IFN‐α/β‐mediated NK2R expression is related to the malignancy of colon cancer cells

Abstract Neurokinin 2 receptor (NK2R), a G protein‐coupled receptor for neurokinin A (NKA), a tachykinin family member, regulates various physiological functions including pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. However, the precise role and regulation of NK2R expression in cancer cells have not been fully elucidated. In this study, we found that high NK2R gene expression was correlated with the poor survival of colorectal cancer patients, and Interferon (IFN‐α/β) stimulation significantly enhanced NK2R gene expression level of colon cancer cells in a Janus kinas 1/2 (JAK 1/2)‐dependent manner. NKA stimulation augmented viability/proliferation and phosphorylation of Extracellular‐signal‐regulated kinase 1/2 (ERK1/2) levels of IFN‐α/β‐treated colon cancer cells and NK2R blockade by using a selective antagonist reduced the proliferation in vitro. Administration of an NK2R antagonist alone or combined with polyinosinic‐polycytidylic acid, a synthetic analog of double‐stranded RNA, to CT26‐bearing mice significantly suppressed tumorigenesis. NK2R‐overexpressing CT26 cells showed enhanced tumorigenesis and metastatic colonization in both lung and liver after the inoculation into mice. These findings indicate that IFN‐α/β‐mediated NK2R expression is related to the malignancy of colon cancer cells, suggesting that NK2R blockade may be a promising strategy for colon cancers.


| INTRODUC TI ON
Tachykinins, such as substance P (SP), neurokinin A (NKA) and B, hemokinin-1, and endokinins, are a family of neuropeptides that act through three types of G-protein coupled receptors (GPCRs), neurokinin-1 receptor (NK1R), neurokinin-2 receptor (NK2R), and neurokinin-3 receptor (NK3R), functioning as neurotransmitters in the peripheral and central nervous systems. [1][2][3] Neuropeptide signaling through the receptors is well known to regulate various physiological functions such as induction of pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. It has been demonstrated that tachykinins and their receptors are expressed in several non-neuronal cells, which connect the nervous and peripheral organ systems to control respiratory, cardiovascular, endocrine, and gastrointestinal activity. 2,4 Furthermore, recent studies revealed that neuropeptide receptors are expressed in various cancer cells in the tumor microenvironment, therefore tachykinin signaling through these receptors possibly regulates pathological functions related to the development of cancer and tumor malignancy. 5,6 Previous research has indicated that SP-NK1R signaling is implicated in the promotion of cancer incidence and facilitates metastatic progression. Various tumor types express NK1R, the main receptor of SP, such as glioblastoma, 7 breast cancer, 8 leukemia, 9 pancreatic ductal adenocarcinoma, 10 hepatoblastoma, 11 and colon cancer, 12,13 which use SP signaling through NK1R to promote the proliferation and survival of cancer cells and to release soluble mediators to promote tumor growth or metastasis. However, the precise role of NK2R expression in cancer cells and the mechanism for tumor malignancy have not been fully demonstrated compared with that of the NK1R-mediated SP signaling cascade.
Colorectal cancer (CRC) is the second leading cause of cancer death worldwide. With increasing mortality observed in recent years, the global burden of CRC is expected to increase by 60% to more than 2.2 million new cases and 1.1 million deaths by 2030. 14 The rise in CRC incidence can be attributed to aging populations, unfavorable daily diet habits, stress, and increased risk factors. 15 Recent studies indicated that the molecular pathobiology of CRC involves viral and/or bacterial infection-induced inflammation, further promoting malignant progression, invasion, and metastasis of the tumor, [16][17][18] suggesting that patients with inflammatory bowel disease (IBD) have a higher risk of CRC. A recent phase II clinical trial using NK2R inhibitor for irritable bowel syndrome (IBS) with diarrhea reported improved overall symptoms, abdominal pain, and stool patterns in female patients. 19 Type I IFNs, IFNα and IFNβ, are the two main IFNs involved in fighting viral and bacterial infections. 20,21 Previous papers revealed that IFNα/β produced in the tumor microenvironment activate anticancer immunity by targeting dendritic cells (DCs) and noncancerous cells 22 and have the capacity to control cancer cell transformation. 23 When IFNα/β bind to the receptor, IFN-AR1, Janus kinase (JAK), signal transducer and activator of transcription (STAT) are activated.
Previous research revealed that activation of IFN-STAT1 signaling triggers apoptosis and cell cycle arrest in various cancer cells. 24,25 In this study, we first found that high expression levels of NK2R gene in tumor tissues were correlated with poor survival of CRC patients. Next, we revealed that in vitro stimulation with type I interferons, IFNα/β, or in vivo administration of a synthetic analog of double-stranded RNA, polyinosinic-polycytidylic acid (poly I:C), to tumor-bearing mice significantly augmented NK2R gene expression levels in colon cancer cells. NK2R antagonist treatment combined with IFNα/β or poly I:C significantly suppressed the proliferation of colon cancer cells in vitro or tumorigenesis of colon cancer-bearing mice, respectively. Furthermore, we confirmed that overexpression of the NK2R gene in murine colon cancer cells promoted tumorigenesis and metastasis in vivo. Here, we report the involvement of IFN-α/β-mediated NK2R expression in the malignancy of colon cancer cells, which may contribute to the development of effective therapy for CRC. An NK2R-selective antagonist (GR 159897) was purchased from TOCRIS Bioscience. Poly I:C (HMW) was purchased from InvivoGen.

