Imbalance in WNT: retinoic acid signaling dysregulates HOX gene expression: implications for cancer stem cell heterogeneity and CRC patient survival.

How HOX transcription factors play a role in the stem cell (SC) origin of cancer is unclear. Our goal is to determine how dysregulation of HOX gene expression promotes colorectal cancer (CRC) development through aberrant SC regulatory mechanisms. We investigated retinoic acid (RA) and WNT signaling because they regulate HOX expression and both pathways are dysregulated in cancer. Previously, we reported WNT and RA pathways are functionally linked, and in CRC, APC mutation generates a WNT:RA imbalance leading to incomplete differentiation and SC overpopulation. We hypothesize that aberrant WNT- and RA-signaling dysregulates HOX expression that contributes to the SC origin of CRC. We discovered a HOX expression signature of eight genes (six-upregulated, two-downregulated) that are significantly altered in CRC, and that predict poor CRC patient survival. NanoString profiling of fluorescence activated cell sorted SC subpopulations, bioinformatics studies, and RNA-seq analysis of fresh colon tissues revealed: (1). The six-upregulated genes are regulated by RA signaling; (2). ATRA-treatment of CRC cells, but not wt-APC induction or RARA knockout, leads to a HOX expression pattern matching the HOX signature; (3). Mid-cluster (group 3) HOXB gene expression characterizes normal colon, but increased group 3 HOXC expression occurs in CRC; (4). LGR5+ SCs selectively express group 3 HOXB genes while ALDH+ SCs express group 3 HOXC genes; (5). HOXA13 and HOXB13 which are downregulated and predict poor patient survival likely possess tumor-suppressor activity and function by retro-inhibition of earlier HOX genes. Given HOX expression predicts survival and identifies SC phenotype, this indicates dysregulated HOX expression contributes to SC origin of CRC.