Research progress of CCL20-CCR6 axis and epithelial-mesenchymal transition in breast cancer / 国际外科学杂志

CCL20 and CCR6 are chemokines produced by a variety of cells. CCL20 and CCR6 combine to stimulate a series of downstream pathways, participate in the occurrence and development of various malignant tumors, and also play an important role in the invasion and metastasis of breast cancer and the process of chemotherapy resistance. Epithelial-mesenchymal transformation (EMT) is a key step in the process of tumor cell metastasis, which is characterized by loss of cell adhesion, down-regulation of E-cherherin expression, up-regulation of mesenchymal markers and fibrinectin expression, and enhancement of cell motor ability and invasion ability. This article reviews the research of CCL20-CCR6 biological axis and EMT on invasion and metastasis of breast cancer cells.

AKT/MMP3 activation, not EMT pathway, participates in CCL20-induced invasion and migration of colon cancer SW480 cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the molecular mechanism of chemokine CCL20/CCR6 in promoting invasion and migration of colon cancer SW480 cells. Methods: Colorectal cancer SW480 cells with high expression of CCR6 receptor were screened by immunochemistry (IHC). After co-culture with recombinant human CCL20, the invasion and migration of SW480 cells were detected by Transwell assay and Wound-Healing assay, respectively. Expressions of EMT markers, AKT signal protein and target protein MMP3 were detected by immunofluorescence (IF) and WB. AKT signaling pathway as the key mechanism was confirmed by MK2206 blocking assay. The expressions of CCL20 and MMP3 in colorectal cancer tissues as well as their correlation were analyzed by TCGAdatabase resources (https://portal.gdc.cancer.gov/). Results: CCL20 promoted the invasion and migration ability of SW480 cells significantly (all P <0.01), and this was induced by activation of AKT signaling and up-regulation of downstream target protein MMP3, instead of EMT. Blocking AKT signaling could significantly inhibit the invasion and migration of SW480 cells, and down-regulate MMP3 expression (P<0.05). TCGA platform data showed that the expressions of CCL20 and MMP3 in colorectal cancer tissues were significantly higher than those in normal mucosa tissues (P<0.05 or P<0.01), and an evidently positive correlation was found between CCL20 and MMP3 (r =0.051, P<0.01). Conclusion: The chemokine CCL20 promotes the invasion and migration of SW480 cells throughAKT/MMP3 signal axis, but not the EMT.

Research advances in the role of CCL20/CCR6 in liver cancer and other tumors / 临床肝胆病杂志

The role of chemokines/receptors in the pathophysiological activities such as inflammation and tissue injury has been clarified .In recent years, there have been increasing studies on their effect on tumors in the tumor microenvironment .This article reviews research advances in C -C motif chemokine ligand 20 (CCL20) and its receptor CCR6 in liver cancer and other tumors in recent years and analyzes the possible mechanism by which CCL20 and CCR6 promote tumor progression and their effect on tumor prognosis .Blocking CCL20/CCR6 interaction may provide a new direction for the targeted therapy for liver cancer.

CCR6 Is a Predicting Biomarker of Radiosensitivity and Potential Target of Radiosensitization in Rectal Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: This study aimed to explore the functions and mechanisms of C-C motif chemokine receptor 6 (CCR6), a gene associated with progression and metastasis of colorectal cancer (CRC), in radiosensitivity of rectal cancer (RC). MATERIALS AND METHODS: RNA sequencing and immunohistochemical analysis on CCR6 expression were performed in pretreatment tissues of RC patients exhibiting different therapeutic effects of radiotherapy. Colonogenic survival assay was conducted in different CRC cell lines to assess their radiosensitivity. And the impact of CCR6 expression on radiosensitivity was validated through RNA interference. The DNA damage repair (DDR) abilities of cell lines with different CCR6 expression were evaluated through immunofluorescence-based γH2AX quantification. RESULTS: The CCR6 mRNA level was higher in patients without pathologic complete remission (pCR) than in those with pCR (fold changed, 2.11; p=0.004). High-level expression of CCR6 protein was more common in the bad responders than in the good responders (76.3% vs. 37.5%, p < 0.001). The CRC cell lines with higher CCR6 expression (LoVo and sw480) appeared to be more radioresistant, compared with the sw620 cell line which had lower CCR6 expression. CCR6 knockdown made the LoVo cells more sensitive to ionizing radiation (sensitization enhancement ratio, 1.738; p < 0.001), and decreased their DDR efficiency. CONCLUSION: CCR6 might affect the RC radiosensitivity through DDR process. These findings supported CCR6 as a predicting biomarker of radiosensitivity and a potential target of radiosensitization for RC patients.

