Triterpenoid ursolic acid regulates the environmental carcinogen benzo[a]pyrene-driven epigenetic and metabolic alterations in SKH-1 hairless mice for skin cancer interception.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens accountable to developing skin cancers. Recently, we reported that exposure to benzo[a]pyrene (B[a]P), a common PAH, causes epigenetic and metabolic alterations in the initiation, promotion and progression of non-melanoma skin cancer (NMSC). As a follow-up investigation, this study examines how dietary triterpenoid ursolic acid (UA) regulates B[a]P-driven epigenetic and metabolic pathways in SKH-1 hairless mice. Our results show UA intercepts against B[a]P-induced tumorigenesis at different stages of NMSC. Epigenomic cytosines followed by guanine residues (CpG) methyl-seq data showed UA diminished B[a]P-mediated differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq revealed UA revoked B[a]P-induced differentially expressed genes (DEGs) of skin cancer-related genes, such as leucine-rich repeat LGI family member 2 (Lgi2) and kallikrein-related peptidase 13 (Klk13), indicating UA plays a vital role in B[a]P-mediated gene regulation and its potential consequences in NMSC interception. Association analysis of DEGs and DMRs found that the mRNA expression of KLK13 gene was correlated with the promoter CpG methylation status in the early-stage comparison group, indicating UA could regulate the KLK13 by modulating its promoter methylation at an early stage of NMSC. The metabolomic study showed UA alters B[a]P-regulated cancer-associated metabolisms like thiamin metabolism, ascorbate and aldarate metabolism during the initiation phase; pyruvate, citrate and thiamin metabolism during the promotion phase; and beta-alanine and pathothenate coenzyme A (CoA) biosynthesis during the late progression phase. Taken together, UA reverses B[a]P-driven epigenetic, transcriptomic and metabolic reprogramming, potentially contributing to the overall cancer interception against B[a]P-mediated NMSC.

An Update on Potential Molecular Biomarkers of Dietary Phytochemicals Targeting Lung Cancer Interception and Prevention.

Since ancient times, dietary phytochemicals are known for their medicinal properties. They are broadly classified into polyphenols, terpenoids, alkaloids, phytosterols, and organosulfur compounds. Currently, there is considerable interest in their potential health effects against various diseases, including lung cancer. Lung cancer is the leading cause of cancer deaths with an average of five-year survival rate of lung cancer patients limited to just 14%. Identifying potential early molecular biomarkers of pre-malignant lung cancer cells may provide a strong basis to develop early cancer detection and interception methods. In this review, we will discuss molecular changes, including genetic alterations, inflammation, signal transduction pathways, redox imbalance, epigenetic and proteomic signatures associated with initiation and progression of lung carcinoma. We will also highlight molecular targets of phytochemicals during lung cancer development. These targets mainly consist of cellular signaling pathways, epigenetic regulators and metabolic reprogramming. With growing interest in natural products research, translation of these compounds into new cancer prevention approaches to medical care will be urgently needed. In this context, we will also discuss the overall pharmacokinetic challenges of phytochemicals in translating to humans. Lastly, we will discuss clinical trials of phytochemicals in lung cancer patients.

Metabolic rewiring and epigenetic reprogramming in leptin receptor-deficient db/db diabetic nephropathy mice.

BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the United States. Emerging evidence suggests that mitochondrial metabolism and epigenetics play an important role in the development and progression of DN and its complications. For the first time, we investigated the regulation of cellular metabolism, DNA methylation, and transcriptome status by high glucose (HG) in the kidney of leptin receptor-deficient db/db mice using multi-omics approaches. METHODS: The metabolomics was performed by liquid-chromatography-mass spectrometry (LC-MS), while epigenomic CpG methylation coupled with transcriptomic gene expression was analyzed by next-generation sequencing. RESULTS: LC-MS analysis of glomerular and cortex tissue samples of db/db mice showed that HG regulated several cellular metabolites and metabolism-related signaling pathways, including S-adenosylmethionine, S-adenosylhomocysteine, methionine, glutamine, and glutamate. Gene expression study by RNA-seq analysis suggests transforming growth factor beta 1 (TGFß1) and pro-inflammatory pathways play important roles in early DN. Epigenomic CpG methyl-seq showed HG revoked a list of differentially methylated regions in the promoter region of the genes. Integrated analysis of DNA methylation in the promoter regions of genes and gene expression changes across time points identified several genes persistently altered in DNA methylation and gene expression. Cyp2d22, Slc1a4, and Ddah1 are some identified genes that could reflect dysregulated genes involved in renal function and DN. CONCLUSION: Our results suggest that leptin receptor deficiency leading to HG regulates metabolic rewiring, including SAM potentially driving DNA methylation and transcriptomic signaling that could be involved in the progression of DN.

