Chemokine Receptor CXCR4 Expression in Colorectal Cancer Patients Increases the Risk for Recurrence and for Poor Survival
http://www.100md.com
《临床肿瘤学》
the Departments of Molecular Oncology, Gastrointestinal Cancer Section, Surgical Oncology, and Surgical Pathology, John Wayne Cancer Institute, Saint John's Health Center, Santa Monica
Department of Biomathematics, University of California Los Angeles School of Medicine, Los Angeles, CA
ABSTRACT
PURPOSE: Liver metastasis is the predominant cause of colorectal cancer (CRC) related mortality. Chemokines, soluble factors that orchestrate hematopoetic cell movement, have been implicated in directing cancer metastasis, although their clinical relevance in CRC has not been defined. Our hypothesis was that the chemokine receptor CXCR4 expressed by CRC is a prognostic factor for poor disease outcome.
METHODS: CRC cell lines (n = 6) and tumor specimens (n = 139) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC were assessed. Microarray screening of select specimens and cell lines identified CXCR4 as a prominent chemokine receptor. CXCR4 expression in tumor and benign specimens was assessed by quantitative real-time reverse transcription polymerase chain reaction and correlated with disease recurrence and overall survival.
RESULTS: High CXCR4 expression in tumor specimens (n = 57) from AJCC stage I/II patients was associated with increased risk for local recurrence and/or distant metastasis (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065). High CXCR4 expression in primary tumor specimens (n = 35) from AJCC stage IV patients correlated with worse overall median survival (9 months v 23 months; RR, 2.53; 95% CI, 1.19 to 5.40; P = .016). CXCR4 expression was significantly higher in liver metastases (n = 39) compared with primary CRC tumors (n = 100; P < .0001).
CONCLUSION: CXCR4, a well-characterized chemokine receptor for T-cells, is differentially expressed in CRC. CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence, survival, and liver metastasis. The CXCR4-CXCL12 signaling mechanism may be clinically relevant for patients with CRC and represents a potential novel target for disease-directed therapy.
INTRODUCTION
The observation of an orderly, systematic targeting of organs by metastatic breast cancer led Paget to hypothesize the "seed and soil" theory of cancer metastasis.1 In this model, organs that provide suitable environmental conditions for cancer growth are the preferential sites of cancer metastasis. Since Paget's original report more than one century ago, others have attempted to test, challenge, or supplement this theory. Recently, a novel mechanism for cancer metastasis has emerged that highlights the role of chemokines. In this signaling/"homing" mechanism, target organs produce and release specific chemokines that attract nearby or distant cancer cells bearing specific corresponding receptors.2 This mechanism, which was originally characterized for organogenesis, hematopoiesis, and inflammation, draws on the principles of the "seed and soil" hypothesis. Signaling results in directional, site-specific cancer cell migration leading to implantation in favorable "soil." Recent studies support this signaling mechanism by demonstrating in numerous cancer models that malignant cells can target specific organs or tissues by select chemokine receptors.3-11
Chemokines are signaling molecules that function in myriad cell trafficking events. They have been classified into four subgroups (C, CC, CXC, and CX3C) based on the positioning of their cysteine residues.12 The binding of chemokines to their G protein–linked receptors on target cells leads to signal transduction events involving the generation of inositol triphosphate, release of intracellular calcium, and activation of protein kinase C and proteins of the Ras and Rho families.13 Chemokine receptor activation can lead to growth, adhesion, and most importantly directional migration.14 This chemokine migratory activation has been well characterized for orchestrating T-cell migration during immune and inflammatory responses.15 For example, when infection or tissue injury occur, immune cells bearing chemokine receptors are drawn from far distances to those sites of insult, where chemokines are released. In hematopoeisis or development, stem cells and progenitor immune cells migrate to and from various organs and tissues under the directional guidance of chemokines.16
Colorectal cancer (CRC) is an appropriate paradigm to evaluate the clinical relevance of chemokine receptor expression and the signaling/homing hypothesis of cancer metastasis. Despite new chemotherapeutic regimens and improved surgical outcomes, CRC remains one of the three leading causes of cancer-related death among men and women worldwide. Although the 5-year survival rate for patients with local CRC approaches 90%, the spread of disease to distant sites decreases the 5-year survival rate to 19%.17 In large part, this mortality is secondary to liver metastasis. Although vascular drainage patterns may contribute to a mechanical process for distant metastasis,18 there is growing evidence for chemokine involvement in this process. Previous studies have demonstrated that cellular extracts from liver parenchyma have relatively high concentrations of CXCL12, the specific ligand to CXCR4.3 The high levels of CXCL12 in the liver could provide a specific homing target for CXCR4 receptor–bearing cells. Here, we examine tumor specimens from patients with CRC to resolve whether this signaling mechanism can be translated to the clinical arena and, thereby, be a significant prognostic factor for patients with CRC.
METHODS
Patient Selection
Patients who underwent surgery for American Joint Committee on Cancer (AJCC) stages I to IV CRC were selected consecutively from a stage-specific institutional patient and specimen gastrointestinal database. We assessed 152 specimens from 138 patients. Thirteen specimens were of poor tumor quality and, thus, excluded from further analysis. The remaining 139 specimens from 125 patients were primary CRC tumors (n = 100) or liver metastases (n = 39) (Table 1). Forty-five matched benign colon and liver specimens served as controls.
Specimens from patients with stage IV CRC were either primary tumors (n = 35) or secondary liver metastases (n = 39); the former were procured from patients who had synchronous colon and metastatic disease. Of the 74 stage IV specimens, a subset of synchronous lesions (primary colon, n = 14; metastatic liver, n = 14) from 14 patients was available for analysis. All eligible patients with stages III and IV CRC received fluorouracil- and leucovorin-based chemotherapy. No patient in this cohort received chemotherapy by hepatic arterial infusion pump. All patients were treated at John Wayne Cancer Institute, Saint John's Health Center (JWCI/SJHC, Santa Monica, CA) between 1996 and 2002, and were initially followed up at JWCI. Patients provided informed consent for tissue procurement, and institutional review board approval was obtained before study initiation.
