Overexpression of glutamine synthetase in human primary liver cancer

doi:10.1016/0016-5085(94)90024-8

Gastroenterology

Volume 106, Issue 5, May 1994, Pages 1312-1320

https://doi.org/10.1016/0016-5085(94)90024-8 Get rights and content

Abstract

Background/Aims: We have identified several clones specifically expressed during malignant cell proliferation by screening a complementary DNA library constructed from a human primary liver cancer with subtractive probes. One clone was identified as the glutamine synthetase (GS) transcript. Its expression is tightly regulated during development, especially in the hepatic lobule. Because this enzyme is involved in nitrogen homeostasis, it might contribute to tumor development/progression in primary liver cancer. Methods: We compared the expression of GS messenger RNA (mRNA) and protein in tumorous and nontumorous liver from 34 patients with primary liver cancers, using a combination of Northern blot, dot blot, western blot, and determination of GS enzyme activity. Results: GS mRNA was higher in tumors versus nontumors in 23 of 34 primary liver cancer samples. GS activity was higher in 6 of 8 selected primary liver cancer samples with high RNA levels. GS protein levels were proportional to enzyme activity. A major GS transcript of 2.8 kilobase was detected by Northern blotting and sequencing. This comprised the minor 1.8-kb transcript and a long 3′ untranslated region; the latter contained an AT-rich zone, fully conserved in the chicken, mouse, and rat, which might be important for stability. Conclusions: Our results show an overexpression of GS in human primary liver cancers and, thus, point to its potential involvement in hepatocyte transformation.

