The Effect Of CXCL5 In Mediating Neutrophil Infiltration And Its Role In Hepatocellular Carcinoma Microenvironment

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. 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. 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.Inflammation has emerged as the seventh hallmark of cancer. 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.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. 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. 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. Recently, CXCL5 (epithelial neutrophilactivating 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. CXCL5 is overexpressed in gastric, prostate, endometrial, squamous cell, and pancreatic cancer. 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. Furthermore, CXCL5 directly induces endothelial cell proliferation and invasion in vitro and promotes tumor angiogenesis in nonsmall cell lung carcinoma and pancreatic cancer. 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 and its receptor, CXCR2, in a series of different metastatic HCC cell lines. We explored how invasive and metastatic ability changed with changes in CXCL5 expression. We also investigated how these changes in CXCL5 expression influenced neutrophil infiltration. 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. Then, we tested the signaling pathways related to CXCL5 and its receptor CXCR2, and explored which signaling pathway(s) was (were) crucial for CXCL5 induced EMT in HCC cells. Finally, we examied the effect of CXCL5 in ICC and revealed the relationship between its expression and neutrophil infiltration and evaluated its prognostic significance.Part oneOverexpression of CXCL5 Mediates Neutrophil Infiltration and Indicates Poor Prognosis for Hepatocellular CarcinomaBACKGROUND: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. This study was designed to investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC).METHODS:CXCL5 mRNA and protein levels were examined in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. Proliferative and invasive ability of HCC cells were analyzed after CXCL5 knockdown or overexpression in cultured cells and in xenograft nude mice models. The correlation between CXCL5 expression and neutrophil infiltration was investigated The role of CXCL5 in neutrophil infiltration was investigated in vitro by chemotaxis assay, and in vivo using xenograft SCID mice models and HCC patients.RESULTS:CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to without. CXCL5 had a direct chemoattractant effect on neutrophils in vitro. Up-regulation of CXCL5 in HCC cells promoted proliferation, invasion, metastasis, and intratumoral neutrophil infiltration. Conversely, down-regulation of CXCL5 in HCC cells reduced proliferation, invasion, metastasis, and intratumoral neutrophil infiltration. CXCL5 could activated PI3K-Akt and ERK1/2 signaling pathways via its receptor, CXCR2. Immunohistochemical analysis 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 in the presence of intratumoral neutrophils, were independent prognostic indicators 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. Part two CXCR2/CXCL5 Axis Contributes to Epithelial-Mesenchymal Transition of HCCCells through Activating PI3K/Akt/GSK-3β/Snail SignalingBACKGROUND:Upregulation of CXCR2 in tumor cells has been documented in several types of cancer. As one of its ligands, CXCL5 is associated with neutrophil infiltration and poor prognosis in hepatocellular carcinoma (HCC). However, little is known about the role of CXCR2/CXCL5 axis in the invasion and metastasis of HCC cells.METHODS:We examined CXCR2 expression in human HCC cell lines and in three independent cohorts of HCC patients. The molecular effects of high expression levels of CXCR2 and CXCL5 in HCC cells were determined using qRT-PCR, western blot analysis, immunofluorescence, matrigel invasion assay, and xenograft mouse models.RESULTS:High levels of CXCR2 correlated with progression and poor prognosis in human HCC. CXCR2/CXCL5 together promoted cell spreading by inducing the epithelial-mesenchymal transition (EMT) through activation of the PI3K/Akt/GSK-30/Snail signaling pathway. In clinical HCC samples, high expression of both CXCR2 and CXCL5 showed a significant correlation with the activation of PI3K/Akt/GSK-3β/Snail signaling and EMT phenotype.CONCLUSION:CXCR2/CXCL5 axis contributes to EMT of HCC cells through activating PI3K/Akt/GSK-3β/Snail signaling, and it may serve as a potential therapeutic target.Part 3CXCL5 contributes to tumour metastasis and recurrence of intrahepatic cholangiocarcinoma by recruiting infiltrative intratumoural neutrophilsBACKGROUND:CXCL5 is a member of the CXC-type chemokine family that may play a role in carcinogenesis and cancer progression. This study investigates the biological function and clinical significance of CXCL5 in intrahepatic cholangiocarcinoma (ICC).METHODS:We examined CXCL5 expression in normal human intrahepatic biliary epithelial cell line, ICC cell lines, and two independent cohorts of ICC patients. The effect of CXCL5 on the progression of ICC was analysed by CXCL5 depletion or overexpression in cultured cells and xenograft SCID mice models. The role of CXCL5 in neutrophil infiltration was investigated in vitro by chemotaxis assay, and in vivo using xenograft SCID mice models and ICC patients.RESULTS:CXCL5 was overexpressed in ICC cell lines and tumour samples compared with paired normal tissues. CXCL5 had a direct chemoattractant effect on neutrophils in vitro through PI3K-Akt and ERK1/2 signalling pathways. In animal studies, CXCL5 promoted tumour growth and metastasis without altering in vitro proliferative and invasive ability of ICC cells, and this effect was mediated by the recruitment of intratumoural infiltrative neutrophils by tumour-derived CXCL5. Immunohistochemical analysis of ICC samples showed that overexpression of CXCL5 correlated strongly with intratumoural neutrophil infiltration, shorter overall survival, and high tumour recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoural neutrophils, was an independent prognostic indicator for ICC.CONCLUSION:CXCL5 promotes ICC growth and metastasis by recruiting intratumoural neutrophils. CXCL5 alone or combined with intratumoural neutrophils is a novel prognostic predictor for ICC patients and a potential therapeutic target.Conclusion1. CXCL5 activats the PI3K-Akt and ERK1/2 signaling pathways in HCC cells and promots proliferation, migration, and invasion.2. CXCL5 activats the PI3K-Akt and p65 signaling pathways in neutrophils and mediates neutrophil intratumoral infiltration.3. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC.4. CXCR2/CXCL5 axis contributes to epithelial-mesenchymal transition of HCC cells through activating PI3K/Akt/GSK-3β/Snail signaling.5. CXCL5 contributes to tumour metastasis and recurrence of intrahepatic cholangiocarcinoma by recruiting infiltrative intratumoural neutrophils.Movelty1. For the first time, we demonstrated and reported the role of CXCL5 in the invasion, metastasis and inflammation-associated microenvironment of HCC.2. For the first time, we demonstrated and reported that CXCR2/CXCL5 axis contributes to epithelial-mesenchymal transition of HCC cells through activating PI3K/Akt/GSK-3β/Snail signaling.3. For the first time, we demonstrated and reported the role of CXCL5 in the metastasis and recurrence of ICC.Potential application1. Our research will be beneficial for revealing the role of chemokines in metastasis, recurrence and microenvironment of HCC.2. CXCL5 and neutrophils were the novel independent predictor for recurrence and metastasis in HCC patients, which is beneficial in predicting which patients are at highest risk of recurrence, thus facilitating patient selection for more aggressive treatment.3. Inhibition PI3K/Akt/GSK-3β/Snail signaling induced by CXCL5/CXCR2 axis can reverse the EMT of HCC cells, providing a new route to further improve the effect of anti-neoplastic agents.4. Depletion of intratumoural neutrophils will be emphasized in the treatment of advanced HCC and prevention of the recurrence after curative resection.