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ELR+ CXC chemokine expression in benign and malignant colorectal conditions
- Claudia Rubie†1Email author,
- Vilma Oliveira Frick†1,
- Mathias Wagner2,
- Jochen Schuld1,
- Stefan Gräber3,
- Brigitte Brittner1,
- Rainer M Bohle2 and
- Martin K Schilling1
© Rubie et al; licensee BioMed Central Ltd. 2008
Received: 23 November 2007
Accepted: 25 June 2008
Published: 25 June 2008
CXCR2 chemokine ligands CXCL1, CXCL5 and CXCL6 were shown to be involved in chemoattraction, inflammatory responses, tumor growth and angiogenesis. Here, we comparatively analyzed their expression profile in resection specimens from patients with colorectal adenoma (CRA) (n = 30) as well as colorectal carcinoma (CRC) (n = 48) and corresponding colorectal liver metastases (CRLM) (n = 16).
Chemokine expression was assessed by microdissection, quantitative real-time PCR (Q-RT-PCR), the enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC).
In contrast to CXCL6, we demonstrated CXCL1 and CXCL5 mRNA and protein expression to be significantly up-regulated in CRC and CRLM tissue specimens in relation to their matched tumor neighbor tissues. Moreover, both chemokine ligands were demonstrated to be significantly higher expressed in CRC tissues than in CRA tissues thus indicating a progressive increase in the transition from the premalignant condition to the development of the malignant status. Although a comparative analysis of the CXCL1/CXCL5 protein expression profiles in CRC patients revealed that the absolute expression level of CXCL1 was significantly higher in comparison to CXCL5, mRNA- and protein overexpression of CXCL5 in CRC and CRLM tissues was much more pronounced (80- and 60- fold in CRC tissues, respectively) in comparison to CXCL1 (5- and 3.5- fold in CRC tissues, respectively).
Our results demonstrate a significant association between CXCL1 and CXCL5 expression with CRC and CRLM suggesting for both chemokine ligands a potential role in the progression from CRA to CRC and thus, in the initiation of CRC.
To date, colorectal cancer (CRC) constitutes one of the leading causes of cancer-related deaths worldwide. In spite of many currently available and continuously improving therapies for early stage colon cancer, the majority of CRC patients develop metastases which worsen the prognosis for survival dramatically [1, 2].
The development of cancer metastases is a complex process consisting of various interacting mechanisms, which involve numerous biochemical and immunological changes like abnormally expressed growth factors and cytokines . One group of cytokines that recently attracted a lot of attention with view to cancer-related mechanisms, are small signaling molecules termed chemokines . Chemokines were originally identified in inflammatory pathways stimulating the migration of leucocytes, such as neutrophils and natural killer cells [5, 6]. Only in recent years chemokines were reported to induce the migration of tumor cells and their expression was correlated with tumor growth, angiogenesis and metastatic potential in various human carcinomas and animal models [7–9].
In the present study, we monitored the expression profiles of several ELR+ CXC chemokines and their corresponding receptor CXCR2 in benign and malign colorectal conditions and in CRLM. We focused on CXCL1 [growth-related oncogene α (GROα)] , CXCL5 [epithelial neutrophil -activating protein 78 (ENA-78)]  and CXCL6 [granulocyte chemotactic protein-2 (GCP2)] , all structurally related members of the CXC chemokine family. This chemokine family is further subdivided into ELR+ and ELR- CXC chemokines, based on the presence or absence of the amino acid sequence Glu-Leu-Arg (ELR) preceding the first conserved cysteine amino acid residue in the primary structure of the CXC chemokines. All three chemokines under investigation contain the ELR motif, which is important for ligand/receptor interactions and the regulation of CXC chemokine induced angiogenesis [13–15].
The angiogenic activity of all ELR+ CXC chemokines is mediated by the G-protein-coupled receptor CXCR2 , although some ELR+ CXC chemokines also signal through CXCR1. While CXCL1 activates and interacts exclusively by CXCR2, CXCL5 and CXCL6 signal through both receptors .
All three genes are structurally related to another member of the ELR+ chemokines, CXCL8 [interleukin-8 (IL-8)], which has recently been associated with CRC pathology and various other tumor types [18–21]. Like CXCL8 and other members of the ELR+ CXC chemokine family, CXCL6 is a neutrophil chemoattractant that was recently demonstrated to promote tumor growth through its angiogenic effects in human tumors and animal models [22, 23].