CRC Resources
Fresh tissues were collected immediately after resection, processed under nucleic acid sterile conditions, and stored at –80°C for future use. Paraffin-embedded archival tissue (PEAT) blocks were retrieved from the Department of Surgical Pathology of SJHC for molecular analysis. The established CRC cell lines DLD1, LoVo, HT-29, SW480, and WiDr were obtained from the American Type Culture Collection (Manassas, VA). The CRC cell line CX-1 was provided by Dr. Peter Thomas (Boston University School of Medicine, Boston, MA). All cell lines were maintained in RPMI-1640 1640 medium (Gibco, Carlsbad, CA) with addition of 10% fetal calf serum and 1% penicillin and streptomycin and incubated at 37°C with 5% CO2.
RNA Isolation and Microarray Analysis
Total RNA from frozen tissues and cell lines was extracted, isolated, and purified using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as previously described.19,20 PEAT total RNA was extracted with the Optimum FFPE RNA Isolation Kit (Ambion, Austin, TX) with modifications.21 RNA was quantified and assessed for purity by ultraviolet spectrophotometry and RIBOGreen detection assay (Molecular Probes, Eugene, OR) as described.22
Total RNA (5 μg) from four CRC specimens (primary, n = 2; liver metastasis, n = 2) and two CRC cell lines (HT-29 and SW480) was screened for chemokine receptors by microarray analysis (Human Chemokine & Receptor Q Series GEArray kit, SuperArray Bioscience Corp, Bethesda, MD). cDNA probes were constructed according to the manufacturer's instructions and hybridized onto GEArray nylon membranes. After incubation with a chemifluorescent substrate (ECF, Amersham Biosciences, San Francisco, CA), signal intensities were measured on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and image analysis was performed using ScanAlyze (http://rana.lbl.gov/EisenSoftware.htm). Of 16 chemokine receptors on the microarray membrane, six were expressed by CRC tissues and cell lines. Only CXCR4 was detected on more than half of the microarray membranes tested (n = 4 of 6), and was selected for further analysis.
Synthesis of Primers and Probes
Primer and probe sequences for quantitative real-time reverse transcription polymerase chain reaction (qRT) were designed and verified by using Human BLAT Search (http://genome.ucsc.edu/cgi-bin/hgBlatcommand=start), Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and NCBI BLAST (www.ncbi.nlm.nih.gov/), all of which are available on the World Wide Web. Specific primers were designed to sequence at least one exon-exon region and to optimally amplify cDNA with amplicons less than 150 bp to account for fragmented RNA in PEAT samples. The CXCR4 (145 bp) primer sequence was: 5'-GGAGGGGATCAGTATATACA- 3' (forward); 5'–GAAGATGATGGAGTAGATGG-3' (reverse). The CXCR4 flourescence resonance energy transfer (FRET) probe sequence was: 5'-FAM-CGAGGAAATGGGCTCAGGGG-BHQ–1-3'. The primer sequence for glyceraldehyde-3-phoshate dehydrogenase (GAPDH; 136 bp) was: 5'-GGGTGTGAACCATGAGAAGT-3' (forward); 5'-GACTGTGGTCATGAGTCCT–3' (reverse). The GAPDH FRET probe sequence was: 5'-FAM-CAGCAATGCCTCCTGCACCACCAA-BHQ-1-3'. PCR products were assessed by gel electrophoresis to confirm amplicon sizes.
qRT
Reverse transcription of total RNA was performed using Moloney murine leukemia virus RT (Promega, Madison, WI) as previously described.20 The qRT assay was performed with the iCycler iQ RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using 250 ng of total RNA for each reaction. Each PCR reaction was subjected to 40 cycles at 95°C for 60 seconds, 60°C for 60 seconds, and 72°C for 60 seconds for CXCR4; and 45 cycles at 95°C for 60 seconds, 55°C for 60 seconds, and 72°C for 60 seconds for GAPDH. Each sample was assayed in triplicate with appropriate positive and negative tissue controls and reagent controls. The expression of the housekeeping gene GAPDH was assessed in each sample to verify mRNA integrity. Only specimens with adequate RNA (positive CXCR4 expression or adequate GAPDH gene expression, ie, copy numbers 1,000) were included in the study.
The qRT RT-PCR assay was initially applied to and optimized from frozen tissues. CXCR4 expression was designated as a ratio of CXCR4/GAPDH mRNA units. To account for low background expression of CXCR4 by normal colon and liver tissues, the CXCR4 ratios for primary and metastatic tumor specimens were normalized with respect to the mean CXCR4/GAPDH expression ratios for normal colon and liver tissues, respectively. The CXCR4 qRT assay was subsequently optimized for PEAT specimens. Values of CXCR4 expression for PEAT specimens were acquired and normalized in the same fashion. Accuracy and reproducibility of the qRT assay and heterogeneity of PEAT tissues were assessed by comparing qRT results from different sections of the same tumor, and from frozen and PEAT sections of the same tumor, respectively.
Normalized CXCR4 expression values were utilized to determine differences in CXCR4 expression between primary CRC tumors and liver metastases. Fourteen pairs (n = 28) of synchronous primary and metastatic lesions were included in this analysis. Finally, CXCR4 gene expression from primary tumors was analyzed to identify correlations with recurrence and/or metastasis and survival.
