A point mutation (G574A) in the chemokine receptor CXCR4 detected ...

[Cell Cycle 8:8, 1228-1237; 15 April 2009]; ©2009 Landes Bioscience

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A point mutation (G574A) in the chemokine receptor CXCR4 detected in human cancer cells enhances migration Caterina Ierano,1 Paola Giuliano,1 Crescenzo D’Alterio,1 Michele Cioffi,1 Valentina Mettivier,1 Luigi Portella,1 Maria Napolitano,1 Antonio Barbieri,2 Claudio Arra,2 Giuseppina Liguori,3 Renato Franco,3 Giuseppe Palmieri,4 Carla Rozzo,4 Roberto Pacelli,5 Giuseppe Castello1 and Stefania Scala1,* 1Clinical Immunology; 2Animal Facility; and 3Pathology; The G. Pascale National Cancer Institute; Fondazione “G. Pascale”; Naples, Italy; 4Istituto di Chimica Biomolecolare— Consiglio Nazionale Ricerche; Traversa La Crucca; Italy; 5Istituto di Biostrutture e Bioimmagini (IBB)—Consiglio Nazionale Ricerche; Naples, Italy

Key words: chemokine receptor, CXC chemokine receptor, CXCR4, CXCL12, AMD3100, ERK, pERK, chemotaxis, chemotactic factor, melanoma, colon cancer, G-protein coupled receptor (GPCR), Gi subfamily

The chemokine receptor CXCR4 is widely expressed in human cancers and regulates cell invasion, proliferation and survival. Because mutations in the CXCR4 gene could regulate its function we sequenced the coding region of the CXCR4 gene in 18 human melanoma and 3 human colon carcinoma cell lines. The same somatic point mutation (G574A; V160I) in the fourth transmembrane region of CXCR4 was detected in one colon cancer cell line (PD) and one melanoma cell line (LB). CXCR4 was expressed and functional in both PD and LB cells, PD and LB cells migrated specifically toward the receptor ligand, CXCL12 and P-Erk was specifically induced by CXCL12. To give insight into the function of the mutant CXCR4 receptor, human A431, epidermoid carcinoma cells, were stably transfected with both mutant and wild type CXCR4. In vitro, A431 cells harboring CXCR4G574A migrated specifically toward CXCL12 and CXCL12 induced ERK phosphorylation. Interestingly, in vivo studies showed that the growth of A431 tumors harboring CXCR4G574A was delayed compared to those harboring WT CXCR4. As expected, treatment with AMD3100, a specific CXCR4 inhibitor, reduced the in vivo growth of CXCR4G574A tumor bG574A but surprisingly, increased the growth of CXCR4G574A A431 cells. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells. While the mutation impairs cell growth in vivo, the CXCR4 inhibitor, AMD3100, stimulated the growth of cells harboring CXCR4G574A.

Introduction The CXC chemokine receptor 4 (CXCR4) plays a role in the recruitment of leukocytes to sites of inflammation and to secondary *Correspondence to: Stefania Scala; The G. Pascale National Cancer Institute; Clinical Immunology; Via Mariano Semola; Naples 80131 Italy; Tel.: +39.0815903797; Fax: +39.0815903289; Email: [email protected] Submitted: 02/12/09; Accepted: 02/23/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/8250

