ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
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CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis You-Chuan Xiao a,b,c,1, Zhi-Bin Yang a,1, Xian-Shuo Cheng a,b,1, Xing-Bao Fang b,c, Tao Shen a, Q2 Cui-Feng Xia a, Ping Liu a, Hai-Hua Qian b, Bin Sun b, Zheng-Feng Yin b,*, Yun-Feng Li a a
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Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University, Kunming 650118, China Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China c Medical Oncology, First People’s Hospital of Qujing, Qujing 655000, China b
A R T I C L E
I N F O
Article history: Received 25 December 2014 Received in revised form 10 February 2015 Accepted 10 February 2015 Keywords: Colorectal cancer Anoikis CXCL-8 TOPK Prognosis
A B S T R A C T
Anoikis is a form of apoptosis which occurs when anchorage-dependent cells either show loss of adhesion or inappropriate adhesion. Only a few cancer cells that detach from the primary site of the tumor acquire the ability to resist anoikis and form metastasis. The mechanism underlying the resistance of colorectal cancer (CRC) cells to anoikis remains unclear. Interleukin-8 (alternatively known as CXCL8) is associated with CRC angiogenesis and progression. Here, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of 186 patients with CRC. A combination of high CXCL8 and high TOPK expression had the worst prognosis. We showed that CXCL8 expression was negatively correlated with anoikis in CRC cells. CXCL8 treatment enhanced the resistance of CRC cells to apoptosis, which was accompanied by the increase of TOPK, and the activation of AKT and ERK. Moreover, we demonstrated that the inhibition of either ERK or AKT by specific chemical inhibitors attenuated the CXCL8-mediated resistance to anoikis. Treatment with AKT inhibitor abolished the effects of CXCL8 on TOPK expression, which suggests TOPK was downstream of AKT in the process of anoikis. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis, and that the AKT, TOPK and ERK pathway may be a potential therapeutic target for CRC. © 2015 Published by Elsevier Ireland Ltd.
41 Introduction
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Colorectal cancer (CRC) is the most common malignant disease worldwide with an estimated 5-year survival rate ranging from 90% for stage 1 patients to 10% for metastatic cases [1]. Despite its substantial clinical importance, the detailed mechanisms underlying metastasis in CRC remain unclear. Cancer cells must make a long journey before they spread successfully [2], and only a few tumor cells that enter the circulatory system can survive after detaching from the primary tumor site and thus form metastatic lesions. This is because most of them will be eliminated by the cellular apoptotic machinery. This elimination process involves a special type of apoptosis called suspension-induced apoptosis or anoikis [3], which is induced by loss of cell adhesion or inappropriate cell adhesion and acts as a barrier for metastasis. Thus, anoikis resistance is a crucial event in the progression of metastasis. The mechanisms underlying CRC cell resistance to anoikis are still not fully understood.
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* Corresponding author. E-mail address: [email protected] (Z-F. Yin). 1 These authors contributed equally to this work.
Interleukin-8 (IL-8), alternatively known as CXCL8, is a proinflammatory CXC chemokine. Growing evidence demonstrates that CXCL8/CXCR1/2 signaling plays a substantial regulatory role in the tumor microenvironment and is important for tumor progression and metastasis. Since Koch et al. firstly found that monocytes and macrophages can secrete CXCL8 to regulate angiogenesis in the rat cornea [4], the effect of CXCL8 on angiogenesis has been demonstrated in bronchogenic carcinoma [5], prostate cancer [6], ovarian cancer, and colon cancer [7]. Recent studies suggest that CXCL8 is associated with the migration, invasion, and proliferation of human cancers cells [8–10]. However, the role of CXCL8 in CRC proliferation and metastasis has not been thoroughly characterized and whether CXCL8 affects anoikis resistance of CRC cells is currently unknown. Increasing evidence suggests that the abnormal expression of growth factor receptors or components of their signaling pathways plays an important regulatory role in the anoikis of carcinoma cells. Over-expression of the epidermal growth factor (EGF) receptor maintains the activation of ERK in cells in suspension and may block anoikis via the suppression of Bim expression in MCF-10A cells [11]. Hepatocyte growth factor (HGF) inhibits anoikis in HNSCC cells via a mechanism that depends on both ERK and AKT signaling pathways [12]. PI3K/AKT and Ras/Raf/ERK pathways are regarded as the
http://dx.doi.org/10.1016/j.canlet.2015.02.021 0304-3835/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: You-Chuan Xiao, et al., CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.021
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key components to determine whether a cell undergoes anoikis [13]. CXCL8 can activate multiple intracellular signaling pathways, including PI3K/AKT and Raf/MEK/ERK [14]. However, little is known about the role of CXCL8, and the involvement of PI3K/AKT and Raf/ MEK/ERK during anoikis in CRC cells. TLAK cell-originated protein kinase (TOPK) is a recently identified MAPKK-like serine/threonine protein kinase. It is barely detectable in normal tissue except in the testis and thymus, where it is believed to be involved in the maturation of germ cells and the activation of immune cells, respectively. Recently, TOPK has been shown to participate in the proliferation, migration, and invasion of cancer cells [15], and its over-expression promotes tumorigenesis by regulating the activation of ERK [16]. Hu et al. report that high levels of PBK/TOPK may contribute to tumor cell development and progression by suppressing p53 function and consequently limiting apoptotic rate [17]. However, whether TOPK can promote cellular resistance to anoikis, particularly in CRC cells, has never been addressed. In the present study, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of CRC patients. CRC subtype with high CXCL8 and high TOPK expression had the worst prognosis. Noteworthy, CXCL8 can inhibit anoikis of human colorectal carcinoma cells. We then evaluated whether PI3K/AKT, ERK and TOPK are involved in the inhibitory effects of CXCL8 on anoikis. Our data demonstrated that CXCL8 induces anoikis resistance in CRC cells via increasing TOPK levels and activating AKT and ERK signaling. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis. The AKT, ERK and TOPK pathway may be a potential therapeutic target for CRC.
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Ethics statement
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The study was approved by the Institutional Review Board of Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University in China. Patient information was anonymized and all samples were obtained with written informed consent reviewed by the ethical board of the corresponding hospital.
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A total of 186 patients with CRC who were treated at the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University (Kunming, China) between February 2006 and May 2008 were enrolled in this study. The median age of patients at the time of admission was 61 years, range 18–82 years. 81 patients were women, and 105 were men. All patients underwent radical resection of the primary tumor with regional lymph node dissection. All diagnoses of adenocarcinoma were confirmed by histopathology. Only patients with sporadic colorectal cancer were selected for our analysis, and patients with a medical history of hereditary non-polyposis colorectal cancer or familial adenomatous polyposis were excluded. The patient demographics and clinicopathological factors were obtained from the patients’ medical and pathological records, which are summarized in Table 1. Primary tumor stage was defined according to the sixth edition of the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system. Tumor tissues and their matched non-cancerous tissues were collected for real-time PCR and immunohistochemistry analyses. Cell lines and culture conditions
Bioinformatics analysis
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Human CRC cell lines SW480, Caco-2, HT29 and Lovo were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HT29, SW480
TCGA RNA-Seq and corresponding clinical data were downloaded from the TCGA website https://tcga-data.nci.nih.gov/tcga/ following approval of this project by the consortium. Data from 262 CRC and 41 adjacent normal tissues were used for RNASeq analysis. To further investigate the biological pathways involved in ovarian cancer pathogenesis through the CXCL8 pathway, we performed a gene set enrichment analysis (GSEA) by using GSEA version 2.0 from the Broad Institute at MIT. The data in question were analyzed in terms of their differential enrichment in a predefined biological set of genes (representing pathways). In this study, GSEA firstly generated an ordered list of all genes according to their correlation with CXCL8 expression, and then a predefined gene set (signature of gene expression upon perturbation of certain cancer-related gene) receives an enrichment score (ES), which is a measure of statistical evidence rejecting the null hypothesis that its members are randomly distributed in the ordered list. The expression level of CXCL8 was used
Materials and methods
Patients and sample collection
Table 1 Correlation of CXCL8 expressing with TOPK in 186 patients with colorectal carcinoma.