In vitro chemokine (C-C motif) receptor 6-dependent non-inflammatory chemotaxis during spermatogenesis

BACKGROUND: Chemokine (C-C motif) receptor 6 (CCR6) is present in sperm and plays a significant role in sperm motility and chemotaxis acting in the reproductive tracts. However, the expression and functional significance of CCR6 in testis are still poorly understood, especially in the process of spermatogenesis. METHODS AND RESULTS: CCR6 was expressed in spermatogenic cell lines and its expression was shown in an age-dependent upregulation manner from puberty to adulthood in mouse testis. Immunostaining results confirmed the localization of CCR 6 in testis. Further chemotaxis assays demonstrated that spermatogenic cells GC-1 and -2 exhibited a directional movement toward CCR6-specific ligand such as CCL20 or Sertoli cells in vitro. CONCLUSIONS: The present findings indicate that CCR6 is involved in the chemotaxis of spermatogenic cells in vitro and promotes chemotaxis under non-inflammatory conditions during normal spermatogenesis.

Unbalanced expression of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of rheumatoid arthritis / Expressão não equilibrada do receptor de hidrocarboneto arílico nos linfócitos T CCR6+ CD4+ e CD4+ CD25+ do sangue periférico na artrite reumatoide

ABSTRACT Objective: The goal of this study was to analyze the role of aryl hydrocarbon receptor in peripheral blood CCR6+CD4+ and CD4+CD25+T cells of patients with rheumatoid arthritis. Methods: Flow cytometry was applied to determine the proportion of AhR positive cells in CCR6+CD4+T, CD4+CD25+T and peripheral blood peripheral mononuclear cells from each subject. AhR mRNA and CYP1A1 mRNA relative expression levels were tested by real-time PCR. Results: The percentage of AhR positive cells in peripheral blood mononuclear cells was higher in RA group than that in healthy cases [(35.23 ± 10.71)% vs. (18.83 ± 7.32)%, p < 0.01]. The expression levels of AhR and CYP1A1 were both increased in patients with RA while compared to controls [(3.71 ± 1.63) vs. (2.00 ± 1.27), p = 0.002; (2.62 ± 2.08) vs. (0.62 ± 0.29), p < 0.01, respectively]. In RA patients, the percentage of AhR positive cells in CD4+CD25+T cells was significantly lower than that from controls [17.90 (6.10 ± 80.10)% vs. (52.49 ± 19.18)%, p < 0.01]; In healthy controls, the percentage of AhR positive cells in CD4+CD25+T cells was significantly higher than that in CCR6+CD4+T cells, and was also significantly higher than that in PBMCs [(52.49 ± 19.18)% vs. (23.18 ± 5.62)% vs. (18.06 ± 7.80)%, X 2 = 24.03, p < 0.01]; in RA patients, the percentage of AhR positive cells in CCR6+CD4+T cells was significantly increased than that in CD4+CD25+T cells and PBMCs [(46.02 ± 14.68)% vs. 17.90 (6.10 ± 80.10)% vs. (34.22 ± 10.33)%, X 2 = 38.29, p < 0.01]; Nevertheless, no statistically significant relationship was found between clinical data and AhR positive cells in CCR6+CD4+T and CD4+CD25+T cells. Conclusion: AhR may participate in the pathological progress of RA by controlling the differentiation of Th17 and Treg cells in peripheral blood.

