RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13

Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis.

Raf激酶抑制蛋白（Raf Kinase Inhibitory Protein，RKIP）最初通过酵母双杂交筛选（yeast 2-hybrid screen）被鉴定为Raf-1结合蛋白。RKIP通过竞争性抑制MEK与Raf-1的结合，阻断Raf-1对MEK的激活磷酸化，从而对Raf-MEK-细胞外调节蛋白激酶（Extracellular Regulated Protein Kinase，Erk）通路发挥抑制作用。RKIP已被鉴定为转移抑制基因。在癌症转移灶中RKIP的表达水平较低。尽管原发肿瘤的生长不受影响，但RKIP的重新表达可抑制癌症转移。从机制上来说，RKIP通过抑制血管生成、局部侵袭、内渗和定植来限制肿瘤转移。目前RKIP如何抑制这些独立步骤的分子机制尚未明确。在本研究中，我们通过无偏倚的基于聚合酶链式反应（Polymerase Chain Reaction，PCR）的筛选方法，并分析DNA微阵列表达数据集，发现在乳腺癌细胞系及临床样本中，包括MMP1、MMP3、MMP10和MMP13在内的多种基质金属蛋白酶（Matrix Metalloproteinase，MMPs）的表达与RKIP的表达呈负相关。由于癌细胞表达基质金属蛋白酶对于癌症转移至关重要，我们提出假说：RKIP可能通过抑制多种基质金属蛋白酶来介导乳腺癌转移的抑制。我们证实，RKIP与基质金属蛋白酶的联合表达特征，相较于单个基因，能更好地预测高转移风险。通过结合功能缺失与功能获得性实验手段，我们发现MMP13是RKIP介导的癌细胞局部侵袭抑制的关键靶点。有趣的是，仅过表达MMP13不足以逆转RKIP表达对乳腺癌细胞肺转移的抑制作用。我们发现RKIP通过Erk2信号通路负调控MMP13的表达，且RKIP对MMP13的抑制不依赖于转录因子激活蛋白1（Activator Protein 1，AP-1）。综上，我们的研究结果表明，RKIP至少部分通过抑制MMP13来抑制癌细胞侵袭。上述数据同时表明RKIP参与调控基质金属蛋白酶的转录，提示了RKIP抑制肿瘤进展与转移的潜在作用机制。