IL‐7–Mediated IL‐7R‐JAK3/STAT5 signalling pathway contributes to chemotherapeutic sensitivity in non–small‐cell lung cancer

Abstract Objectives The chemotherapy drug resistance is a major challenge for non‐small‐cell lung cancer (NSCLC) treatment. Combination of immunotherapy and chemotherapy has shown promise for cancer. The goal of this study was to evaluate the anti‐tumour efficacy of interleukin‐7 (IL‐7) combining cisplatin against NSCLC. Materials and Methods Cell proliferation was analysed using CCK‐8 assay, EdU proliferation assay and colony‐forming assay. Cell apoptosis was evaluated using HOECHST 33342 assay and flow cytometry. The protein expression levels were analysed by Western blot. The blocking antibody against the IL‐7 receptor and the inhibitors of STAT5 and JAK3 were used to investigate the pathway involved. A xenograft model was established to assess the anti‐tumour efficacy of IL‐7 combining cisplatin in vivo. Results Here we found IL‐7R was increased in A549/DDP cells compared with A549 cells. The block of IL‐7R reversed the inhibitory effects of IL‐7 combined with cisplatin and decreased the numbers of apoptosis cells induced by treatment of IL‐7 combined with cisplatin. The JAK3 inhibitor and STAT5 inhibitor were used to identify the pathway involved. The results showed that JAK3/STAT5 pathway was involved in enhancing role of cisplatin sensitivity of NSCLC cells by IL‐7. In vivo, cisplatin significantly inhibited tumour growth and IL‐7 combined with cisplatin achieved the best therapeutic effect. Conclusion Together, IL‐7 promoted the sensitivity of NSCLC cells to cisplatin via IL‐7R‐JAK3/STAT5 signalling pathway.


| INTRODUC TI ON
As one of the most common cancer types in women and men, lung cancer is the leading cause of cancer-related morbidity and mortality worldwide, representing 13% of newly diagnosed new cancer cases. 1,2 NSCLC accounts for about 85% of all lung cancer cases and has a high incidence of cancer recurrence and metastasis, which leads to the failure in treatment of NSCLC. 3,4 Although early diagnosis and therapeutic approaches of NSCLC patients have considerably progressed, the total 5-year survival rate of NSCLC patients is less than 15%. 5 Currently, radiotherapy and chemotherapy for NSCLC are the standard treatment options and have a certain efficacy. However, the toxicity and side effects have limited its implementation in these patients. 6,7 Importantly, the chemotherapy drug resistance limits the clinical efficacy of therapies for NSCLC, which leads to the main recurrence and metastasis of NSCLC. 4,8 The development of novel strategies for lung cancer is still critical.
Platinum-based chemotherapy regimens, particularly cisplatin, are a standard adjuvant therapeutic strategy in advanced stage NSCLC. 9,10 Nowadays, the incidence of cisplatin resistance of NSCLC is up to 63%. In cells, cisplatin binds to DNA in nuclei and mitochondria to form the cisplatin-DNA adducts, which destroys DNA by blocking DNA replication and transcription and induces cell cycle arrest and apoptosis. 11,12 The NSCLC cells develop the chemoresistance to cisplatin by increasing drug detoxification, changes in DNA repair, DNA damage response, DNA damage tolerance, cell cycle checkpoints and reduced cell apoptosis. [13][14][15][16][17][18] Therefore, identifying novel molecular-targeted therapeutic approaches to overcome cisplatin resistance or developing safe methods to reverse drug resistance is essential for the treatment of NSCLC.
Growing evidence has demonstrated that combination of immunotherapy and chemotherapy has shown promise in the treatment of NSCLC. Interleukin-7 (IL-7) plays an important role in affecting T-cell proliferation, development and homeostasis. [19][20][21][22] Previous studies have reported that administration of IL-7 combined with oxaliplatin significantly suppressed the growth of tumours in lung and abdomen metastasis models of colon cancer. 23 For NSCLC, whether the IL-7 combining cisplatin has a better anti-tumour activity and reverses drug resistance, however, is still unclear.
Here, we have investigated whether IL-7 affects the chemotherapeutic sensitivity of NSCLC cells to cisplatin, and showed that IL-7 enhanced the sensitivity of NSCLC cells. We have also showed that IL-7 enhanced the sensitivity of A549/DDP cells to cisplatin. To investigate the roles of IL-7/IL-7R signalling pathway in enhancing the sensitivity of A549 cells to cisplatin, a blocking antibody against the IL-7 receptor was used. We found that the block of IL-7R reversed the inhibitory effects of IL-7 combined with cisplatin and decreased the numbers of apoptosis cells induced by treatment of IL-7 combined with cisplatin.
The JAK3 and STAT5 inhibitors were used to validate the involvement of JAK3/STAT5 pathway in enhancing the role of cisplatin sensitivity of NSCLC cells by IL-7. In addition, a xenograft model was established to confirm the anti-tumour effect of combining IL-7 and cisplatin in vivo.

