Across several cancers, IL18 receptor accessory protein (IL18RAP) is abnormally expressed, and this abnormality is related to tumor immunity and heterogeneous clinical outcomes. In this study, based on bioinformatics analysis, we discovered that IL18RAP is related to the human tumor microenvironment and promotes various immune cells infiltration. Additionally, the multiple immunofluorescence staining revealed that with the increased expression of IL18RAP, the number of infiltrated M1 macrophages increased. This finding was confirmed by coculture migration analysis using three human cancer cell lines  with IL18RAP knockdown. We discovered a positive link between IL18RAP and the majority of immunostimulators, immunoinhibitors, major histocompatibility complex (MHC) molecules, chemokines, and chemokine receptor genes using Spearman correlation analysis. Additionally, functional IL18RAP’s gene set enrichment analysis (GSEA) revealed that it is related to a variety of immunological processes, such as positive regulation of interferon gamma production and positive regulation of NK cell-mediated immunity. Moreover, we used single-cell RNA sequencing analysis to detect that IL18RAP was mainly expressed in immune cells, and HALLMARK analysis confirmed that the INF-γ gene set expression was upregulated in CD8Tex cells. In addition, in human and mouse cancer cohorts, we found that the level of IL18RAP can predict the immunotherapy response. In short, our study showed that IL18RAP is a new tumor biomarker and may become a potential immunotherapeutic target in cancer. Today, cancer is a severe threat to social health and a leading cause of death . GloballDespite significant advancements in therapy, patients still do not feel pleased with cancer care due to ineffective treatment outcomes, substantial drug side effects, drug resistance, and high treatment costs. In the past decade, immunotherapy treatment for cancer has made great breakthroughs, among which the progress of immune checkpoint inhibitors (ICIs) is the most remarkable. Since anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA4) was approved for advanced melanoma treatment in 2011, ICIs have rapidly gained approval and been used to treat various cancers, which has led to an unprecedented increase in survival –5. HowevIL18, an IL1-related cytokine that is crucial for both adaptive and innate immunity, is widely known . InitialUsing the GTEx and TCGA databases, we investigated the IL18RAP expression in 33 different human cancers. Additionally, this study emphasized the connection between pancancer-level IL18RAP expression and clinical prognosis, DNA methylation, tumor mutation burden (TMB), TME, microsatellite instability (MSI), and immunotherapy.We learned from the TIMER2.0 database that there were significant differences between the levels of IL18RAP expression in various cancers and the corresponding normal tissues . For furFirst, we used the “pROC” and “ggplot2” R tools to examine the diagnostic value of IL18RAP in a variety of cancers. As shown in Following that, we investigated the relationship between the mRNA expression levels of IL18RAP and overall survival (OS) in 33 different types of human cancers using single variate Cox regression analysis. Uveal melanoma (UVM), UCEC, THYM, SKCM, sarcoma (SARC), LIHC, LGG, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), BRCA, and ACC all demonstrated significant hazard ratios (HRs) for IL18RAP, with UVM having the highest HR (12.258) . AdditioUsing the cBioPortal platform, we investigated the IL18RAP alteration sites, alteration frequency, and alteration type across cancers. As shown in We next investigated the relationship between DNA methylation and IL18RAP expression in human cancers utilizing the GSCALite platform since DNA methylation frequently impacts gene expression and cancer prognosis. The results demonstrated that the IL18RAP expression levels and DNA methylation were negatively correlated in 15 cancers, including UVM, THCA, TGCT, BRCA, PRAD, KICH, cholangiocarcinoma (CHOL), KIRP, PAAD, THYM, LGG, GBM, LUAD, LAML, and DLBC . AdditioTMB and MSI are well-known characteristics of the TME and are considered to be involved in tumor mutation and epigenetic alterations. Immune cells and stromal cells are two significant components of TME, and it is widely recognized that the TME plays a significant role in the cancer occurrence and development. We assessed the ESTIMATE score, tumor purity, immune score, and stromal score across 33 different types of human cancers using the “ESTIMATE” R package. As shown in The TIMER2.0 database was then analyzed in order to further assess the relationship of IL18RAP expression and immune cells across cancers. Using the TIMER algorithm, we were able to determine the correlations between the mRNA expression of IL18RAP and B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells in BRCA, SARC, and SKCM. As shown in Through the TISCH2 database, we identified the cell subtypes in LAML (GSE154109), glioma (GSE131928_10X), HNSC (GSE103322), OV (GSE130000), and PRAD (GSE150692) and described the IL18RAP expression levels in different clusters of cells –7E. IL18in vitro experiments were used for further investigation. Multiple immunofluorescence staining was utilized to identify the IL18RAP expression and the M1 macrophage markers CD68 and iNOS in BRCA, BLCA, GBM, CESC, KIRC, HNSC, LUAD, LIHC, and LUSC. According to the findings, which are shown in in vitro experiments. The human GBM cell Line U251, the BRCA cell line MDA-MB-231 and the LIHC cell Line HepG2 was transfected with si-IL18RAP-1, si-IL18RAP-2 or si-NC. In contrast to the control group and the si-NC group, Western blotting demonstrated that transfection of si-IL18RAP-1 and si-IL18RAP-2 led in a considerable reduction of IL18RAP protein level  network was constructed using Cytoscape . On thisThe GSEA analysis results of IL18RAP in 12 cancers are shown in Through TIDE database, we explored the predictive power of OS and response outcomes response of IL18RAP as a biomarker for the human immunotherapy cohort. As shown in Through the TISMO database, we discovered that IL18RAP exhibits a great ability to predict the immunotherapy response in 4 murine immunotherapy cohorts. Interestingly, these four cohorts all included anti-CTLA4 therapy, and the level of IL-18RAP in the responders increased significantly . This tein vitro studies were performed. As expected, the results showed that MDA-MB-231 cells in the si-IL18RAP group proliferated faster . For data analysis, we used R software (version 4.2.1). The IL18RAP expression differences between cancer and paracancerous tissues were found using the “Wilcox.test” method. Using Sangerbox (http://vip.sangerbox.com/home.html) and Xiantao Academic (https://www.xiantao.love/products/apply), we then displayed the data and produced violin graphs.We collected the RNA sequencing data of human cancers from the GTEx and TCGA datasets, as well as clinical data including tumor-node metastasis stage and survival time, through the UCSC Xena platform . Then, using Kaplan-Meier curves and forest plots, we examined and displayed the effect of IL18RAP expression on OS, DSS, DFS, and PFI. The impact of IL18RAP on patient survival was assessed using the HR.https://www.cbioportal.org/). Additionally, the correlation of IL18RAP expression and the degree of DNA methylation and gene CNV were investigated using GSCA (https://bioinfo.life.hust.edu.cn/web/GSCALite/). In the GSCA website, we have additionally entered 4 genes closely related to IL18RAP function, including IL18, IL18BP, IL1R1, IL1R2 and IL18R1. Finally, the connection between the expression of IL18RAP and the five methyltransferases  was assessed using the Spearman method across 33 types of cancers.We were able to obtain information about IL18RAP’s genetic alterations  through cBioPortal database . After that, we assessed the relationship between the mRNA expression level of IL18RAP and the infiltration of several immune cell types using Spearman correlation analysis. Additionally, it was examined whether the expression of IL18RAP correlated with MSI or TMB. Finally, we investigated the relationship between the mRNA expression level of IL18RAP and immunostimulators, tumor-infiltrating lymphocytes, MHC molecules, immunoinhibitors, chemokines, and chemokine receptors in the 33 different types of human cancers using the “GSVA” R package.First, the connection between IL18RAP expression and the ESTIMATE score, tumor purity, immunological score, and stromal score across 33 different types of human cancers was calculated. Then, we retrieved the infiltration scores from the TIMER2.0 database (http://discover.nci.nih.gov/cellminer/), and we utilized the R packages “limma” and “ggpubr” to generate and visualize the results. Moreover, using the GDSC and CTRP module, a comprehensive examination of the connection between IL18RAP expression and drug sensitivity was conducted. Then, using the Spearman approach, we determined the association between the mRNA expression level of IL18RAP and drug response sensitivity. The immunotherapy response was also predicted using the TISMO database (http://tismo.cistrome.org), ROC Plotter (http://www.rocplot.org/), and TIDE database (http://tide.dfci.harvard.edu).Briefly, the drug sensitivity data were downloaded from the CellMiner database (https://string-db.org/) was used to download information about the potential protein interactions with IL18RAP, and Cytoscape was used to import all of the information (v3.8.2). Then, using the cytoHubba plugins, we displayed the top 50 nodes and top 10 nodes ranked by MCC. Additionally, we used the Spearman approach to investigate the association of the top 10 genes across cancers.The STRING database (https://david.ncifcrf.gov/summary.jsp). The results were visualized using BioLadder (https://www.bioladder.cn/web/#/chart/28), an online mapping platform. Additionally, using the GSCALite database (https://bioinfo.life.hust.edu.cn/web/GSCALite/), we examined the connection between IL18RAP and well-known cancer-related pathways that were either activated or inhibited across cancers.The top 10 genes screened by Cytoscape included IL18, IL18RAP, IL18R1, IL18BP, STAT4, RELA, NFKB1, MAPK9, MAPK8, and IL37. The top 10 genes’ KEGG enrichment and GO function analysis findings were then retrieved from the DAVID database (https://www.gsea-msigdb.org/gsea/downloads.jsp). The biological pathway variations between the high- and low-IL18RAP groups were identified using the “clusterProfiler” program. False discovery rate (FDR) < 0.25 and an adjusted p-value < 0.05 were regarded as remarkably modified pathways. For each analysis, the Gene set permutation should be run 1,000 times. Finally, Xiantao Academic (https://www.xiantao.love/products/apply) was used to display the results.We obtained gene ontology sets and curated gene sets from the GSEA (http://tisch.comp-genomics.org/home/). In addition, through the “GSEA” section of the “Dataset” module, we also obtained hallmark and single-cell signature analysis results for different cell types.Briefly, we analyzed the correlation of IL18RAP expression and various cell types in a variety of cancers using the TISCH2 website (The HepG2 (LIHC cell line), MDA-MB-231 (BRCA cell line) and U251 (GBM cell line) were cultured in DMEM. In RPMI-1640 medium, the human monocyte cell line