Yi Wu, Feng-Juan Li, Ke Zhang, Qun Lv, Xue-Yuan Yang, Li-Ming Li,?, Ming-Jun Jiang,?

1West China School of Medicine, Sichuan University, Chengdu, Sichuan 610041, China, 2Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, Jiangsu 210042, China.

Introduction

Cutaneous squamous cell carcinoma (CSCC), a keratinocyte-derived skin neoplasm with malignant potential,1represents 20%-50%of skin cancers and currently has an increasing incidence in the United States.2Ultraviolet(UV)solar radiation is the primary risk factor for the development of CSCC, and the cumulative exposure received over a lifetime plays a major role in this development.3Mutations in thep53gene are the most common genetic abnormalities,causing nonfunctional p53 protein production and cells with damaged DNA replicate in CSCC.4

Recent studies have shown that CSCC-free survival was shorter in the group treated with a calcineurin inhibitor compared with the group treated with sirolimus,which is a mammalian target of rapamycin (mTOR) inhibitor,5and withdrawal of the calcineurin inhibitor reduced subsequent nonmelanoma cancers.6Additionally, functional evidence supports a critical role for calcineurin/nuclear factor of activated T-cell (NFAT) signaling in CSCC,specifically in tumor initiation and development and in the tumor microenvironment. Herein, we discuss these findings of the calcineurin/NFAT pathway in CSCC in this review.

General introduction of calcineurin/NFAT pathway

Calcineurin is the only known highly conserved serinethreonine phosphatase under calcium-calmodulin control,and its activation promotes the localization of NFAT to the nucleus.7Calcineurin consists of a catalytic subunit A(CnA)and a regulatory subunit B (CnB). The CnA contains a phosphatase catalytic domain, a CnB-binding domain, a calmodulin-binding domain, and a carboxy-terminal autoinhibitory domain. The CnB has a main latch region with four Ca2+-binding EF-hand motifs, through which calcineurin is recognized by immunosuppressive complexes and competitively binds to the CnA.8

NFAT family includes NFAT1 (NFATc2/NFATp),NFAT2 (NFATc1/NFATc), NFAT3 (NFATc4), NFAT4(NFATc3/NFATx),and NFAT5.NFAT proteins contain an amino-terminal transactivation domain and a carboxyterminal domain.NFAT1-4 has an additional Rel-homology region, similar to the DNA-binding domain of the nuclear factor kappa-B family, and an NFAT homology region,whichisaregulatorydomainwithacalcineurin-bindingsite.8

The calcineurin/NFAT pathway is primarily activated by the increased level of intracellular Ca2+.During the resting phase, the autoinhibitory domain interacts with the calmodulin-binding domain to inhibit its phosphatase activity. In the activated state, an increased cytoplasmic Ca2+concentration assists calmodulin in displacing the autoinhibitory domain and promotes CnB binding to CnA by activating Ca2+-binding EF-hand motifs in the CnB,which ultimately leads to calcineurin activation. The activated calcineurin dephosphorylates multiple phosphoresidues in the NFAT homology region,leading to NFAT cytoplasmic-nuclear trafficking9and the induction of NFAT-mediated gene transcription.8

The roles of calcineurin/NFAT pathway in CSCC

Involvement in tumor initiation and development

Cyclosporine A (CsA), which is a commonly used immunosuppressant, and function as an inhibitor of the calcineurin/NFAT pathway. Transplant recipients receive treatment with CsA and tacrolimus to prevent transplant rejection,which is accompanied by a high incidence of skin cancer, especially CSCC. Notably, other more recently developed immunosuppressive drugs that do not affect calcineurin activity, such as mTOR inhibitors, have a much lower impact on CSCC formation.6,10Thus,although immune suppression is important, it is unlikely to directly account for the selectively increased risk of CSCC development associated with calcineurin inhibit treatments,suggesting an intrinsic role for the calcineurin/NFAT pathway in keratinocyte tumor suppression.

