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[Cancer Research 65, 1800-1807, March 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Epigenetic Up-regulation of C-C Chemokine Receptor 7 and C-X-C Chemokine Receptor 4 Expression in Melanoma Cells

Takuji Mori, Joseph Kim, Tomoki Yamano, Hiroya Takeuchi, Sharon Huang, Naoyuki Umetani, Kazuo Koyanagi and Dave S.B. Hoon

Department of Molecular Oncology, John Wayne Cancer Institute, Santa Monica, California

Requests for reprints: Dave S.B. Hoon, Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404. Phone: 310-449-5267; Fax: 310-449-5282. E-mail: hoon{at}jwci.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Histone deacetylation and DNA methylation establish epigenetic modifications, which through chromatin remodeling may result in gene silencing. We hypothesized that chemokine receptors C-C chemokine receptor 7 (CCR7) and C-X-C chemokine receptor 4 (CXCR4) on melanoma cells undergo epigenetic regulation. We investigated whether a histone deacetylase inhibitor and a demethylating agent influence CCR7 and CXCR4 expression on melanoma cells. Initially, microarray analysis was done to screen changes in chemokine receptor expression on melanoma cells after treatment with trichostatin A (TSA) and 5-Aza-2-deoxycytidine (5-Aza). CCR7 and CXCR4 mRNA expression were uniformly altered and selected for further investigation. Quantitative real-time reverse transcription-PCR assay, immunohistochemistry, and Western blot analysis were used to assess changes in mRNA and protein expression induced by TSA and 5-Aza in melanoma lines. Cell migration assays were conducted to assess the effects of altered CCR7 and CXCR4 expression on cell function. Treatment with TSA or 5-Aza increased gene expression of both CCR7 and CXCR4 in melanoma lines. TSA was the strongest enhancer. With combined treatment, CCR7 and CXCR4 mRNA expression was also up-regulated. Immunohistochemistry after combined treatment showed enhanced staining of both CCR7 and CXCR4 compared with control cells. Melanoma cell migration in TSA- and 5-Aza-treated cells was 7- and 2-fold higher than control cells for CCR7 and CXCR4, respectively. In summary, a histone deacetylase inhibitor and a demethylating agent up-regulated CCR7 and CXCR4 expression on melanoma cells. This increase in chemokine receptor expression correlated with functional activity. Most importantly, we have identified an epigenetic mechanism that may endogenously regulate chemokine receptor expression on melanoma cells.

Key Words: CCR7 • CXCR4 • methylation • histone deacetylase inhibitor • melanoma


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Malignant cutaneous melanomas are characterized by an aggressive pattern of recurrence, resulting in a poor prognosis (1). Early metastasis, most commonly to regional tumor-draining lymph nodes, is a frequent mechanism for these poor outcomes. Several recent studies have implicated the involvement of chemokines and their respective receptors on cancer cells in potential mechanisms for cancer metastasis (2–11). Of note, investigators have reported the association of chemokine receptors C-C chemokine receptor 7 (CCR7) and C-X-C chemokine receptor 4 (CXCR4) expression in several cancers with organ or tissue specific metastasis (3, 4, 7–9, 11).

Chemokines belong to the chemoattractive cytokine family and are grouped into C, CC, CXC, and CX3C chemokines based on four conserved cysteine residues (12, 13). These chemoattractive molecules mediate their effects on target cells by interacting with G protein–linked receptors, which consist of seven-transmembrane spanning domains (13). A chemokine and a receptor of particular interest are C-C ligand 21 (CCL21) and CCR7, respectively. This chemokine pair has been identified as a contributing factor in the metastasis of melanoma, breast, gastric, and esophageal cancers (3, 7, 8, 10, 11). Recently, our group showed the significance of CCR7 expression in melanoma (7). Takeuchi et al. (7) characterized CCR7 expression in melanoma cell lines and patient tumor specimens and assessed its effects on functional activity. They showed that the ligand CCL21 (also called SLC, 6Ckine, or exodus) regulated the migratory behavior of melanoma cells bearing CCR7 from the primary lesion sites to the first regional tumor-draining lymph nodes, the sentinel lymph node.