| Cell lines and mice
The human colorectal cancer cell line DLD-1 (TKG 0379) was obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University.
The murine colon carcinoma cell line CT26 (CRL-2638) was obtained from the American Type Culture Collection. Wild-type BALB/c mice were obtained from Charles River Japan. All mice were maintained in specific pathogen-free conditions and used at 6-8 weeks of age. All mouse experiments were approved by the Animal Ethics Committee of Hokkaido University (No.14-0062 and No.19-0036) and were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the University. intratumorally injected into the CT26-bearing mice at day 5 and then every 4 days thereafter. Tumor size was measured from day 5 and tumor volumes were calculated as described previously. 28 In some experiments, tumor tissues were collected from mice. After collagenase treatment and homogenization, total tumor cells were analyzed by flow cytometry.

| Metastasis mouse models
GFP-transfected CT26-NK2R OE cells or CT26 mock control cells (1 × 10 6 ) were inoculated intravenously into BALB/c mice. Lung tissues were collected from CT26 cell-inoculated mice at day 14 and metastasis were evaluated according to the previous study. 27 In some experiments, GFP-transfected CT26-NK2R OE cells or CT26 mock control cells (2 × 10 5 ) were inoculated intrasplenically into BALB/c mice to evaluate metastatic colonization in the liver tissues on day 14.

| Confocal microscopy
DLD-1 cells cultured on 35-mm culture dishes with glass coverslips (Iwaki) were stained with anti-NK2R mAb and DAPI. Fluorescence signals were detected with the Spectral Confocal Scanning system (FV300; Olympus) through a 633/1.4 Oil DIC I lens. The signals were visualized with standard software (Olympus).

DLD-1 cells were treated with a Fix/Perm Buffer kit (Biolegend)
and stained with anti-NK2R mAb. The expression of NK2R was evaluated by FACSCanto II (BD Biosciences) and analyzed with FlowJo software (Tree Star) according to the previous study. 27 In some animal model experiments, CD45 + CD11c + 7AAD − cells and CD45 − GFP + 7AAD − CT26 cells were isolated as DCs and colon cancer cells by FACSAria II (BD Biosciences), respectively.
Protein expression was visualized as described previously. 27 Tumor tissues obtained from CT26 tumor-bearing mice were fixed in 4% paraformaldehyde phosphate buffer solution (Wako Pure Chemical Industries) and then embedded in paraffin. After deparaffinization, sections were stained with HE.

| Cell proliferation assay
DLD-1 and CT26 cells were plated in 96-well plates and incubated at 37°C for 12, 24, or 48 h. Cell proliferation was determined using a cell counting kit-8 (CCK-8) cellular proliferation assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's protocol.

| Western blotting
Whole cell lysates prepared from DLD-1 cells or CT26 cells using cold RIPA buffer (Thermo Fisher Scientific) were separated on SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were probed with anti-phospho-ERK1/2 (Thr202/ Tyr204, #4370; Cell Signaling Technology) and α-tubulin (clone DM1A; Sigma-Aldrich) antibodies. The protein signals were detected by Image Quant LAS4000 mini (GE Healthcare). The p-ERK1/2 signals were standardized based on those of α-tubulin by using Image J (National Institutes of Health).

| Statistical analysis
In vitro experiments were repeated three to five times. In vivo experiments consisting of three to six mice in each group were independently performed two to three times. Single representative experiments are indicated in the figures. All data were expressed as the mean ± standard deviation (SD). Significant differences in the results were determined by one-way or two-way analysis of variance (ANOVA) and Dunnett's post-test. In some experiments, the twotailed Student's t-test was used for the evaluation of differences between two groups. p values of <0.05 were considered statistically significant. Kaplan-Meier survival analysis was performed, and the log-rank test was used to determine statistically significant differences in survival curves among cancer patients.

