CXC receptor-4 mRNA silencing abrogates CXCL12-induced migration of colorectal cancer cells
- Claudia Rubie†1Email author,
- Vilma O Frick†1,
- Pirus Ghadjar2,
- Mathias Wagner3,
- Christoph Justinger1,
- Sabrina K Faust1,
- Benjamin Vicinus1,
- Stefan Gräber4,
- Otto Kollmar1 and
- Martin K Schilling1
© Rubie et al; licensee BioMed Central Ltd. 2011
Received: 23 August 2010
Accepted: 24 February 2011
Published: 24 February 2011
Interactions between CXCR4 and its ligand CXCL12 have been shown to be involved in cancer progression in colorectal cancer (CRC). We performed a comparative CXCL12/CXCR4 expression analysis and assessed the effect of external CXCL12 stimulation on migration of CRC cells without and with CXCR4 inhibition.
Expression of CXCL12/CXCR4 was assessed by quantitative real-time PCR, ELISA and immunohistochemistry in resection specimens of 50 CRC patients as well as in the corresponding normal tissues and in three human CRC cell lines with different metastatic potential (Caco-2, SW480 and HT-29). Migration assays were performed after stimulation with CXCL12 and CXCR4 was inhibited by siRNA and neutralizing antibodies.
In CRC tissues CXCL12 was significantly down-regulated and CXCR4 was significantly up-regulated compared to the corresponding normal tissues. In cell lines CXCR4 was predominantly expressed in SW480 and less pronounced in HT-29 cells. CXCL12 was only detectable in Caco-2 cells. CXCL12 stimulation had no impact on Caco-2 cells but significantly increased migration of CXCR4 bearing SW480 and HT-29 cells. This effect was significantly abrogated by neutralizing anti-CXCR4 antibody as well as by CXCR4 siRNAs (P < 0.05).
CXCR4 expression was up-regulated in CRC and CXCL12 stimulation increased migration in CXCR4 bearing cell lines. Migration was inhibited by both neutralizing CXCR4 antibodies and CXCR4 siRNAs. Thus, the expression and functionality of CXCR4 might be associated with the metastatic potential of CRC cells and CXCL12/CXCR4 interactions might therefore constitute a promising target for specific treatment interventions.
Colorectal cancer (CRC) represents one of the most frequent malignancies worldwide with distant recurrence primarily affecting the liver as the predominant cause of CRC related mortality. The 5-year survival rate of 90% in patients with tumor restricted to the colon decreases to 10% in the presence of distant metastasis .
Recently, various cancer-related studies demonstrated that specific chemokines and their receptors may be involved in the molecular mechanisms that control metastasis in the early stages of cancer development . In this respect, the homeostatic chemokine CXCL12, a non-ELR+ CXC chemokine, has been implicated in promoting angiogenesis and metastasis [3, 4]. CXCL12, also known as stromal derived factor 1 (SDF-1), is the only chemokine that is essential for survival  and a highly efficacious chemoattractant for T cells and thymocytes [6, 7]. It is expressed by stromal cells such as fibroblasts and endothelial cells and signals exclusively via its G-protein-linked transmembrane receptor CXCR4 . Expression of functional CXCR4 has been reported in various types of cancer cells [9–12], but also in immune cells such as peripheral blood lymphocytes, unprimed T cells, dendritic cells and lymphocytic leukemia B cells, where CXCR4 mediates spontaneous migration beneath bone marrow and stromal cells . In infectious disease CXCR4 is employed by the human immunodefiency virus (HIV) to gain entry to cells  and in stem cell localization CXCR4 plays an important role for B-cell lymphopoiesis and bone marrow myelopoiesis . In breast cancer, CXCR4 signaling was shown to play a crucial role in distant recurrence by mediating actin polymerisation and pseudopodia formation, thus, leading to chemotactic and invasive responses .
Recently, CXCR4 expression was associated with advanced tumor stage and the development and recurrence of lymphatic or distant colorectal liver metastases (CRLM) [15–17]. Thus, it was reported that CXCR4 is differentially expressed in CRC and significantly correlates with survival, recurrence and liver metastasis [15, 18]. Moreover, it was shown that concomitant and high expression of CXCR4 and VEGF is a strong and independent predictor of early distant relapse in CRC  and recent evidence indicates that CXCR4 may also play a role in tumor angiogenesis of CRC .
