Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling are poorly understood. Here, we demonstrate that hepcidin is activated ectopically in CRC. Mice deficient in hepcidin specifically in the colon tumour epithelium, compared with wild-type littermates, exhibit significantly diminished tumour number, burden and size in a sporadic model of CRC, whereas accumulation of intracellular iron by deletion of the iron exporter ferroportin exacerbates these tumour parameters. Metabolomic analysis of three-dimensional patient-derived CRC tumour enteroids indicates a prioritization of iron in CRC for the production of nucleotides, which is recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that iron chelation decreases mitochondrial function, thereby altering nucleotide synthesis, whereas exogenous supplementation of nucleosides or aspartate partially rescues tumour growth in patient-derived enteroids and CRC cell lines in the presence of an iron chelator. Collectively, these data suggest that ectopic hepcidin in the tumour epithelium establishes an axis to sequester iron in order to maintain the nucleotide pool and sustain proliferation in colorectal tumours

Expansion of cat-ELCCA for the Discovery of Small Molecule Inhibitors of the Pre-let-7–Lin28 RNA–Protein Interaction

Dysregulation of microRNA (miRNA) expression has been linked to many human diseases; however, because of the challenges associated with RNA-targeted drug discovery, additional approaches are needed for probing miRNA biology. The emerging regulatory role of miRNA-binding proteins in miRNA maturation presents such an alternative strategy. Exploiting our laboratory’s click chemistry-based high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a modular method by which to discover new chemical tools for manipulating pre-miRNA–miRNA–binding protein interactions. Using the pre-let-7d–Lin28 interaction as proof-of-concept, the results presented demonstrate how HTS using cat-ELCCA can enable the discovery of small molecules targeting RNA–protein interactions

Protocol to generate human liver spheroids to study liver fibrosis induced by metabolic stress

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies

IL-6 Inhibition With MEDI5117 Decreases The Fraction of Head and Neck Cancer Stem Cells and Prevents Tumor Recurrence

Head and neck squamous cell carcinomas (HNSCC) exhibit a small population of uniquely tumorigenic cancer stem cells (CSC) endowed with self-renewal and multipotency. We have recently shown that IL-6 enhances the survival and tumorigenic potential of head and neck cancer stem cells (i.e. ALDHhighCD44high cells). Here, we characterized the effect of therapeutic inhibition of IL-6 with a novel humanized anti-IL-6 antibody (MEDI5117) using three low-passage patient-derived xenograft (PDX) models of HNSCC. We observed that single agent MEDI5117 inhibited the growth of PDX-SCC-M1 tumors (P < .05). This PDX model was generated from a previously untreated HNSCC. In contrast, MEDI5117 was not effective at reducing overall tumor volume for PDX models representing resistant disease (PDX-SCC-M0, PDX-SCC-M11). Low dose MEDI5117 (3 mg/kg) consistently decreased the fraction of cancer stem cells in PDX models of HNSCC when compared to IgG-treated controls, as follows: PDX-SCC-M0 (P < .001), PDX-SCC-M1 (P < .001), PDX-SCC-M11 (P = .04). Interestingly, high dose MEDI5117 (30 mg/kg) decreased the CSC fraction in the PDX-SCC-M11 model (P = .002), but not in PDX-SCC-M0 and PDX-SCC-M1. MEDI5117 mediated a dose-dependent decrease in the number of orospheres generated by ALDHhighCD44high cells cultured in ultra-low attachment plates (P < .05), supporting an inhibitory effect on head and neck cancer stem cells. Notably, single agent MEDI5117 reduced the overall recurrence rate of PDX-SCC-M0, a PDX generated from the local recurrence of human HNSCC. Collectively, these data demonstrate that therapeutic inhibition of IL-6 with low-dose MEDI5117 decreases the fraction of cancer stem cells, and that adjuvant MEDI5117 inhibits recurrence in preclinical models of HNSCC

Hyaluronic acid fuels pancreatic cancer cell growth

Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not preclude the growth of human tumor xenografts in mice, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. We found that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating N-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles

Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling are poorly understood. Here, we demonstrate that hepcidin is activated ectopically in CRC. Mice deficient in hepcidin specifically in the colon tumour epithelium, compared with wild-type littermates, exhibit significantly diminished tumour number, burden and size in a sporadic model of CRC, whereas accumulation of intracellular iron by deletion of the iron exporter ferroportin exacerbates these tumour parameters. Metabolomic analysis of three-dimensional patient-derived CRC tumour enteroids indicates a prioritization of iron in CRC for the production of nucleotides, which is recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that iron chelation decreases mitochondrial function, thereby altering nucleotide synthesis, whereas exogenous supplementation of nucleosides or aspartate partially rescues tumour growth in patient-derived enteroids and CRC cell lines in the presence of an iron chelator. Collectively, these data suggest that ectopic hepcidin in the tumour epithelium establishes an axis to sequester iron in order to maintain the nucleotide pool and sustain proliferation in colorectal tumours

Menin regulates the serine biosynthetic pathway in Ewing sarcoma

Developmental transcription programs are epigenetically regulated by multiâ protein complexes, including the meninâ and MLLâ containing trithorax (TrxG) complexes, which promote gene transcription by depositing the H3K4me3 activating mark at target gene promoters. We recently reported that in Ewing sarcoma, MLL1 (lysine methyltransferase 2A, KMT2A) and menin are overexpressed and function as oncogenes. Small molecule inhibition of the meninâ MLL interaction leads to loss of menin and MLL1 protein expression, and to inhibition of growth and tumorigenicity. Here, we have investigated the mechanistic basis of meninâ MLLâ mediated oncogenic activity in Ewing sarcoma. Bromouridine sequencing (Bruâ seq) was performed to identify changes in nascent gene transcription in Ewing sarcoma cells, following exposure to the meninâ MLL interaction inhibitor MIâ 503. Meninâ MLL inhibition resulted in early and widespread reprogramming of metabolic processes. In particular, the serine biosynthetic pathway (SSP) was the pathway most significantly affected by MIâ 503 treatment. Baseline expression of SSP genes and proteins (PHGDH, PSAT1, and PSPH), and metabolic flux through the SSP were confirmed to be high in Ewing sarcoma. In addition, inhibition of PHGDH resulted in reduced cell proliferation, viability, and tumor growth in vivo, revealing a key dependency of Ewing sarcoma on the SSP. Loss of function studies validated a mechanistic link between menin and the SSP. Specifically, inhibition of menin resulted in diminished expression of SSP genes, reduced H3K4me3 enrichment at the PHGDH promoter, and complete abrogation of de novo serine and glycine biosynthesis, as demonstrated by metabolic tracing studies with 13Câ labeled glucose. These data demonstrate that the SSP is highly active in Ewing sarcoma and that its oncogenic activation is maintained, at least in part, by meninâ dependent epigenetic mechanisms involving trithorax complexes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144706/1/path5085_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144706/2/path5085.pd

GOT1 inhibition promotes pancreatic cancer cell death by ferroptosis

Cancer metabolism is rewired to support cell survival in response to intrinsic and environmental stressors. Identification of strategies to target these adaptions is an area of active research. We previously described a cytosolic aspartate aminotransaminase (GOT1)-driven pathway in pancreatic cancer used to maintain redox balance. Here, we sought to identify metabolic dependencies following GOT1 inhibition to exploit this feature of pancreatic cancer and to provide additional insight into regulation of redox metabolism. Using pharmacological methods, we identify cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. We demonstrate that targeting any of these pathways triggers ferroptosis, an oxidative, iron-dependent form of cell death, in GOT1 knockdown cells. Mechanistically, we reveal that GOT1 inhibition represses mitochondrial metabolism and promotes a catabolic state. Consequently, we find that this enhances labile iron availability through autophagy, which potentiates the activity of ferroptotic stimuli. Overall, our study identifies a biochemical connection between GOT1, iron regulation, and ferroptosis