Optimized Metformin And Trastuzumab Dosing Schedule Based On Circadian Dynamics Of Glucose Metabolism Overcome Trastuzumab Resistance In HER2-positive Gastric Cancer

BackgroundGastric cancer is one of the most common digestive tumors in China,with the second highest incidence rate and the third highest mortality rate.Most patients with gastric cancer in China are in the advanced stage at the time of initial diagnosis.This group of patients mainly undergo palliative treatment,and the median survival time is often less than one year.Encouragingly,the emergence of targeted therapies has brought new hope to patients with advanced gastric cancer.Trastuzumab,which targets human epidermal growth factor receptor 2(HER2),can significantly prolong overall survival(OS)and progression-free survival(PFS)in patients with HER2-positive advanced gastric cancer,is currently the only monoclonal antibody recommended in the NCCN guidelines for first-line treatment of locally advanced or metastatic gastric cancer.However,most patients who were initially effective on trastuzumab developed secondary resistance in the short term after continuous treatment,which greatly limited the patient's clinical benefit.Therefore,further research into the mechanism of trastuzumab resistance is of great significance to improve the drug strategy to solve trastuzumab resistance.High levels of aerobic glycolysis are one of the important causes of secondary resistance to trastuzumab.Lactate dehydrogenase A(LDH-A)expression was up-regulated in trastuzumab-resistant tumor cells,and the level of glycolysis was elevated.The combination of trastuzumab and glycolytic inhibitors effectively reverses the resistance of trastuzumab,thereby inhibiting tumor growth.In addition,trastuzumab inhibits the energy metabolism regulator phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling by restoring PTEN function,thereby exerting an anti-tumor effect.Inhibition of the PI3K pathway enhances the antitumor effect of trastuzumab.Therefore,efficient and specific inhibition of high levels of aerobic glycolysis may be an important therapeutic strategy to overcome trastuzumab resistance.The molecular clock is a group of negative rhythm adjustment loops composed of clock genes(CLOCK,BMAL1,PER,CRY,DEC,etc.)and their encoded proteins,drive a 24-hour change in activities such as metabolism,cell cycle events,and angiogenesis in the body.In addition,the molecular clock also affects the body's sensitivity to drugs.Studies have reported that clock gene CLOCK mutations or BMAL1 deletion mice are more sensitive to cyclophosphamide than wild-type mice,whereas CRY1/2-deficient mice are more resistant to cyclophosphamide.In addition,the study reported that miR-135b promotes gemcitabine resistance in pancreatic cancer by inhibiting the clock gene BMAL1.Therefore,changes in biological rhythm may be one of the important reasons for trastuzumab resistance.Many key enzymes in the metabolic process,such as pyruvate kinase and HMG-CoA reductase,are regulated by the circadian clock mechanism and expressed as circadian rhythm.The rhythm of metabolic genes affects the sensitivity of tumor cells to drugs.Studies have reported that cisplatin resistance is associated with the regulation of glutathione metabolism-related genes by the clock gene CLOCK.In glioblastoma,the sensitivity of tumor cells to bortezomib chemotherapy is related to the circadian rhythm of the redox state regulated and the anabolism of glycerophospholipids by the clock gene BMAL1.Therefore,starting from the circadian rhythm of glycolysis may provide new ideas for reversing the resistance strategy of trastuzumab.Cancer chronotherapy is a method of improving the tolerance and/or efficacy of anti-tumor drugs by adjusting the circadian rhythm system or providing treatment for circadian rhythm systems.Since the efficacy and/or toxicity of many drugs is related to the rhythm of physiological,biochemical,and behavioral processes,administration at the optimal tolerated and/or most effective time point during the day can improve the effectiveness of the drug treatment.A number of studies have shown that the peak expression of circadian rhythm of tumor target genes in one day,the use of target gene inhibitors can play a more significant anti-tumor effect.The study reported that the clock gene CLOCK induced the rhythm expression of the essential cystine transporter xCT,and the administration of the xCT inhibitor sulfasalazine at the peak of xCT expression improved its anti-tumor effect.The number of aldehyde dehydrogenase(ALDH)positive cells in the mouse triple negative breast cancer model showed circadian rhythm changes.The use of the ALDH selective inhibitor DEAB when the number of ALDH positive cells is sufficient can significantly improve the anti-tumor and anti-metastatic effects of ALDH inhibitors on triple-negative breast cancer.Therefore,studying the glycolytic metabolic rhythm of trastuzumab resistanceandbased on this development of a new chronotherapyofglycolytic inhibitor may be an effective means to solve trastuzumab resistance.This study was to investigate the mechanism of circadian rhythm of glycolysis mediates the resistance of trastuzumab.The effect of clock gene on trastuzumab resistance was observed by constructing a 3D culture model of trastuzumab-resistant cell line and a subcutaneous tumor formation model in nude mice in vitro.To elucidate the specific mechanism by which clock genes regulate circadian rhythm of glucose metabolism and mediate resistance to trastuzumab,In order to combine the glycolytic inhibitors from the perspective of chronotherapy and it provides a new strategy for reversing resistance to trastuzumab.Methods and Results:1.HER2-positive trastuzumab-resistant gastric cancer cells have regular circadian rhythms of glucose metabolismHER2 overexpressing N87 cells and SNU216 cells(trastuzumab-sensitive cells,hereinafter referred to as N87 and SNU216),and trastuzumab-resistant cells constructed using a 3D cell culture model were selected(trastuzumab resistant cell line,hereinafter referred