Despite increasing knowledge about the expression of CXCL12/CXCR4 in CRC and its involvement of CXCR4 in the invasion and dissemination of CRC the precise mechanisms of CXCL12/CXCR4 interactions and subsequent metastasis are not entirely known. We therefore performed a comparative CXCL12/CXCR4 expression analysis and investigated how external addition of CXCL12 would promote CXCR4-mediated migration of CRC cell lines with different metastatic capabilities and how inhibition of CXCR4 by either CXCR4 siRNAs or neutralizing anti-CXCR4 antibodies would influence their migration potential.
Materials and patients
Clinical characteristics of patients with colorectal carcinomas
Localization of primary tumor
Hepatitis (A,B or C)
Largest tumor diameter (cm)3
TNM1 stage of primary tumor
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 adenocarcinomas. As corresponding normal tissue we used adjacent unaffected mucosa, 2-3 cm distal to the resection margin from the same resected adenocarcinoma. All tissues obtained were reviewed by an experienced pathologist (M.W.) 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 (>75%) of the tumor sample.
Single-strand cDNA synthesis
Total RNA was isolated using RNeasy columns from 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 (Foster City, CA). The reaction conditions were 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C.
All Q-RT PCR assays containing the primer and probe mix were purchased from Applied Biosystems, (Applied Biosystems, Foster City, CA) 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 and 1 μL gene assay (Applied Biosystems, Foster City, CA), 8 μL Rnase-free water and 1 μL cDNA template (50 mg/L). The theoretical basis of the qRT assays is described in detail elsewhere . All reactions were run in triplicate 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 the signal detection, ABI Prism 7900 sequence detector 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.
Gene expression of all target genes was analyzed in relation to the levels of the slope matched housekeeping genes phosphomannomutase (PMM1) and β2-Microglobulin (β2 M) . Data analysis was performed according to the relative standard curve method. Data are presented in relation to the respective housekeeping genes.
Isolation of total protein
Protein lysates from frozen tissues were extracted with the radioimmunoprecipitation (RIPA) cell lysis and extraction buffer from Pierce (Pierce, Rockford, USA). Total protein content was assessed by using the Pierce BCA protein assay reagent kit (Pierce, Rockford, USA).
Sandwich-Type Enzyme-Linked Immunosorbent Assay
The chemokine protein levels in the different tissue lysates were determined by sandwich-type enzyme-linked immunosorbent assays (ELISA) according to the manufacturer's instructions. Samples were assayed in duplicate with all values calculated as the mean of the two measurements. CXCL12 levels were assayed using a validated commercial ELISA (Duo Set R&D Systems, DY350, Minneapolis, Minn., USA). The absorbance was read at 450 nm in a 96-well microtiter plate reader. The chemokine concentration from each tissue lysate was normalized to the total protein content of each sample.
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 sequential treated with avidin and biotin (Avidin/Biotin blocking kit, Vector Laboratories Inc., Burlingame, CA, USA). In addition the specimens were further blocked for 30 min at room temperature with normal rabbit serum. Overnight incubation at 4°C with primary goat polyclonal anti-human CXCR4 antibody (1:300, AB1671, Abcam, Cambridge, UK) was followed by incubation of secondary 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. Immunocytochemical staining of CRC cells was performed using culture slides from Becton Dickinson (Becton Dickinson, Heidelberg, Germany). After blocking of endogenous peroxidase activity and blocking for 30 min at room temperature with normal rabbit serum, cells were overnight incubated at 4°C with primary goat polyclonal anti-human CXCR4 antibody (1:300, AB1671, Abcam, Cambridge, UK). Following incubation of secondary biotinylated rabbit anti-goat IgG antibody and the avidin-biotin-peroxidase reaction (Vectastain ABC ELITE Kit, Vector Laboratories, Burlingame, CA, USA) and all further steps were carried out as described above for the immohistochemical staining procedure.
Negative controls were conducted in all cases omitting primary antibody. For evaluation of immunocytochemical staining the total number of cells per 5 high-power fields (using ×40 -HPF objective magnification) was determined. Cells were considered positive, when they demonstrated strong and exclusive labelling.
