Brian Osmond

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.

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.

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.

Given that the stem cell (SC) marker ALDH is a key component of the retinoic acid (RA) signaling pathway and since APC mutation leads to increased WNT signaling and overpopulation of ALDH+ SCs in colorectal cancer (CRC), we hypothesized that human CRC evolves due to an imbalance between dysregulated RA and WNT signaling. Herein, we investigated how to target the link between these two pathways to restore proper WNT and RA signaling, and promote differentiation of CSCs along the neuroendocrine cell (NEC) lineage. Accordingly, we conducted in vitro experiments to determine if restoring wt-APC in APC-mutant CRC cells reduces Wnt/β-catenin signaling, increases sensitivity to retinoids, reduces cell proliferation, reduces expression of stem cell markers, reduces the number of ALDH+ cells, and enhances NEC maturation. We utilized Nanostring profiling, TCF reporter assay, western blot, and fluorescence activated cell sorting analyses to ascertain the effects of restoring WNT in HT29 CRC cells. We found that induction of wt-APC expression decreased WNT/β-catenin signaling and reduced protein expression of WNT-target genes. Notably, inducing wt-APC decreased ATRA-induced expression of the WNT target gene CYP26A1 (by 50%), which is predicted to increase RA signaling by lowering the degradation of RA. Indeed, wt-APC increased sensitivity of CRC cells to ATRA-induced inhibition of cell proliferation in a dose-dependent manner. Expression of ALDH1A1 decreased 3-fold by ATRA treatment and inducing wt-APC furthered the decrease by an additional 2-fold. Furthermore, we found an increase in the protein expression of several NEC markers including CHGA, GLP2R, NSE (ENO2), and SSTR1, and an increased number of GLP2R+ NECs upon inducing wt-APC. Thus, the link between WNT and RA signaling at the level of CYP26A1, provides a mechanism that can explain how mutant APC leads to decreased maturation of ALDH+ SCs along the NEC lineage and an increased number of ALDH+ SCs. Therefore, discovering ways to increase differentiation of SCs along the NEC lineage in vivo might lead to new more effective treatment strategies involving retinoids for CRC patients. Citation Format: Caroline O. Facey, Victoria O. Hunsu, Brian T. Osmond, Lynn M. Opdenaker, Bruce M. Boman. Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6068.

HOX genes encode master regulatory transcription factors that regulate stem cells (SCs), but their role in carcinogenesis is not fully understood. SC overpopulation drives colorectal cancer (CRC) development, which is also linked with the aberrant expression of specific HOX genes. One such gene is HOXA9. Our goal is to find out how HOXA9-regulatory mechanisms play a role in the SC origin of CRC. Previous research in our lab reported that HOXA9 as well as other HOX proteins (e.g. HOXA4 & HOXD10) are selectively expressed in human colonic cancer SCs (CSCs). The SC marker aldehyde dehydrogenase (ALDH) and other retinoic acid (RA) pathway components are also selectively expressed in CSCs. Moreover, HOXA9 expression is positively correlated with expression of ALDH genes such as ALDH1A1. These findings suggest that RA signaling regulates HOXA9 gene expression in colonic ALDH+ SCs. Hypothesis: Dysregulated HOXA9 expression leads to ALDH+ SC overpopulation, which contributes to CRC growth and development. We used immunofluorescent (IF) mapping to determine the histologic location of HOXA9+ cells in relation to colonic SCs and other differentiated colonic cell types in normal and neoplastic human colonic tissues. We also measured the effects of all-trans retinoic acid (ATRA) and the ALDH enzyme inhibitor diethylamino benzaldehyde (DEAB) on HOXA9 expression, cell proliferation and differentiation using CRC cell lines. Immunofluorescence mapping indicated that not only is HOXA9 selectively expressed in ALDH-positive SCs, but that HOXA9 expression is increased in colon carcinoma tissue compared to normal colon epithelium. Our results also showed that both ATRA and DEAB treatment decreased the proliferation of HT29 and SW480 CRC cell lines. Western blot analysis showed that HOXA9 has reduced expression in ATRA-treated HT29 CRC cells. Additionally, our independent bioinformatics analyses show that HOXA9 is predominantly expressed in both stem cells and neuroendocrine cells (NECs) in normal human colonic epithelium. Taken together, these findings indicate that regulation of HOXA9 gene expression by RA signaling controls differentiation of ALDH+ SCs along the NEC lineage in human colon tissue. Thus, our results show that HOXA9 is regulated by RA signaling and when dysregulated, contributes to CRC development and growth. These findings provide a mechanism that may be targeted in the design of novel treatments against SC overpopulation in CRC. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. HOXA9 overexpression contributes to stem cell overpopulation that drives development and growth of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6069.