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Carcinogenesis Advance Access originally published online on October 9, 2008
Carcinogenesis 2009 30(2):205-213; doi:10.1093/carcin/bgn228
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Stromal cell-derived factor-1{alpha} (SDF-1{alpha}/CXCL12)-enhanced angiogenesis of human basal cell carcinoma cells involves ERK1/2–NF-{kappa}B/interleukin-6 pathway

Chia-Yu Chu1,2, Shih-Ting Cha2, Wan-Chi Lin2, Po-Hsuan Lu3, Ching-Ting Tan4, Cheng-Chi Chang2, Ben-Ren Lin5, Shiou-Hwa Jee1,{dagger} and Min-Liang Kuo2,{dagger},*

1 Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 100, Taiwan
2 Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine and Angiogenesis Research Center, National Taiwan University, No.1, Sec.1, Jen-Ai Road, Taipei 100, Taiwan
3 Department of Dermatology, Mackay Memorial Hospital, Taipei 100, Taiwan
4 Department of Otolaryngology
5 Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 100, Taiwan

* To whom correspondence should be addressed. Tel: +886 2 2312 3456 8607; Fax: +886 2 2341 0217; Email: kuominliang{at}ntu.edu.tw

Correspondence may also be addressed to Shiou-Hwa Jee. Tel: +886 2 2356 2141; Fax: +886 2 2393 4177; Email: shiouhwa{at}ntu.edu.tw


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Stromal cell-derived factor 1{alpha} (SDF-1{alpha}) (CXCL12) has been observed to enhance tumor angiogenesis. However, the comprehensive role of SDF-1{alpha} (CXCL12)–CXCR4 interaction, exerted during angiogenesis, has not been well understood. We have previously demonstrated that human basal cell carcinoma (BCC) tissues and a BCC cell line (BCC-1/KMC) had significant expression of CXCR4, whose level was higher in invasive than in the non-invasive BCC types. Here, we observed that human BCC tissues with high expression levels of CXCR4 had higher vascularity. Further, among the 71 BCCs diagnosed between the years 2004–2005, BCCs with high CXCR4 expression had concomitantly higher microvessel density, as compared with those with low CXCR4 expression (P < 0.001). We found that SDF-1{alpha} induced angiogenic activity in human BCC cells, both in vitro and in vivo. SDF-1{alpha} significantly upregulated several angiogenesis-associated genes such as interferon-alpha-inducible protein 27, interleukin (IL)-6, bone morphogenetic protein (BMP)-6, SOCS2 and cyclooxygenase 2 (COX)-2 in human BCC cells. Among them, IL-6 was the earliest and highest upregulated gene whose induction was observed within 6 h of the commencement of SDF-1{alpha}–CXCR4 interaction. The mechanisms behind the SDF-1{alpha}-induced time and dose-dependent upregulation of messenger RNA expression and protein secretion of IL-6 were investigated. The transcriptional regulation of IL-6 by SDF-1{alpha} was mediated by phosphorylation of extracellular signal-related kinase 1/2 and activation of the nuclear factor-{kappa}B complex. The identification of the angiogenic profiles induced through SDF-1{alpha}–CXCR4 interactions in human BCC cells may contribute further insights into the mechanisms involved in the angiogenic potential of SDF-1{alpha} (CXCL12).

Abbreviations: AP, activator protein; BCC, basal cell carcinoma; bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; CM, conditioned medium; COX, cyclooxygenase; ERK1/2, extracellular signal-related kinase 1/2; HUVEC, human umbilical vein endothelial cell; IFI27, interferon-alpha-inducible protein 27; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; mRNA, messenger RNA; MVD, microvessel density; NF-{kappa}B, nuclear factor-{kappa}B; RT, reverse transcription; SCC, squamous cell carcinoma; SDF-1{alpha}, stromal cell-derived factor 1{alpha}; VEGF, vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Tumor growth and metastasis depend upon the ability to induce its own blood supply; hence, angiogenesis represents an essential step for the neoplastic growth and also for the expansion of the metastatic colonies (1). A number of studies have shown the association between intratumor microvessel density (MVD) and tumor aggressiveness (2–4).

Chemokines are structurally related, small (8–14 kDa) polypeptide signaling molecules that bind to and activate a family of seven transmembrane G protein-coupled receptors, the chemokine receptors (5). Recently, interaction between the chemokine receptor CXCR4 and its ligand, stromal cell-derived factor 1{alpha} (SDF-1{alpha} or CXCL12), has been found to play an important role in tumorigenicity, proliferation, metastasis and angiogenesis in many cancers such as lung cancer, breast cancer, melanoma, glioblastoma, pancreatic cancer and cholangiocarcinoma (6–14). SDF-1{alpha} (CXCL12) may promote tumor angiogenesis directly by stimulating the formation of capillary-like structures in human vascular endothelial cells or attracting endothelial stem cells (15–18). On the other hand, SDF-1{alpha} (CXCL12) can also enhance tumor angiogenesis indirectly, by inducing the secretion of several angiogenic factors such as vascular endothelial growth factor (VEGF) and interleukin (IL)-8 from tumor cells or infiltrating cells (19–23). However, the overall angiogenesis profiles regulated by SDF-1{alpha} (CXCL12)–CXCR4 interactions have not been comprehensively investigated by a high-throughput microarray approach.

