Loss of zinc‐finger protein 143 contributes to tumour progression by interleukin‐8‐CXCR axis in colon cancer

Abstract Several studies have shown that expression of zinc‐finger protein 143 (ZNF143) is closely related to tumour progression including colon cancer. However, it remains unclear how ZNF143 expression is related to tumour progression within the tumour microenvironment. Here, we investigated whether ZNF143 expression affects the tumour microenvironment and tumour progression by screening molecules secreted by colon cancer cells stably expressing short‐hairpin RNAs against ZNF143 or control RNAs. We observed that secretion of interleukin (IL)‐8 was increased when ZNF143 expression was reduced in two colon cancer cell lines. The mRNA and protein levels of IL‐8 were increased in cells following ZNF143 knockdown, and this effect was reversed when ZNF143 expression was restored. The Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) and extracellular signal‐regulated kinase pathways were also shown to contribute to IL‐8 expression in ZNF143‐knockdown cells. The expression levels of ZNF143 and IL‐8 were inversely correlated with three‐dimensionally grown spheroids and colon cancer tissues. THP‐1 cells were differentiated when cells were incubated with condition media from colon cancer cell with less ZNF143, drastically. Loss of ZNF143 may contribute to the development of colon cancer by regulating intracellular and intercellular signalling for cell plasticity and the tumour microenvironment respectively.

The tumour microenvironments are composed of tumour cells, tumour stromal cells, the extracellular matrix, oxygen levels and so on. Stromal cells including macrophages, endothelial cells and fibroblasts contribute to tumour-supportive environments by expressing growth factors, cytokines and chemokines, which are the main mediators for communication between tumours and stromal cells. 5,6 In colon cancers, macrophages were shown to interact and contribute to colon cancer progression, 7  Chemokines mediate the accumulation of immunocompetent cells and help in shaping a tumour-promoting or -suppressive microenvironment. Although the tumour microenvironment is being increasingly recognized as a key factor in cancer aggressiveness, the underlying mechanisms remain controversial. 8 Cancer cells up-regulate the expression of numerous cytokines by expressing receptors that benefit their own survival. Cytokines are soluble proteins that play an important role in inflammation as well as in the initiation and promotion of carcinogenesis. 9 Interleukin (IL)-8, which is also known as CXCL8, is one of the most significantly up-regulated chemokines in CRC, indicating its potential diagnostic value. 10 IL-8 acts by binding to the cell surface receptors CXCR1/2, and the downstream signalling pathway contributes to tumour growth and invasion, inducing CRC cell proliferation and migration. 11 IL-8 signalling has been associated with activation of the classic mitogen-activated protein kinase (MAPK) signalling cascade, with downstream phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in cancer cells. 12 Furthermore, increased phosphorylation of signal transducer and activator of transcription (STAT)-3 has been detected in patients with colorectal carcinoma and was associated with metastasis and poor prognosis. 13 Zinc-finger protein 143 (ZNF143), a ubiquitously expressed transcriptional activator that belongs to the Kruppel family of zincfinger proteins, has been implicated in the transcriptional regulation of genes associated with the cell cycle and DNA replication. 14 Accumulating evidence has suggested that ZNF143 is involved in a variety of cellular and pathogenic processes. [15][16][17][18][19][20] The role of ZNF143 as a transcriptional regulator has been studied in various cancers, such as lung adenocarcinoma, as well as in colon, prostate, breast, gastric cancers and leukaemia. [20][21][22][23] Recently, we found that the expression of ZNF143 was reduced in invasive ductal carcinoma tissue compared to normal epithelial breast tissue, suggesting a role in cancer cell motility and invasion. 24 In the present study, we demonstrate the role of ZNF143 in regulating IL-8 expression via the crosstalk of MAPK and Janus tyrosine kinase (JAK)/STAT signalling, associated with intercellular communication with immune cells which maintains the tumour microenvironments for enhanced CRC cell invasiveness.

