CCR6/CCL20 Chemokine Expression Profile in Distinct Colorectal Malignancies
V. O. Frick,
Department of General, Visceral, Vascular and Pediatric Surgery, University of the Saarland, Homburg/Saar, Germany
Correspondence to: V. O. Frick, PhD, Labor für Allgemeine Chirurgie, Viszeral-, Gefäß- und Kinderchirurgie, Universität des Saarlandes, Chirurgische Klinik, Gebäude 57, 66421 Homburg/Saar, Germany. E-mail: firstname.lastname@example.org or email@example.com
Originally, chemokines and their G-protein-coupled receptors were described to regulate multiple physiological functions, particularly tissue architecture and compartment-specific migration of white blood cells. Now, it is established that the chemokine/chemokine receptor system is also used by cancer cells for migration and metastatic spread. Here, we examined the relative levels of CC-chemokine CCL20 and its corresponding receptor CCR6 in resection specimens from patients with different malignant and non-malignant colorectal diseases as well as in colorectal liver metastases (CRLM). CCL20/CCR6 mRNA and protein expression profiles were assessed by quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in resection specimens from patients with ulcerative colitis (UC, n = 15), colorectal adenoma (CRA, n = 15), colorectal adenocarcinoma (CRC, n = 61) and colorectal liver metastases (CRLM, n = 16). Corresponding non-diseased tissues served as control. In contrast to UC tissues, the CCL20/CCR6 system showed a distinct upregulation in CRA, CRC and CRLM related to corresponding non-affected tissues (P < 0.05, respectively). Furthermore, CRA, CRC and CRLM tissue samples displayed significantly higher protein amounts of CCL20 in comparison with UC specimens (P < 0.05, respectively). Our results strongly suggest an association between CCL20/CCR6 expression and the induction of CRA, CRC and the development of CRLM. Therefore, CCL20 and CCR6 may provide potential targets for novel treatment strategies of CRC.
Colorectal cancer (CRC) represents one of the three leading causes of cancer-related death among men and women worldwide . The 5-year survival rate for patients with tumours restricted only to the colon decreases dramatically from 90% to 10% in the presence of distant metastases [1-3].
Currently, tumour growth followed by metastatic dissemination is viewed as a result of a complex, dysregulated molecular machinery leading to several phenomena, such as the resistance of tumour cells to apoptosis, tumour cell migration, tumour cell invasion and tumour cell immune escape mechanisms. It is well known that lymphoid cells differentially express a broad range of chemokine receptors, which serve as key regulators of migration in specific compartments and tissues. Regarding these aspects, metastasis in CRC and many other neoplasias is not random but an organ-selective process and recent data suggest that chemokines and chemokine receptors may direct lymphatic and haematogenous spreading and may additionally influence the sites of metastatic growth of different tumours . Originally, chemokines and their G-protein-coupled receptors were reported to mediate different pro- and anti-inflammatory responses . Müller et al.  were the first to suggest that the aberrant expression of chemokine receptors on tumour cells, that is, CXCR4 and CCR7, is involved in the metastasis of human breast cancer cells to distant organs that express and secrete their respective ligands, CXCL12 and CCL21. This concept has been supported by several subsequent investigations. Various surveys demonstrated the impact of CXCR4 for the metastasis of kidney cancer , acute myeloid leukaemia , and prostate cancer, where CXCR4 may enhance migration to the bone marrow . Moreover, recently, CXCR4 has been proposed to be involved in the carcinogenesis and induction of human CRC . Furthermore, CXCR4 is suggested as a prognostic factor for patients with CRC, indicating that CXCR4 expression increases the risk of recurrence and of poor survival . While many studies suggest that CXCR4 is mainly important in metastasis to lung, liver and bone marrow, CCR7 seems to be the most important receptor accounting for dissemination to the lymph nodes in patients with gastric , oesophageal , lung  and breast cancer .
