Background: The metastasis-promoting G-protein-coupled receptor CXC Receptor 4 (CXCR4) is activated by the chemokine CXCL12, also known as stromal cell-derived factor 1 (SDF-1). The CXCL12/CXCR4 pathway in cancer promotes metastasis but the molecular details of how this pathway cross-talks with oncogenes are understudied. An oncogene pathway known to promote breast cancer metastasis in MDA-MB-231 xenografts is that of Mouse Double Minute 2 and 4 (MDM2 and MDM4, also known as MDMX). MDM2 and MDMX promote circulating tumor cell (CTC) formation and metastasis, and positively correlate with a high expression of CXCR4. Interestingly, this MDMX-associated upregulation of CXCR4 is only observed in cells grown in the tumor microenvironment (TME), but not in MDA-MB-231 cells grown in a tissue culture dish. This suggested a cross-talk signaling factor from the TME which was predicted to be CXCL12 and, as such, we asked if the exogenous addition of the cell non-autonomous CXCL12 ligand would recapitulate the MDMX-dependent upregulation of CXCR4. Methods: We used MDA-MB-231 cells and isolated CTCs, with and without MDMX knockdown, plus the exogenous addition of CXCL12 to determine if MDMX-dependent upregulation of CXCR4 could be recapitulated outside of the TME context. We added exogenous CXCL12 to the culture medium used for growth of MDA-MB-231 cells and isogenic cell lines engineered for MDM2 or MDMX depletion. We carried out immunoblotting, and quantitative RT-PCR to compare the expression of CXCR4, MDM2, MDMX, and AKT activation. We carried out Boyden chamber and wound healing assays to assess the influence of MDMX and CXCL12 on the cells’ migration capacity. Results: The addition of the CXCL12 chemokine to the medium increased the CXCR4 cellular protein level and activated the PI3K/AKT signaling pathway. Surprisingly, we observed that the addition of CXCL12 mediated the upregulation of MDM2 and MDMX at the protein, but not at the mRNA, level. A reduction in MDMX, but not MDM2, diminished both the CXCL12-mediated CXCR4 and MDM2 upregulation. Moreover, a reduction in both MDM2 and MDMX hindered the ability of the added CXCL12 to promote Boyden chamber-assessed cell migration. The upregulation of MDMX by CXCL12 was mediated, at least in part, by a step upstream of the proteasome pathway because CXCL12 did not increase protein stability after cycloheximide treatment, or when the proteasome pathway was blocked. Conclusions: These data demonstrate a positive feed-forward activation loop between the CXCL12/CXCR4 pathway and the MDM2/MDMX pathway. As such, MDMX expression in tumor cells may be upregulated in the primary tumor microenvironment by CXCL12 expression. Furthermore, CXCL12/CXCR4 metastatic signaling may be upregulated by the MDM2/MDMX axis. Our findings highlight a novel positive regulatory loop between CXCL12/CXCR4 signaling and MDMX to promote metastasis.
背景：促进转移的G蛋白偶联受体CXC趋化因子受体4（CXCR4）由趋化因子CXCL12（亦称基质细胞衍生因子1）激活。CXCL12/CXCR4通路在癌症中促进转移，但该通路与癌基因相互作用的分子机制尚未被充分研究。已知在小鼠MDA-MB-231异种移植模型中，小鼠双微体2和4（MDM2与MDMX）构成的癌基因通路可促进乳腺癌转移。MDM2与MDMX能促进循环肿瘤细胞形成及转移，并与CXCR4高表达呈正相关。值得注意的是，这种MDMX相关的CXCR4上调仅出现在肿瘤微环境培养的细胞中，而在组织培养皿中生长的MDA-MB-231细胞中未被观察到。这提示肿瘤微环境中存在与MDMX交互作用的信号因子，我们推测该因子为CXCL12。因此，我们探究外源性添加非细胞自主性CXCL12配体能否重现MDMX依赖的CXCR4上调现象。 方法：本研究采用MDA-MB-231细胞及其分离的循环肿瘤细胞，通过设置MDMX敲低组与对照组，并结合外源性CXCL12添加，验证MDMX依赖的CXCR4上调是否能在肿瘤微环境之外重现。我们在培养MDA-MB-231细胞及经基因编辑敲除MDM2或MDMX的同源细胞系时添加外源性CXCL12，通过免疫印迹和定量逆转录聚合酶链反应检测CXCR4、MDM2、MDMX表达水平及AKT活化状态，并采用Boyden小室迁移实验和划痕愈合实验评估MDMX与CXCL12对细胞迁移能力的影响。 结果：培养基中添加CXCL12趋化因子可提升CXCR4细胞蛋白水平并激活PI3K/AKT信号通路。意外发现，CXCL12的添加介导了MDM2和MDMX在蛋白水平（而非mRNA水平）的上调。降低MDMX（而非MDM2）表达会削弱CXCL12介导的CXCR4与MDM2上调效应。此外，同时降低MDM2与MDMX表达会抑制外源性CXCL12促进Boyden小室细胞迁移的能力。CXCL12对MDMX的上调至少部分通过蛋白酶体通路上游环节实现，因为在放线菌酮处理或蛋白酶体通路被阻断后，CXCL12并未增加蛋白稳定性。 结论：本研究证实CXCL12/CXCR4通路与MDM2/MDMX通路之间存在正向前馈激活环路。在原发性肿瘤微环境中，CXCL12表达可能上调肿瘤细胞的MDMX表达；反之，MDM2/MDMX轴也可能上调CXCL12/CXCR4转移信号。我们的发现揭示了CXCL12/CXCR4信号与MDMX之间促进转移的新型正向调控环路。
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