RESUMO Objetivo: Analisar o papel do receptor de hidrocarboneto arílico (AhR) nos linfócitos T CCR6+ CD4+ e CD4+ CD25+ no sangue periférico de pacientes com artrite reumatoide (AR). Métodos: Foi aplicada citometria de fluxo para determinar a proporção de células AhR positivas em linfócitos CCR6+ CD4+ e CD4+ CD25+ do sangue periférico e células mononucleares periféricas de cada indivíduo. Os níveis de expressão relativa de ácido ribonucleico mensageiro (do inglês ribonucleic acid, RNAm,) de AhR e RNAm de enzima de primeiro estágio essencial para o AhR (CYP1A1) foram testados por reação em cadeia de polimerase (do inglês polymerase chain reaction, PCR,) em tempo real. Resultados: A percentagem de células AhR positivas nas células mononucleares do sangue periférico foi maior no grupo com AR do que nos indivíduos saudáveis [(35,23 ± 10,71)% vs. (18,83 ± 7,32)%, (p < 0,01)]. Os níveis de expressão de AhR e CYP1A1 estavam aumentados em pacientes com AR quando comparados com os controles [(3,71 ± 1,63) vs. (2,00 ± 1,27), p = 0,002; (2,62 ± 2,08) vs. (0,62 ± 0,29), p < 0,01, respectivamente]. Em pacientes com AR, a percentagem de células AhR positivas nos linfócitos T CD4+ CD25+ foi significativamente inferior à dos controles [17,90 (6,10 ± 80,10)]% vs. (52,49 ± 19,18)%, p < 0,01]; em controles saudáveis, a percentagem de células AhR positivas nos linfócitos T CD4+ CD25+ foi significativamente mais elevada do que nos linfócitos T CCR6+ CD4+ e também foi significativamente maior do que nas células mononucleares do sangue periférico (do inglês peripheral blood mononuclear cells, PBMC,) [(52,49 ± 19,18)% vs. (23,18 ± 5,62)% vs. (18,06 ± 7,80)%, X 2 = 24,03, p < 0,01]; em pacientes com AR, a percentagem de células AHR positivas nos linfócitos T CCR6+ CD4+ era significativamente maior em comparação com os linfócitos T CD4+ CD25+ e PBMC (46,02 ± 14,68)% vs. [17,90 (6,10 ± 80.10)]% vs. (34,22 ± 10,33)%, X2 = 38,29, p < 0,01]; no entanto, não foi encontrada correlação estatisticamente significativa entre os dados clínicos e células AhR positivas em linfócitos T CCR6+ CD4+ e CD4+ CD25+. Conclusão: O Ahr pode participar do progresso patológico da AR ao controlar a diferenciação de linfócitos Th17 e Treg no sangue periférico.

Relationship between subtypes of T follicular helper cells and unexplained recurrent spontaneous abortion / 上海交通大学学报（医学版）

Objective · To discuss the relationship between unexplained recurrent spontaneous abortion (URSA) and T follicular helper (Tfh) cell subtypes. Methods · Twenty-eight normal early pregnancy women, who had undergone induced abortion, were taken as control, and 28 patients with URSA were enrolled in the abortion group. The mononuclear cells in peripheral blood and decidual tissues were separated in the two groups, and CXCR3+CCR6- Tfh cells, CXCR3-CCR6- Tfh cells, CXCR3-CCR6+ Tfh cells and B cells were tested by flow cytometry. Results · The decidual CXCR3-CCR6- Tfh cells significantly increased in the abortion group compared with control group (P=0.015). And there was a strong association between the decidual CXCR3-CCR6- Tfh cells and B cells in URSA patients (R2=0.779, P=0.025). Conclusion · The up-regulation of decidual CXCR3-CCR6- Tfh cells in early pregnancy women may be related with the occurrence of URSA.