| Cell culture and treatment
NSCLC A549 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in F-12K medium (GIBCO, USA) supplemented with 10% foetal bovine serum (Gibco, USA), 100 U/mL penicillin and streptomycin (Amresco, Solon, OH) at 37°C in a 5% CO 2 humidified incubator.
The cisplatin-resistant A549 cell line (A549/DDP) from A549 cells was incubated with gradually increasing cisplatin concentration and maintained in 10% F-12K medium supplemented with 1 μg/ mL DDP. The relative cisplatin resistance was determined by clonogenic assay.

| CCK8 assay
The cell viability of cells after indicated treatment was measured by the CCK-8 assay using the Cell Counting Kit (Dojindo, Japan) according to the manufacturer's instructions. In Brief, 5 × 10 3 cells/ well were seeded into 96-well plates. A549 cells were treated with 50 ng/mL IL-7, 1 μg/mL DDP or 50 ng/mL IL-7 and 1 μg/mL DDP.
A549/DDP cells were treated with 5 μg/mL DDP or 50 ng/mL IL-7 and 5 μg/mL DDP. After the indicated treatment, 10 μL of CCK-8 reagent was added and incubated with the cells for 2 hours at 37°C.
The absorbance of the converted dye at 450 nm was measured using a microplate reader (Thermo Fisher, Finland).

| Colony-forming assay
A total of 1000 A549 cells or A549/DDP cells per well were seeded into 12-well plate and cultured for 12 days. Then, the colonies were fixed with 20% methanol and stained with 0.1% crystal violet dye.
The representative images were photographed, and the colonies were scored.

| Cell apoptosis assay
After the treatment, the cells were collected, washed, fixed and permeabilized. Then, the cells were stained by Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) (BD, CA, USA) according to the manufacturer's instructions. After 15 minutes, the cell apoptosis was measured using flow cytometry (BD Biosciences).

| Hoechst 33342 staining
The apoptosis of cells was evaluated using a Hoechst 33342 kit (Thermo Fisher Scientific, MA, USA) according to manufacturer's instructions. In brief, following the treatment for 48 hours, the cells were stained with Hoechst 33342 (10 mg/mL) at 37°C for 10 minutes. Five visual fields were randomly selected from each slide, and approximately 200 cells were counted per field. The FV10i confocal microscope (OLYMPUS, Japan) was used to capture the images.

| Immunohistochemical assay
Immunohistochemical assay was performed to analyse the expression of IL-7R and Ki-67 as previously described. 24 The tumour tissue was cut into five μm thick sections and deparaffinized with xylene. IL-7R (1:100 dilution; Santa Cruz Biotechnology, USA) and Ki-67(1:500 dilution; Cell Signaling Technology, MA, USA) antibodies were used in the study. Tissue sections were stained with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA). After IHC staining, the sections were counterstained with haematoxylin.

| Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay
The apoptotic cells were determined using an Apoptag ® Peroxidase In Situ Apoptosis Detection Kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Ten fields were randomly selected for the quantification of apoptotic cells at ×20 magnification, and the average counts of TUNEL-positive cells were calculated.

