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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

CXCL5 (epithelial neutrophil-activating peptide-78) is a member of a proangiogenic subgroup of the CXC-type chemokine family of small, secreted proteins. Recently, evidence that CXCL5 is involved in carcinogenesis and cancer progression has emerged. To investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC), we examined CXCL5 messenger RNA (mRNA) and protein levels in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. We found that CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to controls. CXCL5 activated the PI3K-Akt and ERK1/2 signaling pathways in HCC cells and promoted proliferation, migration, and invasion. Furthermore, we found that CXCL5 had a direct chemoattractant effect on neutrophils in vitro. In animal studies, the up-regulation of CXCL5 in HCC cells promoted tumor growth, lung metastasis, and intratumoral neutrophil infiltration. Conversely, down-regulation of CXCL5 in HCC cells reduced tumor growth, metastasis, and intratumoral neutrophil infiltration. Immunohistochemical analysis in HCC samples showed that overexpression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for overall survival and cumulative recurrence. Conclusion: CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC. (HEPATOLOGY 2012;56:2242–2254)

Hepatocellular carcinoma (HCC) is one of the most prevalent tumor types, and both the incidence and mortality rates of HCC have increased in recent years.1 Although survival of patients with HCC has improved due to advances in surgical techniques and perioperative management, long-term survival after surgical resection remains low due to the high rate of recurrence and metastasis.2, 3 The molecular pathogenesis and complicated signal transduction pathways implicated in HCC are not fully understood. Although several molecular markers for the risk of recurrence and metastatic potential of HCC have been proposed, none have been approved for routine clinical use.4

Inflammation has emerged as the seventh hallmark of cancer.5 Over the last decade it has been established that cancer-related inflammation is involved in many aspects of malignancy, and in particular enhances tumor cell survival, proliferation, and metastasis.6–8 Most HCCs occur in an inflamed liver, often observed in Chinese patients infected with hepatitis B virus and in Western populations with hepatitis C virus, suggesting a possible crosstalk between inflammation and HCC development.9 Therefore, studies on the mechanisms of inflammation-associated progression and prognosis in HCC are urgently needed.

Chemokines and chemokine receptors are downstream of genetic events and are components of cancer-related inflammatory conditions, which predispose one to cancer and promote cancer progression.10 Chemokines and their receptors affect multiple pathways that contribute to tumor progression in both cell autonomous and nonautonomous ways, including: leukocyte recruitment and function, cellular senescence, tumor cell proliferation and survival, and invasion and metastasis.11 Recently, CXCL5 (epithelial neutrophil-activating peptide-78) has been the focus of studies examining the role(s) of chemokines in carcinogenesis and tumor progression. Like other chemokines that recognize and bind the G-protein-coupled receptor CXCR2, CXCL5 is a proangiogenic CXC-type chemokine that is an inflammatory mediator and a powerful attractant for neutrophils.12–14 CXCL5 is overexpressed in gastric,15 prostate,16, 17 endometrial,18 squamous cell,19 and pancreatic cancer.20–22 Its increased expression is associated with advanced tumor stages, local invasion, and metastatic potential. These studies demonstrated that CXCL5 directly stimulates cancer cell proliferation and invasion.17, 19 Furthermore, CXCL5 directly induces endothelial cell proliferation and invasion in vitro23, 24 and promotes tumor angiogenesis in nonsmall cell lung carcinoma and pancreatic cancer.22, 25 However, the role of CXCL5 in HCC and the relationship between CXCL5 and cancer-related inflammation is largely unknown.

In the present study we investigated the expression of CXCL5 in a series of different metastatic HCC cell lines. We then explored how invasive and metastatic ability changed with changes in CXCL5 expression. Cell lines with low metastatic potential (HepG2 and PLC/PRF/5) were transfected with CXCL5 complementary DNA (cDNA) and highly metastatic cell lines (HCCLM3 and MHCC97H) were transfected with specific short hairpin RNAs (shRNAs). We also investigated how these changes in CXCL5 expression influenced neutrophil infiltration. Finally, using tissue microarrays (TMAs) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in HCC samples, we determined the relationship between CXCL5 expression and neutrophil infiltration and evaluated the prognostic significance of CXCL5 expression and neutrophil infiltration.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Cell Lines and Animals.

Six HCC cell lines, one normal liver cell line, and nude mice were used in this study and are described in the Supporting Information.

Patients and Follow-up.

Three independent cohorts totaling 919 HCC patients were enrolled in this study. The detailed information and follow-up procedures are described in the Supporting Information.

Statistical Analysis.

Statistical analyses were done using SPSS 16.0 for Windows. Quantitative data between groups were compared using Student's t test. Categorical data were analyzed by the chi-square test or Fisher's exact test. Correlation analysis was performed between CXCL5 and CD66b. Overall survival (OS) and cumulative recurrence rates were calculated by the Kaplan-Meier method and differences were analyzed by the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. P < 0.05 was considered statistically significant.

For other descriptions of the materials and methods used in this study, see the Supporting Information.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

CXCL5 Expression Level Is Associated with the Metastatic Potential of HCC Cell Lines.