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lymphoid organs.1-3 Its ligand, CXCL12, is a highly efficient chemotactic factor for T cells, monocytes, pre-B cells, dendritic cells and hematopoietic progenitor cells.4 In addition evidence has demonstrated that CXCL12 supports survival or growth of a variety of normal or malignant cell types, including hematopoietic progenitors,5 germ cells,6 leukemia B cells7 and carcinoma cells.8-13 The distinct pattern of chemokine receptor expression by neoplastic cells has a critical role in determining the site(s) of metastatic spread.8 In addition in melanoma and colon cancer CXCR4 expression has been correlated with prognosis.14-17 CXCR4 is a seven-membrane-spanning G-protein coupled receptor (GPCR). The activation of GPCRs leads to the activation of heterotrimeric G proteins, which dissociate from the receptors and initiate second messenger signaling via trimeric G-proteins. Chemokines, in particular, signal via the pertussis toxin-sensitive Gi subfamily, inducing phosphorylation of multiple substrates including Pyk2, AKT and ERK1/2.18-20 Not surprisingly, mutagenesis studies to discern the function of the different CXCR4 domains have shown that CXCR4 function can be affected by mutations.21-24 For example deletions or substitutions in the NH2-terminal extracellular domain (NT), the extracellular loops (ECL), or the transmembrane domains (TMs) alter CXCR4 function. While mutations at two tyrosine residues in the NH2-terminal extracellular domain or a single asparagine residue in the second and third extracellular loop or the second transmembrane domain markedly impair the activity of CXCR4.21 In addition, the ability of CXCR4 mutants to bind CXCL12 and mediate cell signaling is consistent with a two-site model of chemokine-receptor interaction. Site I, involved in CXCL12 binding but not signaling, is located in the NT with Glu14 and/or Glu15 and Tyr21 serving crucial roles. Residues required for both CXCL12 binding and signaling, and thus functionally a part of site II, include Asp187 in ECL2, Asp97 in TMII and Glu288 in TMVII.21 Finally, deletion of the COOH-terminal domain of CXCR4 led to enhanced motility

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Mutations in the CXCR4 receptor

and proliferation accompanied by altered receptor trafficking and changes in gene expression in MCF-7 human breast cancer cells.21 Given the evidence indicating mutations in CXCR4 regulate its function we began by analyzing the coding region of eighteen human melanoma cell lines (6 primary tumors, 8 skin metastases, 4 lymph node metastases) and three colon carcinoma cell lines for mutations in CXCR4.

Results Human colon carcinoma cell line (PD) and human melanoma cell line (LB) harbor a mutant CXCR4. CXCL12 activation of CXCR4 has been shown to affect growth, survival and migration of human cancer cells. Since mutations of CXCR4 could affect its function we determined the coding sequence of CXCR4 in eighteen melanoma cell lines (6 primary tumors, 8 skin metastases, 4 lymph node metastases) and three colon carcinoma cell lines. A G → A transition at nucleotide 574 (G574A) was identified in PD human colon carcinoma and LB human melanoma cell lines. The mutation changes amino acid 160 located within the fourth transmembrane region of CXCR4 from valine to isoleucine (Val160Ile) (Fig. 1A). In both cell lines only the mutant allele (CXCR4G574A) could be detected suggesting only one CXCR4 allele was present since it would be very unlikely the same mutation would have occurred in both alleles. Furthermore, the CXCR4G574A mutation was not detected in normal genomic DNA from 103 unrelated healthy individuals (corresponding to 206 control chromosomes) reducing the likelihood it represented a polymorphism. As shown in Figure 1B, CXCR4 mRNA was expressed in PD and LB cells compared to two other cell lines with high and low levels of expression (ARO and FB1, anaplastic thyroid carcinoma cells). CXCR4 total protein and cell surface expression were also detected in PD and LB cells by immunoblotting and flow cytometry, respectively, as shown in Figure 1C and D. CXCL12 activates CXCR4G574A in LB and PD cells. To evaluate the function of the mutated CXCR4G574A, CXCL12 induced cell migration, proliferation and Erk activation were analyzed in LB and PD cells. As shown in Figure 2A both LB and PD cells migrated toward the CXCR4 ligand, CXCL12 (100 ng/ ml). In LB cells, CXCL12 induced a 1.8-fold increase in migration specifically inhibited by 1 μM AMD3100, a CXCR4 inhibitor. In PD cells 100 ng/ml CXCL12 induced a 1.2-fold increase in the migration, specifically inhibited by 1 μM AMD3100. The migration was compared to the CXCL12 induced migration in MDA-231, human breast cancer cells harboring a WT receptor (3.7-fold induction of migration). In order to study the effect of the CXCR4G574A on cell growth, LB and PD cells were serum starved for 16 hours and then treated with 100 ng/ml CXCL12. Figure 2B shows that CXCL12 moderately increased cell growth in LB (1.2-fold) and PD cells (1.5-fold) compared to the CXCL12 induced proliferation of PES 43 human melanoma cells line bearing a wild type CXCR4 receptor (2-fold). Finally, 100 ng/ ml CXCL12 led to ERK phosphorylation at 20 minutes in LB and PD cells compared to an earlier activation in PES43 cells (Fig. 2C). Thus in LB and PD cells the CXCR4 ligand, CXCL12, induced www.landesbioscience.com

