Integrative analyses of transcriptome, methylome, and metabolome in skin cancer: effect of phytochemicals and their derivatives on regulatory network
Creators: name: Kuo, Hsiao-Chen
Publisher: name: No Publisher Supplied
Year: 2022

description: Non-melanoma skin cancer including basal cell carcinoma and squamous cell carcinoma is one of the most diagnosed cancers in the United States. Ultraviolet (UV) radiation or chemical promoters have been widely applied to understand the mechanisms of skin carcinogenesis. Skin cancer development comprising initiation, promotion, and progression stages is mainly attributed to overwhelmed reactive oxygen species (ROS) and DNA damage, which could be counteracted by anti-oxidative pathways such as nuclear factor E2- related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway. Natural products extracted from fruits and vegetables with low toxicity are emerging strategies to restore redox balance and alleviate carcinogenesis through the modulation of epigenetic modification. The detailed molecular mechanisms remain poorly understood in the context of skin cancer which will be partially addressed in this dissertation.We hypothesized that phytochemicals could regulate epigenetic modifications and metabolism by targeting antioxidative, anti-inflammatory, and immune-modulatory pathways such as Nrf2-ARE, Tumor necrosis factor receptor 2 (TNFR2), and macrophage stimulating protein (MSP)-recepteur d'origine nantais (RON) pathways in non-melanoma skin cancer. The hypothesis was examined in vitro and in vivo skin carcinogenesis models induced by chemicals and UVB irradiation. The DNA methylation regulated by epigenetic modifiers was studied in promoters of pathways of interest as well as at a genome-wide scale. The metabolites and metabolic pathways and their interactions with epigenetic regulation were profiled.In the first chapter, we established a 12-O-tetradecanoylphorbol- 13-acetate (TPA)- induced neoplastic cell transformation in mouse epidermal JB6 P+ cells. Then, we discussed the effect and epigenetic mechanism of delphinidin, one of the most potent and abundant anthocyanidins in berries. 5 to 20 μM of delphinidin pretreatment significantly inhibited the TPA-induced anchorage-independent growth. The effect of delphinidin on the Nrf2-ARE defense mechanism was validated based on observations of enhanced ARE-driven luciferase activity and increased mRNA and protein expression of Nrf2 downstream genes, such as heme oxygenase-1 (Ho-1). Activation of the Nrf2-ARE signaling was correlated with hypomethylation of 15 CpG sites in the mouse Nrf2 promoter region between −1226 and −863 from the transcription start site. Suppression in the protein expression of DNA methyltransferases 1 (DNMT1), DNMT3a, and class I/II histone deacetylases (HDACs) is potentially linked to the reduced CpG methylation ratio in the Nrf2 promoter. Overall, the results suggest that delphinidin, an epigenetic demethylating agent of the Nrf2 promoter, can enhance the Nrf2-ARE pathway as a potential skin cancer prevention agent.In the second chapter, we further the mechanistic study of phytochemicals by genome-wide profiling of gene expression and DNA methylation to capture multi-omics alterations involved in biologically relevant pathways in response to a skin tumor promoter and treatment. Triterpenoid is one of the most potent types of phytochemicals exhibiting anti-oxidative, and anti-inflammatory activities at the nanomolar level. We showed that 12.5 to 100 nM 2-cyano 2,3- dioxoolean-1,9-dien-28-oic acid (CDDO or Bardoxolone) blocks the neoplastic transformation induced by TPA by 41 to 81 percent. Differential expressed analysis was performed between TPA vs. Control as well as TPA+CDDO vs. TPA comparisons to identify novel differential expressed genes (DEGs) involved in the prevention of skin cell transformation. Also, we profiled the CpG methylome and explore the differentially methylated regions (DMRs) in different gene regions. Furthermore, we performed multi-omics analysis to characterize the DMRs showing transcription promotion coupled with promoter hypomethylation or transcription inhibition along with promoter hypermethylation. The correlated DMRs with inverse alterations in DNA methylation status and RNA expression changes were subject to functional analysis. Incorporating the CpG methylome and transcriptome alterations, we showed that CDDO significantly restored gene expression of Nudix Hydrolase 14 (Nudt14), NAD(P)H quinone dehydrogenase 1 (Nqo1), and protein kinase C gamma (Prkcg) dysregulated by TPA by modification of promoter CpG methylation. Functional pathway analysis revealed that CDDO neutralized the effect of TPA through modulating cell cycles, cell migration, inflammatory, and immune response regulatory pathways. Notably, TNFR2 signaling was significantly downregulated by CDDO to prevent TPA-induced cell transformation. Finally, integrating transcriptome, methylome, and significantly regulated pathways, we built a pathway network connecting the genes of interest and characterized the associated molecules and interactions. The multi-omics profiling provided us an insight into the preventive mechanism of CDDO during skin cell transformation. The results could be useful for future human studies and target development for skin cancer.In the third chapter, we extended the in vitro skin transformation model to a two-stage mouse skin carcinogenesis model in hairless SKH1 mice. First, the two-stage skin carcinogenesis mouse