Fluorescence microscopic analysis for CXCL5 expression showed that HCC cells displayed immunostaining in the cytoplasm (Fig. 1A-C). qRT-PCR, enzyme-linked immunosorbent assay (ELISA), and western blot showed that both the mRNA and protein expression levels of CXCL5 in six established HCC cell lines were significantly increased in comparison to the nontransformed hepatic cell line L-02 (P < 0.005; Fig. 1D-F). In addition, we observed that CXCL5 expression in the highly metastatic HCC cell lines (MHCC97-L, MHCC97-H, and HCCLM3) was significantly higher than in the HCC cell lines with low metastatic potential (HepG2, PLC/PRF/5, and Huh7; P < 0.005). These data indicate that CXCL5 is overexpressed in HCC cell lines and its increased expression is positively correlated with the metastatic potential of HCC cells.

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Figure 1. CXCL5 expression in six HCC cell lines (HepG2, PLC/PRF/5, Huh7, MHCC97L, MHCC97H, and HCCLM3) with low and high invasive potentials, and a nontransformed hepatic cell line, L-02. (A-C) Fluorescence microscopic analysis for CXCL5 expression. MHCC97L, MHCC97H, and HCCLM3 cells displayed strong immunostaining, especially in the cytoplasm, compared with HepG2, PLC/PRF/5, Huh7, and L-02, which displayed little fluorescence. (D) qRT-PCR confirmed different CXCL5 mRNA expression in HCC cell lines. Data shown are mean (±SD) from three independent experiments. (E) ELISAs showed significantly increased levels of CXCL5 protein in the highly invasive cells lines MHCC97L, MHCC97H, and HCCLM3, compared with HepG2, PLC/PRF/5, Huh7, and L-02. Data shown are means (±SD) from three independent experiments. (F) Western blot analysis showed a significant increase of CXCL5 in the highly invasive cells lines, MHCC97L, MHCC97H, and HCCLM3.

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CXCL5 Promotes Proliferation, Migration, and Invasion of HCC Cells.

Stable up- or down-regulation of CXCL5 expression in transfected HCC cell lines was confirmed by qRT-PCR and western blot. The results were consistent with those obtained using an ELISA to determine the levels of CXCL5 in cell culture supernatants (Fig. 2A). Down-regulation of CXCL5 by shRNA in MHCC97H and HCCLM3 cells caused significant suppression of cell proliferation after 48 hours (P < 0.05). Similarly, cell proliferation of HepG2-CXCL5 and PLC/PRF/5-CXCL5 cells was significantly higher than HepG2-Mock and PLC/PRF/5-Mock cells (P < 0.05; Fig. 2C). In wound-healing migration assays, microscopic examination at 24 hours postwounding revealed a significant delay in the wound closure rate of HCCLM3-shRNA-CXCL5 and MHCC97H-shRNA-CXCL5 cells compared with the control cell lines, whereas HepG2-CXCL5 and PLC/PRF/5-CXCL5 cells had a significant increase in the wound closure rate compared with control cell lines (P < 0.0005; Fig. 2D; Supporting Fig. S1A,B). In vitro invasive assays showed that the numbers of invasive HCCLM3-Mock and MHCC97H-Mock cells were 42.4 ± 14.7 and 31.9 ± 12.8, respectively, significantly higher than those of HCCLM3-shRNA-CXCL5 and MHCC97H-shRNA-CXCL5 cells (25.5 ± 9.9 and 15.7 ± 9.2, respectively; P < 0.005). Similarly, the numbers of invasive HepG2-CXCL5 and PLC/PRF/5-CXCL5 cells were 25.5 ± 10.4 and 29.7 ± 11.7, respectively, significantly higher than those of HepG2-Mock and PLC/PRF/5-Mock cells (15.1 ± 5.9 and 18.2 ± 8.5, respectively; P < 0.05; Fig. 2E; Supporting Fig. S1C,D).

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Figure 2. Characterization of CXCL5 expression, proliferation, migration, and invasion ability in HCC cells. (A) qRT-PCR, western blot, and ELISAs confirmed CXCL5 expression in stably transfected and parent cells. (B) CXCL5 induced phosphorylation of PI3K-Akt and ERK1/2 in HCC cells in a CXCR2-dependent manner. (C) Cell proliferation was detected by CCK8 assay. (D) Cell monolayers were wounded and then monitored at 0 and 24 hours for wound channel closure. The cleaned area was measured and plotted as the percentage of the original timepoint (0 hours). (E) Invasive behavior tested by transwell Matrigel invasion assays. Data shown are mean (±SD) from three independent experiments.

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CXCL5 Activates PI3K-Akt and ERK1/2 Signaling Pathways in HCC Cells.