However, in recent years, an increasing number of studies have drawn attention to CC-chemokine CCL20 and its physiological sole receptor CCR6 to play a role in the onset, development and metastatic spread of various gastrointestinal malignancies.
While some studies observed an overexpression of CCR6/CCL20 in hepatocellular carcinoma [16, 17], in non-melanoma skin cancer, a downregulation of CCR6 was observed .
However, in pancreatic cancer (PCA), CCL20 and CCR6 were shown to be significantly upregulated compared with the normal pancreatic tissue and CCL20 was significantly associated with advanced T-category in patients with PCA . In this context, CCL20 was also shown to stimulate the migration and growth of pancreatic cancer cell lines in vitro and was therefore suggested to contribute to pancreatic tumour cell growth and invasion . Moreover, CCL20 was shown to promote cancer cell invasion by the upregulation of MMP-9 . CCL20 was also found in the microenvironment of breast cancer, and it was suggested that CCL20 also induces migration and proliferation of breast epithelial cells, thus being a factor involved in the ontogeny of breast carcinoma . While CCR6 was shown to be expressed in various cancer types, the receptor was lately also demonstrated to be significantly overexpressed in colorectal cancer (CRC) and stimulation by its ligand CCL20 has been reported to promote CRC cell proliferation and migration in vitro. Further, CCR6/CCL20 interactions may play a role in organ-selective liver metastasis of CRC [23, 24].
Despite the increasing number of studies indicating a role for CC-chemokines in different cancer types, it still remains unclear whether chemokine expression is related to cancer induction, progression and the metastatic potential in CRC. Therefore, the present study aimed to profile CCR6/CCL20 expression across the axis of inflammatory disease to the adenoma and adenocarcinoma sequence to illuminate the prognostic impact on CRC and CRLM treatment.
Material and methods
Surgical specimens and corresponding normal tissue from the same samples were collected from patients with ulcerative colitis (UC, n = 15), colorectal adenomas (CRA, n = 15), colorectal carcinomas of different tumour categories (CRC, n = 61) and primary colorectal tumours with corresponding synchronous or metachronous liver metastases (CRLM, n = 14 and 16, respectively) who underwent surgical resection at our department between 2002 and 2006. The study was approved by the local ethics commission of the Ärztekammer des Saarlandes, and written informed consent for tissue procurement was obtained from all patients. The clinical data and patient characteristics for the different malignant and non-malignant entities were obtained prospectively from the clinical and pathological records and are summarized in Tables 1 and 2. The data obtained are in accordance with the UICC/TNM classification .
Table 1. Clinical characteristics of patients with colorectal carcinomas and colorectal liver metastases
Tissue specimens were collected immediately after surgical resection, snap frozen in liquid nitrogen and then stored at −80 °C until they were processed under nucleic acid sterile conditions for RNA extraction. Tumour samples were taken from vital areas of histopathologically confirmed colorectal adenocarcinomas and liver metastases, respectively. As corresponding normal tissue, we used adjacent unaffected mucosa, 2–3 cm distal to the resection margin from the same resected adenocarcinoma or liver specimens, respectively. All tissues obtained were reviewed by an experienced pathologist and examined for the presence of tumour cells. As minimum criteria for usefulness for our studies, we only used tumour tissues in which tumour cells constituted at least >80% of the tumour biopsy.
Isolation of total protein
Protein lysates from frozen tissue were extracted with the radioimmunoprecipitation buffer containing Complete, a protease inhibitor cocktail (Roche Diagnostics, Penzberg, Germany). Total protein quantification was performed using the BCA protein assay reagent kit (Pierce, Rockford, IL, USA).
The chemokine protein levels in the different tissue lysates were determined by sandwich-type ELISA according to the manufacturer's instructions. Samples were assayed in duplicate with all values calculated as the mean of the two measurements. CCL20 levels were assayed using a validated commercial ELISA (DuoSet; R&D Systems, Minneapolis, MN, USA). The absorbance was read at 450 nm using a 96-well microtitre plate reader. The chemokine concentration from each tissue lysate was normalized to the total protein content of the sample.
