Transcriptional and post-transcriptional positive regulatory loops between HIF-1α and the RNA-Binding protein hnRNPA18.

Abstract

Hypoxia, the scarcity of oxygen, is a hallmark of solid tumors to which they adapt by activating the hypoxia-inducible transcription factor 1 (HIF-1). HIF activates the transcription of genes bearing hypoxia responsive elements (RE) in their promoter regions including genes related to angiogenesis, glucose metabolism, cell proliferation, survival, invasion, and metastasis. The HIF-1 pathway is thus an attractive target to prevent cancer aggressiveness and improve the effectiveness of cancer therapy. HIF-1α activity is known to be mainly regulated through post-translational modification by prolyl hydroxylase domain (PHD) enzymes, but accumulating evidence indicate that it is also regulated by other mechanisms such as transcriptional initiation, translational initiation, protein-protein interaction, post-translational modifications and post-transcriptionally, primarily through the action of trans-acting factors (noncoding RNAs and RNA-binding proteins) that interact with the HIF-1α mRNA to regulate its decay and translational rates. We recently identified the RNA binding protein hnRNP A18 as a regulator of HIF-1α translation under hypoxic conditions. hnRNP A18 is a nuclear stress responsive protein that translocates to the cytosol in response to cellular stress including hypoxia to bind to a recognition motif in the 3’UTR of its targeted transcripts including HIF-1α to stabilize the transcripts and increase their translation. Using a bicistronic reporter plasmid containing a HIF-1α internal ribosome entry site (IRES) we now show that hnRNP A18 can also regulate HIF-1α through its IRES in the 5’UTR. In fact, cells expressing hnRNP A18 significantly increase translation of a reporter CAT protein under the control of HIF-1α IRES in the presence of the hypoxia mimetic agent CoCl2 . On the other hand, deletion of hnRNP A18 prevents the translation of the CAT reporter protein even in the presence of CoCl2. Moreover, we also identified several HIF-1α REs in hnRNP A18 promoter. Two of the predicted HIF-1α REs, located at - 57 and -226 upstream of the hnRNP A18 start codon were validated as bonafide HIF-1α binding sites by Electromobility Shift Assay with recombinant HIF-1α protein and by Chromatin Immunoprecipitation assay in human pancreatic cancer cells Panc 01. These data thus indicate that a positive regulatory loop between hnRNP A18 and HIF1-1α exist to amplify HIF-1α expression under cellular stress. hnRNP A18 can upregulate HIF1α protein expression by stabilizing its transcript at the 3’UTR and by binding to its IRES. On the other hand, HIF-1α can upregulate hnRNP A18 by binding to its proximal promoter. Targeting hnRNP A18 could thus provide a new mechanism to regulate HIF-1α expression and sensitize cancer cells to therapies.