To explore which signaling pathways contributed to HCC proliferation and invasion induced by CXCL5, the stably transfected and parent cells were assessed for the activation status of multiple pathways. Activation was determined by the analyzing the phosphorylation state of Akt (PI3K-Akt pathway), ERK1/2 (ERK1/2 pathway), p38 MAPK (p38 MAPK pathway), JNK (JNK pathway), p65 (nuclear factor kappa B [NF-κB] pathway), and STAT3 (JAK/STAT pathway). All of these proteins, and the associated pathways, can be activated through chemokine receptors. As shown in Fig. 2B (left panel), CXCL5 did not affect p38 MAPK, JNK, p65, or STAT3 phosphorylation in HCC cells. Intriguingly, down-regulation of CXCL5 by shRNA in MHCC97H and HCCLM3 cells caused a significant decrease in the phosphorylation levels of Akt and ERK1/2. Phosphorylated Akt and ERK1/2 were also obviously increased in HepG2-CXCL5 and PLC/PRF/5-CXCL5 cells when compared with HepG2-Mock and PLC/PRF/5-Mock cells. Furthermore, we confirmed that the effect of CXCL5 on Akt and ERK1/2 phosphorylation in HCC cells was dependent on the CXCL5 receptor, CXCR2 (Fig. 2B, right panel).

CXCL5 Is a Direct Chemoattractant for Neutrophils In Vitro.

The effect of CXCL5 on neutrophil chemotaxis was assayed using a transwell system. As shown in Fig. S2A, CXCL5 induced concentration-dependent migration of neutrophils in vitro. To analyze the mechanism underlying CXCL5-mediated chemotaxis of neutrophils, we performed a phosphoexplorer antibody array on untreated human neutrophils or those treated with 10 nM of CXCL5. Among the 19 phosphorylated proteins that were up-regulated more than 1-fold (Table S1), we found seven that participate in the PI3K/Akt pathway, including p-AKTSer473. In addition, two phospho-proteins (p-IKK-α/β and p-p65) involved in NF-κB signaling were increased when compared with control. To validate these data we performed western blots to detect the phosphorylation of Akt and the p65 subunit of NF-κB in cells stimulated with CXCL5 (Fig. S2B). These data showed that CXCL5 activates several signaling pathways in human neutrophils, including the PI3K/Akt and NF-κB pathways.

CXCL5 Mediates Neutrophil Infiltration and Promotes HCC Progression In Vivo.

After orthotropic transplantation of all cell lines into nude mice, all the groups successfully formed liver tumors. The tumor sizes of HCCLM3-Mock-derived and MHCC97H-Mock-derived xenografts were 5.83 ± 1.29 cm3 and 3.39 ± 0.72 cm3, respectively, significantly larger than the tumor sizes of xenografts derived from HCCLM3-shRNA-CXCL5 and MHCC97H-shRNA-CXCL5 cells (1.30 ± 0.49 cm3 and 0.56 ± 0.16 cm3, respectively, P < 0.0005). Similarly, the tumor sizes of HepG2-CXCL5-derived and PLC/PRF/5-CXCL5-derived xenografts were 1.84 ± 0.65 cm3 and 2.71 ± 0.49 cm3, respectively, markedly larger than those of HepG2-Mock-derived and PLC/PRF/5-Mock-derived tumors (0.53 ± 0.38 cm3 and 0.73 ± 0.40 cm3, respectively; P < 0.0005; Fig. 3A,B; Fig. S3A). Pulmonary metastasis occurred in 100% (8/8) of the HCCLM3-Mock and MHCC97H-Mock mice, a higher rate than observed in the HCCLM3-shRNA-CXCL5 (2/8) and the MHCC97H-shRNA-CXCL5 mice (1/8). The number of metastatic nodules of each grade was also greater in the HCCLM3-Mock and MHCC97H-Mock mice (Fig. 3D; Fig. S3E,F). In the HepG2-CXCL5 and the PLC/PRF/5-CXCL5 mice, pulmonary metastasis occurred in 75% (6/8) and 87.5% of mice (7/8), respectively, but was 0% (0/8) in PLC/PRF/5-Mock and HepG2-Mock mice (Fig. 3C; Fig. S3C,D). Notably, we found that in the HCCLM3-Mock-derived and MHCC97H-Mock-derived xenografts, intratumor Gr1+ neutrophil infiltration was significantly more than that observed in HCCLM3-shRNA-CXCL5-derived and MHCC97H-shRNA-CXCL5-derived xenografts. In the HepG2-Mock-derived and PLC/PRF/5-Mock-derived xenografts, intratumor Gr1+ neutrophil infiltration was significantly less than that observed in HepG2-CXCL5-derived and PLC/PRF/5-CXCL5-derived xenografts (Fig. 4; Figs. S3B, S4).

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Figure 3. CXCL5 promotes HCC progression in a xenograft nude mice model. (A,B) Macrograph of tumors in all groups. (C,D) Hematoxylin and eosin (H&E)-stained images of metastatic nodules in lungs from all groups with magnification of the selected areas.

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Figure 4. CXCL5 mediates neutrophil infiltration in a xenograft nude mice model. (A-D) Representative images from tumor sample serial sections stained with H&E, CXCL5, and Gr1. Magnification, 100× (A-D).

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CXCL5 is Up-regulated in HCC Tissues and Coincides with Increased Intratumoral Neutrophil Infiltration.