| Cell culture
The human colon carcinoma cell lines HCT116 and HT29 were obtained from the American Type Culture Collection (Manassas, VA) and THP-1, human monocytes, were from Korean cell bank (KCLB, 40202, South Korea). All cells were authenticated by short-tandem repeat PCR method in 2017 (HCT116 and HT29) and in 2018 (THP-1).
HCT116 cells were maintained as monolayers in DMEM. HT29 cells were maintained in McCoy 5A media. THP-1 cells were maintained as suspension culture in RPMI 1640. All maintenance media were supplemented with 10% heat-inactivated FBS. All cells were grown at 37°C in a humidified 5% CO 2 atmosphere.

| Short-hairpin RNA-mediated silencing of human ZNF143 in colon cancer cells
To achieve stable lentivirus-mediated expression of short hairpin RNA (shRNA) specific for the gene encoding ZNF143, HCT116 and HT29 cells were grown for 24 hours, incubated with 5 mg/ mL polybrene for 1 hour, and infected with the lentiviral vector (approximately 1 molar ratio of infection) as described previously. 25,26

| Cytokine profile assessment
To screen for cytokine expression and secretion, we used the Proteome Profiler Human Cytokine Array Kit, Panel A (Research & Diagnostic Systems, Inc, Minneapolis, MN). The supernatants isolated from cells (700 µL) were subjected to profiling according to the manufacturer's instructions. The cytokines present were detected by exposing the membrane to X-ray film, which was subsequently developed. The mean luminescence was normalized to reference spots from the same membrane following background correction.

| Immunoblotting
Cells were harvested in lysis buffer containing a protease and phosphatase inhibitor cocktail and quantified using the bicinchoninic assay according to the manufacturer's instructions (Pierce, Thermo Fisher Scientific Ltd). Protein samples were heated at 95°C for 5 minutes and separated by SDS-PAGE using 8-15% acrylamide gels, followed by transfer to polyvinylidene difluoride membranes. The membranes were blocked for 1 hour in Tris-buffered saline with 0.01% Tween-20 (TBST) with 3% bovine serum albumin (BSA), after which they were incubated overnight with primary antibody in TBST with 2% BSA, followed by incubation with horseradish peroxidase-conjugated antimouse or -rabbit antibody. The blots were developed with an enhanced chemiluminescence kit (West-ZOL plus, Western Blot Detection System; Intron Biotechnology, Inc, Daejeon, South Korea), and quantification of band intensity on XAR-5 film (Eastman Kodak Co., Rochester, NY) was measured with Quantity One software (Bio-Rad).

| Cell fractionation
Cells were lysed and separated into cytosolic, membrane, nuclear and cytoskeletal fractions using the Qproteome Cell Compartment kit (Qiagen) according to the manufacturer's instructions. Briefly, harvested cell pellets were resuspended in ice-cold extraction buffers CE1, CE2, CE3 and CE4, followed by different incubation times and centrifugation, ultimately resulting in the separation of cytosolic, membrane, nuclear and cytoskeletal proteins. The soluble protein fractions underwent acetone precipitation for downstream applications. Proteins were quantified, and immunoblotting was performed.

| Immunofluorescence staining
Cells were fixed in 4% paraformaldehyde and permeabilized in 0.25% Triton X-100 in phosphate-buffered saline (PBST). Cells were incubated with 1% BSA diluted in PBST for 30 minutes with gentle rocking to block non-specific binding. Finally, cells were immunostained with the primary antibody (phosphorylated STAT3 at Tyr 705 [p-STAT3 Tyr705 ]) and secondary antibody in PBST with 1% BSA. Cells were mounted with anti-fade Vectashield mounting medium (Vector Laboratories, Inc, Burlingame, CA) and visualized under a confocal microscope.

| Three-dimensional cell cultures
Matrigel was mixed with serum-free media to a final concentration of 3 mg/mL. Hundred-microlitre of the medium was added to 96-well culture plate and allowed to solidify in CO 2 incubator for overnight. Next day, harvested cells were counted, and 100 cells were seeded onto Matrigel followed by 100 μL of growth media over the Matrigel. Cells were grown into spheroids for 14 days.
Further these spheroids were harvested and counted for reseeding and RNA isolation.

