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Cancer research has transformed our view on cellular mechanisms for oxygen sensing. It has been documented that these mechanisms are important for maintaining animal tissues and life in environments where oxygen (O2) concentrations fluctuate. In adult animals, oxygen sensing is governed by the Hypoxia Inducible Factors (HIFs) that are stabilized at low oxygen concentrations (hypoxia). However, the importance of hypoxia itself during development and for the onset of HIF-driven oxygen sensing remains poorly explored. Cellular responses to hypoxia associates with cell immaturity (stemness) and proper tissue and organ development. During mammalian development, the initial uterine environment is hypoxic. The oxygenation status during avian embryogenesis is more complex since O2 continuously equilibrates across the porous eggshell. Here, we investigate HIF dynamics and use microelectrodes to determine O2 concentrations within the egg and the embryo during the first four days of development. To determine the increased O2 consumption rates, we also obtain the O2 transport coefficient (DO2) of eggshell and associated inner and outer shell membranes, both directly (using microelectrodes in ovo for the first time) and indirectly (using water evaporation at 37.5°C for the first time). Our results demonstrate a distinct hypoxic phase (<5% O2) between day 1 and 2, concurring with the onset of HIF-α expression. This phase of hypoxia is demonstrably necessary for proper vascularization and survival. Our indirectly determined DO2 values are about 30% higher than those determined directly. A comparison with previously reported values indicates that this discrepancy may be real, reflecting that water vapor and O2 may be transported through the eggshell at different rates. Based on our obtained DO2 values, we demonstrate that increased O2 consumption of the growing embryo appears to generate the phase of hypoxia, which is also facilitated by the initially small gas cell and low membrane permeability. We infer that the phase of in ovo hypoxia facilitates correct avian development. These results support the view that hypoxic conditions, in which the animal clade evolved, remain functionally important during animal development. The study highlights that insights from the cancer field pertaining to the cellular capacities by which both somatic and cancer cells register and respond to fluctuations in O2 concentrations can broadly inform our exploration of animal development and success.

Background: The aim of this project was to investigate the role of Activator Protein 1 (AP1) in molecular adaptation to hypoxia in colorectal cancer. Colorectal cancer is the 3rd most common cancer worldwide. Low oxygen (hypoxia) found in 30-50% of colorectal tumors is associated with resistance to chemotherapy, radiotherapy and poor patient prognosis. Hypoxia stabilizes the transcription factors HIF1α and HIF2α which enable molecular adaptation to the hypoxic insult at a transcriptional level, in cooperation with additional transcription factors. To identify genes that regulate hypoxic survival in colorectal cancer, we carried out a lentiviral shRNA unbiased screen targeting 6142 genes, in HCT116 cells. We identified AP1 transcription factor subunits as key mediators of hypoxic cell viability that were required in hypoxia and in 3D spheroid culture in colorectal cancer Methods: 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 cJUN, JUNB, JUND, FOSL1 and FOSL2 are upregulated by hypoxia in colorectal cancer in a HIF independent manner. We observed that individual AP1 subunit knockdown in particular FOSL2 and JUND knockdown significantly decreased cancer cell survival in hypoxia. FOSL2 and JUND decreased the cell survival respectively by 40% and 60% in HCT116 and by 45% and 60% in LS174T (n=3, p<0.001). This effect was not observed when the cells were treated with T-5224 (10 µM), an AP-1 inhibitor suggesting a complex modulation of the transcriptional response by AP1 in hypoxia. In hypoxic conditions, by Co-IP studies, we established in HCT116 and LS174T that a selected pattern of AP1 heterodimers in particular FOSL2/JUNB and FOSL2/cJUN 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) and ANGPTL4 (n=3, p<0.05), important modulators of the hypoxic response. Conclusions: 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. Citation Format: Eric Vancauwenberghe, Hannah Bolland, Christopher Carroll, Leonardo Da Motta, Anna Grabowska, Francesca Buffa, Adrian Harris, Alan McIntyre. Defining the role of AP1 in molecular adaptation to hypoxia in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2652.