Ninety-four tumors (cohort 1) were analyzed by immunohistochemistry, qRT-PCR, and western blot. qRT-PCR revealed that CXCL5 was significantly overexpressed in tumors when compared to corresponding peritumoral liver tissue (Fig. 5A). Samples from patients with tumor recurrence (59 of 94) had higher levels of CXCL5 than those without recurrence (35 of 94; Fig. 5B). These results were confirmed by western blot analyses on 54 HCC samples randomly selected from the 94 HCC cases using computer-generated random numbers with SPSS software (Fig. 5C). Tumor tissues that expressed high levels of CXCL5 showed more neutrophil infiltration. A scatterplot of CXCL5 expression and intratumoral neutrophil presence revealed a significant positive correlation between CXCL5 expression (at mRNA and protein levels) and neutrophil infiltration in cancerous tissues (Fig. 5D,E). Similar results were also observed in the other two cohorts of patients (paraffin-embedded tissues, cohort 2, n = 323; cohort 3, n = 502) by immunohistochemistry.

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Figure 5. CXCL5 is up-regulated in HCC tissues (cohort 1, n = 94) and coincides with increased intratumoral neutrophil infiltration. (A) qRT-PCR analysis of CXCL5 expression in 94 HCC tumors and peritumoral liver tissues. (B) qRT-PCR analysis of CXCL5 expression in 94 HCC tumors with recurrence or no recurrence. (C) Western blot analysis showed CXCL5 in 54 HCC samples with recurrence or no recurrence. (D,E) Scatterplots show a positive correlation between CXCL5, at the mRNA or protein level, and intratumoral CD66b+ neutrophils in 94 HCCs.

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Expression of CXCL5 and Combined Expression of CXCL5 and Intratumoral Neutrophils Correlates with Poor Prognosis in HCC Patients.

In the cohort of 94 patients, high CXCL5 level indicated worse patient outcome (Fig. S5). The cumulative recurrence rates were significantly higher among patients with high CXCL5 protein levels (72.3% versus 53.2%, P < 0.05) or high CXCL5 mRNA levels (70.2% versus 55.3%, P < 0.05) when compared to those of HCC patients with low CXCL5 expression. The median OS was substantially reduced among patients with high CXCL5 protein expression (24.4 months versus 48.0 months, P < 0.005) or patients with high CXCL5 mRNA expression (24.4 months versus 48.0 months, P < 0.005).

We investigated the expression of CXCL5 and CD66b (a marker which is thought to be uniquely expressed by human neutrophils) by immunohistochemical staining in a tissue microarray composed of primary tumors from 323 HCC patients in cohort 2. Representative cases of immunohistochemical staining are shown in Fig. 6A-D. We found that CXCL5 expression was significantly correlated with tumor size (P = 0.005), tumor encapsulation (P = 0.017), vascular invasion (P = 0.039), and tumor-node-metastasis staging (P = 0.030). Other clinical characteristics, including age, sex, preoperative serum alpha-fetoprotein, tumor number, and tumor differentiation, were not directly related to the expression of CXCL5 (Table 1).

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Figure 6. Expression and prognostic value of CXCL5 and intratumoral CD66b+ neutrophils in HCC samples (cohort 2, n = 323). (A-D) Representative HCC tumor samples show the expression of CXCL5 (brown staining in the cytoplasm of HCC cells) and intratumoral CD66b (brown staining in the cell membrane of the intratumoral neutrophils). (A,B) High expression of both CXCL5 and intratumoral CD66b. (C,D) Low-level expression of CXCL5 and intratumoral CD66b. (E) Scatterplot indicates a significant positive correlation between CXCL5 and intratumoral CD66b in cancerous tissues. (F) Prognostic values of CXCL5 and intratumoral CD66b using Kaplan-Meier analysis. I, CXCL5Low/intratumoral CD66bLow; II, CXCL5Low/intratumoral CD66bHigh and CXCL5High/intratumoral CD66bLow; III, CXCL5High/intratumoral CD66bHigh.

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Table 1. Correlation Between the Factors and Clinicopathologic Characteristics in HCC (Cohort 2, n = 323)
Clinicopathological IndexesCXCL5CD66b
LowHighPLowHighP
  • AFP, alpha-fetoprotein; GGT, gamma glutamyl transferase; TNM, tumor-node-metastasis.

  • *

    Fisher's exact tests; chi-square tests for all other analyses.