| Western blot assay
Western blot assay was used to detect the protein level in the cells.

| Statistical analyses
All results were represented as the mean ± SD. Statistical significance between the groups was analysed by unpaired t test, and the differences between more than two groups were analysed by one-way ANOVA or Kruskal-Wallis test. P value of <.05 was considered statistically significant. Each experiment was performed in triplicates.

| IL-7 enhanced the sensitivity of NSCLC cells to cisplatin
To determine whether IL-7 affects the chemotherapeutic sensitivity of NSCLC cells, the effect of IL-7 alone and of IL-7 plus cisplatin on A549 cells was determined. As shown in Figure 1A, IL-7 alone exerted no effects on the cell proliferation, but the combination of IL-7 and cisplatin significantly decreased the proliferation of A549 cells compared with cisplatin alone treatment. We also observed that IL-7 decreased the proliferation of A549/DDP cells ( Figure 1B Figure 1D). In addition, colony formation assay showed that the combination of IL-7 and cisplatin resulted in a decrease in the clonogenic survival of A549 cells compared with cisplatin treatment alone, and the numbers of colony in control group, DMSO group, IL-7 group, DDP group and DDP + IL-7 group were 101.33 ± 4.16, 101.00 ± 4.58, 98.00 ± 2.64, 63.67 ± 7.37 and F I G U R E 1 IL-7 enhanced the sensitivity of NSCLC cells to cisplatin. A, B, Cell proliferation analysis using CCK-8 assay was performed to assess the cell viability of A549 and A549/DDP cells after indicated treatment. C, EdU proliferation assays were performed on A549 cells after indicated treatment for 48 h, and the percentage of EdU-positive cells was quantified. DDP group vs DMSO group (**P < .01), IL-7 group vs DDP + IL-7 group (***P < .001), DDP group vs DDP + IL-7 group (#P < .05). D, EdU proliferation assays were performed for A549/ DDP cells after indicated treatment for 48 h, and the percentage of EdU-positive cells was quantified. IL-7 group vs DMSO group (**P < .01). E, F, Colony-forming assay was performed to analyse the colony formation efficiency of A549 and A549/DDP cells after indicated treatment. G, The average numbers of colony formed by A549 cells were counted. DDP group vs DMSO group (**P < .01), IL-7 group vs DDP + IL-7 group (***P < .001), DDP group vs DDP + IL-7 group (#P < .05). H, The average numbers of colony formed by A549/DDP cells were counted. IL-7 group vs DMSO group (**P < .01). I, The A549 cells were treated with indicated treatment for 48 h, and the cell apoptosis was measured by flow cytometry. DDP group vs DMSO group (**P < .01), IL-7 group vs DDP + IL-7 group (***P < .001), DDP group vs DDP + IL-7 group (##P < .01). J, The A549/DDP cells were treated with indicated treatment for 48 h, and the cell apoptosis was measured by flow cytometry. DMSO group vs IL-7 group (**P < .01). K, L, The image of A549 and A549/DDP cells apoptosis treated with indicated treatment. M, N, Cell apoptosis was evaluated using HOECHST 33342 assay. Representative images of different groups are showed. Scale bars, 20 µm. Data represent three independent experiments 36.33 ± 4.51, respectively ( Figure 1E and G). In A549/DDP cells, IL-7 treatment alone also decreased the colony formation, and the numbers of colony in control group, DMSO group and IL-7 group were 80.67 ± 6.03, 80.00 ± 3.61 and 41.33 ± 6.11, respectively ( Figure 1F and H). Next, we assessed cell apoptosis of A549 cells under different treatment conditions. As shown in Figure 1I