62
TOPK IHC score
63 64 65
70 Anoikis assay
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Anoikis was assessed in exponentially growing cells that were trypsinized and resuspended in either Poly-Hema-coated (Sigma-Aldrich) dishes (suspension culture) or uncoated plates (attached culture) at a concentration of 3 × 105 cells/dish. Cells were grown in these conditions for 24–72 h with or without CXCL8 (R&D Systems), in the presence of growth medium. Poly-HEMA-coated plates were prepared as described previously [18]. Cell apoptosis was assessed with the Annexin V–FITC Apoptosis Detection Kit (Beyotime) and by western blot analysis of cleaved caspase-3 (antibody from Cell Signaling Technology). Flow cytometry was performed with the FACSCalibur (Becton Dickinson), and all experiments were performed in triplicate.
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Western blot analysis
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Total cell lysate (30 μg/lane) was prepared and manipulated according to a previous report [19]. Primary antibodies were as follows: Mouse anti-human CXCL8 was from Abcam, while anti-TOPK was from Epitomics. Mouse anti-human AKT (C67E7), rabbit anti-human p-AKT (Ser473), rabbit anti-human p-ERK1/2 (Thr202/Tyr204), rabbit anti-human ERK1/2 (137F5) and rabbit anti-human Bcl-2 were from Cell Signaling. Immunoreactive proteins were visualized with an enhanced chemiluminescence detection system with exposure of X-ray film. The images were analyzed by Quantity One (Bio-Rad).
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Immunofluorescent staining
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Cells were grown on coverslips in 6-well dishes. After washing twice with phosphate-buffered saline (PBS), cells were fixed with 2% (w/v) paraformaldehyde and permeabilized with 1% (v/v) Triton X-100. Coverslips were blocked with 10% (w/v) normal goat serum in PBS at room temperature for 1 hour and then incubated with anti-CXCL8 (Abcam) and anti-TOPK (Epitomics) at 4 °C overnight. Cells were then washed and incubated with Cy3-labeled and FITC-conjugated secondary antibodies (Beyotime) at room temperature for 1 hour, and were co-stained with DAPI (Sigma-Aldrich) to visualize nuclei. Images were obtained with a Nikon Total Internal Reflection Fluorescence microscope (200× magnification) and NIS Elements AR3.10.
103 Immunohistochemistry
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All specimens had been fixed in buffered formalin and embedded in paraffin. The embedded tissues were cut into 4 μm-thick serial sections. The sections were deparaffinized, dehydrated in xylene graded alcohols, rinsed in PBS and probed with primary antibodies (antibodies against CXCL8 and TOPK were from Abcam and Epitomics, respectively) at 4 °C overnight. Next, the sections were incubated with appropriate biotin-conjugated secondary antibodies at room temperature for 1 hour, and immunoreactivity was detected with the ABC kit and DAB substrate. A reddishbrown precipitate in the cell membrane, cytoplasm or nucleus indicated a positive reaction. The optimal antibody concentration was determined for each assay with a titration experiment. Negative controls were performed by replacing the primary antibody with mouse or rabbit isotype control antibody. Counter-staining of the nucleus was performed with hematoxylin. The intensity of immunoreactivity was graded according to a previous report and was as follows: IRS = staining intensity (SI) × percentage of positive cells (PP). The SI was determined as 0, negative; 1, weak; 2, moderate; 3, strong. A Staining index >3 was considered as indicative of overexpression. The assessment of the staining was conducted by two investigators independently.
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and Lovo were maintained in RPMI 1640 (Gibco) and Caco2 were maintained in DMEM medium (Gibco). All culture media were supplemented with 10% fetal calf serum (Gibco), 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco) and cells were grown at 37 °C in a 5% CO2 incubator.
CXCL8 IHC score
>3 (n=114) ≤3 (n = 72)
r
>3 (n=135)
≤3(n = 51)
93 42
21 30
P
3) (Table 1). Furthermore, a positive correlation was observed between the expression of TOPK and CXCL8 based on the data from TCGA (Fig. 1C), our real-time PCR (Fig. 1F) and IHC analysis (Table 1). These results suggest that co-expression of TOPK and CXCL8 may be useful for the diagnosis of CRC.
Overexpression of CXCL8 and TOPK in colorectal cancer (CRC) To explore the expression of CXCL8 and TOPK in CRC, we compared their expression by bioinformatics analysis with the TCGA_COAD_exp_HiSeqV2 dataset. CXCL8 (Fig. 1A) and TOPK (Fig. 1B) expression was significantly higher in CRC tissues than that in the adjacent tissues of patients. We then detected the mRNA levels of CXCL8 and TOPK in tumor and normal tissues from 60 CRC patients who were admitted to the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University. Our real-time PCR results confirmed that the mRNA levels of CXCL8 and TOPK increased in CRC tissues (Fig. 1D and E). To further confirm their overexpression in CRC, we performed immunohistochemistry (IHC) staining of CXCL8 and TOPK in 186
45 Correlation of CXCL8 and TOPK expression in CRC with clinicopathological factors To evaluate the prognostic value of CXCL8 and TOPK in CRC, we then analyzed for the correlations between clinicopathological variables and the expression of CXL8 or TOPK in CRC tissues (Table 2). Chi-square test showed that the corexpression of CXCL8 and TOPK was significantly correlated with tumor grade, lymph node metastasis, and liver metastasis, while there was no significant correlation between the coexpression of these two proteins and sex, age and tumor size (Table 2). These data indicate CXCL8 and TOPK may be associated with CRC progression.
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Fig. 1. Overexpression of CXCL8 and TOPK in CRC tissues. CXCL8 and TOPK mRNA expression analysis by using data downloaded from TCGA_COAD_exp_HiSeqV2 (A–C) and by Real-time PCR with tumor tissues and matched non-cancerous tissues from 60 patients who were admitted to the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University (D, E). The two-tailed Student’s t-test was used to evaluate statistical differences between two groups. *** P value < 0.001.
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Table 2 Relationship between the expression of CXCL8 and TOPK and clinicopathological factors in 186 colorectal cancer patients. Parameters
N
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
TOPK IHC score ≤3 (n = 51)
4 Sex Male Female Years ≤65 >65 Location of tumor Left sided Right sided Rectum Tumor Size (cm) ≥5 3 (n = 114)
105 81
32 19
73 52
0.620
40 32
65 49
0.880
85 101
19 32
66 69
0.188
38 34
47 67
0.133
63 59 64
17 15 19
46 44 45
27 14 31
36 45 33
0.035
79 107
18 33
61 74
37 35
42 72
0.067
108 78
39 12
69 66
0.003
40 32
68 46
0.157
149 37
47 4
102 33
0.013
66 6
83 31
0.002
41 145
13 38
28 109
0.551
28 44
13 101
0.000
106 80
37 13
69 67
0.005
51 21
55 59
0.003
158 28
48 3
110 25
0.038
67 5
91 23
0.019
161 25
49 2
112 23
0.017
58 14
103 11
0.077
0.625 0.865
0.248
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CRC subtypes that express both CXCL8 and TOPK have a poor prognosis We carried out Kaplan–Meier survival analysis of CRC cases to investigate the clinical outcome of each CRC subtype identified based on the expression levels of CXCL8 and TOPK. CXCL8 or TOPK expression was associated with poor overall survival (for CXCL8, logrank = 11.302, P = 0.001; and for TOPK, log-rank = 10.620, P = 0.001) and poor disease-free survival (for CXCL8, log-rank = 9.6, P = 0.02; and for TOPK, log-rank = 10.502, P = 0.001) (Fig. 2). CRC subtypes expressed with both proteins had significantly shorter overall survival and disease-free survival than CRC subtypes with CXCL8 or TOPK expression alone. Multivariate Cox regression analysis demonstrated that the expression of CXCL8 and TOPK affected the overall survival of patients with CRC. CXCL8 and TOPK expressions were independent risk factors (P < 0.05); CXCL8 was associated with a relative risk of 1.386 (CI 95%: 0.328–2.267, P < 0.001), TOPK was associated with a relative risk of 2.547 (CI 95%: 1.583–2.975, P = 0.017), and co-expression of CXCL8 and TOPK was associated with a relative risk of 4.393 (CI 95%: 1.524– 7.058, P < 0.001). The expression of CXCL8 and TOPK affected tumorfree survival in patients with CRC: CXCL8 was associated with a relative risk of 1.147 (CI 95%: 0.929–3.756, P < 0.002), TOPK was associated with a relative risk of 2.57 (CI 95%:1.46–5.81, P < 0.043) and CXCL8 and TOPK co-expression was associated with a relative risk of 3.871 (CI 95%:1.742–6.558, P < 0.018) (Table 3). These data thus indicate that CRC subtypes that express both CXCL8 and TOPK have a poor prognosis. CXCL8 is associated with the sensitivity of CRC cells to anoikis We next investigate the role of CXCL8 on cell apoptosis. The abundance of CXCL8 and TOPK protein was firstly determined by
immunofluorescent staining (Fig. 3A) and western blotting (Fig. 3B) in three CRC cell lines, SW480, Lovo, and Caco2 cells. The protein levels of CXCL8 and TOPK were highest in Caco2 cells and lowest in Lovo cells. The relative mRNA level of CXCL8 in these three CRC cells was consistent with its protein level (Fig. 3C). How CXCL8 plays a role in CRC progression remained unclear. To probe the CXCL8-associated pathways on an unbiased basis, we performed Gene Set Enrichment Analysis (GSEA) using data from the TCGA. GSEA is designed to detect coordinated differences in expression of predefined sets of functionally related genes. Among all the 188 predefined ‘KEGG pathways’ gene sets, the apoptosis and MAPK signaling pathway was identified to be closely related with CXCL8 expression in CRC patients (Fig. 4A), which indicated CXCL8 may play a role in CRC progression through regulating MAPK signaling and apoptosis. We then assessed the apoptosis of CRC cells under attachment and detachment conditions. There was no significant difference in the rate of apoptosis among the three cell lines when they were cultured under attachment conditions (Fig. 4C). Cell–cell adhesion was lost in suspension-cultured cells (Fig. 4B), which resulted in a high apoptotic rate of CRC cells (Fig. 4E). Additionally, the apoptotic rate of CRC cells increased in a time-dependent manner under detachment conditions (Fig. 4E). Taken together, these data indicate that CRC cells undergo apoptosis when cultured in suspension. CXCL8 suppresses anoikis in CRC cells The apoptotic rate and survival of CRC cells were examined after treatment with different concentrations of rhCXCL8 for 48 h. CXCL8 treatment significantly reduced the anoikis of SW480 and Caco2 cells (Fig. 5A). In addition, the apoptotic ratio decreased with the increasing concentration of CXCL8, and reached the lowest level at 50 ng/ml (Fig. 5A). This dose was used for the following assays.