Age(year)≤5084820.86988780.291
 >507879 7483 
SexFemale26200.35123230.982
 Male136141 139138 
HBsAgNegative24210.64620250.409
 Positive138140 142136 
HCVNegative1571590.448*1601560.283*
 Positive52 25 
Alcohol intake historyNo1491440.4331501430.243
 yes1317 1218 
AFP (ng/ml)≤2052440.34853430.237
 >20110117 109118 
GGT (U/L)≤5471600.2369620.455
 >5491101 9399 
Liver cirrhosisNo21150.29821150.298
 yes141146 141146 
Tumor size(cm)≤595690.00584800.698
 >56792 7881 
Tumor numberSingle1361400.4441431330.149
 Multiple2621 1928 
Microvascular invasionabsence97780.03989860.784
 present complete6583 7375 
Tumor encapsulationnone93710.01785790.541
 I + II6990 7782 
Tumor differentiationIII + IV1261290.6051301250.566
 I3632 3236 
TNM stageII + III87670.0386680.051
  7594 7693 

By the last follow-up, in March of 2009, 54.2% (175/323) of the patients had suffered a recurrence and 51.1% (165/323) had died. The 1-, 3-, and 5-year OS rates and cumulative recurrence rates in the group (cohort 2) were 85.4% and 25.4%, 62.2% and 50.2%, and 50.7% and 59.7%, respectively. In addition, we found that the 1-, 3-, and 5-year survival rates of the CXCL5Low patients were significantly higher than the survival rates of those of the CXCL5High group (92.0% versus 79.0%, 73.5% versus 50.9%, and 62.9% versus 38.5%, respectively; Fig. 6F). Similarly, CXCL5High HCC patients had the poorest prognosis at 1-, 3-, and 5-years, with higher cumulative recurrence rates than CXCL5Low patients (32.7% versus 18.4%, 59.8% versus 41.2%, and 72.2% versus 48.4%, respectively; Fig. 6F). The presence of intratumoral neutrophils significantly correlated with OS (P < 0.001, hazard ratio [HR] = 1.896) and time to recurrence (TTR) (P < 0.001, HR = 1.846; Table 2), consistent with previously reported results.9

Table 2. Univariate and Multivariate Analyses of Prognostic Factors in HCC (Cohort 2, n = 323)
VariableTTROS
HR (95% CI)PHR (95% CI)P
  • Cox proportional hazards regression model.

  • *

    I, CXCL5Low/CD66bLow; II, CXCL5Low/CD66bHigh and CXCL5High/CD66bLow; III, CXCL5High/CD66bHigh.

  • AFP, alpha-fetoprotein; GGT, gamma glutamyl transferase; TNM, tumor-node-metastasis.

  • HR, hazard ratio; CI, confidential interval; NA, not adopted.

Univariate analysis    
Age, year (≤50 versus >50)1.023 (0.760-1.377)0.8790.828 (0.610-1.125)0.228
Sex (female versus male)1.863 (1.143-3.036)0.0131.757 (1.049-2.942)0.032
HBsAg (negative versus positive)1.007 (0.659-1.538)0.9741.039 (0.669-1.614)0.866
Alcohol intake history (no versus yes)1.021 (0.619-1.683)0.9361.001 (0.598-1.677)0.996
AFP, ng/ml (≤20 versus >20)1.155 (0.835-1.597)0.3851.548 (1.083-2.211)0.016
GGT, U/L (≤54 versus >54)1.329 (0.978-1.805)0.0691.737 (1.253-2.409)0.001
Liver cirrhosis (no versus yes)1.102 (0.677-1.795)0.6961.308 (0.769-2.224)0.332
Tumor size, cm (≤5 versus >5)1.817 (1.346-2.452)0.0002.482 (1.806-3.412)0.000
Tumor number (single versus multiple)1.362 (0.907-2.044)0.1361.517 (1.025-2.243)0.037
Microvascular invasion (no versus yes)1.915 (1.420-2.583)0.0002.479 (1.815-3.388)0.000
Tumor encapsulation (complete versus none)1.679 (1.246-2.263)0.0011.715 (1.260-2.336)0.001
Tumor differentiation (I + II versus III + IV)1.238 (0.870-1.763)0.2361.581 (1.119-2.233)0.009
TNM stage (I versus II III)2.072 (1.527-2.814)0.0002.683 (1.935-3.720)0.000
CXCL5 (low versus high)1.932 (1.428-2.612)0.0002.100 (1.533-2.877)0.000
Intratumor CD66b (low versus high)1.846 (1.363-2.499)0.0001.896 (1.386-2.593)0.000
Combination of CXCL5 and CD66b* 0.000 0.000
I versus II1.592 (1.070-2.368)0.0221.727 (1.134-2.629)0.011
II versus III1.562 (1.097-2.224)0.0131.556 (1.085-2.233)0.016
I versus III2.503 (1.731-3.620)0.0002.696 (1.836-3.958)0.000
Multivariate analysis    
Sex (female versus male)1.558 (0.965-2.515)0.0701.325 (0.796-2.206)0.279
AFP, ng/ml (≤20 versus >20)NANA1.242 (0.857-1.802)0.252
GGT,U/L (≤54 versus>54)NANA1.434 (1.020-2.015)0.038
Tumor size, cm (≤5 versus >5)1.622 (1.191-2.210)0.0022.035 (1.459-2.839)0.000
Tumor number (single versus multiple)NANA1.311 (0.877-1.959)0.187
Tumor encapsulation (complete versus none)1.614 (1.186-2.197)0.0021.583 (1.148-2.184)0.005
Microvascular invasion (no versus yes)1.513 (1.106-2.070)0.0101.997 (1.445-2.760)0.000
Tumor differentiation (I + II versus III + IV)NANA1.491 (1.040-2.140)0.030
CXCL5 (low versus high)1.697 (1.249-2.304)0.0011.817 (1.323-2.496)0.000
Intratumor CD66b (low versus high)1.938 (1.428-2.629)0.0002.000 (1.455-2.749)0.000
Combination of CXCL5 and CD66b* 0.000 0.000
I versus II1.521 (1.019-2.272)0.0401.623 (1.060-2.485)0.026
II versus III1.470 (1.027-2.105)0.0351.515 (1.052-2.182)0.026
I versus III2.334 (1.611-3.382)0.0002.456 (1.669-3.612)0.000