Basal cell carcinoma (BCC) is one of the most common skin neoplasms in humans and is characterized by local aggressiveness with little metastatic potential (24), though BCC may behave aggressively with deep invasion, recurrence and regional and distant metastasis (25). BCC can be classified as superficial, nodular, micronodular, infiltrative (including morpheaform) or mixed type based on the histology of its growth pattern. The latter three BCC types have been classified as aggressive forms since they more frequently exhibit deep invasion potential (25,26). The tumor vascularization was also found to correlate with the aggressive phenotype in human BCC (3,4). Previously, human BCC tissues and a BCC cell line have shown significant levels of CXCR4 expression, which was higher in invasive than in the non-invasive BCC types (22,27). Although CXCR4 expression was shown to enhance tumorigenesis and angiogenesis of BCC in a previous study, the clinical correlation of CXCR4 expression and tumor vascularization as well as detailed mechanisms of its regulation in angiogenesis were not elucidated (22). In this study, we aimed to examine the expression profiles of SDF-1{alpha}/CXCR4-regulated angiogenesis-associated genes in BCC cells by using a DNA oligonucleotide microarray and also elucidate the underlying mechanism involved.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell origin and cell culture
The human BCC cell line (BCC-1/KMC) was established from human BCC derived from the undifferentiated type of BCC tumor arising from a thermal traumatic scar (28,29). Passages 136–142 of this cell line were used in the present investigation. For all the experiments, if not specifically indicated, BCC-1/KMC cells were cultured in medium containing 10% fetal calf serum first. At confluence, the BCC-1/KMC cells were serum starved for 24 h and then treated with SDF-1{alpha} at various concentrations for indicated duration of time under no serum condition.

Establishment of BCC/CXCR4 transfectants
A constitutive expression vector, pCMV-CXCR4, carrying 1.09 kb full-length human CXCR4 complementary DNA under the control of the cytomegalovirus promoter/enhancer sequence was constructed. BCC cells were transfected with pCMV-CXCR4 or control pcDNA3 vector (GIBCO Invitrogen, Grand Island, NY) containing a cytomegalovirus promoter and a neomycin selection marker, using the TransFastTM transfection reagent (Promega, Madison, WI). Twenty-four hours after transfection, cells were replated in RPMI-1640 (Gibco BRL, Rockville, MD) with 10% (vol/vol) fetal calf serum and 500 µg/ml G418 (Sigma, St Louis, MO). G418-resistant clones were selected and expanded. The messenger RNA (mRNA) and protein levels of CXCR4 in these cells were checked by reverse transcription (RT)–polymerase chain reaction and western blot analysis. BCC cells transfected with control vector (BCC/pcDNA3) served as control. Surface expression of CXCR4 was examined by flow cytometric analysis. Human BCC/CXCR4 and BCC/pcDNA3 cells were stained with isotype control or mouse anti-human CXCR4 monoclonal antibodies followed by fluorescein isocyanate-conjugated secondary goat anti-mouse antibody and quantified (in triplicate) by flow cytometry. Histograms of CXCR4 expression were shown along with the mean fluorescence intensities of stained cells. CXCR4 surface expression was calculated as the relative ratio of the mean fluorescence intensities of cells stained with CXCR4 antibody compared with those stained with the isotype control antibody (supplementary Figure 1 is available at Carcinogenesis Online). BCC/CXCR4 and BCC/pcDNA3 cells were grown at 37°C and 5% CO2 in RPMI-1640 medium supplemented with 10% fetal calf serum and 100 µg/ml G418.

Antibodies and reagents
The anti-CXCR4 (clones 12G5), anti-CXCL12, anti-IL-6 and isotype control antibodies were purchased from R&D Systems (Minneapolis, MN). The anti-CD31 antibody (sc-1506), anti-nuclear factor-{kappa}B (NF-{kappa}B) p65 antibody (sc-8008), anti-I{kappa}B{alpha} antibody (sc-371), as well as the affinity-purified monoclonal mouse anti-Akt, anti-phospho-Akt, anti-c-Jun N-terminal kinase (JNK), anti-phospho-JNK, anti-p38 mitogen-activated protein kinase (MAPK), anti-phospho-p38 MAPK, anti-extracellular signal-related kinase 1/2 (ERK1/2) and anti-phospho-ERK1/2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-c-Jun and anti-c-Jun were from Cell Signaling Technology (Danvers, MA). The CXCR4-specific chemical inhibitor (AMD3100), the MAPK/ERK kinase inhibitor (U0126), selective JNK inhibitor (SP600125) and NF-{kappa}B inhibitor (pyrrolidinethiocarbamate) were purchased from Sigma. I{kappa}B{alpha} phosphorylation inhibitor (Bay 11-7082) was obtained from Calbiochem (La Jolla, CA).

