Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression.

The transcription partners of eyes absent homologs and sine oculis homeobox homologs (EYA-SIX) contribute to tumorigenesis and progression of multiple cancers through mediating the expression of oncogenes and tumor suppressors. This study aimed to determine the roles of individual EYA-SIX partners and their downstream targets in colorectal cancer (CRC). Immunoblot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to measure protein and gene expression levels. Cell Counting Kit-8 (CCK-8) assay, colony formation, cell invasion assays, and a tumor xenograft model were chosen to investigate tumor cell growth. Immunoprecipitation, mass spectrometry, and co-immunoprecipitation (Co-IP) experiments were performed to determine the assembly of the SIX5-associated complex. Chromatin immunoprecipitation (ChIP) assay was used to evaluate the occupancy of SIX5-associated complex on its target gene promoters. We discovered that the hypoxia-induced EYA3 coupled with SIX5 and a histone acetyltransferase p300 to assemble a complex in CRC biopsies. The EYA3-SIX5-p300 complex was required for the transactivation of epidermal growth factor receptor (EGFR), vascular endothelial growth factor D (VEGFD), and five matrix metallopeptidases (MMPs), including MMP3, MMP7, MMP8, MMP21, and MMP26. The results of ChIP revealed that the EYA3-SIX5-p300 complex specifically bound to the promoters of EGFR/VEGFD/MMPs. Disruption of the assembly of EYA3-SIX5-p300 complex decreased the expression of EGFR/VEGFD/MMPs, inhibiting CRC cell growth. Administration of EYA3 inhibitor (benzarone) in mice harboring tumor xenografts significantly inhibited tumor growth. The hypoxia-dependent EYA3-SIX5-p300 complex is involved in the pathogenesis of CRC through mediating EGFR/VEGFD/MMPs and targeting this complex may represent a new therapeutic strategy for CRC treatment.