RARβ Expression Research Articles

Antiproliferative and Morphological Effects of Fenretinide Lipid Nanosystems in Colon Adenocarcinoma Cells

Objective: Colon adenocarcinoma is characterized by the downregulation of the retinoic acid receptor, making natural retinoids such as all-trans retinoic acid, 9-cis retinoic acid and 13-cis retinoic acid effective in treatment and chemoprevention due to their ability to increase RARβ expression. However, major limitations to their use include tolerability and acquired resistance. In this study, we evaluated fenretinide, a semisynthetic derivative of all-trans retinoic acid, in an HT-29 cell line. Fenretinide was evaluated both as a free drug and encapsulated in self-assembling phosphatidylcholine nanosystems with the aim of increasing the aqueous solubility and cell availability of the drug. Methods: Fenretinide was encapsulated in lipid nanosystems obtained in water by the dispersion of an amphiphilic mixture of phospholipids, glyceryl tributyrate and polysorbate 80. The physico-chemical characterization of the nanosystems was carried out by dynamic light scattering and spectrophotometry. The biological activity was evaluated by quantitative phase imaging microscopy, MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Results: Fenretinide in phosphatidylcholine nanosystems was more active than free fenretinide in inhibiting HT-29 cells’ proliferation, as indicated by quantitative phase imaging data. Indeed, encapsulated fenretinide increased duplication time, decreased dry mass and decreased the rate of cell growth more efficiently than fenretinide. Moreover, encapsulated fenretinide effectively decreased the motility of the cells that survived the treatment. Conclusions: The results indicate that the proposed nanosystems can be considered a valuable alternative to natural retinoids in the chemoprevention and treatment of colorectal cancer. This is due to the favorable pharmacologic characteristics of fenretinide in colorectal cancer and the improved drug activity provided by nanoencapsulation.

Unveiling the role of RARs in stomach adenocarcinoma: clinical implications and prognostic biomarkers.

Retinoic acid receptors (RARs) family are known to play a significant role in the occurrence and development of tumors. However, the relationship between RARs and stomach adenocarcinoma (STAD) has not yet been clearly identified. The aim of this study is to evaluate the expression profile and clinical value of the RARs family in STAD. The expression level, clinical characteristics, prognostic value, immunity-related evaluations, genetic alteration and methylation site of RARs in STAD were explored using a series of online databases including gene expression profiling interactive analysis (GEPIA), tumor immune estimation resource (TIMER), University of Alabama at Birmingham cancer data (UALCAN), Human Protein Atlas (HPA), Kaplan-Meier plotter, gene set cancer analysis (GSCA), cBioPortal, MethSurv, GeneMANIA, LinkedOmics, Metascape, Search tool for the retrieval of interacting genes (STRING), tumor immune single-cell hub (TISCH) and cancer cell line encyclopedia (CCLE). We discovered dramatically increased expression of RARA and decreased expression of RARB in STAD tissues, and many clinical variables were closely related to RARs. Notably, higher expressions of RARA and RARB as well as lower expression of RARG correlated with worse overall survival (OS) for STAD patients. The clinical value of prognostic model indicated that RARs were identified to be potential prognostic biomarkers for STAD patients. Moreover, RARB was closely related to immune cell infiltration, which had effect on the role of RARB in STAD prognosis. And the genetic alteration of RARB was significantly associated with the longer disease-free survival (DFS) of STAD patients. Additionally, some CpG sites of the RARs family were related with the prognosis of STAD patients. Functional enrichment analyses indicated that several pathways in STAD might be pivotal pathways regulated by RARs. At the single-cell level, there was some extent of infiltration of tumor microenvironment-related cells in the RARs expression in STAD. Our results evaluated the expression profile and clinical values of RARs in patients with STAD, which provided a basis for future in-depth exploration of the specific mechanisms of each member of RARs in STAD.

RARB associated with MSI, affects progression and prognosis of gastric cancer

Microsatellite instability (MSI) has been widely acknowledged as an important factor regulating tumor intrinsic biological behavior and affecting the survival of gastric cancer patients. Here, we firstly identified the RARB as a gene associated with MSI gastric cancer. RARB was downregulated in human gastric cancer tissues compared to paired paracancerous tissues, Knockdown of RARB accelerated the proliferation, invasion and migration of cancer cells in vitro. Mechanismly, RARB knockdown promoted epithelial-mesenchymal transition (EMT) process of gastric cancer. However, RARBLow patients exhibited better survival compared to RARBHigh patients. Further study revealed that RARB expression was inversely correlated with MSI status and immune infiltrates in vivo. Thus, RARB may be a potential target for the treatment of gastric cancer.

C286, an orally available retinoic acid receptor β agonist drug, regulates multiple pathways to achieve spinal cord injury repair.