In general, as a chemoattractant, CCL21 is involved in recruiting CCR7 receptor (+) naïve T cells, natural killer cells, memory T-cells, and dendritic cells (14–19). It is constitutively expressed in high endothelial venules of lymph nodes, Peyer's patches, thymus, spleen, and mucosal tissue (17, 20). Consequently, the release of CCL21 by high endothelial venules results in the specific recruitment of CCR7 receptor (+) cells to those lymph nodes (15, 19–21).

The C-X-C ligand 12 (CXCL12)-CXCR4 axis is also of considerable interest because of its reported roles in the metastatic mechanism of multiple cancers (4, 6, 10, 22–24). Of note, the CXCR4 receptor was originally characterized as the HIV coreceptor (25). Its specific chemokine ligand was later identified as stromal cell–derived factor-1{alpha}, now designated CXCL12 (25, 26) . It is expressed in many tissues, including lymph node, liver, lung, skin, and bone marrow, and has chemoattractive effects on cells bearing CXCR4 receptors (25). Recent studies have recognized that CXCR4 may play an important role in melanoma metastasis (6, 27, 28). Consequently, intense scrutiny has been placed on both CCR7 and CXCR4. Because variable and heterogeneous expression patterns have been shown on tumor cells, the mechanisms that regulate the expression of the chemokine receptors CCR7 and CXCR4 on tumor cells need to be further elucidated.

It has been established that gene expression can be regulated by chromatin remodeling. Chromatin is intrinsically modified by histone acetyltransferase or histone deacetylase, and alterations in these mechanisms may lead to altered gene expression (29). Additionally, gene expression may be governed by DNA methylation (30). Specifically, CpG island hypermethylation, which occurs by the addition of a series of cytosines within the promoter regions of genes and within heterochromatin, can result in gene silencing (30). These epigenetic modifications are now being recognized as significant contributing factors in human tumorigenesis and cancer progression (30, 31). In short, the deregulation of genes that are keys to cell proliferation and differentiation can result in silencing of those genes and are observed in many cancers (32–34).

We hypothesized that epigenetic changes regulate the expression of CCR7 and CXCR4 receptors on melanoma cells. This study is the first report demonstrating that epigenetic activations of CCR7 and CXCR4 occur in tumor cells and that use of trichostatin A (TSA) and 5-Aza-2-deoxycytidine (5-Aza) results in the up-regulated expression of functional chemokine receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Melanoma Cell Line Treatment. Eleven melanoma cell lines (MA-MK) from metastatic melanoma tumors established at John Wayne Cancer Institute (Santa Monica, CA) were assessed. Cell lines were maintained in RPMI 1640 supplemented with heat-inactivated 10% (v/v) fetal bovine serum, penicillin G, and streptomycin (100 units/mL). Melanoma cell lines and lymphocytes were treated with TSA (Wako Biochemicals, Osaka, Japan) and 5-Aza (Sigma Chemical Co., St Louis, MO). After treatment with TSA and 5-Aza, melanoma cells were washed with physiologic PBS (pH 7.4) and removed with 0.25% (w/v) trypsin-0.53 mmol/L EDTA (Life Technologies, Auckland, NJ).

RNA Isolation. Total cellular RNA from melanoma cell lines was extracted using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH) as described previously (7, 35, 36). Briefly, cells were digested and RNA was solubilized in a guanidinium-based buffer, separated by phenol/chloroform, and precipitated by isopropanol. RNA extraction was done in a designated sterile laminar flow hood using RNase/DNase–free plasticware. The RNA was quantified and assessed for purity by UV spectrophotometry and by the RIBOGreen detection assay (Molecular Probes, Eugene, OR). The expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an internal reference housekeeping gene, was assessed on all RNA samples to verify the integrity of RNA. Sample processing, RNA extraction, and subsequent assays were done in separate designated rooms to prevent cross-contamination as described previously (7).