Immunohistochemistry
Immunohistochemistry (IHC) was performed to confirm the translation of CXCR4 mRNA to protein in PEAT primary and metastatic CRC tissues. Specimens were routinely fixed in 10% formalin in the immediate postoperative period and paraffin-embedded within the first six hours after procurement. After tumor sections (5 μm) were dried overnight at 37°C, they were deparaffinized with xylene. The sections were treated with an antigen retrieval solution (Target Retrieval, Dakocytomation, Carpinteria, CA) at 95°C for 15 minutes, and incubated overnight at 4°C with a monoclonal mouse antihuman CXCR4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200. Isotype control slides were incubated with an antihuman tyrosinase-related protein 2 (TRP-2) polyclonal antibody (Santa Cruz Biotechnology). The next day, sections were labeled with a secondary Link-Streptavidin horseradish peroxidase solution (Dako Corp., Carpinteria, CA) and developed with diaminobenzaminidine (DAB) and counterstained with hematoxylin.
Statistical Analysis
Patient characteristics and CXCR4 expression were summarized using mean, standard deviation, median, and frequency. Due to the distribution pattern of CXCR4 values, a logarithmic transformation was performed. A general linear model was used to compare the CXCR4 values with AJCC stage; pair-wise comparisons were also performed. Clinical factors of stage I and II patients with high- versus low-CXCR4–expressing tumors were compared by Fisher's exact test and t test. Disease-free survival curves for stage I/II patients with high or low CXCR4 expression levels were constructed using the Kaplan-Meier method. The log-rank test was used to compare the equality of the two curves. Univariate analysis of prognostic factors including age, sex, stage, number of lymph nodes, tumor histology, tumor grade, tumor size, obstruction, and lymphovascular invasion was assessed by the log-rank test. A multivariate analysis using the Cox proportional hazards regression model was also performed to evaluate the prognostic significance of CXCR4 expression when clinical prognostic factors were adjusted. A stepwise method was chosen for covariate selection. Because there was no recurrence in patients whose tumors expressed low levels of CXCR4, dichotomized CXCR4 values could not be included in the Cox model; rather, the logarithmically transformed CXCR4 values were assessed. The same methods were used to evaluate prognostic factors for patients with stage IV disease and to compare their overall survival with respect to CXCR4 expression. However, for multivariate analysis of prognostic factors of overall survival, dichotomized CXCR4 values were evaluated in the Cox model. All analyses were performed using SAS (SAS/STAT User's Guide, version 8; SAS Institute Inc, Cary, NC) and tests were two-sided with a significance level < .05.
RESULTS
qRT Assay
To assess potential differences in CXCR4 mRNA levels in frozen versus PEAT tissues, we compared CXCR4 expression for eight pairs of frozen and PEAT sections from the same tumors. qRT assay demonstrated that CXCR4 expression for paired frozen and PEAT tissues was similar (6.6 ± 12.1 v 6.5 ± 7.3 copies, respectively; Spearman correlation coefficient = 0.25; P = .55). To assess concordance and heterogeneity in tumor tissues, nine paired PEAT sections from different areas of the same tumors were analyzed. Significant agreement in CXCR4 expression from the paired specimens was observed ( =.605; P = .0004).
Quantification of CXCR4 mRNA
qRT results of CXCR4 expression for all specimens are presented in Figure 1. Analysis of 100 primary CRC tumors (from all stages of disease) and 39 liver metastases demonstrated significantly higher CXCR4 expression in the liver metastases (mean, 279 ± 373 ratio units v 2 ± 13 ratio units; P < .0001). In 12 (86%) of the 14 paired-tissue specimens from patients with synchronous primary and metastatic lesions, qRT analysis demonstrated that CXCR4 expression was higher in the liver metastasis than the paired primary tumor (mean, 291 ± 331 ratio units v 0.1 ± 0.1 ratio units, respectively; P = .0005).
Correlation of CXCR4 With Disease Recurrence
Patients were dichotomized as having high or low CXCR4 expression based on the median of all normalized stage I/II CXCR4 expression values. In stages I and II, six of 30 patients with tissues expressing high CXCR4 had local (n = 2) or distant (n = 4) recurrence, whereas none of 27 with low-CXCR4–expressing tissues had recurrence during a median follow-up time of 28 months (range, 4 months to 94 months). Comparison by the Kaplan-Meier method revealed a significant decrease in disease-free survival of patients with high CXCR4 expression (log-rank P = .019; Fig 2). When the clinical characteristics of these patients were assessed with respect to CXCR4 expression, a significantly higher number of stage I versus stage II tumors was noted in the low- versus high-CXCR4–expressing tumor groups, respectively (Table 2). By univariate analysis, location of tumor (colon v rectum) was significantly different; by multivariate analysis, only CXCR4 expression remained a significant prognostic factor for disease-free survival in stage I/II patients (Table 3). The hazard of recurrence increased 35% for each unit (log) increase in CXCR4 expression (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065).
Correlation of CXCR4 With Survival
The median normalized CXCR4 expression ratio of the primary CRC specimens from 35 stage IV patients stratified these tumors as high- or low-CXCR4–expressing tumors. Accordingly, 18 tumors had high expression and 17 had low expression. Table 4 compares clinical characteristics with respect to CXCR4 expression. By the Kaplan-Meier method, patients with high CXCR4-expressing tissues had significantly decreased overall survival (median, 9 months v 23 months; range, 1 month to 87 months; log-rank P = .03; Fig 3). To determine the prognostic significance of CXCR4 as a predictor of overall survival in patients with stage IV CRC, univariate and multivariate analyses were conducted (Table 5). CXCR4 expression and tumor location were the only significant factors determining overall survival.
