Isoforms Switch Of Glutaminase-1 Drives Therapeutic Resistance And Progression Of Prostate Cancer

BackgroundsProstate cancer（PCa）is the most commonly diagnosed malignancy and the second leading cause of cancer-related death in western countries.Its incidence is also increasing rapidly in countries such as China where PCa had traditionally been considered relatively uncommon.Generally,early stage of PCa where the lesions are organ-confined could be cured by prostatectomy.For patients with advanced or metastatic PCa where local therapies can no longer be applied,the treatment of choice turns out to be androgen deprivation therapy（ADT）which inhibits androgen production and/or blocks androgen receptor（AR）function.The molecular basis for hormonal therapy is that PCa is a hormonally-regulated disease.However,this therapy is not curative and cancer recurs after an initial response period averaging 18 months which is known as castration-resistant prostate cancer（CRPC）.Although newer agents have been developed to inhibit intratumoral androgen production（e.g.abiraterone acetate）or more effectively block AR function（e.g.enzalutamide）to extend survival in patients who have exhausted therapeutic options,the clinical benefits are limited and patient would eventually progress to therapeutic resistance stage.Therefore,exploring the potent mechanism and seeking new drug targets are urgently needed.As a transcriptional factor,AR plays a critical role in the progression of PCa whose signaling pathway is usually the pharmacological target.However,there are still rare cases that do not express AR,such as neuroendocrine prostate cancer（NEPC）.As a consequence,they have little response to antiandrogen treatment and show poor prognosis.To this regard,it is quite worthy finding other targets which are beyond AR axis.Alteration in cellular metabolism in cancer cells is recognized as a hallmark of malignancy which could be distinguished from normal cells.Glutamine is the most abundant amino acid in organism and serves as a major nutrient and energy source to a variety of tumors.Studies and clinic experience have demonstrated that PCa prefers to take up glutamine rather that glucose.Herein,in this study,we will discover the effect of glutamine metabolism on PCa,especially on the disease development and thus try to identify a novel AR-independent therapeutic target.AimsThis study is to assess the alteration of glutamine metabolism,specifically the rate-limited enzyme glutamiase-1,in different stage of prostate cancer and to explore the mechanism of the therapeutic resistance and thus finding a novel pharmacological target.Methods1.The concentrations of metabolites from culture media and cells were determined byliquid chromatography-mass spectrometry（LC-MS）.2.¹³C₅-glutamine tracing experiment was performed by using ¹³C₅-glutamineisotompomer and determined by LC-MS.3.Cell viability and proliferation were detected by trypan blue staining and MTSassay.4.Protein levels were determined by western blot.5.mRNA levels were determined by qRT-PCR.6.Protein concentrations in tissues were determined by immunohistochemistrystaining.7.Transient knockdown experiment was performed by using siRNAs.8.Stable cell lines were generated by lentivirus infection.9.Invasive ability of cells was determined by Transwell experiment.10.The resistance of GAC-overexpressed LNCaP cells to ADT,the inhibitory efficacyof CB-839 and the switch of the two isoforms in vivo were all tested in animalmodels.11.The association of AR and GLS1 was determined by ChIP.12.TheconstructedplasmidspRL-CMV-GLS1-3’UTR,pRL-CMV-GLS1-3’UTR-mutAREI,pRL-CMV-GLS1-3’UTR-mutAREIIandpRL-CMV-GLS1-3’UTR-mutAREI&II were performed by a series of geneengineering processes.13.The regulation of AR on GLS1 was determined by using luciferase due-reportersystem.14.The inhibitory efficacy of CB-839 on different cell lines was manifested by IC₅₀value.Results1.ADT inhibits ADPC by decreasing metabolites levels.2.ADT renders glutamine accumulated in LNCaP cells.3.ADT-induced glutamine accumulation is due to the disability of glutamineutilization rather than the decreased glutamine up-taking.4.AIPC cells are more dependent on glutamine than ADPC cells.5.AIPC cells consume more glutamine than ADPC cells to replenish TCA cycle fornutrients and energy supplement.6.GLS1 expresses strongly in high pathological grade and advanced clinic stage.7.GLS1 is critical to the invasive ability of AIPC cells.8.KGA,one of the GLS1 isoforms,is the major isoform in ADPC while GAC,anotherisoform of GLS1,dominates in AIPC.9.Isoforms switch of KGA and GAC contributes the development of AIPC.10.AR regulates GLS1 expression through 3’UTR and mediates the splicing of GLS1to different isoforms.11.GAC is the isoform which is beyond androgen regulation and confers cells withresistance to ADT via enhancing glutamine utilization.12.Selective GLS1 inhibitor CB-839 has the inhibitory efficacy on AIPC tumor.Conclusions1.Isoforms switch of KGA to GAC drives therapeutic resistance and diseaseprogression of prostate cancer.2.GLS1（GAC）is a novel pharmacological target of AIPC.