Department of Surgery, City of Hope Comprehensive Cancer Center, Duarte, CA 91010, USA.
J Transl Med. 2012 Apr 2;10:68. doi: 10.1186/1479-5876-10-68.
Previously assumed to be a select ligand for chemokine receptor CXCR4, chemokine CXCL12 is now known to activate both CXCR4 and CXCR7. However, very little is known about the co-expression of these receptors in cancer cells.
We used immunohistochemistry to determine the extent of co-expression in pancreatic cancer tissue samples and immunoblotting to verify expression in pancreatic cancer cell lines. In cell culture studies, siRNA was used to knock down expression of CXCR4, CXCR7, K-Ras and β-arrestin -2 prior to stimulating the cells with CXCL12. Activation of the mitogen-activated protein kinase pathway (MAPK) was assessed using both a Raf-pull down assay and western blotting. The involvement of the receptors in CXCL12-mediated increases in cell proliferation was examined via an ATP-based proliferation assay.
First, we discovered frequent CXCR4/CXCR7 co-expression in human pancreatic cancer tissues and cell lines. Next, we observed consistent increases in ERK1/2 phosphorylation after exposure to CXCL12 or CXCL11, a CXCR7 agonist, in pancreatic cancer cell lines co-expressing CXCR4/CXCR7. To better characterize the receptor-mediated pathway(s), we knocked down CXCR4 or CXCR7, exposed the cells to CXCL12 and examined subsequent effects on ERK1/2. We observed that CXCR7 mediates the CXCL12-driven increase in ERK1/2 phosphorylation. Knockdown of CXCR4 expression however, decreased levels of K-Ras activity. Conversely, KRAS knockdown greatly reduced CXCL12-mediated increases in ERK1/2 phosphorylation. We then evaluated the role of β-arrestin-2, a protein directly recruited by chemokine receptors. We observed that β-arrestin-2 knockdown also inhibited increases in ERK1/2 phosphorylation mediated by both CXCR4 and CXCR7. Finally, we investigated the mechanism for CXCL12-enhanced cell proliferation and found that either receptor can modulate cell proliferation.
In summary, our data demonstrate that CXCR4 and CXCR7 are frequently co-expressed in human pancreatic cancer tissues and cell lines. We show that β-arrestin-2 and K-Ras dependent pathways coordinate the transduction of CXCL12 signals. Our results suggest that the development of therapies based on inhibiting CXCL12 signaling to halt the growth of pancreatic cancer should be focused at the ligand level in order to account for the contributions of both receptors to this signaling pathway.
先前被认为是趋化因子受体 CXCR4 的选择性配体，趋化因子 CXCL12 现在已知可激活 CXCR4 和 CXCR7。然而，关于这些受体在癌细胞中的共表达知之甚少。
我们使用免疫组织化学方法来确定胰腺癌组织样本中共同表达的程度，并使用免疫印迹法来验证胰腺癌细胞系中的表达。在细胞培养研究中，使用 siRNA 敲低 CXCR4、CXCR7、K-Ras 和 β-arrestin-2 的表达，然后用 CXCL12 刺激细胞。使用 Raf 下拉测定法和 Western blot 法评估丝裂原活化蛋白激酶途径 (MAPK) 的激活。通过基于 ATP 的增殖测定法检查受体在 CXCL12 介导的细胞增殖增加中的参与。
首先，我们在人类胰腺癌组织和细胞系中发现了 CXCR4/CXCR7 的频繁共表达。接下来，我们观察到在共表达 CXCR4/CXCR7 的胰腺癌细胞系中，暴露于 CXCL12 或 CXCL11（CXCR7 激动剂）后 ERK1/2 磷酸化持续增加。为了更好地描述受体介导的途径，我们敲低了 CXCR4 或 CXCR7，用 CXCL12 处理细胞，并检查了对 ERK1/2 的后续影响。我们观察到 CXCR7 介导了 CXCL12 驱动的 ERK1/2 磷酸化增加。然而，敲低 CXCR4 表达会降低 K-Ras 活性。相反，KRAS 敲低大大减少了 CXCL12 介导的 ERK1/2 磷酸化增加。然后，我们评估了 β-arrestin-2 的作用，β-arrestin-2 是一种被趋化因子受体直接募集的蛋白质。我们观察到β-arrestin-2 敲低也抑制了由 CXCR4 和 CXCR7 介导的 ERK1/2 磷酸化增加。最后，我们研究了 CXCL12 增强细胞增殖的机制，发现两种受体都可以调节细胞增殖。
总之，我们的数据表明，CXCR4 和 CXCR7 在人类胰腺癌组织和细胞系中经常共表达。我们表明，β-arrestin-2 和 K-Ras 依赖性途径协调了 CXCL12 信号的转导。我们的结果表明，基于抑制 CXCL12 信号以阻止胰腺癌生长的疗法的开发应集中在配体水平上，以考虑到这两种受体对该信号通路的贡献。
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