Retinoic acid receptor β2 (RARβ2) is an emerging therapeutic target for spinal cord injuries (SCIs) with a unique multimodal regenerative effect. We have developed a first-in-class RARβ agonist drug, C286, that modulates neuron-glial pathways to induce functional recovery in a rodent model of sensory root avulsion. Here, using genome-wide and pathway enrichment analysis of avulsed rats' spinal cords, we show that C286 also influences the extracellular milieu (ECM). Protein expression studies showed that C286 upregulates tenascin-C, integrin-α9, and osteopontin in the injured cord. Similarly, C286 remodulates these ECM molecules, hampers inflammation and prevents tissue loss in a rodent model of spinal cord contusion C286. We further demonstrate C286's efficacy in human iPSC-derived neurons, with treatment resulting in a significant increase in neurite outgrowth. Additionally, we identify a putative efficacy biomarker, S100B, which plasma levels correlated with axonal regeneration in nerve-injured rats. We also found that other clinically available retinoids, that are not RARβ specific agonists, did not lead to functional recovery in avulsed rats, demonstrating the requirement for RARβ specific pathways in regeneration. In a Phase 1 trial, the single ascending dose (SAD) cohorts showed increases in expression of RARβ2 in white blood cells correlative to increased doses and at the highest dose administered, the pharmacokinetics were similar to the rat proof of concept (POC) studies. Collectively, our data suggests that C286 signalling in neurite/axonal outgrowth is conserved between species and across nerve injuries. This warrants further clinical testing of C286 to ascertain POC in a broad spectrum of neurodegenerative conditions.

Alteration of Gene Expression in Pathological Keratinization of the Ocular Surface.

To investigate the molecular mechanism of pathological keratinization in the chronic phase of ocular surface (OS) diseases. In this study, a comprehensive gene expression analysis was performed using oligonucleotide microarrays on OS epithelial cells obtained from three patients with pathological keratinization (Stevens-Johnson syndrome [n = 1 patient], ocular cicatricial pemphigoid [n = 1 patient], and anterior staphyloma [n = 1 patient]). The controls were three patients with conjunctivochalasis. The expression in some transcripts was confirmed using quantitative real-time PCR. Compared to the controls, 3118 genes were significantly upregulated by a factor of 2 or more than one-half in the pathological keratinized epithelial cells (analysis of variance P < 0.05). Genes involved in keratinization, lipid metabolism, and oxidoreductase were upregulated, while genes involved in cellular response, as well as known transcription factors (TFs), were downregulated. Those genes were further analyzed with respect to TFs and retinoic acid (RA) through gene ontology analysis and known reports. The expression of TFs MYBL2, FOXM1, and SREBF2, was upregulated, and the TF ELF3 was significantly downregulated. The expression of AKR1B15, RDH12, and CRABP2 (i.e., genes related to RA, which is known to suppress keratinization) was increased more than twentyfold, whereas the expression of genes RARB and RARRES3 was decreased by 1/50. CRABP2, RARB, and RARRES3 expression changes were also confirmed by qRT-PCR. In pathological keratinized ocular surfaces, common transcript changes, including abnormalities in vitamin A metabolism, are involved in the mechanism of pathological keratinization.

THU085 Can Genetic Mutations And Molecular Markers Predict Recurrence In Adamantinomatous Or Papillary Craniopharyngiomas? A Systemic Review.

Abstract Disclosure: M. Nicholas: None. U. Mahfuza: None. A. De Ieva: None. M. Rodriguez: None. V.A. Preda: None. Craniopharyngiomas are rare, but clinically significant intracranial tumors. According to the WHO criteria, two clinicopathological subtypes, adamantinomatous (aCP) and papillary (pCP) (WHO 2021) are distinct entities[1]. They may have a poor prognosis due to their high recurrence rate and anatomical location. The association between surgical procedures and recurrence is well reported, however, a definitive predictive factor for recurrence based on tumor molecular markers has yet to be elucidated. The genetic mutations BRAF V600E and CTNNB1 are mutually exclusive to aCP and pCP respectively and their presence has been well documented (WHO). The expression of molecular markers ki67, p53, VEGF, PD-L1, pATM, RARγ, RARβ, CD166, CD133, E-Cadherins, FABP5, CRAPBII, Vimentins, CXCL12, CXCR4, MCM6 and DNA Topo II alpha have also all been found in populations of craniopharyngiomas, but the level of their expression and proliferation indices have not previously been clearly summarized in the literature as markers of recurrence. Aim: To perform a systematic review of the common genetic and molecular markers found in either adamantinomatous or papillary craniopharyngiomas and assessment of their impact on recurrence rate. Methods: MEDLINE, Embase, PubMed, Cochrane Library and Scopus electronic were searched in December 2022 using a search string with key words “craniopharyngioma”, “adamantinomatous”, “papillary”, “genetic mutation”, “molecular markers” and “recurrence”. The initial search resulted in 1105 studies. These studies were assessed against inclusion and exclusion criteria. Results: The final review included 26 studies that included 905 patients. Ki67, p53, PD-L1, CXCL12, CXCR4, RARγ FABP5, CRABPII, MCM6 and DNA Topo II alpha molecular markers and CTNNB1 and PTTG-1 gene mutations were found to have an association with an increase in recurrence of adamantinomatous craniopharyngiomas, while VEGF, TERTp, pATM, RARβ, CD166, CD133, E-Cadherins and Vimentins were not. Ki67, p53, PD-L1, FABP5, CRABPII, MCM6 and DNA Topo II alpha molecular markers and BRAF and PTTG-1 gene mutations were found to have an association with an increase in recurrence of papillary craniopharyngiomas, while TERTp, pATM were not. The association of other molecular markers and tumour recurrence, despite large scale studies, gave conflicting results. Conclusion: Our systematic review demonstrates that a multitude of molecular markers and genetic mutations are markers of recurrence or worsening clinicopathology. The identification of genetic mutations and expression of molecular markers and their role in the likelihood of recurrence in either adamantinomatous or papillary craniopharyngiomas provides insight into the potential for novel targets in future therapies.

