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Abstract
Nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4) has been implicated in keratinocyte development and several types of cancer. A well-defined role for NFATC4 in cutaneous squamous cell carcinoma (CSCC) has not yet been established. In this study, NFATC4 gene function in CSCC development was examined. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to measure the mRNA expression of NFATC4 in CSCC tissues and controls. A431 and Colo16 cell proliferation, invasion, and apoptosis were measured by CCK-8 assay, transwell invasion, and flow cytometry, respectively, after an NFATC4 expression lentivirus infection. Animal models were applied to validate the function of the NFATC4 gene. (1) CSCC tissues showed a significant decrease in NFATC4 expression compared to controls. (2) Overexpression of NFATc4 suppresses A431 and Colo16 cell proliferation and invasion but promotes cell apoptosis. (3) Mouse models overexpressing NFATC4 showed reduced tumourigenesis. It was suggested that NFATC4 might be a tumour suppressor gene in CSCC.
Keywords: Calcineurin, carcinogenesis, cutaneous squamous cell carcinoma, NFATC4
Introduction 
Up to 9000 deaths are caused annually by cutaneous squamous cell carcinoma (CSCC), the most common form of non-melanoma skin cancer in the United States.[1] Satisfactory results can be obtained in early diagnosis and surgical cases. Nevertheless, the disease may also result in disfigurement and extensive damage to the soft tissue, cartilage, and bone.[2] Besides, incidence of CSCC has been rising rapidly.[3],[4] Therefore, it is still urgent to explore its pathogenesis and early biological markers.
The calcineurin/NFAT pathway refers to activated multiple phosphoric residues in the NFAT homologous region dephosphorylated by calcineurin, leading in NFAT cytoplasmic-nuclear trafficking and induction of NFAT-mediated gene transcription.[5] The NFAT family includes NFAT1 (NFATC2/NFATp), NFAT2 (NFATC1/NFATc), NFAT3 (NFATC4), NFAT4 (NFATC3/NFATx), and NFAT5.[6],[7] Since the calcineurin/NFAT pathway was first described, many reports have shown that this pathway plays an important role in cancer development and cancer treatment. So far, the relationship of the calcineurin/NFAT pathway with CSCC has mainly used immunosuppressive methods and UV irradiation. Recent studies have shown that patients receiving calcineurin inhibitor have a much shorter CSCC-free survival than the group treated with sirolimus, which is a target of rapamycin inhibitor.[8] Additionally, the withdrawal of calcineurin inhibitor reduces non-melanoma incidence.[9] Thus, this suggests that the calcineurin pathway plays an intrinsic role in keratinocytic tumour inhibition, although immunosuppression is important. Activation of NFATC1 is sufficient to initiate CSCC and establish a tumour microenvironment. Notably, the same NFAT isoforms have opposite functions in different cancer types. Therefore, the isoform-specific NFAT function requires further investigation.
NFATC4 is involved in a variety of physiological processes, including immune, cardiovascular, musculoskeletal, and nervous system development,[10–13] as well as in the initiation, development, and prognosis of lung cancer, breast cancer, and ovarian cancer.[14–17] The effects of NFATC4 on CSCC have not been thoroughly studied, despite its functional activity in human keratocytes.[18] This study examined the association between NFATC4 expression in CSCC tissues and adjacent tissues, as well as the role it plays in tumourigenesis in CSCC.
Materials and Methods Ethics statement
In this study, approval was granted by the Medical and Animal Ethics Committee of the Institute of Dermatology of the Chinese Academy of Medical Science and Peking Union Medical College (Institute of Dermatology, CAMS and PUMC). We followed the principles of the Declaration of Helsinki in the conduct of the study. A written and informed consent was obtained from each participant for participation in the study.
Samples and cell lines
Fresh CSCC and adjacent normal tissues from patients with a diagnosis of CSCC admitted at the Institute of Dermatology, CAMS and PUMC, were collected from the scalp and facial skin. As soon as samples were taken, they were placed in liquid nitrogen for analysis.
The Chinese Center for Type Culture Collection cell lines A431 and Colo16 were used for this study. They were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS) under 5% CO2 at 37°C.