Primers and Probes. Primer and probe sequences were designed to optimally hybridize and amplify target cDNA for quantitative real-time reverse transcription-PCR (RT-PCR) assay using Oligo Primer Analysis Software (National Biomedical Systems, Plymouth, MN). To avoid possible amplification of contaminating genomic DNA, primers were designed such that PCR products covered at least one exon-exon junction. The primers and FRET probe sequences used were as follows: CCR7: 5'-AACCAATGAAAAGCGTGCTG-3' (forward), 5'-CGAACAAAGTGTAGTCCACTG-3' (reverse), and 5'-FAM-ATCGTCCGTGACCTCATCTTGACAC-BHQ-1-3' (FRET probe); CXCR4: 5'-GGAGGGGATCAGTATATACA-3' (forward), 5'-GAAGATGATGGAGTAGATGG-3' (reverse), and 5'-FAM-CGAGGAAATGGGCTCAGGGG-BHQ-1-3' (FRET probe); and GAPDH: 5'-GGGTGTGAACCATGAGAAGT-3'(forward), 5'-GACTGTGGTCATGAGTCCT-3' (reverse), and 5'-FAM-CAGCAATGCCTCCTGCACCACCAA-BHQ-1-3' (FRET probe).

Quantitative Real-time RT-PCR Assay. Reverse transcription was done using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) with oligo(dT) (GeneLink, Hawthorne, NY) priming as described previously (36). The quantitative real-time RT-PCR assay was done with the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA); cDNA from total RNA (250 ng) was used for each reaction as described previously (36). The PCR reaction mixture consisted of 1 µmol/L of each primer, 0.3 µmol/L FRET probe, 1 unit AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA), 200 µmol/L of each deoxynucleotide triphosphate, 4.5 mmol/L MgCl2, and AmpliTaq buffer to a final volume of 25 µL. Samples were amplified with 50 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and extension at 72°C for 1 minute and denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute for CCR7 and CXCR4 and for GAPDH, respectively.

Plasmids for individual gene cDNA (CCR7, CXCR4, and GAPDH) were constructed and used as standards in quantitative real-time RT-PCR analysis as described previously (36). Positive (peripheral blood lymphocytes) and negative (mouse muscle) controls were included in each assay. Reagent controls for quantitative real-time RT-PCR assays were included in each assay as described previously (36). Each assay was repeated two to four times to verify the accuracy of the results.

cDNA Microarray Analysis. Gene expression profiling of cDNA changes after drug treatment was done using GEArray kits (SuperArray, Frederick, MD) according to the manufacturer's instructions. A matched chemokine/chemokine receptor expression array (GEArray Q series kit, Human Chemokine and Receptor Array) containing 77 cDNAs was used. First, total RNA was extracted from treated and control melanoma cell lines. Total RNA (10 µg) was converted to biotinylated cDNA probes by reverse transcription with a deoxynucleotide triphosphate mix containing biotin-16-dUTP. Denatured probes were hybridized to gene-specific cDNA fragment spots on the membranes and then washed. Membranes were blocked and incubated with streptavidin conjugated to alkaline phosphate. After incubation with an ECF chemiluminescent substrate (Amersham Biosciences, San Francisco, CA), the relative expression levels were detected as a signal on a Storm 860 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Each cDNA was independently normalized according to the expression of 10 housekeeping genes and positive and negative controls. GAPDH was chosen to serve as an internal hybridization control. In this manner, the relative expression of chemokines was estimated by comparing their signal intensity to that derived from GAPDH through analysis with the SuperArray software. Only cDNA transcripts with a 2-fold increase in expression after TSA and 5-Aza treatment compared with control (untreated) cells were characterized as significantly up-regulated genes. Twenty-five percent (19 of 77) of the chemokines assessed were up-regulated and 18% (14 of 77) were down-regulated after treatment with TSA and 5-Aza. Secondary to alterations in gene expression and their implicated significance in recent studies, CCR7 and CXCR4 were selected for verification.