Effects of Vitamin A on Immune Responses and Vitamin A Metabolism in Broiler Chickens Challenged with Necrotic Enteritis.

Necrotic enteritis (NE) is an important enteric inflammatory disease of poultry, and the effects of vitamin A (VitA) on NE birds are largely unknown. The present study was conducted to investigate the effects of VitA on the immune responses and VitA metabolism of NE broilers as well as the underlying mechanisms. Using a 2 × 2 factorial arrangement, 336 1-day-old Ross 308 broiler chicks were randomly assigned to 4 groups with 7 replicates. Broilers in the control (Ctrl) group were fed a basal diet without extra VitA supplementation. Broilers in the VitA group were fed a basal diet supplemented with 12,000 IU/kg of VitA. Birds in NE and VitA + NE groups were fed corresponding diets and, in addition, co-infected with Eimeria spp. and Clostridium perfringens on days 14 to 20. Samples of the blood, jejunum, spleen and liver were obtained on day 28 for analysis, and meanwhile, lesion scores were also recorded. The results showed that NE challenge increased lesion score in the jejunum and decreased serum glucose, total glyceride, calcium, phosphorus and uric acid levels (p < 0.05). VitA supplementation reduced the levels of serum phosphorus, uric acid and alkaline phosphatase in NE-challenged birds and increased serum low-density lipoprotein content and the activity of aspartate aminotransferase and creatine kinase (p < 0.05). Compared with the Ctrl group, the VitA and NE groups had higher mRNA expression of interferon-γ in the jejunum (p < 0.05). NE challenge up-regulated mRNA expression of interleukin (IL)-13, transforming growth factor-β4, aldehyde dehydrogenase (RALDH)-2 and RALDH-3 in the jejunum, while VitA supplementation increased jejunal IL-13 mRNA expression and hepatic VitA content, but down-regulated splenic IL-13 mRNA expression (p < 0.05). The VitA + NE group had higher serum prostaglandin E2 levels and the Ctrl group had higher splenic RALDH-3 mRNA expression than that of the other three groups (p < 0.05). NE challenge up-regulated jejunal retinoic acid receptor (RAR)-β and retinoid X receptor (RXR)-α as well as splenic RAR-α and RAR-β mRNA expression (p < 0.05). VitA supplementation up-regulated jejunal RAR-β expression but down-regulated mRNA expression of RXR-α, RXR-γ, signal transducers and activators of transcription (STAT) 5 and STAT6 in the spleen (p < 0.05). Moreover, compared with the Ctrl group, the VitA and NE groups had down-regulated mRNA expression of jejunal and splenic Janus kinase (JAK) 1 (p < 0.05). In conclusion, NE challenge induced jejunal injury and expression of Th2 and Treg cell-related cytokines and enhanced RALDH and RAR/RXR mRNA expression, mainly in the jejunum of broilers. VitA supplementation did not alleviate jejunal injury or Th2 cell-related cytokine expression; however, it improved hepatic VitA deposition and inhibited the expression of RALDH-3, RXR and the JAK/STAT signaling pathway in the spleen of broilers. In short, the present study suggested the modulatory effects of vitamin A on the immune responses and vitamin A metabolism in broiler chickens challenged with necrotic enteritis.

Stem Cells as Target for Prostate cancer Therapy: Opportunities and Challenges.