Preparation of conditioned medium
BCC cells were plated in 1 ml of culture medium at 1 x 105 cells per well in 24-well 18 mm culture dishes. After serum starvation for 1 day, the BCC cells were treated with 200 ng/ml SDF-1{alpha} for various time periods. The culture supernatants were collected and centrifuged sequentially at 12 500g with Microcon YM-3 centrifugal filter devices (Millipore Co., Bedford, MA) (with the cutoff of molecules smaller than 3000 Da) for 30 min to obtain a 10-fold concentrate of culture supernatant.

Immunohistochemistry, RT–polymerase chain reaction, western blot analysis of the cell lysate, enzyme immunoassay (enzyme-linked immunosorbent assay), in vitro capillary tube formation on Matrigel and tumorigenicity assay and in vivo angiogenesis potential in nude mice
Details are available as supplementary data (available at Carcinogenesis Online).

Total RNA purification and DNA oligomicroarray for angiogenesis-associated genes
Total RNA was extracted by Trizol® Reagent (Invitrogen, Carlsbad, CA) and followed by RNeasy Mini Kit (Qiagen, Hilden, Germany). Purified RNA was quantified at optical density 260 nm by using a ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and qualitated by using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) with RNA 6000 nano labchip kit (Agilent Technologies). Total RNA (0.5 µg) was amplified by a low RNA input fluor linear amp kit (Agilent Technologies) and labeled with Cy3 or Cy5 (CyDye, PerkinElmer, Waltham, MA) during the in vitro transcription process. Total RNA from BCCs after SDF-1{alpha} treatment for 6, 12 or 24 h was labeled by Cy5 and RNA from BCCs after SDF-1{alpha} treatment for 0 h was labeled by Cy3. Two micrograms of Cy-labeled complementary ribonucleic acid (cRNA) was fragmented to an average size of ~50–100 nucleotides by incubation with fragmentation buffer at 60°C for 30 min. Correspondingly, fragmented labeled cRNA was then pooled and hybridized to the angiogenesis-associated oligo microarray (Agilent Technologies) at 60°C for 17 h. The angiogenesis oligonucleotide microarray contained 561 oligonucleotide probes corresponding to individual angiogenesis-associated genes or expressed sequence tags (supplementary data are available at Carcinogenesis Online). Oligonucleotide probe sequences were chosen to maximize gene specificity and minimize the 3' replication bias inherent in RT of mRNA. In addition, the microarray formats contained ~1000 control probes for quality control purposes. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images were analyzed by Feature extraction 9.1 software (Agilent Technologies), an image analysis and normalization software used to quantify signal and background intensity for each feature, and the data were substantially normalized by rank-consistency-filtering LOWESS method.

Nuclear and cytosolic protein extraction
For nuclear and cytosolic protein extraction, the protein extracts were prepared from SDF-1{alpha}-treated BCC-1/KMC cells using a modified procedure, described previously (27).

NF-kB decoy oligodeoxynucleotide and antisense c-Jun oligonucleotide treatment
The synthetic double-stranded oligodeoxynucleotides for NF-kB decoy and scrambled decoy were synthesized and utilized as described (30). The c-Jun antisense oligonucleotide used in this study was c-Jun antisense, 5'-CGTTTCCATCTTTGCAGT-3', which was used as described before (27).

IL-6 promoter assay and transient transfection
A full-length human IL-6 promoter construct (phIL-6-luc) containing the 1.2 kb 5'-flanking region of the human IL-6 gene was kindly provided by Dr C-H Chou and Dr Jason C-H Cheng (Department of Oncology, National Taiwan University Hospital). The luciferase reporter plasmid pIL-6-luc cloned into the pGL2 basic luciferase reporter vector (Promega) and the control vector were transfected as described (30). In addition, different constructs of luciferase reporter plasmids were kindly provided by Dr Oliver Eickelberg (Department of Medicine II, University of Giessen, Giessen, Germany). The parental plasmid, pIL6-Luc651, contained a 651 bp fragment of the human IL-6 gene promoter, located directly upstream of the transcriptional start site, subcloned into pGL3 basic luciferase reporter gene vector. Within pIL6-luc651, the following consensus sites were inactivated by site-directed mutagenesis. The activator protein (AP)-1 consensus sequence (positions –283 to –276, 5'-TGAGTCAC-3') was changed to 5'-TGCAGCAC-3'; the C/EBP-β consensus sequence (positions –154 to –146, 5'-TTGCACAAT-3') was changed to 5'-CCGTTCAAT-3' and the NF-{kappa}B consensus sequence (positions –72 to –63, 5'-GGGATTTTCC-3') was changed to 5'-CTCATTTTCC-3'. These mutations have previously been shown to inactivate the described consensus sequences (31). All mutant clones were verified by DNA sequencing. The transient transfection experiments were performed as described (27).