Immunohistochemistry. Expression of CCR7 and CXCR4 in cell lines was assessed by immunohistochemistry. Cells were cultured on Lab-Tek II Chamber slides (Nalge Nunc International, Naperville, IL) and treated with TSA and 5-Aza as described above. Specimens were fixed in 4% paraformaldehyde and then incubated overnight with mouse anti-human CCR7 monoclonal IgM antibody (1:200 dilution, BD Biosciences, Bedford, MA) or CXCR4 monoclonal IgG antibody (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C. Control cells were treated with a nonimmunized immunoglobulin fraction under equivalent conditions without primary antibody. Paraffin-embedded human normal spleen tissues with relatively abundant dendritic cells were used as positive controls for CCR7 and CXCR4 staining (37). Biotinylated horseradish peroxidase, streptavidin reagents (Dako Corp., Carpinteria, CA), and Vectastain ABC kit (Vector Laboratories, Burlingame, CA) were used as secondary developing reagents. Slides were counterstained with H&E for reading.

Western Blot Analysis. CXCR4 protein expression in cell lines was assessed by Western blot analysis. Because the anti-CCR7 antibody for Western blot analysis is not specific or commercially available, CCR7 protein expression by Western blot analysis was not evaluated. Cells treated with TSA and 5-Aza were harvested with trypsin-EDTA, washed twice with PBS, and lysed with a radioimmunoprecipitation assay buffer. Appropriate positive (HL-60) and negative (MD line) cell lines were assessed. Twenty micrograms of protein from the cell lysates were separated by 12% SDS-PAGE gel and blotted onto a nitrocellulose membrane. The membrane was blocked for 15 minutes using 2% nonfat milk in TBS-Tween, 10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, and 0.1% Tween and probed overnight at 4°C with 0.4 µg/mL rabbit polyclonal anti-fusin (CXCR4) IgG antibodies (Santa Cruz Biotechnology) or goat polyclonal anti-GAPDH IgG antibodies (Santa Cruz Biotechnology). After washing thrice with TBS-Tween, the membranes were blocked with 2% nonfat milk in TBS-Tween for 15 minutes and then labeled for 1 hour at room temperature with horseradish peroxidase–conjugated antibodies, anti-rabbit IgG (BD Biosciences), or anti-goat IgG (Santa Cruz Biotechnology). After washing four times with TBS-Tween, the blot was developed with enhanced chemiluminescence substrate (Amersham Pharmacia Biotech, Piscataway, NJ) and imaged using a Storm 860 PhosphorImager.

Cell Migration Assay. Cell migration assays were done using a modified Boyden transwell double chamber system with 8 µm pores (Becton Dickinson and Co., Franklin Lakes, NJ) as described previously (7). Initially, the insert was coated with vitronectin (1 µg/100 µL) and incubated overnight at room temperature. After 24 hours, melanoma cells (with and without TSA and 5-Aza treatment) were harvested from the culture dishes using trypsin-EDTA and washed twice with physiologic PBS. Cells (2 x 104) were resuspended in fibroblast basal medium (Clonetics, Walkersville, MD) with 0.1% bovine serum albumin and seeded into the insert of the transwell chamber. The lower chambers contained fibroblast basal medium and recombinant CCL21 (1 µg/mL) or CXCL12 (100 ng/mL, R&D Systems, Minneapolis, MN).

The modified Boyden chambers were incubated at 37°C in 5% CO2 for 8 hours. After this incubation period, nonmigratory cells on the upper membrane were removed with cotton-tipped swabs; cells that had migrated to the lower surfaces of the membranes were fixed in 100% ethanol, washed with PBS, and stained with H&E. The number of migrating cells was counted in five different fields at 200x magnification as described previously (7).

Proliferation Assay. The effect of TSA and 5-Aza on cell proliferation was assessed. Four representative melanoma cell lines (MA, MB, ME, and MG) were examined with or without TSA and 5-Aza treatment as described above. Cells were first counted, treated with TSA and/or 5-Aza, and then harvested with trypsin-EDTA and stained with trypan blue (Sigma Chemical). The number of cells that did not stain were counted using a hemocytometer in addition to the number of unstained control cells. Data were expressed as the mean percentage of control. Each assay was done in triplicate.