| IL-7 enhanced the sensitivity of NSCLC cells to cisplatin by IL-7R-JAK3/STAT5 pathway
As stated earlier, IL-7 enhanced the sensitivity of NSCLC cells to cisplatin. We assessed the expression of IL-7R in A549 and A549/ DDP cells. The mRNA level of IL-7R in A549/DDP cells was higher compared with A549 cells (Figure 2A). The protein level of IL-7R in A549/DDP cells was higher compared with A549 cells (Figure 2B, C).
To investigate the roles of IL-7/IL-7R signalling pathway in enhancing the sensitivity of A549 cells to cisplatin, a blocking antibody against the IL-7 receptor was used. As shown in Figure 2D Figure 2L-O). The blocking of IL-7R decreased the numbers of apoptotic cells induced by treatment of IL-7 combined with cisplatin ( Figure 2L-O). Similar results were observed with by HOECHST 33342 assays ( Figure 2P,Q). It has been showed that IL-7 could activate the tyrosine kinases Jak1 and Jak3 and STAT5 by binding of IL-7R. [25][26][27] Western blot assay results showed that the levels ofpJA-K3and pSTAT5 in both A549 and A549/DDP cells were decreased by cisplatin treatment but increased by the combined use of IL-7 and cisplatin, but not affected the expression of JAK3 and STAT5.
As expected, the blocking of IL-7R markedly decreased the levels of p-JAK3 and p-STAT5 ( Figure 2R). The levels of JAK3 and STAT5 had no significant change under the different treatment conditions ( Figure 2R). We also assessed the expression of apoptosis-related protein. As shown in Figure 2R, the protein levels of caspase-3 and Bax were increased by cisplatin treatment and increased by the combined use of IL-7 and cisplatin, and markedly decreased by the blocking of IL-7R. However, contrasting results were observed for Bcl-2 protein expression ( Figure 2R).
To further investigate the involvement of JAK3/STAT5 pathway in enhancing the role of cisplatin sensitivity of NSCLC cells by IL-7, a novel JAK3 inhibitor was used to inhibit the JAK3 pathway. As shown in Figure 3A Figure 5C). In A549 cell transplanted model, cisplatin significantly inhibited tumour growth and IL-7 combined with cisplatin achieved the best therapeutic effect, while the treatment of IL-7 alone had no effect on tumour growth ( Figure 5A   The combination of IL-7 and IL-12 synergistically enhances the anti-tumour immunity by promoting CD3 + T-cell and CD4 + T-cell proliferation. 33 Also, the combination of IL-7 and IL-12 synergistically enhances the anti-tumour immunity by promoting the proliferation and anti-tumour function of cytotoxic CD8 + T cells. 34 Most of the previous studies on the anti-tumour activity were focused on the immune responses. Our study investigated the anti-tumour role of IL-7 in tumour cells itself and not the immune responses.

