Thioredoxin domain-containing protein 9 protects cells against UV-B-provoked apoptosis via NF-κB/p65 activation in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC), a type of non-melanoma skin cancer (NMSC), is the most common malignancy worldwide. Thioredoxin (TXN) domain-containing protein 9 (TXNDC9) is a member of the TXN family that is important in cell differentiation. However, the biological function of this protein in cancer, particularly cSCC, is still unknown. In the present study, our experiments revealed the protective effects of TXNDC9 on UV-B-irritated cSCC cells. The initial findings showed that TXNDC9 is significantly upregulated in cSCC tissue and cells compared to normal skin tissue and keratinocytes. UV-B radiation robustly induces the expression of TXNDC9, and UV-B-induced cSCC cell death is boosted by TXNDC9 deficiency. Moreover, cSCC cells lacking TXNDC9 displayed attenuated activation of the NF-κB pathway. Additional studies by inhibiting TXNDC9 confirmed this finding, as TXNDC9 deficiency attenuated UV-B radiation-induced translocation of NF-κB p65 from the cytoplasm to the nucleus of cSCC. In conclusion, our work demonstrates the biological roles of TXNDC9 in cSCC progression and may provide a novel therapeutic target to treat cSCC in the future.


Introduction
Cutaneous squamous cell carcinoma (cSCC), a type of nonmelanoma skin cancer (NMSC), is one of the most common malignancies worldwide [1]. It causes 20%-30% of keratinocyte carcinomas, affecting more than 3 million people each year. Although mortality is currently declining, cSCC incidence is increasing [2][3][4]. It has lesions primarily common in the sun-exposure area and would be affected by oxidative stress caused by ultra violet (UV) radiation [5,6]. Over the past 30 years, efforts have been made to understand the numerous interrelated physiological and pathological factors underlying cSCC progression, including genetics, age, oxidative stress, etc. [7][8][9][10][11].
There is a direct relationship between UV radiation from the sun and oxidative damage to skin tissue [12]. Individuals under excessive UV radiation, particularly UV-B (275-320 nm), are at greater risk of skin damage, including solar erythema, photoaging, and even skin cancers [13]. UV-B radiation is known to generate reactive oxygen species (ROS), such as singlet oxygen, superoxide anion, and hydrogen peroxide, leading to a redox imbalance in skin cells [14][15][16]. However, tumor cells can tolerate stressed environments by upregulating the expression of some antioxidant genes.
The TXN system is a central antioxidant system that protects cells from oxidative stress and helps cells maintain cellular redox homeostasis [17]. Dysfunction of the TXN system can lead to many health problems including metabolic syndrome, cancer, cardiovascular disease, and neurodegeneration [17][18][19].
Thioredoxin (TXN) domain-containing protein 9 (TXNDC9) is a member of the TXN family that contains the same redox-active site as TXN. Recent studies reported its roles in cell differentiation and prostate cancer progression [20]. However, the biological function of this protein in cancer, particularly cSCC, is still enigmatic.
In the present study, we determined the protective effects of TXNDC9 against UV-B irradiation in cSCC cells. TXNDC9 is significantly upregulated in cSCC tissue and cells compared to normal skin tissue and keratinocytes. UV-B radiation can robustly induce the expression of TXNDC9 in a dosedependent manner. Moreover, TXNDC9 deficiency was able to augment UV-B-induced cSCC cell death via inhibition of the NF-κB/p65 pathway. In conclusion, our work demonstrates the biological role of TXNDC9 in cSCC progression and may provide a novel therapeutic target to treat cSCC in the future.

Materials and Methods
Patients and tissue specimens Normal human tissue and early cSCC (Stage I and Stage II) sections were obtained from the Department of Dermatology at The First Affiliated Hospital of Fujian Medical University with IRB approval (Suppl. Table 1). This study was approved by the institutional review board of the First Affiliated Hospital of Fujian Medical University (LY2019-013-01). All patients participating in this study received signed written informed consent.

