- Email: [email protected]
Interaction of ligand–receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis
BBRC Biochemical and Biophysical Research Communications 320 (2004) 656–663 www.elsevier.com/locate/ybbrc
Interaction of ligand–receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis Hideki Mochizuki,a Akio Matsubara,b Jun Teishima,b Kazuaki Mutaguchi,b Hiroaki Yasumoto,b Rajvir Dahiya,c,* Tsuguru Usui,b and Kenji Kamiyaa a
Division of Genome Biology, Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan b Programs for Biochemical Research, Division of Frontier Medical School, Department of Urology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan c Department of Urology and Urology Research Center, University of California San Francisco and Veterans Affairs Medical Center, USA Received 24 May 2004 Available online
Abstract Interaction of ligand–receptor systems between stromal-cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) is closely involved in the organ specificity of cancer metastasis. We hypothesized that SDF-1–CXCR4 ligand–receptor system plays an important role in prostate cancer metastasis. To test this hypothesis, expression level of SDF-1 and CXCR4 was analyzed in prostate cancer (PC) cell lines (LNCaP, PC3, and DU145) and normal prostate epithelial cell line (PrEC). We also performed migration assay and MTT assay to investigate the chemotactic effect and growth-promoting effect of SDF-1 on DU145 and PC3 cells, respectively. Furthermore, we performed immunohistochemical analysis of CXCR4 expression in tissues from 35 cases of human prostate cancer. CXCR4 expression was detected in all three prostate cancer cell lines, but not in PrECs. SDF-1 significantly enhanced the migration of PC3 and DU145 cells in a dose-dependent manner, and anti-CXCR4 antibody inhibited this chemotactic effect. However, SDF-1 itself did not significantly stimulate the cell growth rate of prostate cancer cell lines. Positive CXCR4 protein was found in 20 out of 35 clinical PC samples (57.1%). Three patients with lung metastasis showed definitely positive CXCR4 immunostaining. Logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis to Gleason sum (P < 0:05). Furthermore, among PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients with bone metastasis than in those with no bone metastasis (P ¼ 0:017). These findings suggest that the interaction between SDF-1 and CXCR4 ligand–receptor system is involved in the process of PC metastasis by the activation of cancer cell migration. This is the first report to investigate the role of interaction of ligand–receptor systems between SDF-1 and CXCR4 in prostate cancer metastasis. Ó 2004 Elsevier Inc. All rights reserved. Keywords: CXCR4; SDF-1; Prostate cancer; Metastasis
In advanced prostate cancer, the sites most frequently affected by metastasis are the bones and regional lymph nodes [1,2]. Patients with these metastases not only have a poor prognosis but also suffer pain and lower limb edema. It is therefore extremely important to elucidate *
Corresponding author. Fax: 1-415-750-6639. E-mail addresses: [email protected] (R. Dahiya), musui@ hiroshima-u.ac.jp (T. Usui). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.013
the molecular mechanisms underlying prostate cancer metastases to the bones and/or lymph nodes and to explore the avenues preventing such metastases. The molecular processes associated with metastases such as migration of cancer cells, their adhesion to specific organs, and cell proliferation within the microcirculation of these organs have been fully investigated [1,2]. In this regard, it is assumed that the target organ specificity of cancer metastasis is followed by the similar mechanism
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
observed in the homing of hematopoietic stem cells (HSC) to bone marrow in the accepted model of hematopoiesis [3–5]. Bone marrow stromal cells produce a chemokine, stromal-cell-derived factor-1 (SDF-1), especially during the process of fetal hematopoiesis and after bone marrow transplantation. At the same time, HSC significantly expresses its receptor, CXC chemokine receptor 4 (CXCR4) [3–5]. SDF-1 is a member of the CXC subfamily of chemokines, and its chemotactic and growth-promoting effects are mediated by CXCR4. Consequently, SDF-1 acts as a powerful chemo-attractant for HSC, which in turn “homes in” on the bone marrow specifically and efficiently [3–5]. Based on this hematopoiesis model, recent publications have demonstrated that the SDF-1–CXCR4 ligand–receptor system is closely involved in the organ specificity of cancer metastasis. For instance, Muller et al. [6] have reported that (i) CXCR4 is highly expressed in human breast cancer cells and (ii) the highest SDF-1 expression is observed in those organs that are usually the primary destinations of breast cancer metastases. SDF-1 signaling through CXCR4 interaction appears to determine the directional migration of breast cancer cells through the basement membrane. Furthermore in vivo, the interaction between SDF-1 and CXCR4 significantly represses the metastatic potential of breast cancer cells to regional lymph node and lung. These findings indicate that the SDF-1–CXCR4 ligand– receptor system plays a critical role in determining the metastatic destination of cancer cells. Similar experimental results have also been reported in other types of cancer such as malignant melanoma, ovarian, pancreatic, and lung [7–13]. However, such studies are limited in prostate cancer. To investigate whether SDF-1–CXCR4 ligand–receptor system is involved in the metastasis of human prostate cancer, we investigated the gene and protein expression of CXCR4 in the prostate cancer cell lines LNCaP, PC3, and DU145, in comparison with normal prostate epithelial cells. We also investigated the chemotactic activity and the growth-promoting effect of SDF-1 on DU145 and PC3 cells. Furthermore, we performed CXCR4 immunostaining in clinical samples (35 human prostate cancer tissues and nine non-malignant human prostate tissues), and evaluated the relationship between CXCR4 expression and clinicopathological findings with special reference to cancer metastasis.
Materials and methods Prostate tissue samples. Thirty-five prostate cancer samples (32 prostatic biopsy samples, 2 radical prostatectomy samples, and one transurethrally resected sample) and nine non-malignant prostate samples (seven normal prostate obtained at total cystoprostatectomy and two BPH tissues from suprapubic prostatectomy) were obtained with informed consent at the Department of Urology, Hiroshima
657
University School of Medicine, Hiroshima, Japan, between 1998 and 2001. All tissue samples were pathologically diagnosed and confirmed to contain the objective parts. A single pathologist classified each sample according to the Gleason grading system [14]. Staging examination for PC included digital rectal examination, transrectal ultrasound, urethrocystography, computed tomography, magnetic resonance imaging, and bone scanning. The clinicopathological stage of prostate cancer was categorized according to the 1997 International Union Against Cancer (UICC) TNM classification [15]. Serum prostate-specific antigen (PSA) levels were measured by Tandem R assay (Hybritech, San Diego, CA, USA). Cell culture. Human prostate cancer cell lines of LNCaP, PC-3, and DU145 were purchased from the American Type Culture Collection (Drive Rockville, MD, USA) and maintained in OPTI-MEM 1 medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 5% fetal bovine serum (FBS), 1 mM CaCl2 , and 100 lg/mL kanamycin (Life Technologies). LNCaP cells were originally isolated from lymph node in a patient with hormone-dependent prostate cancer involved in bone and lymph node metastases. PC3 and DU145 cells were isolated from the regions of vertebral and brain metastases, respectively, in hormone-independent prostate cancer patients. These cell lines were cultured at 37 °C in a humid atmosphere of 5% CO2 and passaged at approximately 70% confluence. Human normal prostate epithelial cell line, prostate epithelial cell (PrEC), was obtained from Clonetics (San Diego, CA, USA). PrECs were grown in PrECG Bullet Kit medium according to the manufacturer’s instructions and used without passage. Total RNA extraction and reverse transcriptase PCR (RT-PCR). Total RNA was extracted from each human prostate cancer cell line and PrEC line by the acid guanidium–phenol–chloroform method using Isogen (Nippon Gene, Tokyo, Japan). Pelvic lymph nodes taken from a bladder cancer patient (T2N0M0) were homogenized and used as positive controls of SDF-1 and CXCR4 expression [6,16,17]. The RNA pellet obtained after isopropanol and ethanol precipitation was dried and re-suspended in 50 lL RNase-free water. RNA specimens were stored and frozen in aliquots at )80 °C until reverse-transcribed. The concentration of RNA was determined by spectrophotometer, and their integrity was checked by agarose gel electrophoresis. One microgram of RNA was added to 0.5 lg oligodeoxythymidilic acid primer (TaKaRa, Shiga, Japan) and prepared to a final volume of 20 lL. The samples were placed at 55 °C for 5 min and then cooled on ice. The primer–RNA mixture was then combined with 0.25 U Avian myeloblastosis virus reverse transcriptase (TaKaRa, Shiga, Japan) and 0.5 U RNase inhibitor. Finally, the mixture was added to 1 mM dNTP, 50 mM Tris–HCl, 75 mM KCl, and 5 mM MgCl2 . The reverse transcriptase reaction was then carried out at 45 °C for 45 min. The cDNA was then incubated at 95 °C for 5 min to inactivate the reverse transcriptase. Samples were stored at )20 °C until used. Primer sets of PCR for CXCR4 (GenBank Accession No. XM_051225), SDF-1 [10] and b-actin were designed and synthesized (Amersham–Pharmacia Biotech, Tokyo, Japan) as follows: CXCR4 (347 bp): forward 50 -AGC TGT TGG CTG AAA AGG TGG TCT ATG-30 , reverse 50 -GCG CTT CTG GTG GCC CTT GGA GTG TG30 ; SDF-1 (380 bp): forward 50 -CCG CGC TCT GCC TCA GCG ACG GGA AG-30 , reverse 50 -CCT GTT TAA AGC TTT CTC CAG GTA CT-30 ; and b-actin (696 bp): forward 50 -CAC CAA CTG GGA CGA CAT GG-30 , reverse 50 -CCA GGG TAC ATG GTG GTG CC30 . The cDNA samples (2 lL) were diluted into 20 lL solution containing 50 mM dNTP, 500 nM of each primer, 0.5 U of Taq polymerase, and PCR buffer provided by the manufacturer (TaKaRa). Suitable PCR cycles for CXCR4, SDF-1, and b-actin were determined to remain within the linear range of the reaction. Briefly, after predenaturing at 94 °C for 4 min, 35 cycles of amplification comprising denaturing at 94 °C for 30 min, annealing at 50 °C for 1 min and extension at 72 °C for 1 min were performed using a thermal cycler (RoboCycler GRADIENT 96, Stratagene, CA, USA). The PCR products were electrophoresed on 2.0% agarose gels.