cell migration and ERK phosphorylation and minimally affected cell growth. Expression of CXCR4WT and CXCR4G574A in human epithelial carcinoma A431 cells. To further characterize the effect of the (G574A) CXCR4, A431 human epithelial carcinoma cells, were stably transfected with a CXCR4WT and CXCR4G574A. Although CXCR4 expression was detectable also in A431 untransfected cells, overexpression of CXCR4WT and CXCR4G574A was confirmed at both the RNA and the protein levels as shown in Figure 3A–C. CXCL12 activates CXCR4G574A in CXCR4-MUT and WT-A431 cells. To compare the function of the CXCR4WT and CXCR4G574A receptors, transfected A431 cells were evaluated for migration, cell growth and ERK activation—confirming the observations in the LB and PD cells. As shown in the left panel of Figure 4A, CXCR4WT and CXCR4G574A-transfected A431 cells migrated to CXCL12. The basal migration registered in A431-CXCR4G574A cells was higher (180%) than that in A431CXCR4WT as was the migration in CXCL12 treated cells; 20 ng/ ml CXCL12 induced similar increases in migration of 4.2-fold and 3.2-fold in A431-CXCR4WT and A431-CXCR4G574A cells respectively. Interestingly AMD3100 modestly reduced the migration in A431-CXCR4G574A cells compared to A431-CXCR4WT cells. In the right panel of Figure 4A the migration of MDA231, human breast cancer cells, was evaluated in the presence of 20 and 100 ng/ml of CXCL12 plus or minus AMD3100. In Figure 4B the addition of CXCL12 to the upper and lower well (chemokinesis) did not affect migration in A431-CXCR4G574A, A431-CXCR4WT nor in MDA231, suggesting that migration observed in Figure 4A is specifically induced by CXCL12. To further investigate cell motility, an in vitro wound closure assay was performed as shown in Figure 4C. Wounds in the A431-CXCR4G574A closed faster than A431-CXCR4WT at the 20 hours time point suggesting an acquired increased motility of A431-CXCR4G574A cells. The effect of CXCR4G574A on cell growth was also evaluated as shown in Figure 4D. In conditions of serum deprivation, 100 ng/ml CXCL12 increased the growth of A431-CXCR4WTcells but not that of A431-CXCR4G574A cells. Interestingly, the basal growth of A431-CXCR4G574A was slightly increased compared to the A431-CXCR4WT when serum was deprived (1.23-fold). On the contrary, in the presence of 10% of serum the rate of proliferation of A431-CXCR4WT was two fold higher compared to A431-CXCR4G574A cells. Finally looking at ERK phosphorylation, P-ERK was induced by CXCL12 treatment in A431, A431-CXCR4G574A and A431CXCR4WT cells. Thus CXCR4G574A is a functional CXCR4 receptor (Fig. 4E). In vivo growth of CXCR4G574A cells is delayed compared to A431-CXCR4WT. To evaluate the effect of CXCR4G574A in vivo, 2 x 106 A431-CXCR4WT and A431-CXCR4G574A cells were inoculated into the right flank of nude mice. Two other groups of mice inoculated with cells that had been pre-incubated for 30 min with 10 μM AMD3100 were treated twice a day intraperitoneally with 1.25 mg/kg AMD3100 for three weeks. A431-CXCR4G574A cells showed a slight delay in growth in vivo compared to A431CXCR4WT cells, confirming the in vitro results when cells were

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Figure 1. For figure legend, see page 1231.

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Mutations in the CXCR4 receptor Figure 1. PD, human colon carcinoma cell line and LB, human melanoma cell line harbor a mutated CXCR4. (A) Model of CXCR4 showing the site of valine to isoleucine substitution at position 160 (Val160Ile) within the fourth transmembrane region of the receptor. (B) RT-PCR for CXCR4 expression in PD and LB cells compared to ARO, anaplastic thyroid carcinoma cells, a CXCR4 overexpressing cell line, and to the FB-1, anaplastic thyroid carcinoma cells with low CXCR4 expression. (C) Immunoblot analysis for CXCR4 protein expression. Protein lysates from LB and PD cells were resolved by SDSPAGE (10%) gel electrophoresis, transferred to a nitrocellulose membrane. The proteins on the membranes were subjected to immunoblot analysis with anti-CXCR4. (D) FACS analysis for CXCR4 cell surface expression.