Immunofluorescence
Briefly, xylene dewaxed and alcohol-rehydrated paraffin sections were placed in a bottle filled with a 0.01 M trisodium citrate solution and microwaved. After heating, slides were thoroughly rinsed in cool running water for 5 min. Sections were immersed in 3% H 2 O 2 at room temperature for 30 min to block any endogenous peroxidase activity. Add rabbit anti-TXNDC9 (Abcam, Cambridge, UK), diluted 1:200 at an appropriate concentration to the tissue, and incubate at room temperature for 2 h, or in a moist chamber overnight. Adding fluorescently conjugated secondary antibodies Goat anti-rabbit IgG H&L (Alexa Fluor 594) (Abcam, Cambridge, UK, ab150080), and incubate for 30 min at room temperature in the dark. Add a small volume of mounting media containing DAPI (Abcam, Cambridge, USA, ab104139) stain and add a coverslip of 15 μl is typically enough to cover a tissue underneath a coverslip fully.

Immunohistochemistry (IHC)
Briefly, paraffin sections deparaffinized with xylene and rehydrated with alcohol were placed in a bottle containing 0.01 M trisodium citrate solution and heated in a microwave. Then sections were rinsed in cold water for 5 min and soaked in 3% H 2 O 2 for 30 min at room temperature to block endogenous peroxidase activity. Washed with Tris-buffered saline (TBS) pH 7.4. Then incubated at room temperature for 2 h, or in a moist chamber overnight at 4°C with rabbit anti-TXNDC9 (1:200, Abcam, Cambridge, UK). Sections were then treated with biotin labeled goat anti-rabbit secondary antibodies (Abcam, Cambridge, UK) at 37°C for 30 min, followed by horseradish peroxidase-labeled streptavidin solution. Signals were developed by using diaminobenzidine (DAB) chromogen (Dako, Glostrup, Denmark) as substrate. Meyer's hematoxylin solution (Dako, Glostrup, Denmark) was used as a nuclear neutralizing dye. Incubation without specific antibody was used as a negative control.

Western blot
The methods have been described previously with slight modifications [26,28]. Briefly, whole cell lysates were prepared in cell lysis buffer and clarified by centrifugation. Protein samples of 50 μg were separated on 8%-12% denaturing SDS-PAGE gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes by electroblotting. Membranes were blocked with blocking buffer (LI-COR, Lincoln, NE, USA) for 30 min at room temperature and then incubated with primary antibodies overnight at 4°C. Blots were incubated with specific secondary antibodies (Abcam, Cambridge, UK) at room temperature for 1 h the next day. The signals were detected by ECL reagents (Meilun Biological Technology, Dalian, China). β-actin (1:3000, Cell Signaling Technology, Danvers, MA, USA) and Lamin B1 (1:1000, Cell Signaling Technology, Danvers, MA, USA) were equivalent loading controls.
Cell viability assays Methods have been described previously [25]. Cell viability was assessed by Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions (Dojindo Laboratories, Kumamoto, Japan). In brief, When the cells cultured in a 96well plate grew at a density of 2 × 106/mL, the CCK-8 solution (10 μl) added to each well at 24 h. Then, the absorbance of OD 450 nm was measured by a spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA).

Apoptosis assay
The degree of apoptosis in cSCC cells was detected by Western blot analysis of cleaved Caspase-3 and TUNEL assay (Sigma-Aldrich, St Louis, MO, USA). The process has been described [25]. In brief, cells (1 × 106 cells/well) were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.25% Triton X-100 for 20 min at room temperature. Subsequently, after cells were rinsed with PBS, TdT solution and dUTP solution were added and incubated at 37°C for 1 h in the dark. Cells were treated with DAPI (Abcam, Cambridge, USA, ab104139) for nucleus staining for 5 min at 37°C. The cell apoptosis ratio was measured by the TUNEL percentage (TUNEL/Hoechst 33342 × 100%). The results shown were representative of at least 200 cells in 5 random scope fields per treatment.

NF-kB DNA binding assay
The activation of NF-κB used NF-κB (p65) Transcription Factor Assay Kit (Cayman Chemical, Ann Arbor, Michigan, USA) that detects specific NF-κB (p65) DNA binding activity in nuclear extracts. In this study, 10 μg of nuclear fractions extracted from cSCC cells were run in duplicate along with positive and negative controls. After incubation with the primary and secondary antibody, add developing solution and measure sample at an absorbance at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis
All data shown in this study were expressed as the mean values ± SEM of at least three independent experiments. Appropriate statistical tests were performed using GraphPad Prism 7.0 software to calculate p-values (GraphPad Software Inc., San Diego, CA). A significant difference considered to be present at p < 0.05.