| D ISCUSS I ON
Our study showed that IL-7 enhanced the sensitivity of NSCLC cells to cisplatin via IL-7R-JAK3/STAT5 pathway in vitro and in vivo. Sharma S et al reported the anti-tumour activity of lung tumour-derived IL-7 by IL-7 gene transfer in NSCLC cells. 35 However, IL-7 was found to have no effect on tumour growth in mice colon carcinoma and cell resistance to oxaliplatin in CT26 cells. 23 In glioma cells, IL-7 has a pro-tumour role with the enhanced cisplatin resistance and decreased cell apoptosis induced by cisplatin. 36 The pro-tumour roles of IL-7 were also reported in NSCLC for promoting the metastatic process. 37,38 Our results showed the direct effect of IL-7 on the A549 cells. However, further studies are required to validate its role in other cancer cells. IL-7 binds to the IL-7R, a heterodimer consisting of IL-7 receptor α and common γ chain receptor. 39 IL-7R is found to be expressed on F I G U R E 2 IL-7 enhanced the sensitivity of NSCLC cells to cisplatin by IL-7/IL-7R pathway. A, The mRNA level of IL-7R in A549/DDP and A549 cells was analysed by qRT-PCR. B, The levels of IL-7R in A549/DDP and A549 cells were analysed by Western blot. Densitometry plot of results from the blots is showed. The relative expression levels were normalized to GAPDH. C, Immunofluorescence analysed the protein levels of IL-7R in A549/DDP and A549 cells. D, E, The A549 and A549/DDP cells were treated with DMSO, cisplatin, IL-7 or the blocking antibody against the IL-7 receptor (Ab) alone or combined for 24, 48 or 72 h. The concentration of cisplatin for A549 and A549/DDP cells was 1 and 10 μmol/L, respectively. Cell proliferation analysis using CCK-8 assay was performed to assess the cell viability after indicated treatment. F, G, The A549 and A549/DDP cells were treated with DMSO, DDP, IL-7 or the blocking antibody against the IL-7 receptor alone or combined for 48 h. EdU proliferation assays were performed of A549 and A549/DDP cells after indicated treatment, and the percentage of EdU-positive cells was quantified. H-K, Colony-forming assay was performed to analyse the colony formation efficiency of A549 and A549/DDP cells after indicated treatment. The average numbers of colony were counted. L-O, The A549 and A549/DDP cells were treated with indicated treatment for 48 h, and the cell apoptosis was measured by flow cytometry. P, Q, Cell apoptosis was evaluated using HOECHST 33342 assay. Representative images of different groups are showed. Scale bars, 20 µm. R, The levels of JAK3, p-JAK3, STAT5, p-STAT5, caspase-3, Bcl-2 and Bax in A549 and A549/DDP cells after indicated treatment for 48 h were analysed by Western blot. Data represent three independent experiments (average and s.e.m of triplicate samples). The lowercase letters (a, b, c, d) represent statistically significant (P < .05). To compare whether there is a significant difference, same letters marked were considered to have no significant difference between the two groups and different letters marked were considered to have significant difference between the two groups F I G U R E 3 Tofacitinib inhibited the sensitivity of NSCLC cells by IL-7. The A549 and A549/DDP cells were treated with DMSO, DDP, IL-7(50 ng/mL) or tofacitinib (pre-treated, 100 nmol/L) alone or combined as indicated in the figure for 24, 48 or 72 h. The concentration of cisplatin in A549 and A549/DDP cells was 1 and 5 μg/mL, respectively. A, B, Cell proliferation analysis using CCK-8 assay was performed to assess the cell viability after indicated treatment. C, D, EdU proliferation assays were performed for A549 and A549/DDP cells after indicated treatment for 48 h, and the percentage of EdU-positive cells was quantified. E-H, Colony-forming assay was performed to analyse the colony formation efficiency of A549 and A549/DDP cells after indicated treatment for 48 h. The average numbers of colony were counted. I-L, The A549 and A549/DDP cells were treated with indicated treatment for 48 h, and the cell apoptosis was measured by flow cytometry. M, N, Cell apoptosis was evaluated using HOECHST 33342 assay. Representative images of different groups are showed. Scale bars, 20 µm. O, The levels of JAK3, p-JAK3, STAT5, p-STAT5, caspase-3, Bcl-2 and Bax in A549 and A549/DDP cells after indicated treatment for 48 h were analysed by Western blot. Data represent three independent experiments (average and s.e.m of triplicate samples). The lowercase letters (a, b, c, d) represent statistically significant (P < .05). To compare whether there is a significant difference, same letters marked were considered to have no significant difference between the two groups and different letters marked were considered to have significant difference between the two groups various cell types, including naive and memory T cells and many others. It has been known to play a critical role in the development of immune cells. In prostate cancer cells, the IL-7R is found to be upregulated. 