Statistical analysis
The two-tailed Student’s t-test was used for simple comparison of two values wherever appropriate. All data were expressed as mean ± SD from at least three independent experiments. All statistical tests were two sided. The Chi-square test was used for comparison of CXCR4 expression between non-invasive and invasive BCCs. A P-value of <0.05 was considered statistically significant for all tests. All analyses were performed with the use of SAS software (version 8.02, SAS institute, Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Correlation of CXCR4 expression and MVD of human BCC tumors
Immunohistochemical examination of human BCC tissues demonstrated that tumors with high CXCR4 expression had more blood vessels in the stroma surrounding the tumors as compared with BCC tumors with low CXCR4 expression (Figure 1). To examine the relationship of CXCR4 expression with tumor angiogenesis, we studied its expression in different histological types of human BCCs. We collected 71 human BCCs diagnosed during 2004–2005. The MVD of BCCs with high CXCR4 expression was significantly higher (15.9 ± 4.6) compared with that of low CXCR4-expressing BCCs (7.8 ± 3.1, P < 0.001), indicating that CXCR4 may be involved in BCC tumor angiogenesis (Table I).


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  		Table I. Correlation of MVD with the expression of CXCR4 in both non-invasive and invasive BCCs

 


Figure 1
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  		Fig. 1. Human BCCs with high CXCR4 expression also have high CD31 expression (MVD). (A and B) High CXCR4 expression in BCCs. (C and D) Low CXCR4 expression in BCCs. Bar, 10 µm. Arrows indicate vessels with CD31 positive; insert, higher magnification.

 
SDF-1{alpha}–CXCR4 interaction induces angiogenesis potential in BCC-1/KMC cells
The effect of SDF-1{alpha}–CXCR4 interaction on angiogenesis in vitro was examined by tube formation of human umbilical vein endothelial cells (HUVECs) cultured with conditioned medium (CM) collected from BCC-1/KMC cells after stimulation with 200 ng/ml SDF-1{alpha} for 24 h. Formation of the network-like tubular HUVEC structure involves the migration, invasion and differentiation of endothelial cells. The CM obtained from the SDF-1{alpha}-treated BCCs showed increased tube formation per microscopic field as compared with vehicle controls (13.0 ± 1.0 versus 2.3 ± 0.6, P < 0.001). This angiogenic effects could be inhibited by pretreatment of cells with the CXCR4-neutralizing antibody (12G5) or a specific inhibitor, AMD3100 (Figure 2A). In addition, we also examined the HUVEC tube formation ability of CM collected from SDF-1{alpha}-treated BCCs after pretreatment of the HUVEC with the CXCR4-neutralizing antibody (12G5) or pretreatment of the collected CM with the SDF-1{alpha}-neutralizing antibody in order to exclude the possible angiogenic effects of residual SDF-1{alpha} in the CM. The CM obtained from the SDF-1{alpha}-treated BCCs showed significant effects on tube formation as compared with vehicle controls (CXCR4-neutralizing antibody-pretreated group, 17.6 ± 2.1 versus 0.6 ± 0.5, P < 0.001 and CXCL12-neutralizing antibody-pretreated group, 12.1 ± 1.2 versus 3.2 ± 1.4, P < 0.001), confirming the angiogenic potential of CM from BCC-1/KMC cells after SDF-1{alpha} stimulation (Figure 2B–D).


Figure 2
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  		Fig. 2. SDF-1{alpha} treatment in human BCC cells induces tube formation of HUVECs. (A) Ten times concentrated culture medium (CM) collected from human BCC cells after stimulation with 200 ng/ml SDF-1{alpha} for 24 h showed increased tube formation per microscopic field (9.7 mm2) as compared with vehicle controls (13.0 ± 1.0 versus 2.3 ± 0.6, P < 0.001). This angiogenic effects could be inhibited by pretreatment of BCC cells with the CXCR4-neutralizing antibody (12G5) or AMD3100. To exclude the possible angiogenic effects of residual SDF-1{alpha} in the CM, we pretreated the HUVEC with the CXCR4-neutralizing antibody (12G5) or added the SDF-1{alpha}-neutralizing antibody into the collected CM. HUVEC tube formation ability of CM collected from SDF-1{alpha}-treated BCCs after (B and C) pretreatment of HUVECs with the 12G5 (5 µg/ml for 30 min) or (D) pretreatment of the collected CM with the SDF-1{alpha}-neutralizing antibody (120 µg/ml for 30 min) showed significant effects of tube formation as compared with vehicle controls (CXCR4-neutralizing antibody-pretreated group, 17.6 ± 2.1 versus 0.6 ± 0.5, P < 0.001; CXCL12-neutralizing antibody-pretreated group, 12.1 ± 1.2 versus 3.2 ± 1.4, P < 0.001). The number of tube-like structures is expressed as the mean ± SD following a random count of five 9.7 mm2 microscopic fields.

