Abstract P4-08-06: Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line

Objective: To explore the role of HIF2 in growth factor receptor-driven HIF modulation and investigate the relationship between growth factor- and hypoxia-driven HIF activation. HIF-mediated transcriptional activity is known to drive genes involved in various processes which are associated with cancer pathology such as glycolysis, angiogenesis and metastasis. Therefore, understanding the implications of hypoxia-independent HIF regulation for both HIF1 and HIF2, may give new insight into the mechanisms by which HIF drives cancer pathology in vivo and a greater understanding of when HIF inhibitory agents may be effective therapies. Methods: We used an ERBB2 overexpressing MCF7 cell line (MCF7-HER2) to investigate the effect of ERBB2 on the HIF-axis. Western blotting was used to assess protein level in these cell lines. HIF protein expression was compared with and without ERBB stimulation by ERBB3 ligand neuregulin 1beta. Illumina BeadChip analysis was used to compare mRNA levels between these cell lines in normoxia (20% oxygen), acute hypoxia (0.5% oxygen for 24 hours) and chronic hypoxia (0.5% oxygen for 10 weeks). Differentially expressed genes were identified using rank products analysis with a cut-off P-value of 0.01. This allowed an in-depth comparison of hypoxia responses at the level of transcription between the cell lines to ascertain the effect of ERBB2 overexpression on hypoxia driven transcriptional changes. Results: Immunoblotting shows that HIF1 protein level is comparable between MCF7 and MCF7-HER2 cell lines, and is inducible in normoxia by stimulation with neuregulin 1beta. Conversely, HIF2 protein is unaffected, but is constitutively expressed in MCF7-HER2 only. This suggests that both HIF isoforms can be up-regulated in normoxia but by different mechanisms. Microarray data suggests that the constitutively higher HIF2 levels in the MCF7-HER2 cell line may be due, at least in part, to the increased transcription of the HIF2A gene which is higher in normoxia and in response to hypoxia when compared to wild-type MCF7. Overexpression of ERBB2 in MCF7-HER2 cells appears to prime cells for their response to hypoxia, as 14% (N= 591) of the genes which are induced in acute hypoxia are also expressed at significantly higher levels in normoxic MCF7-HER2 cells. However, only 1% are more highly expressed in wild-type MCF7 cells. For chronic hypoxic genes, 18% (N= 514) were more highly expressed in normoxic MCF7-HER2 cells and just 8% in wild-type MCF7 cells. These up-regulated genes include both HIF1 and HIF2 target genes which may have important consequences for glycolysis (ALDOC, PFKFB), tumour cell survival (E4BP4, STC2) and proliferation (FOS, KDM5B). Conclusions: We have demonstrated that both HIF1 and HIF2 can be regulated independently of hypoxia, however these appear to be controlled through distinct mechanisms. Whilst the implications of HIF1 in breast cancer pathology have been appreciated for some time, relatively little is known about the impact of HIF2. Here we show that ERBB2 overexpression can not only increase HIF2 protein levels in normoxia, but may also prime cells for hypoxia by allowing the constitutively higher expression of HIF1 and HIF2 target genes. Citation Format: Jarman EJ, Turnbull AK, Martinez-Perez C, Meehan J, Xintralopoulou C, Ward C, Langdon SP. Modulation of hypoxia-inducible factors and the HIF transcriptional response to hypoxia by ERBB2 overexpression in the MCF7 breast cancer cell line. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-08-06.