Cancer stem cells (CSCs) and cells in a cancer stem cell-like (CSCL) state have proven to be responsible for tumor initiation, growth, and relapse in Prostate Cancer (PCa) and other cancers; therefore, new strategies are being developed to target such cellular populations. TLR3 activation-based immunotherapy using Polyinosinic:Polycytidylic acid (PIC) has been proposed to be used as a concomitant strategy to first-line treatment. This strategy is based on the induction of apoptosis and an inflammatory response in tumor cells. In combination with retinoids like 9cRA, this treatment can induce CSCs differentiation and apoptosis. A limitation in the use of this combination is the common decreased expression of TLR3 and its main positive regulator p53. observed in many patients suffering of different cancer types such as PCa. Importantly, human exposure to certain toxicants, such as iAs, not only has proven to enrich CSCs population in an in vitro model of human epithelial prostate cells, but additionally, it can also lead to a decreased p53, TLR3 and RA receptor (RARβ), expression/activation and thus hinder this treatment efficacy. Therefore, here we point out the relevance of evaluating the TLR3 and P53 status in PCa patients before starting an immunotherapy based on the use of PIC +9cRA to determine whether they will be responsive to treatment. Additionally, the use of strategies to overcome the lower TLR3, RARβ or p53 expression in PCa patients, like the inclusion of drugs that increase p53 expression, is encouraged, to potentiate the use of PIC+RA based immunotherapy in these patients.

Reduced Retinoic Acid Receptor Beta (Rarβ) Affects Pancreatic β-Cell Physiology.

Simple SummaryVitamin A is one of the important micronutrients involved in various biological functions. Numerous studies have suggested a link between vitamin A (retinoic acid) and type 2 diabetes (T2D). However, the functional role of vitamin A receptors (RARα, β, and γ) in β-pancreatic cells is not clear yet. In this study, we performed bioinformatics and functional experiments in human islets and INS-1 cells in order to evaluate the potential role of RARβ on insulin secretion and pancreatic β-cell function.Various studies have suggested a link between vitamin A (VA), all-trans-retinol, and type 2 diabetes (T2D). However, the functional role/expression of vitamin A receptors (Rarα, β, and γ) in pancreatic β-cells is not clear yet. Accordingly, we performed a series of bioinformatics, molecular and functional experiments in human islet and INS-1 cells to evaluate the role of Rarβ on insulin secretion and pancreatic β-cell function. Microarray and RNA-sequencing (RAN-seq) expression analysis showed that RARα, β, and γ are expressed in human pancreatic islets. RNA-seq expression of RARβ in diabetic/hyperglycemic human islets (HbA1c ≥ 6.3%) revealed a significant reduction (p = 0.004) compared to nondiabetic/normoglycemic cells (HbA1c < 6%). The expression of RARβ with INS and PDX1 showed inverse association, while positive correlations were observed with INSR and HbA1c levels. Exploration of the T2D knowledge portal (T2DKP) revealed that several genetic variants in RARβ are associated with BMI. The most associated variant is rs6804842 (p = 1.2 × 10−25). Silencing of Rarβ in INS-1 cells impaired insulin secretion without affecting cell viability or apoptosis. Interestingly, reactive oxygen species (ROS) production levels were elevated and glucose uptake was reduced in Rarβ-silenced cells. mRNA expression of Ins1, Pdx1, NeuroD1, Mafa, Snap25, Vamp2, and Gck were significantly (p < 0.05) downregulated in Rarβ-silenced cells. For protein levels, Pro/Insulin, PDX1, GLUT2, GCK, pAKT/AKT, and INSR expression were downregulated considerably (p < 0.05). The expression of NEUROD and VAMP2 were not affected. In conclusion, our results indicate that Rarβ is an important molecule for β-cell function. Hence, our data further support the potential role of VA receptors in the development of T2D.

Abstract 3051: Commensal microbiota, host DNA methylation and gene expression: A pilot study in colorectal adenomas