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Fig. 2. Cumulative survival rates of the 186 enrolled patients with colorectal cancer (CRC) who underwent curative resection as assessed by the Kaplan–Meier method. The differences in survival rates were analyzed by the log-rank test. Patients were divided into groups according to the abundance of CXCL8 and TOPK in samples of their tumor. (A) The overall and disease-free survival in CRC patients in the ‘high’ CXCL8 group was significantly lower than that of patients in the ‘low’ CXCL8 group; (B) the overall survival of CRC patients in the ‘high’ TOPK group was lower than that of patients in the ‘low’ TOPK group. (C) The overall survival of CRC patients in the ‘high’ CXCL8, ‘high’ TOPK group was lower than that of any other group. (D) Survival analysis of patients from GSE33113 dataset.
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Table 3 Multivariate Cox proportional hazards analysis for the candidate variables. Variables
4 5 6 7 8 9 10 11 12 13
Depth of invasion (T3,4 vs. T1,2) Tumor size (5 cm vs. ≤5 cm) Tumor differentiation (G1,2 vs. G3) CEA (ng/ml) (5 vs. ≤5) Lymphatic metastases (yes vs. no) Liver metastases (yes vs. no) CXCL8( IHC score >3 vs. ≤3) TOPK( IHC score >3 vs. ≤3) CXCL8 and TOPK IHC score3 vs. others
Overall survival
Disease-free survival
HR
95% CI
P
HR
95% CI
P
1.190 0.053 0.726 1.382 1.212 2.308 1.386 2.547 4.393
0.564–2.508 0.179–1.010 0.338–1.562 0.681–2.807 0.480–3.064 1.520–3.289 0.328–2.267 1.583–2.975 1.524–7.058
0.007 0.081 0.414 0.139 0.006 0.001 0.000 0.017 0.041
1.19 0.425 0.56 1.382 1.308 1.272 1.147 1.470 3.871
0.564–2.508 0.179–1.010 0.260–1.207 0.681–2.807 0.520–3.289 0.159–2.466 0.929–3.756 1.212–3.043 1.742–7.058
0.048 0.053 0.013 0.037 0.569 0.004 0.021 0.043 0.018
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Western blot analysis showed that the expression of caspase-3 was lower in cells transiently treated with CXCL8 than that in controltreated cells (Fig. 5B). Moreover, the transient exposure of cells to CXCL8 was also associated with a high abundance of Bcl-2, which
is an important apoptosis suppressor factor (Fig. 5B). CXCL8 treatment did not affect apoptosis when cells were grown in attachment conditions (Fig. 5C). We also determined whether CXCL8 suppressed anoikis in other CRC cell lines. CXCL8 stimulation also
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Fig. 3. The expression level of CXCL8 in various colorectal cancer cell lines. (A) Immunocytochemistry of CXCL8 in SW480, Lovo, and Caco2. (B) Western blot of CXCL8 and TOPK in colorectal cancer cell lines. GAPDH was used as a loading control. (C) The relative abundance of CXCL8 mRNA was determined by real-time PCR in the three colorectal cancer cell lines. The abundance of CXCL8 mRNA was normalized to that of a GAPDH control. Experiments were repeated at least three times.
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Fig. 4. Apoptosis of various colorectal cancer cell lines grown under attachment (anchorage) or detachment (nonanchorage) conditions. (A) GSEA was performed using TCGA dataset. Cell apoptosis and MAPK signaling pathway were identified with the strongest association with CXCL-8-higher expression. (B) Morphology and growth of colorectal cancer cell lines under attachment and detachment conditions. Cells (350,000) were seeded on 6 well plates pre-coated with poly-HEMA (detachment conditions) or were seeded on 6 well plates not coated with poly-HEMA (attachment conditions). (C–E) Annexin V/PI staining was performed to assess cell apoptosis. Colorectal cancer cells were grown in (C, D) attachment conditions, or (E) detachment conditions at 24, 48, 72 h following plating. Data are expressed as the mean of each group with standard error bars. The results were statistically analyzed with a t test (*P < 0 .05).
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suppressed anoikis in HT29, HCT116, and Lovo cells (Fig. 5C). Taken together, our results demonstrate that CXCL8 provides protection against apoptosis when cells are in suspension, but does not affect apoptosis when cells are grown in attachment conditions. PI3K/AKT, TOPK and ERK are involved in the resistance of CRC cells to anoikis mediated by CXCL8 Although the total protein of AKT and ERK was unaffected by CXCL8, the phosphorylation of AKT and ERK was significantly increased by CXCL8 stimulation (Fig. 6A), suggesting that resistance to anoikis mediated by CXCL8 involves PI3K/AKT and ERK signaling. In order to further confirm the involvement of AKT and ERK signaling, cells were pre-treated with specific inhibitors to AKT (MK2206) and ERK (U0126) and then stimulated with 50 ng/ml of CXCL8. The apoptosis of SW480 cells and Caco2 in suspension culture treated with 1 μM MK2206 or 40 μM U0126 was then evaluated. MK2206 and U0126 significantly inhibited the activation of AKT and ERK in CRC cells, respectively (Fig. 6D). Interestingly, Annexin V/PI staining showed that the
apoptotic ratio of SW480 cells treated with both CXCL8 and MK2206 was comparable with that of control cells (Fig. 6B), suggesting that resistance to anoikis mediated by CXCL8 was reversed by MK2206. Consistent with the apoptotic ratio, an increase of cleaved Caspase-3 and a decrease of Bcl-2 protein were observed in cells treated with both CXCL8 and MK2206, compared with that in cells treated with CXCL8 alone (Fig. 6D). Resistance to anoikis mediated by CXCL8 was also can be reversed by U0126. These results further demonstrated that CXCL8 inhibited anoikis through PI3K/AKT and ERK signaling. Additionally, CXCL8 treatment also upregulated the expression of TOPK (Fig. 6A and D), which was significantly inhibited by the exposure of SW480 cells to MK2206. These data indicate that TOPK may be the downstream of PI3K/AKT signaling during the resistance of CRC cells to anoikis mediated by CXCL8. Discussion The properties of cancer cell invasion and migration depend on the interactions between tumor- and host-derived cells in the
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Fig. 5. CXCL8 mediates resistance to apoptosis in colorectal cancer cells and promotes the accumulation of the antiapoptotic protein Bcl2. (A) Percentage of apoptotic cells in cultures grown in the presence of various concentrations of CXCL8. Cells were grown for 48 h in the presence of the indicated concentration of CXCL8, or an equivalent volume of PBS (control). Annexin V/PI staining was used to identify apoptotic cells, and then cells were analyzed by flow cytometry. (B) Colorectal cancer cells were seeded on poly-HEMA-coated 6 well plates for 48 h with or without 50 ng/ml of CXCL8. Total cell lysate was analyzed by western blotting. GAPDH was used as a loading control. (C) Percentage of apoptotic cells in cultures grown in attachment conditions in the presence of CXCL8. Cells were grown for 48 h in the presence of CXCL8 (50 ng/ml), or an equivalent volume of PBS (Control). (D) Another 3 CRC cell lines HCT116, HT29 and Lovo were also used to detect the effect of CXCL8 (50 ng/ml).
