RARβ Gene Research Articles

Retinoic acid receptor alpha expression in endometrioid heterotopias and the endometrium of patients with endometriosis

BACKGROUND: Vitamin A is a fat-soluble compound, which is a biologically inactive food form and undergoes transformation by cytosolic alcohol dehydrogenases and microsomal retinol dehydrogenases into retinaldehyde, followed by oxidation by retinaldehyde dehydrogenases 1, 2 and 3 into retinoic acid. All-trans-retinoic acid has been identified as an active metabolite of retinoic acid, indirectly participating in various processes: differentiation, proliferation and apoptosis. Based on a number of studies, the role of retinoic acid and its receptors in various diseases has been proven. For example, the destruction of the RAR-β gene by the integration of the hepatitis B virus into its loci can be a factor in the development of human hepatocellular carcinoma. Subsequently, it was described that the characteristic point of interruption of translocation t(15:17), observed in acute promyelocytic leukemia, was located at the locus encoding retinoic acid receptor-α. As a result, in patients with acute promyelocytic leukemia, complete remission of the disease could often be achieved by treatment with high doses of all-trans-retinoic acid. Existing data on the ability of retinol to influence the processes of differentiation, proliferation, and apoptosis indicate that retinol may be involved in the pathogenesis of endometriosis, but the mechanisms of its effect on the disease are subject to further study. AIM: The aim of this study was to evaluate the retinoic acid receptor alpha expression in endometrioid heterotopias and the endometrium of patients with different endometriosis stages according to the Revised American Society for Reproductive Medicine classification. MATERIALS AND METHODS: This was a prospective study to determine the retinoic acid receptor alpha expression in the endometrium and endometrioid heterotopias of patients with endometriosis (n = 28) and of the control group (n = 10). RESULTS: The retinoic acid receptor alpha expression in endometrioid heterotopias was lower compared to the eutopic endometrium of patients with endometriosis and the endometrium of patients in the control group by 3.4 and two times, respectively (p 0.001; the Kruskal – Wallis method). CONCLUSIONS: The data obtained indicate the involvement of retinol in the pathogenesis of endometriosis. Despite the relative increase in retinol level in the peripheral blood and peritoneal fluid in patients with endometriosis compared to the control group, shown in our previous studies, the reduced retinoic acid receptor alpha expression in endometrioid heterotopias does not allow for full binding of vitamin A to the receptor and is accompanied by its improper metabolism.

A multi-ancestry genome-wide association study identifies novel candidate loci near the RARB gene associated with hypertensive disorders of pregnancy in the personalized environment and genes study

A multi-ancestry genome-wide association study identifies novel candidate loci near the RARB gene associated with hypertensive disorders of pregnancy in the personalized environment and genes study

Combination of DNA methylation biomarkers with multiparametric magnetic resonance and ultrasound imaging fusion biopsy to detect the local spread of prostate cancer.

This study aimed to investigate the extent of field cancerization adjacent to index lesions in prostate cancer (PCa) by measuring DNA methylation of selected tumor suppressor genes in the perifocal tissue of PCa not visible on multiparametric magnetic resonanse imaging (mpMRI) for the safe zone of focal therapy identification. A total of 272 patients were enrolled in this study, 44 patients' tissue biosamples were included in the field cancerization research, and 272 urine samples were included in the urine-based test development. Targeted biopsies were performed using the mpMRI/ultrasoundimage fusion system. Quantitative analysis revealed significantly higher DNA methylation levels of RARB, RASSF1, GSTP1 &APC genes in the index lesion compared with perifocal tissue samples 10 mm away from it (p < 0.0001). Notably, the RARB, GSTP1 & APC and RARB, RASSF1, GSTP1 & APC biomarker combinations exhibited the highest sensitivity and specificity comparing the extent of DNA methylation in index lesions and noncancerous prostate tissues 20 mm away (both area under the curve [AUC] = 0.98; p < 0.0001). The analysis of the potential urinary biomarkers showed that the combination of all four DNA methylation biomarkers with prostate-specific antigen (PSA) or PSA density (PSAD) in the blood significantly improves the detection of clinically significant PCa (csPCa). The combination of the four-biomarker test with PSAD allowed the identification of csPCa with ≥90% sensitivity and specificity. Thus, this study suggests that for focal therapy by region target hemi-ablation, the safe distance from the index lesion is no less than 10 mm. Noninvasive urine DNA methylation tests in combination with PSAD could be used for further follow-up of the patients, but larger prospective studies with external validation are needed.