| Treatment of monocytes with colon cancer cell conditioned medium (CM)
Colon cancer cells (5 × 10 5 ) were seeded in 6-well plates. Next day, 2 mL of fresh media was replaced and kept for 24 hours to get the conditioned medium (CM) from cancer cells. Cancer cell CM was collected and centrifuged at 3000 rpm for 10 minutes. THP-1 cells were grown in RPMI 1640 medium supplemented with 10% FBS and 50 μmol/L β-mercaptoethanol. For the experiment, THP-1 cells

| Statistical analysis
All data are expressed as percentages of the control and shown as means ± standard error (S.E.). Statistical comparisons between groups were performed with Student's t tests. Values of P < 0.05 were considered significant.

| IL-8 expression is increased following ZNF143 knockdown in colon cancer cells
We examined the effect of ZNF143 knockdown on the cytokine profile of the colon cancer cell line HCT116. After 24 hours of growth, media from control and ZNF143-silenced HCT116 cells were obtained and subjected to proteomic profiling ( Figure 1A,B). IL-8, IL-10 and macrophage inhibitory factor (MIF) were found to be significantly increased in the supernatants of HCT116 sh-ZNF143 cells ( Figure 1A), whereas the levels of IL-6 were low, and SERPINE1/plasminogen activator inhibitor (PAI) levels were only marginally altered.
MIF is a pleiotropic cytokine that is overexpressed in a number of solid tumours, including breast, prostate and colon cancers. 28 PAI-1 initiates several events that promote cancer; clinically, increased levels of PAI-1 in colon cancer epithelia are associated with increased metastasis. 29 IL-8, an important pro-inflammatory chemokine, has been shown to modulate endothelial cell migration and promote angiogenesis, tumourigenesis and metastasis. 30,31 We further investigated whether ZNF143 knockdown had an effect on IL-8 expression in colon cancer cells. HCT116 and HT29 cells expressing sh-ZNF143 were prepared as described previously. 20 Cell lysates and supernatants were harvested to assess IL-8 synthesis and secretion, respectively, by performing a CBA assay ( Figure 1C,D).
The results were normalized by protein quantification or cell number. Increased IL-8 was observed in HCT116 and HT29 sh-ZNF143 cells when compared to control cells. This result was confirmed by immunoblotting ( Figure 1E) and real-time PCR ( Figure 1F

| ERK and JAK/STAT pathways are altered following ZNF143 knockdown and contribute to IL-8 expression in colon cancer cells
As the IL-8 promoter contains consensus binding sites for nuclear factor kappa B (NF-κB), β-catenin/Tcf, hypoxia-inducible factor (HIF)-1 and activator protein (AP)-1, 32-34 , several signalling pathways were investigated by immunoblotting ( Figure 3A). Interestingly, the phosphorylation of ERKs and STAT3 was increased in HCT116 and HT29 sh-ZNF143 cells when compared to control cells. In addition to the phosphorylation of Ser 727 and/or Tyr 705 of STAT3, nuclear translocation of activated STAT3 is required for transcriptional activation. [35][36][37] Thus, translocation of activated p-STAT3 into the nucleus was examined by the subcellular fractionation and immunofluorescence staining of colon cancer cells ( Figure 3B,C). IκBα, epidermal growth factor receptor (EGFR) and c-Myc served as controls for the cytosolic, membrane and nuclear fractions respectively. p-STAT3 Tyr705 was strongly detected in the membrane and nuclear fractions of HCT116 sh-ZNF143 cells, and this was further confirmed by fluorescence staining in the nuclear region of HCT116 sh-ZNF143 cells ( Figure 3C),

| IL-8 may contribute to cell-cell communication in the tumour microenvironment in colon cancer progression
Within the tumour microenvironment, IL-8 signalling is initiated by binding of its G-protein-coupled receptors CXCR1/2.  Figure 5A,B, increased expression of IL-8 receptor, CXCR2 was seen when THP-1 cells was grown in CM-cells with less ZNF143 than control cells ( Figure 6D).
Colon tissues on tissue arrays were stained with haematoxylin and specific antibodies against ZNF143, IL-8, and p-STAT3 Tyr705 and scored by professional pathologists (Figure 7A,B, Tables S2 and S3).
Stained tissues were digitized with the Aperio AT Turbo whole slide scanner. Nuclear expression of ZNF143 was reduced as tumour malignancy increased in colon cancer tissue. Interestingly, IL-8 expression was higher in benign tissue when compared to normal tissue.
In particular, some tissues showed reciprocal expression of ZNF143 and IL-8 ( Figure 7A), supporting a relationship between ZNF143 and IL-8.