658
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
Western blotting. Cultured cells were washed with PBS buffer and lysed by freeze–thawing in ice-cold lysis buffer (50 mM Hepes, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 100 mM NaF, 1.5 mM MgCl2 , 1 mM ECTA, 1 mM Na3 VO4 , 1 mM PMSF, and 10 lg/mL aprotinin). After removal of nuclei and cellular debris by centrifugation at 12,000 rpm for 5 min at 4 °C, normalized lysate (40 lg) was electrophoresed on 12% SDS–polyacrylamide gels and transferred onto nitrocellulose filters. The filters were blocked overnight at 4 °C in TBS-T containing 5% non-fat milk powder and then incubated for 1 h with 1:1000, diluted anti-human CXCR4 mouse monoclonal antibody (R&D Systems, Minneapolis, MN, USA) or 1:1000, diluted antihuman b-actin mouse monoclonal antibody (Sigma, St. Louis, MO, USA). The filters were incubated for 1 h with 1:4000, diluted antimouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and specific proteins were detected using an enhanced chemiluminescence Western blotting analysis system (ECL, Amersham– Pharmacia Biotech, Little Chalfont, UK). CXCR4 immunostaining. Immunohistochemistry of CXCR4 was performed on 5 lm thick consecutive sections obtained from paraffinembedded materials. Briefly, the de-paraffinized and hydrated sections were routinely processed with antigen retrieval (0.01 M sodium citrate buffer (pH 6.0) in a microwave oven for 5 min) before incubating for 12 h with anti-CXCR4 monoclonal antibody (R&D Systems, Minnneapolis, MN, USA) at 4 °C overnight. This process was performed after inhibition of both endogenous peroxidase activities using 3% H2 O2 and of non-specific immunoreactivity using 2% normal bovine serum. The sections were incubated with goat anti-mouse IgG biotinylated secondary antibodies (Vectastain ABC Kit, Burlingame, CA, USA) for 1 h at room temperature and thereafter incubated in streptavidin–peroxidase complex for 30 min. The sections were then developed with diaminobenzidine (DAB) as the substrate for 3 min and were counterstained with hematoxylin. In negative controls, an irrelevant antibody replaced the primary antibody. Positive staining was defined as moderate or strong intensity, while negative staining was considered if immunoreactivity was weak or lost. Migration assay. Cell migration assays were performed in 6.4 mm diameter chambers with 8 lm pore filters (Transwell, 24-well cell cultures; Becton–Dickinson, Bedford, MA). The lower surface of each filter was pre-coated with 50 lg fibronectin (100 lg/mL) and subsequently dried with air blown into a clean ventilator. PC3 and DU145 cells were starved in serum-free RITC 80-7 medium (Kyokuto Pharmaceuticals, Tokyo, Japan) for 24 h. PC3 and DU145 cells were then suspended at 5 104 or 20 104 cells/mL, respectively, in serum-free RITC 80-7 medium, and then 200 lL of the cell suspension was added to the upper chamber. Various concentrations of SDF-1 (0, 1, 10, 50, or 100 ng/mL) in 600 lL serum-free RITC 80-7 medium were applied in the lower chamber. For neutralization studies, cells pre-treated with 20 lL/mL anti-human CXCR4 monoclonal antibody (44717.111, IgG2B) were incubated for 12 h (for DU145) or 6 h (for PC3) at 37 °C in a humid atmosphere of 5% CO2 . After incubation, the upper surface of the filter was scraped with a cotton swab to remove non-migrating cells, and then the lower surface of the membrane containing the cells that had migrated was stained (Diff-Quick Fixative, Kokusai Shinyaku, Tokyo, Japan). The stained cells were counted under a light microscope (200). The experiments were performed in triplicate wells. MTT assay. Effects of SDF-1 on proliferation of prostate cancer cells were analyzed by MTT (Promega, Madison, USA) assay. Cells (1 103 ) of each prostate cancer cell line (PC3 and DU145) were seeded in 96-well plates. After pre-incubation for 1 h with serum-free RITC 80-7 medium, the cells were maintained in RITC 80-7 medium with several concentrations of SDF-1 (0, 1, 10, 50, or 100 ng/mL of SDF-1, or 50 ng/mL) combined with 20 lg/mL anti-human CXCR4 antibody. FBS (5%) was used as a positive control for growth promotion. On days 0, 2, and 4, 15 lg of MTT solution was added and incubated at 37 °C for 4 h. After incubation with MTT solution, solubilization/stop solution of 100 lL was added and incubated at 37 °C
for 1 h to completely dissolve the formazan product. Plates were analyzed using an ELISA plate reader (Bio-Rad Laboratories, Hercules, CA) at 570 nm with the reference wavelength set to 630 nm. Statistical analyses. Results of migration assay and MTT assay were analyzed by Student’s t test. Relationship between CXCR4 immunostaining and clinicopathological findings was analyzed by v2 test or Fisher’s exact test. P value of less than 0.05 was regarded as statistically significant.