Gene expression analysis by RT-qPCR
Using previously described protocol,[19] quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed. Total RNA was extracted from fresh skin tissues using the RNeasy Mini Kit (Qiagen, Germany) and reverse transcribed as cDNA using PrimeScriptTM RT mastermix (TaKaRa, Dalian, China). The AceQ qPCR SYBR Green Mastermix (Vazyme, Nanjing, China) was used for qPCR. This study detected the mRNA for NFATC4 using primers of the forward 5'-ACCCCACAGATGTTCATCGGC-3' and reverse 5' -ATTCCCGCGCAGTCAATGT-3'. Meanwhile, PP2Bβ, the catalytic subunit of calcineurin, has the function of dephosphorylating NFAT to participate in the calcineurin-NFAT signalling cascade, for which mRNA was verified using primers of the forward 5' -GCTGCCATCCTTCGGAGAG-3' and reverse 5'-TCGCCAAGAAAAAGGTATCGTG-3'. β-actin was selected as the reference gene.
Establishment of stable cell lines of NFATC4
GeneChem Biomart (Shanghai, China) created a lentivirus that expressed NFATC4 in both A431 and Colo16 in accordance with the manufacturer's instructions. Cells were treated with puromycin for 14 days for obtaining cell lines with stabilized overexpression of NFATC4.
Western blot
To lyse the infected cells, we used RIPA lysis buffer (Beyotime, Jiangsu, China) containing 1% protease inhibitors (Sigma, USA). BCA protein assay kit (Beyotime, Jiangsu, China) was used to measure the protein concentration. The protein was separated by 10% SDS-PAGE and afterward transferred to Immun-Blot PVDF membranes (BioRad, USA). The membranes were incubated with rabbit antihuman NFATC4 antibody (1:1000, sc-135771, Santa, USA) and anti-β-actin antibody (1:1000, #4967, CST, USA) as an internal control after protein extraction with 10% SDS-PAGE. A LumiGLO® reagent and peroxide (20%, BioRad, USA) were used to develop the images.
Cell proliferation assay
About 5 × 103 cells were added to each well of the 96-well plates and cultured overnight. Cell Counting Kit-8 (CCK-8) was added to each well and incubated at 37°C for four hours. Microplate readers were used to measure absorption at 450 nm after 24, 48, and 72 h of incubation.
Transwell invasion assay
Matrigel Basement Membrane Matrix was placed in each well of the 24-well plate through the 8-mm transwell insert. About 500 μL of 20% FBS containing DMEM medium were added to the bottom chambers and 200 μL of cell suspension in serum-free media were added in the upper chambers. After 48 h of incubation, the upper matrigel and cells were removed. In the bottom wells, phosphate-buffered saline (PBS) was used for washing, followed by pre-cooled methanol fixation and staining with 0.1% crystal violet solution for 30 min. A 400× microscope was used to examine the membrane at the bottom of the insert.
Apoptosis analysis
We digested the cells with 0.25% pancreatic enzymes without EDTA and then suspended them in PBS for flow cytometry analysis. According to the annexin V-FITC apoptosis detection kit protocol (eBioscience, San Diego, CA), the cells were stained with 5 μL annexin V followed by 10 μL propidium iodide (PI) and incubated at room temperature before flow cytometry analysis.
Tumourigenesis in nude mice
Specimens of BALB/c nude mice were randomly divided into two groups (A431-NC and A431-NFATc4) of five each. To each mouse, 200 μL cells with a density of 1 × 107 cells/mL were injected. The tumours were measured every three days. After 28 days, anaesthesia was applied to the mice to sacrifice them.
Results NFATC4 expression in CSCC tissues
An RT-qPCR study was conducted on 30 malignant and adjacent normal tissues of patients with CSCC. NFATC4 expression revealed significantly lower levels in CSCC tissues than in nearby normal controls, as well as PP2Bβ [Figure 1].
Figure 1: Gene expression of calcineurin-NFAT pathway in CSCC tissues. Data are expressed as means ± SD (n = 3) with statistical significance determined by student's t test (*P < 0.05, **P < 0.01, ***P < 0.001)Overexpression of NFATC4 by lentivirus infection
To construct stable NFATC4 overexpressing cell lines, A431 and Colo16 cells were infected with a NFATC4 expression lentivirus. As exhibited in [Figure 2], NFATC4 expression level was upregulated markedly both at the mRNA and at the protein level.