Calcineurin/NFAT pathway has been proved to play a significant role in skin stem cells.11Recent evidence has shown that hair follicle stem cells act as cancer cells of origin for CSCC and cannot initiate tumors during the quiescent phase of the hair cycle.Horsleyet al.12reported that NFATc1 is preferentially expressed by hair follicle stem cells in their niche,where its expression is activated by bone morphogenetic protein (BMP) signaling upstream.NFATc1 acts downstream to repress CDK4 and maintain stem cell quiescence,which can be stopped by pharmacologically suppressing calcineurin/NFATc1 signaling orviaa complete or conditionalNFATc1gene ablation.NFATc1 has been reported to regulate bulge stem cell activity during native hair aging13and pregnancy.14

A recent study found that the expression of NFAT3 was high in A431 cell lines, whereas it was markedly lower in the immortalized skin cell line HaCaT. As expected, compared with normal skin tissue, NFAT3 was highly expressed in tumor tissues. Furthermore, a knockdown of endogenous NFAT3 expression by short hairpin RNA significantly inhibited A431 cell proliferation, colony formation, and anchorage-independent cell growth.15

Antagonistic role of calcineurin and ATF3 in p53-dependent cancer cell senescence

Cancer cell senescence is a failure-protected mechanism against tumor development that can inhibit the cancer stem cell potential.Genetic and pharmacological suppression of calcineurin/NFAT function increased ATF3 expression by abolishing the binding of endogenous NFATc1 to two distinct regions of the ATF3 promoter harboring NFAT-binding sites and subsequently blocked the expression ofp53and senescence-associated genes,thereby reducing senescence and promoting tumor formation in mouse skin and in xenografts.16This process must be independent of the pathway described in Ref.[17]because the oxidative stress induced by CsA was too low to stabilize NRF2,and the UVA radiation doses that induce ATF3 expression do not interfere with NFATc1 activity.

Considering the previously defined role of ATF3 in CSCC development, these findings may provide an explanation and a mechanism for the frequently observed CSCC burden on sun-exposed areas of the skin in organ transplant recipients who are treated with calcineurin inhibitors.However,the mTOR inhibitor sirolimus is able to reverse the expression of ATF3.18

Roles of calcineurin/NFAT pathway in the tumorigenic microenvironment

SCID-beige mice, which are devoid of most innate immunity components that were injected with A431 cells and treated with CsA,developed much larger tumors with enhanced VEGF expression as compared with those treated with a vehicle control. Intriguingly, this trend continued with increased cell proliferation, decreased apoptosis,and reduced cell migration and invasion of the epithelial-mesenchymal transition after the cessation of CsA treatment,which may be due to increased TGF-β and TGF-β-dependent signaling.19Later, paradoxically, another study reported that NFATc1 activation adjacent to neighboring cells without NFATc1 activation was sufficient for initiating CSCC along with a significant upregulation of c-Myc and Stat3 activation.20Moreover,factors secreted by the NFATc1+cells initiated the establishment of an inflammatory and promitogenic tissue environment for both NFATc1+and NFATc1-cells to proliferate and become part of the tumor, which also affected the stem cells/progenitor cells and their niches,leading to their participation in tumor formation. These findings indicate that the activation of NFAT signaling establishes a tumorigenic microenvironment through cell and non-cell autonomous mechanisms.21Goldsteinet al.20reported consistent data about the carcinogenic effect of the constitutive NFATc1 overexpression, but they did not detect overt alterations in skin inflammatory or cytokine gene expression during homeostasis after NFATc1 inhibition.Thesefindingssuggestthatcalcineurinmayinhibitskin tumorigenesis through its regulation of additional NFAT isoforms or other proteins such as ATF3 or p53.

Relationship between CSCC risk factors and calcineurin/NFAT pathway

The main risk factors for CSCC include UV radiation,immunosuppression, and chronic scar formation. The relationship between these three factors and calcineurin/NFAT pathway is discussed in the following sections.