Promoter hypermethylation of RARB and GSTP1 genes in plasma cell-free DNA as breast cancer biomarkers in Peruvian women.

Promoter hypermethylation is one of the enabling mechanisms of hallmarks of cancer. Tumor suppressor genes like RARB and GSTP1 have been reported as hypermethylated in breast cancer tumors compared with normal tissues in several populations. This case-control study aimed to determine the association between the promoter methylation ratio (PMR) of RARB and GSTP1 genes (separately and as a group) with breast cancer and its clinical-pathological variables in Peruvian patients, using a liquid biopsy approach. A total of 58 breast cancer patients and 58 healthy controls, matched by age, participated in the study. We exacted cell-free DNA (cfDNA) from blood plasma and converted it by bisulfite salts. Methylight PCR was performed to obtain the PMR value of the studied genes. We determined the association between PMR and breast cancer, in addition to other clinicopathological variables. The sensitivity and specificity of the PMR of these genes were obtained. A significant association was not found between breast cancer and the RARB PMR (OR = 1.90; 95% CI [0.62-6.18]; p = 0.210) or the GSTP1 PMR (OR = 6.57; 95% CI [0.75-307.66]; p = 0.114). The combination of the RARB + GSTP1 PMR was associated with breast cancer (OR = 2.81; 95% CI [1.02-8.22]; p = 0.026), controls under 50 years old (p = 0.048), patients older than 50 (p = 0.007), and postmenopausal (p = 0.034). The PMR of both genes showed a specificity of 86.21% and a sensitivity of 31.03%. Promoter hypermethylation of RARB + GSTP1 genes is associated with breast cancer, older age, and postmenopausal Peruvian patients. The methylated promoter of the RARB + GSTP1 genes needs further validation to be used as a biomarker for liquid biopsy and as a recommendation criterion for additional tests in asymptomatic women younger than 50 years.

Retinoic acid protects against lipopolysaccharide-induced ferroptotic liver injury and iron disorders by regulating Nrf2/HO-1 and RARβ signaling

Acute liver injury (ALI) can progress to severe liver diseases, making its prevention and treatment a focus of research. Retinoic acid (RA) has been shown to have anti-oxidative and iron-regulatory effects on organs. In this study, we investigated the effect of RA on lipopolysaccharide (LPS)-induced ALI in both in vivo and in vitro experiments. We found that RA significantly reduced LPS-induced serum iron and red blood cell-associated disorders, as well as decreased serum ALT and AST levels. RA also reversed the accumulation of non-heme iron and labile iron in LPS-induced mice and hepatocytes by increasing the expression of FTL/H and Fpn. Furthermore, RA inhibited tissue reactive oxygen species (ROS) and malondialdehyde (MDA) production and improved the expression of Nrf2/HO-1/GPX4 in mice and Nrf2 signaling in hepatocytes. In vitro experiments employing RAR agonists and antagonists have revealed that retinoic acid (RA) can effectively inhibit cell ferroptosis induced by lipopolysaccharide (LPS), erastin, and RSL3. The mechanism underlying this inhibition may involve the activation of retinoic acid receptors beta (RARβ) and gamma (RARγ). Knocking down the RARβ gene in Hepatocytes cells significantly diminished the RA's protective effect, indicating that the anti-ferroptotic role of RA was partially mediated by RARβ signaling. Overall, our study demonstrated that RA inhibited ferroptosis-induced liver damage by regulating Nrf2/HO-1/GPX4 and RARβ signaling.

Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor β (RARβ) Gene in Hepatocytes.

HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of the RARβ gene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely, RARβ promoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increased RARβ promoter activity 15-fold. The human RARβ2 promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, including APOC3 and CYP2C9, while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such as CYP26A1 and RARβ, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.