Results CXCR4 and SDF-1 expression in malignant and normal cultured prostate cells To confirm the expression of mRNA transcript of CXCR4 and its ligand SDF-1, RT-PCR was performed in LNCaP, PC3, DU145, and PrECs. Total RNA extracted from a pelvic lymph node was used as a positive control. As shown in Fig. 1A, expression of mRNA transcript of CXCR4 was clearly detected in all three PC cell lines, whereas it was not detectable in PrECs. On the other hand, mRNA transcript of SDF-1 was not found in either PC or PrEC lines (Fig. 1A). Similarly, Western blotting clearly showed that CXCR4 expression at protein level was detected in all three prostate cancer cell lines, but not in PrECs (Fig. 1B). Effect of SDF-1 on chemotaxis of cultured prostate cancer cells Since expression level of CXCR4 protein in PC3 and DU145 cells was significantly higher than in LNCaP cells (Fig. 1B), chemotactic effect of SDF-1 on PC3 and DU145 cells was analyzed using a migration assay through filters coated with the extracellular matrix protein, fibronectin. SDF-1 at a concentration of 50– 100 ng/mL showed a significant increase in migrated PC3 cell counts, and this effect of SDF-1 on PC3 cell migration was significantly inhibited, by a maximum of 39% (P < 0:05), by the addition of neutralizing antihuman CXCR4 antibody (Fig. 2A). Likewise, SDF-1 also significantly stimulated the migration ability of DU145 cells with a maximal response observed at 50 ng/mL, and this effect was significantly inhibited, by a maximum of 50% (P < 0:05), by neutralizing antihuman CXCR4 antibody (Fig. 2B). Effect of SDF-1 on growth rate of cultured prostate cancer cells To investigate whether SDF-1 stimulates the cell growth rate of PC3 and DU145 cells, MTT assay was performed. Contrary to the positive correlation of SDF1 with chemotactic activity described above, 1–100 ng/ mL of SDF-1 treatment did not affect the OD570 value in either the PC3 or DU145 cells (Figs. 2C and D). This
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
659
Fig. 1. RT-PCR and Western blotting of CXCR4 and SDF-1 in prostate cancer cell lines and normal PrECs. (A) CXCR4 and SDF-1 mRNA expression as determined by RT-PCR. Lane 1, PrEC; lane 2, LNCaP; lane 3, PC3; lane 4, DU145; and lane 5, pelvic lymph node (as a positive control). SDF-1 expression was not detectable in any of the prostate cancer cell lines. DNA ladder was applied in the left lane. (B) CXCR4 protein expression as determined by Western blotting. CXCR4 expression was detected in all prostate cancer cell lines, but not in normal prostate epithelial cells. b-Actin expression was used as an internal standard.
was not the case in the positive control (5% FBS), in which the OD570 value was significantly increased in the presence of 5% FBS (P < 0:05). These findings suggest that SDF-1 alone is not sufficient enough to act as a growth factor in prostate cancer cell lines. CXCR4 protein expression in malignant and non-malignant prostate tissues: relationship between expression of CXCR4 and clinicopathological findings Typical immunostaining of CXCR4 in non-malignant prostate tissue and PC tissues is shown in Fig. 3. Epithelial cells of non-malignant prostate tissues showed negative CXCR4 immunoreactivity (Fig. 3A); however, in prostate cancer tissues, positive CXCR4 immunoreactivity was observed in 20 of 35 samples (57.1%) (Figs. 3B and C). The relationships between positive CXCR4 immunoreactivity and clinicopathological findings are summarized in Table 1. The positive rate of CXCR4 expression in each group with high PSA value, high clinical T stage, high Gleason sum, positive lymph node metastasis, and positive lung metastasis was higher than in the group of each counterpart, although the difference did not reach statistical significance. There was no sig-
nificant correlation of CXCR4 expression with PSA value and Gleason sum. Although PSA value is a strong predictor for bone metastasis, logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis to Gleason sum (P < 0:05) (Table 1). Patients with positive CXCR4 are 4.5 times more likely to have bone metastasis compared with CXCR4 negative patients. In PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients with bone metastasis than in those with no bone metastasis (P ¼ 0:017).
Discussion In this study, CXCR4 expression was detected in three PC cell lines of LNCaP, PC3, and DU145 at both the mRNA and protein levels. This was in contrast to normal human prostate epithelial cells, in which no detectable level of CXCR4 expression was found. In addition, we demonstrated that (i) SDF-1 significantly enhanced the migration of PC3 and DU145 cells in a dose-dependent manner and (ii) neutralizing anti-CXCR4 antibody
660
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
Fig. 2. Migration assay of (A) PC3 and (B) DU145. PC3 or DU145 cells were suspended in the upper chamber, and SDF-1 (0–100 ng/mL) was applied into the lower chamber. Anti-CXCR4 antibody (Ab) was pre-incubated for neutralization study. SDF-1 at 50–100 lg/mL significantly increased the chemotactic ability of PC3 and DU145, and these effects were inhibited by 39% and 50%, respectively, by anti-human CXCR4 neutralizing antibody (P < 0:05). Representative results done in triplicate are shown. Data are presented as mean migration of cells per filter. Bars, SD ðn ¼ 3Þ. *, significant increase over untreated control (P < 0:05, Student’s t test). # , significant inhibition by anti-CXCR4 Ab (P < 0:05, Student’s t test). MTT assay of prostate cancer cell lines (C) PC3 and (D) DU145. We assessed 1 103 cells of each prostate cell line in serum-free RITC medium with SDF-1 (0–100 ng/mL), SDF-1 plus anti-CXCR4 antibody, or 5% FBS (positive control). SDF-1 did not significantly stimulate the growth rate of both prostate cancer cell lines as compared to the positive control. In comparison with untreated cells, a significant increase was observed only in the cells treated with 5% FBS (*P < 0:05). Representative results done in triplicate are shown. Data are presented as means SD of OD at wavelength of 570 nm ðn ¼ 3Þ. Data for 1 and 10 ng/mL SDF-1 are omitted.
inhibited this chemotactic effect of SDF-1. These results indicate that CXCR4 protein, which functions as mediator of SDF-1-involved chemotactic activation, might be de novo expressed during prostatic tumorigenesis. On the other hand, SDF-1 was not expressed in these three prostate cancer cell lines. Therefore, although an autocrine action of SDF-1–CXCR4 ligand–receptor system is supposedly present in neuroblastoma [12], prostate cancer cells might be affected not by endogenous but exogenous SDF-1.
Muller et al. [6] reported that among human organ tissues, SDF-1 mRNA is preferentially expressed in lymph nodes and bone marrow. In bone marrow, osteoblasts, fibroblasts, and endothelial cells constitutively produce SDF-1 [18]. Lymph nodes and bone marrow also represent the most common sites of prostate cancer metastasis. Taking together, our results suggest that PC cells metastasize to specific sites via cell migration mediated by an interaction between SDF-1 and CXCR4 in a paracrine way.
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
661
Fig. 3. Immunohistochemical staining for CXCR4 (100). (A) CXCR4-negative staining. Immunostaining of non-malignant prostate tissue obtained from cystoprostatectomy in a bladder cancer patient is shown. (B) CXCR4-positive staining. Immunostaining of prostate cancer biopsy (PSA 18.6 ng/mL, Gleason grade 3/4 ¼ 7, and linical stage T3N0M0) is shown, where the intensity is moderate. (C) CXCR4-positive staining. Immunostaining of prostate cancer biopsy [PSA 112 ng/mL, Gleason grade 4/5 ¼ 9, and clinical stage T4N1M1 (bone and lung metastasis)] is shown, where the intensity is strong.