| High NK2R expression is correlated with poor prognosis in CRC patients and IFN-α/β augments NK2R expression in human colon cancer cells
To clarify the involvement of NK2R expression in the pathology of patients with CRC, we first evaluated NK2R expression levels in the tumor tissues of 25 CRC patients (Table S2) To address the precise regulation of NK2R-mediated neuropeptide signaling in cancer cells, we next investigated the gene expression levels of NK2R and TAC1, which encodes SP and NKA, in human colon cancer cells. Stimulation with IFNα or IFNβ significantly induced NK2R gene expression in DLD-1 cells, whereas IFNγ, IFN-λ1, IFN-λ2, IFN-λ3, IL-6, or TGFβ stimulation did not alter the expression levels ( Figures 1C and S2). After the IFNα/β treatment of DLD-1 cells, upregulation of NK2R protein expression was observed by confocal microscopy ( Figure 1D) and flow cytometry ( Figure 1E). The upregulation of NK2R gene expression levels was significantly suppressed in the presence of a JAK1/2 inhibitor ( Figure 1F). Furthermore, we found that TAC1 gene expression levels were significantly enhanced by IFNα/β stimulation, and this upregulation was reduced by the JAK1/2 inhibitor ( Figure 1G). Analysis for ISRE and GAS sequences on the promoters of NK2R and TAC1 genes revealed that STAT1:STAT2 or IRF9 binding motifs existed in the promoter regions of NK2R (TACR2, Tacr2) and TAC1 (TAC1, Tac1) genes in both humans and mouse ( Figure S3). These findings suggest that IFNα/β type I interferons may augment neuropeptide signaling in colon cancer cells through the induction of both the NKA ligand and the receptor, NK2R.  (Figure 2A,B). IFNα/β stimulation reduced the viability and proliferation of DLD-1 cells in vitro and this reduction was recovered by treatment with NKA ( Figure 2C,D). We confirmed that stimulation of NKA combined with IFNα/β enhanced phospho-ERK1/2 expression revel in DLD-1 cells compared to stimulation with IFNα/β alone ( Figure 2E,F). These data suggest that NK2R-mediated neuropeptide signaling may be related to the viability and proliferation of human colon cancer cells.

| NK2R-mediated signaling regulates the viability and proliferation of human and murine colon cancer cells in vitro
Furthermore, we found that IFNα/β stimulation significantly enhanced NK2R and TAC1 gene expression levels in CT26 murine colon cancer cells in a JAK1/2-dependent manner ( Figure 3A,B). Treatment  Figure 3E,F). We confirmed that NKA stimulation combined with IFNα/β augmented phospho-ERK1/2 expression levels in CT26 cells compared to stimulation with IFNα/β alone, as in the case of DLD-1 cells ( Figure 3G,H). These data suggest that NK2R-mediated neuropeptide signaling is also involved in the viability and proliferation of murine colon cancer cells.

| Administration of poly I:C augments NK2R expression in murine colon cancer cells and NK2R blockade reduces tumorigenesis in vivo
Next, we investigated the roles of NK2R-mediated neuropeptide signaling in the malignancy of colon cancer cells in vivo. Administration of NK2R antagonist into CT26-bearing mice significantly reduced the tumor growth of CT26 cells ( Figure 4A-C). Furthermore, we found that NK2R blockade augmented the reduction of tumorigenesis of CT26 cells by poly I:C treatment in vivo. We attempted to elucidate the in vivo regulation mechanism of NK2R gene expression in the tumor microenvironment. In tumor tissues of the CT26-bearing model, we found IFNα and IFNβ as well as TAC1 gene expression levels in CD11c + DCs were enhanced after poly I:C injection into the tumor site ( Figure 4D).
The gene expression levels of NK2R and TAC1 in CT26 cancer cells were augmented by the administration of poly I:C ( Figure 4E). In this study, we confirmed that the enhanced NK2R gene expression in CT26 cells after poly I:C injection was not observed in the case of IFNAR1 KO cells ( Figure 4F). Moreover, the induction of NK2R gene expression in CT26 cancer cells after IFNα/β-stimulation in vitro was abolished in the IFNAR1 KO cells ( Figure 4G). These results suggest that neuropeptide signaling through NK2R expression in cancer cells was augmented by IFNα/β produced by host CD11c + DCs and NKA by DCs as well as cancer cells in an IFNAR1-dependent manner. Based on these findings, we speculate that IFNα/β induced by host cells may enhance NK2Rmediated neuropeptide signaling in cancer cells to augment the subsequent tumorigenesis in vivo.