Isolation of total RNA
Total RNA was isolated using RNeasy columns from Qiagen (Qiagen, Hilden, Germany) according to the manufacturer's instructions. All samples were treated with RNase-free DNase to prevent amplification of genomic DNA. The samples were dissolved in RNase-free water and quantified by an average of triplicate spectrophotometric readings at 260 nm (A260). The purity of total RNA was determined by the A260: A280 and A260: A230 ratios. Before cDNA synthesis, the integrity of the RNA samples was confirmed by electrophoresis on 1% agarose gels.
Single-strand cDNA synthesis
For cDNA synthesis, 5 μg of each patient total RNA sample was reverse-transcribed in a final reaction volume of 50 μl containing 1× TaqMan RT buffer, 2.5 μM/l random hexamers, 500 μM/l dNTP, 5.5 mM/l MgCl2, 0.4 U/μl RNase inhibitor and 1.25 U/μl Multiscribe RT. All RT-PCR reagents were purchased from Applied Biosystems (Applied Biosystems, Foster City, CA, USA). The reaction conditions were 10 min at 25 °C, 30 min at 48 °C and 5 min at 95 °C.
All qRT-PCR assays containing the primer and probe mix were purchased from Applied Biosystems (Applied Biosystems) and utilized according to the manufacturer's intructions. PCRs were carried out using 10 μl 2× Taqman PCR Universal Master Mix No AmpErase® UNG (Applied Biosystems) and 1-μl gene assay, 8-μl RNase-free water and 1-μl cDNA template (50 ng/μl). The theoretical basis for the qRT assays is described in detail elsewhere . All reactions were carried out in duplicate along with no template controls and an additional reaction in which reverse transcriptase was omitted to assure absence of genomic DNA contamination in each RNA sample. For signal detection, the ABI Prism 7900 sequence detector (Applied Biosystems) was programmed to an initial step of 10 min at 95 °C, followed by 40 thermal cycles at 15 s at 95 °C and 10 min at 60 °C and the log-linear phase of amplification was monitored to obtain CT values for each RNA sample.
Gene expression of all target genes was analysed in relation to the levels of the slope-matched housekeeping gene cyclophilin C (CycC) . Because reporting of data obtained from raw CT values falsely represents the variations, we converted the individual CT values to the linear form as follows:
Consequently, the normal tissue becomes the 1× sample, and all other quantities are expressed as a n-fold difference relative to this tissue.
We examined 60 operative specimens immunohistochemically with regard to the expression of CCR6 and CCL20. The respective tissue samples comprised UC (n = 10), CRA (n = 10), CRC (n = 30) and CRLM (n = 10) patients along with their corresponding non-affected neighbour tissues.
The tissue samples were routinely fixed in formalin and subsequently embedded in paraffin. Before staining, 4-μm paraffin-embedded tissue sections were mounted on Superfrost Plus slides, deparaffinized and rehydrated in graded ethanol to deionized water. The sections were submitted to microwave antigen retrieval (Target Retrieval Solution; Dakocytomation, Carpinteria, CA, USA), and after blocking of the endogenous peroxidise activity with 3% hydrogen peroxide, the section was further blocked for 30 min at room temperature with normal rabbit serum. Overnight incubation at 4 °C with primary goat polyclonal anti-human CCR6 antibody (C2099-70B, 16 μg/ml; Biomol, Hamburg, Germany) or primary goat polyclonal anti-human CCL20 antibody (AF360, 15 μg/ml; R&D Systems) was followed by incubation with secondary biotinylated rabbit anti-goat IgG antibody and avidin–biotin–peroxidase reaction (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA). After colour reaction with aminoethylcarbazole solution (Merck, Darmstadt, Germany), tissues were counterstained with haematoxylin. Negative controls were performed in all cases, omitting primary antibody.