With regard to the adhesion of prostate cancer cells to a metastatic site, another process is essential for the establishment of metastasis. A recent investigation revealed that during the adhesion of HSC to endothelial cells or bone marrow stromal cells, rapid but transient activation of integrins by SDF-1 is common, that in turn facilitates the binding of HSC to the cells [3]. This adhesion-promoting effect of SDF-l mediated by integrin has also been demonstrated in malignant tumors including neuroblastoma [12] and ovarian tumors [9] and thus, a similar adhesion mechanism can be speculated in bone marrow metastasis of prostate cancer [13]. As a final step in metastasis, growth factors available at the metastatic sites enable cancer cells to thrive and proliferate [12,19]. SDF-1 may also help to establish metastasis as a function of growth factors. To confirm this possibility, we performed MTT assay under serumfree conditions in the presence of various concentrations of SDF-1. However, we could not draw any definite conclusion that SDF-1 drives the growth rate of prostate cancer cells. This was consistent with the observation of Taichman et al. [13], where SDF-1 alone does not affect the growth rate of prostate cancer cells. It has been re-
ported that SDF-1 alone does not stimulate the proliferation of early hematopoietic cells but stimulates synergistically with other growth factors [20]. In a study using the lung cancer cell line NCI-H69, SDF-1 did not exert a growth-promoting effect under serum-free conditions but did in the presence of 10% FBS [11]. Thus, SDF-1 alone is not sufficient enough to act as a growth factor and might stimulate prostate cancer cell growth in combination with some other growth factor. As in the results of the cell culture model described above, we clearly demonstrated that expression of CXCR4 protein was found only in prostate cancer tissues, but not in non-malignant prostate tissues. As shown in Table 1, positive rates of CXCR4 expression were higher in PC patients with worse clinicopathological parameters than in those with better parameters, although the difference was not statistically significant. Logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis compared to Gleason sum (P < 0:05). In addition, among PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients
662
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
Table 1 Relationship between expression of CXCR4 and clinicopathological features Variables Univariate analysis for CXCR4 expression Total PSA (ng/mL) 4–20 20< T category T1-2 T3-4 Cleason sum 2–6 7–10 Lymph node metastasis Negative Positive Bone metastasis Negative Positive Lung metastasis Negative Positive Bone metastasis and PSA>20/ng/mL Negative Positive SE
n
CXCR4 positive (%)
P value
35
20 (57.1)
10 25
5 (50.0) 15 (60.0)
0.71
8 27
4 (50.0) 16 (59.3)
0.70
10 25
5 (50.0) 15 (60.0)
0.71
21 14
10 (47.6) 10 (71.4)
0.30
14 21
5 (35.7) 15 (71.4)
0.08
32 3 25 4 21
17 (53.1) 3 (100.0)
0.24
0 (0.0) 15 (71.4)
0.017a
Coefficient/SE
v2
P value
RR
Logistic regression analysis for bone CXCR4 0.738
2.038
4.16
0.042
4.5
CXCR4 Gleason sum
0.764 0.83
1.969 1.397
3.876 1.952
0.049 0.162
4.5 3.19
CXCR4 PSA Gleason sum
0.975 1.231 1.038
1.732 2.721 1.046
2.998 7.406 1.095
0.083 0.007 0.295
5.41 28.5 2.96
a
Fisher’s exact test, SE, standard error; and RR, relative risk.
with bone metastasis than in those with no metastasis. Patients with lung metastasis showed positive immunostaining for CXCR4. Because lymph node metastasis was clinically diagnosed using an imaging study (CT and MRI), this type of metastasis might have been microscopically overlooked. Thus, pathological examination of lymph nodes could have the possibility to improve the correlation between positive CXCR4 immunoreactivity and lymph node metastasis. These findings suggest that CXCR4 expression in PC tissues is closely associated with cancer progression or metastasis and is a useful tool in predicting metastasis in PC patients. We hypothesize that disruption of the SDF-1– CXCR4 ligand–receptor system could repress metastasis. Our in vitro chemotactic assay demonstrated that anti-CXCR4 antibody blocked SDF-1-induced migration of prostate cancer cells. In a breast cancer metastasis model, Muller et al. [6] reported that administration of an antibody to CXCR4 inhibited metastasis. Similarly, in vivo studies have also shown that a neutralizing antibody to CXCR4 inhibits the homing of human stem cells to the bone marrow in
SCID mice [5]. These findings suggest that interaction between SDF-1–CXCR4 ligand–receptor system is involved in the process of PC metastasis by the activation of cancer cell migration.
Acknowledgments This research was partially supported by the funding from REAP award from Department of Veterans Affairs and Grant-in-Aid for Scientific Research (B) 13470333 and for Encouragement of Young Scientists (B) 14770809 from the Ministry of Education, Science, Sports and culture of Japan.
References [1] G.L. Nicolson, Paracrine and autocrine growth mechanisms in tumor metastasis to specific sites with particular emphasis on brain and lung metastasis, Cancer Metastasis Rev. 12 (1993) 325– 343. [2] J.M. Wang, X. Deng, W. Gong, S. Su, Chemokines and their role in tumor growth and metastasis, J. Immunol. Methods 220 (1998) 1–17.
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663 [3] A. Peled, O. Kollet, T. Ponomaryov, I. Petit, S. Franitza, V. Grabovsky, M.M. Slav, A. Nagler, O. Lider, R. Alon, D. Zipori, T. Lapidot, The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/ SCID mice, Blood 95 (2000) 3289–3296. [4] D.Y. Jo, S. Rafii, T. Hamada, M.A. Moore, Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor-1, J. Clin. Invest. 105 (2000) 101–111. [5] A. Peled, I. Petit, O. Kollet, M. Magid, T. Ponomaryov, T. Byk, A. Nagler, H. Ben-Hur, A. Many, L. Shultz, O. Lider, R. Alon, D. Zipori, T. Lapidot, Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4, Science 283 (1999) 845–848. [6] A. Muller, B. Homey, H. Soto, N. Ge, D. Catron, M.E. Buchanan, T. McClanahan, E. Murphy, W. Yuan, S.N. Wagner, J.L. Barrera, A. Mohar, E. Verastegui, A. Zlotnik, Involvement of chemokine receptors in breast cancer metastasis, Nature 410 (2001) 50–56. [7] M.M. Robledo, R.A. Bartolome, N. Longo, J.M. RodriguezFrade, M. Mellado, I. Longo, G.N. van Muijen, P. SanchezMateos, J. Teixido, Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells, J. Biol. Chem. 276 (2001) 45098–45105. [8] A.S. Payne, L.A. Cornelius, The role of chemokines in melanoma tumor growth and metastasis, J. Invest. Dermatol. 118 (2002) 915– 922. [9] C.J. Scotton, J.L. Wilson, K. Scott, G. Stamp, G.D. Wilbanks, S. Fricker, G. Bridger, F.R. Balkwill, Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer, Cancer Res. 62 (2002) 5930–5938. [10] T. Koshiba, R. Hosotani, Y. Miyamoto, J. Ida, S. Tsuji, S. Nakajima, M. Kawaguchi, H. Kobayashi, R. Doi, T. Hori, N. Fujii, M. Imamura, Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression, Clin. Cancer Res. 6 (2000) 3530–3535. [11] T. Kijima, G. Maulik, P.C. Ma, E.V. Tibaldi, R.E. Turner, B. Rollins, M. Sattler, B.E. Johnson, R. Salgia, Regulation of
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19] [20]
663
cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cancer cells, Cancer Res. 62 (2002) 6304–6311. H. Geminder, O. Sagi-Assif, L. Goldberg, T. Meshel, G. Rechavi, I.P. Witz, A. Ben-Baruch, A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma, J. Immunol. 167 (2001) 4747–4757. R.S. Taichman, C. Cooper, E.T. Keller, K.J. Pienta, N.S. Taichman, L.K. McCauley, Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone, Cancer Res. 62 (2002) 1832–1837. D.F. Gleason, Histologic grading of prostate cancer: a perspective, Human Pathol. 23 (1992) 273–279. L.H. Sobin, I.D. Fleming, TNM Classification of Malignant Tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer, Cancer 80 (1997) 1803–1804. M. D’Apuzzo, A. Rolink, M. Loetscher, J.A. Hoxie, I. ClarkLewis, F. Melchers, M. Baggiolini, B. Moser, The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4, Eur. J. Immunol. 27 (1997) 1788–1793. C.C. Bleul, R.C. Fuhlbrigge, J.M. Casasnovas, A. Aiuti, T.A. Springer, A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1), J. Exp. Med. 184 (1996) 1101– 1109. T. Ponomaryov, A. Peled, I. Petit, R.S. Taichman, L. Habler, J. Sandbank, F. Arenzana-Seisdedos, A. Magerus, A. Caruz, N. Fujii, A. Nagler, M. Lahav, M. Szyper-Kravitz, D. Zipori, T. Lapidot, Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function, J. Clin. Invest. 106 (2000) 1331–1339. R. Radinsky, I.J. Fidler, Regulation of tumor cell growth at organ-specific metastases, In Vivo 6 (1992) 325–331. S.K. Gupta, K. Pillarisetti, Cutting edge: CXCR4-Lo: molecular cloning and functional expression of a novel human CXCR4 splice variant, J. Immunol. 163 (1999) 2368– 2372.
