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Abstract

Human papillomaviruses (HPVs) cause cervical cancer and are also closely linked to other malignancies in the anogenital and oropharyngeal regions. It is widely assumed thatHPV-positive cancer cells are ‘oncogene addicted’ in that they depend on the continuous expression of the two viral oncogenes, E6 and E7, to maintain their malignant phenotype and to avoid tumor-suppressive senescence induction. However, recently it was shown that hypoxic HPV-positive cancer cells efficiently shut down E6/E7 expression and enter a growth arrest without inducing senescence. This process is reversible upon reoxygenation and can be counteracted by high glucose concentrations. These surprising findings shed new light on the crosstalk between the viral oncogenes and the host cell, particularly under hypoxia, a condition frequently found in cervical cancer that is associated with poor patient prognosis. The present work aimed to delineate the regulatory mechanisms underlying hypoxic E6/E7 repression. It was revealed that E6/E7 mRNA and protein expression is impaired in hypoxic HPV-positive cancer cells by a combination of transcriptional repression and reduction of mRNA and protein half-lives. Moreover, hypoxic repression of E6/E7 was shown to be not only glucose-sensitive, but also dependent on intracellular calcium. There was no indication for a role of HIF transcription factors, considered master orchestrators of the cellular response to hypoxia, or for involvement of transcription factors from the glucose-responsive Mlx network. However, the PI3K/AKT signaling pathway was identified as being crucial for the hypoxic repression of E6/E7. AKT is activated by hypoxia which can be counteracted by high glucose concentrations. Blocking of hypoxia-induced AKT activation by chemical inhibitors, including clinically applied drugs that target AKT or its upstream activators PI3K and mTORC2, counteracts repression of E6/E7 under hypoxia. Knockdown and overexpression experiments showed that the AKT1 and AKT2 isoforms act redundantly in hypoxic repression of E6/E7. Reactivation of E6/E7 expression under hypoxia upon inhibition of the PI3K/AKT pathway does not induce proliferation and can sensitize HPV-positive cells to therapeutic effects of chemotherapy in a cell line-dependent manner. Finally, comparison of the proteome composition of HPV-positive cancer cells under normoxia and hypoxia as well as proteome analyses of hypoxic cells treated with an AKT inhibitor or high glucose concentrations were performed. They identified several novel candidate proteins possibly linked to hypoxic repression of E6/E7 providing an experimental basis for future investigations. Collectively, these findings provide new insights into the regulation of the viral oncogenes in hypoxic HPV-positive tumor cells and have implications for the development of novel treatment strategies.