 
Increased tumor growth and tumor angiogenesis in BCC/CXCR4 cells
To confirm the angiogenic effects of CXCR4 in vivo, we performed animal studies by subcutaneously injecting BCC/CXCR4 cells and BCC/pcDNA3 cells (1 x 106 each) into nude mice. The tumor size in BCC/CXCR4-injected mice was higher (1232.22 ± 629.78 mm3) than that of control BCC/pcDNA3-injected mice (182.78 ± 76.45 mm3; P < 0.05) on day 49 (Figure 3A). The xenografts from the BCC/CXCR4 group had higher vascularity (Figure 3B). In addition, the MVD quantified by CD31-positive vessels was higher in BCC/CXCR4 group compared with the BCC/pcDNA3 group (16.9 ± 1.8 versus 5.5 ± 1.5, P < 0.01; Figure 3C).


Figure 3
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  		Fig. 3. Animal study of tumorigenicity and MVD between BCC/CXCR4 and BCC/pcDNA3 cells. In all, 1 x 106 human BCC/pcDNA3 cells and BCC/CXCR4 cells were injected subcutaneously into the nude mice (five mice per group). (A) The gross appearance of the xenograft following the killing of nude mice performed 7 weeks after injection of BCC cells (left panel). The BCC/CXCR4 cells had greater tumorigenicity compared with BCC/pcDNA3 cells. (B) Immunohistochemical staining of sections of xenografts using anti-CXCR4 and anti-CD31 antibodies showed higher MVD in BCC/CXCR4 cells as compared with BCC/pcDNA3 cells. Bar, 10 µm. (C) Higher MVD (calculated by the number of CD31-positive vessels in each high-power field) in BCC/CXCR4 xenografts as compared with that of vector control (BCC/pcDNA3) cells (16.9 ± 1.8 versus 5.5 ± 1.5, P < 0.01, two-tailed Student’s t-test). Data are presented as mean ± SD of three different fields from five xenografts in each group.

 
SDF-1{alpha}–CXCR4 interaction induces angiogenesis profiles in BCC-1/KMC cells
The importance of the interaction between SDF-1{alpha} and CXCR4 for BCC angiogenesis was examined using a DNA oligomicroarray designed for studying the profiles of 561 known angiogenesis-associated genes. Hierarchical clustering analysis by selecting probes with >2-fold change in at least one time point revealed 25 genes (Figure 4A). Among them, significantly upregulated genes with >3-fold change were interferon-alpha-inducible protein 27 (IFI27) (5.84-fold), IL-6 (3.92-fold), bone morphogenetic protein (BMP)-6 (3.67-fold), SOCS2 (3.37-fold) and cyclooxygenase 2 (COX)-2 (3.22-fold); significantly downregulated genes with >3-fold change included PAX3 (0.26-fold), KRT 19 (0.28-fold), ELMO1 (0.30-fold) and NME5 (0.30-fold) (supplementary Table 1 is available at Carcinogenesis Online). We found that IL-6 was the earliest and highest upregulated gene 6 h after the commencement of SDF-1{alpha}–CXCR4 interactions, and these effects persisted up to 24 h (supplementary Table 1 is available at Carcinogenesis Online).


Figure 4
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  		Fig. 4. SDF-1{alpha}–CXCR4 interaction induces upregulation of IL-6 in human BCC cells. (A) Hierarchical clustering analysis of SDF-1{alpha}-induced angiogenesis profiles by selecting probes with >2-fold change in at least one sample revealed 25 genes (with 28 probes). (B) IL-6 mRNA and protein concentrations were upregulated time dependently after SDF-1{alpha} treatment with peaking at 2–6 and 12–24 h. (C) Dose-dependent change of IL-6 mRNA and protein secretion after different concentrations of SDF-1{alpha} treatment for 2 h in human BCC cells. (D) Pretreatment of AMD3100 and CXCR4-neutralizing antibody (12G5) inhibits IL-6 mRNA expressions and IL-6 secretion in human BCC cells. (E) Human BCC cells were cotransfected with pGL2 (control) or pGL2-based full-length IL-6 promoter (phIL-6-luc) constructs along with, as an internal control of transfection efficiency, pRL-TK containing the renilla luciferase gene. The promoter activities were calculated as the firefly:renilla luciferase activity ratios and normalized to the control (vehicle). In BCC cells transfected with phIL-6-luc, treatment of SDF-1{alpha} for 2 h markedly stimulated luciferase activity relative to basal levels (vehicle). SDF-1{alpha}-stimulated luciferase activity was inhibited by AMD3100 (500 ng/ml) or CXCR4-neutralizing antibody (12G5) but not by the isotype control antibody (10 µg/ml). Data are shown as mean ± SD of three independent experiments. (F) Pretreatment of BCC cells with the IL-6-neutralizing antibody prior to adding SDF-1{alpha} inhibits tube formation of HUVEC induced by CM from BCC cells (*P < 0.05; **P < 0.001; ***P < 0.0001; P > 0.05 when isotype control compared with SDF-1{alpha} only group). (G) Human BCC tumors with high expression of CXCR4 had high expression of IL-6, whereas those with low CXCR4 expression had low IL-6 expression.