model was initiated by Benzo[a]pyrene (BaP), followed by TPA with or without ursolic acid (UA) treatment until sacrifice at 5, 20, or 26 weeks. We provide unique insights into the regulation of DNA methylation and gene expression alterations in response to BaP+TPA and BaP+TPA+UA treatment from initiation to progression stages compared to Control and BaP+TPA. The global and local epigenomic and transcriptomic changes were profiled by differential methylation and expression analyses. Global gene expression and methylation profiles present DEGs and DMRs dysregulated by BaP+TPA treatment while restored by UA treatment from 5 to 26 weeks. In addition, comparative transcriptome analysis at 5 weeks showed significantly changed genes with opposite log2 fold changes in two comparisons including C-C motif chemokine ligand 8 (Ccl8), interleukin 17F (Il17f), and lactoperoxidase (Lpo), and the regulation of Il17f continued until the late promotion stage (20 weeks). The result demonstrates that the regulation of inflammatory and oxidative genes by UA started from the early stage of skin carcinogenesis. In addition, we intercompared the CpG methylation level changes and identify the DMRs showing high differences in responses to BaP+TPA and BaP+TPA+UA treatment compared to Control and BaP+TPA groups. Multi-omics analysis revealed molecular markers associated with epigenetic activation or inhibition of gene expression at different stages of skin cancer development. The results showed that BaP+TPA promoted transcription of Kallikrein-related peptidase 13 (Klk13) by promoter demethylation at 5 weeks and continued to 26 weeks, while BaP+TPA+UA treatment suppressed its expression through promoter hypermethylation at 5 weeks, indicating the early intervention of UA. IPA pathway analysis further presented that MSP-RON signaling was significantly upregulated by BaP+TPA at 5 weeks compared to Control, while suppressed by BaP+TPA+UA treatment through regulation of Kallikrein and Creb. The mechanism of the pathway involved several Kallikrein Related Peptidases such as KLK13, KLK12, and KLK6, leading to inhibition of survival, proliferation, invasion, metastasis, and epithelial-mesenchymal transition of tumor cells. The profiled alterations in methylome, transcriptome, and signaling pathway could benefit future skin cancer prevention and treatment research targeting the epigenetically regulated genes and pathways.In the fourth chapter, we profiled the metabolomic, transcriptomic, and epigenomic mechanisms in an established UVB-induced skin cell transformation. Continuous exposure to UV is the primary factor contributing to skin carcinogenesis. Metabolic rewiring and non-mutational epigenetic reprogramming are increasingly discussed as cancer hallmarks. We first established UVB-induced skin cell transformation by exposing human keratinocytes (HaCaT cells) to 10 cycles of 10 mJ/cm2 UVB irradiation. Following that, we cultured cells with or without 10 μM sulforaphane (SFN). Transcriptomic changes and pathway analysis show that UVB irradiation promoted signaling associated with inflammation and ROS imbalance including nuclear factor kappa B (NF-κB), transforming growth factor beta (TGF-β), Interleukin 6 (IL-6), matrix metalloproteinases (MMPs), and hypoxia-inducible factor 1-alpha (HIF-1α) signaling, whereas SFN would block or attenuate MMPs and HOX antisense intergenic RNA (HOTAIR) signaling pathways activated by UVB. SFN can also enhance toll-like receptor and triggering receptor expressed on myeloid cells 1 (TREM1) signaling pathways. DNA methylation profiling characterizes novel DMRs in different gene regions modified by UVB irradiation, while reversed by SFN. These DMRs could be potential epigenetic targets of SFN in skin cell transformation. Integrating RNA-seq and Methyl-seq data, we visualized the correlation of mRNA expression and CpG methylation difference changes in a starburst plot and identified a subset of genes with gene transcription promoted or inhibited by DNA hypomethylation or hypermethylation. Next, we characterized the metabolites determined by Liquid Chromatography-Mass Spectrometry and performed metabolic pathway analysis. We first profiled the short-term (1 cycle) and long-term (10 cycles) UVB effects and identified the significantly changed metabolites and metabolic pathways. The changes in metabolites and metabolic pathways induced by sulforaphane (SFN) in normal or 10-cycle UVB exposed HaCaT cells were also characterized. We observed that the differential regulation of metabolites as cofactors and substrates in the S-adenosyl methionine (SAM) and citric acid cycle including SAM, adenosine triphosphate (ATP), and nicotinamide adenine dinucleotide (NAD) was associated with the expression of epigenetic modifiers such as DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). The interactions potentially influence epigenetic reprogramming and gene expression. The findings offer new insight into the interplay of transcriptomic, epigenomic, and metabolomic regulations during UVB-induced skin cell transformation. We also characterized the protective effect and molecular mechanism of SFN in the prevention of UVB-induced cell transformation.Overall, the multi-omics profiling in this dissertation demonstrates a framework for studying molecular mechanisms during different stages of chemical- and UV-induced skin carcinogenesis and the chemopreventive effect of different classes of phytochemicals including anthocyanidins, triterpenoids, and isothiocyanates.