Abstract Increasing evidence suggests that gut microbiota plays a critical role in colorectal cancer (CRC) development; however, the underlying mechanism is largely unknown. We investigated the relationship of commensal microbiota with host DNA methylation (DNAm) and gene expression in colorectal adenoma, the major precursor of CRC. This study included 72 participants from the Tennessee Colorectal Polyp Study. Microbiome, DNA methylome, and transcriptome data of fresh frozen conventional adenoma samples were generated using 16S rRNA gene sequencing, HumanMethylation450 BeadChip, and RNA-seq, respectively. Among 35 participants with microbiome and matched DNA methylome data, microbial features were evaluated for their associations with DNAm at CpG sites (CpGs) via linear regression. False-discovery rate (FDR) correction was conducted separately for each microbial feature and significant associations were identified at FDR&amp;lt;0.1. DNAm at microbiome-associated CpGs were then tested for their correlations with expression of flanking genes (500Kb) among 45 subjects with DNA methylome and matched transcriptome data. For genes with expression significantly (FDR&amp;lt;0.1) correlated with DNAm at microbiome-associated CpGs, their differential expression between colorectal adenomas and adenocarcinomas were examined using microarray data from Gene Expression Omnibus (GEO, 363 conventional adenoma samples) and CRC Subtyping Consortium (CRCSC, 2,760 adenocarcinoma samples). The R package “limma” was used to identify significantly differentially expressed genes at FDR&amp;lt;0.1. Four alpha diversity indexes, three beta diversity matrices, and 194 taxa were investigated for their associations with DNAm at 28,081 variable CpGs (variance&amp;gt;0.02 across samples). Faith’s phylogenetic diversity index and abundance of 11 taxa were significantly associated with DNAm at 68 CpGs. Among them, the most significant association was observed between Lactococcus and cg03292388 (β=-0.67, P=4.39×10-9). DNAm at 11 of the 68 CpGs were significantly correlated with expression of 12 genes (551 genes tested). Six of these 12 genes were found in data from GEO and CRCSC, and three showed a significant differential expression between adenomas and adenocarcinomas. Integrating these results revealed potential bacteria-DNAm-gene-adenocarcinoma pathways in which Bacteroides ovatus, three CpGs, and three genes were involved. Specifically, increased abundance of B. ovatus was associated with decreased DNAm at cg19003815 (β=-0.28, P=7.64×10-5), which was correlated with increased expression of RARB (rho=-0.65, P=2.18×10-6). These findings are in line with the higher expression of RARB in adenocarcinomas compared to adenomas (fold-change=1.51, P=8.58×10-10). In conventional adenomas, bacterial-related host DNAm changes may affect expression of nearby genes, which might be implicated in adenoma-carcinoma development. Citation Format: Yaohua Yang, Jirong Long, Martha J. Shrubsole, Qiuyin Cai, Zhiguo Zhao, Fei Ye, Zhigang Li, Xingyi Guo, Bingshan Li, Seth R. Bordenstein, Ken S. Lau, Harvey J. Murff, Reid M. Ness, Robert J. Coffey, Wei Zheng. Commensal microbiota, host DNA methylation and gene expression: A pilot study in colorectal adenomas [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 3051.

Aberrant epigenetic regulation of RARβ by TET2 is involved in cutaneous squamous cell carcinoma resistance to retinoic acid

With the growing incidence of cutaneous squamous cell carcinoma (CSCC), the treatment-resistant invasive CSCC should be taken seriously. Retinoic acid receptor β (RARβ) functions as a tumor suppressor gene and is associated with the proliferation inhibition to retinoic acid. Demethylase TET2 directed epigenetic landscape contributes to cell malignant transform and is involved in therapeutic resistance in tumors. Whether aberrant TET2 participated in the deficient RARβ remains largely unknown. Hereby, we identified the aberrant-TET2 directed epigenetic landscape contribute to the deficient RARβ in CSCC. The immunohistochemistry was used to detect the expression of RARβ and TET2. The bisulfite sequencing PCR was used to detect the RARβ promoter methylation. Plasmid transfection was used to upregulate TET2 in CSCC cells. Stable overxpressed TET2 cells were used to detect the effect of TET2 on RARβ and drug sensitivity in the CCSC. We observed RARβ decreased with promoter hypermethylation in CSCC and aberrant TET2 associated with deficient RARβ. We upregulated TET2 could reverse promoter hypermethylation and showed a significantly increased expression of RARβ, which enhanced the sensitivity of tumor cells to retinoic acid treatment. Aberrant TET2 leaded to the hypermethylation of RARβ promoter, which contributed to the deficient RARβ in CSCC. While reversing the hypermethylation of the RARβ promoter by recovering the TET2 could enhance tumor cells to be sensitive to retinoic acid.

The Sin3A/MAD1 Complex, through Its PAH2 Domain, Acts as a Second Repressor of Retinoic Acid Receptor Beta Expression in Breast Cancer Cells

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARβ is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARβ has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARβ expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARβ induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARβ from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARβ expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARβ promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARβ activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARβ expression. These data suggest that SIN3A/MAD1 acts as a second RARβ repressor and may be involved in fine-tuning retinoid sensitivity.