Relationship between subtypes of T follicular helper cells and unexplained recurrent spontaneous abortion / 上海交通大学学报（医学版）

Objective · To discuss the relationship between unexplained recurrent spontaneous abortion (URSA) and T follicular helper (Tfh) cell subtypes. Methods · Twenty-eight normal early pregnancy women, who had undergone induced abortion, were taken as control, and 28 patients with URSA were enrolled in the abortion group. The mononuclear cells in peripheral blood and decidual tissues were separated in the two groups, and CXCR3+CCR6- Tfh cells, CXCR3-CCR6- Tfh cells, CXCR3-CCR6+ Tfh cells and B cells were tested by flow cytometry. Results · The decidual CXCR3-CCR6- Tfh cells significantly increased in the abortion group compared with control group (P=0.015). And there was a strong association between the decidual CXCR3-CCR6- Tfh cells and B cells in URSA patients (R2=0.779, P=0.025). Conclusion · The up-regulation of decidual CXCR3-CCR6- Tfh cells in early pregnancy women may be related with the occurrence of URSA.

Role and recruitment of Th9 cells in liver cirrhosis patients

Objective: To investigate the role of T helper 9 (Th9) cells in liver cirrhosis (LC) patients and whether chemokine receptor type 6 (CCR6)/chemokine ligand 20 (CCL20) axis is involving in the recruitment of Th9 cells into liver. Methods: Peripheral blood and liver tissue from 30 LC patients and 18 normal controls were recruited. The frequency of Th9 cells and CCR4, CCR6 in the peripheral blood was tested by flow cytometry. Serum interleukin (IL)-9 and CCL20 levels were tested by enzyme-linked immunosorbent assay. Immunohistochemical staining was used to detect α-smooth muscle actin, CCR6 and CCL20 expression in liver tissue. Results: The frequency of Th9 cells in LC patients was significantly increased compared with controls (P < 0.05). The serum IL-9 level and CCL20 level increased markedly in LC patients compared with controls (P < 0.05), and IL-9 was positively correlated to Th9 cells and CCL20. Furthermore, the frequency of Th9 cells was correlated to prothrombin time, total bilirubin level, hyaluronic acid and type IV collagen in LC patients. We also found that Th9 cells in LC patients expressed higher frequency of CCR4

Expression and its clinical significance of chemokine receptor 6/chemokine ligand 20 axis in hepatocellular carcinoma / 中华消化杂志

Objective To explore the expression and clinical significance of chemokine receptor 6 (CCR6)/chemokine ligand 20(CCL20) axis in hepatocellular carcinoma (HCC).Methods From March 2003 to December 2005,48 patients with HCC were selected,and one specimen of HCC tissue and one of corresponding adjacent tissue were taken from every patient.Another eight patients with benign liver lesions were selected,and one specimen of surgical sectioned normal liver tissue of each was taken.The relative expression quantity of CCR6 and CCL20 at mRNA level was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR).And the expression of CCR6 and CCL20 at protein level was determined by immunohistochemisty methods.One-way analysis of variance (ANOVA) was performed for comparison among groups of measurement data.Chi-square test was used for rate comparison.The correlation coefficient was calculated by Spearman's method.Survival curves were plotted by Kaplan-Meier method and the survival rate was compared by Log-rank test.Results The relative expression quantity of CCR6/CCL20 at mRNA level in HCC tissues (0.99±0.21 and 0.46± 0.11) were significantly higher than those of para carcinoma tissues (0.33 ± 0.09 and 0.31 ± 0.07) and normal liver tissues (0.22±0.06 and 0.28±0.05),and the differences were statistically significant (F=127.43 and 21.10,both P＜0.05).The positive percentage of CCR6 protein expression in HCC tissues (54.17％,26/48) was significantly higher than that in para carcinoma tissues (16.67％,8/48) and normal liver tissues (0/8),and the difference was statistically significant (x2 =19.55,P＜0.05).There was no statistically significant difference in the positive percentage of CCL20 protein expression among HCC tissues (50.00％,24/48),paracarcinoma tissues (33.33％,16/48),normal liver tissues (2/8) (all P＜0.05).There was correlation between the positive percentage of CCR6 protein expression and that of CCL20 protein expression in HCC tissues (r=0.42,P＜0.05).The positive percentage of CCR6 protein expression was correlated with the pTNM stage of HCC,vascular tumor thrombosis,intrahepatic metastasis and lung metastasis (x2 =5.48,4.02,5.07 and 5.19,all P＜0.05).The positive percentage of CCL20 expression was significantly correlated to tumor maximum diameter and pTNM stage (x2 =4.09 and 4.00,both P＜0.05).Both the disease-free survival (DFS) rate and overall survival (OS) rate of CCR6-positive group were significantly lower than those of negative group (x2 =4.57 and 6.57,both P＜ 0.05).There were no significant differences in DFS rate and OS rate between CCL20-positive group and negative group (both P＞0.05).Conclusion CCR6/CCL20 axis may be related with the malignant behavior and the prognosis of HCC.