HCC patients with high CXCL5 expression had more intratumoral neutrophil infiltration than patients with low CXCL5 expression (n = 323, r = 0.569, P < 0.001) (Fig. 6E). When evaluating the combined effect of CXCL5 expression and intratumoral neutrophil presence on HCC prognosis, we found that the 1-, 3-, and 5-year OS rates in the CXCL5high/intratumoral neutrophilshigh patients were 78.9%, 46.5%, and 33.3%, respectively; significantly lower than that in CXCL5low/intratumoral neutrophilslow patients (92.2%, 79.1%, and 67.8%, respectively; Fig. 6F). The 1-, 3-, and 5-year cumulative recurrence rates in the CXCL5high/intratumoral neutrophilshigh patients were 34.9%, 65.0%, and 77.2%, respectively, significantly higher than those in the CXCL5low/intratumoral neutrophilslow patients (17.0%, 36.0%, and 43.2%, respectively; Fig. 6F). Univariate and multivariate analyses revealed that, along with tumor size, encapsulation, microvascular invasion, and tumor-node-metastasis stage, CXCL5 expression and the coindex (CXCL5/intratumoral neutrophils) were independent prognostic factors for both OS (P < 0.001, HR = 1.817 and P < 0.001, HR = 2.456) and TTR (P = 0.001, HR = 1.697 and P < 0.001, HR = 2.334; Table 2).

Independent Validation.

The prognostic value of CXCL5 protein expression and intratumoral neutrophil presence was validated in an independent cohort of 502 HCC patients by immunohistochemical staining. Similarly, the expression of tumor-derived CXCL5 had a significant positive correlation with intratumoral neutrophil presence (n = 502, r = 0.632, P < 0.001). Univariate and multivariate analyses revealed that CXCL5 and the coindex (CXCL5/intratumoral neutrophils) were independent prognostic factors for both OS (P < 0.001, HR = 1.895 and P < 0.001, HR = 2.557) and TTR (P < 0.001, HR = 1.688 and P < 0.001, HR = 2.068) (Fig. S6, Table S6).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

In this study we confirmed that the expression of CXCL5 was markedly increased in HCC tissues compared with that in adjacent nontumor liver tissues. Moreover, our results indicate that overexpression of CXCL5 contributes to the invasion and metastasis of HCC cells, and the activation of PI3K-Akt and ERK1/2 signaling pathways by way of its receptor CXCR2. Although CXCL5 has been found to participate in various cellular functions, including activation, proliferation, migration, invasion,16, 17, 19, 22 and pathological angiogenesis,22, 25 to the best of our knowledge, this is the first study to demonstrate that expression of CXCL5 is positively related to the metastatic potential of HCCs. In line with recent studies on CXCL5 in prostate, squamous, and pancreatic cancer,17, 19, 22 our results revealed that HCC cells in which CXCL5 was down-regulated have decreased proliferation, invasion, and metastasis in vitro and in vivo. The above results support the notion that CXCL5 makes a substantial contribution to tumor progression.

CXCL5 was first identified as a neutrophil-activating inflammatory peptide with homology to interleukin 8.12 In a study of rheumatoid arthritis14 and chronic obstructive pulmonary disease,13 CXCL5 was shown to have a strong effect on neutrophil recruitment. However, its relationship to infiltrating neutrophils in tumors, especially in HCC, is largely unknown. In this study we revealed a striking correlation between CXCL5 production and neutrophil infiltration in HCC. First, we found that CXCL5 acts as a direct chemoattractant on neutrophils in vitro. Then, through CXCL5 overexpression or knockdown in HCC cells, we further investigated the role of CXCL5 in recruiting neutrophils to the tumor site in vivo. Finally, we validated this relationship between CXCL5 and neutrophil infiltration using clinical HCC samples. In 94 tumors (cohort 1), we found a strong correlation between CXCL5 expression (both at protein and mRNA levels) and intratumoral neutrophils; in two other independent cohorts of patients (paraffin-embedded tissues, cohort 2, n = 323; cohort 3, n = 502), similar results were obtained. The migration of neutrophils across the tumor vasculature is mainly mediated by CXC chemokines, especially glutamic acid-leucine-arginine motif positive-CXC chemokines that bind to and activate CXCR1 and/or CXCR2.26 In this study we demonstrated that CXCL5 activates several signaling pathways in human neutrophils, including the PI3K-Akt and NF-κB signaling pathways. Activation of Akt is crucial for the chemotaxis and migration of neutrophils induced by chemoattractants.27, 28 Recent studies have also revealed that NF-κB activation contributes to the recruitment of neutrophils that is induced by chemotactic cytokines.29 Thus, we suggest that CXCL5 plays a crucial role in recruiting neutrophils to the tumor site of HCCs, potentially through the activation of AKT and NF-κB.