Abstract 4289: Long-read, assembly-based characterization of rearranged cancer karyotypes

Abstract Introduction: Recent pan-cancer whole-genome sequencing studies revealed the rich landscape of structural variants (SV), from simpler indels to complex events involving multiple breakpoints and sequence gain/loss. SVs may contribute to tumorigenesis through direct modification of coding sequence or deregulation from copy number alterations, enhancer hijacking, or topological domain modification. A substantial part of the variation in the human genome is not accessible to short reads due to mapping ambiguities. Recent benchmarking studies reported that the best short-read methods only have 30-70% SV sensitivity. Long-read sequencing (such as PacBio or Oxford Nanopore) can overcome the limitations of short reads, however the current methods were not designed for the analysis of rearranged cancer genomes with complex copy number profiles. Methods: We developed BGA (Breakpoint Graph Assembler), a method that combines the ideas from long-read assembly and breakpoint graph frameworks. BGA detects abnormally mapped reads and builds a breakpoint graph that characterizes the structure of derived cancer karyotypes. Complex events with multiple breakpoints form connectivity clusters and are classified based on the subgraph properties. BGA also takes advantage of phased haplotypes and can incorporate multiple related datasets (such as in tumor-normal comparison or multi-site tumor sampling). BGA is freely available at: https://github.com/KolmogorovLab/BGA. Results: We first analyzed three cancer cell lines and corresponding matching normal DNAs (HCC1954, H2009, and COLO829). In each cell line, we identified 8-56 somatic rearrangement clusters involving more than two breakpoints and at least 1kb of sequence. In H2009, we identified a chromoplexy event involving chr13 and chr1, consistent with previous FISH experiments. COLO829 showed the lowest number of somatic rearrangement clusters (n=8), including translocation and inversion events between chr3, chr10, and chr12 within the RARB, BICC1, and TRHDE genes. Homologous recombination deficient HCC1954 has the highest number of complex events including the chr17q arm which hosts ERBB2, chromoplexy between chr8, chr5, and chromothripsis in chr21. In addition, we analyzed three other HPV-infected cell lines (CaSki, SCC152, SNU1000). In each of them, we observed complex clusters of HPV-HPV and HPV-human breakpoints that formed cycles, suggesting extrachromosomal amplification. The HPV fragments had many interactions with chromosomal DNA in CaSki and SC152, but not in mostly episomal SNU1000 cells. We also observed karyotype-scale changes that did not involve HPV sequences, such as the simultaneous exchange of six chromosome arms of chr2, chr7, and chr17 in CaSki. Citation Format: Ayse Keskus, Tanveer Ahmad, Ataberk Donmez, Yi Xie, Isabel Rodriguez, Rose Milano, Nicole Rossi, Hong Lou, Laksh Malik, Kimberley Billingsley, Cornelis Blauwendraat, Michael Dean, Mikhail Kolmogorov. Long-read, assembly-based characterization of rearranged cancer karyotypes. [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 4289.

Genomic, epigenomic, and transcriptomic signatures of prostate cancer between African American and European American patients.

Prostate cancer is the second most common cancer in men in the United States, and racial disparities are greatly observed in the disease. Specifically, African American (AA) patients have 60% higher incidence and mortality rates, in addition to higher grade and stage prostate tumors, than European American (EA) patients. In order to narrow the gap between clinical outcomes for these two populations, genetic and molecular signatures contributing to this disparity have been characterized. Over the past decade, profiles of prostate tumor samples from different ethnic groups have been developed using molecular and functional assays coupled with next generation sequencing or microarrays. Comparative genome-wide analyses of genomic, epigenomic, and transcriptomic profiles from prostate tumor samples have uncovered potential race-specific mutations, copy number alterations, DNA methylation, and gene expression patterns. In this study, we reviewed over 20 published studies that examined the aforementioned molecular contributions to racial disparities in AA and EA prostate cancer patients. The reviewed genomic studies revealed mutations, deletions, amplifications, duplications, or fusion genes differentially enriched in AA patients relative to EA patients. Commonly reported genomic alterations included mutations or copy number alterations of FOXA1, KMT2D, SPOP, MYC, PTEN, TP53, ZFHX3, and the TMPRSS2-ERG fusion. The reviewed epigenomic studies identified that CpG sites near the promoters of PMEPA1, RARB, SNRPN, and TIMP3 genes were differentially methylated between AA and EA patients. Lastly, the reviewed transcriptomic studies identified genes (e.g. CCL4, CHRM3, CRYBB2, CXCR4, GALR1, GSTM3, SPINK1) and signaling pathways dysregulated between AA and EA patients. The most frequently found dysregulated pathways were involved in immune and inflammatory responses and neuroactive ligand signaling. Overall, we observed that the genomic, epigenomic, and transcriptomic alterations evaluated between AA and EA prostate cancer patients varied between studies, highlighting the impact of using different methods and sample sizes. The reported genomic, epigenomic, and transcriptomic alterations do not only uncover molecular mechanisms of tumorigenesis but also provide researchers and clinicians valuable resources to identify novel biomarkers and treatment modalities to improve the disparity of clinical outcomes between AA and EA patients.

