The role of HIF-1α in tumour growth and metastasis at sub-cutaneous and orthotopic sites

Natalie Burrows, Muhammad Babur, Brian Telfor, Kaye Williams. University of Manchester, Manchester, Lanchashire, United Kingdom.

Background: Intratumoural hypoxia is associated with aggressive and metastatic phenotypes. The transcription factor HIF-1α is a key regulator of hypoxia, inducing the expression of genes which collectively, promote tumour cell growth and metastasis. We have generated a dominant negative variant of HIF-1α (DN-HIF) that blocks HIF-1 dependent gene transcription. The DN-HIF variant, along with an empty vector control (EV), contains an EGFP tag for experimental analysis. The dominant-negative and empty vector was stably transfected into HT1080 (fibrosarcoma) and MDA-231 (breast carcinoma) cell lines.

Aims: a) To develop and compare models of tumour growth and metastasis from sub-cutaneous and orthotopic sites and b) to explore the effect of DN-HIF expression on tumour growth and metastasis.

Results: In vitro luciferase reporter assays showed HIF-1α activity was inhibited in cells expressing DN-HIF. Scratch wound migration assays revealed migration was inhibited in DN-HIF expressing cells. In vivo analysis showed that EV and DN-HIF cells did not grow uniformly at the sub-cutaneous site compared to WT cells and cells implanted at orthotopic sites. Orthotopic tumours established quicker and displayed a higher metastatic potential than sub-cutaneous tumours. Orthotopic DN-HIF tumours showed a lower level of lung metastasis compared to control tumours. Orthotopic DN-HIF tumours established slower and displayed lower levels of hypoxic markers e.g. CA-9, LOX compared to control tumours. Orthotopic DN-HIF tumours were less dense and highly necrotic compared to controls.

Conclusions: Orthotopic tumours are a more reliable and realistic model of cancer growth and metastasis compared to routinely used sub-cutaneous models. HIF-1α is a vital component of tumour growth and metastasis, shown by the ability of DN-HIF to inhibit migration, reduce metastasis and reduce expression of hypoxic markers. DN-HIF represents.