Figure 2. CXCL12 activated CXCR4 (G574A) in LB and PD cells. (A) Migration was assayed in LB and PD cells in 24-well plates. LB and PD cells were placed in the upper chamber (2.0 x 105 cells/well) in migration media, and 100 ng/ml CXCL12 was added to the lower chamber in triplicate. MDA-231 cells were used as positive control. The cells were counted in ten different fields (original magnification x40). Representative data from three individual experiments. (B) Proliferation of LB and PD cells (20–50 x 104) seeded in a six-multiwell in complete medium. After 24 hours, the medium was replaced with serum-deprived medium and CXCL12 (100 ng/ml) was added. (C) CXCL12 mediated P-Erk induction was evaluated in LB and PD cells in comparison to human melanoma cell line, PES 43. Total lysates were obtained from LB and PD cells that had been serum starved and treated with CXCL12 plus and minus AMD3100 at the indicated times and concentration. The cell lysates were resolved by SDS-PAGE (10%) gel electrophoresis, transferred to a nitrocellulose membrane and incubated with anti-PERK and anti-ERK2. Representative data from three individual experiments are shown.

grown in the presence of serum. Interestingly, while in vivo treatment with AMD3100 reduced the growth of A431-CXCR4WT cells, it increased the growth of A431-CXCR4G574A cells (Fig. 5). These data indicate that the CXCR4G574A is detrimental to A431 cell growth in vivo. However AMD3100 enhances cell growth behaving as an agonist more than antagonist with regards to cell growth. www.landesbioscience.com

Discussion A somatic point mutation (G574A) was detected in the CXCR4 coding region of PD and LB, human colon cancer and melanoma cells respectively. The mutation resulted in a valine to isoleucine amino acid substitution (V160L), in the fourth transmembrane region (TM4) of CXCR4. The mutant receptor was

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Figure 3. Overexpression of CXCR4-WT and CXCR4G574A in human epidermoid A431 cells. (A) RT-PCR for CXCR4 expression in A431 cells transfected with either CXCR4-WT or CXCR4G574A in comparison to a high or low expressing cell line (ARO and FB-1, anaplastic thyroid carcinoma cells). (B) CXCR4 total protein determined by immunoblot analysis (C) CXCR4 cell surface expression assessed by FACS analysis.

expressed and functional in PD and LB cells as demonstrated by ligand specific migration and P-Erk induction. These findings were confirmed in A431 epidermoid cancer cells, stably transfected with either the WT (A431-CXCR4WT ) or the mutant (A431-CXCR4G574A) CXCR4 receptor. In vivo experiments demonstrated that the growth of A431-CXCR4G574A tumors was delayed compared to A431-CXCR4WT and that AMD3100 enhanced cell growth behaving as an agonist and not as an antagonist. Previous studies of CXCR4 mutations designed to elucidate the mechanism(s) of receptor interaction with agonist and

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antagonists, succeeded in identifying several domains. The third intracellular loop and the C terminus domain of CXCR4 play key roles in CXCL12-mediated signaling and are specifically involved in G(i)-dependent signals such as calcium mobilization and ERK activation, but do not trigger CXCR4 internalization after CXCL12 binding, indicating ERK phosphorylation is independent of CXCR4 endocytosis. However, the second and third intracellular loops, as well as the C terminus of CXCR4, are needed to transduce CXCL12-mediated chemotaxis, suggesting this event involves multiple activation pathways and/or cooperation of several cytoplasmic domains of CXCR4.22