Interaction of ligand–receptor system between stromal-cell-derived factor-1 and CXC chemokine receptor 4 in human prostate cancer: a possible predictor of metastasis Hideki Mochizuki,a Akio Matsubara,b Jun Teishima,b Kazuaki Mutaguchi,b Hiroaki Yasumoto,b Rajvir Dahiya,c,* Tsuguru Usui,b and Kenji Kamiyaa a
Division of Genome Biology, Department of Experimental Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan b Programs for Biochemical Research, Division of Frontier Medical School, Department of Urology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan c Department of Urology and Urology Research Center, University of California San Francisco and Veterans Affairs Medical Center, USA Received 24 May 2004 Available online
Abstract Interaction of ligand–receptor systems between stromal-cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) is closely involved in the organ specificity of cancer metastasis. We hypothesized that SDF-1–CXCR4 ligand–receptor system plays an important role in prostate cancer metastasis. To test this hypothesis, expression level of SDF-1 and CXCR4 was analyzed in prostate cancer (PC) cell lines (LNCaP, PC3, and DU145) and normal prostate epithelial cell line (PrEC). We also performed migration assay and MTT assay to investigate the chemotactic effect and growth-promoting effect of SDF-1 on DU145 and PC3 cells, respectively. Furthermore, we performed immunohistochemical analysis of CXCR4 expression in tissues from 35 cases of human prostate cancer. CXCR4 expression was detected in all three prostate cancer cell lines, but not in PrECs. SDF-1 significantly enhanced the migration of PC3 and DU145 cells in a dose-dependent manner, and anti-CXCR4 antibody inhibited this chemotactic effect. However, SDF-1 itself did not significantly stimulate the cell growth rate of prostate cancer cell lines. Positive CXCR4 protein was found in 20 out of 35 clinical PC samples (57.1%). Three patients with lung metastasis showed definitely positive CXCR4 immunostaining. Logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis to Gleason sum (P < 0:05). Furthermore, among PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients with bone metastasis than in those with no bone metastasis (P ¼ 0:017). These findings suggest that the interaction between SDF-1 and CXCR4 ligand–receptor system is involved in the process of PC metastasis by the activation of cancer cell migration. This is the first report to investigate the role of interaction of ligand–receptor systems between SDF-1 and CXCR4 in prostate cancer metastasis. Ó 2004 Elsevier Inc. All rights reserved. Keywords: CXCR4; SDF-1; Prostate cancer; Metastasis
In advanced prostate cancer, the sites most frequently affected by metastasis are the bones and regional lymph nodes [1,2]. Patients with these metastases not only have a poor prognosis but also suffer pain and lower limb edema. It is therefore extremely important to elucidate *
Corresponding author. Fax: 1-415-750-6639. E-mail addresses: [email protected] (R. Dahiya), musui@ hiroshima-u.ac.jp (T. Usui). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.013
the molecular mechanisms underlying prostate cancer metastases to the bones and/or lymph nodes and to explore the avenues preventing such metastases. The molecular processes associated with metastases such as migration of cancer cells, their adhesion to specific organs, and cell proliferation within the microcirculation of these organs have been fully investigated [1,2]. In this regard, it is assumed that the target organ specificity of cancer metastasis is followed by the similar mechanism
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
observed in the homing of hematopoietic stem cells (HSC) to bone marrow in the accepted model of hematopoiesis [3–5]. Bone marrow stromal cells produce a chemokine, stromal-cell-derived factor-1 (SDF-1), especially during the process of fetal hematopoiesis and after bone marrow transplantation. At the same time, HSC significantly expresses its receptor, CXC chemokine receptor 4 (CXCR4) [3–5]. SDF-1 is a member of the CXC subfamily of chemokines, and its chemotactic and growth-promoting effects are mediated by CXCR4. Consequently, SDF-1 acts as a powerful chemo-attractant for HSC, which in turn “homes in” on the bone marrow specifically and efficiently [3–5]. Based on this hematopoiesis model, recent publications have demonstrated that the SDF-1–CXCR4 ligand–receptor system is closely involved in the organ specificity of cancer metastasis. For instance, Muller et al. [6] have reported that (i) CXCR4 is highly expressed in human breast cancer cells and (ii) the highest SDF-1 expression is observed in those organs that are usually the primary destinations of breast cancer metastases. SDF-1 signaling through CXCR4 interaction appears to determine the directional migration of breast cancer cells through the basement membrane. Furthermore in vivo, the interaction between SDF-1 and CXCR4 significantly represses the metastatic potential of breast cancer cells to regional lymph node and lung. These findings indicate that the SDF-1–CXCR4 ligand– receptor system plays a critical role in determining the metastatic destination of cancer cells. Similar experimental results have also been reported in other types of cancer such as malignant melanoma, ovarian, pancreatic, and lung [7–13]. However, such studies are limited in prostate cancer. To investigate whether SDF-1–CXCR4 ligand–receptor system is involved in the metastasis of human prostate cancer, we investigated the gene and protein expression of CXCR4 in the prostate cancer cell lines LNCaP, PC3, and DU145, in comparison with normal prostate epithelial cells. We also investigated the chemotactic activity and the growth-promoting effect of SDF-1 on DU145 and PC3 cells. Furthermore, we performed CXCR4 immunostaining in clinical samples (35 human prostate cancer tissues and nine non-malignant human prostate tissues), and evaluated the relationship between CXCR4 expression and clinicopathological findings with special reference to cancer metastasis.
Materials and methods Prostate tissue samples. Thirty-five prostate cancer samples (32 prostatic biopsy samples, 2 radical prostatectomy samples, and one transurethrally resected sample) and nine non-malignant prostate samples (seven normal prostate obtained at total cystoprostatectomy and two BPH tissues from suprapubic prostatectomy) were obtained with informed consent at the Department of Urology, Hiroshima
657
University School of Medicine, Hiroshima, Japan, between 1998 and 2001. All tissue samples were pathologically diagnosed and confirmed to contain the objective parts. A single pathologist classified each sample according to the Gleason grading system [14]. Staging examination for PC included digital rectal examination, transrectal ultrasound, urethrocystography, computed tomography, magnetic resonance imaging, and bone scanning. The clinicopathological stage of prostate cancer was categorized according to the 1997 International Union Against Cancer (UICC) TNM classification [15]. Serum prostate-specific antigen (PSA) levels were measured by Tandem R assay (Hybritech, San Diego, CA, USA). Cell culture. Human prostate cancer cell lines of LNCaP, PC-3, and DU145 were purchased from the American Type Culture Collection (Drive Rockville, MD, USA) and maintained in OPTI-MEM 1 medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 5% fetal bovine serum (FBS), 1 mM CaCl2 , and 100 lg/mL kanamycin (Life Technologies). LNCaP cells were originally isolated from lymph node in a patient with hormone-dependent prostate cancer involved in bone and lymph node metastases. PC3 and DU145 cells were isolated from the regions of vertebral and brain metastases, respectively, in hormone-independent prostate cancer patients. These cell lines were cultured at 37 °C in a humid atmosphere of 5% CO2 and passaged at approximately 70% confluence. Human normal prostate epithelial cell line, prostate epithelial cell (PrEC), was obtained from Clonetics (San Diego, CA, USA). PrECs were grown in PrECG Bullet Kit medium according to the manufacturer’s instructions and used without passage. Total RNA extraction and reverse transcriptase PCR (RT-PCR). Total RNA was extracted from each human prostate cancer cell line and PrEC line by the acid guanidium–phenol–chloroform method using Isogen (Nippon Gene, Tokyo, Japan). Pelvic lymph nodes taken from a bladder cancer patient (T2N0M0) were homogenized and used as positive controls of SDF-1 and CXCR4 expression [6,16,17]. The RNA pellet obtained after isopropanol and ethanol precipitation was dried and re-suspended in 50 lL RNase-free water. RNA specimens were stored and frozen in aliquots at )80 °C until reverse-transcribed. The concentration of RNA was determined by spectrophotometer, and their integrity was checked by agarose gel electrophoresis. One microgram of RNA was added to 0.5 lg oligodeoxythymidilic acid primer (TaKaRa, Shiga, Japan) and prepared to a final volume of 20 lL. The samples were placed at 55 °C for 5 min and then cooled on ice. The primer–RNA mixture was then combined with 0.25 U Avian myeloblastosis virus reverse transcriptase (TaKaRa, Shiga, Japan) and 0.5 U RNase inhibitor. Finally, the mixture was added to 1 mM dNTP, 50 mM Tris–HCl, 75 mM KCl, and 5 mM MgCl2 . The reverse transcriptase reaction was then carried out at 45 °C for 45 min. The cDNA was then incubated at 95 °C for 5 min to inactivate the reverse transcriptase. Samples were stored at )20 °C until used. Primer sets of PCR for CXCR4 (GenBank Accession No. XM_051225), SDF-1 [10] and b-actin were designed and synthesized (Amersham–Pharmacia Biotech, Tokyo, Japan) as follows: CXCR4 (347 bp): forward 50 -AGC TGT TGG CTG AAA AGG TGG TCT ATG-30 , reverse 50 -GCG CTT CTG GTG GCC CTT GGA GTG TG30 ; SDF-1 (380 bp): forward 50 -CCG CGC TCT GCC TCA GCG ACG GGA AG-30 , reverse 50 -CCT GTT TAA AGC TTT CTC CAG GTA CT-30 ; and b-actin (696 bp): forward 50 -CAC CAA CTG GGA CGA CAT GG-30 , reverse 50 -CCA GGG TAC ATG GTG GTG CC30 . The cDNA samples (2 lL) were diluted into 20 lL solution containing 50 mM dNTP, 500 nM of each primer, 0.5 U of Taq polymerase, and PCR buffer provided by the manufacturer (TaKaRa). Suitable PCR cycles for CXCR4, SDF-1, and b-actin were determined to remain within the linear range of the reaction. Briefly, after predenaturing at 94 °C for 4 min, 35 cycles of amplification comprising denaturing at 94 °C for 30 min, annealing at 50 °C for 1 min and extension at 72 °C for 1 min were performed using a thermal cycler (RoboCycler GRADIENT 96, Stratagene, CA, USA). The PCR products were electrophoresed on 2.0% agarose gels.