Analysis of CXCR4 Expression by IHC
IHC for CXCR4 protein expression was performed on PEAT specimens that were included in the qRT assay. Primary and metastatic CRC specimens, and normal colon and liver were evaluated. Thirty tumor specimens with varied CXCR4 mRNA expression were stained for CXCR4 protein expression. Immunostaining was observed in both tumor and benign-appearing tissue sections. Weak to equivocal patterns of staining were observed in disease-free cells from representative normal colon and liver specimens (Figs 4A and 4B) and tumor-associated stromal cells from an area adjacent to a representative liver metastasis (Fig 4C) and from a primary colon cancer specimen (Fig 4D). In contrast, malignant cells demonstrated more focal immunoreactive staining in both the cytoplasm and cell membrane (Fig 4D). This finding substantiates cancer cells as the primary source of CXCR4 gene expression.
DISCUSSION
CXCR4, originally named LESTR or fusin, gained prominence with its identification as an essential coreceptor for T-tropic HIV-1 and HIV-2 entry into CD4+ cells.23-25 Recognition of CXCL12 (previously termed stromal cell–derived factor 1 [SDF-1]) as the specific ligand to CXCR426,27 was followed by intense research to elucidate the varied chemotactic properties of this chemokine and its receptor. As a result, studies now implicate chemokine receptors in numerous cancer-specific metastases.3-12 However, most of these studies have been conducted in in vitro models or in animals. Few have reported clinical relevance or significance for patients with malignancies; none currently apply to CRC.28-30
As the initial report of clinical utility of CXCR4 gene expression, our study may serve as a basis for determining ranges of normal and abnormal CXCR4 gene expression values. Here we stratified CXCR4 expression values into high and low groups according to the stage-specific median for our study cohort. Our findings suggest that high expression of CXCR4 in patients with stage I/II CRC is an independent risk factor for developing locoregional recurrence and/or liver metastasis. Though the number of stage I versus stage II patients was different with respect to low versus high CXCR4 expression, respectively, two of six recurrence and/or metastasis events were diagnosed in patients with T1 tumors. The absolute number of recurrences was small but consistent with the rate (10%) reported after "curative" treatment in patients with early-stage CRC.17,20 Although independent prognostic factors such as perforation, CEA blood levels, or histologic type are well supported in the literature,31-37 they were either not obtained (ie, carcinoembryonic antigen levels) or were not detected (perforation and signet cell variant) in our study cohort.
Expression of CXCR4 by primary stage IV CRC tumors was inversely related to duration of survival. Of the 35 primary CRC tissues from stage IV patients, 18 expressed high levels of CXCR4. Survival was significantly worse in this group than in stage IV patients whose primary tumors expressed low levels of CXCR4. At a median follow-up of 13 months, only three patients (all with low CXCR4 expression) were alive, whereas 32 patients had died from cancer-related causes (n = 29) or cardiovascular complications (n = 3). A multivariate analysis demonstrated that CXCR4 expression and tumor location were the only significant factors for overall survival in this group of 35 stage IV patients. Large-cohort studies in a multicenter setting will be necessary to validate these findings and examine potential mechanisms for decreased survival.
CXCR4 expression was upregulated in liver metastases compared with primary CRC specimens. The specific timing of this upregulation is unclear; both colon and hepatic factors likely contribute to this process. The 14 patients with available synchronous primary and metastatic tumor specimens were perhaps the optimal subgroup to assess changes in CXCR4 expression after liver metastasis. The finding that CXCR4 expression was higher in the liver metastases than the primary tumors in 12 (86%) of 14 patients supports the theory that the liver, with a selectively high production of CXCL12, may be a nonrandom target organ for cells with high expression of CXCR4. It is plausible that CRC cells with elevated CXCR4 have a higher potential to spread to local and distant tissues that produce CXCL12 by an alteration to a migratory phenotype.38,39
Because the mechanisms of cancer metastasis defy simplification, the "seed and soil" hypothesis has been supplemented by more recent discoveries on the events of metastasis. These events have been attributed to the same complex signaling mechanism responsible for development, organogenesis, and immunity. In a manner similar to the specific targeting of sites of infection and injury by immune cells, cancer cells likely exploit these benign pathways to promote insidious distant organ metastasis. Indeed, the putative role for chemokine receptors in cancer cell metastasis is based on these benign mechanisms. We would be remiss, however, to propose that the signaling/homing theory fully accounts for the mechanisms and events that underlie cancer metastasis. It is more likely that elements from several mechanisms are involved in cancer metastasis. For CRC, the signaling mechanism along with a mechanical drainage pattern may facilitate specific metastasis to the liver. After metastatic cells have passed through vascular channels and implanted in the liver, a favorable "soil" may be responsible for further growth and proliferation. Frequently, these colon metastases to the liver occur in multiple foci. Little is understood regarding these patterns. Furthermore, the concept that cancer implantation enacts an immune response, thus accentuating chemoattraction of CXCR4-positive tumor and immune cells, cannot be discounted. All of these theories may be relevant for cancer metastasis.40,41
In summary, the clinical evidence of this study lends credence to a homing or signaling mechanism for CRC cancer metastasis. In human CRC, the functional design of the hematopoetic chemokine receptor CXCR4 may facilitate metastasis to the liver, where the ligand is abundantly produced. Additionally, we report the potential clinical value of chemokine receptor expression in patients with CRC. If validated by prospective studies, CXCR4 expression could be a potential predictive factor for recurrence or liver metastasis. This could improve the current staging of CRC by defining additional criteria for administration of systemic therapy in patients without overt signs of advanced disease. Perhaps most important is the identification of the CXCR4 receptor as a novel target for clinical therapy. Recent use of antagonist peptides to the CXCR4 receptor have shown promise for future therapeutic strategies that may allow control of tumor spread by blocking the CXCR4 receptor.42-45
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by R01-CA90848-02, NCI, NIH; Rod Fasone Memorial Cancer Fund, Indianapolis, IN; and Roy E. Coats Research Laboratories, John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA.