| D ISCUSS I ON
Increased cell motility has been observed in colon and breast cancer cells following a decrease in ZNF143 expression. 20,24 Another study demonstrated that overexpression of ZNF143 in gastric cancer cell lines enhanced metastatic potential. 43 Strong expression of ZNF143 in lung adenocarcinomas was shown to predict shorter disease-specific survival with increased Ki-67 labelling. 21 Gonzalez and colleagues showed that ZNF143 as an important regulator for CCAAT/enhancer-binding protein alpha (C/EBPα) in myeloid cells, 23  45 Recently, IL-8 has been shown to increase the mobilization of immature myeloid cells, resulting in increased tumourigenesis. 46 In this study, we investigated the key factors altered following knockdown of ZNF143 by proteomic profiling in HCT116 cell supernatants. Among cytokines, IL-8 attracted attention, with its significantly increased levels, as a secreted soluble factor in ZNF143-knockdown colon cancer cells (Figure 1). In addition to IL-8, we also found that expression of the chemokines MIF and IL-10 ( Figure 1A,B), which have been shown to mediate tumour-promoting crosstalk between tumours and the tumour microenvironment, was receptor α and β chain. 49 Therefore, it is likely that dysregulation of ZNF143 also promotes formation of the tumourigenic microenvironment in a paracrine manner to modulate the initiation and progression of colon cancer.
IL-8 signalling is mediated through the G-protein coupled receptors CXCR1/2 and has been associated with a poor prognosis in colon cancer. 50,51 It can also activate STAT3, AKT, ERK and EGFR signalling, which can enhance migration and inhibit anoikis of tumour cells. 52 Our results demonstrated the involvement in STAT3 signalling ( Figure 3) with increased expression of CXCR1/2 ( Figure 5A,B), resulting in the overexpression of IL-8 in ZNF143-silenced cells ( Figure 5C). The IL-8 promoter containing binding sites for NF-κB and TNFα is a well-known inducer of NF-κB. 53, 54 We also observed that TNFα significantly induced the phosphorylation of p65 in . Some cells were treated with 100 nmol/L phorbol 12-myristate13-acetate (PMA) or DMSO for 24 hours. All data were analysed with the data from media as control, C. Results shown are representative of at least three independent experiments. Data are expressed as means ± S.E. of at least three independent experiments. Statistical significance was assessed using unpaired student's t test and one-way ANOVA (***P < 0.0001; **P < 0.001 and *P < 0.01) CXCR1/2 signalling axis ( Figure 5C). Finally, we identified a negative correlation between ZNF143 and IL-8 in the tissues of colon cancer patients, suggesting it may be of diagnostic importance (Figure 7).
Tumour-associated macrophages are one of the widely found host immune cells in the tumour microenvironment involved in different cellular events to facilitate metastasis. 6 In our study, we found ZNF143 reduced colon cancer cells secrete some factors including IL-8, potentially differentiated monocytes into macrophages with increased expression of macrophage markers IL-6 and CD206 (Figure 6), suggesting its role in intercellular communication with immune cells to set up an environment for tumour progression to metastasis.
Hence, this is the first study focused on ZNF143 and the regulation of IL-8, which may be important for maintaining the tumour microenvironment and facilitating metastasis in colon cancer cells.

ACK N OWLED G EM ENTS
We thank Tae

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interests.
F I G U R E 7 ZNF143-IL-8 cascade contributes to colon cancer progression. (A, B) Expression of ZNF143 and IL-8 in colon cancer and normal tissue was examined by immunohistochemistry and scored by a professional pathologist. (A) The stained tissues on the tissue microarray were digitized at 20× magnification using the Aperio AT Turbo whole slide scanner equipped with a clinical grade RGB camera. (B) The results were categorized as normal, benign, malignant or metastatic according to information from the tissue microarray suppliers. Data are expressed as means ± S. E. of at least three independent experiments. Statistical significance was assessed using unpaired Student's t tests (*P < 0.006 and **P < 0.05)

AUTH O R S' CO NTR I B UTI O N S
VV, AP and HY performed the research; HY designed the research study; VV, AP and BC contributed essential reagents or tools; VV and EH analysed the data; VV and HY wrote the manuscript.