Involvement of CXCL5 has also been reported in different neoplastic processes with major focus on non small cell lung cancer (NSCLC), where CXCL5 was shown to be an important angiogenic factor . Accordingly, in pancreatic cancer cell lines angiogenic activity was demonstrated  and with view to CRC elevated protein quantities have been reported .
The third ELR+ chemokine under investigation, CXCL1, originally identified as melanoma growth stimulating activity, has recently been adressed a role in HIV-infection  and correlated with tumorigenic and angiogenic effects and metastasis in squamous cell carcinoma and melanoma [28, 29]. Recently, prostaglandin E2 (PGE2) was shown to induce the expression of CXCL1 in human CRC cells and to induce microvascular endothelial cell migration and tube formation in vitro . Moreover, elevated CXCL1 expression has recently been associated with an invasive phenotype in human colon carcinoma cells  and down-regulation of the matrix protein fibulin-1 .
Despite the increasing number of studies indicating a role for CXCL1, CXCL5 and CXCL6 in different cancer types and processes, their relevance with view to the development of CRC is still rather limited. Thus, we were prompted to investigate their expression profiles in colorectal adenoma (CRA) specimens as premalignant stages in the development of CRC, further in CRC tissues of different tumor categories and corresponding colorectal liver metastases (CRLM), to track potential differences in the expression levels between the transition from a premalignant condition to a colorectal malignancy.
Clinical characteristics of patients with colorectal carcinomas and colorectal liver metastases
CRC2 n = 48
CRLM3 n = 164
Localization of primary tumor
Largest tumor diameter (cm)5
TNM1 category of primary tumor
Chemotherapy before operation
Radiotherapy before operation
Clinical characteristics of patients with colorectal adenomas
CRA1 n = 30
Localization of colorectal adenoma, respectively
Tissue samples were collected immediately after resection, snap frozen in liquid nitrogen and then stored at -80°C until they were processed under nucleic acid sterile conditions for RNA and protein extraction. Tumor samples were taken from vital areas of histopathologically confirmed (R.M.B. and M.W.) 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 specimen, respectively. All tissues obtained were reviewed by a minimum of two experienced pathologists (M.W. et al.) and examined for the presence of tumor cells. As minimum criteria for usefulness for our studies we only chose tumor tissues in which tumor cells occupied a major component (> 80%) of the tumor sample.
Single-strand cDNA synthesis
Total RNA was isolated using RNeasy columns from Qiagen (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA integrity was confirmed spectrophotometrically and by electrophoresis on 1% agarose gels. For cDNA synthesis 5 μg of each patient total RNA sample were reverse-transcribed in a final reaction volume of 50 μl containing 1× TaqMan RT buffer, 2.5 μM/l random hexamers, 500 μM/l each 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). The reaction conditions were 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C.
Expression profiles of CXCL1, CXCL5 and CXCL6 were monitored in CRA and CRC tissue specimens and corresponding CRLM by Q-RT-PCR. Adjacent, disease and tumor free colorectal epithelium and liver samples served as control groups, respectively. All Q-RT PCR assays containing the primer and probe mix were purchased from Applied Biosystems and utilized according to the manufacturer's instructions. PCR reactions 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 mg/l). The theoretical basis of the Q-RT 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 of 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.
Consequently, the normal tissue becomes the 1 × sample, and all other quantities are expressed as an n-fold difference relative to this tissue.
Isolation of total protein
Protein lysates from frozen tissues were extracted with a radioimmunoprecipitation (RIPA) cell lysis and extraction buffer (Pierce, Rockford, USA). Total protein content was assessed by using the Pierce BCA protein assay reagent kit (Pierce).
Enzyme-linked immunosorbant assay (ELISA)
Tissue concentrations of CXCL1, CXCL5 and CXCL6 in the lysates were determined by sandwich-type ELISA according to the manufacturer's protocol: R&D systems (DuoSet, R&D Systems Inc. Minneapolis, Minnesota, USA). The absorbance was read at 450 nm.
Operative specimens were routinely fixed in formalin and subsequently embedded in paraffin. Before staining, 4-μm thick paraffin-embedded tissue sections were mounted on Superfrost Plus slides, deparaffinized and rehydrated in graded ethanol to deionized water. The sections were microwaved with an antigen retrieval solution (Target Retrieval, Dakocytomation, Carpinteria, CA, USA) and after blocking of endogenous peroxidase activity with 3% hydrogen peroxide, the sections were further blocked for 30 minutes with normal rabbit serum. Overnight incubation at 4°C with primary goat polyclonal anti-human CXCL5 antibody (15 μg/ml, AF254, R&D Systems, Minneapolis, Minn., USA) or primary goat anti-human CXCL1 antibody (3.5 μg/ml, sc-1374, Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was followed by incubation of secondary biotinylated biotinylated rabbit anti-goat IgG antibody and the avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit, Vector Laboratories, Burlingame, CA, USA). After colour reaction with aminoethylcarbazol solution (Merck, Darmstadt, Germany), tissues were counterstained with haematoxylin. Negative controls were performed in all cases omitting primary antibody. All pictures were taken using a digital camera (Olympus DP71, Olympus Optical Co, Ltd, Tokyo, Japan).