Comparative evaluation of ZMYND-8 and RARβ2 genes promoters’ methylation changes in tumor and tumor margin tissues of patients with lung cancer

BackgroundLung cancer remains one of the most lethal carcinomas worldwide because of its late diagnosis. One of the DNA modifications is methylation, one of the primary alterations of tumor development, consisting of fascinating indicators for cancer diagnosis. This study investigated ZMYND-8 and RARβ2 gene methylation in NSCLC as a new epigenetic tool.MethodsFirst, to find out the potential diagnostic capability of ZMYND-8 and RARβ2 genes methylation, we entirely surfed DNA methylation microarrays from the Cancer Genome Atlas (TCGA) data of NSCLC samples. Additionally, we took advantage of using q-MSP in several pieces comprising NSCLC tumors and neighboring normal tissues; ZMYND-8 and RARβ2 genes methylation grades were acquired.ResultsOur finding displayed significant hypomethylation of ZMYND-8 and hypermethylation of RARβ2 in NSCLC samples compared to neighboring standard specimens, which significantly correlated with the clinical stage of malignancy. In addition, the incredible precision of ZMYND-8 and RARβ2 methylations as reliable cancer diagnosis indicators in NSCLC was confirmed, drawing the ROC curve analysis with an AUC value of 0.751 and 0.8676, respectively, for ZMYND-8 and RARβ2. Additional studies of other dominant cancer entities in TCGA displayed that RARβ2’s higher methylation degree and ZMYND-8 lower methylation degree are prevalent changes in tumor evolution which could be possibly considered as a potential diagnostic biomarkers for lung cancer.ConclusionBased on this study, ZMYND-8 and RARβ2 methylation are reliable biomarkers for lung cancer.

The synergistic effect of retinyl propionate and hydroxypinacolone retinoate on skin aging.

Aging is responsible for the majority of skin and soft tissue remolding in humans. Retinol and its derivatives or retinoids effectively intervene skin aging process. Nevertheless, retinoids usually induce skin intolerance, especially among the Chinese, and thus, their application to prevent skin aging is yet to be well accepted. The study of optimal composition and concentration of retinoids is necessary to offer strong antiaging efficacies with minimum irritations. Therefore, a better understanding of retinol and its derivatives is acutely needed to develop strategies to combat skin aging. In this study, we aimed to determine the optimal ratio of two retinol derivatives-hydroxypinacolone retinoate (HPR) and retinyl propionate (RP) in terms of dermal remodeling and skin aging prevention-and to investigate their synergistic antiaging effects both in vitro and in vivo. An in vitro human foreskin fibroblast (HFF-1) cell model was established to evaluate the cell viability of HPR and/or RP treatment. In addition, the antiaging and retinol receptor genes expressions in HFF-1 cells cotreated with HPR and RP were quantified. The in vivo adverse reaction evaluation of skincare serums containing various levels of retinol or the optimal HPR and RP combination termed Gravi-A was performed by 24 h patch tests in 33 subjects prior to the clinical research. Last but not the least, clinical research with 42 Chinese urban women was conducted to assess the in vivo antiaging efficacy of the skincare serum containing this optimal retinoid combination. The combination of HPR and RP at the weight ratio of 5:9 was shown to achieve the optimal in vitro antiaging performance. Coadministration of 5μg/mL HPR and 9μg/mL RP to HFF-1 cells promoted their proliferation at 24 h and synergistically enhanced the expressions of type IV collagen, CRBP-I, and RARB genes. In addition, the skincare serum containing HPR and RP combination at 5:9 weight ratio demonstrated superior in vivo anti-wrinkle and skin elasticity improvement benefits without any adverse reactions, while retinol in the same concentration exerted much higher adverse effect. Skin wrinkles, skin smoothness, TEWL, skin elasticity R2 and R5 were improved by 8.3%, 11.9%, 25.7%, 14.5%, and 22.6%, respectively, after 8-week use. Our results indicated the advanced antiaging effect of HPR and RP combination both in vitro and in vivo. In addition, little adverse effect was observed in this study, in comparison with retinol. This combination named as Gravi-A is a potential therapeutic strategy to prevent skin aging, especially for Chinese women.