Cell migration assays
Cell migration assays were performed in triplicate using BD Falcon cell culture inserts containing polyethylene terephthalate membranes (8 μm pore size) from BD Biosciences (Bedford, MA, USA). Caco-2, HT-29 and SW480 cells at a concentration of 50000 cells per ml in 500 μl medium with antibiotics but without FCS were placed in the top of a two-chamber assay system and incubated for 48 hours with and without CXCL12 (350-NS-10, R&D Systems, Wiesbaden, Germany) in 800 μl medium without FCS placed in the lower chamber. CXCL12 concentrations in the receiver wells were 10 ng, 50 ng and 100 ng per ml, respectively. After the incubation period, the non-migrated cells on the upper surface of the filters were removed using a cotton swab. Migrated cells, which are adherent to the lower surface of the filters, were stained with a 0.1% crystal violet solution (Merck, Darmstadt, Germany). The number of these migrated cells was quantified microscopically by counting them in 10 high-power microscopic fields. Medium with 20% FCS placed in the lower chamber of the two-chamber assay system served as a positive control and reference in the migration assays.
Cell proliferation assays
Cell proliferation assays (Roche Diagnostics GmbH, Mannheim, Germany) were performed in triplicate using flat-bottomed 96-well microtiter plates for culturing Caco-2, HT-29 and SW480 cells in the presence of CXCL12 at 37°C for 48 hours. Subsequently, bromodeoxyuridine (BrdU) was added to the cells and the cells were reincubated for 24 hours. During this labeling period the pyrimidine analogue BrdU is incorporated in place of thymidine into the DNA of proliferating cells. After removing the culture media, cells were fixed and anti-BrdU-POD was added to bind to the BrdU incorporated in newly synthesized cellular DNA. Immune complexes were detected by the subsequent substrate reaction and the reaction product was quantified by photometrically measuring the absorbance of the developed color.
Cell inhibition assays
Inhibition assays were performed in analogy to the cell migration assays using BD Falcon cell culture inserts from BD Biosciences. Similar to the migration assays Caco-2, HT-29 and SW480 cells in 500 μl medium without FCS were mixed with anti-CXCR4 antibodies (MAB171, R&D systems) applied in a final concentration of 6 μg per ml and placed in the upper chamber of a two-chamber assay system. After incubation for 48 hours with and without CXCL12 in 800 μl medium without FCS placed in the lower chamber steps were continued as mentioned above for the migration assays and migrated CRC cells were quantified microscopically by counting the cells that had migrated into the filters.
Applied RNAi control experiments - overview
Positive control siRNA
siRNA that is known to provide high knockdown of its target gene
Hs_MAPK1 siRNA (Qiagen)
Negative control siRNA
a nonsilencing siRNA with no homology to any known mammalian gene
AllStars Negative control siRNA (Qiagen)
Transfection control siRNA
siRNA that is used to measure the transfection efficiency, e.g. by siRNA
AllStars Hs Cell Death Control siRNA (Qiagen)
Mock transfection control
control experiment where cells go through the transfection process without addition of siRNA
Untransfected cells control
control experiment where gene expression analysis is carried out on cells that have not gone through the transfection process
Western Blot Analysis
Gene silencing was monitored 72 hours after transfection at the protein level using western blotting. Total protein (25 μg/lane) was separated by SDS-PAGE using a 10% gel and blotted onto nitrocellulose membranes (Hybond ECL, Amersham Biosciences, Piscataway, NJ, USA). Membranes were blocked by incubation in Tris-buffered saline (TBS) containing 5% non fat dry milk and 0.1% Tween 20 for 2 h at room temperature and then incubated overnight at 4°C with primary goat polyclonal anti-human CXCR4 antibody (1:300, AB1671, Abcam, Cambridge, UK). Blots were then washed and incubated at room temperature for 1 h with donkey anti-goat HRP antibody (diluted 1:5000, sc-2056, Santa Cruz Biotechnology, Santa Cruz, CA USA). Bands were visualized by ECL Western blotting analysis systems (Amersham Biosciences, Piscataway, NJ, USA). The human cell lysate HL-60 (sc-2209, Santa Cruz Biotechnology, Santa Cruz, CA, USA) served as positive control. Quantification of band intensities has been performed on three independent samples using image J software.
