Defining the role of AP-1 in molecular adaptation to hypoxia in colorectal cancer


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Eric VANCAUWENBERGHE1,Hannah Bolland2,Christopher Carroll2,Leonardo Da Motta2,Anna Grabowska3,Francesca Buffa4,Adrian Harris5,Alan McIntyre2
1Hypoxia and Tumour Microenvironment Group, Cancer Biology, Division of Cancer and Stem Cells, University of Nottingham,2Hypoxia and Tumour Microenvironment Group, Cancer Biology, Division of Cancer and Stem Cells, University of Nottingham,3Cancer Biology, Division of Cancer and Stem Cells, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham, UK,4Computational Biology and Integrative Genomics, Department of Oncology, University of Oxford, Oxford, UK,5Molecular Oncology Laboratories, Department of Oncology, The Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

Abstract

Background

Colorectal cancer is the 3rd most common cancer worldwide. Low oxygen (hypoxia) found in roughly one third of colorectal cancer patients is associated with therapy resistance and poor patient outcome. Hypoxia stabilizes the transcription factors HIF1a and HIF2a which enable molecular adaptation at a transcriptional level to the hypoxic insult, in cooperation with additional transcription factors. We identified FOSL2 (FRA2), a subunit of the Activator protein 1 (AP-1) transcription factor, as a key mediator of hypoxic cell viability in an induced essentiality lentiviral shRNA screen in colorectal cancer. The aim of this project was to investigate the role of FOSL2 in molecular adaptation to hypoxia in colorectal cancer.

 

Method

Our experiments were conducted in a panel of cancer and normal colorectal cell lines (HCT116, LS174T, SW620, HT29 and CCD 841 CoN) in normoxia and hypoxia (1%O2) and in 3D spheroid cultures. The association of the different AP1 subunits and the impact of AP1 subunits on hypoxia-regulated expression and cell phenotypes was investigated using a variety of cell and molecular biology approaches.

Results

Our results identify that AP1 subunits are upregulated by hypoxia in colorectal cancer in a HIF independent manner. We observed that individual AP1 subunit knockdown  significantly decreased cancer cell survival in hypoxia. In hypoxic conditions, by Co-IP studies, we established in HCT116 and LS174T that a selected pattern of AP1 heterodimers were induced to mediate the transcriptional response to hypoxia. siRNA knockdown demonstrated that AP1 subunits modulate the expression of a specific set of hypoxia-regulated genes such as CA9 (Carbonic Anhydrase 9), important modulator of the hypoxic response.

Conclusion

These data identify AP1 as a key mediator of the molecular adaptation to the hypoxic insult in the colorectal tumour microenvironment. Targeting AP1 subunits is a likely therapeutic approach for the treatment of the therapy resistant hypoxic regions of colorectal cancers.