CYP26A1 Expression Research Articles

Development and validation of CYP26A1 inhibition assay for high-throughput screening.

All-trans retinoic acid (atRA) is an endogenous ligand of the retinoic acid receptors, which heterodimerize with retinoid X receptors. AtRA is generated in tissues from vitamin A (retinol) metabolism to form a paracrine signal and is locally degraded by cytochrome P450 family 26 (CYP26) enzymes. The CYP26 family consists of three subtypes: A1, B1, and C1, which are differentially expressed during development. This study aims to develop and validate a high throughput screening assay to identify CYP26A1 inhibitors in a cell-free system using a luminescent P450-Glo assay technology. The assay performed well with a signal to background ratio of 25.7, a coefficient of variation of 8.9%, and a Z-factor of 0.7. To validate the assay, we tested a subset of 39 compounds that included known CYP26 inhibitors and retinoids, as well as positive and negative control compounds selected from the literature and/or the ToxCast/Tox21 portfolio. Known CYP26A1 inhibitors were confirmed, and predicted CYP26A1 inhibitors, such as chlorothalonil, prochloraz, and SSR126768, were identified, demonstrating the reliability and robustness of the assay. Given the general importance of atRA as a morphogenetic signal and the localized expression of Cyp26a1 in embryonic tissues, a validated CYP26A1 assay has important implications for evaluating the potential developmental toxicity of chemicals.

Gonadal Transcriptome Analysis Reveals that SOX17 and CYP26A1 are Involved in Sex Differentiation in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

The Chinese soft-shelled turtle (Pelodiscus sinensis) is an important aquaculture animal in China and exhibits growth dimorphism. Single-male cultures are often selected for higher economic efficiency. However, the mechanism of sex differentiation in P. sinensis is not well-known. In this study, a comparative transcriptome analysis of male (ZZ)- and 17β-oestradiol (E2)-induced pseudo-female (ZZ + E2)-stage embryonic gonads of P. sinensis was performed. A total of 420 differentially expressed genes (DEGs), which included 271 upregulated genes and 149 downregulated genes, were identified. These DEGs were mainly involved in several sex-related pathways, such as "ovarian steroidogenesis", "steroid hormone biosynthesis", "PPAR signalling pathway", and "metabolism of xenobiotics by cytochrome P450". In addition, 50 known and novel candidate genes involved in sex differentiation, such as the male-biased genes AMH, DMRT1, TBX1, and CYP26A1 and the female-biased genes CYP1A1, RASD1, and SOX17, were investigated and identified. For further verification, the full-length cDNAs of SOX17 and CYP26A1 were obtained. SOX17 contains a 1218-bp ORF and encodes 405 amino acids containing an HMG functional domain unique to the Sox superfamily. CYP26A1 contains a 1485-bp ORF and encodes 494 amino acids. Different expression levels of SOX17 and CYP26A1 could be detected in all the tested tissues of males and females. Notably, the expression of CYP26A1 was markedly greater in the gonads of male embryos (P < 0.05) than in those of female embryos, whereas the expression of SOX17 showed the opposite trend (P < 0.05). Taken together, the RNA-seq and qRT‒PCR results suggested potential roles for SOX17 and CYP26A1 in promoting female and male gonadal development, respectively, in P. sinensis. Our results provide new evidence for the mechanism of sex differentiation in P. sinensis.