CXCR4 Methylation Analysis. Methylation of the CpG islands in the promoter region of the CXCR4 gene was assessed in melanoma cell lines. Because CpG islands are absent in the CCR7 promoter region, methylation of CCR7 in melanoma cells was not assessed. In brief, cultured cells were digested with proteinase K at 50°C overnight, denatured at 95°C for 10 minutes, treated with sodium bisulfite as described previously (38), and used as direct templates for methylation-specific PCR. SssI methylase-treated cells and untreated normal control DNA were used as positive and negative controls, respectively. PCR amplification was done in a 10 µL reaction volume with 1 µL DNA template for 40 cycles consisting of 30 seconds at 94°C, 30 seconds at 60°C, 30 seconds at 72°C, and ending with a 7-minute final extension at 72°C and 30 seconds at 94°C, 30 seconds at 55°C, 30 seconds at 72°C, and ending with a 7-minute final extension at 72°C for methylated CXCR4 and unmethylated CXCR4, respectively. Forward primers were labeled with WellRED dye–labeled phosphoramidites (Genset Oligos, Boulder, CO). The primer sequences are as follows: methylated CXCR4, 5'-GGAGTATTTAGGTTTTCGGCGT-3' (forward); methylated CXCR4, 5'-ACGTATTTTTATAAAAATCCGACCG-3' (reverse); unmethylated CXCR4, 5'-GGAGTATTTAGGTTTTTGGTGT-3' (forward); and unmethylated CXCR4, 5'-CAAAACATATTTTTATAAAAATCCAAC-3' (reverse). PCR product separation was done using capillary array electrophoresis (CEQ 8000XL, Beckman Coulter, Inc., Fullerton, CA). Peak signal intensity and relative size were generated by fragment analysis software (Beckman Coulter).

Statistical Analysis. Statistical analysis of the data was done using the unpaired Student's t test and Mann-Whitney U test. Two-sided P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Treatment with TSA and 5-Aza. Baseline CCR7 and CXCR4 mRNA expression levels in 11 melanoma cell lines, normalized to GAPDH, were assessed by quantitative real-time RT-PCR. Briefly, four study treatments were established. The groups consisted of treatment with TSA, 5-Aza, TSA and 5-Aza, or culture medium alone. In the TSA treatment, cells were treated with TSA and then assessed after 24 hours. In the 5-Aza treatment, cells were treated with 5-Aza and then assessed after 48 hours. In the TSA and 5-Aza treatment arm, cells were initially treated with 5-Aza; then, 24 hours later, 5-Aza and TSA were both applied. The cell lines were treated with varying concentrations of TSA and 5-Aza to empirically determine optimal dosages for CCR7 and CXCR4 gene up-regulation. Control cells were not treated with either drug. Treatment of the MA cell line with TSA and/or 5-Aza resulted in variable changes of CC R7 and CXCR4 mRNA expression (Fig. 1A and B). Similar findings were noted with other melanoma cell lines (data not shown). The optimal up-regulation of both CCR7 and CXCR4 mRNA expression was induced by concentrations of 1,000 and 500 nmol/L for TSA and 5-Aza, respectively, and were used throughout the remainder of the study.



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  		Figure 1. A, change in expression of CCR7 mRNA in a representative melanoma MA cell line induced by various concentrations of TSA (T) and 5-Aza (A). For example, T1 represents TSA at 1 x 102 nmol/L. B, change in expression of CXCR4 mRNA in a representative melanoma MA cell line induced by various concentrations of TSA and 5-Aza.

 
Melanoma cell lines overall showed limited increase in CCR7 expression when treated with 5-Aza alone (Table 1); MI cell line had a slight decrease in CCR7 expression. Three of 11 cell lines showed >2 times increase in CCR7 after 5-Aza treatment. When cell lines were treated with TSA alone, the increase in CCR7 gene expression was considerably higher than with 5-Aza alone for all cell lines. The combination of 5-Aza and TSA for all cell lines enhanced CCR7 expression above the individual drugs. The exception was the MJ cell line for TSA treatment. When 5-Aza alone was given, there was no observed doubling of CXCR4 expression over controls (Table 2). TSA treatment, however, enhanced CXCR4 expression (>2 times) in all but two melanoma cell lines (MD and MI). When TSA and 5-Aza were combined, a significant synergistic increase in CXCR4 expression was noted above individual drugs in three cell lines (Table 2). There seemed to be limited synergy of TSA and 5-Aza action for up-regulation of CXCR4 gene expression (Table 2), indicating that TSA as the most effective agent for CXCR4 gene up-regulation. Assays were done ≥2 times for verification.


