In Colorectal Cancer Cells With Mutant KRAS, SLC25A22-Mediated Glutaminolysis Reduces DNA Demethylation to Increase WNT Signaling, Stemness, and Drug Resistance

Abstract

BACKGROUND & AIMS: Mutant KRAS promotes glutaminolysis, a process that uses steps from the tricarboxylic cycle to convert glutamine to a-ketoglutarate and other molecules via glutaminase and SLC25A22. This results in inhibition of demethylases and epigenetic alterations in cells that increase proliferation and stem cell features. We investigated whether mutant KRAS-mediated glutaminolysis affects the epigenomes and activities of colorectal cancer (CRC) cells. METHODS: We created Apc min Kras G12D mice with intestine-specific knockout of SLC25A22 (Apc min Kras G12D SLC25A22 fl/fl mice). Intestine tissues were collected and analyzed by histology, immunohistochemistry, and DNA methylation assays; organoids were derived and studied for stem cell features, along with organoids derived from 2 human colorectal tumor specimens. Colon epithelial cells (1CT) and CRC cells (DLD1, DKS8, HKE3, and HCT116) that expressed mutant KRAS, with or without knockdown of SLC25A22 or other proteins, were deprived of glutamine or glucose and assayed for proliferation, colony formation, glucose or glutamine consumption, and apoptosis; gene expression patterns were analyzed by RNA sequencing, proteins by immunoblots, and metabolites by liquid chromatography-mass spectrometry, with [U-13 C 5 ]-glutamine as a tracer. Cells and organoids with knocked down, knocked out, or overexpressed proteins were analyzed for DNA methylation at CpG sites using arrays. We performed immunohistochemical analyses of colorectal tumor samples from 130 patients in Hong Kong (57 with KRAS mutations) and Kaplan-Meier analyses of survival. We analyzed gene expression levels of colorectal tumor samples in The Cancer Genome Atlas. RESULTS: CRC cells that express activated KRAS required glutamine for survival, and rapidly incorporated it into the tricarboxylic cycle (glutaminolysis); this process required SLC25A22. Cells incubated with succinate could proliferate under glutamine-free conditions. Mutant KRAS cells maintained a low ratio of a-ketoglutarate to succinate, resulting in reduced 5-hydroxymethylcytosine-a marker of DNA demethylation, and hypermethylation at CpG sites. Many of the hypermethylated genes were in the WNT signaling pathway and at the protocadherin gene cluster on chromosome 5q31. CRC cells without mutant KRAS, or with mutant KRAS and knockout of SLC25A22, expressed protocadherin genes

Vitamin C Cytotoxicity and Its Effects in Redox Homeostasis and Energetic Metabolism in Papillary Thyroid Carcinoma Cell Lines

“…Indeed, vitamin C is known to act as an essential cofactor to numerous monooxygenases and dioxygenases, including the ten-eleven translocation (TET) enzyme family, which catalyzes the hydroxylation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the active process of DNA demethylation [34,35]. This mechanism leads to the reactivation and upregulation of proapoptotic genes, which are conversely downregulated in those cells harboring KRAS and BRAF mutations, which are in turn known to limit DNA demethylation [36][37][38].…”

Constructing Tumor Immune Microenvironment and Identifying the Immune-Related Prognostic Signatures in Colorectal Cancer Using Multi-omics Data

“…Interestingly, the mutation frequency of APC, TP53, TTN and KRAS were accounted for the top four positions in both immunity cohorts, indicating that the four genes are less regulated the process related to immune infiltration but mainly participated in tumorigenesis and progression [35][36][37] . It is worth noting that gene mutations in different immune levels may have different effects on prognosis.…”

“…For example, M1 macrophages participate in the inflammatory response and anti-tumor immune process by secreting pro-inflammatory cytokines such as IL-1, IL-12, TNF-α and many chemokine ligands 34 , which is consistent with our findings that M1 macrophages accounted for a significantly higher proportion in the high-immune group. Interestingly, the mutation frequency of APC, TP53, TTN and KRAS were accounted for the top four positions in both immunity cohorts, indicating that the four genes are less regulated the process related to immune infiltration but mainly participated in tumorigenesis and progression [35][36][37] . It is worth noting that gene mutations in different immune levels may have different effects on prognosis.…”

The Crosstalk Between Signaling Pathways and Cancer Metabolism in Colorectal Cancer

“…Knockdown of the glutamine transporter in KRAS mutant CRC cells reduces the proliferation rate and inhibits other oncogenic activities, including migration, invasion, and metastasis ( Wong et al, 2016 ). A recent study by Wong et al also reveals that glutamine metabolism in mutant KRAS CRC cells contributes to the activation of Wnt signaling, cancer stemness, and drug resistance by reducing DNA methylation through SLC25A22, a mitochondrial glutamine transporter ( Wong et al, 2020 ). SLC25A22 expression is associated with poor prognosis in advanced-stage CRC with mutant KRAS ( Wong et al, 2020 ).…”