Abstract 266: The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer

Abstract Understanding how to therapeutically target colonic stem cells (SCs) is important because SC overpopulation drives CRC growth and development. Our goal is to therapeutically sensitize cancer SCs (CSCs) to the differentiation-inducing effects of retinoid agents. We previously found that ALDH specifically marks normal and malignant colonic SCs. We also found that retinoic acid (RA) receptors and other RA signaling components are selectively expressed in ALDH+ SCs, indicating that RA signaling occurs through ALDH+ SCs. Because all-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia, which is caused by a genetic translocation involving retinoic acid receptor alpha (RARA), we conjecture that the response of RA agents may be effective against solid tumors depending on their pathway genotype. Our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that most of the RA pathway genes are altered in human CRCs. Therefore, we hypothesize that the ability of retinoid agents and retinoid metabolism blocking agents to induce differentiation of CSCs depends on the RA pathway genotype of CRC cells.Testing the effect of various retinoid drugs on cell proliferation shows that RA pathway genotype affects the response of RA agents in CRC cell lines. HCT116 and SW480 CRC cell lines, which have RA receptor mutations, displayed resistance to ATRA. In contrast, HT29 CRC cells that have wild-type RA receptors are sensitive to ATRA. All three cell lines however showed similar dose responses to the ATRA metabolizing enzyme, CYP26A1 inhibitor Liarozole. This finding indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism can increase intracellular RA levels in a similar manner on all three cell lines to induce anti-proliferative effects. We used CRISPR-Cas9 gene editing to knock out the RARA gene in HT29 cells and evaluated its response to ATRA compared to HT29 cells. The RARA knockout derived lines showed reduced sensitivity to ATRA treatment and a slower rate of cell division compared to HT29 control cells. Additionally, Nanostring mRNA profiling analysis shows that ATRA treatment of HT29 cells increases expression of RARA, CYP26A1, and KRT20 (differentiation marker); and decreased decreases expression of CSC markers LGR5 and ALDH1A1. Altogether, these findings indicate that upregulated RA signaling can reduce cell proliferation, increase differentiation, and decrease CSC numbers in CRCs. Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Thus, therapeutically sensitizing CSCs to differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Bruce M. Boman. The effects of retinoid agents and retinoic acid receptor genotype on proliferation, differentiation, and stem cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 266.

Abstract 5430: Discovery of CYP26A1 as a link between WNT and retinoic acid signaling pathways that modulates retinoid-induced differentiation of APC-mutant colorectal cancer cells

Abstract 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. We hypothesize that 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 and retinoid agents. We found that 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. NanoString profiling and bioinformatics also identified a novel CYP26A1-based network that links WNT and RA signaling. Moreover, in an effort to translate manipulation of the WNT/RA imbalance as a therapeutic approach in vitro, we treated HT29, SW480, and HCT116 CRC cells with inhibitors against CYP26A1 enzyme activity and WNT signaling. We found that CYP26A1 inhibitors sensitized CRC cells to the anti-proliferative effect of drugs that downregulate WNT signaling. Furthermore, decreased CYP26A1 expression in tumor vs normal tissue was determined to be a predictor of patient survival in patients who have wt-APC. Thus, by inducing wt-APC expression and exogenously activating RA signaling, we found a way to increase differentiation of SCs along the NEC (and other) lineage(s). Translating this strategy in vivo might lead to new, more effective treatments involving retinoids for CRC patients. Citation Format: Caroline O. Facey, Victoria O. Hunsu, Chi Zhang, Brian T. Osmond, Lynn M. Opdenaker, Bruce M. Boman. Discovery of CYP26A1 as a link between WNT and retinoic acid signaling pathways that modulates retinoid-induced differentiation of APC-mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5430.

Expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF in the developing rhesus monkey retina

The fovea centralis (fovea) is a specialized region of the primate retina that plays crucial roles in high-resolution visual acuity and color perception. The fovea is characterized by a high density of cone photoreceptors and no rods, and unique anatomical properties that contribute to its remarkable visual capabilities. Early histological analyses identified some of the key events that contribute to foveal development, but the mechanisms that direct the specification of this area are not understood. Recently, the expression of the retinoic acid-metabolizing enzyme CYP26A1 has become a hallmark of some of the retinal specializations found in vertebrates, including the primate fovea and the high-acuity area in avian species. In chickens, the retinoic acid pathway regulates the expression of FGF8 to then direct the development of a rod-free area. Similarly, high levels of CYP26A1, CDKN1A, and NPVF expression have been observed in the primate macula using transcriptomic approaches. However, which retinal cells express these genes and their expression dynamics in the developing primate eye remain unknown. Here, we systematically characterize the expression patterns of CYP26A1, FGF8, CDKN1A, and NPVF during the development of the rhesus monkey retina, from early stages of development in the first trimester until the third trimester (near term). Our data suggest that some of the markers previously proposed to be fovea-specific are not enriched in the progenitors of the rhesus monkey fovea. In contrast, CYP26A1 is expressed at high levels in the progenitors of the fovea, while it localizes in a subpopulation of macular Müller glia cells later in development. Together these data provide invaluable insights into the expression dynamics of several molecules in the nonhuman primate retina and highlight the developmental advancement of the foveal region.