THP-1 was cultured. First, THP-1 cells were stimulated for 6 hours with 320 nM PWA  to differentiate into M0 macrophages. Lipopolysaccharide  was used to stimulate M0 macrophages for 48 hours in order to polarize them into M1 macrophages.The siRNA was purchased from OBiO Technology (Shanghai). For siRNA transfection, the siRNA sequence used to knock down IL18RAP (si-IL18RAP) was si-IL18RAP-1 (forward sequence: 5’-AAAAUAAGACAAAUUCCUCUU-3’ and reverse sequence: 5’- GAGGAAUUUGUCUUAUUUUGU-3’) and si-IL18RAP-2 (forward sequence: 5’-AUAGCUUUUCCUAAUGUCCUC-3’ and reverse sequence: 5’- GGACAUUAGGAAAAGCUAUCC-3’). According to the manufacturer’s protocol, the U251, MDA-MB-231 and HepG2 were transfected with si-NC, si-IL18RAP-1 or si-IL18RAP-2 using Lipofectamine 2000 .At Renmin Hospital of Wuhan University, tissue samples from hospitalized patients were collected and the pathology department provided all sample wax blocks.The produced tissue sections were given a 3% H2O2 treatment for 10 minutes and then incubated with the primary antibody at 4° C after being blocked in 3% bovine serum albumin and 0.3% Triton X-100. Following that, a corresponding fluorescent secondary antibody was incubated with the samples. DAPI was used to counterstain nuclei. We processed multiplex immunofluorescence staining using TSA fluorescent kits  according the manufacturer’s instructions. Finally, we acquired images using a fluorescence microscope (Olympus BX51). Anti-IL18RAP , anti-CD68 , and anti-iNOS  were the primary antibodies employed.In a word, we obtained the cell samples and lysed them in RIPA buffer  with 0.1 mM PMSF . After centrifuging the lysate, the supernatant was gathered. The proteins were then transferred to PVDF membranes through electrophoresis. The membranes and the primary antibodies were then incubated together for an overnight period at 4° C after being blocked with 5% nonfat milk and being washed with TBS-T. The membranes were then exposed for an hour to the secondary antibodies. In the end, we used the ChemiDocTM Touch Imaging System to take the images . The outcomes were examined with the use of ImageJ software.5) to the lower chamber and M1 macrophages (5 × 105) to the upper chamber to conduct cell migration tests. Nonmigrated cells were removed after coculturing for 24 hours, and migrated M1 macrophages were then stained with 0.5% crystal violet solution. Finally, we used an inverted microscope to obtain the images (Olympus). Five areas were randomly selected to count the cells and calculate the mean value. The outcomes were examined with the use of ImageJ software.We added si-NC and si-IL18RAP groups of MDA-MB-231, U251 and HepG2  was added to the lower chamber. Nonmigrated cells were removed after coculturing for 24 hours, and migrated MDA-MB-231 cells were then stained with 0.5% crystal violet solution for 15 min. An inverted microscope was used to obtain the images. Five areas were randomly selected to count the cells and calculate the mean value. For invasion assays, Matrigel and serum-free medium were mixed at a ratio of 1:8 and 80μl mixed solution was then added to the upper chamber. The following steps of the experiment were identical to those in the migration experiment. The outcomes were evaluated using ImageJ software.MDA-MB-231 cells transfected with siRNA were digested and resuspended; 200 μl of serum-free medium containing 2× 10MDA-MB-231 cells were plated in 6-well plates until cell confluence reached 80% after si-IL18RAP or si-NC transfection. Cells were scraped with a sterile pipette tip to form a straight line. The culture was then continued with serum-free RPMI-1640 media after the floating cells had been removed with PBS. At 0, 24, and 48 hours, pictures were taken with an inverted microscope. Finally, ImageJ software was used to measure and calculate the percentage of wound healing area at different time points.For statistical analysis, we utilized R software (version 4.2.1), and to determine the connection between different variables, we used the Spearman method. Using the SPSS 19.0 program, a Student’s t test or a one-way ANOVA was employed to compare the differences . The graphs were produced using GraphPad Prism 5.0 software. All information is displayed as means ± standard deviations (SD). P < 0.05 indicates a significant difference.The datasets generated for this study are available on request to the corresponding authors.Supplementary Figures


In this review, we briefly outline the roles, molecular mechanisms and the latest advances in ZFPs in GC, including eight principal aspects, such as cell proliferation, epithelial–mesenchymal transition (EMT), invasion and metastasis, inflammation and immune infiltration, apoptosis, cell cycle, DNA methylation, cancer stem cells (CSCs) and drug resistance. Intriguingly, the myeloid zinc finger 1 (MZF1) possesses reversely dual roles in GC by promoting tumor proliferation or impeding cancer progression via apoptosis. Therefore, a thorough understanding of the molecular mechanism of ZFPs on GC progression will pave the solid way for screening the potentially effective diagnostic indicators, prognostic biomarkers and therapeutic targets of GC.According to the 2020 global cancer data released by the World Cancer Research Fund (WCRF) International, gastric cancer (GC) is the fifth most common cancer worldwide, with yearly increasing incidence and the second-highest fatality rate in malignancies. Despite the contemporary ambiguous molecular mechanisms in GC pathogenesis, numerous in-depth studies have demonstrated that zinc finger proteins (ZFPs) are essential for the development and progression of GC. ZFPs are a class of transcription factors with finger-like domains that bind to Zn Helicobacter pylori (Hp) infection, etc. [Gastric cancer (GC) is one of the most frequently diagnosed gastrointestinal malignancies worldwide with insidious onset and poor prognosis, and among which stomach adenocarcinoma (STADs) is the most common ,2. With on, etc. .Comprehensive treatment based on surgical resection is still the primary choice for the clinical treatment of GC; however, the eventual postoperative mortality and survival rates remain unfavorable . For insAs canonical transcription factors, zinc finger (ZNF) proteins play precisely defined regulatory roles in physiological processes, such as transcription translation, cell differentiation and embryonic growth, by binding specific nucleotide sequences upstream of genes . Recent 2+) to stabilize the structure [ZFPs are a family of proteins with short self-folding finger-like domains that share the common feature of binding a zinc ion , DAZ-interacting zinc finger protein 1 (DZIP1), E3 ubiquitin ligase ring finger protein 114 (RNF114) and pleomorphic adenoma gene like-2 (PLAGL2). Ke et al. found that the proliferation of GC cells could be suppressed when using a CRISPR/CAS803 system to knock out ZNF852 due to the downregulated epidermal growth factor receptor (EGFR) expression by ZNF852 deficiency . A colonMultiple C2H2-type ZFPs can also be suppressed, thus promoting GC development, specifically ZNF545, ZNF479, ZNF471, ZNF331 and Krüppel-like factors (KLF4 and KLF6). Wang et al. found that restoring ZNF545 expression could inhibit GC cell proliferation, validating the fact that ZNF545 can act as a tumor suppressor to repress the transcription of ribosomal RNA (rRNA) and recruit corepressor and heterochromatin protein 1β . As an oSeveral ZFPs with special structures can also participate in the proliferation of GC cells, such as zinc finger CCCH-type containing 15 (ZC3H15), speckle-type POZ protein (SPOP), microrchidia family CW-type zinc finger 2 (MORC2) and zinc and ring finger 3 (ZNRF3). ZC3H15 is a highly functional and evolutionarily conserved protein . ConsideA significant process in the invasion and metastasis of malignant tumors is EMT, in which epithelial cells undergo a loss of polarity and cell–cell adhesions and change into motile mesenchymal-like cells . Both E-Amounts of C2H2-type ZNFs with a high expression which can trigger EMT, invasion and metastasis were found in GC, particularly ZNF521, ZNF460, ZNF143, zinc finger protein 139 (ZNF139), zinc finger and BTB domain-containing 20 (ZBTB20), Snail, DZIP1, Slug, RNF114, PLAGL2, GLI family zinc finger 1 (GLI1), Krüppel-like factor 8 (KLF8), CCCTC-binding factor (CTCF) and the E-box-binding protein ZEBs (ZEB1 and ZEB2), as illustrated in Certain C2H2-type ZNFs, notably ZNF471, ZNF331, ZNF24, KLF4, Krüppel-like factor 9 (KLF9), ZNF382 and ring finger protein 180 (RNF180), can be suppressed to inhibit EMT, migration and metastasis, thus halting gastric tumorigenesis . The ectSome ZNFs with unique structures can also participate in inducing EMT and the motility and invasion of GC cells, namely highly expressed ZNFs including ZC3H15, Twist family bHLH transcription factor 1 (TWIST1) and MORC2 . ZC3H15 H. pylori infection, and its colonization in GC epithelial cells can result in a series of inflammatory premalignant events [+ T cells and several immunosuppressive cytokines such as CXCL10, IL-1α and CSF1, while decreasing IFN-γ and IL-2 to trigger GC progression [Tumor cells, inflammatory cells and adjacent cellular stroma in chronic and recurrent inflammation can create an inflammatory tumor microenvironment (TME) conducive to tumor progression . The prit events . A recent events . Thus, wt events . Additiogression . Elevategression . Additiogression . In concApoptosis, also known as programmed cell death, is tightly regulated by genes and proteins. Its dysfunction may result in uncontrolled growth and tumor formation . At presr = −0.686; p < 0.01) and the underlying molecular mechanism was that ZNF139 can attenuate apoptosis in GC tissues by inhibiting Caspase-3 and Bax while promoting Bcl-2 expression [Many C2H2-type ZFPs are closely associated with apoptosis, such as ZNF143, GLI1, ZNF139 and ZFX, and all of them are upregulated. A recent study showed that ZNF143 is associated with apoptosis triggered by ROS-induced oxidative damage in vivo . Moreovepression . Thereafpression . In GC, pression .Ubiquitination is one of the most common post-translational modifications in proteomics, which covalently binds ubiquitin molecules to target proteins and regulates metabolic reprogramming in cancer . In GC, Under normal circumstances, cell-cycle-protein-dependent kinases (CDKs) and CDKIs precisely orchestrate the cell cycle, and CDKIs prevent CDKs from functioning . CarcinoIn GC, the upregulation of some ZNFs can increase the malignancy of GC via cell-cycle-related regulation such as ZNF139, ZFX, MORC2, GLI1, ZNF460, Krüppel-like factor 12 (KLF12) and Ring finger protein 2 (RNF2). Fan discovered that G0/G1 cells were greatly elevated but G2/M cells were dramatically decreased when suppressing ZNF139, indicating that ZNF139 can intervene in the cell cycle to block the apoptosis of GC cells . The ZFXp = 0.001), while no ZNF545 methylation was detected in 20 normal gastric mucosa tissues [p < 0.001) [DNA methylation is the most dominant form of epigenetic inheritance, characterized by unchanged nucleic acid sequence and heritable gene expression . Gene si tissues . This mi< 0.001) . Mechani< 0.001) . The met< 0.001) . Moreove< 0.001) . NonetheUnder appropriate circumstances, stem cells may self-renew or differentiate, giving rise to daughter cells with precise genotypes and phenotypes, as