UV radiation

UV-induced DNA damage plays a key role in the initiation phase of skin cancer as a result of photochemical reactions,in which DNA bases absorb UV energy to form characteristic photonic layers, such as cyclobutane pyrimidine dimers and reactive oxygen species,producing mutagenic base damage and then leading to the activation of proto-oncogenes or the inactivation of tumor suppression genes.22Irradiation of keratinocytes in culture with UVA-1 was reported to result in a dose-dependent decrease in calcineurin activity.23Similarly, an increase in NFAT DNA-binding activity was found in the group with a low UVA dose (＜45kJ/m), whereas a reduction to 60% was found in the group with a higher dose(135kJ/m)of UVA.However,another protein phosphatase 2A,closely related to calcineurin, was insensitive to inactivation by UVA-1 irradiation.24Notably,UVB irradiation caused a decrease in the calcineurin activity in one of two fibroblast cultures but was ineffective in keratinocytes,even though UVB was demonstrated to be the most effective type of light for inducing skin cancer in animals.25Upon UVA irradiation and reactive oxygen species formation, the nuclear factor erythroid 2-related factor 2(NRF2)protein is activated in cultured primary keratinocytes as well as in the epidermis of normal skin, which leads to binding of nuclear factor erythroid 2-related factor 2 to the two predicted antioxidant response element motifs in theATF3gene promoter and induces ATF3 expression.26A recent study revealed that calcineurin activity and NFATc1 in HaCaT cells may be necessary for UV-induced gasdermin-C expression,27which can form pore channels to help inflammatory molecules such as interleukin-1(IL-1) to be actively released across cell membranes and cause tissue inflammation.28

Immunosuppression

The tumor promotion effect of calcineurin inhibitory drugs is generally attributed to an inhibition of the immune system, particularly the suppression of T-cell function.Previous work showed that transplant-associated CSCCs have a higher ratio of regulatory T cells to cytotoxic CD8+T cells,a lower percentage of interferon-γ(IFN-γ)-producing CD4+T cells,and a lower level of IFNγ-producing CD8+T cells compared with immune-competent CSCC.29CsA treatment results in enhanced IL-22 production,which can increase the proliferative,migratory,and invasive capacity of CSCC.30Calcineurin inhibitors can not only directly inhibit p53 and E-cadherin tumor-suppressive activity,thus allowing a tumorigenic upregulation of proinflammatory and mitogenic nuclear factor kappa-B, extracellular regulated protein kinases, and activator protein-1 signaling,31but also reduce the repair of cyclobutane pyrimidine dimers induced in DNA by inhibiting NFAT expression.32Another aspect of immunosuppression refers to viral infections associated with skin cancers, such as β-human papillomavirus(β-HPV).17β-HPV oncoproteins have been reported to destabilize the UV-repair protein p300 histone deacetylase, thus disabling the repair of sun-exposed lesions.33A recent study reported that HPV16 oncoproteins E6 and E7 increased the NFAT2 expression levels and DNA-binding activity,thus facilitating cell proliferation in cervical cancer.34At present, the relationship between β-HPV and the calcineurin/NFAT pathway still remains uncharacterized in CSCC.

Chronic scar formation

Risk factors for developing CSC in patients of color include chronic inflammatory diseases, such as discoid lupus,and chronic scars resulting from burns,skin ulcers,or radiation sites.35Most skin cancers develop on the periphery of the decolorized discoid lupus erythematosus plaques, colocalized with active inflammatory regions.FK506,a macrolide antibiotic isolated fromStreptomyces,was able to lock cutaneous lupus inflammation to prevent skin cancer development in MRL/lpr mice accompanied by a significant reduction of CD4+T cells and Foxp3+regulatory T cells in the skin,36but its correlation with the calcineurin/NFAT pathway is unknown.

Conclusion

Since the calcineurin/NFAT pathway was originally described,many reports have indicated that this pathway plays an important role in cancer development and treatment. To date, studies on the relationship of the calcineurin/NFAT pathway with CSCC have mainly used immunosuppressive approaches and UV irradiation,which provide strong evidences for the discussion mentioned above. The calcineurin/NFAT pathway has been shown to function in keratinocyte differentiation,migration, and DNA repair. Furthermore, dysregulation of this signaling pathway has been reported to play a significant role in CSCC formation,abnormal growth,and tumorigenic microenvironment. Despite its positive function of maintaining skin stem cell quiescence, NFATc1 activation is sufficient for initiating CSCC and establishing a tumor microenvironment. Notably, the same NFAT isoform has opposite functions in different cancer types.Hence, the isoform-specific NFAT function requires further investigation in CSCC.

Acknowledgements

Our work was supported by Grants from the Chinese Academy Medical Sciences Initiative for Innovative Medicine (No. 2016-I2M-3-021),National Natural Science Foundation of China(No.31470274), Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs (No. 2012ZD006), and Jiangsu provincial SixTalent Peaks (No. 2013-WSW-060).