Aluminum inhibits non-amyloid pathways via retinoic acid receptor

BackgroundAluminium neurotoxicity has been widely confirmed and mainly manifests as cognitive impairment. Al3+ can inhibit the expression of ADAM10, a key enzyme of the nonamyloid pathway, but its mechanism of toxicity has not been fully elucidated. Studies have shown that RARs can regulate ADAM10 expression. MethodsWe explored whether Al3+ affects the expression of ADAM10 through RARs, thereby affecting the nonamyloid pathway. ResultsAl3+ reduced the expressions of RARα, RARβ and ADAM10. The expression levels of the RARα, RARβ and ADAM10 proteins were upregulated in the RA group compared with the control group. In the RA + 200 μmol Al(mal)3 group, the downregulation of RARα, RARβ and ADAM10 was weaker than that of the 200 μmol Al(mal)3 group, which indicated that RA participated in and upregulated the expression of ADAM10 through RARα and RARβ. ConclusionAl3+ inhibits ADAM10 expression through RARα and RARβ and results in a decrease in the nonamyloid pathway.

RARβ Expression in Keratinocytes from Potentially Malignant Oral Lesions: The Functional Consequences of Re-Expression by De-Methylating Agents.

Simple SummaryPatients may develop white or red patches of the lining of the mouth with an increased risk of developing oral cancer. Treatment with Vitamin A derivatives (retinoids) results in some improvement in these lesions, but this is not maintained, and there are side effects. We know that the cells of the mouth lose cellular receptors for retinoids as these lesions develop, initially by a reversible alteration to the DNA (DNA methylation). Drugs, such as 5-AZA-CdR, which reduce DNA methylation, may restore sensitivity to the effects of retinoids. Treatment of a panel of cells from mouth precancer white patches with retinoids, 5-AZA-CdR and a combination results in varied responses: some cells re-sensitise to retinoids, whereas in others, the main effects on cell division rate and cell lifespan seem related to the effects of 5-AZA-CdR alone. These findings help us to understand the varied responses to retinoids in the clinical setting.Loss of RARβ2 expression by promoter methylation is an early event in oral carcinogenesis. Understanding the mechanisms and consequences of RARβ loss may aid in understanding the disappointing results of retinoid chemoprevention trials. This study aimed to describe the effects of all-trans retinoic acid (ATRA) and the de-methylating agent 5-Aza-2′ deoxycytidine (5-AZA-CdR) on a panel of immortal potentially malignant oral lesion (PMOL) cell cultures. RARβ expression was assessed in PMOL tissues by immunohistochemistry. Cells were treated with ATRA ± 5-AZA-CdR, and the effects on the cell cycle and senescence were assessed. In PMOL tissues, RARβ expression was variable, but lower in biopsies which gave rise to immortal cell cultures. Treatment of iPMOL cells with ATRA resulted in little change in RARβ expression, but the addition of 5-AZA-CdR resulted in significant increases. The effects on the cell cycle and senescence were variable and may be related to 5-AZA-CdR, as this has wider effects on the cell cycle. Overall, the response of iPMOL cells to ATRA and 5-AZA-CdR treatment was variable and is dependent on several factors, including RARβ-promoter methylation. These findings may help to explain the lack of consistent effect of retinoids in PMOLs seen in chemoprevention trials.

Human prostate epithelial cells and prostate-derived stem cells malignantly transformed in vitro with sodium arsenite show impaired Toll like receptor -3 (TLR3)-associated anti-tumor pathway

A therapeutic strategy for prostate cancer (PCa) involves the use of 9-cis-retinoic acid (9cRA) to induce cancer stem cells (CSCs) differentiation and apoptosis. Polyinosinic:polycytidylic acid (PIC) is a Toll-like receptor 3 (TLR3) agonist that induces tumor cells apoptosis after activation. PIC+9cRA combination activates retinoic acid receptor β (RARβ) re-expression, leading to CSC differentiation and growth arrest. Since inorganic arsenic (iAs) targets prostatic stem cells (SCs), we hypothesized that arsenic-transformed SCs (As-CSCs) show an impaired TLR3-associated anti-tumor pathway and, therefore, are unresponsive to PIC activation. We evaluated TLR3-mediated activation of anti-tumor pathway based in RARβ expression, on As-CSC and iAs-transformed epithelial cells (CAsE-PE). As-CSCs and CAsE-PE showed lower TLR3 and RARβ basal expression compared to their respective isogenic controls WPE-Stem and RWPE-1. Also, iAs transformants showed reduced expression of mediators in TLR3 pathway. Importantly, As-CSCs were irresponsive to PIC+9cRA in terms of increased RARβ and decreased SC-markers expression, while CAsE-PE, a heterogeneous cell line having a small SC population, were partially responsive. These observations indicate that iAs can impair TLR3 expression and anti-tumor pathway activated by PIC+9cRA in SCs and prostatic epithelial cells. These findings suggest that TLR3-activation based therapy may be an ineffective therapeutic alternative for iAs-associated PCa.

Normalization of Enzyme Expression and Activity Regulating Vitamin A Metabolism Increases RAR-Beta Expression and Reduces Cellular Migration and Proliferation in Diseases Caused by Tuberous Sclerosis Gene Mutations.

