Trastuzumab Reverses Chemotherapy Resistance Induced By ABCG2 In HER2-positive Gastric Cancer Through PI3K-AKT

Background and purpose:Gastric cancer is the fifth most commonly diagnosed cancer and the third leading cause of cancer-related death worldwide.The median overall survival for patients with locally advanced disease or distant metastasis is 10-12 months.Chemotherapy remains the cornerstone of gastric cancer treatment,which has low efficiency and high adverse effects.The addition of anti-HER2 drugs to chemotherapy regimens in HER2-positive advanced gastric cancer significantly improved PFS,OS.Trastuzumab was approved as first-line treatment for patients with HER2-positive metastatic gastric adenocarcinoma,and trastuzumab deruxtecan was approved as second-line treatment.However,much fewer drugs have been approved for HER2-positive gastric cancer than for HER2-positive breast cancer.Gastric cancer is a heterogeneous solid tumor,which makes drug development difficult,and the stomach,as an important digestive organ,is closely related to patients' quality of life.Therefore,research on drug therapy for gastric cancer needs to be strengthened.We searched for the best advantageous combination of trastuzumab and chemotherapeutic drugs by investigating which chemotherapeutic drugs were enhanced by the combination of trastuzumab.And the chemotherapeutic drug we selected could also be candidates for ADC.Methods:Part ?:HER2 mRNA expression levels and clinical data were downloaded from the TCGA database.We have obtained data which contain 252 HER2-negative and 50 HER2-positive gastric cancers.The genes with |log2(FC)|>1 and-log10(Padj)>1.30103 were taken as differentially expressed genes in HER2-positive versus negative gastric cancers and heat map was plotted.PPi analysis was performed using the R package STRING,and the results were visualized and plotted using cytoscape.Then we select a HER2-associated sub-network.We performed functional enrichment analysis,which called GO and KEGG analysis,on differentially expressed genes.To investigate the relationship between HER2 expression and clinicopathological parameters in gastric cancer patients,common clinical information such as age,gender,tumor site,TNM stage,lymph node metastasis and depth of tumor infiltration were counted in HER2-positive and negative patients.Western blot was applied to detect HER-2 protein expression in common gastric cancer cell lines MKN28,MGC803,AGS,NCI-N87 and SGC7901,and HER-2-positive and negative cell lines were screened.Part ?:MTT was used to determine the IC50 of cisplatin(cDDP),fluorouracil(5-Fu),docetaxel(TXT)and epirubicin(EPI)on the screened cell lines MKN28,SGC7901 and NCI-N87.The inhibition rate of trastuzumab on the cell lines at different concentrations was also detected by MTT.Gastric cancer cell lines MKN28,SGC7901 and NCI-N87 were incubated with single drug concentrations of epirubicin 1 ?M,cisplatin 10 ?M,5-fluorouracil 1000 ?M and docetaxel 5 ?M for 24 h,then the sensitivity of different cell lines to chemotherapeutic drugs was determined.To screen the advantageous combination of drug combination,the IC50 of each chemotherapeutic drug combined with trastuzumab(100 mg/L)was used to co-treat the screened cell lines.The inhibition rate was detected by MTT to compare which drug has synergistic effect with trastuzumab by comparing with the inhibition rate of chemotherapeutic drugs alone.Part ?:Western blot was applied to detect the expression of pAkt,AKT,pERK and ERK proteins in common gastric cancer cell lines MKN28,MGC803,AGS,NCI-N87,SGC7901.The screened cell lines were treated with four treatment groups(control,epirubicin alone,trastuzumab alone,trastuzumab combined with epirubicin)were set up with three time points(0h,12h,24h),and the expression of key kinases pAKT and pERK in PI3K/AKT and MAPK pathways were detected by western blot.Part ?:Epirubicin can produce visible light under excitation light,so it can autofluoresce.The difference of intracellular epirubicin accumulation was detected by fluorescence microscopy after 12 hours by treating with epirubicin alone and epirubicin combined with Herceptin in MKN28 and NCI-N87 cell lines.Western blot was used to detect the difference of ABCG2 protein expression in