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microenvironment during the process of metastasis [20]. In patients with solid tumors, thousands of cancer cells break away from the primary tumor into the circulation every day, but only a handful of cells can resist apoptosis and survive [21]. Normal epithelial cells undergo apoptosis after losing contact with neighboring cells or the extracellular matrix (ECM), which is termed anoikis, a particular type of apoptosis due to loss of cell adhesion or inappropriate cell adhesion [22]. Metastasis to vital organs is the major cause of mortality among colon cancer patients [23]. Chemokines, mediators of leukocyte trafficking and homing, are important components of the tumor microenvironment and have been shown to participate in tumor progression and metastasis [24,25]. CXCL8, belonging to the CXC subfamily of chemokines, is overexpressed in many types of human tumor, including CRC [26]. In the present study, we investigated the interplay between CXCL8 and TOPK in a clinical setting. An overexpression of CXCL8 and TOPK was observed in CRC tissues by bioinformatics analysis, real-time PCR (Fig. 1) and IHC analysis (Table 1). The expression of TOPK was positively correlated with that of CXCL8 (Fig. 1 and Table 1). Moreover, the expression of CXCL8 and
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TOPK was associated with tumor cell differentiation, tumor invasion depth, lymph node metastasis, and liver metastasis. These data suggest the critical role of CXCL8 and TOPK in the promotion of CRC metastasis. In addition, the overexpression of CXCL8 or TOPK indicated poor prognosis (Fig. 2). The CRC subtype overexpressed with TOPK and CXCL8 had significantly shorter overall survival and disease-free survival than CRC subtypes with CXCL8 or TOPK expression alone, which implies that coexpression of CXCL8 and TOPK may be a marker of poor prognosis in CRC. Moreover, the expression of CXCL8 is associated with the aggressiveness of human colon carcinoma cells [26]. It is reported that CXCL8 can regulate a variety of tumor cell functions, including proliferation, migration, angiogenesis, and chemosensitivity in colon cancer models [10]. However, the precise action of CXCL8 in CRC metastasis is unclear. Detachment of cancer cells from primary tumors is a prerequisite for metastasis. In the present study, we cultured three CRC cell lines under detachment conditions and evaluated the apoptosis. We showed that cell apoptosis stimulated by the loss of adhesion was reverse correlated with CXCL8 and TOPK
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Fig. 6. Inhibition of AKT or ERK suppresses anoikis resistance in CRC cells treated with CXCL8. (A) CXCL8 induces the activation of ERK and AKT in SW480 and Caco2 cells. SW480 and Caco2 cells were serum-starved for one night, and then trypsinized and suspended in the presence of CXCL8 (50 ng/ml) for another 48 h. The phosphorylation of AKT and ERK was detected by western blotting. (B, C) Percentage of apoptotic cells was determined by the Annexin V/PI staining assay under various conditions. SW480 and Caco2 cells grown in detachment conditions were pretreated with (B) MK2206 or (C) U0126 inhibitor for 1 h, and then treated with CXCL8 (50 ng/ml) for 48 h. Values are means ± S.D. (n = 4); *p < 0.05 vs. the control cells. (D)The levels of p-AKT, AKT, p-ERK, ERK, Bcl-2, Caspase-3, and TOPK in SW480 cells were detected by Western blotting. GAPDH was used as a loading control.
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1 expression. Interestingly, there was no significant difference in the 2 rate of apoptosis among the three cell lines cultured in attach3 ment conditions (Fig. 4). These results indicate CXCL8 signaling is important for the sensitivity of CRCs cells to anoikis. 4 The anoikis was significantly lower in CRC cells treated with CXCL8 5 than in CRC cells without treatment (Fig. 5), which further suggests that 6 the expression of CXCL8 in CRC cells may play an important role in 7 tumor growth and metastasis. Extensively studies have reported that 8 the mitochondrial pathway plays a key role in cell apoptosis and cleaved 9 Caspase-3 is a marker of apoptosis mediated by the mitochondrial 10 pathway [27–29]. Bcl-2 is a well-known regulator to suppress cell apop11 tosis [30]. In the present study, Bcl-2 expression was significantly 12 increased and Caspase-3 was notably decreased in CRC cells treated with 13 CXCL8 (Fig. 5), which suggests that CXCL8 inhibits anoikis of CRC cells 14 through regulating the mitochondrial apoptotic pathway. 15 Many recent studies have investigated the molecular mecha16 nisms underlying the resistance of tumor cells to anoikis. The PI3K/ 17 AKT pathway is activated by a variety of chemokines, and 18 consequently stimulates cell migration and invasion in various 19 human cancers [12,31–33]. AKT is believed to play a key role on the 20 resistance to anoikis in many types of cancer cell. The importance 21 of ERK signaling on cell apoptosis has been demonstrated in several 22 cell types [34,35]. CXCL8 has been reportedly correlated with PI3K/ 23 AKT and Raf/MEK/ERK signaling [36]. Our data showed that CXCL8 24 treatment resulted in a significant activation of AKT and ERK (Fig. 6). 25 We then inhibited AKT and ERK signaling by using inhibitors MK2206 26 and U0126, respectively, and found that these two inhibitors notably 27 reverse the inhibitory effects of CXCL8 treatment on cell anoikis 28 (Fig. 6). Our data suggest that CXCL8 exerts its anti-anoikis role in 29 CRC cells through activating AKT and ERK signaling. 30 TOPK has recently been shown to regulate the proliferation, migra31 tion, and invasion of cancer cells [15], and its over-expression promotes 32 tumorigenesis by regulating ERK activation [16]. CXCL8 was shown to 33 increase the expression of TOPK (Fig. 6), while CXCL8 did not stimu34 late the expression of TOPK when AKT was inhibited by MK2206, which 35 indicates the regulation of CXCL8 on TOPK is through AKT signaling. 36 In conclusion, CXCL8 is highly involved in anoikis resistance of 37 CRCs. We further show that the resistance to anoikis mediated by 38 CXCL8 is through AKT, TOPK and ERK pathways. Histopathological 39 analysis of clinical samples suggests a correlation between the ex40 pression of CXCL8 and the clinical outcome of CRC cases. However, 41 whether CXCL8 is a novel target for therapies designed to prevent 42 CRC metastasis requires further investigation. The signaling via CXCL8 43 is an important regulator for the sensitivity of CRC cells to apop44 tosis, specifically under detachment conditions. 45 46 Acknowledgments 47 48 This work was supported by grants from the National Natural 49Q4 Q5 Science Foundation of China. The authors apologize for the inabil50 ity to cite all related and important literature due to space limitations. 51 We thank the Department of Molecular Oncology (Eastern 52 Hepatobiliary Surgical Hospital Institute, The Second Military Medical 53 University, Shanghai, China) and Center Library (Changhai Hospi54 tal, The Second Military Medical University, Shanghai, China) for 55 experimental technical help. We also thank professor Su-Changqing 56 for helpful comments regarding of the experiment coordination. 57 58 Conflict of interest 59 60 The authors disclose no potential conflicts of interest. 61 62 Appendix: Supplementary material 63 64 Supplementary data to this article can be found online at 65 doi:10.1016/j.canlet.2015.02.021. 66
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Contents lists available at ScienceDirect
Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
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CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis You-Chuan Xiao a,b,c,1, Zhi-Bin Yang a,1, Xian-Shuo Cheng a,b,1, Xing-Bao Fang b,c, Tao Shen a, Q2 Cui-Feng Xia a, Ping Liu a, Hai-Hua Qian b, Bin Sun b, Zheng-Feng Yin b,*, Yun-Feng Li a a
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Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University, Kunming 650118, China Molecular Oncology Laboratory, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China c Medical Oncology, First People’s Hospital of Qujing, Qujing 655000, China b
A R T I C L E
I N F O
Article history: Received 25 December 2014 Received in revised form 10 February 2015 Accepted 10 February 2015 Keywords: Colorectal cancer Anoikis CXCL-8 TOPK Prognosis
A B S T R A C T
Anoikis is a form of apoptosis which occurs when anchorage-dependent cells either show loss of adhesion or inappropriate adhesion. Only a few cancer cells that detach from the primary site of the tumor acquire the ability to resist anoikis and form metastasis. The mechanism underlying the resistance of colorectal cancer (CRC) cells to anoikis remains unclear. Interleukin-8 (alternatively known as CXCL8) is associated with CRC angiogenesis and progression. Here, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of 186 patients with CRC. A combination of high CXCL8 and high TOPK expression had the worst prognosis. We showed that CXCL8 expression was negatively correlated with anoikis in CRC cells. CXCL8 treatment enhanced the resistance of CRC cells to apoptosis, which was accompanied by the increase of TOPK, and the activation of AKT and ERK. Moreover, we demonstrated that the inhibition of either ERK or AKT by specific chemical inhibitors attenuated the CXCL8-mediated resistance to anoikis. Treatment with AKT inhibitor abolished the effects of CXCL8 on TOPK expression, which suggests TOPK was downstream of AKT in the process of anoikis. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis, and that the AKT, TOPK and ERK pathway may be a potential therapeutic target for CRC. © 2015 Published by Elsevier Ireland Ltd.