 
SDF-1{alpha}–CXCR4 interaction induces transcriptional upregulation of IL-6 in BCC-1/KMC cells
Previous studies have shown significant angiogenic potential of IL-6 in human BCC cells (29). We, therefore, hypothesized that IL-6 may be involved in SDF-1{alpha}/CXCR4-induced BCC angiogenesis. RT–polymerase chain reaction analysis showed that SDF-1{alpha} induced mRNA expression and protein secretion of IL-6 in both time- and dose-dependent manners (Figure 4B and C). Time kinetic changes of IL-6 mRNA expression without SDF-1{alpha} treatment were not significantly changed during the period of 0–24 h (supplementary Figure 2 is available at Carcinogenesis Online). The upregulation of IL-6 mRNA expression was abolished by AMD3100 or CXCR4-neutralizing antibody (12G5), whereas the isotype control antibody had no effect (Figure 4D), confirming CXCR4 involvement in IL-6 regulation.

Human BCC cells were cotransfected with pGL3 (control) or pGL3-based IL-6 promoter constructs along with, as an internal control of transfection efficiency, pRL-TK containing the renilla luciferase gene. The promoter activities were calculated as the firefly:renilla luciferase activity ratios and normalized to the control (vehicle). In BCC cells transfected with a full-length IL-6 promoter construct, SDF-1{alpha} after 2 h markedly stimulated luciferase activity relative to basal levels (vehicle). SDF-1{alpha}-induced luciferase activity was inhibited by AMD3100 (500 ng/ml) or CXCR4-neutralizing antibody (12G5; 10 µg/ml) but not by the isotype control antibody (10 µg/ml) (Figure 4E). Further, the in vitro HUVEC tube formation activity of the CM from SDF-1{alpha}-treated BCC was inhibited by pretreatment of BCC with an IL-6-neutralizing antibody but not by the isotype control antibody (Figure 4F). Human BCC tumors with high expression of CXCR4 also had high expression of IL-6, whereas those with low CXCR4 expression had concomitant low IL-6 expression (Figure 4G). We also found that human BCCs with high IL-6 expression (n = 18) had a higher MVD as compared with BCC with low IL-6 expression (n = 19) (17.6 ± 4.7 versus 9.4 ± 4.5, P < 0.001; supplementary Table 2 is available at Carcinogenesis Online).

ERK1/2- and NF-{kappa}B-signaling pathways are involved in SDF-1{alpha}-mediated IL-6 upregulation
Since SDF-1{alpha}–CXCR4 interaction has been shown to activate several signaling pathways, including phosphatidyl inosital 3 (PI3)-kinase/Akt and MAPK, in various cancer cell lines (9,12,27,32,33), we performed western blot analysis to elucidate the signal transduction mechanisms involved in the SDF-1{alpha}-induced upregulation of IL-6. SDF-1{alpha} activated the ERK1/2 pathway in BCC cells, as evidenced by the increase in phosphorylated p42 and p44 (p-ERK1/2) between 5–30 min, which then rapidly declined to basal levels (Figure 5A). Other signaling pathways including JNK, p38 MAPK and Akt were not activated up to 4 h after treatment (Figure 5A). SDF-1{alpha}-induced mRNA expression and protein secretion of IL-6 were greatly reduced by treatment with U0126, a specific MEK inhibitor, but was not affected by SP600125 (a JNK inhibitor), LY294002 or wortmannin (both are PI3-kinase inhibitors) treatments (Figure 5B).


Figure 5
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  		Fig. 5. ERK1/2- and NF-{kappa}B-signaling pathways are involved in SDF-1{alpha}-mediated IL-6 upregulation. (A) SDF-1{alpha} induced transient upregulation of ERK1/2 phosphorylation, which peaked at 5–15 min, without significant changes of JNK, p38 MAPK or Akt phosphorylation. (B) SDF-1{alpha}-induced upregulation of mRNA expression of IL-6 in human BCC cells was abolished by MEK inhibitor U0126 (10 and 20 µM) but not by JNK inhibitor SP600125 (10 µM). (C) SDF-1{alpha}-stimulated luciferase activity was completely abolished by site-directed mutagenesis of NF-{kappa}B-binding sites, but not AP-1 or C/EBP-β site mutation. The promoter activities were calculated as the firefly:renilla luciferase activity ratios and normalized to the control [phosphate-buffered saline (PBS)]. Data are shown as mean ± SD of three independent experiments. (D) SDF-1{alpha}-activated NF-{kappa}B was evidenced by accumulation of p65 in the nuclei of human BCC cells, starting at 30 min and peaking at 120 min. (E) Pretreatment of AMD3100 (500 ng/ml), CXCR4-neutralizing antibody (12G5; 10 µg/ml) or MEK inhibitor (U0126; 20 µM) for 30 min inhibited SDF-1{alpha}-induced time-dependent nuclear accumulation p65 protein in nuclei of human BCC cells. (F) Pretreatment of pyrrolidinethiocarbamate (100 µM), Bay 11-7085 (50 µM), NF-kB decoy or U0126 (20 µM), but not NF-kB scramble, c-Jun antisense or SP600125 (10 µM), inhibited SDF-1{alpha}-induced IL-6 promoter activity in human BCC cells. The promoter activities were calculated as the firefly:renilla luciferase activity ratios and normalized to the control (vehicle). Data are shown as mean ± SD of five independent experiments.

