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  • It is generally accepted that angiogenesis

    2019-07-30

    It is generally accepted that angiogenesis is a fundamental step in the transition of tumors from a benign state to a malignant one [28]. Therefore, targeting the angiogenic pathway has become a choice therapeutic strategy for advanced CRC [29]. Interestingly, we observed that overexpressed CCR6 enhanced the ability of tube formation in CRC. We constructed CRC cell lines that overexpressed or knocked down CCR6. Tubular formation ability was impaired in CCR6- knockdown groups, while overexpression of CCR6 enhanced the ability of tube formation. Adding recombinant human CCL20 (rhCCL20) increased the pro-angiogenesis effect of CtrlshRNA-SW480-CM and CCR6-SW1116-CM and the effect could be blocked by adding Nifurtimox against rhCCL20 and CCR6 (Supplementary Fig. 1), suggesting that CCR6 promoted angiogenesis in a CCL20-dependent manner, which is secreted in an autocrine or paracrine manner. In addition, up-regulation of CCR6 promoted tumor angiogenesis in vivo. Neovascularization is a complicated progress consisting of the degradation of the basement membrane, activation of endothelial cells (ECs), and the proliferation and migration of ECs [30]. Further investigations were conducted and we found that high expression of CCR6 facilitated the proliferation and migration of HUVECs, which contributed to angiogenesis in vivo and in vitro in CRC. To explore the molecular mechanisms involved in CCR6-mediating tumor angiogenesis, we conducted an angiogenesis array by semi-quantitative RT-PCR, and VEGF-A was identified as a potential target due to its highest up-regulation alteration at the mRNA level. VEGF is expressed in various tumor cells and cancer cell lines [31] and has been recognized as an important regulator of tumor angiogenesis, binding to cell surface tyrosine kinase receptor-VEGFR [32] to induce and positively regulate a physiological and pathological angiogenic cascade [33], [34], [35]. Cancer cells can secrete VEGF to bind to its receptor in endothelial cells, which promotes the proliferation and migration of ECs and induces tumor angiogenesis [36], [37]. In our study, we investigated the level of VEGF-A in CRC cells and CRC cell supernatant and discovered that the expression of VEGF-A was decreased under conditions of CCR6 knockdown, while overexpression of CCR6 increased the level of VEGF-A. In vivo studies of H. Chen et al. [44], matrigel mixed with recombinant humanVEGF (rhVEGF) was implanted subcutaneously in mice. And they found that neovessel formation was notably stimulated by rhVEGF in the matrigel plugs compared with the control group (without VEGF). Therefore human VEGF could act on mouse cells, which could be further confirmed in other studies [11], [38]. Therefore, up-regulation of CCR6 promoted tumor angiogenesis in vivo in our study. CCR6 is a G protein-coupled receptor involved in a variety of pathways, such as the ERK, PI3K/AKT, and JNK pathways [19]. We revealed in our study that both the ERK and AKT pathways can be activated by CCR6. To verify which pathway is related with CCR6-induced up-regulation of VEGF-A, the tumor cells were treated with chemical inhibitors against ERK and PI3K pathways (U0126 and LY294002). We discovered that VEGF-A expression was down-regulated when treated with LY294002 but not U0126, indicating that the PI3K/AKT pathway plays a crucial role in CCR6-mediated angiogenesis. Furthermore, p-p65 declined after preincubation with LY294002 and inhibiting the PI3K and NF-κB pathway by LY294002 and TPCA-1 decreased tube formation. Therefore, we conclude that CCR6 promotes tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer. To date, drugs against angiogenesis, such as bevacizumab, have been used in clinical treatment for metastatic CRC [39]. However, many doctors are beset with tumor regrowth and disease progression after an initial response because cancer cells evade therapeutic inhibition of angiogenesis [40], [41], [42]. The deeper mechanism of tumor angiogenesis requires further study. Wen Li et al. indicated that vasculogenic mimicry formation may play a role in CRC [43]. The relationship between CCR6 and vasculogenic mimicry remains unclear and require further research.