41 Introduction
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Colorectal cancer (CRC) is the most common malignant disease worldwide with an estimated 5-year survival rate ranging from 90% for stage 1 patients to 10% for metastatic cases [1]. Despite its substantial clinical importance, the detailed mechanisms underlying metastasis in CRC remain unclear. Cancer cells must make a long journey before they spread successfully [2], and only a few tumor cells that enter the circulatory system can survive after detaching from the primary tumor site and thus form metastatic lesions. This is because most of them will be eliminated by the cellular apoptotic machinery. This elimination process involves a special type of apoptosis called suspension-induced apoptosis or anoikis [3], which is induced by loss of cell adhesion or inappropriate cell adhesion and acts as a barrier for metastasis. Thus, anoikis resistance is a crucial event in the progression of metastasis. The mechanisms underlying CRC cell resistance to anoikis are still not fully understood.
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* Corresponding author. E-mail address: [email protected] (Z-F. Yin). 1 These authors contributed equally to this work.
Interleukin-8 (IL-8), alternatively known as CXCL8, is a proinflammatory CXC chemokine. Growing evidence demonstrates that CXCL8/CXCR1/2 signaling plays a substantial regulatory role in the tumor microenvironment and is important for tumor progression and metastasis. Since Koch et al. firstly found that monocytes and macrophages can secrete CXCL8 to regulate angiogenesis in the rat cornea [4], the effect of CXCL8 on angiogenesis has been demonstrated in bronchogenic carcinoma [5], prostate cancer [6], ovarian cancer, and colon cancer [7]. Recent studies suggest that CXCL8 is associated with the migration, invasion, and proliferation of human cancers cells [8–10]. However, the role of CXCL8 in CRC proliferation and metastasis has not been thoroughly characterized and whether CXCL8 affects anoikis resistance of CRC cells is currently unknown. Increasing evidence suggests that the abnormal expression of growth factor receptors or components of their signaling pathways plays an important regulatory role in the anoikis of carcinoma cells. Over-expression of the epidermal growth factor (EGF) receptor maintains the activation of ERK in cells in suspension and may block anoikis via the suppression of Bim expression in MCF-10A cells [11]. Hepatocyte growth factor (HGF) inhibits anoikis in HNSCC cells via a mechanism that depends on both ERK and AKT signaling pathways [12]. PI3K/AKT and Ras/Raf/ERK pathways are regarded as the
http://dx.doi.org/10.1016/j.canlet.2015.02.021 0304-3835/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: You-Chuan Xiao, et al., CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.021
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key components to determine whether a cell undergoes anoikis [13]. CXCL8 can activate multiple intracellular signaling pathways, including PI3K/AKT and Raf/MEK/ERK [14]. However, little is known about the role of CXCL8, and the involvement of PI3K/AKT and Raf/ MEK/ERK during anoikis in CRC cells. TLAK cell-originated protein kinase (TOPK) is a recently identified MAPKK-like serine/threonine protein kinase. It is barely detectable in normal tissue except in the testis and thymus, where it is believed to be involved in the maturation of germ cells and the activation of immune cells, respectively. Recently, TOPK has been shown to participate in the proliferation, migration, and invasion of cancer cells [15], and its over-expression promotes tumorigenesis by regulating the activation of ERK [16]. Hu et al. report that high levels of PBK/TOPK may contribute to tumor cell development and progression by suppressing p53 function and consequently limiting apoptotic rate [17]. However, whether TOPK can promote cellular resistance to anoikis, particularly in CRC cells, has never been addressed. In the present study, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of CRC patients. CRC subtype with high CXCL8 and high TOPK expression had the worst prognosis. Noteworthy, CXCL8 can inhibit anoikis of human colorectal carcinoma cells. We then evaluated whether PI3K/AKT, ERK and TOPK are involved in the inhibitory effects of CXCL8 on anoikis. Our data demonstrated that CXCL8 induces anoikis resistance in CRC cells via increasing TOPK levels and activating AKT and ERK signaling. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis. The AKT, ERK and TOPK pathway may be a potential therapeutic target for CRC.
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Ethics statement
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The study was approved by the Institutional Review Board of Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University in China. Patient information was anonymized and all samples were obtained with written informed consent reviewed by the ethical board of the corresponding hospital.
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A total of 186 patients with CRC who were treated at the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University (Kunming, China) between February 2006 and May 2008 were enrolled in this study. The median age of patients at the time of admission was 61 years, range 18–82 years. 81 patients were women, and 105 were men. All patients underwent radical resection of the primary tumor with regional lymph node dissection. All diagnoses of adenocarcinoma were confirmed by histopathology. Only patients with sporadic colorectal cancer were selected for our analysis, and patients with a medical history of hereditary non-polyposis colorectal cancer or familial adenomatous polyposis were excluded. The patient demographics and clinicopathological factors were obtained from the patients’ medical and pathological records, which are summarized in Table 1. Primary tumor stage was defined according to the sixth edition of the American Joint Committee on Cancer (AJCC) tumor-node-metastasis (TNM) staging system. Tumor tissues and their matched non-cancerous tissues were collected for real-time PCR and immunohistochemistry analyses. Cell lines and culture conditions
Bioinformatics analysis
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Human CRC cell lines SW480, Caco-2, HT29 and Lovo were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HT29, SW480
TCGA RNA-Seq and corresponding clinical data were downloaded from the TCGA website https://tcga-data.nci.nih.gov/tcga/ following approval of this project by the consortium. Data from 262 CRC and 41 adjacent normal tissues were used for RNASeq analysis. To further investigate the biological pathways involved in ovarian cancer pathogenesis through the CXCL8 pathway, we performed a gene set enrichment analysis (GSEA) by using GSEA version 2.0 from the Broad Institute at MIT. The data in question were analyzed in terms of their differential enrichment in a predefined biological set of genes (representing pathways). In this study, GSEA firstly generated an ordered list of all genes according to their correlation with CXCL8 expression, and then a predefined gene set (signature of gene expression upon perturbation of certain cancer-related gene) receives an enrichment score (ES), which is a measure of statistical evidence rejecting the null hypothesis that its members are randomly distributed in the ordered list. The expression level of CXCL8 was used
Materials and methods
Patients and sample collection
Table 1 Correlation of CXCL8 expressing with TOPK in 186 patients with colorectal carcinoma.
