In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.
The two subtypes of retinoid Z receptor (RZR alpha and beta) and the three splicing variants of retinoid orphan receptor (ROR alpha 1, alpha 2, and alpha 3) form a subfamily within the superfamily of nuclear hormone receptors. Very recently we found that the pineal gland hormone melatonin is a natural ligand of RZR alpha and RZR beta. Ligand-induced transcriptional control is therefore proposed to mediate physiological functions of melatonin in the brain where RZR beta is expressed, but also in peripheral tissues, where RZR alpha was found. However, no natural RZR responding genes have been identified yet. Here, we report that a response element in the promoter of 5-lipoxygenase binds specifically RZR alpha and ROR alpha 1, but not ROR alpha 2 and alpha 3. 5-Lipoxygenase is a key enzyme in the biosynthesis of leukotrienes, which are known to be allergic and inflammatory mediators. We could show that the activity of the whole 5-lipoxygenase promoter as well as of the RZR response element fused to the heterologous thymidine kinase promoter could be repressed by melatonin. The hormone down-regulated the expression of 5-lipoxygenase about 5-fold in B lymphocytes, which express RZR alpha. In contrast, 5-lipoxygenase mRNA levels were not affected in differentiated monocytic and granulocytic cell lines, which do not express RZR alpha. This indicates that 5-lipoxygenase is the first natural RZR alpha responding gene. Furthermore, our results open up a new perspective in understanding the involvement of melatonin in inflammatory and immunological reactions.
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