Mechanism of thyroid hormone and its structurally similar contaminant bisphenol S exposure on retinoid metabolism in zebrafish larval eyes

The photoreceptor necessitates the retinoids metabolism processes in visual cycle pathway to regenerate visual pigments and sustain vision. Bisphenol S (BPS), with similar structure of thyroid hormone (TH), was reported to impair the light-sensing function of zebrafish larvae via disturbing TH-thyroid hormone receptor β (TRβ) signaling pathway. However, it remains unknown whether TRβ could modulate the toxicity of BPS on retinoid metabolism in visual cycle. This study showed that BPS diminished the optokinetic response of zebrafish larvae and had a stimulative effect on all-trans-retinoic acid (atRA) metabolism, like exogenous T3 exposure. By modulating CYP26A1 and TRβ expression, it was found that CYP26A1 played a crucial role in catalyzing oxidative metabolism of atRA and retinoids regeneration in visual cycle, and TRβ mediated cyp26a1 expression in zebrafish eyes. Similar with 10 nM T3 treatment, cyp26a1 expression could be induced by BPS in the presence of TRβ. Further, in CYP26A1 and TRβ- deficient eyes, 100 μg/L BPS could no longer promote atRA metabolism, or decrease the all-trans-retinol and 11-cis retinal contents in visual cycle, demonstrating that BPS exposure disturbed CYP26A1-mediated visual retinoids metabolism via TRβ. Overall, this study highlights the role of TRβ in mediating the retinoids homeostasis disruption caused by BPS, and provides new clues for exploring molecular targets of visual toxicity under pollutants stress.

Hyperglycemia alters retinoic acid catabolism in embryos exposed to a maternal diabetic milieu.

Pregestational diabetes is highly associated with increased risk of birth defects. We previously reported that the expression of Cyp26a1, the major catabolizing enzyme for controlling retinoic acid (RA) homeostasis, is significantly down-regulated in embryos of diabetic mice, thereby increasing the embryo's susceptibility to malformations caused by RA dysregulation. However, the underlying mechanism for the down-regulation of Cyp26a1 remains unclear. This study aimed to investigate whether elevated maternal blood glucose in the diabetic milieu is a critical factor for the altered Cyp26a1 expression. Streptozotozin-induced diabetic pregnant mice were treated with phlorizin (PHZ) to reduce blood glucose concentrations via induction of renal glucosuria. Embryonic Cyp26a1 expression level, RA catabolic activity and susceptibility to various RA-induced abnormalities were examined. To test the dose-dependent effect of glucose on Cyp26a1 level, early head-fold stage rat embryos of normal pregnancy were cultured in vitro with varying concentrations of D-glucose, followed by quantification of Cyp26a1 transcripts. We found that Cyp26a1 expression, which was down-regulated in diabetic pregnancy, could be normalized under reduced maternal blood glucose level, concomitant with an increase in RA catabolic activity in embryonic tissues. Such normalization could successfully reduce the susceptibility to different RA-induced malformations including caudal regression, cleft palate and renal malformations. The expression level of Cyp26a1 in the embryo was inversely correlated with D-glucose concentrations. Diabetic patients suffer from retinopathy, dermopathy, male infertility and increased cancer risk. Coincidentally, RA dysregulation is also associated with these health problems. Our results provided evidence that elevated glucose can down-regulate Cyp26a1 expression level and disturb RA homeostasis, shedding light on the possibility of affecting the health of diabetic patients via a similar mechanism.

Evidence for RA-dependent meiosis onset in a turtle embryo.