well as progenitor cells with particular differentiated objectives . UnfortuDrug resistance leads to metastasis or recurrence in advanced cancer, which is one of the major obstacles to successful cancer treatment . It deveRight now, findings on GC prove that some C2H2-type ZFPs, for example, ZNF852, ZNF139, ZFP64, GLIS family zinc finger 2 (GLIS2), GLI1, GLI2 and ZEB2, and their expressions are all upregulated. ZNF852 deficiency can enhance oxaliplatin-induced GC cell death, suggesting that ZNF852 increases Sox2, Oct4 and Nanog transcription, which may boost carcinogenesis and enhance drug resistance . ZNF139 Certain unique ZFPs can affect the development of GC via distinct pathways besides the eight routes indicated above. In this part, we mainly focus on C2H2-type ZNF479 and RNF43, which are both upregulated in GC . Jin et Generally, certain kinds of ZFPs only play either carcinogenic or antitumor roles in GC. A growing body of evidence has suggested that myeloid zinc finger 1 (MZF1) constitutes dimers via highly conserved SCAN motifs and may have distinct functions in GC . MZF1 waThe potential of MZF1 in clinical therapy and the prognosis prediction of GC may not be underestimated. The inhibitory effect of the MZF1-SMAD4 axis may provide new evidence for the discovery of the novel molecular target therapy of GC . AdditioOver the last few years, there has been a remarkable advancement in the identification of novel GC compounds ,173. ResFirstly, many ZFPs exert prognostic effects via regulating proliferation, EMT, invasion and metastasis. Given that the ZNF460-APOC1 axis promotes GC progression in vivo , ZNF460 Secondly, certain ZFPs function as prognostic or diagnostic markers via DNA methylation. In comparison to GC patients with unmethylated or hypomethylated ZNF545 promoters, those with hypermethylated ZNF545 promoters have a substantially shorter median OS . The proPivotally, current research has shown that the RNF43 gene commonly exhibits mutations in GC and RNF43 displays a crucial independent prognostic role in GC ,93,94,95Recently, preclinical and clinical studies on targeted therapy have also been carried out, and the novel targets researched in GC involve Claudin18.2 (CLDN18.2) , FGFR2b A large number of miRs are aberrantly expressed in GC, affecting the expression of multiple ZFPs, specifically ZNF521, ZNF139, ZNF24, zinc finger and BTB domain-containing 4 (ZBTB4), Snail, KLF6 and MZF1. The negative link between miR-204-5p and its target gene ZNF521 suggests that downregulating ZNF521 can enhance cell death and inhibit GC proliferation, migration and invasion . miR-195It is worth mentioning that other molecules, as important regulators of GC, can target miRs, affect the expression of ZFPs and in turn control the progression of GC, including cyclic RNAs (circRNAs) and long noncoding RNAs (lncRNAs) . circCSNThe above provides a brief overview of the research progress on the relationship between ZFPs and GC nowadays, as well as the basic molecular mechanisms as seen through multiple biological processes, including cell proliferation, EMT, invasion and metastasis, inflammation and immune infiltration, apoptosis, cell cycle, DNA methylation, CSCs, drug resistance, etc. Significantly, we highlight the dual inverse role of MZF1 in GC. Despite extensive previous studies in this area, there are still substantial gaps in the complete knowledge of a large number of ZFPs, including ZNF146, ZNF281 and ZDHHC2, wherein their mechanisms of action in GC are still not fully elucidated ,203.As a wide-ranging family of eukaryotic transcription variables, ZFPs have exhibited promising potential in several biological fields, such as disease treatment, on account of their specific transcriptional regulation roles . GC rema


Natronoccoccus sp. TC6 and Halorubrum tebenquichense SU10 was not previously described, being the aim of this work. The carotenoids profile was performed by UV-Vis spectrophotometry, thin-layer chromatography, nuclear magnetic resonance spectroscopy, and high-resolution mass spectrometry (UPLC-ESI-MS/MS). Antioxidant capacity was determined for DPPH, ABTS, and FRAP. In addition, MMP-9 inhibition was studied using docking simulations. The carotenoid profile of studied strains was composed of bacterioruberin, some derivatives like mono, bis, and tris anhydrobacterioruberin, and also some bacterioruberin cis isomers. The carotenoid pools showed antioxidant capacity for DPPH > ABTS > FRAP; Natronococcus sp. TC6 carotenoid pool was better for ABTS and DPPH, while Halorubrum tebenquichense SU10 carotenoid pool was better for FRAP. Additionally, docking and molecular dynamics suggest that bacterioruberin inhibits MMP-9 through hydrophobic interactions near the catalytic site. Bacterioruberin shows the higher binding energy of −8.3 . The carotenoids profile of both strains was elucidated, their antioxidant activity and singular participation of each carotenoid on MMP-9 in silico inhibition were evaluated.Natural pigments from haloarchaea are of great interest; bacterioruberin is the major pigment, it shows higher antioxidant power when compared with β-carotene. However, characterization of bacterioruberin and its isomers along with its antioxidant and the matrix metallopeptidase 9 (MMP-9) inhibition activities in extracts from  Halobacteriaceae and Haloferacaceae families), extreme microorganisms that grow in hypersaline environments, up to 4.5 M of NaCl, can produce and accumulate red pigments in their cell membrane + m/z 740.5, 741.5, 742.5 (45H64O) at m/z 620.3 and monoanhydrobacterioruberin MABR (C50H74O3) at m/z 722.47 (40H52O4) at m/z 598.39, β-carotene (C40H56) at m/z 537. 2, phytoene (C40H64) at m/z 543.50, and lycopersene (C40H66) at 545.96, some of these found in TLC analysis . In the case of ferric ion-reducing antioxidant power FRAP  in the inhibition between the strains from which the pigments were extracted, Natronococcus sp. TC6 was better in ABTS and DPPH radicals and Halorubrum tebenquichense SU10 was better in FRAP. In in the case of FRAP, there are significant differences (p < 0.05) for extract pigment concentration   E–G.cis-26-bacterioruberin fragment has the lowest binding energy value, and its affinity is not in the catalytic cavity of MMP-9, but near Glu402  derivatives found in the pigment extracts like monoanhydrobacterioruberin (MABR), bisanhydrobacterioruberin (BABR), and 2-isopentenyl-3,4 dihydrorhodopin (DIR) are reported in strains like ismortis ,38. BAR p. TBZ16 . A majorrotenoid ,40. In toxanthin ,42.Natronococcus sp. TC6 and Halorubrum tebenquichense SU10) were higher (above 90%) compared with pigments extracted from Aquisalibacillus elongatus MB592, Haloarcula japonica HM1, and Halobacterium salinarum HM2 that were able to inhibit 20, 30, and 80% percent, respectively [Halorubrum tebenquichense, Haloarcula japonica HM1, and Halobacterium salinarum HM2 have been reported as good FRAP inhibitors (above 70%) [Natronococcus sp. showed the best results when compared with Halorubrum tebenquichense SU10 and other strains of the same genus [Natronococcus sp. TC6 and Halorubrum tebenquichense SU10 have a major antioxidant capacity of DPPH > ABTS > FRAP dominating the capacity of hydrogen and single electron transfer  as the typical “free radical scavenger” mechanism [Halorubrum tebenquichence SU10 on FRAP that shows more antioxidant capacity than Natronococcus sp. TC6, related to the presence of astaxanthin in Halorubrum pigment extract and its participation in a synergy with the other pigments.It has been shown that carotenoid extracts from several haloarchaea have antioxidant activities and are dose- and profile-dependent, particularly of isomers of bacterioruberin and other carotenoids . DPPH scectively . Other pove 70%) ,43. Contme genus . Accordiechanism ,45. In aechanism . Additioechanism . This ca2+) and calcium (Ca2+) dependent, present intracellularly and membrane-bound. Their importance is their participation in physiological processes including development, wound, healing, remodeling of tissues, angiogenesis, and particularly participation in the tumor invasion, metastasis, and angiogenesis acting in the degradation of gelatin and type IV, V, XI, and XII collagens. The overexpression of MMP-9 is associated with different cancers, such as lung, prostate, gastric, pancreatic, and colon and with other diseases, such as autoimmune and cardiovascular diseases [The MMP-9 is an enzyme of metalloproteinase group (MMP’s) particularly zinc  [Natronococcus sp. TC6 and Halorubrum tebenquichense SU10 extracts showed the highest binding energies.Docking studies point towards the importance of the archaeal carotenoids for MMP-9 inhibition because of their interaction in the catalytic pocket or around of active site. The inhibitory effect of archaeal carotenoids on MMP-9 has been studied in vitro using a carotenoid extract of 5 µg/mL) . HoweverThere are few reports about the molecular dynamics of bacterioruberin and other related carotenoids. The potential with molecular dynamics studies for other enzymes has been reported, like acetylcholinesterase (ACE) and butyrylcholinesterase using fragments of the carotenoids and showing binding energies in a range of ΔG  −7.42 to −10.35 ΔG . MoleculMolecular dynamics results suggest that bacterioruberin blocks the entry to both Binding Site 1 and Binding Site 2. The selection of the Allosteric Binding Site was based on bacterioruberin large size. Previous crystallographic studies have confirmed MMP-9 interactions with large molecules, such as antiMMP-9 antibodies, making it plausible for bacterioruberin to interact in that area as well. As mentioned earlier, most of the residues involved in these interactions are primarily hydrophobic, indicating hydrophobic interactions as the primary mode, with some hydrogen bond formation see D. AdditiThe carotenoids profile of both strains was elucidated, and their antioxidant activity and MMP-9 inhibition in silico were evaluated, future research must be focused on elucidating the mechanism to enhance bacterioruberin and derivates production at a large scale and their incorporation in functional foods and ingredients.Halorubrum tebenquichense SU10, isolated form Uyuni saltern in Bolivia and Natronococcus sp. isolated from El Golea, a salt lake in Argelia were characterized in their carotenoids pigments. Bacterioruberin was identified in both Halorubrum tebenquichense SU10 and Natronococcus sp. TC6 pigment extracts, also, bacterioruberin derivates were identified, 2-isopentenyl-3,4 dihydrorhodopin, and monoanhydrobacterioruberin. Other pigments and intermediaries were identified, astaxanthin, β-carotene, phytoene, and lycopersene. The pigment extracts showed considerable antioxidant activity on DPPH, ABTS, and FRAP. The inhibitory effect of the haloarchaeal pigments over MMP-9 was suggested by docking and molecular dynamics assays, monoahydrobacterioruberin and trisanhydrobacterioruberin display the best binding energy values. Molecular docking suggests that bacterioruberin has hydrophobic interactions with the tested MMP-9 sites, and the ligand remains stable during molecular dynamics simulations. Future research must be focused on large scale bacterioruberin production, also in the purification of pigments on the pool and their study on health benefits and also to verify their effect on MMP-9 inhibition on in vitro assays.