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Figure 4. CXCL12 activates CXCR4 in A431-CXCR4WT and A431-CXCR4G574A cells. (A) Migration was assayed in A431-CXCR4WT and A431CXCR4G574A cells as described above. MDA-231 cells, in the same conditions, were used as positive control. Representative data from three individual experiments. (B) Chemokinesis was assayed in A431-CXCR4G574A , A431-CXCR4WT and in MDA-231. Cells were placed in the upper chamber (2.0 x 105 cells/well) in migration media, and 100 ng/ml CXCL12 was added to the upper and lower chamber. The cells were counted in ten different fields (original magnification x40). Representative data from three individual experiments. (C) Wound closure cell motility assay in A431-CXCR4WT and A431CXCR4G574A. Cells were allowed to reach confluence in complete medium in six well plates and then scratched with a pipette tip to make wounds. The closure of the wounds was monitored by microscopy after 20 hours. Representative data from three individual experiments.

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Figure 4. CXCL12 activates CXCR4 in A431-CXCR4WT and A431-CXCR4G574A cells. (D) Proliferation of A431-CXCR4WT and A431-CXCR4G574A (20–50 x 104) seeded in a six-multiwell in complete medium. After 24 hours the cells were serum starved and CXCL12 (100 ng/ml) was added. Cells were counted using a hemocytometer. (E) CXCL12-induced ERK activation in A431-CXCR4WT and A431-CXCR4G574A was evaluated in comparison to human melanoma cell line, PES 43. Total lysates obtained from A431-CXCR4WT and A431-CXCR4G574A cells, serum starved, and treated with CXCL12 at the indicated concentration for the indicated times. Representative data from one of three experiments.

Figure 5. In vivo growth of A431-CXCR4G574A is delayed when compared to that of A431CXCR4WT. Female BALB/c athymic (nu+/nu+) mice, 8 to 10 weeks of age, weighing 24 to 32 g, were injected s.c. with 5 x 105 cells suspended in 50 μl phosphatebuffered saline (PBS). Mice were divided in four groups. Ten mice per group were inoculated with (A) A431CXCR4WT cells; (B) A431-CXCR4WT cells pretreated with AMD3100; (C) A431-CXCR4G574A cells; or (D) A431-CXCR4G574A cells pretreated with AMD3100. The two groups of mice inoculated with A431-CXCR4WT and A431-CXCR4G574A pre-incubated for 30 min with AMD 3100 (10 μM) were then treated twice a day intraperitoneally with AMD 3100 (1.25 mg/kg for three weeks). Tumor size were measured twice a week. The animals were sacrificed 21 days after the tumor cell injection. 1234

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Chemokine receptor CXCR4 is known to form homodimers. It has previously been shown that a synthetic peptide of the CXCR4 TM4 can inhibit CXCL12-induced actin polymerization and can block chemotaxis of malignant cells. The TM4 region peptide has potent effects on ligand-directed migration of cancer and normal cells, partially by altering the interactions between monomers of CXCR4.24 The CXCR4 mutation described in the present manuscript, a transition (G574A), results in an amino acid substitution (V160L), in the fourth trans-membrane region of CXCR4. Although the mutation did not impair receptor function, A431-CXCR4G574A tumors grew slower than A431-CXCR4WT in agreement with in vitro experiments in the presence of serum. Thus the CXCR4 (G574A) mutation seems detrimental to in vivo tumor growth. The finding that treatment with AMD3100 increased the growth of A431-CXCR4G574A cells in vivo suggests the mutation may affect the AMD3100 binding site such that upon binding to the CXCR4 receptor AMD3100 has agonist and not antagonist effects. TM4 of CXCR4 has been previously reported to be involved in the binding of the receptor antagonist AMD3100.26 The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against the human immunodeficiency viruses, HIV-1 and HIV-2. 2009; Vol. 8 Issue 8