Copy number variations analysis in a cohort of 47 fetuses and newborns with congenital diaphragmatic hernia.

The congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and lung hypoplasia, is a common and severe birth defect that affects around 1 in 4000 live births. However, the etiology of most cases of CDH remains unclear. The aim of this study was to perform a retrospective analysis of copy number variations (CNVs) using a high-resolution array comparative genomic hybridization (array-CGH) in a cohort of fetuses and newborns with CDH. Forty seven fetuses and newborns with either isolated or syndromic CDH were analyzed by oligonucleotide-based array-CGH Agilent 180K technique. A mean of 10.2 CNVs was detected by proband with a total number of 480 CNVs identified based on five categories: benign, likely benign, of uncertain signification, likely pathogenic, and pathogenic. Diagnostic performance was estimated at 19.15% (i.e., likely pathogenic and pathogenic CNVs) for both CDH types. We identified 11 potential candidate genes: COL25A1, DSEL, EYA1, FLNA, MECOM, NRXN1, RARB, SPATA13, TJP2, XIRP2, and ZFPM2. We suggest that COL25A1, DSEL, EYA1, FLNA, MECOM, NRXN1, RARB, SPATA13, TJP2, XIRP2, and ZFPM2 genes may be related to CDH occurrence. Thus, this study provides a possibility for new methods of a positive diagnosis.

Size and Methylation Index of Cell-Free and Cell-Surface-Bound DNA in Blood of Breast Cancer Patients in the Contest of Liquid Biopsy.

Aberrantly methylated circulating DNA (cirDNA) has proven to be a good cancer marker, but its detection is limited by low concentrations, fragmentation, and insufficiency. Since the methylated cirDNA was shown to be more stable in circulation than the unmethylated one and was shown to bind with the blood cell surface, we studied the concentration, representation, and fragmentation of tumor-derived methylated DNA in cell-free and cell-surface-associated DNA. We found that long DNA fragments (more than 10 kb) are mainly associated with the surface of blood cells. However, in plasma short DNA fragments (100–1000 bp) were also found along with long DNA fragments. Isolation of short fragments after separation of cirDNA in 6% PAGE followed by quantitative PCR (L1 element) has shown that short DNA fragments in healthy females represent 22% versus 0.5–4.4% in breast cancer patients. The methylated form of the RARβ2 gene was detected only in long DNA fragments by Real-time TaqMan PCR of bisulfite-converted DNA. The methylation index of cirDNA from healthy women was estimated at 0%, 9%, and 7% in plasma, PBS-EDTA, and trypsin eluates from the surface of blood cells, respectively. The methylation index of breast cancer patients’ DNA was found to be 33%, 15%, and 61% in the same fractions confirming the overrepresentation of methylated DNA in csbDNA.

Combining phenotypic profiling and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: Retinoic acid as an example

The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.

GLAD-PCR assay of DNA methylation sites in regulatory regions of some tumor-suppressor genes in breast cancer

Hypermethylation of the RcgY sites is shown for many cancer diseases. such aberrant methylation, suppressing the gene activity, occurs at early stages of carcinogenesis. Recently, using glad-pcR assay, we have detected aberrantly methylated RcgY sites, which can be considered to be epigenetic markers of colorectal, lung, and gastric cancers. in breast cancer, methylation of the regulatory regions of ALX4, BMP2, CCND2, CDH13, CDX1, FOXA1, GALR1, GATA5, GREM1, HIC1, HMX2, HS3ST2, HOXC10, ICAM5, LAMA1, RARB, RASSF1A, RUNX3, RXRG, RYR2, SFRP2, SOX17, TERT, and ZNF613 tumor-suppressor genes is reported. in the present work, we determined aberrantly methylated RcgY sites in the regulatory regions of these genes in dNa preparations from breast cancer tissues. the study of dNa samples from 30 tumor and 22 normal mammary tissue samples demonstrates a high diagnostic potential of selected R(5mc)gY sites in regulatory regions of CCND2, BMP2, GALR1, SOX17, HMX2, and HS3ST2 genes with total index of sensitivity and specificity for R(5mc)gY detection in tumor dNa 90.0 % and 100.0 %, respectively.