| Overexpression of the NK2R gene in murine colon cancer cells augments tumorigenesis and metastatic colonization in vivo
To investigate the effects of NK2R expression in cancer cells on tumorigenesis in vivo, we established NK2R gene-overexpressing (NK2R OE) CT26 cell lines. The tumor growth was significantly enhanced by the overexpression of the NK2R gene in CT26 cells compared with the mock control cells (Figure 5A-C). We further evaluated the effect of NK2R expression on metastasis in vivo.
The lung metastasis of NK2R OE cells was significantly augmented compared with the mock control cells (Figure 5D-G). NK2R OE cells significantly augmented the metastatic colonization in the liver of the mice ( Figure 5H-K). Furthermore, we performed wound healing assay using NK2R OE cells in vitro. As a result, we found that overexpression of NK2R augmented the mobility of cancer cells compared to mock control cells. E-cadherin expression levels were reduced and N-cadherin was increased in NK2R OE cells compared to the mock control cells, suggesting that NK2R overexpressing cells showed epithelial to mesenchymal transition ( Figure S4). Based on these data, we speculated that overexpression of the NK2R gene in colon cancer cells was not only involved in the tumorigenesis but also related to the metastatic colonization in vivo.

| DISCUSS ION
In this study, we revealed that NK2R, a receptor of NKA, was overex-  29 It has been reported that the SP-NK1R cascade activates the PI3K/AKT and MAPK signaling pathways, which contribute to the suppression of apoptosis and tumor metastasis of cancer cells. 30,31 Many researchers have reported that activation of GPCR-mediated ERK/MAPK signal pathways in cancer cells caused malignant alteration such as tumorigenesis and metastasis. [32][33][34][35] In the present study, we found that NKA stimulation significantly enhanced the phosphorylation levels of ERK and the proliferation of IFNα/β-treated colon cancer cells. Therefore, we speculated that the augmented NK2R gene expression might contribute to cancer cell malignancy through the activation of the ERK signal pathway.
Previous studies indicated that NK2R expression was increased in the inflammatory cells of intestinal tissue from patients with Crohn's disease and ulcerative colitis, and in the distal ileum of rats with acute necrotizing pancreatitis. 36,37 Chronic inflammation in the tumor microenvironment occasionally promotes cancer development rather than protecting against it. 19,38,39 In our previous study, we found that NK2R expression and NKA production levels of murine DCs were significantly elevated after stimulation with IFNγ. 27 Furthermore, we revealed that IFNα/β stimulation augmented NK2R expression levels of human DCs in a STAT1-dependent manner. 40 Generally, F I G U R E 4 The effects of poly I:C and NK2R antagonist on tumorigenesis and NK2R expression levels of CT26 cells in vivo. GFPtransfected CT26 cells (1 × 10 6 ) were intradermally injected into BALB/c mice (day 0). Poly I:C (50 μg/mouse) or control PBS combined with or without NK2R antagonist (2.4 μg/mouse) was injected intratumorally into the mice at day 5 and then every 4 days thereafter. (A, B) Tumor growth was monitored for 13 days and tumor size was evaluated at 13 days after injection. Means and SDs (n = 5) of the tumor volumes are indicated. *P < 0.05 by Tukey's multiple comparisons test after ANOVA test. (C) Representative images of tumors at day 13. (D) Tumorinfiltrating CD45 + CD11c + dendritic cells were collected from tumor tissues at day 13. Mean and SD (n = 4) of the relative IFNα, IFNβ, and TAC1 gene expression levels are indicated. *P < 0.05 by Student's t test. (E) CD45 − GFP + CT26 cancer cells were collected from tumor tissues at day 13. Mean and SD (n = 4) of relative NK2R and TAC1 gene expression levels are indicated. *P < 0.05 by Student's t test. (F) Mock control and IFNAR1 KO CT26 cells (1 × 10 6 ) were intradermally injected into BALB/c mice. Poly I:C (50 μg/mouse) or vehicle control (PBS control) were injected intratumorally into the mice at day 5. Mean and SD (n = 4) of relative NK2R gene expression levels of colon cancer cells 24 h after the poly I:C treatment are indicated. *P < 0.05 by Student's t test. (G) Mock control and IFNAR1 KO CT26 cells were treated with IFNα (50 ng/ml) and IFNβ (50 ng/ml) for 6 h in vitro. Mean and SD (n = 4) of relative NK2R gene expression levels are indicated. *P < 0.05 by Student's t test administration of poly I:C, a TLR 3 agonist, promoted the maturation of DCs, followed by release of type I IFNs. 41 We confirmed that poly I:C treatment induced NK2R and TAC1 gene expressions in DCs and augmented NKA signaling through NK2R expression. 27,40 In this study, we revealed that NK2R overexpressing colon cancer cells augmented metastatic colonization in lung and liver tissues in vivo  Figure 5D-K). Furthermore, we found that overexpression of NK2R augmented the mobility of cancer cells compared to mock control cells in vitro ( Figure S4). E-cadherin expression levels were reduced and N-cadherin was increased in NK2R-overexpressing CT26 cells compared to the mock control cells, suggesting that NK2R overexpressing cells showed epithelial-mesenchymal transition (EMT).
Another study demonstrated that not only did NK1R activate the ERK1/2 signaling cascade but also that activation of NK1R promoted proliferation, EMT, and migration of lung cancer cells. 42 Based on these findings, we speculate that activation of NK2R as well as NK1R regulates ERK/MAPK-mediated malignant alteration of colon cancer cells ( Figure S5).
Previous research revealed that activation of IFN-STAT1 signaling triggers apoptosis and cell cycle arrest in various cancer cells. 24,25 In this study, we found that type I IFNs not only inhibited the growth of tumor cells but also induced NKA expression of cancer cells. The augmented NK2R-NKA signal reversed the inhibitory effects of the IFN on tumor growth, whereas NKA stimulation enhances further the IFN-induced phosphorylation of ERK1/2 kinase (Figures 2 and   3). Therefore, we speculate that IFNα/β-mediated induction of NK2R in cancer cells is a kind of feedback activation, suggesting one of the immune escape mechanisms to survive with malignant alteration. Previously our studies revealed that IFNα/β, or IFNγ, induced by host immune cells in IL-6-deficient mice augmented PD-L1 expression levels of cancer cells. 27,28 As a result, we confirmed that PD-L1 blockade under the IL-6-deficient condition effectively suppressed tumor growth of cancer cells in vivo. In this study, poly I:C treatment combined with NK2R antagonist showed a more effective antitumor effect (Figure 4). Based on these findings, we speculate that blockade of IFNα/β-mediated NKA-NK2R signaling may be a promising target for regulation in the tumorigenesis of colon cancer