Podophyllotoxin (PPT) has shown strong antitumor effects on various types of cancers. However, the non-specific toxicity and poor solubility severely limits its clinical transformation. In order to overcome the adverse properties of PPT and explore its clinical potential, three novel PTT−fluorene methanol prodrugs linked by different lengths of disulfide bonds were designed and synthesized. Interestingly, the lengths of the disulfide bond affected the drug release, cytotoxicity, pharmacokinetic characteristics, in vivo biodistribution and antitumor efficacy of prodrug NPs. To be more specific, all three PPT prodrugs could self-assemble into uniform nanoparticles (NPs) with high drug loading (>40%) via the one-step nano precipitation method, which not only avoids the use of surfactants and cosurfactants, but also reduces the systemic toxicity of PPT and increases the tolerated dose. Among the three prodrug NPs, FAP NPs containing α-disulfide bond showed the most sensitive tumor-specific response and fastest drug release rate, thus demonstrating the strongest in vitro cytotoxicity. In addition, three prodrug NPs showed prolonged blood circulation and higher tumor accumulation. Finally, FAP NPs demonstrated the strongest in vivo antitumor activity. Our work will advance the pace of podophyllotoxin towards clinical cancer treatment. Cancer is a serious threat to human health . At presPodophyllotoxin (PPT) has been widely reported in many studies because of its potent antitumor activity ,15,16,17Nano-drug delivery systems (Nano-DDSs) have attracted much attention in recent years ,25,26,27Numerous previous studies have demonstrated that the antitumor effect of prodrug self-assembled nanodrug delivery systems depends on high tumor accumulation and rapid drug activation. In our previous study, it was found that the stronger the ability of the prodrugs to assemble, the longer the circulation and then the higher the tumor accumulation. Therefore, how to improve the self-assembly capacity of small molecule prodrugs has been the direction of our efforts. Many studies have shown that the introduction of 9-fluorenylmethoxycarbonyl (Fmoc) into nanocarriers can improve the drug loading capacity, which is attributed to the fact that Fmoc can be used as a functional building block to interact with drug molecules. Hence, we wonder if the introduction of Fmoc into small-molecule prodrugs can facilitate the nanoassembly of a prodrug. In addition to excellent assembly capacity, rapid drug activation also determines the ultimate antitumor effect. Disulfide bonds have been shown to respond specifically to high GSH at tumor sites, thus accelerating drug release. More importantly, the position of disulfide bonds in the linker also affects the redox responsiveness of the prodrug. To verify the above hypothesis, we designed three novel PPT-Fmoc prodrugs  linked by various lengths of disulfide bonds, abbreviated as FAP, FBP and FGP, respectively. Surprisingly, the three prodrugs could self-assemble into uniform NPs  by introducing the Fmoc group, demonstrating the critical role of the Fmoc group in prodrug self-assembly . In addi2k was purchased from AVT (Shanghai) Pharmaceutical Technology Co., Ltd., Shanghai, China. Penicillin–streptomycin and fetal bovine serum were obtained from GIBCO, Invitrogen Corp., Waltham, MA, USA. Other chemicals and solvents were of analytical or high-performance liquid chromatography (HPLC) grade.PPT, RPMI 1640 cell culture medium, DMEM-F12 cell culture medium, 3--2,5-dipphenyl-2H-terazolium bromide (MTT) and Hoechst 33342 were purchased from Dalian Meilun Biotech Co., Ltd., Dalian, China. 9-Fluorene methanol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China. Dithiodiglycolic acid, 3,3′-dithiobispropionic acid and 4,4′-dithiodibutyric acid were obtained from TCI (Shanghai) Chemical Industry Development Co., Ltd., Shanghai, China. DSPE-PEGFAP, FBP and FGP were obtained through two-step esterifications. In order to promote the esterification activity of dithioglycolic acid, 3,3′-dithioglycolic acid and 4,4′-dithioglycolic acid, they were stirred at room temperature for 2 h in the system of acetic anhydride, and then, dithiodiacetic anhydride, 3,3′-dithiodipropionic anhydride and 4,4′-dithiodibutyric anhydride were obtained. The esterification of 9-fluorenyl methanol with dithiodiacetic anhydride, 3,3′-dithiodipropionic anhydride and 4,4′-dithiodibutyric anhydride, respectively, was carried out overnight at room temperature under the condition of DMAP catalysis. Then, the intermediate products purified via column chromatography were activated with EDCI, HOBt and DMAP through an ice bath for 4 h. PPT was then added to the system and stirred for 48 h at room temperature to obtain three kinds of prodrugs. The purity of products could reach more than 97% after purification via pre-HPLC . The mobile phase was acetonitrile/water = 80:20. 2k (0.25 mg) were dissolved in tetrahydrofuran . Then, the mixed solution was added dropwise into deionized water (4 mL) under stirring (1000 rpm). Finally, the THF in the system was removed under vacuum conditions at 30 °C. The preparation of the other two PEGylated prodrug NPs (FBP NPs and FGP NPs) was similar. The prodrug NPs were all prepared via the one-step nano-precipitation method. Briefly, FAP (1 mg) and DSPE-PEGThe size and zeta potential of prodrug NPs were determined with a Zetasizer . Transmission electron microscopy  was used to observe the morphology of FAP NPs, FBP NPs and FGP NPs stained with 2% phosphotungstic acid. The assembly situations of three prodrug structures were simulated through molecular docking , and the interactions between prodrug molecules were analyzed.To monitor the colloid stability of prodrug NPs, FAP NPs, FBP NPs and FGP NPs were incubated in PBS and RPMI 1640 medium ), respectively, in a 37 °C shaker, and samples were taken at 1, 2, 4, 6, 8 and 12 h to measure the particle size. In addition, the sizes of FAP NPs, FBP NPs and FGP NPs were measured at 0, 3, 7, 15, 30, 45, 60 and 90 days, storing them in 4 °C atmospheres. The sizes of NPs were measured with a Zetasizer .The release behaviors of prodrugs in the presence of 0, 2, 5 and 10 mM DTT were investigated, respectively. As for the release medium, 30% ethanol was added to PBS (pH = 7.4) to make sure that hydrophobic PPT released from prodrug NPs could be fully dissolved in the system for easy detection. The released PPT was used for detection via HPLC  for 1, 2, 4, 6, 8 and 12 h after the addition of prodrugs. The proportion of binary eluents was methanol/water = 55:45. Meanwhile, the absorption wavelength was set at 295 nm.2).The mouse breast cancer cell line (4T1) and mouse embryo fibroblasts cell line (3T3) were provided by the American Type Culture Collection (ATCC). 4T1 cells were cultured in RPMI 1640 medium, and 3T3 cells were cultivated in DMEM-F12; both of them were mixed with 10% FBS, penicillin (100 units/mL) and streptomycin (100 μg/mL). Further, the cells were incubated at 37 °C in a proper atmosphere (5% CO5 cells/well). After cultivation for 12 h, fresh culture media containing FAP@C-6 NPs, FBP@C-6 NPs, FGP@C-6 NPs or C-6 solution  were added to replace the precedent culture medium. After incubation for 0.5 or 2 h, the drug-containing culture medium was discarded and cells were washed with cold PBS three times. Then, the cells were collected, and the intracellular fluorescence signals were estimated via flow cytometry .Coumarin-6 (C-6)-labeled prodrug NPs were used to investigate the cellular uptake. The 4T1 tumor cells were planted in 24-well plates. After incubating with C-6 solution and C-6-labeled prodrug NPs with an equivalent concentration of C-6 (250 ng/mL) for 0.5 or 2 h, the medium containing drugs was abandoned. The cells were washed three times using cold PBS and then fixed with 4% paraformaldehyde. Moreover, the nucleus was stained with Hoechst 33342, and the samples were observed via confocal laser scanning microscopy . In addition, 4T1 cells were seeded in 12-well plates (1 × 103 cells/well) were cultured in 96-well plates for 12 h. The precedent medium was replaced with fresh culture medium containing a series of concentrations of prodrug NPs and PPT solution and sequentially cultured for 48 h and 72 h. Then, 25 μL of MTT (5 mg/mL) was added to the culture media and incubated for another 4 h at 37 °C. Eventually, the media with MTT were gingerly discarded, and 200 μL of DMSO was added. After shaking for 10 min, absorbance was measured using a Varioskan LUX multimode microplate reader .4T1 tumor cells were used to investigate the cytotoxicity of FAP NPs, FBP NPs, FGP NPs and PPT solutions by performing an MTT assay. 4T1 cells  rats were used according to the requirements and regulations of the Animal Ethics Committee of Shenyang Pharmaceutical University. n = 6) were randomly assigned and given an i.v. dose of FAP NPs, FBP NPs, FGP NPs or PPT solution at 5 mg/kg  in sequence. At 0.03, 0.083, 1, 2, 4, 8, 12 and 24 h, blood was withdrawn from the eye socket, and the plasma was separated for analyses. Concentrations of prodrugs and PPT were quantified using LC-MS (Waters Xevo-TQD), respectively.SD rats . At 2, 4, 6, 8, 12 and 24 h after administration, the anesthetized tumor-bearing mice were photographed using an IVIS spectrum small-animal in vivo imaging system. In order to observe the distribution of drugs in major organs, 24 h after administration, the tumor-bearing mice were sacrificed, and the heart, liver, spleen, lung, kidney and tumor were dissected and photographed using an IVIS spectrum small-animal in vivo imaging system.DiR-labeled prodrug NPs were prepared to investigate the biodistribution of prodrug NPs in 4T1-tumor-bearing Balb/C mice. When the tumor volume reached 500 mmIn order to investigate the biosafety of prodrug NPs, two doses of 10 mg/kg, 20 mg/kg and 30 mg/kg  were set, and healthy mice were administered through tail intravenous injection every two days , The body weight change and survival curve were recorded during the treatment. In addition, the hemolysis ability of NPs and the solution was detected. We added prodrug NPs and PPT solution  to the same amount of red blood cell suspensions, using normal saline and deionized water as a negative control and positive control, respectively. After 2 and 4 h of incubation in 37 °C shakers, the absorbance value at 545 nm was measured using a Varioskan LUX multimode microplate reader . The hemolysis rate (HR) was calculated according to the following formula: HR = (ODsample − ODnegative)/(ODpositive − ODnegative) × 100%.7 cells/mL) was injected subcutaneously into the right rear of BALB/C mice. When the tumor volume reached 100 mm3, the mice were randomly divided into 5 groups (n = 5): FAP NPs 20 mg/kg, FBP NPs 20 mg/kg, FGP NPs 20 mg/kg, PPT solution 10 mg/kg and control . These formulations were administrated into the 4T1 tumor-bearing mice via tail intravenous injections at an interval of two days for four injections total, respectively. The tumor volume and body weight were measured and recorded once a day. Two days after the last dose, all mice were sacrificed, tumors were collected and soaked in 4% paraformaldehyde to preserve them, and they were prepared for staining with H&E. Tumor sections stained using the terminal deoxynucleoitidyl transferase dUTP nick end labeling (TUNEL) assay were obtained to evaluate damage caused by drugs to tumor cells.4T1 tumor-bearing female Balb/C mice were modeled to investigate the in vivo antitumor activity of prodrug NPs. For this, 100 μL of 4T1 cell suspensions , and p < 0.05 was considered statistically significant.All data were calculated and presented as the mean ± SD. The significant differences between groups were identified by performing a 1H nuclear magnetic resonance (1H NMR) and mass spectra (MS) analysis  in 37 °C shakers were investigated to assess the stability of NPs by simulating an in vivo microenvironment. The particle size of the NPs did not change significantly within 12 h . In addiThe reduction-responsive ability of a disulfide bond is a vital theoretical basis for our design of tumor-specific prodrugs. The introduction of a disulfide bond endowed the prodrugs tumor-specific response ability. In order to investigate the tumor-specific response release ability of three prodrug NPs, prodrug NPs were added into release medium containing DTT to mimic the high GSH in tumor cells, and the content of PPT in the system at different time points was detected via HPLC to calculate the percentage of prodrug release . To our Based on our team’s previous work ,45, the 4T1 cells were utilized to investigate the cellular uptake behavior of three prodrug NPs. C-6 was chosen to label the prodrug NPs for tracing. 