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  		Table 1. CCR7 expression in human melanoma cell lines

 

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  		Table 2. CXCR4 expression in human melanoma cell lines

 
Treatment of the melanoma cell lines with TSA and 5-Aza maintained enhancement of CCR7 mRNA expression 24 hours after treatment but then had significantly dropped at 36 hours (data not shown). The increase in CXCR4 mRNA expression persisted beyond 24 hours up to 48 hours. At 72 hours, CXCR4 mRNA levels decreased, returning close to baseline (data not shown). These findings indicate that the induced enhancement of CCR7 and CXCR4 mRNA expression after treatment was transient and not permanent.

Immunohistochemistry Assessment of CCR7 and CXCR4 Expression. CCR7 and CXCR4 protein expression in melanoma cells treated with TSA and 5-Aza was assessed by immunohistochemistry. Melanoma cell lines showed variations in CCR7 and CXCR4 staining after treatment with TSA and 5-Aza (Fig. 2A and B). The intensity of the immunoreactivity significantly increased in the TSA and 5-Aza and TSA alone treatment groups compared with the 5-Aza alone group and control group. Comparison of the TSA and 5-Aza versus TSA alone groups showed similar intensities in staining. The increase in immunohistochemical staining was consistent with the up-regulation of mRNA expression of both CCR7 and CXCR4.



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  		Figure 2. A, representative immunohistochemical staining at 200x magnification for CCR7 protein expression in melanoma MA cell line. B, representative immunohistochemical staining for CXCR4 protein expression in melanoma MA cell line. a, no treatment; b, 5-Aza treatment; c, TSA treatment; d, combined treatment with TSA and 5-Aza.

 
Western Blot Assessment of CXCR4 Expression. CXCR4 protein expression in melanoma cells treated with TSA and 5-Aza was assessed by Western blot analysis. CXCR4 protein was detected in cell lines after treatment with TSA and 5-Aza (Fig. 3). Additionally, the MD cell line, which does not express CXCR4 mRNA, showed appropriately negligible CXCR4 protein expression. The results of the Western blot analysis corroborate the immunohistochemistry results and are concordant with the results of the quantitative real-time RT-PCR assays.



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  		Figure 3. A, Western blot analysis of CXCR4 expression in melanoma lines MH, MA, MC, and MD after treatment with TSA and 5-Aza. HL60 (myelobastic cell line) served as a positive control. B, Western blot analysis of GAPDH expression in melanoma lines.

 
Assessment of Cell Migration with CCL21 and CXCL12. The functional activity of CCR7 and CXCR4 receptor (+) melanoma cells to their respective ligands was assessed by applying recombinant human CCL21 and CXCL12 in a cell migration assay. The cell lines MA, MC, and ME and cells lines MA, MC, and MH were assessed for CCR7/CCL21 and CXCR4/CXCL12 responses, respectively. Cells treated with combined TSA and 5-Aza had significantly increased numbers of migratory cells in response to CCL21 and CXCL12 compared with untreated cells (P < 0.05 and P < 0.05, respectively; Fig. 4A and B). These results show the correlation between increased CCR7 and CXCR4 mRNA expression resulting from TSA and 5-Aza treatment and a corresponding increase in the number of migrating cells in response to CCL21 and CXCL12, respectively. In short, these chemotaxis studies showed that the increased CCR7 and CXCR4 mRNA expression resulting from TSA and 5-Aza treatment correlated with a subsequent functional change for these melanoma cells.



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  		Figure 4. A, representative studies of melanoma cell migration after CCL21 treatment. Number of migrating cells in three randomly selected fields was counted 8 hours after seeding. Columns, mean ({blacksquare}, no CCL21 treatment; {square}, CCL21 treatment); bars, SD. B, representative studies of melanoma cell migration after CXCL12 treatment. Number of migrating cells in three randomly selected fields was counted 8 hours after seeding. Columns, mean ({blacksquare}, no CXCL12 treatment; {square}, CXCL12 treatment); bars, SD. *, P < 0.05, statistically significant compared with untreated cells (Student's t test).

