HOX proteins are transcription factors that regulate stem cell (SC) function, but their role in the SC origin of cancer is under-studied. Aberrant expression of HOX genes occurs in many cancer types. Our goal is to ascertain how retinoic acid (RA) signaling and the regulation of HOXA9 expression might play a role in the SC origin of human colorectal cancer (CRC). Previously, we reported that aldehyde dehydrogenase (ALDH) and other RA pathway components are co-expressed in colonic cancer SCs (CSCs) and that overpopulation of ALDH-positive CSCs occurs during colon tumorigenesis. Our hypothesis is RA signaling regulates HOXA9 expression, and dysregulated RA signaling results in HOXA9 overexpression, which contributes to CSC overpopulation in CRC. Immunostaining showed that HOXA9 was selectively expressed in ALDH-positive SCs, and HOXA9 expression was increased in CRCs compared to normal epithelium. Modulating RA signaling in CRC cells (HT29 and SW480) with ATRA and DEAB decreased cell proliferation and reduced HOXA9 expression. Bioinformatics analyses identified a network of proteins that functionally interact with HOXA9, and the genes that encode these proteins, as well as HOXA9, contain RA receptor binding sites. These findings indicate that the expression of HOXA9 and its functional network is regulated by RA signaling in normal colonic SCs, and, when dysregulated, HOXA9 may contribute to CSC overpopulation that drives CRC development and growth. Our study provides a regulatory mechanism that might be useful in developing treatments against CSC overpopulation in CRC.

Significant changes in growth potential and feed conversion have been bred into the modern broiler chicken for well over 60 yr. These metabolic changes have had significant effects on the immune performance as well. To better understand these genetic differences in immunometabolism we studied the immune response of the modern broiler and the Athens Canadian Random Bred (ACRB) heritage broiler strain. We injected newly hatched modern broiler and ACRB chicks intraabdominally with CpG oligonucleotide, an immunostimulatory synthetic oligonucleotide. We conducted species-specific kinome array analysis and gene expression analysis on jejunum and cecal tonsil tissue. We also performed metabolic analysis of blood cells. In the modern birds, there is an initial inflammatory response to the injection at d 3 post-hatch with activation of PI3K-Akt, JAK-STAT, and NF-κB signaling, and IL-1β and IL-6 mRNA expression. By d 15 post-hatch this response changed to deactivation and downregulation of these immune responses in modern but not heritage broilers. Metabolic analysis showed an increase in glycolysis in peripheral blood mononuclear cells from modern birds given CpG, but no difference in ACRB. These results show that the ACRB birds may have a less inflammatory and more stable immune profile in response to immune stimulation than the modern broilers, possibly resulting in a more disease resistant phenotype overall.

APC mutation is the main driving mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. Hypotheses: A functional link exists between the WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induce wt-APC expression in APC-mutant CRC cells, and we assess the ability of all-trans retinoic acid (ATRA) to induce differentiation. We found that ATRA increased expression of the WNT target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, the RA and WNT pathways crosstalk to modulate CYP26A1, which metabolizes retinoids. Moreover, inducing wt-APC augments ATRA-induced cell differentiation by: (i) decreasing cell proliferation; (ii) suppressing ALDH1A1 expression; (iii) decreasing ALDH+ SCs; and (iv) increasing neuroendocrine cell differentiation. A novel CYP26A1-based network that links WNT and RA signaling was also identified by NanoString profiling/bioinformatics analysis. Furthermore, CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Notably, in wt-APC-CRCs, decreased CYP26A1 improved patient survival. These findings have strong potential for clinical translation.

APC mutation is the main driver mechanism of CRC development and leads to constitutively activated WNT signaling, overpopulation of ALDH+ stem cells (SCs), and incomplete differentiation. We previously reported that retinoic acid (RA) receptors are selectively expressed in ALDH+ SCs, which provides a way to target cancer SCs with retinoids to induce differentiation. Hypotheses: A functional link exists between WNT and RA pathways, and APC mutation generates a WNT:RA imbalance that decreases retinoid-induced differentiation and increases ALDH+ SCs. Accordingly, to restore parity in WNT:RA signaling, we induced wt-APC expression in APC-mutant CRC cells and assessed the ability of all-trans-retinoic acid (ATRA) to induce differentiation. Notably, ATRA substantially increased expression of the WNT-target gene, CYP26A1, and inducing wt-APC reduced this expression by 50%. Thus, RA and WNT pathways crosstalk to modulate CYP26A1 metabolism of retinoids. We found that inducing wt-APC augments ATRA-induced cell differentiation by: i) decreasing cell proliferation; ii) suppressing ALDH1A1 expression; iii) decreasing ALDH+ SCs; iv) increasing neuroendocrine cell differentiation. Furthermore, NanoString profiling/bioinformatics identified a novel CYP26A1-based network that links WNT and RA signaling. Moreover, CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Notably. in wt-APC-CRCs, decreased CYP26A1 improves patient survival. These findings have strong potential for clinical translation.

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.