4T1 cells incubated with C-6-labeled prodrug NPs or C-6 solution for 0.5 and 2 h were observed via CLMS A,B. The 50 values were calculated and are displayed in The remarkable system-cytotoxicity of PPT has been reported in many studies, which is also a huge obstacle limiting its clinical application. We modified PPT with strong cytotoxic activity using a prodrug strategy and prepared self-assembled NPs. In order to investigate the cytotoxicity of prodrug NPs towards tumor cells, 4T1 cells were selected as the model for cytotoxicity tests, and the survival rate of 4T1 cells was measured by performing an MTT assay. ICSD rats were used to evaluate the pharmacokinetic behavior of prodrug NPs in vivo. Blood samples were collected at different time points within 24 h after i.v. administration, and plasma samples were analyzed to detect the contents of prodrug and PPT. Pharmacokinetic parameters were calculated and are presented in 4T1 tumor-bearing mice were constructed as the biodistribution model. After injecting DiR-labeled prodrug NPs or DiR solution, DiR fluorescence could be detected and observed through the in vivo imaging of small animals to trace the biodistribution of drugs in the body A. The flHealthy Balb/c female mice were used to investigate the maximum tolerated dose (MTD) of prodrug NPs and PPT solution. The doses of 10, 20 and 30 mg/kg formulations were administered through the tail vein once every two days, four times A. The su3, administration was started, and it was repeated once every two days for a total of four times (4T1 tumor-bearing mice were constructed to assess the antitumor activity of the preparations in vivo, and the same administration regimen was adopted as above. When the tumor volume reached about 100 mmur times A. Changeur times B,C. As cur times D. Comparur times E. In accur times . As can ur times . There wIn summary, three novel Fmoc-PPT prodrugs were designed by utilizing different lengths of disulfide bonds. As we hypothesized, the introduction of Fmoc improved the assembly ability, and the three PPT prodrugs could self-assemble into stable nano-systems. Thanks to the one-step nano precipitation method, the self-assembled prodrug NPs avoided the use of surfactants and cosurfactants. Then, prodrug NPs showed higher cell uptake efficiency than solution. In addition, the position of disulfide bonds in the linker also affected the reduction responsiveness of the prodrugs, thereby influencing the cytotoxicity. What makes sense is that our nano-delivery system mitigates the severe side effects of PPT solution by reducing the systemic toxicity and boosting the maximum tolerated dose. Finally, FAP NPs with an α-disulfide bond showed more-potent antitumor effects than FBP NPs and FGP NPs, which could be ascribed to higher reduction-responsiveness and faster tumor-specific drug release. We expect our study to make PPT a new step toward clinical application in cancer treatment.


ALDH2 results in a substantial reduction in enzymatic activity, carriers of this variant allele may have a higher energy demand when consuming alcohol than non-carriers. However, no studies have evaluated this phenomenon to date.According to recent reports, individuals with reduced aldehyde dehydrogenase activity may require more energy for the detoxification of aldehydes. Aldehyde dehydrogenase 2 (ALDH2), an ALDH isozyme, is responsible for detoxifying acetaldehyde, an intermediate metabolite of ethanol. Because the variant allele of the rs671 polymorphism of To test the hypothesis, we statistically examined the interactive effects between the rs671 and ethanol consumption on energy intake using cross-sectional data from a population-based cohort study, the Japan Multi-Institutional Collaborative Cohort Study, which was conducted in Saga city between 2005–2007 .p = 0.03 for interaction between rs671 and ethanol consumption). Energy intake excluding energy from alcoholic beverages, carbohydrate intake, protein intake, and fat intake, showed similar tendencies .General linear regression models adjusted for age, sex, ethanol consumption, current smoking status, years of education, dietary restriction, medical history, and physical activity level revealed that energy intake was higher in variant allele carriers than in non-carriers among individuals with alcohol drinking habits, whereas no such correlation was observed among those without drinking habits (≤2 g ethanol/day) (ALDH2 rs671 polymorphism.These findings support the hypothesis that increased energy intake is required for the detoxification of aldehydes in individuals with low ALDH activity. This epidemiological evidence provides a possible scientific basis for understanding aldehyde detoxification mechanisms and suggests a novel phenotype of the https://doi.org/10.1265/ehpm.22-00276.The online version contains supplementary material available at  ALDH2, only the rs671 has a major effect on ALDH2 enzymatic activity , immunoenotypes , 11. Theenotypes , 19. Bec[ALDH2*2 , 20, 21.ALDH2*2 carriers is a great risk for alcohol-related cancers, such as esophageal cancer, because accumulated acetaldehyde is highly carcinogenic [ALDH2*2 carriers may have been more easily overlooked by medical professionals, because indicators of overdrinking such as serum liver transaminase levels, blood pressure, serum triglyceride levels, and visceral fat content, are reported to be lower in ALDH2*2 carriers [ALDH2*2 carriers have not been revealed; however, the accumulation of aldehydes is a primary possible reason, considering the role of ALDH2.Naturally, habitual drinking in inogenic , 22–25. carriers , 26–36 ecarriers , 35, 36.Aldh2 knockout mice than in wild-type mice after single ethanol administration [ALDH2 rs671 variant allele (ALDH2*2) who consume alcohol habitually, consume higher amounts of foods to compensate for the increased energy demands for aldehyde detoxification, which possibly explains both the unexpected tolerance to aldehydes and the low risk of drinking-related metabolic disorders, that is, the indicators of overdrinking, in this population.Using an animal model, we previously reported unexpected and protective effects of ALDH2 deficiency against ethanol loading , 37–39, stration  reportedstration . This inALDH2 rs671 polymorphism and ethanol consumption on energy and macronutrient intake, we tried to clarify if ALDH2*2 allele is associated with higher energy intake among individuals who routinely consume alcohol.Using a Japanese population-based cohort database, we aimed to examine our hypothesis epidemiologically. By assessing the interactions between the We used baseline survey data from a population-based cohort study, the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study, conducted between 2005–2007 in Saga City, Japan. Among the 61,447 individuals who were invited, 12,068 completed the baseline survey (19.7% participation rate) , 42. ParALDH2 rs671 polymorphism was determined using real-time polymerase chain reaction and the TaqMan® SNP Genotyping Assay  as per the manufacturer’s protocol.All J-MICC Study participants completed a self-administered questionnaire which was used to collect information regarding ethanol consumption, current smoking status, dietary habits, medical history , years of education , and dietary restrictions . In addition to weight measurements and venous blood sampling, physical activity was measured using a uniaxial accelerometer  worn on the waist for 10 days, except during sleeping and bathing. The physical activity level was calculated as the total energy expenditure divided by the basal metabolic rate as reported elsewhere , 44. DNADaily total ethanol consumption was estimated based on the ethanol concentration and consumption frequency  of sake, shochu, chuhai, beer, whiskey, and wine. Consumption was standardized to 60 kg body weight (g ethanol/day/60 kg body weight), the average body weight of the cohort, because ethanol diffuses in many tissues including adipose tissue, liver, and brain , 46; theDietary assessment was conducted as shown in a previous report . BrieflyALDH2*2/*2 from the present analysis, because most carriers of ALDH2*2/*2 do not have alcoholic drinking habits [We excluded 1,010 J-MICC participants with the homozygous genotype g habits , 20, 50.The data of 10,910 individuals were subjected to general linear regression analysis with α = 0.05. Outcomes were logarithmically transformed to approximate a normal distribution. All missing data for covariates, such as years of education (N = 30), current smoking status (N = 1), hyperlipidemia (N = 5), hypertension (N = 1), ischemic disease (N = 4), stroke (N = 1), and physical activity level (N = 160), were assumed to have occurred at random. All analyses were performed using SAS 9.4 . Statistical methods are shown with figures and tables.ALDH2*1/*1 or ALDH2*1/*2) and/or ethanol consumption level . Variables considered to be associated with energy intake or the rs671 polymorphism, including age (continuous variable), sex, years of education , current smoking status , medical history , dietary restrictions , and physical activity level (continuous variable), were used as covariates. Ethanol consumption level (continuous variable) was further included as a covariate when the differences in the amounts of ethanol consumption between genotypes needs to be adjusted.The primary outcomes were 1) total energy intake, 2) energy intake excluding energy from alcoholic beverages, 3) carbohydrate intake, 4) protein intake, and 5) fat intake, and explanatory variables were the rs671 genotype , which include an interactive term of genotype × ethanol consumption (continuous variable). Model A included the ALDH2 rs671 genotype, ethanol consumption, and interaction term as explanatory variables, with sex and age as covariates. Model B included explanatory variables and covariates in model A and additional covariates of years of education, current smoking status, dietary restrictions, medical history, and physical activity level.To examine the hypothesis that the relationships between ethanol consumption and energy or macronutrient intake differ based on the Sensitivity analyses were performed using body height or body fat percentage as an additional covariate because the rs671 polymorphism may affect physique. Additional analysis with stratification by sex was performed because alcohol consumption was substantially higher in men than in women.ALDH2 genotypes in terms of age, sex, years of education, body weight, and current smoking status. ALDH2*1/*2 carriers had significantly lower levels of ethanol consumption than ALDH2*1/*1 carriers, and the significance remained after stratification by ethanol consumption level, that is, 0.0 vs. 0.0 g/day in non-drinkers  (Table p for interaction = 0.043–0.072)  Table , althoug2) Table . To visu2) Table  or body 2) Table , reveale2) Table  and S4.ALDH2*1/*2 carriers among moderate and heavy drinkers had higher