DNA hypermethylation of CADM1, PAX5, WT1, RARβ, and PAX6 genes in oropharyngeal cancer associated with human papillomavirus

ABSTRACT Recently, an increasing incidence of HPV-induced oropharyngeal squamous cell carcinoma (OPSCC) has been observed. Moreover, locoregionally advanced stages require a combined modal approach, and the prognosis is poor. Therefore, it is essential to find early diagnostic and prognostic biomarkers. DNA methylation changes play a crucial role in the process of carcinogenesis and are often investigated as promising biomarkers in many types of cancer. For analysis of DNA methylation levels of selected tumour suppressor genes in HPV-positive and HPV-negative samples (including primary tumours and corresponding metastases of metastasizing OPSCCs, primary tumours of non-metastasizing OPSCCs, and control samples), methylation-specific MLPA and methylation-specific high-resolution melting analyses were used. A significant difference in methylation between OPSCCs and the control group was observed in WT1, PAX6 (P < 0.01) and CADM1, RARβ (P < 0.05) genes. CADM1 and WT1 hypermethylation was detected mostly in HPV-positive samples; all but one HPV-negative samples were unmethylated. Moreover, hypermethylation of PAX5 gene was observed in metastases compared with control samples and was also associated with shorter overall survival of all patients (P < 0.05). Associations described herein between promoter methylation of selected genes and clinicopathological data could benefit OPSCC patients in the future by improvement in screening, early detection, and prognosis of the disease.

Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.

Aberrant promoter hypermethylation of RAR-β in endometrial carcinoma- an Indian study.

Endometrial cancer is the seventh most common cancer in women worldwide with an age-standardized rate of 8.4 per 100,000 women. Epigenetic alterations such as promoterhypermethylation of TSGs are known to be early events in carcinogenesis. The aim of the presentstudy, we assessed the aberrant promoter hypermethylation pattern of RAR-β in 78 endometrialcancer samples. Methods: DNA was isolated from endometrial carcinoma samples and normaltissues and aberrant promoter hypermethylation was assessed using nested and methylation-specificPCR. The Chi-square test was used for statistical analysis and a p-value&lt;0.05 was considered to bestatistically significant. Results: 40 of the 78 (51.28%) endometrial carcinoma samples showedaberrant hypermethylation of the RAR-β gene. Methylation status in each histological subtype, gradeand stage of the disease was also assessed. Conclusion: Aberrant hypermethylation is an importantearly epigenetic alteration that occurs during tumorigenesis. The Data shown here reports thatpromoter hypermethylation of RAR-β occurs in endometrial carcinoma and therefore could be usedas a potential marker for early diagnosis and prognosis of the disease.

Promoter Hypermethylation Analysis of Host Genes in Cervical Cancer Patients With and Without Human Immunodeficiency Virus in Botswana.

