Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse
Dontscho Kerjaschki, … , Dieter Steinhilber, Georg Krupitza
Dontscho Kerjaschki, … , Dieter Steinhilber, Georg Krupitza
Published April 11, 2011
Citation Information: J Clin Invest. 2011;121(5):2000-2012. https://doi.org/10.1172/JCI44751.
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Research Article Oncology

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

Abstract

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.

Authors

Dontscho Kerjaschki, Zsuzsanna Bago-Horvath, Margaretha Rudas, Veronika Sexl, Christine Schneckenleithner, Susanne Wolbank, Gregor Bartel, Sigurd Krieger, Romana Kalt, Brigitte Hantusch, Thomas Keller, Katalin Nagy-Bojarszky, Nicole Huttary, Ingrid Raab, Karin Lackner, Katharina Krautgasser, Helga Schachner, Klaus Kaserer, Sandra Rezar, Sybille Madlener, Caroline Vonach, Agnes Davidovits, Hitonari Nosaka, Monika Hämmerle, Katharina Viola, Helmut Dolznig, Martin Schreiber, Alexander Nader, Wolfgang Mikulits, Michael Gnant, Satoshi Hirakawa, Michael Detmar, Kari Alitalo, Sebastian Nijman, Felix Offner, Thorsten J. Maier, Dieter Steinhilber, Georg Krupitza

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Figure 5

shRNA-mediated knockdown and rescue of lipoxygenase in MCF7 cells.

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shRNA-mediated knockdown and rescue of lipoxygenase in MCF7 cells. (A) T...
(A) The expression of mRNAs of ALOX15 and ALOX12 was determined by real-time PCR in MCF7 and MDA-MB231 mammary carcinoma cells and in controls (noncancerous breast epithelial cells MCF-10A and HLFs). MCF7 cells express only ALOX15 mRNA, but not ALOX12 mRNA, whereas all other cells fail to produce any of the tested ALOXs. (B) mRNA levels of ALOX15 were determined in unmodified MCF7 cells, in control MCF7 cells transduced with scrambled shRNA, and in MCF7/ALOX15– cells. Knockdown of ALOX15 reduced the expression of ALOX15 mRNA significantly (*P = 0.0009 compared with vector control) when compared with unmodified or control transduced MCF7 cells. (C) Production of 12(S)-HETE and 15(S)-HETE, the arachidonic acid metabolites of ALOX15, is reduced by more than 90% in MCF7/ALOX15– cells when compared with control MCF7 cells that were transduced with scrambled shRNA (*P > 0.0001). (D) shRNA-mediated knockdown of ALOX15 in MCF7 cells (blue line) causes a size reduction similar to that of baicalein in CCID (compare to Figure 3D). This is further aggravated by coincubation with 20 μM of the pan-metalloprotease inhibitor GM6001 (yellow line), which had a similar effect (green line) on controls (MCF7 cells transduced with scrambled shRNA, red line). (E) Reconstitution of CCID-forming activity of MCF7/ALOX15– cells by transfection with ALOX12. MCF7 spheroid–induced CCID formation is analyzed in the presence or absence of 100 μM baicalein. There is a significant difference in CCID size between MCF7/control versus MCF7/ALOX15– spheroids (*P = 0.0017), MCF7/ALOX15– versus MCF7/ALOX15–/ALOX12+ spheroids (#P = 0.0249), and MCF7/ALOX15–/ALOX12+ spheroids ± baicalein treatment (†P = 0.0331). All data are presented as mean ± SEM.