Presented in part at the American Society of Clinical Oncology Annual Meeting, June 5-8, 2004, New Orleans, LA.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Department of Biomathematics, University of California Los Angeles School of Medicine, Los Angeles, CA
ABSTRACT
PURPOSE: Liver metastasis is the predominant cause of colorectal cancer (CRC) related mortality. Chemokines, soluble factors that orchestrate hematopoetic cell movement, have been implicated in directing cancer metastasis, although their clinical relevance in CRC has not been defined. Our hypothesis was that the chemokine receptor CXCR4 expressed by CRC is a prognostic factor for poor disease outcome.
METHODS: CRC cell lines (n = 6) and tumor specimens (n = 139) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC were assessed. Microarray screening of select specimens and cell lines identified CXCR4 as a prominent chemokine receptor. CXCR4 expression in tumor and benign specimens was assessed by quantitative real-time reverse transcription polymerase chain reaction and correlated with disease recurrence and overall survival.
RESULTS: High CXCR4 expression in tumor specimens (n = 57) from AJCC stage I/II patients was associated with increased risk for local recurrence and/or distant metastasis (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065). High CXCR4 expression in primary tumor specimens (n = 35) from AJCC stage IV patients correlated with worse overall median survival (9 months v 23 months; RR, 2.53; 95% CI, 1.19 to 5.40; P = .016). CXCR4 expression was significantly higher in liver metastases (n = 39) compared with primary CRC tumors (n = 100; P < .0001).
CONCLUSION: CXCR4, a well-characterized chemokine receptor for T-cells, is differentially expressed in CRC. CXCR4 gene expression in primary CRC demonstrated significant associations with recurrence, survival, and liver metastasis. The CXCR4-CXCL12 signaling mechanism may be clinically relevant for patients with CRC and represents a potential novel target for disease-directed therapy.
INTRODUCTION
The observation of an orderly, systematic targeting of organs by metastatic breast cancer led Paget to hypothesize the "seed and soil" theory of cancer metastasis.1 In this model, organs that provide suitable environmental conditions for cancer growth are the preferential sites of cancer metastasis. Since Paget's original report more than one century ago, others have attempted to test, challenge, or supplement this theory. Recently, a novel mechanism for cancer metastasis has emerged that highlights the role of chemokines. In this signaling/"homing" mechanism, target organs produce and release specific chemokines that attract nearby or distant cancer cells bearing specific corresponding receptors.2 This mechanism, which was originally characterized for organogenesis, hematopoiesis, and inflammation, draws on the principles of the "seed and soil" hypothesis. Signaling results in directional, site-specific cancer cell migration leading to implantation in favorable "soil." Recent studies support this signaling mechanism by demonstrating in numerous cancer models that malignant cells can target specific organs or tissues by select chemokine receptors.3-11
Chemokines are signaling molecules that function in myriad cell trafficking events. They have been classified into four subgroups (C, CC, CXC, and CX3C) based on the positioning of their cysteine residues.12 The binding of chemokines to their G protein–linked receptors on target cells leads to signal transduction events involving the generation of inositol triphosphate, release of intracellular calcium, and activation of protein kinase C and proteins of the Ras and Rho families.13 Chemokine receptor activation can lead to growth, adhesion, and most importantly directional migration.14 This chemokine migratory activation has been well characterized for orchestrating T-cell migration during immune and inflammatory responses.15 For example, when infection or tissue injury occur, immune cells bearing chemokine receptors are drawn from far distances to those sites of insult, where chemokines are released. In hematopoeisis or development, stem cells and progenitor immune cells migrate to and from various organs and tissues under the directional guidance of chemokines.16
Colorectal cancer (CRC) is an appropriate paradigm to evaluate the clinical relevance of chemokine receptor expression and the signaling/homing hypothesis of cancer metastasis. Despite new chemotherapeutic regimens and improved surgical outcomes, CRC remains one of the three leading causes of cancer-related death among men and women worldwide. Although the 5-year survival rate for patients with local CRC approaches 90%, the spread of disease to distant sites decreases the 5-year survival rate to 19%.17 In large part, this mortality is secondary to liver metastasis. Although vascular drainage patterns may contribute to a mechanical process for distant metastasis,18 there is growing evidence for chemokine involvement in this process. Previous studies have demonstrated that cellular extracts from liver parenchyma have relatively high concentrations of CXCL12, the specific ligand to CXCR4.3 The high levels of CXCL12 in the liver could provide a specific homing target for CXCR4 receptor–bearing cells. Here, we examine tumor specimens from patients with CRC to resolve whether this signaling mechanism can be translated to the clinical arena and, thereby, be a significant prognostic factor for patients with CRC.
METHODS
Patient Selection
Patients who underwent surgery for American Joint Committee on Cancer (AJCC) stages I to IV CRC were selected consecutively from a stage-specific institutional patient and specimen gastrointestinal database. We assessed 152 specimens from 138 patients. Thirteen specimens were of poor tumor quality and, thus, excluded from further analysis. The remaining 139 specimens from 125 patients were primary CRC tumors (n = 100) or liver metastases (n = 39) (Table 1). Forty-five matched benign colon and liver specimens served as controls.
Specimens from patients with stage IV CRC were either primary tumors (n = 35) or secondary liver metastases (n = 39); the former were procured from patients who had synchronous colon and metastatic disease. Of the 74 stage IV specimens, a subset of synchronous lesions (primary colon, n = 14; metastatic liver, n = 14) from 14 patients was available for analysis. All eligible patients with stages III and IV CRC received fluorouracil- and leucovorin-based chemotherapy. No patient in this cohort received chemotherapy by hepatic arterial infusion pump. All patients were treated at John Wayne Cancer Institute, Saint John's Health Center (JWCI/SJHC, Santa Monica, CA) between 1996 and 2002, and were initially followed up at JWCI. Patients provided informed consent for tissue procurement, and institutional review board approval was obtained before study initiation.