Specific sites, including negatively charged aspartate residues at positions 171 and 262, in TM4 and TM6 of CXCR4, respectively, are crucial in effecting electrostatic interactions with positive charges on the bicyclams.26 Previous studies have shown that inhibitors such as T140 are inverse agonists whereas inhibitors such as AMD3100 and ALX40-4C are also weak partial agonists.27 In addition, recent studies with AMD3465, a novel CXCR4 antagonist confirmed that the single cyclam ring of AMD3465 binds in the pocket around Asp 171, in a manner similar to AMD3100.28 Ongoing nuclear magnetic resonance (NMR) studies are analyzing specific interactions between the mutated receptor and possible agonists or antagonists. Preliminary studies conducted by us demonstrate that peptides synthesized to be CXCR4 inhibitors and tested as modulators of receptor function have antagonist or agonist properties that depend on the output that is assessed—migration, induction of P-ERK or CXCL12-dependent calcium increase. Our observations give pause to the use of inhibitors that could potentially act as agonists, while also giving more insight into the AMD3100 interaction. CXCR4 mutations have been previously described in patients with the WHIM syndrome. The WHIM syndrome is a rare combined immunodeficiency characterized by warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis and is currently the only immunological disease associated with mutations of a chemokine receptor. The mutations identified to date (one frameshift and three nonsense mutations) all truncate the C-terminal tail of CXCR4 eliminating 10 to 19 of the distal tail aminoacids, including a number of potential phosphorylation sites. This leads to the expression of a receptor with altered regulation. Following activation, there is a lack of desensitization, enhanced chemotaxis, an increase in F-actin polymerization, enhanced calcium mobilization, and a decrease in CXCL12 promoted internalization, although one report found no difference in calcium mobilization or internalization.29-32 CXCR4 mutations were also described in human medulloblastoma.33 One germline and one somatic mutation were detected resulting in amino acid substitutions in the first (Ile53Leu) and second (Asp97Asn) transmembrane regions, respectively, but no further studies on function were described.34 In summary, in the present study we describe the same acquired mutation in the CXCR4 chemokine receptor in two cancer cell lines established from metastatic cancers. While the finding of an acquired mutation does not per se make it significant in the evolution of cancer, the fact that the same mutation was found in two independent cell lines and in both cell lines the mutation was accompanied by loss of the wild type allele, suggests this mutation may have been advantageous. Our data suggests the acquired mutation found in both LB and PD cells may have conferred an advantage in terms of migration that during the process of tumor invasion and possibly metastases may have been valuable. That the mutation does not enhance cell growth is not surprising given the large number of other pathways that impact cell proliferation. This suggests that in vivo, the role of the CXCR4 is likely more important in migration (invasion and metastases).9,35-37 And while antagonists may interfere with www.landesbioscience.com

igration, as we note, our data suggests the need for caution m in targeting this receptor with agents that may function both as agonists and antagonists. This is the first report of a spontaneously occurring, functionally active CXCR4 mutation in human cancer cells that seems to retain function and confer migration advantages. It is hoped further studies will lead to the identification of additional mutations.

Materials and Methods Cell culture. All cell lines were grown at 37°C with 5% CO2 in DMEM with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, 50 g/ml penicillin, 50 g/ml streptomycin. Transfected A431 cells were grown in DMEM media containing 0.1 mg/ml Geneticin (G418). DNA amplification and mutational analysis of the CXCR4 gene. Genomic DNA from each tumor and blood sample was amplified using 5 overlapping primer pairs. PCR was carried out in a final volume of 10 μl with 10–50 ng of genomic DNA in a buffer containing 1.0–1.5 mM MgCl2 (Invitrogen), 200 mM of each deoxynucleoside triphosphate, 5 pmol of each forward and reverse primer and 0.25 U Taq polymerase (Invitrogen). After an initial denaturation step of 94°C for 5 minutes, 35 cycles of PCR were performed with all primer pairs on a Thermoblock Cycler (Biometra, Gottingen, Germany) with a denaturation step of 94°C for 35 seconds, an annealing step of 57°–59°C for 40 seconds and an extension step of 72°C for 40 seconds. Primers sequence for CXCR4 were: F1 = AGA AAG CAA GCC TGA ATT GG R1 = TCC ATC ATC TTC TTA ACT GGC A F2 = GGC TCA GGG GAC TAT GAC TC R2 = CTT CAT CAG TCT GGA CCG CT F3 = ATG CCG TGG CAA ACT GGT A R3 = TTT CAG CAC ATC ATG GTT GG F4 = GTC AGT GAG GCA GAT GAC AGA R4 = AAG TGG ATT TCC ATC ACC GA F5 = GGA TCA GCA TCG ACT CCT TC R5 = CAG TTT TTA TTG CTT GTT GGA TTT Mutational analysis was performed using the SSCP method. PCR products were loaded onto 10% or 14% polyacrylamide (acrylamide:bisacrylamide ratio 1:29, 1:59 or 1:79) gels with or without 10% glycerol and run at room temperature (60 V) or at 4°C (80 V) for 18–24 hr. PCR products showing aberrantly migrating bands were re-amplified at least twice in independent experiments. Bands were then excised and eluted, and the DNA was re-amplified. The resulting PCR products were purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, CA). Sequencing was performed with the PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit and the GeneAmp PCR system 9600 (Perkin-Elmer, Foster City, CA), using 20 ng of PCR product as a template. Sequencing reaction products were separated on an Applied Biosystems (Foste City, CA) 377 sequencer. Genomic DNA from 103 unrelated healthy individuals (corresponding to 206 control chromosomes) were used as controls and screened for the identified sequence variation.