Neutrophils, initially recognized as short-lived effector cells providing the first line of defense against invading pathogens, are the most abundant subpopulation of leukocytes.30 Neutrophils can potentiate cancer cell migration, invasion, and dissemination by secreting immunoreactive molecules such as hepatocyte growth factor,31 oncostatin M,32 b2-integrins,33 or neutrophil elastase.34 Imai et al.35 reported that neutrophils enhance the invasion activity of the HepG2 cell line in vitro by way of the paracrine regulation of hepatocyte growth factor. McDonald et al.36 reported that neutrophils mediate cancer cell adhesion to hepatic sinusoids, which could promote the implantation of circulating tumor cells and formation of hepatic metastases. In our study the HCCLM3-Mock-derived and MHCC97H-Mock-derived xenografts with high expression of CXCL5 had more intratumoral neutrophils, larger tumors, and more pulmonary metastases compared to HepG2-Mock-derived and PLC/PRF/5-Mock-derived xenografts with low CXCL5 expression. After CXCL5 expression was inhibited by shRNA in the HCC cells, the number of intratumoral neutrophils, tumor volume, and pulmonary metastasis decreased, and vice versa. These results suggest that tumor-derived CXCL5 and the accompanying neutrophil infiltration substantially contribute to HCC progression in nude mice models.

To determine whether CXCL5 could be an important factor related to clinical outcome of HCC patients after curative resection, we examined CXCL5 mRNA expression in a cohort of 94 HCC patients by qRT-PCR. Our results indicated that patients with high CXCL5 mRNA levels have a higher recurrence rate and shorter survival time. Subsequently, we detected the protein expression of CXCL5 in TMAs containing this cohort of 94 HCC patients and two other cohorts (323 HCC patients and 502 HCC patients) by immunohistochemistry. Multivariate analysis confirmed that CXCL5 (either at the protein or mRNA level) is a significant independent predictor for OS and TTR. Because CXCL5 acted as a strong chemotactic cytokine for neutrophils in our xenograft model and there was a significant positive correlation between CXCL5 expression and intratumoral neutrophils in clinical HCC samples, we next investigated the prognostic significance of intratumoral neutrophils in TMA by immunohistochemistry. In accordance with our previous study,9 the presence of intratumoral neutrophils was a poor prognostic indicator for HCC after resection. Thereafter, we evaluated the prognostic value of both CXCL5 expression and intratumor neutrophils in HCC patients. We made direct comparisons of prognosis among three subgroups (CXCL5low/intratumor neutrophilslow, CXCL5low/intratumor neutrophilshigh or CXCL5high/intratumor neutrophilslow, and CXCL5high/intratumor neutrophilshigh) in the two cohorts (323 HCC patients and 502 HCC patients). In the TMA analysis, HCC patients who had both high CXCL5 expression and high levels of intratumor neutrophil infiltration were more prone to recurrence of HCC following curative resection. Conversely, HCC patients who expressed both low CXCL5 and had low intratumor neutrophil infiltration had the best prognosis. Although the expression of CXCL5 was also an independent predictor for OS and TTR, the predictive range of CXCL5high/intratumor neutrophilshigh was more sensitive than that of CXCL5high or intratumor neutrophilshigh alone.

Tumor growth and dissemination is the result of dynamic interactions between tumor cells themselves and with components of the tumor inflammatory environment.37 The inflammatory environment includes inflammatory cells and inflammatory mediators (for example, chemokines, cytokines, and prostaglandins).6 Chemokines are emerging as key mediators not only in the homing of cancer cells to metastatic sites but also in the recruitment of a number of different cell types to the tumor microenvironment. Recent studies suggest that chemokines are produced by epithelial cancer cells, leading to the recruitment of tumor-associated macrophages, tumor-associated neutrophils, lymphocytes, cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells to the tumor microenvironment. These infiltrating cells provide a secondary source of chemokines that could affect tumor growth, cell survival, senescence, angiogenesis, and metastasis.37 Considering that the inflammatory cells in HCC are usually mixed with intratumoral lymphocytes accounting for a significant portion, we investigated the relationship between intratumoral infiltration of lymphocytes and neutrophils. As shown in Fig. S7 and Table S8, intratumoral neutrophils positively correlated with most types of intratumoral lymphocytes, including CD3+, CD4+, CD8+, and Foxp3+ lymphocytes. However, we did not observe a significant correlation between tumor-derived CXCL5 and intratumoral infiltration of lymphocytes, indicating that CXCL5 is a strong and specific chemotactic cytokine for neutrophils in HCC. Tumor-derived CXCL5 and accompanying intratumoral neutrophils may cooperatively contribute to HCC growth, invasion, and metastasis. HCC cells with higher expression of CXCL5 are prone to recruit more neutrophils to the tumor site, establishing a tumor-promoting microenvironment and amplifying the inflammatory response, thus facilitating HCC invasion and metastasis.