40 In our study, both the mRNA and protein levels of IL-7R in A549/DDP cells were increased compared with A549 cells. After the IL-7 binds to IL-7R, the tyrosine kinases Jak1 and Jak3 are activated, which leads to the activation of STAT5. [25][26][27]41 The JAK/STAT signalling pathway is a crucial pathway that involves in tumour cell was a key signalling molecule that functions to inhibit cell migration in breast cancer by negating JAK/STAT signalling pathway. 46 In our present study, in both A549 and A549/DDP cells, the levels of p-JAK3 and p-STAT5 were decreased by cisplatin treatment and increased by the combined use of IL-7 and cisplatin. The blocking of IL-7R markedly decreased the levels of p-JAK3 and p-STAT5, with no significant F I G U R E 5 Combination with IL-7 enhanced the anti-tumour efficacy of cisplatin in vivo. A549 and A549/DDP cells were used to establish a xenograft model. In A549 mouse tumour model, the mice received the following treatments: DMSO (5%); IL-7 (5 μg/day); and cisplatin (5 mg/kg) and IL-7 (5 mg/kg) combined with cisplatin (5 mg/kg) as indicated in the Figure 5A, and in A549/DDP mouse tumour model, the mice were received the following treatments: cisplatin (5 mg/kg); DMSO (5%) combined with cisplatin (5 mg/kg); and IL-7 (5 μg/day) combined with cisplatin (5 mg/kg) as indicated in Figure 5C. A, B, Representative images of tumours from A549 established tumours after indicated treatment. The changes in tumour volume were monitored and shown (N = 4 per group). C, D, Representative images of tumours from A549/DDP established tumours after indicated treatment. The changes in tumour volume were monitored and shown (N = 4 per group). E, F, Immunohistochemistry analysis of Ki-67 protein levels in xenograft tumour tissues; TUNEL apoptosis assay analysis of cell apoptosis in tumour tissues. Immunohistochemistry analysis of IL-7R protein levels in xenograft tumour tissues. Scale bar, 50 μm. G, H, The levels of IL-7R, JAK3, p-JAK3, STAT5, p-STAT5 and GAPDH in tumour tissues were analysed by Western blot. The lowercase letters (a, b, c, d) represent statistically significant (P < .05). To compare whether there is a significant difference, same letters marked were considered to have no significant difference between the two groups and different letters marked were considered to have significant difference between the two groups change in JAK3 and STAT5. The inhibition of JAK3 and STAT5 pathway reversed the inhibitory effects of IL-7 combined with cisplatin and decreased the number of apoptotic cells induced by the treatment of IL-7 combined with cisplatin. In squamous cell carcinoma of the head and neck, STAT5 activation was involved in the resistance to cisplatin-mediated apoptosis and inhibiting growth induced by the epidermal growth factor receptor tyrosine kinase inhibitor. 47 Other studies have reported that miR-10a silence enhanced the sensitivity of lung cancer to cisplatin via TGFβ/Smad2/STAT3/STAT5 pathway. 48 In the JAK/STAT signalling pathway, the ligand-receptor internalization and trafficking to the early endosome were reported to be associated with the signalling intensity, 49 suggesting that IL-7/IL-7R may function to promote ligand-receptor generate, causing the activation of JAK/STAT signalling. Additionally, C-terminal truncated STAT5 a/b isoforms, generated by protein processing, 50 inhibition represents a more promising strategy than PI3K-mTOR inhibition for treatment in the chemoresistance setting in NSCLC. 59 In the present study, we only analysed the JAK3/STAT5 pathway involved in IL-7-mediated chemotherapeutic sensitivity in non-small-cell lung cancer, and whether any other possible mechanisms or cell signalling pathway also contribute to IL-7-mediated chemotherapeutic sensitivity in NSCLC needs to be further investigated.
Our results showed that IL-7 promoted the sensitivity of NSCLC cells to cisplatin via IL-7R-JAK3/STAT5 signalling pathway. We highlighted the potential of the combination of IL-7 and chemotherapy to overcome the cisplatin resistance of NSCLC and delineated the underlying molecular mechanism.

ACK N OWLED G EM ENTS
The research is supported by grants from National Natural Science Foundation of China (No. 81774175, 81874381) and Natural Science Foundation of Guangdong Province (2018A0303130121).

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

AUTH O R S' CO NTR I B UTI O N S
LS and ZX contributed equally to this work; LS and BK designed the research study; LS, ZX, QY and YH performed the assays in this research; ZX, YG, FW and BK analysed the data; and LS and BK wrote and revised the manuscript. All authors have read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
Animal experiments were reviewed and approved by the Animal Research Ethics Committee of the Zhujiang Hospital of Southern Medical University.