The Role Of Glutamine Metabolism In Pituitary Adenoma

Background:Pituitary adenomas(PAs)are the second most common primary intracranial tumor with an overall prevalence of about 115 cases per 100,000.According to hormone secretion,PAs are grouped into functional adenomas and clinical nonfunctioning(NF)adenomas.Prolactin(PRL)-secreting adenomas,growth hormone(GH)-secreting adenomas,adrenocorticotropic hormone(ACTH)-secreting adenomas along with NF adenomas are the four most common subtypes of PA.Although PAs are benign tumors,they significantly disturb hormones and affect the quality of life and life span.Of the four most common PA subtypes,those associated with Cushing's disease(ACTH-secreting),acromegaly(GH-secreting),and NF adenomas are generally treated with transsphenoidal surgery as first-line therapy.However,because some PAs can invade the cavernous sinus or surrounding bone in the sellar region,it is sometimes difficult to achieve total surgical resection.Moreover,currently available pharmacotherapies are not satisfactory.Thus,novel approaches for treating PA are needed.Metabolic abnormality is considered as one of the hallmarks of tumor cells.Although benign,PAs have also been shown to undergo metabolic remodeling.Several studies have confirmed differences in metabolites between pituitary tumor and healthy tissues or among different tumor subtypes by using mass spectrometry or nuclear magnetic resonance spectrometry.However,despite changes in metabolites,the specific therapeutic targets are generally metabolite-related enzymes.The expression levels of these enzymes alter metabolic flux and thus can be detected at the transcriptional level.We previously reported that lactate dehydrogenase A,a key glycolysis enzyme,was overexpressed in invasive PAs and is a promising therapeutic target.Abundant available transcriptomic data enable us to systematically investigate the various metabolic reprograming of tumors.However,no omics study has been conducted to identify metabolic-related genes and the mechanisms underlying PA development.Methods:Published studies of the expression profile of human pituitary adenomas were identified by searching “pituitary” “pituitary adenoma” “pituitary tumor” “prolactinoma” “growth hormone” and “Cushing disease” in the Gene Expression Omnibus database(GEO,http://www.ncbi.nlm.nih.gov/geo/).After global normalization and batch effect adjustment,expression data of the four subtypes of PAs were compared with the NPs to identify subtype-associated DEGs.Then we drew Venn diagrams to identify specific DEGs in the four PA subtypes.Then the PRL-and NF-specific and common DEGs were evaluated by KEGG pathways and GO terms enrichment analyses.At last,the human protein-protein interaction(PPI)network was conducted to explore the relations among the common DEGs and metabolic enzymes.Results:1.Microarray data integration and DEG identification1.1 Nine gene expression profiles were obtained from the GEO database.A total of 160 samples were included: 52 ACTH-secreting PAs,38 GH-secreting PAs,40 NF PAs,9 PRL-secreting PAs,and 21 normal pituitary(NP)samples.1.2 512 DEGs in GH-secreting PAs,392 DEGs in ACTH-secreting PAs,1581 DEGs in PRL-secreting PAs,and 1149 DEGs in NF PAs were identified.2.Identification of subtype-specific DEGs and KEGG pathway analysis2.1 Venn diagrams found 0 ACTH-specific DEGs,22 GH-specific DEGs,1081 PRL-specific DEGs,437 NF-specific DEGs,and 217 DEGs in all four subtypes,which we referred to as the “common DEGs”.2.2 PRL DEGs were enriched mostly in osteoclast differentiation,toxoplasmosis,inflammatory bowel disease,and apoptosis.NF DEGs were primarily enriched in glycosphingolipid biosynthesis-globo and isoglobo series,cAMP signaling pathway,proteasome,and serotonergic synapse(Figure 1C).Common DEGs were enriched mostly in the PI3K-Akt signaling pathway,extracellular matrix-receptor interaction,TGF-? signaling pathway,and cytokine-cytokine receptor interaction.3.Metabolism-related KEGG pathway and GO terms3.1 Common DEGs showed enrichment in only two KEGG metabolism pathways: metabolism of xenobiotics by cytochrome P450 and sulfur metabolism.Go terms showed enrichment in glucose homeostasis and retinoid metabolic process.3.2 PRL-specific DEGs were enriched in 6 metabolism pathways: pentose phosphate pathway,fructose and mannose metabolism,fatty acid degradation,tryptophan metabolism,beta-alanine metabolism,and carbon metabolism.3.3 NF DEGs were enriched in 12 metabolism pathways: steroid biosynthesis,arginine biosynthesis,purine metabolism,cysteine and methionine metabolism,tyrosine metabolism,glycosaminoglycan biosynthesis-heparan sulfate/heparin,arachidonic acid metabolism,linoleic acid metabolism,alpha-linolenic acid metabolism,glycosphingolipid biosynthesisglobo and isoglobo series,pantothenate and CoA biosynthesis,and sulfur metabolism.4.PPI construction of common DEGs and modular analysisPDK4 and PCK1 were the top two metabolic enzymes with the highest connectivity(network degree)to other human proteins.Moreover,PDK4 connected to STAT5 b,one of the hub proteins in the network.PRL and NF tumor specific DEGs were both involved in glutamate metabolism,fatty acid and nucleotide metabolism.Conclusions:1.We identified specific DEGs in different hormone-secreting PA subtypes and found that several biological functions related to metabolism had changed.2.Metabolism of xenobiotics by the cytochrome P450 pathway,sulfur metabolism,retinoid metabolism,and glucose homeostasis were abnormal in all subtypes of PA.3.PDK4 and PCK1 might be considered as targets for development of anticancer strategies and therapies.4.PRL and NF tumor specific DEGs were mainly involved in glutamate metabolism,fatty acid and nucleotide metabolism,redox reaction,and gluconeogenesis.Background: The first part suggested that glutamate metabolism was abnormal in pituitary adenomas.Under normal conditions,glutamate binds to toxic free ammonia to form glutamine and transports to the whole body.However,the large amounts of glutamate in tumor cells is believed to be the result of the deaminize from the excessive intaking exogenous glutamine(Gln).Gln is the most abundant amino acid in the human body and is a non-essential amino acid in the physiological state as it is endogenously synthesized.However,most cancer cells die when they lack exogenous Gln,a phenomenon termed as “Gln addiction”.Gln constitutes a major metabolic fuel for some types of tumor cells and is a precursor for various biological macromolecules,such as amino acids,proteins,lipid,and nucleotides.Therefore,it is not surprising that Gln depletion induces severe metabolic stress that may lead cells to undergo apoptosis.Therapeutic agents targeting Gln transporters or glutaminase have been shown to be effective.However,not all cancer types rely on exogenous Gln to survive,such as those that express Gln synthetase(GS).The gene GLUL codes for GS,which is an enzyme that catalyzes Gln synthesis from glutamate and NH4+.The presence or absence of GS expression is an important survival factor after exogenous Gln depletion.The glutamine-or glutamate-related pathways have also been reported to be altered in pituitary gland tumorigenesis using mass spectrometry or nuclear magnetic resonance spectrometry.These data indicate that glutamine/glutamate metabolism is altered in pituitary tumorigenesis and that glutamine/glutamate metabolism processes may differ for the different clinical types of PA.However,the specific role of Gln in the pathophysiology of PA is still unknown.Hence,in this study,we aimed to determine the main role of Gln in human PAs as well as GS expression.Methods: At T20,MMQ and GH3 pituitary cells were cultured in medium with or without glutamine,CCK-8 test was used to get the growth curve of the three cell lines.L-asparaginase was used as a glutamine decomposition agent to confirm the glutamine withdraw effect.Then we tested the expression of GS in the three cell lines and primary pituitary cells derived from 6 pituitary adenoma patients.After that,we analyzed GS expression of 24 human PA tissues using immunohistochemistry(IHC).In total,four hormone types of PAs were included: 5 GH-secreting PAs,5 ACTH-secreting PAs,5 PRL-secreting PAs,and 9 NFPAs.Then GS was blocked by si RNA or pharmacology in GH3 cells.At last,we used liquid chromatograph mass spectrometer(LC-MS)analysis for metabolites to analyze the main metabolic role of glutamine in pituitary adenoma cells.Results: 1.Sensitivity to Gln starvation is correlated with GS expression level 1.1 Pituitary adenoma cell lines showed different sensitivities to Gln starvation,Gln withdrawal showed no significant effect on proliferation of GH3 cells;however,it inhibited the proliferation of MMQ and At T20 cells at 64% and 20%,respectively.1.2 GS was significantly higher expressed in GH3 cells compared to At T20 or MMQ cells(p< 0.001).Furthermore,Gln starvation significantly increased GS expression in GH3(p< 0.001)and At T20(p < 0.05)cells but showed no significant effect on MMQ cells.1.3 Five out of the six patients? primary cells(83.3%)also significantly increased GS expression levels under Gln starvation.1.4 Cells of GS knock-down by si RNA were more sensitive to exogenous glutamine starvation but no significant effect was observed in the presence of exogenous glutamine 1.5 A combination of L-asparaginase and MSO significantly suppressed the proliferation of all PA cell lines 2.GS expression in human PA Tissues 2.1 We divided the observed expression into 4 grades from low to high(score 1-4).Results from IHC staining indicated that GS was expressed in the vast majority(87.5%)of patients and was only absent in 3 patients(level 1).2.2 A positive ki-67 index(ki-67 > 3%)and positive p53 both correlated with a higher GS expression(p < 0.05).2.3 GH-secreting PAs had the highest GS expression level,consistent that observed in the cell lines.3.Removal of both endogenous and exogenous Gln from GS-expressing PA cells resulted in blockage of nucleotide metabolism and cell cycle arrest 3.1 Blocking endogenous and exogenous Gln did not kill GH3 cells,and that replenishing exogenous Gln could still restart their proliferation after Gln starving for 72 hours.3.2 Blocking endogenous and exogenous Gln had no significant difference in the percentage of apoptotic cells.However,the cell cycle distribution showed that Gln deprivation arrested cell cycle progression at the S phase.3.3 The levels of tricarboxylic acid(TCA)cycle intermediates derived from Gln anaplerosis,such as ?-KG and its downstream metabolites succinate and fumarate remain unchanged under both endogenous and exogenous Gln deprivation.3.4 We found a significant decrease under Gln deprivation in the intracellular level of inosine monophosphate(IMP),the key metabolite of the nucleotide de novo synthesize pathway,as well as deoxycytidine triphosphate(d CTP),deoxycytidine monophosphate(d CMP),cytidine triphosphate(CTP),adenosine triphosphate(ATP),and guanosine diphosphate(GDP).3.5 Pathway enrichment of the changed metabolites also indicated significant changes in the purine and pyrimidine metabolic pathways Conclusions: 1.GS is needed for PA cells to survive Gln deprivation.2.Most PAs expressed GS,and positive p53 and ki-67 index was correlated with higher GS expression.3.The proliferation of GS-expressing cells will completely be suppressed after application of a combination of L-asparaginase(a glutamine scavenger)and MSO(an irreversible GS inhibitor)due to blocking of nucleotide metabolism and cell cycle arrest.