Cells were considered positive, when they demonstrated strong and exclusive labelling for the specific antibody.
Calculations and statistical analysis
The expression profiles of CCL20 and CCR6 in the different malignant and non-malignant entities are presented as mean and standard error of the mean (SEM). All statistical calculations were performed with the MedCalc (MedCalc software, Mariakerke, Belgium) software package . The parametric Student's t-test was applied, if normal distribution was given; otherwise, the Wilcoxon's rank-sum test was used. P-values <0.05 at an α < 0.05 were considered significant.
CCL20/CCR6 expression in benign, premalignant and malignant colorectal diseases
We detected CCR6/CCL20 mRNA expression in all histopathologically distinct diseases under investigation. As shown in Fig. 1A, UC tissues showed no significant difference in CCR6 and CCL20 mRNA expression with respect to corresponding unaffected tissue samples. However, CRA and CRC tissue samples revealed significant upregulation of CCR6 and CCL20 mRNA expression compared with the corresponding unaffected tissue samples (P < 0.05, respectively). With regard to the matched normal tissues, CCL20 showed a more pronounced upregulation, ranging from a 14- and 16-fold increase with respect to a 3- and 5-fold CCR6 overexpression in CRA and CRC tissue samples. In consistence with the results obtained on the RNA level, CCL20 protein expression, as assessed by ELISA, revealed also substantial upregulation in CRA and CRC tissues compared with matched normal tissues (Fig. 1B). Thus, we observed a 7- and 8-fold CCL20 overexpression in the CRA and CRC tissue specimens in relation to normal mucosa (P < 0.05, respectively).
CCL20/CCR6 expression in primary CRC and corresponding CRLM
Next, we analysed CCR6/CCL20 expression in CRLM of synchronous and metachronous origin in context with the corresponding primary CRC tissues where the metastases originated from. In both tissue types, CCL20 and CCR6 mRNA expression was significantly elevated relative to corresponding normal liver tissues and non-affected mucosa samples (P < 0.05, respectively, Fig. 2A). On the mRNA level, we observed on average a significant 16- and 5-fold increase in CCL20 expression and a significant 6- and 7-fold increase in CCR6 expression in primary CRC tissues and paired CRLM samples in comparison with corresponding normal mucosa or non-affected liver.
In consistence with the results obtained on the RNA level, CCL20 protein expression, as assessed by ELISA, revealed also substantial upregulation in CRC and corresponding CRLM tissues compared with matched normal tissues (Fig. 2B). Thus, we observed an 8- and 29-fold CCL20 overexpression in CRC and corresponding CRLM tissue specimens in relation to normal mucosa and unaffected liver (P < 0.05, respectively).
Several clinicopathological factors such as TNM stages, grading, age and gender were compared with the mRNA and protein expression of CCL20 as well as its corresponding receptor CCR6. Clinical validation of CCR6 expression showed no significant association with any of the clinicopathological factors tested. However, we observed a significant correlation between the protein expression of CCL20 and premalignant and malignant colorectal diseases. Thus, we observed in CRA, CRC and CRLM tissues significantly elevated protein expression levels for CCL20 with respect to UC specimens (P < 0.05, respectively). Comparing CCL20 protein expression in benign, premalignant and malignant colorectal diseases revealed on average a significantly 3-fold higher CCL20 protein expression in CRA, CRC and CRLM tissue samples with respect to patients with UC (P < 0.05, respectively) (Fig. 3). While in UC CCL20 protein concentration amounts to 10,000 pg/ml per mg total protein, CCL20 protein concentration in CRA, CRC and CRLM tissues ranged from 29,000 to 35,000 pg/ml per mg total protein (Fig. 3).