BackgroundMutation in a tuberous sclerosis gene (TSC1 or 2) leads to continuous activation of the mammalian target of rapamycin (mTOR). mTOR activation alters cellular including vitamin A metabolism and retinoic acid receptor beta (RARβ) expression. The goal of the present study was to investigate the molecular connection between vitamin A metabolism and TSC mutation. We also aimed to investigate the effect of the FDA approved drug rapamycin and the vitamin A metabolite retinoic acid (RA) in cell lines with TSC mutation.MethodsExpression and activity of vitamin A associated metabolic enzymes and RARβ were assessed in human kidney angiomyolipoma derived cell lines, primary lymphangioleiomyomatosis (LAM) tissue derived LAM cell lines. RARβ protein levels were also tested in primary LAM lung tissue sections. TaqMan arrays, enzyme activities, qRT-PCRs, immunohistochemistry, immunofluorescent staining, and western blotting were performed and analysed. The functional effects of retinoic acid (RA) and rapamycin were tested in a scratch and a BrDU assay to assess cell migration and proliferation.ResultsMetabolic enzyme arrays revealed a general deregulation of many enzymes involved in vitamin A metabolism including aldehyde dehydrogenases (ALDHs), alcohol dehydrogenases (ADHs) and Cytochrome P450 2E1 (CYP2E1). Furthermore, RARβ downregulation was a characteristic feature of all TSC-deficient cell lines and primary tissues. Combination of the two FDA approved drugs -RA for acute myeloid leukaemia and rapamycin for TSC mutation- normalised ALDH and ADH expression and activity, restored RARβ expression and reduced cellular proliferation and migration.ConclusionDeregulation of vitamin A metabolizing enzymes is a feature of TSC mutation. RA can normalize RARβ levels and limit cell migration but does not have a significant effect on proliferation. Based on our data, translational studies could confirm whether combination of RA with reduced dosage of rapamycin would have more beneficial effects to higher dosage of rapamycin monotherapy meanwhile reducing adverse effects of rapamycin for patients with TSC mutation.

Quantifying the Protein Levels of All Nuclear Hormone Receptors by Mass Spectrometry

Nuclear Receptors (NRs) are a family of ligand-activated transcription factors that control the expression of genes involved in a wide range of physiological processes. An atlas detailing the expression of all NRs at the mRNA level was completed in 2006 using quantitative PCR [Bookout et al. Cell 2006]. The comparative measurement of NRs at the protein level, however, has been hindered by the poor quality of commercially available antibodies, as well as the absence of a high throughput method for quantitation. To address this need, we are developing a mass spectrometry-based targeted proteomic assay to quantify the absolute amounts of NR protein in a panel of mouse tissues. NRs were overexpressed in HEK293 cells by transient transfection and protein was isolated. The cell lysates were digested with a combination of trypsin and Lys-C following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The peptides were desalted using an in-house made C18 tip, separated on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF in Top20 data-dependent acquisition mode. Protein identifications were made using MaxQuant software, and the identifications were mined for members of the NR family. The NR peptides detected were searched against an in silico generated list of optimal NR peptides (filtered for uniqueness, length, absence of post translational modifications, and conservation between human and mouse). The matching peptides were validated by parallel reaction monitoring (PRM) and purchased as synthetic isotopes with a heavy terminal arginine or lysine. Peptide linearity, and lower limits of detection (LLOD) were estimated by spiking digests from a C57Bl/6 mouse liver lysate with increasing amounts of the labeled peptides and analyzing by PRM. Peptides that displayed non-linear behavior were excluded for quantitation. The LLOD were between 100 amol and 1.5 fmol on column. A test panel of tissues (cerebrum, hippocampus, cerebellum, liver, spleen, brown/white adipose, and kidney) showed that we could detect endogenous expression of NRs. To date, we have purchased and validated peptides for 44 of the 49 receptors. We used this assay to quantify the changes in NR protein expression in mouse livers in response to 16 hours of fasting. We found significant changes in the nuclear expression of CAR (3.1-fold increase), RXRβ (1.8-fold increase), SHP (3.9-fold decrease) and RARβ (2.0-fold decrease) in the fasted vs. fed state. Increased CAR activity with fasting was further supported by label-free quantitative proteomics on the same lysates which revealed 210 differentially expressed proteins (2-fold change, p<0.05), with 61 (29%) identified as known CAR target genes. Once complete, this assay will provide researchers with a robust quantitative tool to investigate changes in NR protein expression that will be widely applicable to endocrine research.