Calculations and Statistical Methods
Chemokine receptor expression profiles in the different groups are shown as mean and standard error of the mean (SEM). Statistical calculations were done with the MedCalc software package. 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 considered significant.
CXCL12/CXCR4 expression in CRC tissues
Organ-specific expression of CXCL12
CXCL12/CXCR4 expression in CRC cell lines
CXCR4 protein inhibition abrogates migration of colorectal cancer cells
When we added neutralizing anti-CXCR4 antibody prior to performance of cell migration assays, the CXCL12 stimulated migration of HT-29 and SW480 cells was significantly abrogated at all CXCL12 concentrations under investigation (Figure 4B and 4C, respectively) (P < 0.05). Application of anti-CXCR4 antibodies had no impact on the migration potential of Caco-2 cells (Figure 4A).
CXC receptor-4 gene silencing abrogates migration of colorectal cancer cells
Proliferative rate of colorectal cancer cells after CXCR4 blockage by mRNA silencing and inhibition antibodies
The importance of the CXCL12/CXCR4 axis in tumor progression and metastasis has been emphasized. Recent evidence also suggests that the CXCL12/CXCR4 system is involved in the progression and metastasis of CRC [15, 20, 28].
Thus, the aim of this study was to conduct a comparative CXCL12/CXCR4 expression analysis and to investigate the functional role of CXCR4 in metastatic and non-metastatic CRC derived cell lines. For this purpose the migration potential of these cell lines was tested under conditions where CXCR4 was inhibited on the mRNA as well as on the protein level. On the mRNA level, CXCR4 silencing was achieved by CXCR4 RNA interference while on the protein level inhibition of CXCR4 protein activity was achieved using an anti-CXCR4 antibody.
As shown initially, CXCL12 protein is significantly higher expressed in the liver in relation to other intestinal organs or glands, thus supporting potential CXCL12 involvement in the homing of CRC cells to the liver. Subsequently, we observed significant up-regulation of CXCR4 and significant down-regulation of CXCL12 in CRC tissues. This inverse expression pattern is supported by previous studies, where CXCR4 was shown to be highly up-regulated in all T-stages of CRC tissues  and CXCL12 expression levels were shown to be low at the base of the crypts and increased in the more differentiated apical intestinal epithelial cells [29, 30]. In compliance with the low CXCL12 expression status in tissues we observed low or no CXCL12 expression, respectively, in three human CRC derived cell lines, non-metastatic cell line Caco-2 and metastatic cell lines SW480 and HT-29. While low CXCL12 expression was detected in Caco-2 cells on the mRNA and on the protein level, CXCL12 was below the detection limit in HT-29 and SW480. It was hypothesized that changes in epithelial CXCL12 expression might contribute to CRC disease progression by allowing CRC cells to more readily sense CXCL12 from exogeneous sources hereby promoting metastasis . Wendt et al. have shown that the reduced CXCL12 expression pattern in CRC tissues and cells is due to DNA hypermethylation in primary CRC and carcinoma-derived cell lines. Thus, it was demonstrated that inhibition or ablation of DNA methyltransferases prevent promoter methylation and restore CXCL12 expression. In addition, re-expression of functional, endogeneous CXCL12 in CRC cells was shown to reduce metastatic tumor formation significantly while silencing CXCL12 greatly enhanced the metastatic potential of CRC cells . Moreover, endogeneous CXCL12 was shown to provide a barrier to metastasis by increasing anoikis via activation of a Bim-mediated intrinsic apoptotic pathway [32, 33]. These results may constitute a plausible explanation for our observation of significantly reduced CXCL12 expression rates in CRC tissues and carcinoma-derived cell lines. Concordantly, an elevated migratory signaling response to ectopic CXCL12 was also shown to contribute to the metastatic potential of CXCR4-expressing mammary carcinoma cells, subsequent to epigenetic silencing of autocrine CXCL12 .