 
Cell Proliferation. Treatment of the melanoma MA cell line with TSA, 5-Aza, or TSA and 5-Aza resulted in a 55 ± 6%, 57 ± 8%, and 27 ± 5% inhibition of cell proliferation compared with controls, respectively. Treatment of representative melanoma lines MA, MB, ME, and MG with TSA and 5-Aza resulted in 27 ± 5%, 56 ± 17%, 66 ± 4%, and 31 ± 9% inhibition of cell proliferation compared with controls, respectively. These results indicate that TSA and 5-Aza treatment had a suppressive effect on cell proliferation, which is consistent with previous reports of growth suppression with these agents in melanoma cells (39).

Methylation Status of CXCR4 Promoter Region. A promoter region with CpG islands was not identified for CCR7. Therefore, methylation-specific PCR was not done for CCR7. CpG islands of the CXCR4 gene promoter region were identified and assessed for aberrant methylation in melanoma cell lines. In 8 of 11 melanoma cell lines, methylation of the CpG islands by capillary array electrophoresis analysis was shown; there was accordingly different levels of CXCR4 mRNA expression. However, degree of CpG island methylation varied in individual cell lines, demonstrating heterogeneity in cell lines and/or monoallelic hypermethylation of the promoter region. Only in MD cell line was complete hypermethylation of the CXCR4 gene promoter region detected.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Much has been reported recently regarding the potential role of chemokines and their corresponding receptors in cancer metastasis for numerous cancers (3, 7, 8, 10, 11). However, reports that detail the mechanisms, which regulate the expression of these chemokine receptors, are still lacking. The present study shows a novel mechanism by which a histone deacetylase inhibitor and a demethylating agent can alter CCR7 and CXCR4 mRNA expression.

In this study, we showed that CCR7 mRNA expression was predominantly up-regulated after treatment with TSA alone or in combination with 5-Aza and TSA. The increase in CCR7 mRNA expression was appreciably higher when 5-Aza was added to the TSA treatment, demonstrating a synergistic effect in regulating CCR7 gene expression. This synergy has been verified by other groups with other proteins (40–41). The mechanism of synergy seems to occur primarily through a common link with DNA methylation. Previous reports have indicated that DNA methylation can modify histone acetylation, which can subsequently impair gene transcription (40, 41). 5-Aza acts to deplete methyltransferase, resulting in generalized demethylation (42). Thus, epigenetic alterations, which may seem distinct, may actually share synergistic activity as illustrated by a more robust reactivation of silenced genes by TSA and 5-Aza in combination than by either one alone (40, 41).

5-Aza induced increased CCR7 mRNA expression in any cell lines MB, MD, and MG despite an absence of CpG islands in the gene promoter region. Similar findings of 5-Aza-mediated mRNA up-regulation in genes without promoter region CpG islands have been reported (43). Although the mechanisms remain to be fully clarified, there are potential explanations for these findings. The up-regulated expression of these loci after treatment with 5-Aza may occur because of reactivation of an upstream transcriptional factor, up-regulation of a transcriptional regulatory factor, increase in post-transcriptional processing, and/or regulation of target genes in an induced transduction pathway (43, 44). Methylation of CpG-poor promoter regions may affect gene expression through mechanisms that have yet to be fully elucidated (43, 44). In summary, TSA treatment induced a robust up-regulation of CCR7 gene expression, and 5-Aza was synergistic to TSA in the up-regulation of CCR7 gene expression.

The mechanisms that regulate CXCR4 gene expression seem to differ to some extent from those of CCR7. Although gene expression of CXCR4 was increased by the use of TSA, it was not as strong as with CCR7. The treatment with 5-Aza plus TSA did not show a uniform synergistic effect on CXCR4 up-regulation on melanoma cell lines. However, there was a strong synergistic effect of the two drugs on CCR7 up-regulation. Similar to CCR7, the mechanisms of TSA action on the CXCR4 gene are unclear. One explanation may be that histone acetylation is the major regulating mechanism independent of methylation to some extent for CXCR4 expression in melanoma cell lines. Even when the gene promoter region contains CpG islands, methylation may not be complete or absolute (29). Interestingly, CpG islands may reside in chromatin with widely and irregularly spaced nucleosomes, which contain highly acetylated histones. This structural conformation may render the promoter region susceptible to histone acetylation, thus identifying a role for TSA action (29).