CRC Resources
Fresh tissues were collected immediately after resection, processed under nucleic acid sterile conditions, and stored at –80°C for future use. Paraffin-embedded archival tissue (PEAT) blocks were retrieved from the Department of Surgical Pathology of SJHC for molecular analysis. The established CRC cell lines DLD1, LoVo, HT-29, SW480, and WiDr were obtained from the American Type Culture Collection (Manassas, VA). The CRC cell line CX-1 was provided by Dr. Peter Thomas (Boston University School of Medicine, Boston, MA). All cell lines were maintained in RPMI-1640 1640 medium (Gibco, Carlsbad, CA) with addition of 10% fetal calf serum and 1% penicillin and streptomycin and incubated at 37°C with 5% CO2.
RNA Isolation and Microarray Analysis
Total RNA from frozen tissues and cell lines was extracted, isolated, and purified using Tri-Reagent (Molecular Research Center, Cincinnati, OH) as previously described.19,20 PEAT total RNA was extracted with the Optimum FFPE RNA Isolation Kit (Ambion, Austin, TX) with modifications.21 RNA was quantified and assessed for purity by ultraviolet spectrophotometry and RIBOGreen detection assay (Molecular Probes, Eugene, OR) as described.22
Total RNA (5 μg) from four CRC specimens (primary, n = 2; liver metastasis, n = 2) and two CRC cell lines (HT-29 and SW480) was screened for chemokine receptors by microarray analysis (Human Chemokine & Receptor Q Series GEArray kit, SuperArray Bioscience Corp, Bethesda, MD). cDNA probes were constructed according to the manufacturer's instructions and hybridized onto GEArray nylon membranes. After incubation with a chemifluorescent substrate (ECF, Amersham Biosciences, San Francisco, CA), signal intensities were measured on a Storm 840 PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and image analysis was performed using ScanAlyze (http://rana.lbl.gov/EisenSoftware.htm). Of 16 chemokine receptors on the microarray membrane, six were expressed by CRC tissues and cell lines. Only CXCR4 was detected on more than half of the microarray membranes tested (n = 4 of 6), and was selected for further analysis.
Synthesis of Primers and Probes
Primer and probe sequences for quantitative real-time reverse transcription polymerase chain reaction (qRT) were designed and verified by using Human BLAT Search (http://genome.ucsc.edu/cgi-bin/hgBlatcommand=start), Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and NCBI BLAST (www.ncbi.nlm.nih.gov/), all of which are available on the World Wide Web. Specific primers were designed to sequence at least one exon-exon region and to optimally amplify cDNA with amplicons less than 150 bp to account for fragmented RNA in PEAT samples. The CXCR4 (145 bp) primer sequence was: 5'-GGAGGGGATCAGTATATACA- 3' (forward); 5'–GAAGATGATGGAGTAGATGG-3' (reverse). The CXCR4 flourescence resonance energy transfer (FRET) probe sequence was: 5'-FAM-CGAGGAAATGGGCTCAGGGG-BHQ–1-3'. The primer sequence for glyceraldehyde-3-phoshate dehydrogenase (GAPDH; 136 bp) was: 5'-GGGTGTGAACCATGAGAAGT-3' (forward); 5'-GACTGTGGTCATGAGTCCT–3' (reverse). The GAPDH FRET probe sequence was: 5'-FAM-CAGCAATGCCTCCTGCACCACCAA-BHQ-1-3'. PCR products were assessed by gel electrophoresis to confirm amplicon sizes.
qRT
Reverse transcription of total RNA was performed using Moloney murine leukemia virus RT (Promega, Madison, WI) as previously described.20 The qRT assay was performed with the iCycler iQ RealTime PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using 250 ng of total RNA for each reaction. Each PCR reaction was subjected to 40 cycles at 95°C for 60 seconds, 60°C for 60 seconds, and 72°C for 60 seconds for CXCR4; and 45 cycles at 95°C for 60 seconds, 55°C for 60 seconds, and 72°C for 60 seconds for GAPDH. Each sample was assayed in triplicate with appropriate positive and negative tissue controls and reagent controls. The expression of the housekeeping gene GAPDH was assessed in each sample to verify mRNA integrity. Only specimens with adequate RNA (positive CXCR4 expression or adequate GAPDH gene expression, ie, copy numbers 1,000) were included in the study.
The qRT RT-PCR assay was initially applied to and optimized from frozen tissues. CXCR4 expression was designated as a ratio of CXCR4/GAPDH mRNA units. To account for low background expression of CXCR4 by normal colon and liver tissues, the CXCR4 ratios for primary and metastatic tumor specimens were normalized with respect to the mean CXCR4/GAPDH expression ratios for normal colon and liver tissues, respectively. The CXCR4 qRT assay was subsequently optimized for PEAT specimens. Values of CXCR4 expression for PEAT specimens were acquired and normalized in the same fashion. Accuracy and reproducibility of the qRT assay and heterogeneity of PEAT tissues were assessed by comparing qRT results from different sections of the same tumor, and from frozen and PEAT sections of the same tumor, respectively.
Normalized CXCR4 expression values were utilized to determine differences in CXCR4 expression between primary CRC tumors and liver metastases. Fourteen pairs (n = 28) of synchronous primary and metastatic lesions were included in this analysis. Finally, CXCR4 gene expression from primary tumors was analyzed to identify correlations with recurrence and/or metastasis and survival.