658
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
Western blotting. Cultured cells were washed with PBS buffer and lysed by freeze–thawing in ice-cold lysis buffer (50 mM Hepes, 1% Triton X-100, 10% glycerol, 150 mM NaCl, 100 mM NaF, 1.5 mM MgCl2 , 1 mM ECTA, 1 mM Na3 VO4 , 1 mM PMSF, and 10 lg/mL aprotinin). After removal of nuclei and cellular debris by centrifugation at 12,000 rpm for 5 min at 4 °C, normalized lysate (40 lg) was electrophoresed on 12% SDS–polyacrylamide gels and transferred onto nitrocellulose filters. The filters were blocked overnight at 4 °C in TBS-T containing 5% non-fat milk powder and then incubated for 1 h with 1:1000, diluted anti-human CXCR4 mouse monoclonal antibody (R&D Systems, Minneapolis, MN, USA) or 1:1000, diluted antihuman b-actin mouse monoclonal antibody (Sigma, St. Louis, MO, USA). The filters were incubated for 1 h with 1:4000, diluted antimouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and specific proteins were detected using an enhanced chemiluminescence Western blotting analysis system (ECL, Amersham– Pharmacia Biotech, Little Chalfont, UK). CXCR4 immunostaining. Immunohistochemistry of CXCR4 was performed on 5 lm thick consecutive sections obtained from paraffinembedded materials. Briefly, the de-paraffinized and hydrated sections were routinely processed with antigen retrieval (0.01 M sodium citrate buffer (pH 6.0) in a microwave oven for 5 min) before incubating for 12 h with anti-CXCR4 monoclonal antibody (R&D Systems, Minnneapolis, MN, USA) at 4 °C overnight. This process was performed after inhibition of both endogenous peroxidase activities using 3% H2 O2 and of non-specific immunoreactivity using 2% normal bovine serum. The sections were incubated with goat anti-mouse IgG biotinylated secondary antibodies (Vectastain ABC Kit, Burlingame, CA, USA) for 1 h at room temperature and thereafter incubated in streptavidin–peroxidase complex for 30 min. The sections were then developed with diaminobenzidine (DAB) as the substrate for 3 min and were counterstained with hematoxylin. In negative controls, an irrelevant antibody replaced the primary antibody. Positive staining was defined as moderate or strong intensity, while negative staining was considered if immunoreactivity was weak or lost. Migration assay. Cell migration assays were performed in 6.4 mm diameter chambers with 8 lm pore filters (Transwell, 24-well cell cultures; Becton–Dickinson, Bedford, MA). The lower surface of each filter was pre-coated with 50 lg fibronectin (100 lg/mL) and subsequently dried with air blown into a clean ventilator. PC3 and DU145 cells were starved in serum-free RITC 80-7 medium (Kyokuto Pharmaceuticals, Tokyo, Japan) for 24 h. PC3 and DU145 cells were then suspended at 5 104 or 20 104 cells/mL, respectively, in serum-free RITC 80-7 medium, and then 200 lL of the cell suspension was added to the upper chamber. Various concentrations of SDF-1 (0, 1, 10, 50, or 100 ng/mL) in 600 lL serum-free RITC 80-7 medium were applied in the lower chamber. For neutralization studies, cells pre-treated with 20 lL/mL anti-human CXCR4 monoclonal antibody (44717.111, IgG2B) were incubated for 12 h (for DU145) or 6 h (for PC3) at 37 °C in a humid atmosphere of 5% CO2 . After incubation, the upper surface of the filter was scraped with a cotton swab to remove non-migrating cells, and then the lower surface of the membrane containing the cells that had migrated was stained (Diff-Quick Fixative, Kokusai Shinyaku, Tokyo, Japan). The stained cells were counted under a light microscope (200). The experiments were performed in triplicate wells. MTT assay. Effects of SDF-1 on proliferation of prostate cancer cells were analyzed by MTT (Promega, Madison, USA) assay. Cells (1 103 ) of each prostate cancer cell line (PC3 and DU145) were seeded in 96-well plates. After pre-incubation for 1 h with serum-free RITC 80-7 medium, the cells were maintained in RITC 80-7 medium with several concentrations of SDF-1 (0, 1, 10, 50, or 100 ng/mL of SDF-1, or 50 ng/mL) combined with 20 lg/mL anti-human CXCR4 antibody. FBS (5%) was used as a positive control for growth promotion. On days 0, 2, and 4, 15 lg of MTT solution was added and incubated at 37 °C for 4 h. After incubation with MTT solution, solubilization/stop solution of 100 lL was added and incubated at 37 °C
for 1 h to completely dissolve the formazan product. Plates were analyzed using an ELISA plate reader (Bio-Rad Laboratories, Hercules, CA) at 570 nm with the reference wavelength set to 630 nm. Statistical analyses. Results of migration assay and MTT assay were analyzed by Student’s t test. Relationship between CXCR4 immunostaining and clinicopathological findings was analyzed by v2 test or Fisher’s exact test. P value of less than 0.05 was regarded as statistically significant.
