The Study Of Glucose And Fatty Acid Metabolism For Prostate Cancer Diagnosis And Its Role In Cancer Progression

Background In recent years,prostate cancer(PCa)has become the tumor with the second highest incidence in elderly males.It is difficult for the early-diagnosis and the treatment in the advanced stage.As one of the most important cancer hallmarks,the tumor metabolism should be paid more attention to.In order to provide enough energy and to supply small molecules to numerous cellular activities,cancer cells tend to present a metabolic reprogramming.Glucose and lipid metabolism rewiring has been accepted as one of the main characteristics of cancer cells.The heterogeneities of prostate cancer include the different metabolic characters between PCa and other cancers and the switches involved in different stages during cancer development.Thus,the study of glucose and lipid metabolism is of great significance for the early diagnosis and advanced treatment of prostate cancer.Aim To investigate the changes of glucose and lipid metabolism in prostate cancer and to explore the new form of glycolysis of prostate cancer-"glycolytic potential" which might provide possible new targets for early diagnosis and adjuvant treatment method.To study the fatty acid chain elongation in advanced prostate cancer and to find new target for the treatment of advanced prostate cancer which is resistant to enzalutamide.Materials and methods Metabolomic profiling is used for the detection of metabolic changes between different tissue and cell samples.Metabolic tracings are to detect the source of metabolites in glycolysis and glucose tricarboxylic acid(TCA)cycle.The Seahorse XF system is to monitor the energy production and the metabolic pathways.ATP colorimetric detection is to measure the ATP level in cells.RNA-Seq analysis measures the transcription level of each gene.Tissue microarray(TMA)confirmed the differential expression detected at m RNA levels.In terms of bioinformatics analysis,combined with public databases,such as TCGA,genomic variation analysis(GSVA)and gene set enrichment(GSE)analysis are utilized.Western blotting and QPCR was to detect protein and RNA levels respectively.The CHIP method performs nucleic acid binding analysis.We also used immunofluorescence to observe mitochondrial dynamics.The use of testing kits was to detect changes in cell proliferation,apoptosis and mitochondrial membrane potential.Wound healing and transwell tests were used to observe the migration ability of tumor cells.For growth curve,we used methods such as cell counting or cell staining.The medium components were adjusted according to different experiments and the conditioned medium was used for cell culture.The corresponding fatty acids were externally subjected to the conditioned medium accordingly.In order to study the effect of up-or down-regulation of genes,we developed plasmids,lentivirus and CRISPR-CAS9.In vivo experiments were performed using nude mice and Nod-Scid mice.Mouse tumor imaging and 18F-FDG-PET/CT were used to observe tumor sizes and radiotherapy as well as intraperitoneal injection were used for tumor treatment.Results The RNA-Seq data show that glycolysis is significantly down-regulated in prostate cancer compared with the non-carcinoma tissue.Mitochondria-related genes are significantly increased in PCa tissues.Metabolomic profiling show that glucose is decreased,while pyruvate,malate,and succinate levels raised.Further analysis reveals that the mitochondria pyruvate carrier2(MPC2)elevated most significantly in PCa tissue.Tissue micro-assay(TMA)suggests that MPC1 significantly decreased with the increase of Gleason score,while MPC2 has no significant difference.There are also close relationships between MPC1 and invasion,incision edge invasion.Both of MPC1 and MPC2 are shown related with the biochemical recurrence and oncogenes related proteins,such as p-AKT,bcl2,and ERG.In order to further observe the changes in cell biological behavior after MPC inhibition or overexpression,we applied MPC inhibitor UK5099 as an inhibitory treatment and MPC1/2 overexpression lentivirus treatment for overexpressed stable cell lines.It shows that highdose UK5099(100 ?M)inhibits growth and migration of all cell lines.Low-dose UK5099(10 ?M)promotes tumor cell proliferation,migration and ATP production within 24 hours,while in benign prostate cell lines all of the above are inhibited.In metabolic profiling analysis,low concentration of UK5099 causes a significant increase in glucose uptake in C4-2B cell lines,while no similar phenotype is observed with BPH-1 cells.Interestingly,although profiling analysis presents a reduced TCA level both in low/high doses of UK5099,metabolic tracer shows that in the C4-2B cell line,the carbon atoms of citric acid and ?-KG are more derived from glucose,which is not observed in BPH-1 cells.This also suggests that prostate cancer cells might have the "glycolytic potential".Since then,immunofluorescence results showed that high doses of UK5099 can promote mitochondrial fission,while the protein levels of VDAC1 vary the most significantly between prostate cancer cells and nontumor prostate cells.Animal experiments suggest that the use of UK5099 increases the sensitivity of 18FDG-PET / CT and radiation therapy for prostate cancer.In lipid metabolism,cells treated with ADT or enzalutamide significantly increased the level of long-chain unsaturated fatty acids(PUFA)according to the profiling data.RNA-Seq data also present a significant increase in ELOVL5 in NEPC(neuroendocrine prostate cancer)cell lines.After knockout or knockdown of ELOVL5 expression,there is no significant change in cell growth.The enzalutamide-sensitive cells are resistant to enzalutamide when ELOVL5 is overexpressed.The proliferation reduction caused by enzalutamide is up regulated after ELOVL5 knocked out in ENZA-resistant cells.After that,the WB results suggested that the AKT-m TOR pathway might mediate its biological effects after ELOVL5 knockout or PUFA treatment.Conclusions This study was the first to put forward the hypothesis of "glycolytic potential" induced by MPC inhibition.It may be provided for the clinical work.VDAC1 may play an important role in glycolytic potential.Glycolytic potential can contribute to the increased sensitivity of 18F-FDG-PET/CT and radiotherapy treatment of prostate cancer.Overall,glycolytic potential is a new model for energy metabolism in prostate cancer and its key enzyme MPC may be a key target for early diagnosis and treatment.This model complements the existing tumor metabolic reprogramming system.Fatty acid elongation plays an important role in the development and drug resistance of NEPC.Among them,the key enzyme ELOVL5 and one of its products,arachidonic acid,are of great significance during the disease development.More importantly,it plays a key role in the tumor enzalutamide resistance.This also provides new possible targets for further clinical treatment of enzalutamide-resistant prostate cancer.