Immunohistochemical evaluation of CCR6 and CCL20
To identify the subset of cells expressing CCR6 and CCL20, we evaluated the respective chemokine receptor/chemokine expression by immunohistochemistry in human CRA, CRC and CRLM tissue samples and in the corresponding non-affected neighbour tissues. Cells were considered positive for CCR6 or CCL20, when they demonstrated strong and exclusive labelling for the specific antibody.
Immunohistochemical staining with CCR6-specific antibodies displayed very weak basal epithelial signals in the non-diseased areas of CRA and CRC tissue samples in 7 of 10 cases (Fig. 4A). In CRA specimens, CCR6 staining was unequal. The range for CCR6 reactivity spans from very weak dot-like, cytoplasmic signals to no substantial CCR6 immunostaining, with 3 of 10 being positive for CCR6 (Fig. 4B). Within CRC specimens, 25 of 30 showed weak to intense CCR6 staining in epithelium of CRC as well as in mesenchymal cells (Fig. 4C). In the adjacent non-affected neighbour tissues of CRLM, we detected in 7 of 10 cases weak to dot-like CCR6 signals in hepatocytes (Fig. 4D). In contrast, we observed accumulated intense CCR6 reactivity in a streak of hepatocytes along the tumour invasion front in 7 of 10 CRLM specimens (Fig. 4E).
CCL20 immunostaining of non-diseased areas of CRA and CRC tissues displayed a specific staining pattern, with 8 of 10 specimens revealing immunoreactivity for CCL20 in the perinuclear regions of mucosal epithelial cells (Fig. 4F). In contrast, only weak to no substantial CCL20 staining signals were found in the epithelium of CRA tissues with 2 of 10 showing positive signals for CCL20 (Fig. 4G). All 30 immunohistochemical analysed CRC specimens displayed immunoreactive signals for CCL20 in mesenchymal elements (e.g. macrophages or lymphocytes), but in 3 of these 30 specimens, also a positive reactivity against CCL20 was observed in the adenocarcinoma cells of CRC tumour sample (Fig. 4H). In the corresponding non-diseased neighbour tissues of CRLM, we observed very weak to no substantial CCL20 staining signals, with 1 of 10 being positive for CCL20 (Fig. 4I), while all analysed CRLM specimens showed accumulated intense CCL20 reactivity in a streak of hepatocytes along the tumour invasion front (Fig. 4J).
Chemokines play an important role in the process of leucocyte trafficking and homing, especially at sites of inflammation, cell damage and malignant tumour growth. To evaluate the potential contribution of chemokines in disease pathophysiology, we analysed the expression profile of the CCR6/CCL20 system in patients with colorectal cancer and colorectal liver metastasis and distinct colorectal diseases like UC and CRA, which often precede the formation of colorectal malignancies.
Our main findings describe significant upregulation of CCR6/CCL20 mRNA and protein expression in premalignant and malignant colorectal diseases as well as in matched CRLM of synchronous and metachronous origin with respect to corresponding unaffected tissue samples.
These results have been in parts confirmed by several other studies. Thus, a targeted array of inflammatory cytokine and receptor genes, validated by RT-PCR, was used to assess inflammatory gene expression and CCL20 upregulation in adenocarcinoma was shown to be significantly upregulated with respect to normal colonic mucosa . Moreover, CCL20 upregulation was also observed in adenoma tissues and McLean et al.  suggested that this upregulation may be caused by the inflammatory phenotype of the microenvironment within premalignant human colonic adenomas. On the contrary, CCL20 expression was also found to be significantly downregulated or lost in the majority of CRC samples . However, CCL20 was also shown to be significantly upregulated in inflammatory diseases of the colon and rectum , suggesting a correlation with the induction and early stages of CRC. To investigate such a correlation, we examined our patient samples according to T-stage and clinical staging and observed no significant differences with respect to CCL20 and CCR6 expression concerning different clinicopathological features. However, other studies reported that tumours of higher TNM stage exhibit significantly lower CCL20 expression . These results contrast our findings that indicated no significant differences in CCL20 expression levels with respect to tumour grading. Thus, we observed no significant differences in CCL20 expression between tumours of low grade (G1 + G2) and tumours of high grade (G3 + G4) (data not shown). These observations show that the expression of CCR6 and CCL20 is not affected by the differentiation state of the tumour.
