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. 2019 Sep;8(11):5327-5340.
doi: 10.1002/cam4.2426. Epub 2019 Jul 26.

Chemokine receptor 7 targets the vascular endothelial growth factor via the AKT/ERK pathway to regulate angiogenesis in colon cancer

Xiang Li  1 , Xuemei Wang  1 , Zitao Li  2 , Zhen Zhang  1 , Yixia Zhang  1
Affiliations

Chemokine receptor 7 targets the vascular endothelial growth factor via the AKT/ERK pathway to regulate angiogenesis in colon cancer

Xiang Li et al. Cancer Med. 2019 Sep.

Abstract

Background: Studies have shown that CXCR7 is expressed in many tumors. The aim of the present study was to investigate the function of CXCR7 in colon cancer. Although evidence indicates that CXCR7 promotes angiogenesis in colon cancer, the mechanism involved in this process remains unclear.

Methods: The expression of CXCR7 in colon cancer was evaluated by quantitative reverse-transcription polymerase chain reaction and western blotting. After transfection, cell proliferation, migration, and lumen formation were measured in vitro. Immunohistochemistry and western blotting were used to identify the functional target of CXCR7 in vivo and in vitro.

Results: In this study, CXCR7 was differentially expressed in four colon cancer cell lines. The proliferation and migration experiments showed that overexpression of CXCR7 enhanced cell growth and migration. Moreover, the tube formation assays showed that co-culture of colon cancer cells overexpressing CXCR7 with human umbilical vein endothelial cells significantly promoted tube formation in the latter cells. Conversely, the stable knockdown of CXCR7 significantly reduced this malignant activity. In addition, we found that CXCR7 activates the AKT and ERK pathways in colon cancer cells. The phosphorylation of AKT and ERK, as well as the expression of the vascular endothelial growth factor, can be inhibited using the LY294002 and U0126 inhibitors. Furthermore, the angiogenic ability of CXCR7-induced colon cancer cells was eliminated.

Conclusion: Expression of CXCR7 contributes to colon cancer growth and angiogenesis, by activating the AKT and ERK pathways. CXCR7 provides a potential therapeutic target against colon cancer.