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Flow cytometry for CXCR4 expression. To evaluate the expression of CXCR4 (CD184), adherent cancer cells at subconfluency (50–70% confluent) were detached with 2 mmol/L EDTA in PBS, washed, re-suspended in ice-cold PBS and incubated for 30 minutes at 4°C with anti-CD184-PE antibody (FAB 173P, clone 44717, R&D Systems, Minneapolis, MN, USA) or mouse IgG2a PE conjugated as negative control. After three washes in PBS, the cells were analysed by FACScan cytofluorometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA, USA). Growth curve viability and cell growth assay. Cells (20–50 x 104) were seeded in a sixmulti-well plates in complete medium culture containing 10% fetal bovine serum. After 24 hours, the medium was replaced as indicated and CXCL12 (R&D, Minneapolis, MN) was added in the presence or absence of AMD3100 (Sigma Aldrich, Inc.,). Cells were counted using a hemocytometer. Cell motility/chemotaxis and wound closure cell motility assay. Migration was assayed in 24-well Transwell chambers (Corning Inc., Corning, NY) using inserts with an 8-μm pore membrane. Membranes were pre-coated with collagen (human collagen type I/III) and fibronectin (10 g/ml each). Test cells were placed in the upper chamber (2.0 x 105 cells/well) in DMEM containing 0.5% BSA (migration media), and 20 or 100 ng/ml CXCL12 was added to the lower chamber or to the upper and lower chamber (chemokinesis). After 16 hours incubation, cells on the upper surface of the filter were removed using a cotton wool swab. MDA-231 cells were used as positive control. The cells were counted in ten different fields (original magnification x40). For the wound closure assay, tested cells were allowed to reach confluence in six-well plates containing serum depleted growth medium and scratched with pipette tips to make wounds. The wound closure was observed microscopically 18 hours post-wounding (Axiovert 40 Cell Zeiss). Construction of CXCR4 mutant. A mutant of CXCR4, in which valine at position 160 was replaced by isoleucine, was constructed using QuikChange XL site-directed mutagenesis kit (Stratagene, Europe) in accordance with the manufacturer’s instructions. The mutation was confirmed by sequencing. Transfection of adherent A431cells. Wild-type or site mutagenized CXCR4 was transfected into A431cells using FuGENE 6 transfection reagent (Roche Diagnostics) according to the manufacturer’s instructions. Selective medium containing G418 (0.1 mg/ml) was used to isolate stably transfected cells. Each stablytransfected cell line was cloned from a single colony. Attempts were made to isolate colonies with comparable wild-type CXCR4 expression. DNA extraction, RNA extraction and cDNA preparation. DNA was isolated from peripheral blood, and from cell lines by standard Proteinase K/SDS digestion followed by phenol/chloroform extraction. Total cellular RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. To remove any contaminating DNA, samples were digested with DNase I (Promega, Madison, WI). Subsequently, DNase was removed by an additional Trizol extraction. Reverse transcription was performed using the Superscript II 1236

