CTP synthase forms the cytoophidium in human hepatocellular carcinoma
Introduction
Cytidine nucleotides are not only precursors for DNA/RNA synthesis, but are also involved in phospholipid synthesis and protein sialylation [1], [2]. As the nucleotide at the lowest concentration in cells, the precise control of cytidine nucleotide production is particularly important for regular cell metabolism [3]. In mammalian cells, cytidine nucleotides can be generated from the de novo synthetic pathway and the salvage pathway. Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step in the de novo CTP synthetic pathway, in which a UTP is converted into CTP with the consumption of glutamine and ATP.
CTPS can aggregate into a filamentous macrostructure termed the cytoophidium (plural: cytoophidia) in prokaryotes and eukaryotes including mammals [4], [5], [6], [7], [8]. Recent studies have demonstrated that assembly of the cytoophidium may function to modulate CTPS activity [9], [10]. While polymerization of E. coli CTPS inhibits its catalytic activity, assembly of the human CTPS1 polymer, which is the building block of the cytoophidium, can upregulate CTPS activity [11]
In many organisms, such as E. coli, S. pombe and some tissues of the fruit fly Drosophila, CTPS cytoophidia are present in most cells. However, the CTPS cytoophidium is rarely observed in mammalian cells cultured under normal conditions [4], [7], [10], [12]. It has been proposed that formation of the cytoophidium is an adaptive action in response to a particular cellular status or stress [13]. Previously, CTPS inhibitors, such as 6-diazo-5-oxo-L-norleucine (DON) and deazauridine (DAU), have been shown to effectively stimulate CTPS aggregation in Drosophila and mammalian cells, and have often been used for investigating the characteristics of the CTPS cytoophidium [5], [14]. In addition, the CTPS cytoophidium could also be induced under glutamine-deprived culture conditions, suggesting that formation of the cytoophidium reflects a cellular stress caused by glutamine deficiency [4].
In the neuroblasts and cells in the imaginal disc tissue of Drosophila larvae, the presence of CTPS cytoophidia is correlated with developmental stage, cell cycle progression and nutrient uptake, suggesting that the formation of cytoophidia is dynamic and may reflect instant cell metabolic status [13]. It has also been shown that the expression level of c-Myc is positively correlated with the presence and the size of CTPS cytoophidia in Drosophila cells, and the inhibition of an E3-ligase, Cbl, which is a proto-oncogene, disrupts the cytoophidium structure in the Drosophila egg chamber [15], [16]. In addition, elevated CTPS activity has been found in various cancers such as lymphoma, renal carcinoma and hepatoma [17], [18], [19]. The possible association between the CTPS cytoophidium and cancer metabolism is particularly intriguing but has not yet been elucidated.
Herein, we firstly surveyed the presence of CTPS cytoophidia in 81 human cancer tissue samples including 11 cancer types on a tumor tissue array. Among them, 36 samples (44%) were found to contain a massive amount of cytoophidia. However, assembly of the cytoophidium does not exclusively occur in cancer as we also observed cytoophidia in some non-cancerous tissues. Subsequently, we focused on hepatocellular carcinoma (HCC) and collected tissues from 203 patients for further characterization. While none of the tumor adjacent hepatocyte population was found to exhibit CTPS cytoophidia, many cytoophidia were observed in 28% of the HCC tumor samples. In those samples with the greatest amount of cytoophidia, the expression level of heat shock protein 90 (HSP90) was significantly higher than in ones without cytoophidia. Taken together, our findings not only prove that assembly of the CTPS cytoophidium is a natural phenomenon in human tissue, but also suggest that the presence of CTPS cytoophidia may correlate with a certain environmental or cell metabolic status in HCC tissues.
Section snippets
Cell culture
Huh7 cells were cultured in DMEM (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific) and 1% Gibco® Antibiotic-Antimycotic (Thermo Fisher Scientific) and with or without L-glutamine as described. L-methionine sulfoximine (Sigma-Aldrich) is dissolved in water and added in medium as indicated. Cells were cultured in a 37 °C humid incubator with 5% CO2.
Tissue sample collection
Human tumor tissue microarray slide was purchased from Origene (CT565858). For screening the presence of CTPS cytoophidia in HCC
CTPS forms cytoophidia in various human tumor tissues
In our previous study, we have demonstrated that human CTPS can aggregate into the cytoophidium in both the cytoplasm and nucleus when cells are cultured in glutamine-deprived medium [4]. Formation of the CTPS cytoophidium is reversible: in the human Huh7 cell, which is a HCC cell line, the proportion of cells with cytoophidia rose from 4% to 28% after culture in glutamine-free medium for 1 day, and subsequently decreased to 14% after 1 day of culture with glutamine supplementation (Fig. 1A and
Discussion
Purinosome and the cytoophidium are two large complexes related with intracellular nucleotide metabolism in mammalian cells found in the last decade [29], [30]. While Purinosome is composed by multiple enzymes in de novo purine synthetic pathway and may facilitate the generation of purine by protein-protein interactions within the complex, the cytoophidium is mainly composed by a bundle of polymers of IMPDH or/and CTPS, which are independent to each other in any ways, except the colocalization
Acknowledgement
We thank the Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University for the confocal microscope facility.
Competing interests
The authors declare no competing interest.
Author contributions
C.C.C L.Y.S and J.L.L conceived this project; C.C.C. designed and performed experiments with assistance from M.P.; Y.M.J. helped with collection of HCC tissue samples. C.C.C, Y.M.J., M.P., G.D.K., L.Y.S and J.L.L. analyzed the data; C.C.C wrote the manuscript with input from G.D.K, L.Y.S and J.L.L.
Funding
This work was supported by Ministry of Science and Technology, Taiwan, ROC #104-2313-B-002-035-MY3 granted to LYS and #106-2917-I-002-043 granted to CCC; and
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