Laser Capture Microdissection
Laser microbeam microdissection (LMM) was employed for obtaining pure tumor cell and pure normal cell samples for subsequent genetic analysis. LMM was performed on three samples for each tissue type for CXCL1, CXCL5 and CXCL6. Histochemical staining was used on cryo sections before microdissection. Specimen preparation, microdissection and catapulting were performed following a laser pressure catapulting protocol according to the manufacturer's instructions (P.A.L.M. Microlaser Technologies, Bernried, Germany). RNA was extracted using the P.A.L.M. RNA extraction kit and for reverse transcription the invitrogen reverse transcription kit (Invitrogen Life Technologies, Karlsruhe, Germany) was applied. Subsequently quantitative PCR analysis was performed.
Calculations and Statistical Methods
Expression profiles of CXCL1, CXCL5 and CXCL6 in the different groups are shown as mean and standard error of the mean (SEM). Statistical calculations were done with the MedCalc software package (MedCalc software, Mariakerke, Belgium) . Where appropriate, either the Student's t-test or the Wilcoxon's rank sum test was applied to test for group differences of continuous variables. A P value of 0.05 or less was deemed significant. To measure how variables are related we performed a bivariate correlation analysis using the spearman ρ correlation coefficient.
CXCL1, CXCL5 and CXCL6 mRNA expression
To verify that up-regulation of CXCL1 and CXCL5 is generated exclusively by tumor cells rather than inflammatory cells or other non-malignant cells in the tissue blocks, we routinely analysed Q-RT-PCR expressions of several sections of microdissected tumor and normal cells from three specimens of all tissue types under investigation. Moreover, we generally determined the differences between gene expressions from matched normal/cancer samples constituting the basis for all our calculations.
CXCL1, CXCL5 and CXCL6 protein expression
Cell-specific expression of CXCL1 and CXCL5 by IHC
In tumor neighbor tissues of CRLM we detected no substantial CXCL5 reactivity. Only insular dotlike, cytoplasmic signals were randomly interspersed in some hepatocytes (Fig. 7E). In contrast, we observed intensive staining signals in epithelial and mesenchymal cells within tumor areas of CRLM specimens (Fig. 7F). Well confined from the positive signals in the tumor areas we also observed unspecific staining of bile pigments (left upper image border in Fig. 7F).
Comparative CXCR2 expression in different colorectal conditions
This study provides evidence that significantly increased CXCL1 and CXCL5 expression associates with the transition from a premalignant condition to colorectal malignancies. In contrast, CXCL6 showed no association with colorectal malignancies in our studies. CXCR2, the corresponding receptor, respectively, showed also significant up-regulation in CRC tissues and no significant up-regulation in the CRA tissues thus matching the expression profile of CXCL1 and CXCL5.
As we have shown previously (21) CXCL8 mRNA and protein expression is significantly up-regulated in CRC specimens in relation to CRA and UC tissues. Moreover, CXCL8 expression revealed a close correlation with tumor grading. Most interestingly, CXCL8 up-regulation was most enhanced in synchronous and metachronous CRLM, if compared with the corresponding primary CRC tissues thus suggesting an association between CXCL8 expression, induction and progression of colorectal carcinoma and the development of colorectal liver metastases. Thus, we observed for the expression levels of CXCL8, CXCL1 and CXCL5 a significant association with the development of colorectal carcinoma and also CRLM. For CXCL8 we also observed on the protein level that the expression was significantly higher in tumor stage 4 in comparison to the lower tumor stages. Also Q-RT-analysis revealed a similar effect for CXCL8.
However, in recent years a number of studies have emerged demonstrating CXCL6 involvement in tumor development and angiogenesis in various animal models and human tumors [22, 23]. Thus, van Coillie et al provided evidence that mouse CXCL6 gene transfer enhanced the development of Bowes melanoma tumors coupled with a higher neutrophil influx and significantly increased angiogenesis . Moreover, CXCL6 production by endothelial cells within gastrointestinal tumors was shown to contribute to tumor development and neovascularization  and constitutive CXCL6 secretion was recently demonstrated in a panel of small cell lung cancer (SCLC) cell lines and in clinical SCLC specimens .