Figure 2: NFATC4 expression in A431 and Colo16 cells was effectively upregulated by lentivirus method. (a) NFATC4 overexpression at mRNA level was detected by RT-qPCR, (b) NFATc4 overexpression at protein level was detected by western blot (P < 0.05) compared with LV-NC* group. *LV: Lentivirus, NC: Negative controlProliferation, invasion of CSCC cells reduced by NFATC4 overexpression, but apoptosis increased
We showed that the proliferation of A431 and Colo16 cells with high NFATC4 expression were lower compared with the control group, which indicated that high NFATC4 expression could reduce cell proliferation of CSCC [Figure 3]a. The Transwell assay results showed the number of cells with high NFATC4 expression crossing the Transwell insert was significantly less than that of the control group in A431 and Colo16 cell lines, indicating that high NFATC4 expression significantly reduced cell invasion in CSCC cells [Figure 3]b. A significant increase in apoptosis was observed in cells treated with NFATC4 lentivirus in comparison to cells that were not transfected with NFATC4 [Figure 3]c.
Figure 3: A431 and Colo16 cells overexpressing NFATC4 were analysed for proliferation, invasion, and apoptosis. (a) CCK-8 was used to determine cell proliferation, (b) Transwell assays evaluated NFATC4 involvement in invasion, (c) NFATC4 overexpressed cell lines underwent apoptosis analysis. We stained the cells with annexin V and propidium iodide doubleNFATC4 overexpression reduced CSCC tumorigenesis in vivo
To further understand the relationship between NFATC4 and tumour development in vivo, we expressed NFATC4 overexpression in mouse models of CSCC. Cell lines expressing high NFATC4 expression displayed a 4- to 9-day delay in tumour development, along with smaller tumour size compared to the control group 24 days after transplantation [Figure 4]. The results of our study showed that in mouse CSCC models, NFATC4 overexpression reduced tumourigenesis.
Discussion We previously performed RNA-Seq transcriptomics on eight pairs of malignant and adjacent normal tissues of patients with CSCC.[19] We found that in human CSCC samples, the expression of genes from the calcineurin/NFAT pathway, including PP2Bβ and NFATC4, was reduced, which was validated in 30 pairs of tissue samples subsequently. On a functional level, we found that CSCC cells overexpressing NFATC4 exhibited reduced proliferation, increased apoptosis, and reduced migration.
Moreover, overexpression of NFATC4 suppresses tumourigenesis in mouse models of skin cancer. These data suggest that NFATC4 is a tumour suppressor gene in CSCC.
NFATC4 expression has been found in many tissues, including the immune system,[20] and several cancerous tissues and cell lines.[21],[22],[23] Furthermore, certain tumours display relatively low NFATC4 mRNA levels.[24] However, the function of NFAT3 in cell transformation and cancer progression is still controversial. In contradiction to our findings, a study reported increased NFATC4 expression in A431 cells compared with HaCaT immortalized skin cell line and in CSCC tissues in comparison to normal samples.[25] This contradiction may be due to selection in reference objects or limitations in the number of samples, which needs to be evidenced by further studies.
As a member of the NFAT family, NFATC4 can be dephosphorylated by calcineurin and translocated to nuclear, thus exerting a transcriptional role.[7] The interaction proteins of NFATC4, such as GATA4, SOX10, and PGC-1α also regulate its transcriptional activity in addition to post-translational modifications.[26],[27],[28] There is a lack of knowledge about the regulation of NFATC4 compared with other NFAT proteins.
Compared with other NFAT proteins, the regulation of NFATC4 has not been deeply investigated. Since the expression and function of NFATC4 have been detected in CSCC, its downstream genes deserve further investigation.
As a result of our study, we identified NFATC4 as a tumour suppressor gene that may be useful for identifying and treating CSCC.
Financial support and sponsorship
Our work was supported by grants from the Qingdao West Coast New Area 2020 (2020-61).
Conflicts of interest
There are no conflicts of interest.
References 
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