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TOPK IHC score
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70 Anoikis assay
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Anoikis was assessed in exponentially growing cells that were trypsinized and resuspended in either Poly-Hema-coated (Sigma-Aldrich) dishes (suspension culture) or uncoated plates (attached culture) at a concentration of 3 × 105 cells/dish. Cells were grown in these conditions for 24–72 h with or without CXCL8 (R&D Systems), in the presence of growth medium. Poly-HEMA-coated plates were prepared as described previously [18]. Cell apoptosis was assessed with the Annexin V–FITC Apoptosis Detection Kit (Beyotime) and by western blot analysis of cleaved caspase-3 (antibody from Cell Signaling Technology). Flow cytometry was performed with the FACSCalibur (Becton Dickinson), and all experiments were performed in triplicate.
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Western blot analysis
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Total cell lysate (30 μg/lane) was prepared and manipulated according to a previous report [19]. Primary antibodies were as follows: Mouse anti-human CXCL8 was from Abcam, while anti-TOPK was from Epitomics. Mouse anti-human AKT (C67E7), rabbit anti-human p-AKT (Ser473), rabbit anti-human p-ERK1/2 (Thr202/Tyr204), rabbit anti-human ERK1/2 (137F5) and rabbit anti-human Bcl-2 were from Cell Signaling. Immunoreactive proteins were visualized with an enhanced chemiluminescence detection system with exposure of X-ray film. The images were analyzed by Quantity One (Bio-Rad).
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Immunofluorescent staining
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Cells were grown on coverslips in 6-well dishes. After washing twice with phosphate-buffered saline (PBS), cells were fixed with 2% (w/v) paraformaldehyde and permeabilized with 1% (v/v) Triton X-100. Coverslips were blocked with 10% (w/v) normal goat serum in PBS at room temperature for 1 hour and then incubated with anti-CXCL8 (Abcam) and anti-TOPK (Epitomics) at 4 °C overnight. Cells were then washed and incubated with Cy3-labeled and FITC-conjugated secondary antibodies (Beyotime) at room temperature for 1 hour, and were co-stained with DAPI (Sigma-Aldrich) to visualize nuclei. Images were obtained with a Nikon Total Internal Reflection Fluorescence microscope (200× magnification) and NIS Elements AR3.10.
103 Immunohistochemistry
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All specimens had been fixed in buffered formalin and embedded in paraffin. The embedded tissues were cut into 4 μm-thick serial sections. The sections were deparaffinized, dehydrated in xylene graded alcohols, rinsed in PBS and probed with primary antibodies (antibodies against CXCL8 and TOPK were from Abcam and Epitomics, respectively) at 4 °C overnight. Next, the sections were incubated with appropriate biotin-conjugated secondary antibodies at room temperature for 1 hour, and immunoreactivity was detected with the ABC kit and DAB substrate. A reddishbrown precipitate in the cell membrane, cytoplasm or nucleus indicated a positive reaction. The optimal antibody concentration was determined for each assay with a titration experiment. Negative controls were performed by replacing the primary antibody with mouse or rabbit isotype control antibody. Counter-staining of the nucleus was performed with hematoxylin. The intensity of immunoreactivity was graded according to a previous report and was as follows: IRS = staining intensity (SI) × percentage of positive cells (PP). The SI was determined as 0, negative; 1, weak; 2, moderate; 3, strong. A Staining index >3 was considered as indicative of overexpression. The assessment of the staining was conducted by two investigators independently.
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and Lovo were maintained in RPMI 1640 (Gibco) and Caco2 were maintained in DMEM medium (Gibco). All culture media were supplemented with 10% fetal calf serum (Gibco), 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco) and cells were grown at 37 °C in a 5% CO2 incubator.
CXCL8 IHC score
>3 (n=114) ≤3 (n = 72)
r
>3 (n=135)
≤3(n = 51)
93 42
21 30
P
3) (Table 1). Furthermore, a positive correlation was observed between the expression of TOPK and CXCL8 based on the data from TCGA (Fig. 1C), our real-time PCR (Fig. 1F) and IHC analysis (Table 1). These results suggest that co-expression of TOPK and CXCL8 may be useful for the diagnosis of CRC.
Overexpression of CXCL8 and TOPK in colorectal cancer (CRC) To explore the expression of CXCL8 and TOPK in CRC, we compared their expression by bioinformatics analysis with the TCGA_COAD_exp_HiSeqV2 dataset. CXCL8 (Fig. 1A) and TOPK (Fig. 1B) expression was significantly higher in CRC tissues than that in the adjacent tissues of patients. We then detected the mRNA levels of CXCL8 and TOPK in tumor and normal tissues from 60 CRC patients who were admitted to the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University. Our real-time PCR results confirmed that the mRNA levels of CXCL8 and TOPK increased in CRC tissues (Fig. 1D and E). To further confirm their overexpression in CRC, we performed immunohistochemistry (IHC) staining of CXCL8 and TOPK in 186
45 Correlation of CXCL8 and TOPK expression in CRC with clinicopathological factors To evaluate the prognostic value of CXCL8 and TOPK in CRC, we then analyzed for the correlations between clinicopathological variables and the expression of CXL8 or TOPK in CRC tissues (Table 2). Chi-square test showed that the corexpression of CXCL8 and TOPK was significantly correlated with tumor grade, lymph node metastasis, and liver metastasis, while there was no significant correlation between the coexpression of these two proteins and sex, age and tumor size (Table 2). These data indicate CXCL8 and TOPK may be associated with CRC progression.
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Q6
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Fig. 1. Overexpression of CXCL8 and TOPK in CRC tissues. CXCL8 and TOPK mRNA expression analysis by using data downloaded from TCGA_COAD_exp_HiSeqV2 (A–C) and by Real-time PCR with tumor tissues and matched non-cancerous tissues from 60 patients who were admitted to the Colorectal Cancer Clinical Research Center, Third Affiliated Hospital, Kunming Medical University (D, E). The two-tailed Student’s t-test was used to evaluate statistical differences between two groups. *** P value < 0.001.
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Table 2 Relationship between the expression of CXCL8 and TOPK and clinicopathological factors in 186 colorectal cancer patients. Parameters
N
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
TOPK IHC score ≤3 (n = 51)
4 Sex Male Female Years ≤65 >65 Location of tumor Left sided Right sided Rectum Tumor Size (cm) ≥5 3 (n = 114)
105 81
32 19
73 52
0.620
40 32
65 49
0.880
85 101
19 32
66 69
0.188
38 34
47 67
0.133
63 59 64
17 15 19
46 44 45
27 14 31
36 45 33
0.035
79 107
18 33
61 74
37 35
42 72
0.067
108 78
39 12
69 66
0.003
40 32
68 46
0.157
149 37
47 4
102 33
0.013
66 6
83 31
0.002
41 145
13 38
28 109
0.551
28 44
13 101
0.000
106 80
37 13
69 67
0.005
51 21
55 59
0.003
158 28
48 3
110 25
0.038
67 5
91 23
0.019
161 25
49 2
112 23
0.017
58 14
103 11
0.077
0.625 0.865
0.248
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
CRC subtypes that express both CXCL8 and TOPK have a poor prognosis We carried out Kaplan–Meier survival analysis of CRC cases to investigate the clinical outcome of each CRC subtype identified based on the expression levels of CXCL8 and TOPK. CXCL8 or TOPK expression was associated with poor overall survival (for CXCL8, logrank = 11.302, P = 0.001; and for TOPK, log-rank = 10.620, P = 0.001) and poor disease-free survival (for CXCL8, log-rank = 9.6, P = 0.02; and for TOPK, log-rank = 10.502, P = 0.001) (Fig. 2). CRC subtypes expressed with both proteins had significantly shorter overall survival and disease-free survival than CRC subtypes with CXCL8 or TOPK expression alone. Multivariate Cox regression analysis demonstrated that the expression of CXCL8 and TOPK affected the overall survival of patients with CRC. CXCL8 and TOPK expressions were independent risk factors (P < 0.05); CXCL8 was associated with a relative risk of 1.386 (CI 95%: 0.328–2.267, P < 0.001), TOPK was associated with a relative risk of 2.547 (CI 95%: 1.583–2.975, P = 0.017), and co-expression of CXCL8 and TOPK was associated with a relative risk of 4.393 (CI 95%: 1.524– 7.058, P < 0.001). The expression of CXCL8 and TOPK affected tumorfree survival in patients with CRC: CXCL8 was associated with a relative risk of 1.147 (CI 95%: 0.929–3.756, P < 0.002), TOPK was associated with a relative risk of 2.57 (CI 95%:1.46–5.81, P < 0.043) and CXCL8 and TOPK co-expression was associated with a relative risk of 3.871 (CI 95%:1.742–6.558, P < 0.018) (Table 3). These data thus indicate that CRC subtypes that express both CXCL8 and TOPK have a poor prognosis. CXCL8 is associated with the sensitivity of CRC cells to anoikis We next investigate the role of CXCL8 on cell apoptosis. The abundance of CXCL8 and TOPK protein was firstly determined by
immunofluorescent staining (Fig. 3A) and western blotting (Fig. 3B) in three CRC cell lines, SW480, Lovo, and Caco2 cells. The protein levels of CXCL8 and TOPK were highest in Caco2 cells and lowest in Lovo cells. The relative mRNA level of CXCL8 in these three CRC cells was consistent with its protein level (Fig. 3C). How CXCL8 plays a role in CRC progression remained unclear. To probe the CXCL8-associated pathways on an unbiased basis, we performed Gene Set Enrichment Analysis (GSEA) using data from the TCGA. GSEA is designed to detect coordinated differences in expression of predefined sets of functionally related genes. Among all the 188 predefined ‘KEGG pathways’ gene sets, the apoptosis and MAPK signaling pathway was identified to be closely related with CXCL8 expression in CRC patients (Fig. 4A), which indicated CXCL8 may play a role in CRC progression through regulating MAPK signaling and apoptosis. We then assessed the apoptosis of CRC cells under attachment and detachment conditions. There was no significant difference in the rate of apoptosis among the three cell lines when they were cultured under attachment conditions (Fig. 4C). Cell–cell adhesion was lost in suspension-cultured cells (Fig. 4B), which resulted in a high apoptotic rate of CRC cells (Fig. 4E). Additionally, the apoptotic rate of CRC cells increased in a time-dependent manner under detachment conditions (Fig. 4E). Taken together, these data indicate that CRC cells undergo apoptosis when cultured in suspension. CXCL8 suppresses anoikis in CRC cells The apoptotic rate and survival of CRC cells were examined after treatment with different concentrations of rhCXCL8 for 48 h. CXCL8 treatment significantly reduced the anoikis of SW480 and Caco2 cells (Fig. 5A). In addition, the apoptotic ratio decreased with the increasing concentration of CXCL8, and reached the lowest level at 50 ng/ml (Fig. 5A). This dose was used for the following assays.