For CXCR4 we observed an inverse expression pattern in the three CRC-derived cell lines. Thus, expression in non-metastatic cell line Caco-2 was significantly lower compared to the metastatic cell lines under investigation. Also drug-resistant invasive HT-29 cells with a metastatic behaviour in immunodeficient mice were shown to exhibit high CXCR4 expression . Responsible for promoting invasion in drug-resistant colon carcinoma cells is the autocrine CXCR4 ligand macrophage migration-inhibitory factor (MIF) as shown by Dessein et al. Impairing the MIF-CXCR4 signaling pathway, e.g. by silencing CXCR4, abolished this aggressive phenotype. To date, in various cancer entities aberrantly expressed chemokine receptors were demonstrated to contribute directly to the development of organ selective distant metastasis by interaction with their organ selectively expressed chemokine ligands. As a consequence of the low CXCR4 expression, Caco-2 cells showed no increase in migration in response to CXCL12 stimulation. In contrast, CXCL12 significantly increased cell migration in the CXCR4 expressing metastatic cell lines SW480 and HT-29. While a stimulative effect of CXCL12 on migration of CRC cell lines was described before , these effects were not significant and refer only to one CXCL12 concentration (100 ng/ml). Moreover, these tests were performed on CRC cell lines LS174T, SW620 and SW480 but not on cell lines HT-29 and Caco-2. To further investigate the functional role of CXCR4 we performed inhibition assays with anti-CXCR4 antibodies added previously to CXCL12 stimulation. The addition of the inhibition antibodies significantly blocked the CXCL12-dependent stimulation of HT-29 and SW480 cell migration but had no impact on Caco-2 migration. Our results correlate with inhibition studies performed on SW480 cells , where chemotaxis induced by CXCL12 was also shown to be blocked, although not as complete as we have shown for both SW480 and HT-29 cells. Likewise, Kim et al  reported on a partial inhibitory effect of anti-CXCR4 antibodies on CXCL12 stimulated melanoma and CRC cell migration (38%). However, to date no study analyzed the effect of CXCR4 gene silencing on CXCL12 mediated cell migration of CRC cells. Thus, we investigated if the chemotactic effect of CXCL12 on the migration of HT-29 and SW480 cells could be counteracted by the down-regulation of the CXCR4 mRNA. Applying four different CXCR4 siRNAs we achieved a mean percent CXCR4 knockdown of 78-84% and a significant abrogation of CXCL12 stimulated migration of HT-29 and SW480 for all four different CXCR4 siRNAs at all CXCL12 concentrations under investigation. On the other hand, application of CXCR4 siRNAs had no impact on the migration potential of Caco-2 cells. Thus, we have demonstrated the functional status of the CXCR4 receptor on CRC cell lines in response to CXCL12 on the mRNA level.
In CRC patients, no significant difference in CXCR4 expression was detected according to the metastatic behaviour. Based on our in vitro results CRC patients with metastasis may be expected to express more CXCR4 in their tumor cells compared to CRC patients without metastasis. However, many other important factors contribute to the metastatic properties of tumor tissues and influence their metastatic potential. When a CRC cell is turned metastatic, CXCR4 may be an important factor for the homing of such a tumor cell to its favourite organ destination, the liver.
Our results are well in line with recent findings demonstrating a role of CXCL12 in promoting cell migration and tumor growth of CRC metastasis in vivo in a murine model . However, while we observed no CXCR4-dependent proliferation of CRC cells, Shen et al. demonstrated CXCR4-induced proliferation for pancreatic cancer cells, where it was linked to AKT and ERK dependent pathways . Moreover, CXCR4 knockdown by small interfering RNA was shown to inhibit cell proliferation and invasion of oral squamous cell carcinoma cells . A recent study demonstrated that CXCL12/CXCR4 interactions may also promote early extravasation of liver metastatic epithelial tumor cells which determines a critical step in formation of organ-specific metastases . Currently, various small-molecule chemokine receptor antagonist compounds are undergoing development in phase I to III studies in infectious and autoimmune diseases and a CCR5 inhibitor is already in the clinic for the treatment of HIV-infected patients.
In conclusion, our results provide evidence that CXCR4 is up-regulated in CRC and stimulation of CXCR4 bearing cancer cells with CXCL12 led to increased migration, an effect which could be inhibited both by CXCR4 siRNA and neutralizing CXCR4 antibodies. Interestingly, CXCR4 was predominantly expressed in cell lines with metastatic potential and consequently, these cell lines showed increased migration after external CXCL12 stimulation. Our results suggest that the metastatic potential of CRC cells may be associated with the aberrant expression of CXCR4 and subsequently the ability of cells to interact with CXCL12 via autocrine and/or paracrine mechanisms.
We thank B. Kruse for excellent technical assistance.
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