Results CXCR4 and SDF-1 expression in malignant and normal cultured prostate cells To confirm the expression of mRNA transcript of CXCR4 and its ligand SDF-1, RT-PCR was performed in LNCaP, PC3, DU145, and PrECs. Total RNA extracted from a pelvic lymph node was used as a positive control. As shown in Fig. 1A, expression of mRNA transcript of CXCR4 was clearly detected in all three PC cell lines, whereas it was not detectable in PrECs. On the other hand, mRNA transcript of SDF-1 was not found in either PC or PrEC lines (Fig. 1A). Similarly, Western blotting clearly showed that CXCR4 expression at protein level was detected in all three prostate cancer cell lines, but not in PrECs (Fig. 1B). Effect of SDF-1 on chemotaxis of cultured prostate cancer cells Since expression level of CXCR4 protein in PC3 and DU145 cells was significantly higher than in LNCaP cells (Fig. 1B), chemotactic effect of SDF-1 on PC3 and DU145 cells was analyzed using a migration assay through filters coated with the extracellular matrix protein, fibronectin. SDF-1 at a concentration of 50– 100 ng/mL showed a significant increase in migrated PC3 cell counts, and this effect of SDF-1 on PC3 cell migration was significantly inhibited, by a maximum of 39% (P < 0:05), by the addition of neutralizing antihuman CXCR4 antibody (Fig. 2A). Likewise, SDF-1 also significantly stimulated the migration ability of DU145 cells with a maximal response observed at 50 ng/mL, and this effect was significantly inhibited, by a maximum of 50% (P < 0:05), by neutralizing antihuman CXCR4 antibody (Fig. 2B). Effect of SDF-1 on growth rate of cultured prostate cancer cells To investigate whether SDF-1 stimulates the cell growth rate of PC3 and DU145 cells, MTT assay was performed. Contrary to the positive correlation of SDF1 with chemotactic activity described above, 1–100 ng/ mL of SDF-1 treatment did not affect the OD570 value in either the PC3 or DU145 cells (Figs. 2C and D). This
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
659
Fig. 1. RT-PCR and Western blotting of CXCR4 and SDF-1 in prostate cancer cell lines and normal PrECs. (A) CXCR4 and SDF-1 mRNA expression as determined by RT-PCR. Lane 1, PrEC; lane 2, LNCaP; lane 3, PC3; lane 4, DU145; and lane 5, pelvic lymph node (as a positive control). SDF-1 expression was not detectable in any of the prostate cancer cell lines. DNA ladder was applied in the left lane. (B) CXCR4 protein expression as determined by Western blotting. CXCR4 expression was detected in all prostate cancer cell lines, but not in normal prostate epithelial cells. b-Actin expression was used as an internal standard.
was not the case in the positive control (5% FBS), in which the OD570 value was significantly increased in the presence of 5% FBS (P < 0:05). These findings suggest that SDF-1 alone is not sufficient enough to act as a growth factor in prostate cancer cell lines. CXCR4 protein expression in malignant and non-malignant prostate tissues: relationship between expression of CXCR4 and clinicopathological findings Typical immunostaining of CXCR4 in non-malignant prostate tissue and PC tissues is shown in Fig. 3. Epithelial cells of non-malignant prostate tissues showed negative CXCR4 immunoreactivity (Fig. 3A); however, in prostate cancer tissues, positive CXCR4 immunoreactivity was observed in 20 of 35 samples (57.1%) (Figs. 3B and C). The relationships between positive CXCR4 immunoreactivity and clinicopathological findings are summarized in Table 1. The positive rate of CXCR4 expression in each group with high PSA value, high clinical T stage, high Gleason sum, positive lymph node metastasis, and positive lung metastasis was higher than in the group of each counterpart, although the difference did not reach statistical significance. There was no sig-
nificant correlation of CXCR4 expression with PSA value and Gleason sum. Although PSA value is a strong predictor for bone metastasis, logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis to Gleason sum (P < 0:05) (Table 1). Patients with positive CXCR4 are 4.5 times more likely to have bone metastasis compared with CXCR4 negative patients. In PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients with bone metastasis than in those with no bone metastasis (P ¼ 0:017).
Discussion In this study, CXCR4 expression was detected in three PC cell lines of LNCaP, PC3, and DU145 at both the mRNA and protein levels. This was in contrast to normal human prostate epithelial cells, in which no detectable level of CXCR4 expression was found. In addition, we demonstrated that (i) SDF-1 significantly enhanced the migration of PC3 and DU145 cells in a dose-dependent manner and (ii) neutralizing anti-CXCR4 antibody
660
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
Fig. 2. Migration assay of (A) PC3 and (B) DU145. PC3 or DU145 cells were suspended in the upper chamber, and SDF-1 (0–100 ng/mL) was applied into the lower chamber. Anti-CXCR4 antibody (Ab) was pre-incubated for neutralization study. SDF-1 at 50–100 lg/mL significantly increased the chemotactic ability of PC3 and DU145, and these effects were inhibited by 39% and 50%, respectively, by anti-human CXCR4 neutralizing antibody (P < 0:05). Representative results done in triplicate are shown. Data are presented as mean migration of cells per filter. Bars, SD ðn ¼ 3Þ. *, significant increase over untreated control (P < 0:05, Student’s t test). # , significant inhibition by anti-CXCR4 Ab (P < 0:05, Student’s t test). MTT assay of prostate cancer cell lines (C) PC3 and (D) DU145. We assessed 1 103 cells of each prostate cell line in serum-free RITC medium with SDF-1 (0–100 ng/mL), SDF-1 plus anti-CXCR4 antibody, or 5% FBS (positive control). SDF-1 did not significantly stimulate the growth rate of both prostate cancer cell lines as compared to the positive control. In comparison with untreated cells, a significant increase was observed only in the cells treated with 5% FBS (*P < 0:05). Representative results done in triplicate are shown. Data are presented as means SD of OD at wavelength of 570 nm ðn ¼ 3Þ. Data for 1 and 10 ng/mL SDF-1 are omitted.
inhibited this chemotactic effect of SDF-1. These results indicate that CXCR4 protein, which functions as mediator of SDF-1-involved chemotactic activation, might be de novo expressed during prostatic tumorigenesis. On the other hand, SDF-1 was not expressed in these three prostate cancer cell lines. Therefore, although an autocrine action of SDF-1–CXCR4 ligand–receptor system is supposedly present in neuroblastoma [12], prostate cancer cells might be affected not by endogenous but exogenous SDF-1.
Muller et al. [6] reported that among human organ tissues, SDF-1 mRNA is preferentially expressed in lymph nodes and bone marrow. In bone marrow, osteoblasts, fibroblasts, and endothelial cells constitutively produce SDF-1 [18]. Lymph nodes and bone marrow also represent the most common sites of prostate cancer metastasis. Taking together, our results suggest that PC cells metastasize to specific sites via cell migration mediated by an interaction between SDF-1 and CXCR4 in a paracrine way.
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
661
Fig. 3. Immunohistochemical staining for CXCR4 (100). (A) CXCR4-negative staining. Immunostaining of non-malignant prostate tissue obtained from cystoprostatectomy in a bladder cancer patient is shown. (B) CXCR4-positive staining. Immunostaining of prostate cancer biopsy (PSA 18.6 ng/mL, Gleason grade 3/4 ¼ 7, and linical stage T3N0M0) is shown, where the intensity is moderate. (C) CXCR4-positive staining. Immunostaining of prostate cancer biopsy [PSA 112 ng/mL, Gleason grade 4/5 ¼ 9, and clinical stage T4N1M1 (bone and lung metastasis)] is shown, where the intensity is strong.
