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E4BP4 promotes thyroid cancer proliferation by modulating iron homeostasis through repression of hepcidin.

ABSTRACT: Iron homeostasis is critical to mammals, and dysregulation in iron homeostasis usually leads to severe disorders including various cancers. Massive hepcidin secretion is an indicator of thyroid cancer, but the molecular mechanisms responsible for this dysregulation are unknown. Hepcidin secretion from thyroid cancer cells also leads to decreased expression of the iron exporter, ferroportin (FPN), and increased intracellular iron retention, which promote cancer proliferation. In this study, we examined the role of hepcidin in thyroid cancer (TC) and the molecular bases of its signaling. Synthesis of hepcidin is regulated by the BMP4/7 agonist SOSTDC1, which was downregulated in TC; SOSTDC1 downregulation was correlated with G9a-mediated hypermethylation in its promoter. The binding of G9a to the SOSTDC1 promoter requires E4BP4, which interacts with G9a to form a multi-molecular complex that contributes to SOSTDC1 silencing. Silencing of E4BP4 or G9a has similar effects to SOSTDC1 overexpression, which suppresses secretion of hepcidin and inhibits TC cell proliferation. Furthermore, our in vivo xenograft data indicated that depletion of E4BP4 also inhibits cancer growth, reduces hepcidin secretion, and reduces G9a nuclear transportation. Iron homeostasis and tumor growth in TC may be regulated by an E4BP4-dependent epigenetic mechanism. These findings suggest a new mechanism of cellular iron dysfunction through the E4BP4/G9a/SOSTDC1/hepcidin pathway, which is an essential link in TC.

SUBMITTER: Zhou Q

PROVIDER: S-EPMC6155336 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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E4BP4 promotes thyroid cancer proliferation by modulating iron homeostasis through repression of hepcidin.

Zhou Qinyi Q Chen Jun J Feng Jialin J Wang Jiadong J

Cell death & disease 20180924 10

Iron homeostasis is critical to mammals, and dysregulation in iron homeostasis usually leads to severe disorders including various cancers. Massive hepcidin secretion is an indicator of thyroid cancer, but the molecular mechanisms responsible for this dysregulation are unknown. Hepcidin secretion from thyroid cancer cells also leads to decreased expression of the iron exporter, ferroportin (FPN), and increased intracellular iron retention, which promote cancer proliferation. In this study, we ex ...[more]

PMID: 30250199

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Project description:Background & aimsHepatic gluconeogenesis provides fuel during starvation, and is abnormally induced in obese individuals or those with diabetes. Common metabolic disorders associated with active gluconeogenesis and insulin resistance (obesity, metabolic syndrome, diabetes, and nonalcoholic fatty liver disease) have been associated with alterations in iron homeostasis that disrupt insulin sensitivity and promote disease progression. We investigated whether gluconeogenic signals directly control Hepcidin, an important regulator of iron homeostasis, in starving mice (a model of persistently activated gluconeogenesis and insulin resistance).MethodsWe investigated hepatic regulation of Hepcidin expression in C57BL/6Crl, 129S2/SvPas, BALB/c, and Creb3l3-/- null mice. Mice were fed a standard, iron-balanced chow diet or an iron-deficient diet for 9 days before death, or for 7 days before a 24- to 48-hour starvation period; liver and spleen tissues then were collected and analyzed by quantitative reverse-transcription polymerase chain reaction and immunoblot analyses. Serum levels of iron, hemoglobin, Hepcidin, and glucose also were measured. We analyzed human hepatoma (HepG2) cells and mouse primary hepatocytes to study transcriptional control of Hamp (the gene that encodes Hepcidin) in response to gluconeogenic stimuli using small interfering RNA, luciferase promoter, and chromatin immunoprecipitation analyses.ResultsStarvation led to increased transcription of the gene that encodes phosphoenolpyruvate carboxykinase 1 (a protein involved in gluconeogenesis) in livers of mice, increased levels of Hepcidin, and degradation of Ferroportin, compared with nonstarved mice. These changes resulted in hypoferremia and iron retention in liver tissue. Livers of starved mice also had increased levels of Ppargc1a mRNA and Creb3l3 mRNA, which encode a transcriptional co-activator involved in energy metabolism and a liver-specific transcription factor, respectively. Glucagon and a cyclic adenosine monophosphate analog increased promoter activity and transcription of Hamp in cultured liver cells; levels of Hamp were reduced after administration of small interfering RNAs against Ppargc1a and Creb3l3. PPARGC1A and CREB3L3 bound the Hamp promoter to activate its transcription in response to a cyclic adenosine monophosphate analog. Creb3l3-/- mice did not up-regulate Hamp or become hypoferremic during starvation.ConclusionsWe identified a link between glucose and iron homeostasis, showing that Hepcidin is a gluconeogenic sensor in mice during starvation. This response is involved in hepatic metabolic adaptation to increased energy demands; it preserves tissue iron for vital activities during food withdrawal, but can cause excessive iron retention and hypoferremia in disorders with persistently activated gluconeogenesis and insulin resistance.

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived thyroid cancer cell line k1 transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: mRNA profiles of shCtrl and shTBX3 cells were generated by deep sequencing, in duplicate, using BGISEQ-500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods:Bowtie2 to map clean reads to reference gene and use HISAT to reference genome.Bowtie2 parameters forSE reads:-q--phred64--sensitive--dpad0--gbar and HISTAparameters forSE reads:-p8--phred64--sensitive-I1-X 1000. qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 24 million sequence reads per sample to the human genome (build hg19) and identified 20511 transcripts in K1-shCtrl and K1-shTBX3 cells with RSEM workflow. Approximately 5% of the transcripts showed differential expression between K1-shCtrl and K1-shTBX3 cells, with a fold change ≥1.5 and p value <0.01. Altered expression of 16 candidate genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to thyroid cancer function. Conclusions: Our study represents the first detailed analysis of thyroid cancer cell line k1 transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

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E4BP4 promotes thyroid cancer proliferation by modulating iron homeostasis through repression of hepcidin.

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E4BP4 promotes thyroid cancer proliferation by modulating iron homeostasis through repression of hepcidin.

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