Doença enxerto contra hospedeiro crônica em mucosa bucal: relação da concentração de células de Langerhans com a expressão da quimiocina CCL20 e de seu receptor CCR6 / Chronic graft versus host disease in the oral mucosa: concentration of Langerhans cells and its relationship with the chemokine CCL20 and its receptor CCR6

A doença enxerto contra hospedeiro (GVHD) é uma complicação comum nos pacientes submetidos ao transplante de células-tronco hematopoiéticas (TCTH), sendo considerada a maior causa de morbidade e mortalidade nesses pacientes. O principal objetivo do presente estudo foi relacionar a concentração de células de Langerhans em mucosa bucal de pacientes com GVHDc bucal com a expressão da quimiocina CCL20 e de seu receptor CCR6 no epitélio bucal, a fim de elucidar os mecanismos biológicos envolvidos no recrutamento das células de Langerhans na GVHDc. Foram selecionados fragmentos obtidos por biópsia de mucosa bucal de 60 pacientes onco-hematológicos e hematológicos submetidos previamente ao transplante de células tronco hematopoiéticas no Hospital Amaral Carvalho, Jaú SP, onde 30 pacientes desenvolveram GVHDc em mucosa bucal (Grupo 1) e 30 não desenvolveram GVHDc (Grupo 2). Amostras obtidas a partir de 30 biópsias de lesões não inflamatórias em mucosa bucal constituíram o Grupo Controle (Grupo 3). Cortes microscópicos foram avaliados em coloração de rotina Hematoxilina e Eosina, e submetidos à técnica imuno-histoquímica, utilizando-se anticorpos monoclonais anti-CD1a e anti-CCR6, e anticorpos policlonais anti-CCL20. As células de Langerhans CD1a+ foram quantificadas no epitélio da mucosa bucal, e os resultados demonstraram um maior número destas células nos pacientes com GVHDc quando comparados àqueles sem GVHDc e ao Grupo Controle (p<0,001). A análise da imunomarcação das moléculas CCR6 e CCL20 foi subjetiva com aplicação de escores. Quanto à molécula CCR6, houve maior expressão no Grupo 1 (p<0,001) em comparação aos outros Grupos; porém, quanto à expressão de CCL20, não houve diferença estatística entre os três Grupos (p=0,108). Estes resultados sugerem que o aumento das células de Langerhans, na doença enxerto contra hospedeiro crônica, em mucosa bucal, pode estar associado a maior expressão do receptor CCR6. Possivelmente, o maior recrutamento de células de...

