Hypoxia promotes invasion and metastasis of breast cancer cells by increasing lysyl oxidase expression

Background

All solid tumors, including breast cancer, contain areas of low oxygen tension (hypoxia). Hypoxic cells are highly aggressive and metastatic, although the underlying processes remain unclear. We have found lysyl oxidase (LOX) expression to be increased by hypoxia in a variety of human cancer cell types. Lysyl oxidase (LOX) plays an essential role in the formation and maintenance of the extracellular matrix, and has previously been linked to increased in vitro invasion of breast cancer cells [1].

Methods

Human breast and cervical cancer cells were deprived of oxygen for 18 hours and the expression levels of LOX were examined by RT-PCR (including quantitative), northern blotting and western blotting. LOX activity was inhibited by antisense or siRNA treatment or by addition of a chemical inhibitor, and the effect on in vitro invasion was examined using Boyden chambers. The in vivo metastatic potential of these cells was also examined in air and hypoxia via tail-vein injection of mice subsequently housed for 4 weeks under 20% or 10% oxygen and assessment of lung micro metastases. Previously published micro-array datasets were examined for correlation between LOX expression and metastasis in human breast cancer patients [2, 3].

Results

Incubation of human breast and cervical cancer cells in oxygen-deprived conditions resulted in elevated levels of LOX due to a hypoxia inducible factor 1-dependent increase in mRNA levels. Oxygen-deprived cells demonstrated enhanced in vitro invasion that could be blocked by transfection with LOX antisense oligonucleotides or LOX-specific siRNA, or by treatment with an inhibitor of LOX activity. Cells stably expressing LOX siRNA grew slightly faster in air but demonstrated non-invasive and non-metastatic phenotypes in three-dimensional culture, and formed dramatically fewer lung micro metastases in vivo when injected into mouse tail veins, particularly those housed in hypoxic conditions. Analysis of expression data from breast cancer patients revealed a good correlation between LOX and lymph node status (Pearson correlation value of 0.78).

Conclusion

Our data reveal that hypoxia-induced LOX plays a key role in invasion and metastasis in human breast (and cervical) cancer, and that inhibition of LOX blocks these processes and may enhance effectiveness of therapy. These novel findings suggest that LOX may represent a novel marker of patient prognosis, particularly as an indicator of lymph node status in breast cancer.