Meiotic entry is one of the earliest sex determination events of the germ cell in higher vertebrates. Although advances in meiosis onset have been achieved in mammals, birds and fish, how this process functions in reptiles is largely unknown. In this study, we present the molecular analysis of meiosis onset and the role of retinoic acid (RA) in this process in the red-eared slider turtle. Our results using Stra8 as a pre-meiosis indicator show that in the female embryonic gonad, meiosis commitment starts around stage 19. Additionally, signals of the meiosis marker Sycp3 could be detected at stage 19 and become highly expressed by stage 23. No expression of these genes was detected in male embryonic gonads, suggesting the entry into meiosis prophase I was restricted to female embryonic germ cells. Notably, RA activity in fetal gonads is likely to be elevated in females than that in males, as evidenced by the higher expression of RA synthase Aldh1a1 and lower expression of RA-degrading enzyme Cyp26a1 in female gonads prior to meiotic entry. In addition, exogenous RA treatment induced the expression of Stra8 and Sycp3 in both sexes, whether in vivo or in vitro. Together, these results indicate that high levels of RA in the embryonic female gonads can lead to the initiation of meiosis in the turtle.

Abstract 2457: Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer

Abstract Dysregulated WNT signaling caused by mutation in the APC gene occurs in 80-90% of all colorectal cancer (CRC) cases. Progressive overpopulation of ALDH+ stem cells occurs during development of CRC in familial adenomatous polyposis (FAP) patients. Given that ALDH is a key member of the retinoic acid signaling (RA) pathway, we hypothesize that human CRC is caused by an imbalance between dysregulated WNT and RA signaling. Herein, we aim to restore proper WNT and RA signaling and identify a common link or target to induce differentiation of CSCs along the neuroendocrine cell (NEC) lineage. Accordingly, to determine the effects of restoring wt-APC in APC-mutant HT29 CRC cells, we conducted in vitro experiments to examine Wnt/β-catenin signaling, sensitivity to retinoids, cell proliferation, expression of stem cell markers, quantification of ALDH+ stem cells, and NEC maturation. We utilized Nanostring profiling, TCF reporter assay, western blot, and fluorescence activated cell sorting analyses to ascertain these effects. 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, in turn, increase RA signaling by lowering degradation of RA. Indeed, inducing wt-APC increased sensitivity of CRC cells to ATRA-induced apoptosis and inhibition of cell proliferation in a dose-dependent manner. Expression of ALDH1A1 decreased (3-fold) with ATRA treatment and inducing wt-APC extended the decrease by an additional 2-fold. Furthermore, upon inducing wt-APC, 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. We also discovered a very highly regulated protein network of 248 proteins and identified a cell signaling mechanism that links the WNT and retinoic acid pathways via CYP26A1. Thus, by inducing wt-APC expression and exogenously activating RA signaling, we found a way to increase differentiation of SCs along the NEC (and other) lineage(s). Translating this strategy in vivo might lead to new, more effective treatments involving retinoids for CRC patients. Citation Format: Caroline O. B. Facey, Victoria O. Hunsu, Brian T. Osmond, Chi Zhang, 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 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2457.

Retinoic acid metabolism in cancer: potential feasibility of retinoic acid metabolism blocking therapy.

Retinoic acid (RA) is an active metabolite of vitamin A, which is an essential signaling molecule involved in cell fate decisions, such as differentiation, proliferation, and apoptosis, in a wide variety of cell types. Accumulated data have demonstrated that expression of RA-metabolizing enzymes, CYP26A1, B1, and C1 (cytochrome P450, family 26A1, B1, and C1, respectively), protects cells and tissues from exposure to RA through restriction of RA access to transcriptional machinery by converting RA to rapidly excreted derivatives. CYP26 enzymes play similar but separate roles in limiting the consequences of fluctuations in nutritional vitamin A. Recently, we found that RA depletion caused by expression of CYP26A1 promotes malignant behaviors of tumor cells derived from various tissues, implicating CYP26A1 as a candidate oncogene. We also showed that the expression levels of CYP26 enzymes are elevated in various types of cancer. We have provided evidence for oncogenic and cell survival properties of CYP26 enzymes, indicating that these molecules are possible therapeutic targets for CYP26-expressing malignancies.

In Vitro Transfection of Up-Regulated Genes Identified in Favorable-Outcome Neuroblastoma into Cell Lines.

