Tumor cell endogenous HIF-1alpha activity induces aberrant angiogenesis and interacts with TRAF6 pathway required for colorectal cancer development

Hypoxia and inflammation are key factors for colorectal cancer tumorigenesis. The colonic epithelium belongs to the tissues with the lowest partial pressure of oxygen in the body, and chronic inflammation is associated with an increased chance to develop colon cancer. How the colonic epithelium responds to hypoxia and inflammation during tumorigenesis remains to be elucidated. Here we show, that murine colon adenocarcinoma cells with attenuated response to hypoxia, due to a knock-down (KD) of HIF-1alpha produce smaller and less hypoxic tumors in an orthotopic mouse model when compared to tumors induced with control cells. HIF-1alpha-KD tumors showed more functional perfused vasculature associated with increased levels of vessel-stabilizing factors and reduced levels of pro-angiogenic factors, including extracellular matrix protein Cyr61/CCN1. Intratumoral injection of Cyr61 in HIF-1alpha-KD tumors revealed an in increased vessel permeability and tumor hypoxia. Further bioinformatics analysis identified a possible interaction between HIF-1alpha and TRAF6, an upstream effector of the NF-kappaB pathway that was confirmed by co-immunoprecipitation in MC-38 and CT26 colon adenocarcinoma cells. Down-regulation of TRAF6 resulted in virtual abrogation of orthotopic tumor growth. Subcutaneous TRAF6-KD tumors were smaller and contained reduced vessel size and differently polarized macrophages. These data demonstrate that the tumor cell response to increased hypoxia in the colon leads to promotion of non-functional angiogenesis, regulated by both hypoxia and TRAF6 pathways. The study objective was to compare the transcriptomes of murine colon adenocarcinoma cells, MC-38 cells with attenuated hypoxia response, knock-down of HIF-1alpha (HIF-1alpha-KD) versus control (Mock) cells, which were isolated (sorted) from an orthotopic tumor grown in cecum wall of a mouse. Downregulation of HIF-1alpha expression resulted in smaller tumors, characterized by reduced tumor hypoxia and increased functional (perfused) vasculature when compared to control (Mock) tumors. To identify factors leading to this phenotype, we performed deep RNAseq (Illumina HiSeq 4000) and searched for genes that are either up-regulated or down-regulated in sorted HIF-1alpha-KD MC-38 cells versus Mock-control cells). All samples were analyzed in triplicates (HIF-KD sorted 1-3, Mock-sorted 1-3).