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Fig. 2. Cumulative survival rates of the 186 enrolled patients with colorectal cancer (CRC) who underwent curative resection as assessed by the Kaplan–Meier method. The differences in survival rates were analyzed by the log-rank test. Patients were divided into groups according to the abundance of CXCL8 and TOPK in samples of their tumor. (A) The overall and disease-free survival in CRC patients in the ‘high’ CXCL8 group was significantly lower than that of patients in the ‘low’ CXCL8 group; (B) the overall survival of CRC patients in the ‘high’ TOPK group was lower than that of patients in the ‘low’ TOPK group. (C) The overall survival of CRC patients in the ‘high’ CXCL8, ‘high’ TOPK group was lower than that of any other group. (D) Survival analysis of patients from GSE33113 dataset.
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Table 3 Multivariate Cox proportional hazards analysis for the candidate variables. Variables
4 5 6 7 8 9 10 11 12 13
Depth of invasion (T3,4 vs. T1,2) Tumor size (5 cm vs. ≤5 cm) Tumor differentiation (G1,2 vs. G3) CEA (ng/ml) (5 vs. ≤5) Lymphatic metastases (yes vs. no) Liver metastases (yes vs. no) CXCL8( IHC score >3 vs. ≤3) TOPK( IHC score >3 vs. ≤3) CXCL8 and TOPK IHC score3 vs. others
Overall survival
Disease-free survival
HR
95% CI
P
HR
95% CI
P
1.190 0.053 0.726 1.382 1.212 2.308 1.386 2.547 4.393
0.564–2.508 0.179–1.010 0.338–1.562 0.681–2.807 0.480–3.064 1.520–3.289 0.328–2.267 1.583–2.975 1.524–7.058
0.007 0.081 0.414 0.139 0.006 0.001 0.000 0.017 0.041
1.19 0.425 0.56 1.382 1.308 1.272 1.147 1.470 3.871
0.564–2.508 0.179–1.010 0.260–1.207 0.681–2.807 0.520–3.289 0.159–2.466 0.929–3.756 1.212–3.043 1.742–7.058
0.048 0.053 0.013 0.037 0.569 0.004 0.021 0.043 0.018
14 15 16 17 18
Western blot analysis showed that the expression of caspase-3 was lower in cells transiently treated with CXCL8 than that in controltreated cells (Fig. 5B). Moreover, the transient exposure of cells to CXCL8 was also associated with a high abundance of Bcl-2, which
is an important apoptosis suppressor factor (Fig. 5B). CXCL8 treatment did not affect apoptosis when cells were grown in attachment conditions (Fig. 5C). We also determined whether CXCL8 suppressed anoikis in other CRC cell lines. CXCL8 stimulation also
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Fig. 3. The expression level of CXCL8 in various colorectal cancer cell lines. (A) Immunocytochemistry of CXCL8 in SW480, Lovo, and Caco2. (B) Western blot of CXCL8 and TOPK in colorectal cancer cell lines. GAPDH was used as a loading control. (C) The relative abundance of CXCL8 mRNA was determined by real-time PCR in the three colorectal cancer cell lines. The abundance of CXCL8 mRNA was normalized to that of a GAPDH control. Experiments were repeated at least three times.
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Fig. 4. Apoptosis of various colorectal cancer cell lines grown under attachment (anchorage) or detachment (nonanchorage) conditions. (A) GSEA was performed using TCGA dataset. Cell apoptosis and MAPK signaling pathway were identified with the strongest association with CXCL-8-higher expression. (B) Morphology and growth of colorectal cancer cell lines under attachment and detachment conditions. Cells (350,000) were seeded on 6 well plates pre-coated with poly-HEMA (detachment conditions) or were seeded on 6 well plates not coated with poly-HEMA (attachment conditions). (C–E) Annexin V/PI staining was performed to assess cell apoptosis. Colorectal cancer cells were grown in (C, D) attachment conditions, or (E) detachment conditions at 24, 48, 72 h following plating. Data are expressed as the mean of each group with standard error bars. The results were statistically analyzed with a t test (*P < 0 .05).
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suppressed anoikis in HT29, HCT116, and Lovo cells (Fig. 5C). Taken together, our results demonstrate that CXCL8 provides protection against apoptosis when cells are in suspension, but does not affect apoptosis when cells are grown in attachment conditions. PI3K/AKT, TOPK and ERK are involved in the resistance of CRC cells to anoikis mediated by CXCL8 Although the total protein of AKT and ERK was unaffected by CXCL8, the phosphorylation of AKT and ERK was significantly increased by CXCL8 stimulation (Fig. 6A), suggesting that resistance to anoikis mediated by CXCL8 involves PI3K/AKT and ERK signaling. In order to further confirm the involvement of AKT and ERK signaling, cells were pre-treated with specific inhibitors to AKT (MK2206) and ERK (U0126) and then stimulated with 50 ng/ml of CXCL8. The apoptosis of SW480 cells and Caco2 in suspension culture treated with 1 μM MK2206 or 40 μM U0126 was then evaluated. MK2206 and U0126 significantly inhibited the activation of AKT and ERK in CRC cells, respectively (Fig. 6D). Interestingly, Annexin V/PI staining showed that the
apoptotic ratio of SW480 cells treated with both CXCL8 and MK2206 was comparable with that of control cells (Fig. 6B), suggesting that resistance to anoikis mediated by CXCL8 was reversed by MK2206. Consistent with the apoptotic ratio, an increase of cleaved Caspase-3 and a decrease of Bcl-2 protein were observed in cells treated with both CXCL8 and MK2206, compared with that in cells treated with CXCL8 alone (Fig. 6D). Resistance to anoikis mediated by CXCL8 was also can be reversed by U0126. These results further demonstrated that CXCL8 inhibited anoikis through PI3K/AKT and ERK signaling. Additionally, CXCL8 treatment also upregulated the expression of TOPK (Fig. 6A and D), which was significantly inhibited by the exposure of SW480 cells to MK2206. These data indicate that TOPK may be the downstream of PI3K/AKT signaling during the resistance of CRC cells to anoikis mediated by CXCL8. Discussion The properties of cancer cell invasion and migration depend on the interactions between tumor- and host-derived cells in the
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Fig. 5. CXCL8 mediates resistance to apoptosis in colorectal cancer cells and promotes the accumulation of the antiapoptotic protein Bcl2. (A) Percentage of apoptotic cells in cultures grown in the presence of various concentrations of CXCL8. Cells were grown for 48 h in the presence of the indicated concentration of CXCL8, or an equivalent volume of PBS (control). Annexin V/PI staining was used to identify apoptotic cells, and then cells were analyzed by flow cytometry. (B) Colorectal cancer cells were seeded on poly-HEMA-coated 6 well plates for 48 h with or without 50 ng/ml of CXCL8. Total cell lysate was analyzed by western blotting. GAPDH was used as a loading control. (C) Percentage of apoptotic cells in cultures grown in attachment conditions in the presence of CXCL8. Cells were grown for 48 h in the presence of CXCL8 (50 ng/ml), or an equivalent volume of PBS (Control). (D) Another 3 CRC cell lines HCT116, HT29 and Lovo were also used to detect the effect of CXCL8 (50 ng/ml).