The environmental carcinogen benzo[a]pyrene regulates epigenetic reprogramming and metabolic rewiring in a two-stage mouse skin carcinogenesis model.

Non-melanoma skin cancer (NMSC) is the most common cancer in the world. Environmental exposure to carcinogens is one of the major causes of NMSC initiation and progression. In the current study, we utilized a two-stage skin carcinogenesis mouse model generated by sequential exposure to cancer-initiating agent benzo[a]pyrene (BaP) and promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), to study epigenetic, transcriptomic and metabolic changes at different stages during the development of NMSC. BaP/TPA caused significant alterations in DNA methylation and gene expression profiles in skin carcinogenesis, as evidenced by DNA-seq and RNA-seq analysis. Correlation analysis between differentially expressed genes and differentially methylated regions found that the mRNA expression of oncogenes leucine rich repeat LGI family member 2 (Lgi2), kallikrein-related peptidase 13 (Klk13) and SRY-Box transcription factor (Sox5) are correlated with the promoter CpG methylation status, indicating BaP/TPA regulates these oncogenes through regulating their promoter methylation at different stages of NMSC. Pathway analysis identified that the modulation of macrophage-stimulating protein-recepteur d'origine nantais and high-mobility group box 1 signaling pathways, superpathway of melatonin degradation, melatonin degradation 1, sirtuin signaling and actin cytoskeleton signaling pathways are associated with the development of NMSC. The metabolomic study showed BaP/TPA regulated cancer-associated metabolisms like pyrimidine and amino acid metabolisms/metabolites and epigenetic-associated metabolites, such as S-adenosylmethionine, methionine and 5-methylcytosine, indicating a critical role in carcinogen-mediated metabolic reprogramming and its consequences on cancer development. Altogether, this study provides novel insights integrating methylomic, transcriptomic and metabolic-signaling pathways that could benefit future skin cancer treatment and interception studies.

Metabolomic, DNA Methylomic, and Transcriptomic Profiling of Suberoylanilide Hydroxamic Acid Effects on LPS-Exposed Lung Epithelial Cells.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor with anticancer effects via epigenetic and non-epigenetic mechanisms. The role of SAHA in metabolic rewiring and epigenomic reprogramming to inhibit pro-tumorigenic cascades in lung cancer remains unknown. In this study, we aimed to investigate the regulation of mitochondrial metabolism, DNA methylome reprogramming, and transcriptomic gene expression by SAHA in lipopolysaccharide (LPS)-induced inflammatory model of lung epithelial BEAS-2B cells. LC/MS was used for metabolomic analysis, while next-generation sequencing was done to study epigenetic changes. The metabolomic study reveals that SAHA treatment significantly regulated methionine, glutathione, and nicotinamide metabolism with alteration of the metabolite levels of methionine, S-adenosylmethionine, S-adenosylhomocysteine, glutathione, nicotinamide, 1-methylnicotinamide, and nicotinamide adenine dinucleotide in BEAS-2B cells. Epigenomic CpG methyl-seq shows SAHA revoked a list of differentially methylated regions in the promoter region of the genes, such as HDAC11, miR4509-1, and miR3191. Transcriptomic RNA sequencing (RNA-seq) reveals SAHA abrogated LPS-induced differentially expressed genes encoding proinflammatory cytokines, including interleukin 1α (IL1α), IL1ß, IL2, IL6, IL24, and IL32. Integrative analysis of DNA methylome-RNA transcriptome displays a list of genes, of which CpG methylation correlated with changes in gene expression. qPCR validation of transcriptomic RNA-seq data shows that SAHA treatment significantly reduced the LPS-induced mRNA levels of IL1ß, IL6, DNA methyltransferase 1 (DNMT1), and DNMT3A in BEAS-2B cells. Altogether, SAHA treatment alters the mitochondrial metabolism, epigenetic CpG methylation, and transcriptomic gene expression to inhibit LPS-induced inflammatory responses in lung epithelial cells, which may provide novel molecular targets to inhibit the inflammation component of lung carcinogenesis. PREVENTION RELEVANCE: Inflammation increases the risk of lung cancer and blocking inflammation could reduce the incidence of lung cancer. Herein, we demonstrate that histone deacetylase inhibitor suberoylanilide hydroxamic acid regulates metabolic rewiring and epigenetic reprogramming to attenuate lipopolysaccharide-driven inflammation in lung epithelial cells.

