Abstract
BackgroundHIF-1α and CXCR4/CXCL12 have crucial roles in the metastatic process of colorectal cancer. Our aim was to study the significance of targeting HIF-1α and the CXCR4/CXCL12 axis in colorectal cancer to prevent the dissemination process in vitro.MethodsWe investigated CXCR4 and CXCR7 mRNA and protein expression in human colon carcinomas and the modulation of their expression by hypoxia and HIF-1α 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.ResultsAnalysis 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-1α, 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).ConclusionWe 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-1α 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.
Highlights
Tumor progression is associated with intratumoral hypoxia, which leads to an increase in vascular density
We found that inhibition of CXCR4 with siRNA or with the CXCR4/CXCL12 neutraligand chalcone 4 significantly decreased the migration of these cells in vitro, an effect that was amplified by concomitant inhibition of HIF-1α
Concerning CXCR7, a weak expression was found in the normal mucosa and stages I to IV, which increased in the liver metastases due to increased number of cells expressing CXCR7 (Figure 2I-K)
Summary
Tumor progression is associated with intratumoral hypoxia, which leads to an increase in vascular density. The expression of chemo-attractant molecules and their receptors (such as CXCL12-SDF1/CXCR4 and VEGF/ VEGFRs) induces tumor cell dissemination from primitive tumor sites to metastatic niches. 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. The role of CXCL12/CXCR7 signaling is not yet fully described, this receptor seems to be essential for the survival and growth of tumor cells [11,12,13,14]. HIF-1α and CXCR4/CXCL12 have crucial roles in the metastatic process of colorectal cancer. Our aim was to study the significance of targeting HIF-1α and the CXCR4/CXCL12 axis in colorectal cancer to prevent the dissemination process in vitro
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