energy intake than ALDH2*1/*1 carriers, whereas there was no such association among non-drinkers  [As hypothesized, energy and macronutrient intakes in individuals with habitual drinking habits were higher in H2*1/*1) –53. HoweAldh2−/− mice after consumption of 5 g/kg ethanol [2, which lowers the respiratory quotient. However, sympathetic stimulation by acetaldehyde may influence with the energy balance as epinephrine enhances energy consumption [ALDH2*1/*2 carriers after alcohol consumption [Detoxification by GSH  represen ethanol  is consisumption . Increassumption –60, in wsumption .ALDH2*1/*2 carriers, stronger associations may have been masked. Furthermore, a larger population sample is required to examine ALDH2*2/*2 carriers, who were excluded from this study. Fourth, the rs1229984 polymorphism of ADH1B gene, which strongly affects the production rate of acetaldehyde from ethanol, is not in the database; therefore, we could not adjust for the impact. Fifth, since we used data from approximately 20% of the total subjects recruited in the J-MICC study, participant bias may have affected the study outcomes. For example, compared with the parent population, the present cohort included higher proportions of women and the elderly [The current study validated for the first time the hypothesis that individuals with inhibited ALDH activity require more energy when they consume ethanol, possibly to activate the GSH detoxification system, an alternative detoxification pathway of ALDH. However, several limitations should be addressed. First, since this is an observational and cross-sectional study, our hypothesis should be confirmed by other methods, including intervention studies. Second, the accuracy in the amount of alcohol consumption and energy and macronutrient intake was limited because the calculations were performed using self-administered questionnaires. Bongers et al. pointed out discrepancies between self-reported and actual alcohol consumption levels; alcohol consumption in heavy drinkers tends to be overreported in men and underreported in women, particularly in older women with lower educational attainment . Third,  elderly . Lastly,ALDH2 rs671 variant allele, ALDH2*2, carriers who routinely consume alcoholic beverages. Although the mechanism needs to be further verified, this finding supports the hypothesis that individuals with low ALDH activity require induced GSH detoxification upon aldehyde overload. This study provides a possible scientific basis for understanding the mechanism of homeostasis associated with aldehyde loading, and suggests a novel phenotype of a common genetic polymorphism in East Asia.In conclusion, we performed an epidemiological analysis to examine a hypothesis based on previous animal research, and the present results suggested that increased energy and macronutrient intake are required in


Human colorectal cancer (CRC) is a global health burden, most notably for minority populations. While screening tools have helped improve disease detection and surveillance, CRC is a complex disease with a diverse set of molecular features that are linked to the location of the primary tumor. These features provide challenges to treatment and improving patient outcome. In addition, tumor metastases to liver and other organ systems are a main cause of CRC-related mortality and represent a substantial obstacle to improving patient outcomes. In this review, we summarize CRC tumor subgroups, their molecular features, treatments and outcomes with attention to health disparities and tumor metastases. Human colorectal cancer (CRC) is one of the most common malignancies in men and women across the globe, albeit CRC incidence and mortality shows a substantial racial and ethnic disparity, with the highest burden in African American patients. Even with effective screening tools such as colonoscopy and diagnostic detection assays, CRC remains a substantial health burden. In addition, primary tumors located in the proximal (right) or distal (left) sides of the colorectum have been shown to be unique tumor types that require unique treatment schema. Distal metastases in the liver and other organ systems are the major causes of mortality in CRC patients. Characterizing genomic, epigenomic, transcriptomic and proteomic (multi-omics) alterations has led to a better understanding of primary tumor biology, resulting in targeted therapeutic advancements. In this regard, molecular-based CRC subgroups have been developed that show correlations with patient outcomes. Molecular characterization of CRC metastases has highlighted similarities and differences between metastases and primary tumors; however, our understanding as to how to improve patient outcomes based on metastasis biology is lagging and remains a major obstacle to improving CRC patient outcomes. In this review, we will summarize the multi-omics features of primary CRC tumors and their metastases across racial and ethnic groups, the differences in proximal and distal tumor biology, molecular-based CRC subgroups, treatment strategies and challenges for improving patient outcomes. Human colon and rectal cancers are worldwide health burdens, with over 1.9 million new combined cases and 935,000 deaths in 2020, accounting for approximately 9–10% of all cancer incidence and mortality ,5. HowevAs with every form of human cancer, racial disparities in incidence and outcome are present for CRC patients. African Americans followed by American Indians display increased CRC incidence, advanced disease stage and mortality as compared to Caucasian Americans . AfricanWith respect to treatment disparities, one report showed that while African American and Caucasian CRC patients were examined by oncologists at similar or equal rates, Caucasian patients were more likely to receive chemotherapy ,15. HoweTreatment disparities are also related to those physicians treating African American versus Caucasian patients, such that medical personnel treating African American patients are more likely to be trained outside of the United States and are less familiar with current screening methods, use older technologies, are understaffed, have overall less funding and have overall higher patient volumes. Since minority groups are generally geographically segregated, the access to local quality care is more limited, thereby resulting in critical overdemand of medical access that is inherently compromised . Indeed,A study led by Warren Anderson  investigSimilar conclusions were reached by Kane et al., who showed that while all minority groups showed lower CRC screening rates compared to Caucasians, this disparity was reduced after adjustment for socioeconomic and behavioral factors . ImproveEarly-onset CRC is becoming increasingly prevalent in the US population over the past 20 years, such that the recommended age for colonoscopy-based screening for polyps, adenomas and adenocarcinomas is now 45 years of age for the general population ,25. EarlMLH1, MSH2 and MSH6 [CRC progresses from the development of precancerous polyps from colonic epithelium to the formation of adenomas, followed by the development of adenocarcinomas that may ultimately invade the colonic epithelium and metastasize to liver and other tissues. Most CRCs develop sporadically, while only 5% of CRCs are derived from germline transmission of mutated driver genes. Among the heritable CRCs, Lynch syndrome represents less than 4% of all CRCs and occurs due to germline mutations in several mismatch repair genes, including and MSH6 .APC alterations (mutations or deletions) represent the first events in CRC tumorigenesis as normal colonic epithelial cells convert into hyperproliferative cells. APC alterations occur in up to 90% of all CRCs [KRAS mutations (KRAS-mut), chromosome 18 deletions that include the DCC locus, and inactivation of the TGF-β response by SMAD2/SMAD4 changes are key steps in the development of adenomas. Finally, TP53 alterations (mutations and/or deletions) represent the gatekeeper events in the development of adenocarcinomas  colon, display the BRAF (V600E) point mutation (BRAF-mut) and are underscored by increased size with a flattened and serrated appearance [While approximately two thirds of CRCs develop from the traditional adenoma pathway, one third of CRCs develop from the serrated pathway ,35. Thespearance ,35  and chromosomal instability (CIN), in which key tumor suppressor genes are deleted and/or mutated. This contrasts with serrated CRCs located in the proximal colon, which are enriched in the BRAF mutation, are diploid (non-CIN) but are hypermutated, display microsatellite instability (MSI) due to MLH1 silencing by DNA methylation and are more prevalent in older women. Interestingly, proximal tumors present with a more diverse panel of somatic mutations compared to distal (non-hypermutated) tumors, namely with enrichment for mutations in ACVR2A, TGFBR2, the mismatch repair genes MSH3 and MSH6 and the DNA polymerase POLE [These findings establish differences in tumor etiology and molecular features between CRCs that originate in the left  versus the right  region of the colon. The normal colonic tissues on the left and right sides originate from unique developmental origins . Most CRase POLE . From a APC, TP53, KRAS and PIK3CA in left-sided tumors, while mutations in ACVR2A, TGFBR2, the mismatch repair genes MSH3 and MSH6 and the DNA polymerase POLE were enriched in right-sided tumors. Giannakis and colleagues performed WES on 619 archived CRC tumors and normal-adjacent tissue pairs to identify novel mutations in BCL9L, CTCF, KLF5 and RBM10. In addition, the extent of somatic mutation correlates with the formation of neoantigen peptides that invoke anti-tumor immunity and is tied to lymphocytic infiltrations and patient survival [The Cancer Genome Atlas (TCGA) presented whole genome sequencing (WGS) and whole exome sequencing (WES) on nearly 300 CRCs to show mutations in survival .KRAS mutation rates in African American patients, as well as mutations of three genes  only in African American CRC patients, although these mutations have a low frequency (3–6%) [An analysis of genetic alterations in primary CRCs between African American and Caucasian patients showed elevated y (3–6%) ,38. Guday (3–6%) .TP53 alterations showed that while TP53 mutation profiles are similar between African American and Caucasian CRC patients, the Pro/Pro polymorphism in codon 72 occurs at a higher frequency in African American patients and is linked to poor patient outcomes [CNV analyses on primary CRCs from African American and Caucasian patients showed African American-specific aberrancies on chromosomes 11, 17p 20q and X ,41,42, aoutcomes . A compaoutcomes .ATRX mutations as well as significantly different mutation frequencies in ATRX, APC, FBXW7, LRP1B, PIK3CA and RNF43 across racial groups, suggesting that somatic mutation profiles occur as a function of race within early-onset tumors [APC and other known CRC driver genes compared to Caucasians but harbored loss-of-function mutations in BCL9L, a negative regulator of B-catenin. Interestingly, Wnt regulatory genes were silenced by promoter DNA hypermethylation in these tumors, suggesting distinct molecular routes for CRC tumorigenesis  as well as other mutations in genes in the EGFR and TGF-B pathways that influence treatment success [AKT, BRCA2, FAT1, FGF1, KDR and NOTCH1 that may be therapeutic targets for disease treatment.Since CRC mortality is driven by metastasis, examining the genetic profiles of CRC metastases may identify novel molecular alterations to identify the changes that occur during metastasis and those that can be exploited for patient treatment and surveillance. Vermaat performed targeted sequencing of over 1200 cancer-related genes across 21 primary CRC/liver metastasis pairs to show that liver metastases are genetically