In conclusion, CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 expression alone or in combination with intratumoral neutrophils serves as a novel prognostic indicator for HCC patients undergoing curative resection. These features of CXCL5 make it a potential therapeutic target as well.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25907_sm_SuppFig1.tif1735KSupporting Information Figure 1. CXCL5 promotes migration and invasion of HCC cells in vitro. (A) In wound-healing migration assays, microscopic examination at 24 hours post-wounding revealed a significant delay in the wound closure rate of HCCLM3-shRNA-CXCL5 and MHCC97H-shRNA-CXCL5 cells compared with the control cell lines, whereas, HepG2-CXCL5 and PLC/PRF/5-CXCL5 cells had a significant increase in the wound closure rate compared with control cell lines (B). (C) In vitro invasive assays showed that the numbers of invasive HCCLM3-Mock and MHCC97H-Mock cells were significantly higher than those of HCCLM3-shRNA-CXCL5 and MHCC97H-shRNA-CXCL5 cells. (D) The numbers of invasive HepG2-CXCL5 and PLC/PRF/5-CXCL5 cells were significantly higher than those of HepG2-Mock and PLC/PRF/5-Mock cells. Magnification, 100× (A-B) and 200× (C-D).
HEP_25907_sm_SuppFig2.tif197KSupporting Information Figure 2. CXCL5 attracts neutrophils in vitro. (A) In vitro transwell assays show that CXCL5 induces concentration-dependent migration of neutrophils. (B) Western blot analysis of p-Akt and p-p65 in response to CXCL5.
HEP_25907_sm_SuppFig3.tif378KSupporting Information Figure 3. Statistical analysis of tumor volume, intratumoral neutrophil number, and pulmonary metastases in xenografts from nude mice. (A) The tumor volume of the xenograft in each nude mouse group. (B) The number of intratumoral neutrophils in each group of xenograft nude mice. (C-F) All grades of metastatic nodules in lungs of all xenograft nude mice groups.
HEP_25907_sm_SuppFig4.tif9004KSupporting Information Figure 4. Representative images of Gr1+ intratumoral neutrophils with magnification of the selected areas in Fig. 4. Magnification, 200×.
HEP_25907_sm_SuppFig5.tif271KSupporting Information Figure 5. Kaplan-Meier analysis of OS rates (A-B) and cumulative recurrence rates (C-D) in 94 HCCs (cohort 1) with different CXCL5 expression levels.
HEP_25907_sm_SuppFig6.tif315KSupporting Information Figure 6. The correlation and prognostic value of CXCL5 and intratumoral CD66b+ neutrophils in HCC samples (cohort 3, n=502). (A-C) Prognostic value of CXCL5 expression and intratumoral CD66b by Kaplan-Meier analysis. I, CXCL5Low/intratumoral CD66bLow; II, CXCL5Low/intratumoral CD66bHigh and CXCL5High/intratumoral CD66bLow; III, CXCL5High/intratumoral CD66bHigh. (D) Significant positive correlation between CXCL5 expression and intratumoral CD66b in cancerous tissues, shown with a scatter plot.
HEP_25907_sm_SuppFig7.tif1900KSupporting Information Figure 7. Representative images of intratumoral lymphocytes. (A) CD3+, (B) CD4+, (C) CD8+, (D) CD45RO+, (E) Foxp3+, and (F) granzyme B+. Magnification, 200×.
HEP_25907_sm_SuppTab1.doc41KSupporting Information Table 1. Up-regulated at least one-fold of protein phosphorylation after CXCL5 treatment in human neutrophils.
HEP_25907_sm_SuppTab2.doc43KSupporting Information Table 2. Clinicopathologic characteristics of three cohorts of patients with hepatocellular carcinoma.
HEP_25907_sm_SuppTab3.doc51KSupporting Information Table 3. Correlation between the factors and clinicopathologic characteristics in HCC (cohort 1, n=94).
HEP_25907_sm_SuppTab4.doc50KSupporting Information Table 4. Univariate and multivariate analyses of prognostic factors in HCC (cohort 1, n=94).
HEP_25907_sm_SuppTab5.doc48KSupporting Information Table 5. Correlation between the factors and clinicopathologic characteristics in HCC (cohort 3, n=502).
HEP_25907_sm_SuppTab6.doc60KSupporting Information Table 6. Univariate and multivariate analyses of prognostic factors in HCC (cohort 3, n=502).
HEP_25907_sm_SuppTab7.doc42KSupporting Information Table 7. Univariate analyses of intratumoral lymphocytes in HCC (cohort 2, n=323 and cohort 3, n=502).
HEP_25907_sm_SuppTab8.doc40KSupporting Information Table 8. Descriptive statistics of immunohistochemical variables of intratumoral lymphocytes in HCC and correlations with CD66b/CXCL5 (cohort 2, n=323 and cohort 3, n=502).
HEP_25907_sm_SuppInfo.doc97KSupporting Information

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