Despite the increasing number of studies suggesting a cancer-related role for CXCL6, we observed no significant CXCL6 up-regulation in CRC tissues or CRLM, neither on the RNA nor on the protein level. Moreover, we measured no significant difference in CXCL6 expression between CRA, CRC or CRLM tissues in our patient cohort. In contrast, both other chemokines under investigation, CXCL1 and CXCL5, showed significant RNA and protein up-regulation in all colorectal malignancies comprising CRC and CRLM tissues. Accordingly, our IHC results confirmed that this up-regulation resulted from tumor cells as we detected intense CXCL1 and CXCL5 staining signals in CRC and CRLM specimens mainly concentrated in the epithelial cells of these tissues.
These findings are supported by a number of recent studies adressing a cancer-related role to these chemokines.
Thus, elevated CXCL5 protein levels have been reported in CRC patients  and in human pancreatic cancer cell lines . The mechanistic involvement of CXCL5 in the promotion of NSCLC was recently investigated in a study by Pöld et al , who demonstrated that enhanced tumor growth of COX-2 over-expressing tumors in a immunodeficient mouse model was associated with enhanced CXCL5 expression and inhibited by neutralizing anti-CXCL5 sera.
Also CXCL1 has recently been shown to promote tumor growth and angiogenesis in different tumor types. In human CRC cells, Wang et al  demonstrated recently, that PGE2 induced the expression of CXCL1 in the human CRC cells and also microvascular endothelial cell migration and tube formation in vitro. Moreover, the authors observed that PGE2 promoted tumor growth in vivo by induction of CXCL1 expression resulting in increased tumor microvessel formation. In murine models of squamous cell carcinoma and Lewis lung cancer CXCL1 expression was shown to parallel tumor growth [28, 39] and in adrenocortical and prostate carcinoma [40, 41] as well as in melanoma [29, 42, 43] CXCL1 was shown to mediate tumorigenicity. Evidence supporting a role for CXCL1 in CRC pathology was recently provided by a study demonstrating an association between increased expression of CXCL1 and down-regulation of the matrix protein fibulin-1, which is known to suppress the motility of various cancer cells . Accordingly, Li et al  investigated CXCL1 expression in human colon carcinoma cells with different metastatic potentials and demonstrated that constitutive expression of CXCL1 was associated with metastatic potential and modulation of colon cancer cell proliferation .
In line with these findings we also observed significant up-regulation of CXCL1 in neoplastic colorectal tissues. Moreover, we found significantly elevated CXCL1 expression in the CRA tissues of our patient cohort. However, both chemokines CXCL1 and CXCL5 were shown to be significantly higher expressed in the CRC tissues in comparison to the CRA tissues. Since CRA constitutes a premalignant condition often preceding the development of colorectal malignancies, we hypothesized that a significant increase of chemokine expression in the CRC tissues with respect to the CRA tissues may indicate a transition from the premalignant condition to the development of the malignancy. This conclusion is supported by previous studies showing elevated GROa expression in the intestinal mucosa of patients with ulcerative colitis and Crohn's disease which are known risk factors for the development of CRC [44, 45]. Although the basic expression level of CXCL1 was generally higher in both disease entities in comparison to CXCL5, we detected that CXCL5 overexpression was significantly more pronounced compared to CXCL1. Moreover, we observed a significant clinicopathological association between CXCL5 expression and early tumor categories of CRC which matched the expression status of the corresponding primary tumors of the CRLM in our investigation.
In our study, we outlined a prominent expression profile for both chemokines, CXCL1 and CXCL5 with respect to CRC pathology. On the basis of the current literature CXCL1 appears to have a role in CRC related malignancies. However, CXCL5 exhibited a much more pronounced significant overexpression in the CRC and CRLM tissues with respect to the CRA tissues thus indicating a vast progressive increase in the transition from the premalignant condition to the development of the malignant status and thus, in the initiation of CRC. Since our results are based merely on descriptive expression data, future functional tests will be needed for further evaluation of the precise pathophysiological role. However, the CXCL1 and CXCL5 expression profiles outlined in this study strongly recommend monitoring the CXCL1 and CXCL5 expression in patients with CRA and CRC. Thus, we suggest that targeting CXCL1 and CXCL5 signaling may be a useful future tool in CRC pathology.
We thank C. Weber, C. Blinn and B. Kopp for excellent technical assistance and HOMFOR (MW) for support with a digital camera (Olympus DP71).
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