cells in vivo.
A clinical study reported that NKA levels predicted the survival of patients with stage IV well-differentiated small bowel neuroendocrine neoplasms. 43 In midgut carcinoid tumors, patients with persistent plasma NKA levels had significantly worse survival than patients with stabilized or downregulated plasma NKA levels. 44 Moreover, a previous paper indicated that NK2R gene polymorphisms predicted lymph node metastasis in CRC patients. 45 In our study, we found that NK2R was overexpressed in the tumor tissues of patients with CRC and high expression was associated with poor survival from the analysis based on TCGA public dataset. 46,47 Based on these findings, we speculate not only that NKA signaling through NK2R may be related to the development and malignancy of CRC, but also that NKA-NK2R expression levels may be useful biomarkers to evaluate the malignancy of colon cancers.
A recent phase II clinical trial using an NK2R inhibitor for IBS with diarrhea reported improved overall symptoms, abdominal pain, and stool patterns in female patients. 25 In addition, the dual tachykinin NK1/NK2 antagonist DNK333 inhibited NKA-induced bronchoconstriction in asthma patients. 48 An NK2R antagonist suppressed cellular proliferation in metastatic breast carcinoma cells independent of SP effects. 49 In colon cancer, targeting NK1R inhibits the proliferation of human colon cancer cells, as previously described. 50 In this study, we confirmed that the blockade of neuropeptide signaling by in vivo injection of an NK2R antagonist alone or combined with poly I:C significantly suppressed the tumor growth of colon cancer cells, suggesting that NK2R may be a novel target to control the tumorigenesis of colon cancers.
We found that NK2R expression was induced by IFNα/βstimulation in colon cancer cells and related to the subsequent malignant alteration, and confirmed that blockade of NK2R-mediated signaling with an antagonist in colon cancer cells significantly reduced proliferation in vitro and tumorigenesis in vivo. Based on these data, we speculate that NK2R expression is related to the malignancy of colon cancer cells, suggesting that regulation of NK2Rmediated signaling will be a promising strategy for the development of more effective therapies for patients with CRC.

AUTH O R CO NTR I B UTI O N S
H. Kitamura, A.T., and H. Kobayashi designed the study; H.X., Y.T.,