Background: Epidemics of human immunodeficiency virus (HIV) and cervical cancer are interconnected. DNA hypermethylation of host genes' promoter in cervical lesions has also been recognized as a contributor to cervical cancer progression.Methods: For this purpose we analyzed promoter methylation of four tumor suppressor genes (RARB, CADM1, DAPK1 and PAX1) and explored their possible association with cervical cancer in Botswana among women of known HIV status. Overall, 228 cervical specimens (128 cervical cancers and 100 non-cancer subjects) were used. Yates-corrected chi-square test and Fisher's exact test were used to explore the association of promoter methylation for each host gene and cancer status. Subsequently, a logistic regression analysis was performed to find which factors, HIV status, high risk-HPV genotypes, patient's age and promoter methylation, were associated with the following dependent variables: cancer status, cervical cancer stage and promoter methylation rate.Results: In patients with cervical cancer the rate of promoter methylation observed was greater than 64% in all the genes studied. Analysis also showed a higher risk of cervical cancer according to the increased number of methylated promoter genes (OR = 6.20; 95% CI: 3.66–10.51; P < 0.001). RARB methylation showed the strongest association with cervical cancer compared to other genes (OR = 15.25; 95% CI: 6.06–40.0; P < 0.001). Cervical cancer and promoter methylation of RARB and DAPK1 genes were associated with increasing age (OR = 1.12; 95% CI: 1.01-1.26; P = 0.037 and OR = 1.05; 95% CI: 1.00-1.10; P = 0.040). The presence of epigenetic changes at those genes appeared to be independent of HIV status among subjects with cervical cancer. Moreover, we found that cervical cancer stage was influenced by RARB (χ2= 7.32; P = 0.002) and CADM1 (χ2=12.68; P = 0.013) hypermethylation, and HIV status (χ2= 19.93; P = 0.001).Conclusion: This study confirms the association between invasive cervical cancer and promoter gene methylation of tumor suppressing genes at the site of cancer. HIV infection did not show any association to methylation changes in this group of cervical cancer patients from Botswana. Further studies are needed to better understand the role of HIV in methylation of host genes among cancer subjects leading to cervical cancer progression.

Polymethoxyflavones from citrus inhibited gastric cancer cell proliferation through inducing apoptosis by upregulating RARβ, both in vitro and in vivo

In order to discover the active anti-tumor ingredients during the flavonoids separation process of Ougan (Citrus reticulata cv. Suavissima), gastric cancer cell lines including AGS, BGC-823, and SGC-7901 were employed to evaluate the proliferation inhibition abilities of Ougan extracts, flavanone components, polymethoxyflavone components, neohesperidin, nobiletin, tangeretin, and 5-demethylnobiletin. Quantitative real-time PCR was used to detect the expression of three retinoic acid receptor genes, including RARA, RARB, and RARG. Western blot and immunohistochemistry were used to detect protein expressions. The results showed that the polymethoxyflavone components and the PMFs monomers inhibited the proliferation of three gastric cancer cell lines and induced apoptosis. The mechanism exploration found that PMFs up-regulated the expression of the RARB gene selectively and activated the Caspase3, 9, and PARP1 proteins. In addition to 5-demethylnobiletin, other PMFs also upregulated the expression of cleaved Caspase8. The mechanism was preliminarily verified by a RARβ inhibitor AGN 193109. Moreover, a nude mice tumor xenograft model confirmed the tangeretin could exhibit in vivo anti-tumor effect through inducing apoptosis and upregulating RARβ protein. All result suggested that tangeretin may be a potentially novel, safe and effective drugs with less toxicity and lesser side effects for gastric cancer therapeutics.

DNA methylation patterns of RAR-β2 and RASSF1A gene promoters in FNAB samples from Greek population with benign or malignant breast lesions.

Promoter hypermethylation is common in Breast Cancer (BC) with studies mainly in histological specimens showing frequent methylation of tumor suppressor genes (TSGs) compared with normal tissues. The aim of this study was to estimate the frequency of promoter methylation of RAR-β2 and RASSF1A genes in breast FNAB material aiming to evaluate the methylation status of these two genes as biomarker for detecting BC in Greek population. FNAB material from 104 patients was collected for cytological evaluation and epigenetic analysis. DNA was extracted and subjected to bisulfite conversion. A methylation-specific PCR was carried out and the final products were separated with electrophoresis in 2% agarose gels. From 104 samples, RASSF1A hypermethylation was observed in 78 (75%) and RAR-β2 hypermethylation in 64 (61.6%). 84% and 78% of the cases diagnosed with breast malignancy (n = 50) were methylated for RASSF1A and RAR-β2, respectively. Methylated RASSF1A and RAR-β2 were also detected in 88.3% and 76.5% in samples diagnosed as suspicious for malignancy (n = 17) and in 57.2% of samples diagnosed with atypia (n = 14). The Odds Ratio for breast malignancy was 4.545 in patients with RASSF1A hypermethylation and 9.167 in patients with RAR-β2 hypermethylation underlying their promoter's methylation positive correlation with breast malignancy. To optimize the sensitivity and specificity of this epigenetic setting, more TSGs related to BC should be gradually imported in our evaluated methylation panel and be validated in a larger study sample with the aim that the obtained epigenetic profiles will provide clinicians with valuable tools for management of BC patients in Greece.