Protective role of All Trans Retinoic Acid on B16F10 melanoma cell line metastasis in C57BL/6 mice by enhancing RAR- β protein and homeostasis maintenance

ABSTRACT Lung cancer is the leading cancer according to the World Health Organization (WHO), resulting in highest death rate worldwide due to the high level of metastasis. Hence, the drugs that protect from metastasis either as an adjuvant or a primary therapeutic agent may help to reduce the death rate. In this study, All Trans Retinoic Acid (ATRA) was tested for its action against metastatic lodging of B16F10 melanoma cells in the lung and liver of the C57BL/6 mouse model. Serum, lung and liver were evaluated biochemically for the cancer associated changes. Metastatic cancer development was confirmed by tumor nodule formation and histopathological analysis. RAR-β protein expression was analyzed by immunohistochemistry and histopathology. ATRA treated mice showed a percentage of inhibition on metastatic tumor growth in lung and liver and a corresponding protection against pathological changes in these organs. Cholesterol and γ-Glutamyl Transferase (GGT) levels found in cancer induced mice were reduced in the ATRA treated group. As compared to the normal group, lung tissue from cell line induced cancer control group had less RAR-β protein expression while the ATRA treated group showed enhanced RAR-β protein expression. This indicates that the anti-metastasis effects of ATRA might have shown the induction of RAR-β expression and subsequent molecular signaling pathways to regulate the homeostasis of biochemical changes. This study demonstrated the capability of ATRA to prevent the establishment of metastasis by the melanoma cell line into the lung and liver of experimental mice.

L-carnitine exerts a nutrigenomic effect via direct modulation of nuclear receptor signaling in adipocytes, hepatocytes and SKMC, demonstrating its nutritional impact

L-carnitine is an indispensable metabolite facilitating the transport of fatty acids into the mitochondrial matrix and has been previously postulated to exert a nutrigenomic effect. However, the underlying molecular mechanisms remain mostly unclear. We hypothesized that L-carnitine interacts with nuclear receptors involved in metabolic regulation, thereby modulating downstream targets of cellular metabolism. Therefore, we investigated the effect of L-carnitine supplementation on protein activity, mRNA expression, and binding affinities of nuclear receptors as well as mRNA expression of downstream targets in skeletal muscle cells, hepatocytes, and differentiated adipocytes. L-carnitine supplementation to hepatocytes increased the protein activity of multiple nuclear receptors (RAR, RXR, VDR, PPAR, HNF4, ER, LXR). Diverging effects on the mRNA expression of PPAR-α, PPAR-δ, PPAR-γ, RAR-β, LXR-α, and RXR-α were observed in adipocytes, hepatocytes, and skeletal muscle cells. mRNA levels of PPAR-α, a key regulator of lipolysis and β-oxidation, were significantly upregulated, emphasizing a role of L-carnitine as a promoter of lipid catabolism. L-carnitine administration to hepatocytes modulated the transcription of key nuclear receptor target genes, including ALDH1A1, a promoter of adipogenesis, and OGT, a contributor to insulin resistance. Electrophoretic mobility shift assays proved L-carnitine to increase binding affinities of nuclear receptors to their promoter target sequences, suggesting a molecular mechanism for the observed transcriptional modulation. Overall, these findings indicate that L-carnitine modulates the activity and expression of nuclear receptors, thereby promoting lipolytic gene expression and decreasing transcription of target genes linked to adipogenesis and insulin resistance.

A tumour suppressive relationship between mineralocorticoid and retinoic acid receptors activates a transcriptional program consistent with a reverse Warburg effect in breast cancer

BackgroundThe role of nuclear receptors in both the aetiology and treatment of breast cancer is exemplified by the use of the oestrogen receptor (ER) as a prognostic marker and treatment target. Treatments targeting the oestrogen signalling pathway are initially highly effective for most patients. However, for the breast cancers that fail to respond, or become resistant, to current endocrine treatments, the long-term outlook is poor. ER is a member of the nuclear receptor superfamily, comprising 48 members in the human, many of which are expressed in the breast and could be used as alternative targets in cases where current treatments are ineffective.MethodsWe used sparse canonical correlation analysis to interrogate potential novel nuclear receptor expression relationships in normal breast and breast cancer. These were further explored using whole transcriptome profiling in breast cancer cells after combinations of ligand treatments.ResultsUsing this approach, we discovered a tumour suppressive relationship between the mineralocorticoid receptor (MR) and retinoic acid receptors (RAR), in particular RARβ. Expression profiling of MR expressing breast cancer cells revealed that mineralocorticoid and retinoid co-treatment activated an expression program consistent with a reverse Warburg effect and growth inhibition, which was not observed with either ligand alone. Moreover, high expression of both MR and RARB was associated with improved breast cancer-specific survival.ConclusionOur study reveals a previously unknown relationship between MR and RAR in the breast, which is dependent on menopausal state and altered in malignancy. This finding identifies potential new targets for the treatment of breast cancers that are refractory to existing therapeutic options.