Abstract 5112: In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation

Abstract

Abstract Hypoxia is a common feature of many solid tumors that promotes tumor progression and leads to radiation and chemotherapy resistance. Hypoxia-inducible factor 1 (HIF-1), as one of the major mediators of the hypoxic response, has been shown to activate hypoxia-responsive genes, which are involved in multiple aspects of tumorigenesis and cancer progression. Previously, we have described an interaction between septin 9 isoform 1 (SEPT9_i1) protein and HIF-1α, the oxygen regulated subunit of HIF-1. SEPT9_i1 is a member of the conserved family of septins, which are GTP-binding proteins that form cytoskeleton-like filaments essential for many functions in eukaryotic organisms including neoplasia. The interaction of SEPT9_i1 with HIF-1α increases protein stability and HIF-1 transcriptional activity in vitro and promotes proliferation, tumor growth and angiogenesis in vivo. Recent data indicated that SEPT9_i1 was also involved in the trafficking of cytoplasmic HIF-1α into the nucleus by direct interaction to the nuclear transporter importin-α. Herein, we utilized split YFP bimolecular fluorescence complementation (BiFC) methodology in order to monitor HIF-1α/SEPT9_i1 interactions in vivo. First, we generated split YFP protein chimeras fused to HIF-1α and to SEPT9_i1 on both their amino and carboxyl termini. Different pairs of HIF-1α and SEPT9_i1 chimeras were tested to functional complementation of the split YFP fragments using transient transfection in HEK293 cells. The complemented proteins capable of fluorescence were identified as SEPT9_i1-YN (the amino terminus of YFP fused to the carboxyl terminal of SEPT9_i1) and YC-HIF-1α (the carboxyl terminus of YFP fused to the N-terminal of HIF-1α). This pair was selected for further studies in PC-3 human prostate cancer cells. YFP complementation fluorescence derived from these chimeras was increased in the presence of hypoxia mimicking agents, such as CoCl2 and the iron chelators deferoxamine (DFO) and dibenzoylmethane (DBM). The chimera, YCΔHLH lacking the HLH domain that is essential for the interaction with SEPT9_i1 gave a significant reduction in YFP complementation fluorescence. Furthermore, expression of SEPT9_i1 252-379aa fragment, the domain essential for the interaction with HIF-1α, abolished almost completely the YFP complementation fluorescence. These results reconfirm our previous studies on HIF-1α/SEPT9_i1 interactions and make this system attractive for identifying new compounds capable of disrupting this complex in order to inhibit tumor growth and angiogenesis. Citation Format: Maya Golan, Nicola J. Mabjeesh. In vivo molecular imaging of HIF-1α and septin 9 interaction by bimolecular fluorescence complementation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5112. doi:10.1158/1538-7445.AM2015-5112