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microenvironment during the process of metastasis [20]. In patients with solid tumors, thousands of cancer cells break away from the primary tumor into the circulation every day, but only a handful of cells can resist apoptosis and survive [21]. Normal epithelial cells undergo apoptosis after losing contact with neighboring cells or the extracellular matrix (ECM), which is termed anoikis, a particular type of apoptosis due to loss of cell adhesion or inappropriate cell adhesion [22]. Metastasis to vital organs is the major cause of mortality among colon cancer patients [23]. Chemokines, mediators of leukocyte trafficking and homing, are important components of the tumor microenvironment and have been shown to participate in tumor progression and metastasis [24,25]. CXCL8, belonging to the CXC subfamily of chemokines, is overexpressed in many types of human tumor, including CRC [26]. In the present study, we investigated the interplay between CXCL8 and TOPK in a clinical setting. An overexpression of CXCL8 and TOPK was observed in CRC tissues by bioinformatics analysis, real-time PCR (Fig. 1) and IHC analysis (Table 1). The expression of TOPK was positively correlated with that of CXCL8 (Fig. 1 and Table 1). Moreover, the expression of CXCL8 and
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TOPK was associated with tumor cell differentiation, tumor invasion depth, lymph node metastasis, and liver metastasis. These data suggest the critical role of CXCL8 and TOPK in the promotion of CRC metastasis. In addition, the overexpression of CXCL8 or TOPK indicated poor prognosis (Fig. 2). The CRC subtype overexpressed with TOPK and CXCL8 had significantly shorter overall survival and disease-free survival than CRC subtypes with CXCL8 or TOPK expression alone, which implies that coexpression of CXCL8 and TOPK may be a marker of poor prognosis in CRC. Moreover, the expression of CXCL8 is associated with the aggressiveness of human colon carcinoma cells [26]. It is reported that CXCL8 can regulate a variety of tumor cell functions, including proliferation, migration, angiogenesis, and chemosensitivity in colon cancer models [10]. However, the precise action of CXCL8 in CRC metastasis is unclear. Detachment of cancer cells from primary tumors is a prerequisite for metastasis. In the present study, we cultured three CRC cell lines under detachment conditions and evaluated the apoptosis. We showed that cell apoptosis stimulated by the loss of adhesion was reverse correlated with CXCL8 and TOPK
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Fig. 6. Inhibition of AKT or ERK suppresses anoikis resistance in CRC cells treated with CXCL8. (A) CXCL8 induces the activation of ERK and AKT in SW480 and Caco2 cells. SW480 and Caco2 cells were serum-starved for one night, and then trypsinized and suspended in the presence of CXCL8 (50 ng/ml) for another 48 h. The phosphorylation of AKT and ERK was detected by western blotting. (B, C) Percentage of apoptotic cells was determined by the Annexin V/PI staining assay under various conditions. SW480 and Caco2 cells grown in detachment conditions were pretreated with (B) MK2206 or (C) U0126 inhibitor for 1 h, and then treated with CXCL8 (50 ng/ml) for 48 h. Values are means ± S.D. (n = 4); *p < 0.05 vs. the control cells. (D)The levels of p-AKT, AKT, p-ERK, ERK, Bcl-2, Caspase-3, and TOPK in SW480 cells were detected by Western blotting. GAPDH was used as a loading control.
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1 expression. Interestingly, there was no significant difference in the 2 rate of apoptosis among the three cell lines cultured in attach3 ment conditions (Fig. 4). These results indicate CXCL8 signaling is important for the sensitivity of CRCs cells to anoikis. 4 The anoikis was significantly lower in CRC cells treated with CXCL8 5 than in CRC cells without treatment (Fig. 5), which further suggests that 6 the expression of CXCL8 in CRC cells may play an important role in 7 tumor growth and metastasis. Extensively studies have reported that 8 the mitochondrial pathway plays a key role in cell apoptosis and cleaved 9 Caspase-3 is a marker of apoptosis mediated by the mitochondrial 10 pathway [27–29]. Bcl-2 is a well-known regulator to suppress cell apop11 tosis [30]. In the present study, Bcl-2 expression was significantly 12 increased and Caspase-3 was notably decreased in CRC cells treated with 13 CXCL8 (Fig. 5), which suggests that CXCL8 inhibits anoikis of CRC cells 14 through regulating the mitochondrial apoptotic pathway. 15 Many recent studies have investigated the molecular mecha16 nisms underlying the resistance of tumor cells to anoikis. The PI3K/ 17 AKT pathway is activated by a variety of chemokines, and 18 consequently stimulates cell migration and invasion in various 19 human cancers [12,31–33]. AKT is believed to play a key role on the 20 resistance to anoikis in many types of cancer cell. The importance 21 of ERK signaling on cell apoptosis has been demonstrated in several 22 cell types [34,35]. CXCL8 has been reportedly correlated with PI3K/ 23 AKT and Raf/MEK/ERK signaling [36]. Our data showed that CXCL8 24 treatment resulted in a significant activation of AKT and ERK (Fig. 6). 25 We then inhibited AKT and ERK signaling by using inhibitors MK2206 26 and U0126, respectively, and found that these two inhibitors notably 27 reverse the inhibitory effects of CXCL8 treatment on cell anoikis 28 (Fig. 6). Our data suggest that CXCL8 exerts its anti-anoikis role in 29 CRC cells through activating AKT and ERK signaling. 30 TOPK has recently been shown to regulate the proliferation, migra31 tion, and invasion of cancer cells [15], and its over-expression promotes 32 tumorigenesis by regulating ERK activation [16]. CXCL8 was shown to 33 increase the expression of TOPK (Fig. 6), while CXCL8 did not stimu34 late the expression of TOPK when AKT was inhibited by MK2206, which 35 indicates the regulation of CXCL8 on TOPK is through AKT signaling. 36 In conclusion, CXCL8 is highly involved in anoikis resistance of 37 CRCs. We further show that the resistance to anoikis mediated by 38 CXCL8 is through AKT, TOPK and ERK pathways. Histopathological 39 analysis of clinical samples suggests a correlation between the ex40 pression of CXCL8 and the clinical outcome of CRC cases. However, 41 whether CXCL8 is a novel target for therapies designed to prevent 42 CRC metastasis requires further investigation. The signaling via CXCL8 43 is an important regulator for the sensitivity of CRC cells to apop44 tosis, specifically under detachment conditions. 45 46 Acknowledgments 47 48 This work was supported by grants from the National Natural 49Q4 Q5 Science Foundation of China. The authors apologize for the inabil50 ity to cite all related and important literature due to space limitations. 51 We thank the Department of Molecular Oncology (Eastern 52 Hepatobiliary Surgical Hospital Institute, The Second Military Medical 53 University, Shanghai, China) and Center Library (Changhai Hospi54 tal, The Second Military Medical University, Shanghai, China) for 55 experimental technical help. We also thank professor Su-Changqing 56 for helpful comments regarding of the experiment coordination. 57 58 Conflict of interest 59 60 The authors disclose no potential conflicts of interest. 61 62 Appendix: Supplementary material 63 64 Supplementary data to this article can be found online at 65 doi:10.1016/j.canlet.2015.02.021. 66
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Please cite this article in press as: You-Chuan Xiao, et al., CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.02.021
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