Immunohistochemistry
Immunohistochemistry (IHC) was performed to confirm the translation of CXCR4 mRNA to protein in PEAT primary and metastatic CRC tissues. Specimens were routinely fixed in 10% formalin in the immediate postoperative period and paraffin-embedded within the first six hours after procurement. After tumor sections (5 μm) were dried overnight at 37°C, they were deparaffinized with xylene. The sections were treated with an antigen retrieval solution (Target Retrieval, Dakocytomation, Carpinteria, CA) at 95°C for 15 minutes, and incubated overnight at 4°C with a monoclonal mouse antihuman CXCR4 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200. Isotype control slides were incubated with an antihuman tyrosinase-related protein 2 (TRP-2) polyclonal antibody (Santa Cruz Biotechnology). The next day, sections were labeled with a secondary Link-Streptavidin horseradish peroxidase solution (Dako Corp., Carpinteria, CA) and developed with diaminobenzaminidine (DAB) and counterstained with hematoxylin.
Statistical Analysis
Patient characteristics and CXCR4 expression were summarized using mean, standard deviation, median, and frequency. Due to the distribution pattern of CXCR4 values, a logarithmic transformation was performed. A general linear model was used to compare the CXCR4 values with AJCC stage; pair-wise comparisons were also performed. Clinical factors of stage I and II patients with high- versus low-CXCR4–expressing tumors were compared by Fisher's exact test and t test. Disease-free survival curves for stage I/II patients with high or low CXCR4 expression levels were constructed using the Kaplan-Meier method. The log-rank test was used to compare the equality of the two curves. Univariate analysis of prognostic factors including age, sex, stage, number of lymph nodes, tumor histology, tumor grade, tumor size, obstruction, and lymphovascular invasion was assessed by the log-rank test. A multivariate analysis using the Cox proportional hazards regression model was also performed to evaluate the prognostic significance of CXCR4 expression when clinical prognostic factors were adjusted. A stepwise method was chosen for covariate selection. Because there was no recurrence in patients whose tumors expressed low levels of CXCR4, dichotomized CXCR4 values could not be included in the Cox model; rather, the logarithmically transformed CXCR4 values were assessed. The same methods were used to evaluate prognostic factors for patients with stage IV disease and to compare their overall survival with respect to CXCR4 expression. However, for multivariate analysis of prognostic factors of overall survival, dichotomized CXCR4 values were evaluated in the Cox model. All analyses were performed using SAS (SAS/STAT User's Guide, version 8; SAS Institute Inc, Cary, NC) and tests were two-sided with a significance level < .05.
RESULTS
qRT Assay
To assess potential differences in CXCR4 mRNA levels in frozen versus PEAT tissues, we compared CXCR4 expression for eight pairs of frozen and PEAT sections from the same tumors. qRT assay demonstrated that CXCR4 expression for paired frozen and PEAT tissues was similar (6.6 ± 12.1 v 6.5 ± 7.3 copies, respectively; Spearman correlation coefficient = 0.25; P = .55). To assess concordance and heterogeneity in tumor tissues, nine paired PEAT sections from different areas of the same tumors were analyzed. Significant agreement in CXCR4 expression from the paired specimens was observed ( =.605; P = .0004).
Quantification of CXCR4 mRNA
qRT results of CXCR4 expression for all specimens are presented in Figure 1. Analysis of 100 primary CRC tumors (from all stages of disease) and 39 liver metastases demonstrated significantly higher CXCR4 expression in the liver metastases (mean, 279 ± 373 ratio units v 2 ± 13 ratio units; P < .0001). In 12 (86%) of the 14 paired-tissue specimens from patients with synchronous primary and metastatic lesions, qRT analysis demonstrated that CXCR4 expression was higher in the liver metastasis than the paired primary tumor (mean, 291 ± 331 ratio units v 0.1 ± 0.1 ratio units, respectively; P = .0005).
Correlation of CXCR4 With Disease Recurrence
Patients were dichotomized as having high or low CXCR4 expression based on the median of all normalized stage I/II CXCR4 expression values. In stages I and II, six of 30 patients with tissues expressing high CXCR4 had local (n = 2) or distant (n = 4) recurrence, whereas none of 27 with low-CXCR4–expressing tissues had recurrence during a median follow-up time of 28 months (range, 4 months to 94 months). Comparison by the Kaplan-Meier method revealed a significant decrease in disease-free survival of patients with high CXCR4 expression (log-rank P = .019; Fig 2). When the clinical characteristics of these patients were assessed with respect to CXCR4 expression, a significantly higher number of stage I versus stage II tumors was noted in the low- versus high-CXCR4–expressing tumor groups, respectively (Table 2). By univariate analysis, location of tumor (colon v rectum) was significantly different; by multivariate analysis, only CXCR4 expression remained a significant prognostic factor for disease-free survival in stage I/II patients (Table 3). The hazard of recurrence increased 35% for each unit (log) increase in CXCR4 expression (risk ratio, 1.35; 95% CI, 1.09 to 1.68; P = .0065).
Correlation of CXCR4 With Survival
The median normalized CXCR4 expression ratio of the primary CRC specimens from 35 stage IV patients stratified these tumors as high- or low-CXCR4–expressing tumors. Accordingly, 18 tumors had high expression and 17 had low expression. Table 4 compares clinical characteristics with respect to CXCR4 expression. By the Kaplan-Meier method, patients with high CXCR4-expressing tissues had significantly decreased overall survival (median, 9 months v 23 months; range, 1 month to 87 months; log-rank P = .03; Fig 3). To determine the prognostic significance of CXCR4 as a predictor of overall survival in patients with stage IV CRC, univariate and multivariate analyses were conducted (Table 5). CXCR4 expression and tumor location were the only significant factors determining overall survival.