5-Aza treatment decreased CXCR4 gene expression in select melanoma cell lines (MF, MI, and MJ). The mechanism of this inhibitory effect of 5-Aza is unclear. In the MD cell line, CXCR4 gene expression could not be detected by quantitative real-time RT-PCR and complete hypermethylation of the CpG island promoter region was noted. Treatment of the MD cell line with 5-Aza did produce a slight increase in CXCR4 gene expression, indicating that DNA methylation may have some role in regulating CXCR4 gene expression in this line.

The effects of TSA and 5-Aza treatment in lymphocytes are different in comparison with the effects in cancer cells. Baseline gene expression levels of CCR7 and CXCR4 in lymphocytes were closer in magnitude to expression levels of the housekeeping gene GAPDH. Treatment with TSA and 5-Aza did not change CCR7 and CXCR4 gene expression in a fashion similar to that observed in the melanoma cell lines. Hence, the mechanism of regulation of CCR7 and CXCR4 in lymphocytes seems different from what was observed in melanoma cells. In lymphocytes, expression of these chemokine receptors is essential for their physiologic immune properties, which is perhaps the reason why CCR7 and CXCR4 expression levels are relatively high. Apart from immune cells, most other cells have relatively low expression levels of these chemokine receptors. In tumor cells, the expression of these deregulated chemokine receptors seems to result from a transformation to recapitulate the genotype of embryonic cells, which are known to express chemokine receptors and migrate to specific anatomic sites (45).

Up-regulation of gene expression with TSA and 5-Aza has been reported for non-chemokine-related genes. Shi et al. (46) showed that methylation and histone acetylation may act in parallel. Treatment with TSA or 5-Aza resulted in up-regulation in 1.1% or 1.9% of the genes analyzed, respectively. Combined treatment, however, resulted in the synergistic reactivation of a greater number of genes. Based on either primary or secondary responses to the treatment, genes were identified as methylation dependent or independent. These types of treatment approaches may be useful for identifying and more fully understanding the mechanisms underlying epigenetic gene silencing.

Recent studies have noted that histone deacetylase inhibitors, including TSA, may be categorized into a new family of anticancer drugs. Histone deacetylase inhibitors induce cell cycle arrest, differentiation, and apoptosis in cancer cells and thus transform cells (34). These characteristics are directly relevant for their use as anticancer drugs. Several clinical trials have been instituted to test the efficacy of histone deacetylase inhibitor for the treatment of different malignant tumors (30). 5-Aza may also exhibit anticancer properties. It inhibits DNA methylation by reducing DNA methyltransferase enzymatic activity via the formation of a stable complex between the enzyme and the 5-Aza-substituted DNA. Subsequently, 5-Aza is being tested in clinical trials as a chemotherapeutic agent for treatment of leukemia and solid tumors (47).

In this study, TSA and 5-Aza treatment inhibited the growth of cells, a result that is consistent with other reports. Despite these growth inhibitory effects, TSA and 5-Aza individually or alone treatment enhanced gene expression of both CCR7 and CXCR4 and stimulated the in vitro migration of melanoma cell lines. It is of great interest, therefore, to note that the use of TSA in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of chemokine receptors. The outcomes of patients treated with TSA and 5-Aza may be determined by tumor suppressor gene reactivation (47) and enhancement of cancer cell migration. These potentially adverse effects of TSA and 5-Aza or other related drugs need to be carefully evaluated (48).

This is the first study to show in melanoma that CCR7 and CXCR4 are epigenetically regulated and can be targeted by histone deacetylase inhibitors and demethylating agents. Furthermore, the mechanisms by which these agents up-regulate chemokine receptor expression require further investigation. Future studies will be aimed at identifying host factors, which can trigger up-regulation of these chemokine receptors in tumor cells.


   Acknowledgments
 
Grant support: NIH, National Cancer Institute grants P01 CA29605 and P01 CA12582, and Martin H. Weil Research Laboratories.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/30/04. Revised 12/20/04. Accepted 12/22/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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