Long term exposure of cigarette smoke condensate (CSC) mediates transcriptomic changes in normal human lung epithelial Beas-2b cells and protection by garlic compounds.

Chronic cigarette smoke condensate (CSC) exposure is one of the preventable risk factors in the CS-induced lung cancer. However, understanding the mechanism of cellular transformation induced by CS in the lung remains limited. We investigated the effect of long term exposure of CSC in human normal lung epithelial Beas-2b cells, and chemopreventive mechanism of organosulphur garlic compounds, diallyl sulphide (DAS) and diallyl disulphide (DADS) using Next Generation Sequencing (NGS) transcriptomic analysis. CSC regulated 1077 genes and of these 36 genes are modulated by DAS while 101 genes by DADS. DAS modulated genes like IL1RL1 (interleukin-1 receptor like-1), HSPA-6 (heat shock protein family A, member 6) while DADS demonstrating ADTRP (Androgen-Dependent TFPI Regulating Protein), ANGPT4 (Angiopoietin 4), GFI1 (Growth Factor-Independent 1 Transcriptional Repressor), TBX2 (T-Box Transcription Factor 2), with some common genes like NEURL-1 (Neuralized E3-Ubiquitin Protein Ligase 1), suggesting differential effects between these two garlic compounds. They regulate genes by influencing pathways including HIF-1alpha, STAT-3 and matrix metalloproteases, contributing to the chemoprotective ability of organosulfur garlic compounds against CSC-induced cellular transformation. Taken together, we demonstrated CSC induced global gene expression changes pertaining to cellular transformation which potentially can be delayed with dietary chemopreventive phytochemicals like DS and DADS influencing alterations at the transcriptomic level.

PTEN-knockout regulates metabolic rewiring and epigenetic reprogramming in prostate cancer and chemoprevention by triterpenoid ursolic acid.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is one of the most frequently mutated/deleted tumor suppressor genes in many human cancers. Ursolic acid (UA) is a natural triterpenoid possessing antioxidant, anti-inflammatory, and anticancer effects. However, how PTEN impacts metabolic rewiring and how UA modifies PTEN-driven metabolic and epigenetic reprogramming in prostate cancer (PCa) remains unknown. In the current study, we found that UA protects against PTEN knockout (KO)-induced tumorigenesis at different stages of PCa. Epigenomic CpG methyl-seq revealed UA attenuated PTEN KO-induced differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq showed UA abrogated PTEN KO-induced differentially expressed genes (DEGs) of PCa-related oncogenes' Has3, Cfh, and Msx1 overexpression, indicating UA plays a crucial role in PTEN KO-mediated gene regulation and its potential consequences on cancer interception. Association analysis of DEGs and DMRs identified that the mRNA expression of tumor suppressor gene BDH2, and oncogenes Ephas, Isg15, and Nos2 were correlated with the promoter CpG methylation status in the early-stage comparison groups indicating UA could regulate the oncogenes or tumor suppressor genes by modulating their promoter methylation at an early stage of prostate tumorigenesis. The metabolomic study showed UA attenuated PTEN KO-regulated cancer-associated metabolisms like purine metabolism/metabolites correlating with RNAseq findings, glycolysis/gluconeogenesis metabolism, as well as epigenetic-related metabolites pyruvate and lactate indicating UA plays a critical role in PTEN KO-mediated metabolic and epigenetic reprogramming and its consequences on cancer development. In this context, UA impacts metabolic rewiring causing epigenetic and transcriptomic reprogramming potentially contributing to the overall protection against prostate-specific PTEN KO-mediated PCa.

Butyrate Drives Metabolic Rewiring and Epigenetic Reprogramming in Human Colon Cancer Cells.

SCOPE: Butyrate (B) is a short-chain fatty acid produced by dietary fiber, known to inhibit histone deacetylases (HDACs) and possess cancer-preventive/anticancer effects. However, the role of B in metabolic rewiring, epigenomic reprogramming, transcriptomic network, NRF2 signaling, and eliciting cancer-preventive effects in colorectal cancer (CRC) HCT116 cell remains unclear. METHODS AND RESULTS: Sodium butyrate (NaB) dose-dependently inhibits the growth of CRC HCT116 cells. NaB inhibits NRF2/NRF2-target genes and blocks NRF2-ARE signaling. NaB increases NRF2 negative regulator KEAP1 expression through inhibiting its promoter methylation. Associative analysis of DEGs (differentially expressed genes) from RNA-seq and DMRs (differentially methylated regions) from CpG methyl-seq identified the tumor suppressor gene ABCA1 and tumor promote gene EGR3 are correlated with their promoters' CpG methylation indicating NaB regulates cancer markers through modulating their promoter methylation. NaB activated the mitochondrial tricarboxylic acid (TCA) cycle while inhibited the methionine metabolism which are both tightly coupled to the epigenetic machinery. NaB regulates the epigenetic enzymes/genes including DNMT1, HAT1, KDM1A, KDM1B, and TET1. Altogether, B's regulation of metabolites coupled to the epigenetic enzymes illustrates the potential underlying biological connectivity between metabolomics and epigenomics. CONCLUSION: B regulates KEAP1/NRF2 signaling, drives metabolic rewiring, CpG methylomic, and transcriptomic reprogramming contributing to the overall cancer-prevention/anticancer effect in the CRC cell model.