Keywords: AKT; CXCR7; ERK; angiogenesis; colon; vascular endothelial growth factor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Expression of CXCR7 in colon cancer cells Notes: A, Expression of CXCR7 in RKO, HCT116, SW480, and Caco‐2 cells. B, Detection of decreased expression of CXCR7 in SW480 and Caco‐2 cells. C, Detection of increased expression of CXCR7 in SW480 and Caco‐2 cells. Data from each group are expressed as the mean ± standard deviation of three independent experiments. *P < .05, **P < .01, and ***P < .001 vs the respective NC group. Abbreviations: NC, negative control; ns, no statistical significance; CXCR7, chemokine receptor 7; CXCR7OE, overexpression of CXCR7
Figure 2
Figure 2
Effect of CXCR7‐mediated tumor cells on proliferation of HUVECs Notes: A, EdU assay showed that SW480 and Caco‐2 overexpressing CXCR7 promoted the proliferation of HUVECs, while inhibitors LY294002 and U0126 reduced their proliferation. B, EdU assay showed that SW480 and Caco‐2 silencing CXCR7 inhibited the proliferation of HUVECs. C, CCK‐8 was used to detect the proliferation of HUVECs in culture supernatants at different concentrations (0%, 20%, 40%, 60%, 80% and 100%). * P < .05, ** P < .01 and *** P < .001 vs each NC group. #<.05, ## P < .01 and ### P < .001 vs overexpression group. Magnification 200×. Data in each group are expressed as mean ± SD from three independent experiments. Abbreviations: CCK‐8, Cell Counting Kit‐8; EdU, 5‐ethynyl‐20‐deoxyuridine; HUVECs: human umbilical vein endothelial cells; NC, negative control. ns: No statistical significance
Figure 3
Figure 3
Effect of CXCR7‐mediated tumor cells on the migration of HUVECs Notes: A, Transwell migration results showed that SW480 and Caco‐2 cells overexpressing CXCR7 promoted the migration of HUVECs. Treatment with the inhibitors LY294002 and U0126 reduced the migration of HUVECs. B, Silencing of CXCR7 in SW480 and Caco‐2 cells inhibited the migration of HUVECs. * P < .05 and ** P < .01 vs the respective NC group. # P < .05 and ## P < .01 vs the respective overexpression group. Magnification 200×. Data in each group are presented as the mean ± standard deviation of three independent experiments. Abbreviations: HUVECs, human umbilical vein endothelial cells; NC, negative control; ns, no statistical significance; CXCR7, chemokine receptor 7; CXCR7OE, overexpression of CXCR7
Figure 4
Figure 4
Effect of CXCR7‐mediated tumor cells on tube formation in HUVECs Notes: A, SW480 and Caco‐2 cells overexpressing CXCR7 promoted tube formation in HUVECs. Treatment with the inhibitors LY294002 and U0126 reduced tube formation in HUVECs. B, Silencing of CXCR7 in SW480 and Caco‐2 cells inhibited tube formation in HUVECs. * P < .05 and ** P < .01 vs the respective NC group. ### P < .001 vs the respective overexpression group. Magnification 200×. Data from each group are presented as the mean ± standard deviation of three independent experiments. Abbreviations: HUVECs, human umbilical vein endothelial cells; NC, negative control; ns, no statistical significance; CXCR7, chemokine receptor 7; CXCR7OE, overexpression of CXCR7
Figure 5
Figure 5
ELISA: Expression of CXCR7 regulates VEGF secretion Notes: A, The levels of VEGF were significantly increased in SW480 and Caco‐2 cells overexpressing CXCR7. Treatment with inhibitors LY294002 and U0126 reduced the levels of VEGF. B, The levels of VEGF in CXCR7‐silencing SW480 and Caco‐2 cells were significantly reduced. * P < .05 and ** P < .01 vs the respective NC group. # P < .05 vs the respective overexpression group. Data from each group are presented as the mean ± standard deviation of three independent experiments. Abbreviations: ELISA, enzyme‐linked immunosorbent assay; VEGF, vascular endothelial growth factor; NC, negative control; ns, no statistical significance; CXCR7, chemokine receptor 7; CXCR7OE, overexpression of CXCR7
Figure 6
Figure 6
Effect of CXCR7 on tumor growth and angiogenesis in vivo Notes: A, Picture of RKO, HCT116, SW480 and Caco‐2 tumor tissue. B, Tumor volume of RKO, HCT116, SW480 and Caco‐2 groups. C, Tumor weight of RKO, HCT116, SW480 and Caco‐2 groups after sacrifice in mice. D, Left panel: Tumor picture of SW480 cells silencing CXCR7, right panel: Tumor picture of Caco‐2 cells overexpressing CXCR7. E, Left panel: Tumor volume of SW480 cells silencing CXCR7, right panel: tumor volume of Caco‐2 cells overexpressing CXCR7. F, Left panel: Tumor weight of SW480 cells silencing CXCR7 groups after sacrifice in mice, right panel: Tumor weight of Caco‐2 cells overexpressing CXCR7 after sacrifice in mice. G, Immunohistochemical analysis of CXCR7 in RKO, HCT116, SW480 and Caco‐2 tumor tissues. H, Tumor tissue sections were harvested for immunohistochemical staining of CXCR7, Ki67, VEGF and CD34 in each group. I. The count of CD34 microvessels reflected the MVD values. * P < .05, ** P < .01 and *** P < .001 vs overexpression groups. magnification 200×. Data in each group are presented as mean ± SD from three independent experiments. Abbreviations: VEGF: vascular endothelial growth factor; MVD: microvessel density NC, negative control. ns: No statistical significance
Figure 7
Figure 7
CXCR7 activates the AKT and ERK pathways to mediate angiogenesis Notes: A, Expression of total ERK, p‐ERK, total AKT, p‐AKT, VEGF, and GAPDH in RKO, HCT116, SW480, and Caco‐2 tumor tissues. B, Expression of total ERK, p‐ERK, total AKT, p‐AKT, VEGF, and GAPDH in CXCR7‐overexpressing or ‐silencing tissues. C, The expression levels of VEGF mRNA and total ERK, p‐ERK, total AKT, p‐AKT, VEGF, and GAPDH protein expression were significantly reduced in CXCR7‐silencing SW480 and Caco‐2 cells. D, The expression levels of VEGF mRNA and total ERK, p‐ERK, total AKT, p‐AKT, VEGF, and GAPDH protein expression were significantly increased in CXCR7‐overexpressing SW480 and Caco‐2 cells. Treatment with the inhibitors LY294002 and U0126 inhibited the mRNA expression of VEGF and protein expression of total ERK, p‐ERK, total AKT, p‐AKT, VEGF, and GAPDH. * P < .05, ** P < .01, and *** P < .001 vs the respective NC group. # P < .05, ## P < .01, and ### P < .001 vs the respective overexpression group. Abbreviations: VEGF, vascular endothelial growth factor; NC, negative control; ns, no statistical significance; CXCR7, chemokine receptor 7; CXCR7OE, overexpression of CXCR7
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