The graft versus host disease (GVHD) is a common complication in patients undergoing hematopoietic stem cell transplantation (HSCT), and considered a major cause of morbidity and mortality in these patients. The main objective of this study was to compare the concentration of Langerhans cells in oral mucosa of patients with oral chronic GVHD (GVHDc) with the expression of the chemokine CCL20 and its receptor CCR6 in oral epithelium, in order to clarify the biological mechanisms involved in the recruitment of Langerhans cells in GVHDc. We selected 60 biopsies of oral mucosa from onco-hematological and hematological patients submitted to prior hematopoietic stem cell transplantation at Hospital Amaral Carvalho, Jaú - SP from which 30 patients developed GVHDc in the oral mucosa (Group 1) and 30 did not develop GVHDc (Group 2). The Control Group (Group 3) was obtained from 30 biopsies of non-inflammatory lesions of oral mucosa. Microscopic sections were evaluated in routine Hematoxylin and Eosin staining, and submitted to immunohistochemistry using anti-CD1a and anti-CCR6 monoclonal antibodies, and anti-CCL20 polyclonal antibody. The Langerhans cells (CD1a+) were quantified in the epithelium of the oral mucosa, and the results showed a greater number of these cells in patients with GVHDc compared to those without GVHDc and the Control Group (p<0.001). Analysis of immunostaining of molecules CCL20 and CCR6 were subjective with application of scores. The expression of CCR6 molecule was more significant in Group 1 (p<0.001) compared to other groups, but in relation to CCL20 expression, there was no statistical difference between the three groups (p=0.108). These results suggest that the increase of Langerhans cells in GVHDc affecting oral mucosa may be associated with increased expression of the receptor CCR6. We suggest that the increased recruitment of Langerhans cells to the oral mucosa in patients with transplanted bone marrow contributes...

Clinical significance of chemokine CCL20 and its receptor CCR6 expression in patients with gastric cancer / 国际外科学杂志

Objective To investigate the expression of CCL20 and CCR6 in the patients with gastric cancer and To examine the relationship between chemokine expression and the occurrence and development of Gastric Cancer. Methods Real-time PCR , flow cytometry and ELISA are used to measure the gene transcription and protein expression levels of chemokine CCL20 and CCR6 in the serum of 50 patients with Gastric Cancer and 30 normal controls. Results The gene expression levels CCL20 and CCR6 in Gastric Cancer group are significantly higher than that in healthy controls. The level protein of CCL20 and CCR6 in peripheral blood of patients with gastric cancer are significantly higher than that in healthy peep le[ (45.4 ±10.9) pg/mL vs (18.6±4.7) pg/mL; (7.11 ±1.03%) vs (1.83±0.43%), P＜0.01. respectively],and the increase significantlyassociated with the clinical stage of Gastric Cancer. Conclusions The method for detecting the expression of CCL20 and CCR6 in patient with Gastric Cancer has been successfully established, and their expression levels were found to be correlated with the occurrence and development of Gastric Cancer. Thus, CCL20 and CCR6 may be involved in the regulatory mechanisms associated with the development of Gastric Cancer, and may be valuable in its diagnosis and prevention.

Expression of chemokine MIP-3α and its receptor CCR6 in ulcerative colitis and its significance / 第二军医大学学报

Objective: To investigate the expression of chemokine MIP-3α and its receptor CCR6 in ulcerative colitis(UC) and to explore its relationship with the involvement and severity of UC, so as to asses their expression in the pathogenesis of UC. Methods: The expression of MIP-3α and CCR6 protein in the colon mucosa was detected by immunohistochemistry method in 35 UC patients and 20 normal controls. Results: MIP-3α and CCR6 were both positive in the UC group and negative or only weakly expressed in the normal control group. The expression of MIP-3α and CCR6 in the UC group was significantly higher than that in normal controls (P<0.01). The expression of MIP-3α and CCR6 was related to the severity and involvement of UC. The expression of MIP-3α was significantly correlated with that of CCR6(r=0.765,P<0.01). Conclusion: The expression of MIP-3α and CCR6 is positively correlated with each other, and both of them participate in the development and progression of UC. The interaction between the 2 may play an important role in the local damage and pathological changes in UC.