 
The promoter region of human IL-6 contains five known cis-regulatory elements including AP-1, cyclic adenosine 3',5'-monophosphate-responsive element, nuclear factor IL-6 (C/EBP-β), Sp-1 and NF-{kappa}B-binding sites (34). Three different IL-6 promoter constructs containing mutations at NF-{kappa}B, AP-1 or C/EBP-β sites, respectively, were generated by site-directed mutagenesis. We found that SDF-1{alpha}-stimulated luciferase activity was completely abolished by NF-{kappa}B-binding site mutations, but not by AP-1 or C/EBP-β site mutations (Figure 5C). SDF-1{alpha}-mediated activation of NF-{kappa}B was evinced by nuclear accumulation of p65 in human BCC cells, starting at 30 min and peaking at 120 min; the cytosol levels of I{kappa}B{alpha} were also decreased (Figure 5D). Activation of ERK1/2 may cause phosphorylation and degradation of I{kappa}B{alpha}, leading to nuclear translocation of p65. Furthermore, pretreatment of AMD3100, CXCR4-neutralizing antibody (12G5) or MEK inhibitor (U0126) inhibited SDF-1{alpha}-induced time-dependent accumulation p65 protein in nuclei of human BCC cells, confirming the involvement of CXCR4 and ERK1/2 in SDF-1{alpha}-induced NF-{kappa}B activation (Figure 5E). SDF-1{alpha}-enhanced IL-6 promoter activity was also inhibited by pretreatments of NF-{kappa}B inhibitors (pyrrolidinethiocarbamate and Bay 11-7085), NF-{kappa}B decoy oligodeoxynucleotides or U0126 but not by control decoy deoxyoligonucleotides, c-Jun antisense oligonucleotides or SP600125 (Figure 5F).


   Discussion
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Supplementary material
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Previously, we have found that human BCCs express CXCR4 but not SDF-1{alpha}, whereas SDF-1{alpha} is expressed by stromal cells (27). The paracrine effects of SDF-1{alpha} on human BCCs may induce the expression and secretion of several angiogenesis-associated factors and lead to higher MVD, which has been found to correlate with the aggressive phenotype of human BCC (3,4). This, however, could not predict the metastasis potential of non-melanoma skin cancers since increased MVD was also observed in many benign skin tumors or Bowen’s disease [squamous cell carcinoma (SCC) in situ] (35,36). A study showed that the formation of blood vessels could be found in the body of SCCs, but not in the nodular BCCs. In contrast, BCCs had higher stromal vessel counts compared with the normal skin. The stromal angiogenic response could explain the appearance of telangiectasia around the tumor. The authors concluded that the invasive growth strongly correlated with an angiogenic response in the stroma, whereas metastatic potential correlated with MVD in the body of the tumor, which may explain why SCCs have greater metastatic potential than BCCs (37). Our results also showed that the majority of BCCs had stromal angiogenic response. Although the expression of VEGF has been found to correlate with MVD (4,36), most of the BCCs expressed low levels of VEGF, except at the invasive border of certain tumors (38,39). Therefore, the expression of CXCR4 has significantly better correlation to the formation of MVD in human BCCs than VEGF does. In view of its strong correlation with invasive (aggressive) histological types, CXCR4 expression in human BCCs may have potential clinical and pathophysiological significance.

Previous studies have shown that SDF-1{alpha} may regulate or induce the expression of several angiogenic factors such as VEGF and IL-8 in various cancer cells (19–23). In addition, some proangiogenesis cytokines or soluble factors, such as IL-6, matrix metalloproteinase-2 and matrix metalloproteinase-9, were also induced by SDF-1{alpha} in head and neck SCC, prostate cancer or mouse hepatocarcinoma cells (20,40,41). We found that SDF-1{alpha}–CXCR4 interactions in human BCC cells induce several angiogenic or angiogenesis-related genes including IFI27, IL-6, BMP-6, SOCS2 and PTGS2 (COX-2), indicating that SDF-1{alpha}-induced angiogenic potential is a complex network of cascades involving multiple factors. These changes in gene expression after SDF-1{alpha} treatment in BCC-1/KMC cells may not represent a direct effect of SDF-1{alpha}–CXCR4 interactions, but reflect a secondary effect of SDF-1{alpha} treatment. In this context, we presume that the genes regulated after 12 h might have actually been resulted from the changes occurred in the genes expressed at 6 h.

Among these genes regulated by SDF-1{alpha}, the most pronounced effect was the upregulation of IFI27 (~5.84-fold). The IFI27 (also known as ISG12 or p27) belongs to a family of small interferon-alpha-inducible genes of unknown function that is highly upregulated in lesional psoriatic epidermis, chronic eczema and in some skin cancers (42). A recent study using a tissue microarray also demonstrated the increased expression of IFI27 genes in human BCCs (43). These studies suggest that IFI27 might be a novel marker of epithelial proliferation and cancer (42). It may also play some roles in cutaneous angiogenesis because psoriasis and BCC are both angiogenesis-related skin disorders.