With regard to the adhesion of prostate cancer cells to a metastatic site, another process is essential for the establishment of metastasis. A recent investigation revealed that during the adhesion of HSC to endothelial cells or bone marrow stromal cells, rapid but transient activation of integrins by SDF-1 is common, that in turn facilitates the binding of HSC to the cells [3]. This adhesion-promoting effect of SDF-l mediated by integrin has also been demonstrated in malignant tumors including neuroblastoma [12] and ovarian tumors [9] and thus, a similar adhesion mechanism can be speculated in bone marrow metastasis of prostate cancer [13]. As a final step in metastasis, growth factors available at the metastatic sites enable cancer cells to thrive and proliferate [12,19]. SDF-1 may also help to establish metastasis as a function of growth factors. To confirm this possibility, we performed MTT assay under serumfree conditions in the presence of various concentrations of SDF-1. However, we could not draw any definite conclusion that SDF-1 drives the growth rate of prostate cancer cells. This was consistent with the observation of Taichman et al. [13], where SDF-1 alone does not affect the growth rate of prostate cancer cells. It has been re-
ported that SDF-1 alone does not stimulate the proliferation of early hematopoietic cells but stimulates synergistically with other growth factors [20]. In a study using the lung cancer cell line NCI-H69, SDF-1 did not exert a growth-promoting effect under serum-free conditions but did in the presence of 10% FBS [11]. Thus, SDF-1 alone is not sufficient enough to act as a growth factor and might stimulate prostate cancer cell growth in combination with some other growth factor. As in the results of the cell culture model described above, we clearly demonstrated that expression of CXCR4 protein was found only in prostate cancer tissues, but not in non-malignant prostate tissues. As shown in Table 1, positive rates of CXCR4 expression were higher in PC patients with worse clinicopathological parameters than in those with better parameters, although the difference was not statistically significant. Logistic regression analysis revealed that positive expression of CXCR4 protein was an independent and superior predictor for bone metastasis compared to Gleason sum (P < 0:05). In addition, among PC patients with PSA greater than 20 ng/mL, the positive rate of CXCR4 protein was significantly higher in patients
662
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663
Table 1 Relationship between expression of CXCR4 and clinicopathological features Variables Univariate analysis for CXCR4 expression Total PSA (ng/mL) 4–20 20< T category T1-2 T3-4 Cleason sum 2–6 7–10 Lymph node metastasis Negative Positive Bone metastasis Negative Positive Lung metastasis Negative Positive Bone metastasis and PSA>20/ng/mL Negative Positive SE
n
CXCR4 positive (%)
P value
35
20 (57.1)
10 25
5 (50.0) 15 (60.0)
0.71
8 27
4 (50.0) 16 (59.3)
0.70
10 25
5 (50.0) 15 (60.0)
0.71
21 14
10 (47.6) 10 (71.4)
0.30
14 21
5 (35.7) 15 (71.4)
0.08
32 3 25 4 21
17 (53.1) 3 (100.0)
0.24
0 (0.0) 15 (71.4)
0.017a
Coefficient/SE
v2
P value
RR
Logistic regression analysis for bone CXCR4 0.738
2.038
4.16
0.042
4.5
CXCR4 Gleason sum
0.764 0.83
1.969 1.397
3.876 1.952
0.049 0.162
4.5 3.19
CXCR4 PSA Gleason sum
0.975 1.231 1.038
1.732 2.721 1.046
2.998 7.406 1.095
0.083 0.007 0.295
5.41 28.5 2.96
a
Fisher’s exact test, SE, standard error; and RR, relative risk.
with bone metastasis than in those with no metastasis. Patients with lung metastasis showed positive immunostaining for CXCR4. Because lymph node metastasis was clinically diagnosed using an imaging study (CT and MRI), this type of metastasis might have been microscopically overlooked. Thus, pathological examination of lymph nodes could have the possibility to improve the correlation between positive CXCR4 immunoreactivity and lymph node metastasis. These findings suggest that CXCR4 expression in PC tissues is closely associated with cancer progression or metastasis and is a useful tool in predicting metastasis in PC patients. We hypothesize that disruption of the SDF-1– CXCR4 ligand–receptor system could repress metastasis. Our in vitro chemotactic assay demonstrated that anti-CXCR4 antibody blocked SDF-1-induced migration of prostate cancer cells. In a breast cancer metastasis model, Muller et al. [6] reported that administration of an antibody to CXCR4 inhibited metastasis. Similarly, in vivo studies have also shown that a neutralizing antibody to CXCR4 inhibits the homing of human stem cells to the bone marrow in
SCID mice [5]. These findings suggest that interaction between SDF-1–CXCR4 ligand–receptor system is involved in the process of PC metastasis by the activation of cancer cell migration.
Acknowledgments This research was partially supported by the funding from REAP award from Department of Veterans Affairs and Grant-in-Aid for Scientific Research (B) 13470333 and for Encouragement of Young Scientists (B) 14770809 from the Ministry of Education, Science, Sports and culture of Japan.
References [1] G.L. Nicolson, Paracrine and autocrine growth mechanisms in tumor metastasis to specific sites with particular emphasis on brain and lung metastasis, Cancer Metastasis Rev. 12 (1993) 325– 343. [2] J.M. Wang, X. Deng, W. Gong, S. Su, Chemokines and their role in tumor growth and metastasis, J. Immunol. Methods 220 (1998) 1–17.
H. Mochizuki et al. / Biochemical and Biophysical Research Communications 320 (2004) 656–663 [3] A. Peled, O. Kollet, T. Ponomaryov, I. Petit, S. Franitza, V. Grabovsky, M.M. Slav, A. Nagler, O. Lider, R. Alon, D. Zipori, T. Lapidot, The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/ SCID mice, Blood 95 (2000) 3289–3296. [4] D.Y. Jo, S. Rafii, T. Hamada, M.A. Moore, Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor-1, J. Clin. Invest. 105 (2000) 101–111. [5] A. Peled, I. Petit, O. Kollet, M. Magid, T. Ponomaryov, T. Byk, A. Nagler, H. Ben-Hur, A. Many, L. Shultz, O. Lider, R. Alon, D. Zipori, T. Lapidot, Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4, Science 283 (1999) 845–848. [6] A. Muller, B. Homey, H. Soto, N. Ge, D. Catron, M.E. Buchanan, T. McClanahan, E. Murphy, W. Yuan, S.N. Wagner, J.L. Barrera, A. Mohar, E. Verastegui, A. Zlotnik, Involvement of chemokine receptors in breast cancer metastasis, Nature 410 (2001) 50–56. [7] M.M. Robledo, R.A. Bartolome, N. Longo, J.M. RodriguezFrade, M. Mellado, I. Longo, G.N. van Muijen, P. SanchezMateos, J. Teixido, Expression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cells, J. Biol. Chem. 276 (2001) 45098–45105. [8] A.S. Payne, L.A. Cornelius, The role of chemokines in melanoma tumor growth and metastasis, J. Invest. Dermatol. 118 (2002) 915– 922. [9] C.J. Scotton, J.L. Wilson, K. Scott, G. Stamp, G.D. Wilbanks, S. Fricker, G. Bridger, F.R. Balkwill, Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer, Cancer Res. 62 (2002) 5930–5938. [10] T. Koshiba, R. Hosotani, Y. Miyamoto, J. Ida, S. Tsuji, S. Nakajima, M. Kawaguchi, H. Kobayashi, R. Doi, T. Hori, N. Fujii, M. Imamura, Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression, Clin. Cancer Res. 6 (2000) 3530–3535. [11] T. Kijima, G. Maulik, P.C. Ma, E.V. Tibaldi, R.E. Turner, B. Rollins, M. Sattler, B.E. Johnson, R. Salgia, Regulation of
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19] [20]
663
cellular proliferation, cytoskeletal function, and signal transduction through CXCR4 and c-Kit in small cell lung cancer cells, Cancer Res. 62 (2002) 6304–6311. H. Geminder, O. Sagi-Assif, L. Goldberg, T. Meshel, G. Rechavi, I.P. Witz, A. Ben-Baruch, A possible role for CXCR4 and its ligand, the CXC chemokine stromal cell-derived factor-1, in the development of bone marrow metastases in neuroblastoma, J. Immunol. 167 (2001) 4747–4757. R.S. Taichman, C. Cooper, E.T. Keller, K.J. Pienta, N.S. Taichman, L.K. McCauley, Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone, Cancer Res. 62 (2002) 1832–1837. D.F. Gleason, Histologic grading of prostate cancer: a perspective, Human Pathol. 23 (1992) 273–279. L.H. Sobin, I.D. Fleming, TNM Classification of Malignant Tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer, Cancer 80 (1997) 1803–1804. M. D’Apuzzo, A. Rolink, M. Loetscher, J.A. Hoxie, I. ClarkLewis, F. Melchers, M. Baggiolini, B. Moser, The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4, Eur. J. Immunol. 27 (1997) 1788–1793. C.C. Bleul, R.C. Fuhlbrigge, J.M. Casasnovas, A. Aiuti, T.A. Springer, A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1), J. Exp. Med. 184 (1996) 1101– 1109. T. Ponomaryov, A. Peled, I. Petit, R.S. Taichman, L. Habler, J. Sandbank, F. Arenzana-Seisdedos, A. Magerus, A. Caruz, N. Fujii, A. Nagler, M. Lahav, M. Szyper-Kravitz, D. Zipori, T. Lapidot, Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function, J. Clin. Invest. 106 (2000) 1331–1339. R. Radinsky, I.J. Fidler, Regulation of tumor cell growth at organ-specific metastases, In Vivo 6 (1992) 325–331. S.K. Gupta, K. Pillarisetti, Cutting edge: CXCR4-Lo: molecular cloning and functional expression of a novel human CXCR4 splice variant, J. Immunol. 163 (1999) 2368– 2372.