Analysis of CXCR4 Expression by IHC
IHC for CXCR4 protein expression was performed on PEAT specimens that were included in the qRT assay. Primary and metastatic CRC specimens, and normal colon and liver were evaluated. Thirty tumor specimens with varied CXCR4 mRNA expression were stained for CXCR4 protein expression. Immunostaining was observed in both tumor and benign-appearing tissue sections. Weak to equivocal patterns of staining were observed in disease-free cells from representative normal colon and liver specimens (Figs 4A and 4B) and tumor-associated stromal cells from an area adjacent to a representative liver metastasis (Fig 4C) and from a primary colon cancer specimen (Fig 4D). In contrast, malignant cells demonstrated more focal immunoreactive staining in both the cytoplasm and cell membrane (Fig 4D). This finding substantiates cancer cells as the primary source of CXCR4 gene expression.
DISCUSSION
CXCR4, originally named LESTR or fusin, gained prominence with its identification as an essential coreceptor for T-tropic HIV-1 and HIV-2 entry into CD4+ cells.23-25 Recognition of CXCL12 (previously termed stromal cell–derived factor 1 [SDF-1]) as the specific ligand to CXCR426,27 was followed by intense research to elucidate the varied chemotactic properties of this chemokine and its receptor. As a result, studies now implicate chemokine receptors in numerous cancer-specific metastases.3-12 However, most of these studies have been conducted in in vitro models or in animals. Few have reported clinical relevance or significance for patients with malignancies; none currently apply to CRC.28-30
As the initial report of clinical utility of CXCR4 gene expression, our study may serve as a basis for determining ranges of normal and abnormal CXCR4 gene expression values. Here we stratified CXCR4 expression values into high and low groups according to the stage-specific median for our study cohort. Our findings suggest that high expression of CXCR4 in patients with stage I/II CRC is an independent risk factor for developing locoregional recurrence and/or liver metastasis. Though the number of stage I versus stage II patients was different with respect to low versus high CXCR4 expression, respectively, two of six recurrence and/or metastasis events were diagnosed in patients with T1 tumors. The absolute number of recurrences was small but consistent with the rate (10%) reported after "curative" treatment in patients with early-stage CRC.17,20 Although independent prognostic factors such as perforation, CEA blood levels, or histologic type are well supported in the literature,31-37 they were either not obtained (ie, carcinoembryonic antigen levels) or were not detected (perforation and signet cell variant) in our study cohort.
Expression of CXCR4 by primary stage IV CRC tumors was inversely related to duration of survival. Of the 35 primary CRC tissues from stage IV patients, 18 expressed high levels of CXCR4. Survival was significantly worse in this group than in stage IV patients whose primary tumors expressed low levels of CXCR4. At a median follow-up of 13 months, only three patients (all with low CXCR4 expression) were alive, whereas 32 patients had died from cancer-related causes (n = 29) or cardiovascular complications (n = 3). A multivariate analysis demonstrated that CXCR4 expression and tumor location were the only significant factors for overall survival in this group of 35 stage IV patients. Large-cohort studies in a multicenter setting will be necessary to validate these findings and examine potential mechanisms for decreased survival.
CXCR4 expression was upregulated in liver metastases compared with primary CRC specimens. The specific timing of this upregulation is unclear; both colon and hepatic factors likely contribute to this process. The 14 patients with available synchronous primary and metastatic tumor specimens were perhaps the optimal subgroup to assess changes in CXCR4 expression after liver metastasis. The finding that CXCR4 expression was higher in the liver metastases than the primary tumors in 12 (86%) of 14 patients supports the theory that the liver, with a selectively high production of CXCL12, may be a nonrandom target organ for cells with high expression of CXCR4. It is plausible that CRC cells with elevated CXCR4 have a higher potential to spread to local and distant tissues that produce CXCL12 by an alteration to a migratory phenotype.38,39
Because the mechanisms of cancer metastasis defy simplification, the "seed and soil" hypothesis has been supplemented by more recent discoveries on the events of metastasis. These events have been attributed to the same complex signaling mechanism responsible for development, organogenesis, and immunity. In a manner similar to the specific targeting of sites of infection and injury by immune cells, cancer cells likely exploit these benign pathways to promote insidious distant organ metastasis. Indeed, the putative role for chemokine receptors in cancer cell metastasis is based on these benign mechanisms. We would be remiss, however, to propose that the signaling/homing theory fully accounts for the mechanisms and events that underlie cancer metastasis. It is more likely that elements from several mechanisms are involved in cancer metastasis. For CRC, the signaling mechanism along with a mechanical drainage pattern may facilitate specific metastasis to the liver. After metastatic cells have passed through vascular channels and implanted in the liver, a favorable "soil" may be responsible for further growth and proliferation. Frequently, these colon metastases to the liver occur in multiple foci. Little is understood regarding these patterns. Furthermore, the concept that cancer implantation enacts an immune response, thus accentuating chemoattraction of CXCR4-positive tumor and immune cells, cannot be discounted. All of these theories may be relevant for cancer metastasis.40,41
In summary, the clinical evidence of this study lends credence to a homing or signaling mechanism for CRC cancer metastasis. In human CRC, the functional design of the hematopoetic chemokine receptor CXCR4 may facilitate metastasis to the liver, where the ligand is abundantly produced. Additionally, we report the potential clinical value of chemokine receptor expression in patients with CRC. If validated by prospective studies, CXCR4 expression could be a potential predictive factor for recurrence or liver metastasis. This could improve the current staging of CRC by defining additional criteria for administration of systemic therapy in patients without overt signs of advanced disease. Perhaps most important is the identification of the CXCR4 receptor as a novel target for clinical therapy. Recent use of antagonist peptides to the CXCR4 receptor have shown promise for future therapeutic strategies that may allow control of tumor spread by blocking the CXCR4 receptor.42-45
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by R01-CA90848-02, NCI, NIH; Rod Fasone Memorial Cancer Fund, Indianapolis, IN; and Roy E. Coats Research Laboratories, John Wayne Cancer Institute at Saint John's Health Center, Santa Monica, CA.
Presented in part at the American Society of Clinical Oncology Annual Meeting, June 5-8, 2004, New Orleans, LA.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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