We previously used microarrays to show that high expression of DHRS3, NROB1, and CYP26A1 predicts favorable NB outcomes. Here, we investigated whether expression of these genes was associated with suppression of NB cell (SK-N-SH, NB12, and TGW) growth. We assessed morphology and performed growth, colony-formation, and migration assays, as well as RNA sequencing. The effects of the transient expression of these genes were also assessed with a tetracycline-controlled expression (Tet-On) system. Gene overexpression reduced cell growth and induced morphological senescence. Gene-expression analysis identified pathways involving cellular senescence and cell adhesion. In these cells, transduced gene dropout occurred during passage, making long-term stable gene transfer difficult. Tet-On-induced gene expression caused more pronounced cell-morphology changes. Specifically, DHRS3 and NROB1 led to rapid inhibition and arrest of cell growth, though CYP26A1 did not affect cell-growth rate or cell cycle. DHRS3 arrested the cell cycle by interacting with the all-trans-retinol pathway and drove differentiation and senescence in tumors. Overexpression of these genes reduced the malignant grade of these cells. A new therapeutic strategy might be the induction of these genes, as they suppress the growth of high-risk neuroblastoma and lead to differentiation and senescence.

Abstract 6067: Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines

Abstract Background: Our overarching goal is to therapeutically sensitize human colorectal cancer stem cells to differentiation-inducing effects of retinoid agents. Tumorigenesis in colorectal cancer (CRC) results from the acquisition of mutations in a colonic stem cell which transforms it into a cancer stem cell (CSC). There are no curative treatments for advanced CRC due to the resistance of CSCs to chemotherapy and/or radiation. All-trans retinoic acid (ATRA) is known to be a greatly effective treatment for acute promyelocytic leukemia (APL) which is caused by translocation in the retinoic acid receptor alpha (RARA) gene. We conjecture that the response of retinoic acid (RA) agents may have effects on solid tumors depending on their pathway genotype. Results from our bioinformatics analysis on mutations and overexpression of RA signaling component genes in CRC showed that the majority of RA pathway genes are overexpressed in CRC. Therefore, we hypothesize that the ability of retinoid and retinoid metabolism blocking agents to induce differentiating effects in CSCs depends on the RA pathway genotype expressed in CRC cells. Methods: The ability of RA agents (ATRA, 13-cis retinoic acid, 9-cis retinoic acid) and RA metabolism blocking agents (Liarozole and Talarozole) to inhibit proliferation and induce differentiation on CRC cell lines with different mutations in RA signaling were evaluated. Additionally, Nanostring Profiling was used to measure the effects of the agents on mRNA expression of CSC markers, indicators of cell differentiation, and RA receptors. Results: Our results show that the RA pathway genotype affects the response of RA agents in CRC cell lines. Both HCT116 and SW480 CRC cell lines, which are both mutant in RARA and RARA and RXRG respectively displayed resistance to ATRA. HT29 CRC cells express wild- type RA receptors and are sensitive to ATRA. All three cell lines showed similar dose responses to the CYP26A1 inhibitor Liarozole. This indicates that regardless of RA receptor mutations, inhibition of ATRA metabolism increases intracellular RA levels in a similar manner on all 3 cell lines. Nanostring Profiling shows that ATRA treatment of HT29 cells induces: an increase in the expression of RARA, CYP26A1 (ATRA metabolizing enzyme) and KRT20 (differentiation marker); and a decrease in the expression of CSC markers LGR5 and ALDH1A1. This indicates an active RA signaling pathway, induced differentiation, and a decrease in stem cells. Discussion: Overall, our findings indicate that colon SCs are regulated by RA signaling mechanisms and dysregulation of RA signaling contributes to the SC overpopulation that drives CRC growth. Therefore, further investigation on how to therapeutically sensitize human colonic CSCs to the differentiation-inducing effects of retinoid agents should provide insight into designing new SC-targeted therapies for CRC. Citation Format: Victoria O. Hunsu, Caroline O. Facey, Lynn M. Opdenaker, Bruce M. Boman. Studying the anti-proliferating and differentiating effects of retinoids based on pathway genotype of colorectal cancer cell lines [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 6067.

Abstract 6068: Restoring the balance between WNT and retinoic acid signaling to promote differentiation of cancer stem cells in treatment of colorectal cancer

Abstract 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.

