Article

Overexpression of Both CXC Chemokine Receptor 4 and Vascular Endothelial Growth Factor Proteins Predicts Early Distant Relapse in Stage II-III Colorectal Cancer Patients

University of Naples Federico II, Napoli, Campania, Italy
Clinical Cancer Research (Impact Factor: 8.72). 05/2006; 12(9):2795-803. DOI: 10.1158/1078-0432.CCR-05-2142
Source: PubMed

ABSTRACT

CXC chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) are implicated in the metastatic process of malignant tumors. However, no data are currently available on the biological relationship between these molecules in colorectal cancer. We studied whether CXCR4 and VEGF expression could predict relapse and evaluated in vitro the contribution of CXCR4 in promoting clonogenic growth, VEGF secretion, and intercellular adhesion molecule-1 (ICAM-1) expression of colorectal cancer cells.
CXCR4 and VEGF were studied in colorectal cancer tissues and in Lovo, HT29, and SW620 colorectal cancer cell lines by immunohistochemistry. Correlations with baseline characteristics of patients and tumors were analyzed by chi2 test. VEGF secretion induced by CXCL12 was measured by ELISA. The effect of CXCL12 on ICAM-1 expression was evaluated by flow cytometry. Clonogenic growth induced by CXCL12 was determined by clonogenic assays. Functional effects induced by CXCL12 were prevented by the administration in vitro of AMD3100, a bicyclam noncompetitive antagonist of CXCR4.
Seventy-two patients, seen between January 2003 and January 2004, were studied. CXCR4 was absent in 16 tumors (22.2%); it was expressed in < or = 50% of cells in 25 (34.7%) tumors and in >50% of cells in 31 (43.0%) tumors. VEGF was absent in 17 (23.6%) tumors; it was expressed in < or = 50% of cells in 16 (22.2%) tumors and in >50% of cells in 39 (54.2%) tumors. There was a significant association between CXCR4 expression and lymph nodal status (P = 0.0393). There were significant associations between VEGF and tumor invasion (P = 0.0386) and lymph nodal involvement (P = 0.0044). American Joint Committee on Cancer stage (P = 0.0016), VEGF expression (P = 0.0450), CXCR4 expression (P = 0.0428), and VEGF/CXCR4 expression (P = 0.0004) had a significant prognostic value for disease-free survival with univariate analysis. The predictive ability of the American Joint Committee on Cancer stage and of the concomitant and high expression of VEGF and CXCR4 was confirmed by multivariate analysis. Prognosis is particularly unfavorable for patients whose primary tumors express CXCR4 and VEGF in >50% of cells (median disease-free survival in relapsed patients, 5.8 months; hazard ratio of relapse, 8.23; 95% confidence interval, 7.24-14.29). In clonogenic assays, CXCL12 (20 ng/mL/d) significantly increased the number of clones in SW620, HT29, and Lovo cells at 7 and 14 days. Again, CXCL12 was able to stimulate VEGF secretion in SW620, HT29, and Lovo cells as well as up-regulated ICAM-1. These effects were prevented by the administration of AMD3100 (1 micromol/L).
We have shown that concomitant and high expression of CXCR4 and VEGF is a strong and independent predictor of early distant relapse in colorectal cancer. CXCR4 triggers a plethora of phenomena, including stimulation of clonogenic growth, induction of VEGF release, and ICAM-1 up-regulation. These data support the inhibition of CXCR4 to prevent the development of colorectal cancer metastasis.

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    • "In many tumor models, these molecules permit tumor cell survival in the metastatic microenvironment and the recruitment of hematopoietic and endothelial progenitors for neovascularization [5]. Interestingly, recent studies have shown that overexpressions of the chemokine receptor CXCR4 and of VEGF were predictive of early distant relapse in stages II and III colorectal cancers [6]. CXCR4 is a highly conserved G protein coupled receptor (GPCR) that binds CXCL12. "
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    ABSTRACT: HIF-1alpha and CXCR4/CXCL12 have crucial roles in the metastatic process of colorectal cancer. Our aim was to study the significance of targeting HIF-1alpha and the CXCR4/CXCL12 axis in colorectal cancer to prevent the dissemination process in vitro. We investigated CXCR4 and CXCR7 mRNA and protein expression in human colon carcinomas and the modulation of their expression by hypoxia and HIF-1alpha in colon cancer cell lines. The migration of tumor cells in a Boyden chamber was studied after CXCR4 inhibition with siRNA or the CXCR4/CXCL12 neutraligand, chalcone 4. Analysis of a cohort of colon polyps and chromosome-unstable carcinomas showed that the expression of CXCR4 and CXCR7 was similar to that of the normal mucosa in the polyps and early-stage carcinomas but significantly increased in late stage carcinomas. Our data demonstrate that hypoxia strongly induced the expression of CXCR4 transcript and protein at the cell membrane, both regulated by HIF-1alpha, whereas CXCR7 expression was independent of hypoxia. After transient hypoxia, CXCR4 levels remained stable at the cell membrane up to 48 hours. Furthermore, reducing CXCR4 expression impaired CXCL12-induced Akt phosphorylation, whereas Erk activation remained unchanged. In contrast, reducing CXCR7 expression did not affect Akt nor Erk activation. In the presence of CXCR4 or CXCR7 siRNAs, a significant reduction in cell migration occurred (37% and 17%, respectively). Although irinotecan inhibited cell migration by 20% (p <0.001), the irinotecan and chalcone 4 combination further increased inhibition to 40% (p <0.001). We demonstrated, for the first time, that hypoxia upregulated CXCR4 but not CXCR7 expression in tumor cells and that the CXCR4 receptor protein level remains high at the cell membrane when the tumor cells return to normoxia for up to 48 hours. In addition we showed the interest to inhibit the CXCR4 signaling by inhibiting both the HIF-1alpha and CXCR4/CXCL12 pathway. CXCR4 seems to be a relevant target because it is continuously expressed and functional both in normoxic and hypoxic conditions in tumor cells.
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    • "Statistical correlation was not found with Dukes' stage and lymph node involvement which is in agreement with other reports [37] [44] [46]. However most of the studies have demonstrated significant correlation between VEGF immunoreactivity and nodal metastases [21] [36] [39] [40] [42] [45] or advanced Dukes' stage [36]. The patients' characteristics (age at diagnosis, gender and ethnicity) were not significantly associated with VEGF expression and similar observation was also noted by other investigators [21] [35] [36] [38] [39] [41] [42] [45] [46]. "
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    • "Statistical correlation was not found with Dukes' stage and lymph node involvement which is in agreement with other reports [37] [44] [46]. However most of the studies have demonstrated significant correlation between VEGF immunoreactivity and nodal metastases [21] [36] [39] [40] [42] [45] or advanced Dukes' stage [36]. The patients' characteristics (age at diagnosis, gender and ethnicity) were not significantly associated with VEGF expression and similar observation was also noted by other investigators [21] [35] [36] [38] [39] [41] [42] [45] [46]. "

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