unique from those of the primary tumor, with 817 coding region alterations exclusively in the liver metastases . This an success . An anal success  identifi success . Ishaque success  performeAPC intron splicing alterations as well as CTNNB1 deletions. As shown by other reports, patients with right-sided primary metastatic disease were of older age at the time of diagnosis and had reduced survival, increased mutation burden, and enrichment of AKT1, KRAS, PIK3CA, PTEN, RNF43 and SMAD4 mutations. In contrast, left-sided tumors exhibit the following characteristics: (1) display mutations in APC, NRAS and TP53; (2) display amplification enrichment in receptor tyrosine kinase signaling genes ERBB2, ERBB3, EGFR and FGFR; (3) do not display mutations or CNV in genes related to cell division; and (4) may be more prone to fluctuations in intestinal microbiomes, suggesting that patients with left- and right-sided CRCs have unique molecular pathways of metastasis. Comparing these data to a separate data cohort of metastatic tumors, as well as to The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortia (ICGC) data for primary CRC mutations, Mendelaar et al. [LINC00672 mutations are associated with treatment response. In addition, the presence of FBXW7 mutations signaled poor patient response to EGFR-targeted therapies.Yaeger et al.  analyzedr et al.  identifiEpigenetic alterations are hallmarks of CRC and every other tumor type. The main facets of epigenetics, DNA methylation, chromatin modifications of histone tail proteins, nucleosome occupancy and non-coding RNAs, namely microRNA (miRNA) and long non-coding RNA (lncRNA), are key for gene regulation and higher-order organization of chromatin. These epigenetic facets work in symphony to compose multiple levels of gene regulation. In human cancers, epigenetic aberrancies function to silence tumor suppressor genes and activate oncogenes. However, epigenetic marks are reversible via passive or active mechanisms, making them attractive therapeutic targets for disease treatment in reverting epigenome profiles to a more normal-like state. For the purposes of epigenetic alterations in CRC, we will focus on DNA methylation in this review.3) group to the C-5 position of cytosine nucleotides in the 5′-CG-3′ or CpG sequence context. Methyl marks are placed by the enzymatic activities of DNA methyltransferases (DNMTs) that use S-adenosylmethionine as a required co-factor. In human cells, DNMT1, DNMT3A and DNMT3B are largely responsible for DNA methylation catalysis, in which DNMT1 is involved in immediately copying the DNA methylation patterns from the parental strand to the nascent daughter strand during DNA replication, while DNMT3A and DNMT3B are responsible for placing methyl-marks at CpGs that were originally unmethylated. However, DNMT1 and DNMT3B are thought to work together to maintain DNA methylation profiles. DNMT3B also serves as an accessory scaffold protein in recruiting DNMT3A to specific CpG sites and is a key DNMT enzyme for catalyzing DNA methylation of gene body (transcribed) regions, in which DNMT3B is recognized by SETD2 for generating histone H3 lysine 36 trimethylation (H3K36me3) marks that are correlated with active gene expression.DNA methylation in human and other mammalian tissues is generally defined as the addition of a methyl , 5-aza-2′-deoxycytidine  and S110, enzymatic DNA demethylation is catalyzed in a stepwise manner through oxidative conversion of 5-methylcytosine (5mC) to 5-hydroxycytosince (5mC) by the TET family of enzymes. After TETs convert 5mC to 5hmC, 5hmC marks are further converted to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), after which both 5fC and 5caC marks are ultimately replaced with unmethylated cytosines through base excision repair ,52,53.MLH1, CDKN2A, MGMT, MLH1, RUNX3, the SFRP gene family, TPEF and VIM . More riewed in  analyzedALX4, BMP3, CDH1, CDKN2A, MGMT, MLH1, NRDG4, PRIMA1, SDC2, SEPT9, SFPR2, TFPI2, TMEFF2 and VIM in blood, stool and/or urine specimens [Most cancer-specific DNA methylation changes do not result in gene expression changes but are biomarkers of disease. DNA methylation-based biomarkers are highly effective in disease monitoring and predicting patient outcome and response to treatment. DNA methylation biomarkers can be found in tumor-derived cell-free DNA (cfDNA) in patient blood serum or plasma, urine and fecal matter. DNA methylation markers include pecimens ,56,57.SEPT9 in tissue and blood plasma, marketed as Epi proColon, and (2) BMP3 and NDRG4 DNA methylation with KRAS mutation screening in stool, marketed as Cologuard. It should be noted that SDC2 DNA methylation testing in stool samples is approved in Korea (EarlyTect-Colon Cancer) and China (Colosafe) for CRC detection, and a yet unapproved test described as ColoDefense includes screening for both SDC2 and SEPT9 DNA methylation (reviewed in [SEPT9 DNA methylation in plasma shows high sensitivity (60–80%) and specificity (80–99%) in identifying stage I and II disease and outperforms both carcinoembryonic antigen (CEA) and fecal occult blood tests for CRC detection. However, SEPT9 DNA methylation shows low sensitivity in detecting adenomas (summarized in [Two DNA methylation biomarker panels are FDA approved for early detection of CRC: (1) iewed in ). SEPT9 rized in ).B3GNT7, CHN2, MUC12 and TBX20 that is linked to gene expression and poor patient outcome [BRD4, FBXL18, KCNQ1 and MET, among others, and the DNA methylation score of this panel was positively correlated with patient survival [NR1H2, SCRIB and UACA) DNA methylation signature that correlated with patient OS irrespective of patient age and gender [ARHGEF4, AV3, BMP3, CHST2, DOCK10, IKZF1, LRRC4, NDRG4, OPLAH, PPP2R5C, PDGFD, QKI, SFMBT2 and ZNF625). The panel detected metastatic CRC with 90% sensitivity and 90% sensitivity and is superior to CEA measurements. (5) Deng and colleagues [BCAN, BCAT, IKZF1, VAV3 and two regions of SEPT9 that were predictive of disease recurrence and outperformed CEA expression to detect CRCs across all four disease stages. (7) Muthamilselvan et al. [FBN1), stage-II (FOXG1), stage-III (HCN1) and stage-IV . (8) Chen and colleagues also analyzed TCGA DNA methylation data to identify a panel of five immune-related genes—FGF5, LTBP4, PIK3CD and PLXNC1 and SCTR—the composite DNA methylation signature of which correlated with prognosis for stage II and III CRC patients [The large extent of DNA methylation changes in CRC has proven fruitful for the development of DNA methylation-based marker panels that predict patient outcome and response to treatment, as well as those that are linked to tumor stage and grade. Several reports have used DNA methylation data to build models related to patient outcome, which include the following: (1) Zhang and colleagues identified differential DNA methylation in  outcome . (2) An  outcome . This clsurvival . (3) Gond gender . (4) Xied gender  used tumlleagues  analyzedlleagues  evaluaten et al.  surveyedpatients . (9) As patients ,25, in cpatients , and a rpatients  used IllCACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1. CIMP is highly concordant with BRAF (V600E) point mutations, MLH1 epigenetic silencing, MSI and a hypermutation profile but not TP53 mutations or chromosomal instability, and it occurs in right-sided tumors and is enriched in female patients with poor clinical outcome [KRAS mutations, chromosomal instability and microsatellite stability and are present in the right side of the colon in male patients [In addition to being used for predictive and prognostic purposes, cancer-specific DNA methylation profiles can be categorized to identify tumor subgroups. Indeed, Toyota and colleagues  first de outcome ,71,72. O outcome  subsequepatients ,72,73. Dpatients ,72,74,75BRAF mutant (BRAF-mut), KRAS wild-type (KRAS-wt), high microsatellite instability (MSI-H); (2) CIMP-positive, BRAF-mut, KRAS-wt, microsatellite stable (MSS) or low microsatellite instability (MSI-low); (3) CIMP-negative, BRAF-wt, KRAS-mut, MSS/MSI-low; (4) CIMP-negative, BRAF-wt, KRAS-wt, MSS/MSI-low; and (5) CIMP-negative, BRAF-wt, KRAS-wt, MSS/MSI-low. Group 2 patients displayed the worst outcome, while MSI-H status was significantly associated with improved patient outcome, especially in cases harboring CIMP and BRAF-mut. In addition, Group 1 and Group 2 patients had significant associations with disease-specific survival (DSS), while Group 3 patients had the worst DSS, most notably in Group 3 patients with stage I-III disease. Indeed, KRAS-mut patients showed poor survival overall, specifically in MSS/MSI-low cases. These findings were also shown by Murcia and colleagues [KRAS-mut and MSS status or CIMP-negative, BRAF-wt, KRAS-wt and MSS status.Stratifying CRCs by CIMP status and other molecular co-variates has been shown to result in a profound dissemination of CRC patient outcome and response to therapy. In pooling data for over 5000 CRC patients, Phipps et al.  categorilleagues , who perBRAF-mut, DNA hypermutation, immune infiltration and activation and are linked to poor patient survival after relapse. CMS2 tumors are non-CIMP, with extensive CNV and activated WNT and MYC signaling. CMS3 tumors display CIMP-L DNA methylation, MSI/MSS, nearly diploid copy number, KRAS-mut and metabolic dysregulation. CMS4 tumors are non-CIMP but display CNVs, stromal cell infiltration, activated TGF-β and angiogenic signaling, and CMS4 patients show poor survival. To simplify CMS classification, CMS1 status can be effectively determined by measuring MSI status, CMS2 and CMS3 subgroups can be stratified by differential DNA methylation of specific loci, and CMS4 tumors can be determined by immunohistochemistry and gene expression classifiers [Given these complicated aspects of CRC biology, a set of four consensus molecular subgroups (CMS) were developed that include RNA expression signatures : CMS1 M. CMS1 tussifiers ,80,81,82LINE-1 repetitive element DNA methylation levels between primary CRCs and liver metastases, Hur et al. [LINE-1 DNA methylation in metastases compared to primary CRCs, also resulting in the activation of proto-oncogenes, including MET, CHRM3 and RAB3IP. (2) Ili et al. [BDNF, FIGN, HCN4, HTRA3 and STAC2, in which DNA methylation status of the panel was significantly associated with poor patient outcome. (3) Orjuela et al. [LBX2 as a biomarker of metastasis that outperforms CEA and imaging testing. (5) An analysis of DNA methylation in 59 early- and late-stage CRCs, normal-adjacent tissues and metastases identified DNA methylation signatures of metastases in early-stage tumors that predicted outcome and the possibility of metastasis [IMPA2 correlated with gene silencing in primary tumors and metastases and predicted poor patient outcome and advanced stage. (7) Bisulfite pyrosequencing of CDH1, CDH13, CDKN2A (p16), CDKN2A (p14), ESR1, HPP1, MGMT, MINT1, MINT2, MINT31, MLH1, THBS1 and TIMP3 across paired primary tumor-liver metastasis pairs, as well as CRCs with or without metastases, showed stochastic variability as a function of metastasis and suggest that cancer-specific DNA changes mostly occur prior to metastasis [While molecular features of the primary tumor are expected to be represented in tumor metastases, there is an increasing body of evidence that metastases are characterized by novel molecular features that are not present in the corresponding primary tissue. Epigenetic profiling of liver metastases is limited compared to the primary tumor; however, several reports over the past decade have painted interesting portraits as to the role of DNA methylation changes in CRC. (1) Global DNA hypomethylation is common in CRC, in which non-CpG rich sequences in single copy loci and repetitive elements lose DNA methylation compared to normal tissues (reviewed in ). In comr et al.  detectedi et al.  