BMAL1-CLOCK-PER1 Regulates Glycolytic Biorhythms To Mediate Trastuzumab Resistance In HER2-positive Gastric Cancer

BackgroundGastric cancer has become a global disease.The population of gastric cancer in China remains high.At present,China is also known as a large country with gastric cancer.Patients with gastric cancer are often characterized by "three highs and three lows".High incidence rate,High metastasis rate,High death rate;low early diagnosis rate,Low radical resection rate and low 5-year survival rate,which leads to high mortality rate of gastric cancer.The treatment of advanced gastric cancer has always been a clinical problem that requires urgent solution.In the treatment of advanced gastric cancer,the efficacy of first-line chemotherapy is still poor.The median survival(mOS)is hardly exceeded 2 years.Among the first-line treatment options for advanced gastric cancer patients,only Trastuzumab has shown improved efficacy in HER2-positive advanced gastric cancer patients,while other targeted drugs have failed in clinical studies.Although Trastuzumab improves overall survival in HER2-positive gastric cancer patients,many patients exhibit primary resistance,and those who initially respond to treatment may eventually become secondary resistant.Therefore,it is of great clinical value to study the mechanisms of resistance to targeted drugs and reversal of targeted drug treatment strategies in gastric cancer.Biological rhythms,also known as molecular clocks or biological clocks,are part of chronobiology,which enables the understanding of organisms in the fourth dimension of time.Three American geneticists were awarded the 2017 Nobel Prize for the occurrence of genes regulating biological rhythms.In molecular terms,complex transcription-translation networks comprise biological rhythms,including the core biological clock genes CLOCK,BMAL1,PER,CRY,and others.These genes are expressed cyclically and constitute a regulatory network driving the rhythmic expression of thousands of downstream target genes,resulting in the rhythmic activity of biological functions and cellular metabolism.The current study found that biorhythms are associated with the development of tumors in vivo.Biorhythms not only maintain environmental homeostasis in the human body,but also affect various important biological functions.As the fourth dimension of time is an important part of the world,clinical research on the temporal dimension is not very common at present and its importance has been neglected.With the progress of medicine and technology,biorhythms can also be used to predict or treat diseases.Currently,in the field of oncology,studies on biorhythms have focused on the core biological clock genes in relation to tumorigenesis and prognosis.We speculate that biorhythms are also associated with Trastuzumab resistance in gastric cancer.In this paper,we investigate the association of core biological clock genes with gastric cancer and Trastuzumab resistance,explore the mechanism of reversing Trastuzumab resistance in gastric cancer,and find the treatment strategy to improve the prognosis of patients with advanced gastric cancer.Methods and Results:1.Altered biorhythms and metabolic rhythms after Trastuzumab resistance in HER2-positive gastric cancerWe have successfully constructed HER2-positive gastric cancer resistant cell lines SNU216-TR and N87-TR by using 3D cell culture method,and verified the resistance by various methods including cell proliferation assay and protein assay.After using dexamethasone to synchronize intracellular biorhythms,cells were collected at 4-hour intervals starting at ZT0 for 48 hours.Core biological clock gene RNA levels were measured in two HER2 overexpressing gastric cancer cell lines,N87 and SNU216,in both sensitive and drug-resistant cell lines.We found that tumor cell biorhythms have been reset in the drug-resistant strains,and a new biorhythm change was formed.By Western Blotting as well as mice experiments,we found that the glycolytic related enzyme HK2 expression cycle was changed and the tumor metabolic rhythm was reset after Trastuzumab resistance in HER2-positive gastric cancer.In this part,we found that the biological rhythm was reset and the oscillatory cycle was changed in tumor cells after tumor cell resistance,while the glycolytic metabolic rhythm of tumor cells was changed and the tumor microenvironment was subsequently altered.It is suggested that after tumor drug resistance,genes are not only structurally altered such as mutations,but also changes in gene expression cycles and biological rhythms.This enables us to explore tumor treatment strategies from the fourth dimension of time,not only limited to genomic chromatin sequence alterations.2.BMAL1-CLOCK-PER1 causes Trastuzumab resistance via HK2In two HER2-positive gastric cancer cell lines,N87-TR and SNU216-TR,we used small interfering RNA to silence 11 core biological clock genes,including:BMAL1,CLOCK,PER1,PER2,PER3,CRY1,CRY2,REV-ERB?,REV-ERB?,ROR?,DBP.Three key clock genes,BMAL1,CLOCK,and PER1,were significantly associated with the expression of glycolysis-related enzyme HK2.The activity of glycolysis-related enzyme HK2,ATP levels,and lactate content of glycolysis products were assayed to further validate our findings.Next,the cells were collected every 6 hours for 48 hours starting from ZT0,and in two drug-resistant cell lines,which BMAL1,CLOCK,and PER1 had been knocked down,the RNA expression of the glycolysis-related enzyme HK2 was measured.The expression rhythms of BMAL1,CLOCK,and PER1,the core biological clock genes,were inhibited.The expression cycle of the glycolysis-related enzyme HK2 was reset,and the glycolytic metabolic rhythms were altered in HER2-positive gastric cancer drug resistance.Using MTT cell proliferation assay and CCK8 cell proliferation assay to conduct the reply experiment.We found that the effect of key biological clock gene BMAL1-CLOCK-PER1 in HER2-positive gastric cancer was affected on Trastuzumab resistance by disrupting the expression rhythm of glycolysis-related enzyme HK2 in tumor cells.In this section,we found that the key biological clock gene expression cycle affects the metabolism of tumor glycolysis-related and its products,as well as tumor metabolic rhythms,by disrupting the expression rhythm of tumor cell glycolysis-related enzyme HK2.3.PER1-PPARy directly regulates HK2 expression rhythmIn the classical mechanism of biological rhythm,BMAL1-CLOCK initiates downstream gene transcription by forming a heterodimer that binds to the E-box(Enhancer box)of the downstream gene promoter region.We speculate that in HER2-positive gastric cancer,BMAL1-CLOCK may similarly regulate HK2 transcription directly by binding to the E-box in the promoter of the target gene.However,we were unable to find the E-box classical binding region in the HK2 promoter region.As for the non-classical region of E-box,we found that BMAL1's antibody did not pull down the non-classical region of E-box in HK2 promoter region by ChIP experiments.We performed CoIP experiments and found that PER1 binds directly to PPARy at the protein level and that this binding action occurs rhythmically in the organism,which in turn regulates downstream HK2 expression and the biological clock rhythm of glucose metabolism within the tumor.ConclusionWe observed changes in gene expression cycles,metabolic cycles,and biorhythms within tumors at the level of the fourth dimension of time.We found that the expression cycle of key biological clock genes in the tumor was changed,the metabolic rhythm of glycolysis in the tumor was altered,and the biological rhythm in the tumor was reset after HER2-positive gastric cancer resistance to Trastuzumab.Three key biological clock genes,BMAL1,CLOCK,and PER1,were significantly associated with the expression rhythm of the glycolysis-related enzyme HK2.Further investigation of the mechanism revealed that BMAL 1-CLOCK caused PER1 and PPARy to bind each other at the protein level,and this binding action occurred rhythmically in the organism,which regulates the metabolic rhythm of HK2 and ultimately makes the tumor resistant to the drug.This project explores the mechanism of tumor targeted therapy resistance from the perspective of molecular biological clock and provides a new idea to improve Trastuzumab resistance in advanced gastric cancer patients.