The glucocorticoid receptor represses, whereas C/EBPβ can enhance or repress CYP26A1 transcription

SummaryRetinoic acid (RA) counters insulin’s metabolic actions. Insulin reduces liver RA biosynthesis by exporting FoxO1 from nuclei. RA induces its catabolism, catalyzed by CYP26A1. A CYP26A1 contribution to RA homeostasis with changes in energy status had not been investigated. We found that glucagon, cortisol, and dexamethasone decrease RA-induced CYP26A1 transcription, thereby reducing RA oxidation during fasting. Interaction between the glucocorticoid receptor and the RAR/RXR coactivation complex suppresses CYP26A1 expression, increasing RA’s elimination half-life. Interaction between CCAAT-enhancer-binding protein beta (C/EBPβ) and the major allele of SNP rs2068888 enhances CYP26A1 expression; the minor allele restricts the C/EBPβ effect on CYP26A1. The major and minor alleles associate with impaired human health or reduction in blood triglycerides, respectively. Thus, regulating CYP26A1 transcription contributes to adapting RA to coordinate energy availability with metabolism. These results enhance insight into CYP26A1 effects on RA during changes in energy status and glucocorticoid receptor modification of RAR-regulated gene expression.

The multiciliated cells in Rathke's cleft express CYP26A1 and respond to retinoic acid in the pituitary.

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.

Development of an adenovirus-mediated reporter assay system to detect a low concentration of retinoic acid in MCF-7 cells.

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.

Expression of retinoic acid signaling pathway in uterus injury induced by procymidone in adolescent mice

To investigate the expression of key genes and proteins of retinoic acid signaling pathway in procymidone-induced uterine injury in adolescent mice, and analyze the relationship between the signaling pathway and female reproductive damage. The 3-week age ICR mice were randomly divided into low, medium, and high-dose groups and one control group with 8 mice in each group by weight. The low, medium and high dose groups were respectively given 50, 100 and 200 mg/(kg·d) procymidone orally for 21 days continuously, while the control group was given equal volume of soybean oil. After the mice were sacrificed, the uterus was taken from both sides for observing the histological changes in the cross-sectional slices of the uterus, the detection of the expression abundance of genes which related to the retinoic acid signaling pathway by the real-time fluorescent quantitative PCR, and the measurement of ALDH2 and CYP26 a1 proteins expression by Western blot. The body weight of mice in low-dose, medium-dose and high-dose groups were(27.50±1.49) g, (27.72±1.40) g and(26.89±1.19) g, respectively, which were lower than those in control group(31.48±1.14) g(P&lt;0.05). The density of uterine lining monolayer columnar epithelium and lamina propria tubular uterine glands gradually decreases, at the same time the uterine folds become less with the dose of procymidone increases. adh1, ad/2, aldh1a1 in each experimental group were higher than those in the control group(P&lt;0.05); the expression levels of aldh1a2 and aldh1a3 genes in the middle and high dose groups were higher than those in the control group(P&lt;0.05); the expression levels of retinoic acid nuclear receptor rarα, rarγ, rxrα and rxrβ genes in the high-dose group were higher than those in the control(P&lt;0.05); yet the expression levels of cyp26a2 and cyp26a3 in the high-dose group were lower than those in the control group(P&lt;0.05); the jnk family in medium and high dose groups were higher than the control(P&lt;0.05). The expression of ALDH2 in each experimental group was higher than that in the control group, and increased with the increase of the dose(P&lt;0.05); the expression of CYP26 a1 in each experimental group was not significantly different from that of the control group. The retinoic acid signal pathway is activated in procymidone-induced uterine injury in mice, then regulates the increase of the expression of jnk family, leading to the damage.

Low retinoic acid levels mediate regionalization of the Sertoli valve in the terminal segment of mouse seminiferous tubules

In mammalian testes, undifferentiated spermatogonia (Aundiff) undergo differentiation in response to retinoic acid (RA), while their progenitor states are partially maintained by fibroblast growth factors (FGFs). Sertoli valve (SV) is a region located at the terminal end of seminiferous tubule (ST) adjacent to the rete testis (RT), where the high density of Aundiff is constitutively maintained with the absence of active spermatogenesis. However, the molecular and cellular characteristics of SV epithelia still remain unclear. In this study, we first identified the region-specific AKT phosphorylation in the SV Sertoli cells and demonstrated non-cell autonomous specialization of Sertoli cells in the SV region by performing a Sertoli cell ablation/replacement experiment. The expression of Fgf9 was detected in the RT epithelia, while the exogenous administration of FGF9 caused ectopic AKT phosphorylation in the Sertoli cells of convoluted ST. Furthermore, we revealed the SV region-specific expression of Cyp26a1, which encodes an RA-degrading enzyme, and demonstrated that the increased RA levels in the SV region disrupt its pool of Aundiff by inducing their differentiation. Taken together, RT-derived FGFs and low levels of RA signaling contribute to the non-cell-autonomous regionalization of the SV epithelia and its local maintenance of Aundiff in the SV region.