Preamplification System (Invitrogen) with random hexamers as primers in a final volume of 20 μl. RNA Isolation and RT-PCR. Total cellular RNA was extracted from cell lines. Dnase treated RNA (2 μg) was reversed transcribed with Superscript II RNase H reverse transcriptase according to manufacturer’s instructions (Invitrogen Life Technologies). RTPCR was carried out using 2 ml of cDNA in a 20 ml final reaction mixture. A Robocycler gradient 96 (Stratagene, CA) was used for the amplification. Cycling conditions were as follows: initial denaturation (4' at 94°C) followed by 32 cycles of denaturation (1' at 94°C), annealing (1'15' at 56°C) and elongation (3' at 72°C). 10 μl of the products were run on a 2% agarose gel and analyzed under UV light. The gene-specific primers used for the amplification were as follows: CXCR4 (forward): 5'-TTCTACCCCAATGAC TTG TG-3' CXCR4 (reverse): 5'-ATGTAGTAAGGCAGCCAACA-3' GAPDH (forward): 5'-ACATGTTCCAATATGATTCCA-3' GAPDH (reverse): 5'-TGGACTCCACGACGTACTCAG-3' Immunoblotting. Cells were homogenized in lysis buffer (40 mM Hepes pH 7.5, 120 mM NaCl, 5 mM MgCl2, 1 mM EGTA, 0.5 mM EDTA, 1% Triton X-100) containing protease (Complete Tablets- EDTA-free, Roche) and phosphatase (20 mM a-glycerolphosphate, 2.5 mM Na-pyrophosphate) inhibitors. The following primary antibodies were used: anti-CXCR4 (Abcam, ab2074), anti-tubulin (Santa Cruz Biotech, CA), anti-phosphorylated p44/42 MAPK and anti-p44/42 MAPK (New England Biolabs). Anti-mouse and antirabbit IgG coupled to peroxidase were used as secondary antibodies (Santa Cruz Biotech, CA) and the signal was revealed through chemoluminescent detection kit (ECL detection kit, Amersham Biosciences, Freiburg, Germany). Animal experiments. Female BALB/c athymic (nu+/nu+) mice, 8 to 10 weeks of age, weighing 24 to 32 g, were purchased fromCharles River Laboratories (Milan, Italy). The research protocol was approved, and mice were housed and maintained under specific pathogen-free conditions in the Animal Care Facility of National Cancer Institute G. Pascale in accordance with the institutional guidelines of the Italian Ministry of Health Animal Care and Use Committee. Mice were acclimatized for 1 week before being injected with cancer cells. Mice were injected s.c. with 2 x 106 cells suspended in 250 μl phosphate-buffered saline (PBS). Mice were divided in four groups. Ten mice per group were inoculated with (a) A431-CXCR4WT cells; (b) A431-CXCR4WT cells pretreated with 10 μg/ml AMD3100 for 30 minutes and after inoculation, mice were treated twice a day with 1.25 mg/kg AMD3100; (c) A431CXCR4G574A; and (d) A431-CXCR4G574A cells pretreated with AMD3100 as above. Tumor sizes were measured twice weekly by the modified ellipsoid formula: (π/6) x AB2, where A is the longest and B is the shortest perpendicular axis of an assumed ellipsoid corresponding to tumor mass.25 Body weight was measured twice weekly as control for treatment toxicity. Two-sided Student’s t test was used to compare the volume of xenograft tumors. Survival analysis was computed by the Kaplan-Meiermethod and statistical analysis was carried out by the χ2 test. The animals were sacrificed 21 days after the tumor cell injection.

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Acknowledgements

This work was supported by grant AIRC n 1107 from the Associazione Italiana per La Ricerca sul Cancro. References 1. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med 1997; 185:111-20. 2. Gupta SK, Lysko PG, Pillarisetti K, Ohlstein E, Stadel JM. Chemokine receptors in human endothelial cells. Functional expression of CXCR4 and its transcriptional regulation by inflammatory cytokines. J Biol Chem 1998; 273:4282-7. 3. Ma Q, Jones D, Borghesani PR, Segal RA, Nagasawa T, Kishimoto T, et al. Impaired B-lymphopoiesis, myelopoiesis and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc Natl Acad Sci USA 1998; 95:9448-53. 4. Baggiolini M. Chemokines and leukocyte traffic. Nature 1998; 392:556-65. 5. Lataillade JJ, Clay D, Bourin P, Herodin F, Dupuy C, Jasmin C. 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