Results
TXNDC9 expression is upregulated in cSCC tissue and cell lines TXNDC9 is a critical regulator in cell proliferation in some types of cancers [20,29]. To identify the role of TXNDC9 in cSCC, we first conducted a series of experiments to examine the expression of TXNDC 9 in cSCC tissues and cell lines. Our results demonstrated that TXNDC9 protein levels were significantly higher in cSCC tissue samples than in standard skin samples (Figs. 1A-1E). Moreover, TXNDC9 mRNA levels were then assessed by RT-PCR and observed to be upregulated (Fig. 1H). Consistent with the results of tissue samples, TXNDC9 expression was robustly elevated in two cSCC cell lines, A431 and SCL-1, as well (Figs. 1F, 1G and  1I). These results revealed that TXNDC9 expression is significantly higher in cSCC than in normal keratinocytes and skin tissues, suggesting a positive correlation between TXNDC9 and cSCC progression. In addition, we analyzed the expression of TXNDC9 in head and neck squamous cell carcinoma (HNSC) and normal tissues using datasets from The Cancer Genome Atlas (TCGA) database (Suppl. Fig. 1).
UV-B radiation stimulates the induction of TXNDC9 expression Next, we tested the potential effect of UV-B in TXNDC9 expression. A431 and SCL-1 cells were subjected to different UV-B radiation intensities (0, 5, 10, 15 mJ/cm 2 ). After 24 h, TXNDC9 protein levels were examined by Western blots analysis. Results revealed that protein levels of TXNDC9 were robustly induced by UV-B radiation in cSCC cells in a dose-dependent manner ( Figs. 2A and 2B). Subsequent analysis by RT-PCR in both cell lines showed that TXNDC9 expression was also induced mainly in response to UV-B radiation (Figs. 2C and 2D). These results suggest that TXNDC9 expression is positively correlated with UV-B radiation.

TXNDC9 deficiency augments UV-B-provoked cell death in cSCC cells
To further define the biological effect of TXNDC9 in cSCC cell progression and its underlying mechanism, the shRNA method was applied to knock down TXNRD9 in cSCC cells. Western blot analysis showed that TXNDC9 shRNA effectively downregulated TXNDC9 in both cSCC cell lines compared to NTC shRNA (Fig. 3A). Next, we examined the degree of UV-B-induced cell apoptosis using TXNDC9knockdown cSCC cells. UV-B-provoked cell apoptosis was tested by TUNEL staining assay (Figs. 3B, Suppl. Figs. 2A and 2B) and Western blot analysis of cleaved caspase-3 levels (Figs. 3C and 3D). Results from these assays supported that UV-B radiation-induced apoptosis in cSCC cells and that this effect was significantly provoked by TXNDC9 deficiency. By these results, MTT cell survival assay revealed that UV-B radiation decreased cSCC cell survival, which was promoted mainly when TXNDC9 was silenced (Figs. 3E and 3F). The evidence suggests that TXNDC9 mediates a cytoprotective effect in cSCC cells against UV-B.

NF-κB/p65
activation mediates TXNDC9-induced cytoprotection against UV-B radiation Our previous results above showed that TXNDC9 promotes cell progression in cSCC cells. To address the underlying mechanism of TXNDC9-mediated cSCC cell survival, we performed a set of studies to verify whether this biological function is related to NF-κB activation since the NF-κB pathway plays a critical role in many biological signs of progress. TXNDC9-deficient A431 and SCL-1 cells were challenged with UV-B radiation, and examined NF-κB DNA binding activity. TXNDC9-deficient cSCC cells showed significantly lower NF-κB activation (Figs. 4A and 4B). In addition, proteins extracted from the cytoplasm (Figs. 4C and 4D), nucleus (Figs. 4E and 4F), and whole cells were also subjected to western blotting to detect proteins of the NF-κB-p65 signaling pathway. Loss of TXNDC9 significantly attenuates UV-B radiation-induced translocation of NF-κB p65 from the cytoplasm to the    nucleus of cSCC (Figs. 4G-4I). Moreover, phosphorylation of NF-κB p65 and IκBα induced by UV-B radiation were also attenuated by TXNDC9 deficiency (Figs. 4G, 4H and 4I). In addition, Immunofluorescence of NF-κB p65 and DAPI in the nucleus after UV-B has evidenced the relationship between TXNDC9 and NF-κB signaling (Suppl. Fig. 3). These results show that NF-κB/p65 activation is critical for TXNDC9-induced cytoprotection against UV-B radiation in cSCC cells.