CYP26A1 Is a Novel Biomarker for Betel Quid-Related Oral and Pharyngeal Cancers.

Betel quid (BQ) has been classified as a Group I human carcinogen in light of evidence demonstrating an association with an elevated risk of oral and pharyngeal cancers. To date, the incidence rate of oral and pharynx cancers among Taiwanese men ranks the highest worldwide. However, no study has yet confirmed variants of CYP26A1 was associated with the risks of oral and pharyngeal cancers. A case-control study was conducted (n = 339). CYP26A1 polymorphism was performed using SNP assay. Real-time qRT-PCR and Western blotting were used to determine the levels of CYP26A1 expression. The cancer cell model involved treatment with arecoline. Our findings showed that the downregulation of CYP26A1 mRNA and protein expression are more frequently observed in cancerous tissues than adjacent normal tissues in patients with oral and pharynx cancers (p < 0.01). We found that CYP26A1 was downregulated as the arecoline dose increased. We hypothesized that lower levels of CYP26A1 mRNA expression can be utilized a clinically biomarker causes oral and pharynx cancers. Arecoline appears to modulate CYP26A1 expression through specific pathways. Carriers of CYP26A1 SNP, rs2068888 (G/G)/rs4418728 (G/G) and who have lower levels of CYP26A1 expression are associated with an increased risk of oral and pharyngeal cancers.

Overlapping Vitamin A Interventions with Provitamin A Carotenoids and Preformed Vitamin A Cause Excessive Liver Retinol Stores in Male Mongolian Gerbils

Vitamin A (VA) deficiency is a public health problem in some countries. Fortification, supplementation, and increased provitamin A consumption through biofortification are efficacious, but monitoring is needed due to risk of excessive VA intake when interventions overlap. Two studies in 28-36-d-old male Mongolian gerbils simulated exposure to multiple VA interventions to determine the effects of provitamin A carotenoid consumption from biofortified maize and carrots and preformed VA fortificant on status. Study 1 was a 2×2×2 factorial design (n=85) with high-β-carotene maize, orange carrots, and VA fortification at 50% estimated gerbil needs, compared with white maize and white carrot controls. Study 2 was a 2×3 factorial design (n=66) evaluating orange carrot and VA consumption through fortification at 100% and 200% estimated needs. Both studies utilized 2-wk VA depletion, baseline evaluation, 9-wk treatments, and liver VA stores by HPLC. Intestinal scavenger receptor class B member 1 (Scarb1), β-carotene 15,15'-dioxygenase (Bco1), β-carotene 9',10'-oxygenase (Bco2), intestine-specific homeobox (Isx), and cytochrome P450 26A1 isoform α1 (Cyp26a1) expression was analyzed by qRT-PCR in study 2. In study 1, liver VA concentrations were significantly higher in orange carrot (0.69±0.12 μmol/g) and orange maize groups (0.52±0.21 μmol/g) compared with baseline (0.23±0.069 μmol/g) and controls. Liver VA concentrations from VA fortificant alone (0.11±0.053 μmol/g) did not differ from negative control. In study 2, orange carrot significantly enhanced liver VA concentrations (0.85±0.24 μmol/g) relative to baseline (0.43±0.14 μmol/g), but VA fortificant alone (0.42±0.21 μmol/g) did not. Intestinal Scarb1 and Bco1 were negatively correlated with increasing liver VA concentrations (P <0.01, r2=0.25-0.27). Serum retinol concentrations did not differ. Biofortified carrots and maize without fortification prevented VA deficiency in gerbils. During adequate provitamin A dietary intake, preformed VA intake resulted in excessive liver stores in gerbils, despite downregulation of carotenoid absorption and cleavage gene expression.