compareda et al.  used meta et al.  used bistastasis . (6) DNAtastasis , which iESR1 CpG island [Cancer is a disease of aging since increased age correlates with cancer risk, and age-related DNA methylation alterations have been described . G island . SubsequG island ,94,95,96G island . Using IG island . The HorG island  surveyedG island ,101.Epigenetic clocks have been important contributors to better understanding age-related diseases such as Alzheimer’s disease, human cancer and cardiovascular disease, as well as how epigenetic age is linked to inherited syndromes and other diseases . EpigeneALU, LINE-1 and SAT2 repetitive elements have been linked to environmental exposures as well as education, occupation, social status, wealth and family income status . With tiewed in  performeiewed in  identifiARHGEF4, CHL1, CHL4, GDF, ITGA4, NELL and several microRNAs including miR-9-3p and miR-124-3p in African American CRC patients [In contrast to cancer risk, there are only a handful of examples of CRC DNA methylation data that are linked to racial health disparities. A global meta-analysis of CIMP prevalence showed that it was highest in Africa and lowest in Asia, Europe and Australia . In addipatients .KRAS-mut, (C4) cancer stem cell, (C5) CIN with Wnt pathway upregulation and (C6) CIN with a normal-like expression profile [BRAF-mut, but groups C2, C3 and C4 were enriched for CIMP and proximal primary tumor location. Alternatively, C1, C5 and C6 subgroups were enriched for CIN, non-CIMP and TP53-mut. The C4 subgroup was enriched for metastatic disease; however, there were no associations between subgroup and tumor staging. It should be noted that subgroups C4 and C6 were associated with shortened relapse-free survival.Much like genetic (mutations and CNV) and epigenetic (DNA methylation) alterations, gene expression changes are common in CRC. From a tumor subgrouping perspective, CRC mRNA gene expression subgrouping generally follows the DNA methylation-based grouping with three subgroups: CIMP/MSI, invasive and chromosomal instability (CIN) . In cont profile . The C2 OR7E104P, GATA1, NPHP3-AS1, POM121L12, ZNF843, NYX, SLC22A25, C20orf203 and SLC4A1, and the enriched pathways are related to cell cycle, mitosis, M phase, membrane targeting and translation. Third, Su et al. [ADH1B, CLEC3B, GCNT2, GLDN, GLP2R, GREM2, JCHAIN, LYVE1, MAMDC2, PLP1, SCARA5 and TMEM100. In addition, individual expression levels of SULT1B1, PTGDR2, GPR15, BMP5 and CPT2 were significantly associated with OS. Pathway analyses showed enrichment of the curated expression panel for processes including metabolic processes and small molecule transport.Cancer-specific gene expression profiles are important in identifying functional networks important for cancer development and progression. Gene networks based on large expression data sets help to identify unique interactions within and between individual networks and expression pathways. Three recent examples examine this. First, Kim and colleagues analyzed CRC gene expression data sets  from uniu et al.  mined thBGN, FAP, GADD45B, INHBA, MK167, MYBL2, MYC and five reference normalization genes [ADAL, ANKRD36B, ARHGAP6, CRYBB2, C17orf81 PSPH, TRNT1, VSIG10L, WDR8 and ZNF835 [Based on whole transcriptome data, the Oncotype DX Colon Cancer Assay panel of 12 genes was developed to predict recurrence after resection of stage II/III disease ,115,116.on genes . Intereson genes . These don genes . This wad ZNF835 .AHSG, APOA2, CP, FGA, F2, HPX, HRG, ITIH2, PLG and SERPINC1. Among these, expression of ITIH2 was statistically significantly associated with survival, suggesting that it is important for CRC metastasis [APC, TP53, KRAS and PIK3CA did not correlate with patient outcome. The gene expression analyses, however, identified 97 genes with aberrant expression, including significant differences in DSCAML1, FGF18, NEUROD1, PLAC1, SAA2, SFTA2 and OTOP3 that were associated with patient outcome. FGF18 and DSCAML1 expression had a protective effect, while the expression of the other genes in the panel was positively associated with outcome risk [Gene expression data can also be used in profiling CRC metastases. A meta-analysis of gene expression data sets of primary CRCs and liver metastases  unveiledtastasis . In comptastasis . Interesome risk .AXIN2 super-enhancer DNA hypomethylation in immune-modulating and antigen-presenting genes to provide MSI-H CIMP tumors with an advantage of immune escape [Increasing amounts of evidence indicate that the tumor microenvironment (TME) is a substantial contributor to CRC tumorigenesis and disease progression. The TME is composed of several cell types surrounding the colorectum, including adipocytes, endothelial cells, fibroblasts, immune cells and inflammatory cells ,121. Tume escape .It is becoming clear that immune-related gene expression differences play a role in CRC health disparities. For instance, African Americans display reduced antitumor cytotoxic immunity . Second,AURKA, AURKB, BUB1, CCNA2, CDK1, CENPF, MAD2L1, TPX2 and UBE2C) are enriched in microtubule and tubule binding and were negatively associated with the presence of fusobacteria and positively associated with blautia, suggesting that these bacteria are interacting partners for modulating gene expression [The gut microbiome consists of microorganisms required for digestive health and is also influential in CRC progression , indicatpression .Therapies using 5-fluorouracil (5-FU) have been the backbone of CRC treatments for several decades. Used as an inhibitor of DNA synthesis by preventing thymidine production, 5-FU has demonstrated efficacy in treating CRC patients and is now generally combined with (1) folinic acid (leucovorin) and oxaliplatin to form the FOLFOX chemotherapy regimen; (2) folinic acid and irinotecan to form the FOLFIRI regimen; (3) folinic acid, oxaliplatin and irinotecan in the FOXFOXIRI regimen ; or (4) Folinic acid is a vitamin B derivative that exacerbates the 5-FU cytotoxicity, irinotecan is a topoisomerase inhibitor that prevents DNA uncoiling and replication, and oxaliplatin is a platinum-based chemotherapy agent that inhibits DNA synthesis and DNA repair. Capecitabine (Xeloda) is an oral fluoropyrimidine that enters the cell and is converted to 5-FU in three steps , in whicMost targeted CRC treatment strategies have focused on the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways (summarized in ). The acRAS-wt metastatic CRC patients resulted in improved patient OS and PFS. However, these agents are only effective in RAS-wt tumors. In fact, only RAS-mutation status and, to a lesser extent, BRAF-mutation status, guide CRC therapeutic decisions [Several monoclonal antibodies, such as cetuximab and panitumumab, have been developed that target the EGFR pathway by binding to the EGFR extracellular domain to block the binding of EGF ligands ,139. Twoecisions ,143, Appecisions .ERBB2 amplification occurs in 3% of all metastatic CRCs and in 5% of CRC patients with RAS-wt tumors [Other agents effective in treating CRC patients include nivolumab, a monoclonal antibody that targets the PD-1 receptor of lymphocytes. Ipilimumab is a monoclonal antibody that inhibits CTLA-4 and subsequently activates the immune response, while pembrolizumab is a monoclonal antibody that also targets PD-1 and is FDA approved for mismatch repair-deficient metastatic CRC . Pertruzt tumors , thus ret tumors ).NTRK1) gene, and NTRK1 alterations have been observed in several human cancers, including colorectal cancer. Interestingly, NTRK1 is involved in multiple chromosomal rearrangements in human cancers that have oncogenic activity, including TPM3-TRK in colorectal cancer as well as MPRIP-NRTK1 and CD74 in non-small cell lung cancer [APC and TP53 alterations as well as DNA hypermutation, MSI-H, enrichment in POLE/POLD1 mutations and location on the right side of the colorectum [Entrectinib is a small-molecule competitive inhibitor of tropomyosin neurotropic tyrosine receptor kinases A, B and C , the proto-oncogene tyrosine protein kinase ROS1 and anaplastic lymphoma kinase (ALK) . Similarg cancer . The CRCg cancer  and co-olorectum . NTRK-allorectum ,153, sugKRAS-wt metastatic colon cancer patients who were treated with either FOLFIRI or FOLFOX before treatment with bevacizumab or cetuximab showed significant differences in OS and PFS after stratification by tumor location [RAS-wt patients with left-sided tumors had improved PFS and OS compared to patients with right-sided tumors. Patients with left-sided RAS-wt tumors showed a significantly improved outcome when given cetuximab + FOLFIRI compared to right-sided, RAS-wt CRC patients [CIMP-H tumors are generally found in the proximal (right) region of the colon; therefore, tumor location is an important determinant of CRC patient outcome (summarized in ). Even llocation ,158, evepatients . Recentlpatients  performeKRAS-wt CRC patients who were given cetuximab demonstrated a shortened PFS compared to non-CIMP patients . InS and OS . InteresS and OS .BRAF-mut CRCs [CIMP-positive tumors are highly enriched for the BRAF V600E point mutation and are resistant to EGFR-based therapies. In addition, treatments with BRAF inhibitors, including vemurafenib, as single agents have not demonstrated clinical efficacy ,170. Intmut CRCs ,172,173.mut CRCs ,174 or tmut CRCs ), suppormut CRCs ,176.BRAF-mut CRC patients. BRAF-mut and BRAF-wt CRC patients with microsatellite instability (MSI-H) given the anti-PD-1 antibody pembrolizumab showed improved outcomes [BRAF-mut, MSI-H CRC patients who were administered nivolumab (anti-PD-1) and ipilimumab (anti-T lymphocyte antigen-4) showed overall response rates similar to those for BRAF-wt CRC patients [BRAF-mut patients than for BRAF-wt patients. Finally, treatment of microsatellite-instable CRC patients with pembrolizumab (anti-PD-1) showed improved PFS compared to standard chemotherapy as a first-line approach [Clinical trials have also evaluated the efficacy of immune checkpoint inhibitors in treating outcomes ,169. Trepatients , althougapproach .As described above, DNA methylation changes are pervasive in CRC and all other forms of human cancer and have roles in gene silencing and activation. DNA methylation is a dynamic and pharmacologically reversible epigenetic mark, making it an attractive therapeutic target summarized in . Indeed,BRAF-wt, KRAS-wt, NRAS-wt and metastatic CRCs have a median survival with treatment of 30 months [BRAF-wt, RAS-wt patients also show modest survival improvement of 2–4 additional months when given cetuximab and panitumumab with backbone chemotherapy compared to chemotherapy alone [Metastatic CRC represents a substantial therapeutic challenge, especially since the survival rates for patients with metastatic disease are dismal. Biller and Schrag  performe0 months . However0 months . These Bpy alone . Left-sipy alone .BRAF-mut patients saw a survival period of 9.3 months when given BRAF- and EGFR-based inhibitors compared to 5.9 months for those given standard chemotherapy [otherapy . Finallyotherapy . Patientotherapy  and showotherapy .KRAS/BRAF mutation, MSI and tumor location complicate treatment response and patient outcomes. CRC treatments historically target DNA synthesis and EGFR/VEGFR signaling; however, new treatment schemes have combined these with immunotherapies. Multi-omics profiling has provided new windows into CRC tumorigenesis and metastasis, but profiling minority populations with adenomas or adenocarcinomas is important for reducing health disparities and improving patient outcomes.In summary, CRC is a worldwide health burden, and CRC outcomes encapsulate socio-economic and racial disparities that affect patient diagnosis, treatment and outcome. From a biological standpoint, CRC is a heterogeneous disease in which biological covariates such as CIMP,