Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell–cell or cell–matrix interactions) and the extended tumour microenvironment (for example vascularization)1. Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear2. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells3. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status4,5,6,7,8,9. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.
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We thank P. Chu (immunohistochemistry), D. Menke (tail-vein injections), R. Nacamuli (quantitative polymerase chain reaction), R. R. Balise (statistical programming) and C. Davis (board-certified veterinary pathologist); N. Quach (FAK, phospho-FAK and F-actin antibodies), A. Cress (α6 antibody), P. Marinkovich (β1 integrin antibody) and D. Mosher (FN-null cells) for supplying reagents; and P. Friedl, Z. Werb and P. Steeg for discussions. All animal work was performed in accordance with the Stanford University Administrative Panel for Laboratory Animal Care. This research was supported by funds from the NIH (J.T.E. and A.J.G.), the Canadian Institutes of Health Research (K.L.B.), the Else Kröner-Fresenius-Foundation (N.D.) and the California Breast Cancer Research Program (S.S.J.). Author Contributions J.T.E., A.J.G. and S.S.J. conceived and designed the experiments. J.T.E. performed the experiments with the assistance of K.L.B. and N.D. for in vivo work. J.T.E., A.J.G., M.N., J.T.C. and S.S.J. analyzed the data. J.T.E. wrote the paper with the assistance of K.L.B., S.S.J. and A.J.G. The head and neck cancer study was performed by Q.T.L. and C.K.
Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.
This table shows associations between LOX and clinical criteria in breast cancer patients10, determined by Chi Square. (PDF 51 kb)
This figure demonstrates hypoxic induction of LOX mRNA expression through increased levels and stability. This is shown by semi- and fully-quantitative RT–PCR in human cervical, breast and renal cancer cell lines, and is shown to be dependent on HIF-1 through an identified and tested functional HRE in the LOX promoter. (PDF 283 kb)
This figure displays a heatmap of analysed micro-array data showing the genes most strongly correlated to LOX expression in human breast cancer patients from the whole NKI dataset10. Genes of particular interest are highlighted. (PDF 537 kb)
This file shows the full list of genes from Supplementary Figure 2. (XLS 84 kb)
As Supplementary Figure 2 except dataset is limited to ER-negative breast cancer patients only. (PDF 776 kb)
This file shows the full list of genes from Supplementary Figure 3. (XLS 84 kb)
This figure shows the generation of human cervical and breast cancer cells that stable express LOX shRNA. Assays to show reduced mRNA and protein levels are displayed, as well as in vitro and in vivo growth curves showing no significant differences with control cells. (PDF 122 kb)
This figure demonstrates a role for LOX in the increased in vitro invasion of anoxic cervical and breast cancer cells lines. Reduced invasion through LOX inhibition is additionally demonstrated in human head and neck, melanoma, colon, lung, pancreatic and renal cells. Inhibition of in vitro invasion is shown using LOX antibody. (PDF 78 kb)
This figure shows a schematic representation of invasive migration, and demonstrates LOX’s independence from FN expression to phosphorylate FAK and increase invasion of hypoxic cells. Data is additionally shown to demonstrate impaired adhesion of SiHa cells to matrix with LOX shRNA expression, and restoration with forced LOX expression. (PDF 692 kb)
This figure demonstrates impaired metastatic growth of MDA231 cells expressing LOX shRNA. This is assessed by tail vein injection, growth in soft agar, and growth in Matrigel. Additionally, photographs of the metastatic lesions formed in the lungs from orthotopic studies show reduced size/elimination with genetic/chemical inhibition of LOX. (PDF 455 kb)
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Erler, J., Bennewith, K., Nicolau, M. et al. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 440, 1222–1226 (2006). https://doi.org/10.1038/nature04695
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