Langdon, Simon

Ward, Carol

Jarman, Edward Joseph

Medical Research Council (MRC)

2018-04-23T09:59:59Z

2018-04-23T09:59:59Z

2018-06-30

Areas of hypoxia caused by poor perfusion are a common occurrence in breast cancer. Hypoxia-inducible factors-1 and 2 (HIF1/2) drive the cellular response to hypoxia in such areas, resulting in the upregulation of genes which facilitate the survival of cancer cells and promote growth, invasion, metastasis and angiogenesis, generally leading to more aggressive tumour characteristics. Previous research has demonstrated that growth factor signalling, such as the ligand-mediated activation of HER receptors, can promote the action of HIFs in normoxia, and correlation between HER2 expression and HIFα proteins has been demonstrated in clinical samples of breast cancer. Despite this, little research has been conducted on how the growth factor-driven regulation of HIFα subunits might modify the cellular response to hypoxia. In this thesis, the role of HER2 overexpression in HIFα modulation was assessed in breast cancer cell lines and publically available clinical datasets for breast cancer with the aim of further understanding the implications of hypoxia and HIFα expression in the context of HER2-positive breast cancer. The upregulation of HIF1α and HIF2α by hypoxia was observed across breast cancer cell lines, and the role of HER2 in this process was assessed using an isogenic MCF7 cell line model overexpressing HER2. This demonstrated an increased hypoxic upregulation of HIF2α but not HIF1α when HER2 was overexpressed. The increased upregulation was shown to be facilitated by an increase in normoxic HIF2α, which is driven by a higher transcriptional rate of the EPAS1 (HIF2) gene as a direct result of HER2 overexpression. HER2 overexpression also resulted in the increased hypoxic upregulation of known hypoxia response genes in 2D and 3D culture models. This demonstrates a novel mechanism for growth-factor mediated HIFα regulation in the context of HER2 overexpression, with an important role for HIF2α. Microarray analysis of MCF7 and MCF7-HER2 cells was used to compare the global transcriptional response to acute (24 hrs) and chronic (>10 weeks) hypoxia (0.5% O2) and demonstrated a broadly increased upregulation of hypoxic response genes in the HER2 overexpressing cell line when compared to wild-type MCF7. This included an increase in previously described HIF1 and HIF2 target genes. MCF7-HER2 also illustrated an increased expression of hypoxia response genes in normoxia, and an analysis of the genes involved showed the promotion of a number of pathological processes including proliferation, invasion, angiogenesis and epithelial to mesenchymal transition. Large-scale, publically available expression datasets for breast cancer cell lines and clinical patient data were used to investigate the expression of HIF2α and hypoxia response genes in relation to HER2 expression. A set of pathologically important genes which were primed for hypoxia in MCF7-HER2 were also demonstrated to correlate with HER2 across breast cancer cell lines, suggesting that HER2 may more broadly promote a readiness to respond to hypoxia in breast cancer cells. Assessment of HIF2α in clinical samples has shown its increased expression in the HER2-positive subtype, and HIF2α was shown to be associated with worse disease-specific survival in the context of HER2-positive samples only. To investigate whether HIF2α is a potential target in HER2 overexpressing breast cancer, the effect of HIF2α inhibition through siRNA or HIF2-specific chemical inhibitors was assessed in cell lines with high or low HER2 expression, and this demonstrated an increased sensitivity of HER2 overexpressing cell lines to HIF2α inhibition. This work highlighted HER2 as an important modulator of the cellular response to hypoxia in breast cancer, demonstrating a previously overlooked role for HIF2α in this process. HIF2α expression can be directly driven by HER2 and this differs mechanistically from that previously reported for HIF1α. Finally, further work into the potential for HIF2α as a target for anti-cancer therapy is suggested, as an increased sensitivity of HER2-positive cell lines to anti-HIF2α agents was shown, as well as a HER2-specific relationship between HIF2α expression and worse prognosis. More generally, this work has shown an important interplay between growth factor receptor expression and the cellular response to hypoxia, suggesting that HER2 may promote a stronger response to hypoxia in breast cancer, which may contribute to the increased aggressiveness of HER2-positive tumours.

http://hdl.handle.net/1842/29610

en

The University of Edinburgh

hypoxia-inducible factor HIF2

hypoxia-inducible factor HIF1

HER2-HIF interactions

HER2 overexpression

HIF activity

breast cancer

HIF2α inhibition

Investigation into the role of HER2 receptor signalling in Hypoxia-inducible Factor Regulation in breast cancer

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy