFATc4 and SOX10 synergistically enhanced KROX20 activity and induced P0 transcription to regulate the cell proliferation. When overexpressing SOX10 in Merlin-null Schwannoma cells in which the SOX10 level was reduced to restore Merlin tumor suppressor, NFATc4 was activated with nuclear translocation and the cell growth was alleviated [23, 53].

Besides from the above mentioned, the involvement of NFATc4 in cancer was gradually discovered in some other types of cancer, including hepatic cell carcinoma, colon cancer, cervical cancer, and leukemia.

It is well-known that certain patients with non-alcoholic fatty liver disease, the most common chronic liver disorder, may also develop non-alcoholic steatohepatitis (NASH). NASH is a disease associated with hepatocyte injury and inflammation, which could then lead to liver fibrosis, and subsequently cirrhosis and/or hepatic cell carcinoma (HCC). NFATc4 was activated and translocated into nucleus during methionine-choline diet-induced NASH, while knockdown of NFATc4 inhibited both methionine-choline diet-induced NASH and obesity-related NASH in mice. NFATc4 directly bound to PPARα and interfered with lipid metabolism through negatively regulating PPARα transcriptional activity, and then contributed to the development of NASH [24, 61]. These findings provided a potential therapeutic target in preventing NASH progression through inhibiting NFATc4 expression and activation. Remarkably, it was found that the inhibition of calcineurin/NFATc4 signaling mediated by RCAN1.4 could retard liver fibrosis [62].

NFATc4 is also involved in colon cancer, another digestive system neoplasm. It has been reported that arsenic sulfide (As₄S₄) and cyclosporine A (CsA) synergistically decreased colon cancer cell proliferation through the regulatory PML and p53 mediated NFAT signal pathway. CsA is a well-known NFAT inhibitor, meanwhile, As₄S₄ could inhibit the transcriptional expression of NFATc1, NFATc3, and NFATc4, except NFATc2 [63].

Accumulating bioinformatical analyses about immune-related genes (IRGs) indicated that NFATc4 was recognized as the key immune gene to predict the poor prognosis in cervical cancer [64, 65] and acute myelocytic leukemia (AML) [66]. In AML, NFATc4, an immune-related transcriptional factor, was co-expressed with immune gene set of T cell co-stimulation and was involved in Tregs recruitment. Simultaneously, NFATc4 was co-expressed with ATP-binding cassette transporter signaling pathway [66]. In addition, NFATc4 played a role in primary myelofibrosis (PMF), a stem cell-derived clonal myeloproliferative neoplasm. In PMF megakaryocytic cells, NFATc4 was up-regulated by activation of FL/Flt3 axis, while the Flt3 inhibition reinforced the Flt3/p38-MAPK axis effect on PMF dysmegakaryopoiesis [67].

In summary, these emerging observations in the past ten years emphasized the significant roles of NFATc4 in the progression of varieties of human malignancies (Table 5). NFATc4 may perform a dual-function during tumor progression like other NFAT proteins (Fig. 5). In the aspect of oncogenesis, NFATc4 activation promoted the EGFR-stimulated process of acinar-to-ductal metaplasia in pancreas and NFATc4 was activated in the NASH and liver fibrosis. NFATc4 also provided as a novel potential molecular target for cancer therapies, since NFATc4 participated in cell proliferation, migration, invasion, colony formation, 3D spheroid formation, and cell apoptosis. Furthermore, NFATc4 was related to the tumor immune microenvironment through recruiting macrophage and Tregs. Finally, the expression of NFATc4 could effectively evaluate the risk and outcome of the patients. Hence, the further study of NFATc4 function in various cancers could help to prevent the precancerous development and improve the individualized cancer therapy.