Discussion
The prevalence of cSCC is rising rapidly and broadly in correlation to excessive UV radiation and photooxidative stress [12][13][14]. A growing interest exists in novel strategies for the diagnosis, treatment, and prevention of cSCC. We demonstrate TXNDC9 as a potent antioxidant protein that protects cSCC cells from UV-B-induced oxidative damage.
Excessive UV-B radiation is the fundamental environmental risk factor that underlies skin damage [30][31][32]. However, in tumorous microenvironments, cancer cells will develop their counterplan to confront different stress and survive. Particularly under chronic oxidative stress, increased levels of antioxidant proteins are detected and are observed to promote cell growth and tumor progression [33]. TXNs contains a redox-active site (CXXC) and are kept in reduced form to regulate redox homeostasis [34]. TXN system is implicated in many biological processes, including cell proliferation and apoptosis [35]. Therefore, this redox system has become a focus of interest in various cancer types [36]. TXN1 has been found to be upregulated in several cancers, including prostate, colon and gastric cancer, to promote cancer progression and predict poor prognosis [37][38][39]. Thioredoxin domain containing 5 (TXNDC5) is also implicated in the regulation of cancer progression of hepatocellular carcinoma, prostate cancer, and colorectal cancer [40][41][42]. TXNDC9 is involved in cancer progression as well. Feng et al. reported that TXNDC9 is a crucial regulator of cell survival and proliferation in prostate cancer and is associated with advanced clinical stages [43]. In hepatocellular carcinoma, recent studies have demonstrated that TXNDC9 promotes cancer progression by mediating the gene regulation networks [29]. However, the role of TXNDC9 in cSCC remains unknown. In our study, we first found that TXNDC9 is significantly upregulated in cSCC tissue and cells. Moreover, UV-B radiation can robustly induce the expression of TXNDC9. The UV-B-induced photooxidative damage is significantly augmented by the inhibition of TXNDC9. Our findings establish TXNDC9 as a positive regulator in cSCC development and demonstrate the potentially use of TXNDC9 as a therapeutic target or biomarker in cSCC patients.
To further investigate the association between TXNDC9 and cSCC, we performed a series of studies using TXNDC9 deficient cSCC cells to assess potential affected cellular signaling pathways under UV-B radiation. An enormous amount of literature has recently accumulated indicating that many thioredoxin family members are critical to regulating the DNA-binding activity of transcription factors, including NF-κB [44,45]. TXNs activate NF-κB signaling by reducing cysteine residues in p50/p65 through its active redox site [44,45]. Our present studies add more evidence in support of this notion. Herein, we demonstrated TXNDC9 regulation of NF-κB DNA binding activity induced by UV-B radiation. The NF-κB/p65 activation is documented to be critical for many biological processes, such as inflammation, cell survival and proliferation, neuronal survival, metastasis, and so on [46,47]. Regarding its specific role in cell cycle regulation, NF-κB activation is observed in many cancers and is potent in promoting cell survival and proliferation [46][47][48]. In the present study, we verified that TXNDC9 deficiency inhibits NF-κB/p65 activation and translocation from cytoplasm to nucleus induced by UV-B-radiation. The data presented herein add to an accumulating body of evidence demonstrating that NF-κB activation is a critical regulatory pathway in cancer development. More importantly, our study has linked TXNDC9 with the NF-κB signaling pathway for the first time and determined the underlying mechanism of TXNDC9-mediated cSCC progression. Therefore, TXNDC9-regulated NF-κB/p65 activation could be a prospective target in the treatment of cSCC.
Our results show that TXNDC9 is upregulated in cSCC tissues and protects cSCC cells from UV-B radiationmediated apoptosis via activating NF-κB signaling. TXNDC9 could serve as a potential and promising therapeutic target in cSCC treatment. Author Contributions: ZXX and QYX performed experiments and contributed to writing the manuscript. HQW and XTZ performed part of experiments. YTZ contributed to acquiring the data. CBB and LHC contributed to the study design. PZ and ML contributed to analyzing the data. TG and CJ designed the study and wrote the manuscript. All authors have read and approved the final manuscript.
Availability of Data and Materials: The data and materials used in the current study are available from the corresponding author on reasonable request.
Ethics Approval: The study was approved by the institutional review board of the First Affiliated Hospital of Fujian Medical University (LY2019-013-01). None of the cell lines used in the present study required ethics approval for their use. Signed, written informed consent was obtained from all patients who participated in this study.

Conflicts of Interest:
The authors declare that they have no conflicts of interest to report regarding the present study.