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Glutamine synthetase (GS) is an enzyme that converts ammonia and glutamate to glutamine using adenosine triphosphate. GS is expressed in the brain, kidney, and liver tissues under normal physiological conditions. GS is involved in abnormal lipid metabolism of the liver by catalyzing de novo synthesis of glutamine, thereby inducing liver inflammation. Metabolic dysfunction-associated steatotic liver diseases (MASLD), such as Metabolic Associated Fatty Liver Disease and Metabolic Associated Steato Hepatitis, are considered risk factors for HCC. GS may also be involved in the development and progression of hepatocellular carcinoma (HCC) through other signaling pathways, including the rapamycin (mTOR) and Wnt/β-catenin signaling pathways. Furthermore, the correct combination of HSP70, GPC3, and GS can improve the accuracy and precision of HCC diagnosis. However, the prognostic value of GS in different HCC populations remains controversial. The expression of GS affects the sensitivity of HCC cells to radiotherapy and chemotherapy. In addition, immunotherapy has been approved for the treatment of advanced HCC. This article delves into the development and application of GS in HCC, laying a theoretical foundation for the subsequent exploration of GS as a potential target for treating HCC.
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Besides fast glucose catabolism, many types of cancers are characterized by elevated glutamine consumption. Medical oncology pursuits to block specific pathways, mainly glycolysis and glutaminolysis, in tumor cells to arrest cancer development. This strategy frequently induces adaptive metabolic resistance that must be countered. Combination therapy is an anticancer synergistic tool to overcome both cancer growth and resistance mechanisms. Dysregulation of glutaminase and glutamine synthetase are key events that allow anabolic adaptation of tumors. Several specific drugs that inhibit metabolic enzymes dealing with glutamine metabolism have been able to eliminate some neoplasms. Targeting the tumor microenvironment can be also another essential factor to be taken into account when single or combined cancer metabolic therapy fails.
Diagnostic Value of Glypican3, Heat Shock Protein 70 and Glutamine Synthetase in Hepatocellular Carcinoma Arising in Cirrhotic and Non-Cirrhotic Livers
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Histopathological distinction of various nodular lesions in liver with sufficient sensitivity and specificity is a challenge even in an expert set up. The panel of immunohistochemical markers composed of glutamine synthetase (GS), Glypican3 (GPC3) and heat shock protein 70 (HSP70) was recommended by the International Consensus Group for Hepatocellular Neoplasia group for the differentiation of high grade dysplastic nodule and early hepatocellular carcinoma (HCC). The panel has been extensively validated in the western population. This study aims to test this panel on Indian population on resected, explanted and autopsy cirrhotic and non-cirrhotic liver specimens of HCC.
This study was conducted on 39 such liver specimens (12 cirrhotic, 12 pre-cirrhotic and 11 non-cirrhotic, non-fibrotic livers), including 35 cases of HCC over a period of 12 years. Immunohistochemistry was performed with antibodies against GS, GPC3 and HSP70 on the sections containing both malignant and dysplastic nodules.
The diagnostic yield depended upon the nature of background liver pathology and was found to be high for only those HCCs arising in cirrhotic background, when positivity of any two markers was taken to be in favor of HCC (sensitivity—58.33%; specificity—100%). GS had a sensitivity and Negative predictive value of 100% for HCCs arising in cirrhotic livers.
Strong positivity for GS is a highly sensitive marker for HCC in a cirrhotic background regardless of the differentiation of the tumor in Indian population. This may be due to preferential activation of Wnt pathway in Indian patients with cirrhosis. The sensitivity of the panel was too low for detecting HCCs arising in non-cirrhotic livers, even in the pre-cirrhotic chronically inflamed livers, even though the specificity was high. GPC3 and HSP70 appear to be useful as individual markers for HCCs arising in non-cirrhotic livers.
Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani
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Citation Excerpt :
Evolution of GS, one of the most ancient genes is considered a good molecular clock [11,12]. It is a ubiquitous enzyme, which is involved in nitrogen metabolism, recycling of neurotransmitter glutamate, and the synthesis of glutamine for the production of amino acids, sugars and glucosamine-6-phosphate [13]. Amino acids are essential components of metabolism in kinetoplastids.
Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L. donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed K_m value of 26.3 ± 0.4 mM for l- glutamate and V_max value of 2.15 ± 0.07 U mg⁻¹. Present study confirms the presence of glutamine synthetase in L. donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target.
Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon
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Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4^CRBN. Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4^CRBN and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4^CRBN that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.
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The current guidelines for the diagnosis of hepatocellular carcinoma (HCC) recommend liver biopsy for hepatic nodules which do not demonstrate the typical features of HCC on imaging. Thus, while not all HCCs are biopsied for histological confirmation, the nodules that pathologists now encounter on biopsy specimens are frequently well-differentiated early HCCs. This paper reviews the pathological features of HCC and its precursor lesions on liver biopsy specimens, with special emphasis on the differential diagnosis between well-differentiated HCCs and high-grade dysplastic nodules, and discusses the different roles of liver biopsy in diagnosis and management of early and progressed HCC. The potential role of liver biopsy for the development of molecular markers to predict prognosis and response to targeted therapy is also discussed.

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: Supported by Institut National de la Santé et de la Recherche Médicale, Association de la recherche contre le cancer, Ligue national contre le cancer, Fondation de France, Caisse nationale contre le cancer, and the Commission of the European communities.

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Overexpression of glutamine synthetase in human primary liver cancer

Abstract

Mol Brain Res

Pharmacol Ther

J Biol Chem

Biochim Biophys Acta

J Mol Biol

Gene

Cell

Identification of a novel gene HIP expressed in primary liver cancer

Cancer Res

Heterogeneous distribution of glutamine synthetase during rat liver development

J Histochem Cytochem

Colocalization in pericentral hepatocytes in adult mice and similarity in developmental expression pattern of ornithine aminotransferase and glutamine synthetase mRNA

Localization of amonia-metabolizing enzymes in human liver: ontogenesis of heterogeneity

Hepatology

Hepatocytes heterogeneity in the metabolism of amino acids and ammonia

Enzyme

Evidence that interaction of hepatocytes with the collecting (hepatic) veins triggers position-specific transcription of the glutamine synthetase and ornithine aminotransferase genes in the mouse liver

Mol Cell Biol

Cell-cell interactions: clues to hepatocyte heterogeneity and beyond?

Hepatology