Both IL-6 and BMP-6 were noted to be the earliest genes upregulated significantly (>3-fold change) in this study, and IL-6 was induced to highest level at 6 h after SDF-1{alpha} treatment (Figure 4). The role of BMP-6 in cutaneous angiogenesis is unknown, while a few studies showed that BMP-6/SMAD5-signaling pathway may have important roles during endothelial cell differentiation and in the organization of capillary-like structures (44). Since IL-6 plays an important role in BCC angiogenesis (29), we further examined its involvement in SDF-1{alpha}/CXCR4-mediated angiogenic potential. We found that SDF-1{alpha} transcriptionally regulated the expression of IL-6 in BCC-1/KMC cells, leading to significantly increased secretion of IL-6 into the CM in both time- and dose-dependent manners. Neutralization of IL-6 in BCC cells prior to SDF-1{alpha} treatment inhibited the angiogenesis activity exerted by CM. This result indicated that IL-6 is an important key regulator of SDF-1{alpha} and various downstream angiogenic factors, since through its indirect response, IL-6 induces other angiogenic factors such as VEGF, basic fibroblast growth factor (bFGF) and COX-2 (29,45). The proangiogenic activity of IL-6 might be due to its autocrine effects on BCC cells or paracrine effects on endothelial cells. Previously, we have shown that bFGF and COX-2 were the downstream effectors of IL-6-induced angiogenic activity in BCC cells (29). However, we could not find significant changes in bFGF expression in the current study. This might be due to other possible SDF-1{alpha}-induced inhibitory pathways that may have counterbalanced the effects of IL-6 on bFGF upregulation. In contrast, COX-2 expression was significantly upregulated following the changes of IL-6 expression, indicating that it may be the downstream effector of IL-6 in SDF-1{alpha}/CXCR4-mediated angiogenesis. Increased COX-2 expression has been found to correlate with MVD in human BCCs (36). Our previous study also confirmed that COX-2-overexpressing human BCC cells exhibited higher angiogenic activity (46).

SDF-1{alpha}–CXCR4 interactions have been reported to enhance VEGF expression in a previous study (22). However, we could not find similar results in the current study. This discrepancy may have resulted from different culture conditions and experimental systems used in the two studies. In the previous study, the upregulation of VEGF was more pronounced in CXCR4-overexpressing BCC cells (CXCR4-BCC), whereas the change of VEGF expression in retroviral vector control cells (pLNCX2-BCC) was not significant. We surmise that SDF-1{alpha} may simultaneously activate stimulatory and also a few inhibitory factors for regulation of VEGF expression, leading to insignificant changes of VEGF levels in wild-type BCC cells. However, in CXCR4-overexpressing BCC cells, the stimulatory effects would be enhanced by selection process or enhanced CXCR4-signaling pathway.

Upregulation of IL-6 is reportedly controlled by the activity of several transcription factors with known consensus sequences in the IL-6 promoter region, including AP-1, C/EBP-β and NF-{kappa}B (31). Several observations from other cell systems indicated that different kinase pathways including the Raf-1/MEK1/ERK1/2 pathway and the JNK signaling cascade are involved in activating the transcription factor NF-{kappa}B (34). Our previous study found that SDF-1{alpha}–CXCR4 interaction activated ERK1/2 and subsequently c-Jun in human BCC cells (27); in this study, however, we found ERK1/2–NF-{kappa}B pathway, rather than AP-1 complex, is involved in SDF-1{alpha}-enhanced BCC angiogenesis.

In conclusion, we present here a comprehensive approach in studying SDF-1{alpha}/CXCR4-induced angiogenesis activity using microarray analysis, which identified several novel angiogenic or proangiogenic factors influencing the angiogenic response of human BCCs. In addition, we also delineated the mechanisms involved in transcriptional regulation of IL-6 by SDF-1{alpha}–CXCR4 interactions in human BCC cells through the activation of ERK1/2–NF-{kappa}B pathway. The identification of SDF-1{alpha}–CXCR4 interaction as an important factor in BCC angiogenesis and aggressiveness may lead to potential improvements in the treatment of aggressive BCC.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Tables 1–2, Figures 1–2 and data can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
National Science Council of Taiwan (NSC 96-2314-B-002-171-MY3 and 97-2314-B-002-120-MY3 to S.-H.J. and NSC 96-2314-B-002-104 to C.-Y.C.); National Taiwan University Hospital (96M010) to C.-Y.C.


   Footnotes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Jason C-H Cheng (Department of Oncology, National Taiwan University Hospital) for providing the reporter plasmid pIL-6-luc and Dr Oliver Eickelberg (Department of Medicine II, University of Giessen) for providing different constructs of pIL6-luc651 reporter plasmids.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received May 17, 2008; revised September 22, 2008; accepted September 28, 2008.


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