Nfe2l2 Regulates Metabolic Rewiring and Epigenetic Reprogramming in Mediating Cancer Protective Effect by Fucoxanthin.

Fucoxanthin (FX) is a carotenoid with many pharmaceutical properties due to its antioxidant/anti-inflammatory and epigenetic effects. NFE2L2 is involved in the defense against oxidative stress/inflammation-mediated diseases, like anticancer effects elicited by phytochemicals including FX. However, the role of FX and NFE2L2 in metabolic rewiring, epigenomic reprogramming, and transcriptomic network in blocking pro-tumorigenic signaling and eliciting cancer-protective effects remains unknown. Herein, we utilized multi-omics approaches to evaluate the role of NFE2L2 and the impact of FX on tumor promoter TPA-induced skin cell transformation. FX blocked TPA-induced ROS and oxidized GSSG/reduced GSH in Nfe2l2wild-type(WT) but not Nfe2l2-knockdown (KD) cells. Both Nfe2l2 KD and TPA altered cellular metabolisms and metabolites which are tightly coupled to epigenetic machinery. The suppressive effects of FX on TPA-enhancedSAM/SAH was abrogated by Nfe2l2 KD indicating Nfe2l2 plays a critical role in FX-mediated metabolic rewiring and its potential consequences on epigenetic reprogramming. Epigenomic CpG methyl-seq revealed that FX attenuated TPA-induced differentially methylated regions (DMRs) of Uhrf1 and Dnmt1 genes. Transcriptomic RNA-seq showed that FX abrogated TPA-induced differentially expressed genes (DEGs) of Nfe2l2-related genes Nqo1, Ho1, and Keap1. Associative analysis of DEGs and DMRs identified that the mRNA expressions of Uhrf1 and Dnmt1 were correlated with the promoter CpG methylation status. Chromatin immunoprecipitation assay showed that FX restored Uhrf1 expression by regulating H3K27Me3 enrichment in the promoter region. In this context, FX/Nfe2l2's redox signaling drives metabolic rewiring causing epigenetic and transcriptomic reprogramming potentially contributing to the protection of TPA-induced JB6 cellular transformation skin cancer model. Graphical abstract.

Epigenetics/Epigenomics and Prevention of Early Stages of Cancer by Isothiocyanates.

Cancer is a complex disease and cancer development takes 10-50 years involving epigenetics. Evidence suggests that approximately 80% of human cancers are linked to environmental factors impinging upon genetics/epigenetics. Because advanced metastasized cancers are resistant to radiotherapy/chemotherapeutic drugs, cancer prevention by relatively nontoxic chemopreventive "epigenetic modifiers" involving epigenetics/epigenomics is logical. Isothiocyanates are relatively nontoxic at low nutritional and even higher pharmacologic doses, with good oral bioavailability, potent antioxidative stress/antiinflammatory activities, possess epigenetic-modifying properties, great anticancer efficacy in many in vitro cell culture and in vivo animal models. This review summarizes the latest advances on the role of epigenetics/epigenomics by isothiocyanates in prevention of skin, colon, lung, breast, and prostate cancers. The exact molecular mechanism how isothiocyanates modify the epigenetic/epigenomic machinery is unclear. We postulate "redox" processes would play important roles. In addition, isothiocyanates sulforaphane and phenethyl isothiocyanate, possess multifaceted molecular mechanisms would be considered as "general" cancer preventive agents not unlike chemotherapeutic agents like platinum-based or taxane-based drugs. Analogous to chemotherapeutic agents, the isothiocyanates would need to be used in combination with other nontoxic chemopreventive phytochemicals or drugs such as NSAIDs, 5-α-reductase/aromatase inhibitors targeting different signaling pathways would be logical for the prevention of progression of tumors to late advanced metastatic states.