to as N87-TR and SNU216-TR).N87-TR cells were found to be less sensitive to trastuzumab by MTT,immunofluorescence,and colony formation experiments.Real-time quantitative polymerase chain reaction(qPCR)and western blot(WB)assays were used to detect dexamethasone-synchronized N87 and N87-TR cells in vitro.Compared with N87 cells,the expression of HK2 in N87-TR cells was significantly increased and exhibited circadian rhythm oscillation,reaching the highest level at ZT6 and ZT30,and dropping to the lowest level at ZT22 and ZT42.N87-TR cells showed stronger HK2 activity and produced more ATP at the time of ZT6 and ZT30(Zeitgeber time,ZT,0 point after biorhythm synchronization is defined as ZTO).The yield of lactic acid in ZT0?12 and ZT24?36 were higher.The above experiment was repeated using dexamethasone-synchronized SNU216 and SNU216-TR cells in vitro,and consistent results were also obtained.The subcutaneous tumor-bearing nude mice reared under a 12:12 hour light-dark cycle were inoculated with N87 cells on the left side and N87-TR cells on the right side and examined by PET-CT showed that the glucose metabolism level of N87-TR tumor was higher than that of N87 tumor.The level of glucose metabolism of N87-TR tumor in the morning is higher than at night.2.The mechanism of clock gene PER1 regulating HK2 circadian rhythm oscillation mediated trastuzumab resistanceIn N87-TR and SNU216-TR cells synchronized with dexamethasone,PER1 mRNA and protein levels increased and were consistent with the trend of HK2 circadian rhythm oscillation.Next,siRNA knockdown of PER1 expression in N87-TR and SNU216-TR cells revealed a significant decrease in HK2 protein expression,HK2 activity,lactic acid and ATP production.Furthermore,PER1 was knocked down in dexamethasone-synchronized N87-TR and SNU216-TR cells,and it was found that the expression of HK2 lost the circadian rhythm oscillation characteristics.This indicates that the clock gene PER1 is involved in the circadian rhythm of HK2 in N87-TR and SNU216-TR cells.TCGA and Kaplan-Meier Plotter database analysis of high expression of PERI in gastric cancer patients is associated with poor prognosis of gastric cancer.MTT,flow apoptosis assay and live/dead cell staining experiments all suggested that silencing PER1 resulted in enhanced growth inhibition of trastuzumab in N87 cells and significantly reversed the resistance of N87-TR and SNU216-TR cells to trastuzumab.This indicates that PER1 is associated with the sensitivity of trastuzumab.Further,MTT and flow results indicate that the glycolysis inhibitor 2-DG or metformin reverses the resistance of trastuzumab caused by PER1 overexpression.The above data indicate that PER1 overexpression mediates trastuzumab resistance by regulating circadian rhythms of cellular glucose metabolism.Finally,we have a preliminary discussion on the mechanism by which PER1 regulates the expression of HK2 rhythm.Silencing PER1 does not affect the expression of PPARy.CoIP results showed that PER1 interacts with PPARy at the protein level.WB results showed that knockdown of PER1 in N87-TR cells and addition of peroxisome proliferators-activated receptor gamma(PPARy)agonist Pioglitazone or Rosiglitazone reversed the inhibitory effect of PER1 deletion on HK2.At the same time,silencing PPAR? in N87 cells reversed the up-regulation of HK2 expression by PER1 overexpression,suggesting that PERI may regulate HK2 expression through the PPARy pathway.3.Combined use of metformin and trastuzumab based on circadian rhythm of glycolysis metabolism to overcome the resistance ofHER2-positive gastric cancer to trastuzumabTo investigate the influence ofadministration time of metformin on the reversal effect of trastuzumab resistance when combined with trastuzumab.N87-TR tumor-bearing mice were treated with PBS,metformin,trastuzumab,and metformin in combination with trastuzumab at ZT6 or ZT18.The tumor volume of mice treated with metfonnin and trastuzumab was significantly reduced at the peak of HK2 expression at ZT6 compared to mice treated at ZT18.The immunofluorescence results of tumor tissues also showed that the expression of cleaved caspase-3 was the highest and the expression of ki67 was the lowest in the combination of metformin and trastuzumab at ZT6.These data reveal the most appropriate dosing time for the selection of metformin plus trastuzumab based on the circadian rhythm of glycolysis metabolismcan completely reverse the resistance of trastuzumab and improve the efficacy of the drug.During this study,we unexpectedly found that in addition to inhibiting the level of glycolysis,metformin also down-regulated the expression of PER1.Furthermore,we found in the WB experiment that metformin can activate the AMPK pathway in N87-TR cells and down-regulate the expression of PER1,but it can not reduce the expression of PERI in the shRNAK group N87-TR cells.The time mRNA expression profile of dexamethasone-synchronized N87-TR cells treated with metformin showed a decrease in PERI amplitude,an increase in the amplitude of BMAL1 and CLOCK,and the peak of both goes forward.Therefore,the above results suggest that metformin can down-regulate the expression of PER1,and the circadian clock of N87-TR cells can partially reset.ConclusionThis study was the first to find a circadian rhythm oscillation in the expression of the key glycolytic enzyme HK2 in trastuzumab-resistant HER2-positive gastric cancer.Optimize the use time of metformin combined with trastuzumab based on changes in circadian rhythm of glycolysis.A chronotherapy strategy of metformin for more effective reversal of trastuzumab resistance in HER2-positive gastric cancer was proposed.Mechanism,the clock gene PER1 is the driving reason for the HK2 circadian rhythm oscillation.PERI may regulate the expression of HK2 by the PPARy pathway.In conclusion,this study revealed the presence of circadian rhythm oscillations in glycolysis in HER2-positive trastuzumab-resistant gastric cancer.The HK2 circadian rhythm can be used as a novel molecular target ofchronotherapy for reversing the resistance to trastuzumab.