Other studies also observed CCR6 expression in colorectal tissue under constitutive and inflammatory conditions as well as in hepatocellular carcinoma . Various in vitro and animal studies provide evidence that chemokines selectively expressed in certain tissues can promote metastasis by attracting specific CCR-expressing tumour cells and/or by providing growth-stimulatory signals [6, 33]. On the basis of this concept, increased expression levels of CCR6 in CRC cells could facilitate chemoattraction of CRC cells by CCL20 expressed in the periportal area of the liver . We performed statistical tests to find correlations between clinical/pathologic characteristics, for example, TNM stage, grading and the expression of CCL20 and CCR6 data, but no significant associations were found except CRA, CRC and CRLM tissues displaying significantly elevated protein expression levels for CCL20 with respect to UC specimens (P < 0.05, respectively).
Thus, we demonstrated heterogeneous CCL20 expression in the tumour and inflammatory microenvironment of CRC tissues. We observed CCL20 expression predominantly in immune cells like macrophages and lymphocytes. Recently, it was shown in a murine model that upregulation of CCL20 by immune cells is involved in recruitment of CCR6-positive T cells and the development of CRC . Here, we also observed CCL20 expression in epithelial cells of normal colorectal mucosa, whereas the majority of CRC cells have shown no CCL20 expression. In compliance with our findings, also Brand et al. observed differential CCL20 expression intensities in different CRC tissue specimens .
Thus, CCL20 expression correlates clinicopathologically with the transition of an inflammatory disease to the adenoma and adenocarcinoma sequence. These data are well in line with a number of recent findings concerning a potential role for the CCR6/CCL20 system in gastrointestinal malignancies. Thus, in PCA, CCL20 and CCR6 were shown to be significantly upregulated compared with the normal pancreatic tissue , findings that correspond with the results of this study with respect to CRC. Moreover, CCL20 was previously shown to be significantly associated with advanced T-category in patients with PCA . These findings could not be verified for colorectal malignancies. However, we have shown that CCL20 expression increases significantly on the mRNA and protein level from the transition of UC, an inflammatory bowel disease to premalignant and malignant colorectal malignancies. Thus, significantly elevated CCL20 protein expression levels were detected in CRA, CRC and CRLM tissues with respect to UC specimens, which may suggest a prognostic impact for CCL20 in colorectal malignancies. Likely, serum CCL20 was recently suggested as an independent predictive factor for liver metastasis correlating high levels of serum CCL20 with poor prognosis . Moreover, the CCR6/CCL20 system was described to predict poor clinical prognosis in human gliomas . In previous studies, we have also investigated the expression of CCL20 in various organs and it was found that CCL20 exhibits peak levels of expression in the liver, thus indicating that an increased production of CCL20 may contribute to the selective recruitment of CCR6-expressing cancer cells in CRC . Furthermore, it was demonstrated that patients with CRC who developed liver metastases express significantly more CCL20 in the liver in comparison with an unaffected control group suggesting an association between CCR6/CCL20 expression in human CRC and the promotion of colorectal liver metastasis .
The identification of key targets promoting disease progression and metastasis is of great interest for the development of specific treatment strategies. Attempt to inhibit metastasis by interfering with chemokine receptor/chemokine interactions is a promising new treatment strategy . Various small-molecule chemokine receptor antagonist compounds are currently undergoing development in phase I to III studies in infectious and autoimmune diseases and more recently also in cancer. Targeting the CCR6/CCL20 axis may therefore represent a potential treatment strategy to be developed for the prevention of disease progression in colorectal malignancies and the occurrence of liver metastases.
We thank B. Kruse for excellent technical assistance.