NCI-N87,MKN28 and SGC7901 cell lines.Epirubicin alone,epirubicin+Herceptin,and epirubicin+Herceptin+740Y-P were treated with HER2-positive NCI-N87 cell line for 24h to detect whether pAKT,AKT,and ABCG2 protein expressions were correlated.And 740Y-P is a PI3K activator.Results:Part ?:HER2 mRNA expression levels and clinical data were downloaded from the TCGA database to obtain 252 HER2-negative and 50 HER2-positive gastric cancer samples.1059 differentially expressed genes,including 307 up-regulated differentially expressed genes and 752 down-regulated differentially expressed genes,were obtained from HER2-positive and HER2-negative samples.PPI analysis of 1059 differentially expressed genes was performed by STRING,and the sub-network map of ERBB2 association was selected,including 21 up-regulated genes and 36 down-regulated genes,of which ABCG2 gene encoding drug transport protein was one of the up-regulated genes.Functional enrichment analysis of differentially expressed genes of HER2 showed that,the main molecular functions are receptor ligand activity,signal receptor activator,monovalent cation transmembrane transporter,the main biological processes are T cell activation,cell adhesion regulation,and calcium ion homeostasis,which are all related to HER2 gene function.The percentage of positive patients among gastric cancer patients obtained from the database was 16.5%,similar to domestic and international studies,but there was no significant relationship between patients' HER2 expression with common clinicopathological data such as gender,age,tumor site,TNM stage,lymph node metastasis and depth of tumor infiltration.Among the common gastric cancer cell lines MKN28,MGC803,AGS,NCI-N87,and SGC7901,NCI-N87 had high expression of HER2 protein relative to other cell lines,SGC7901 had moderate expression,and MKN28 had the lowest expression.NCI-N87 and SGC7901 could be selected as HER2 positive expression gastric cancer cell lines and M28 as HER2 negative expression gastric cancer cell line.Part ?:The single-drug IC50 of cisplatin,5-fluorouracil,docetaxel,and epirubicin for NCI-N87 gastric cancer cell line was 4.08 mg/L,49.45 mg/L,3.00 mg/L,and 0.76 mg/L by MTT,and for MKN28 gastric cancer cell line was 3.44 mg/L,146.16 mg/L,2.54 mg/L,and 0.39 mg/L,and for SGC7901 gastric cancer cell line was 3.62mg/L,198.36mg/L,3.23 mg/L,1.05mg/L.Trastuzumab alone had no significant inhibitory effect on three gastric cancer cell lines,and the inhibition rate of trastuzumab at high concentration of 1000 mg/L on HER2 high expressing cell line N87 was only 10.12%.The inhibition rate of HER2-positive cell line SGC7901 and NCI-N87 was significantly different compared with that of HER2-negative cell line MKN28 at the same concentration of epirubicin,which was statistically significance(p<0.05),suggesting that HER2-positive cell lines were epirubicin-resistant.At the same time,HER2-positive and negative cell lines showed no significant difference in sensitivity to cisplatin,fluorouracil,and docetaxel.In Her-2-positive N87 and SGC7901 cell lines,trastuzumab combined with epirubicin inhibited significantly more than chemotherapy alone(p<0.05).And Herceptin combined with cisplatin,Herceptin combined with 5-fluorouracil,and Herceptin combined with docetaxel showed no significant change than chemotherapy alone.In Her-2-negative M28 cell line,no significant change was seen in the inhibition rate of trastuzumab combined with four chemotherapeutic agents compared with chemotherapy alone.Part ?:Among the common gastric cancer cell lines MKN28,MGC,AGS,NCI-N87,and SGC,the expression of pAkt and pERK in HER2-positive gastric cancer cell lines NCI-N87 and SGC7901 was significantly higher than the other three cell lines,which was statistically significant(P<0.05),suggesting that the expression of MAPK,PI3K-AKT pathway were significantly activated.In Her-2-positive N87 and SGC7901 cell lines,trastuzumab alone and trastuzumab combined with epirubicin significantly decreased pAKT(p<0.05)after treating cells for 12 and 24 h.These trends were not found in the Her-2 negative M2 8 cell line,suggesting that the PI3K/AKT pathway may be involved in the synergistic effect of trastuzumab and epirubicin.In Her-2-positive cell lines N87 and SGC7901,pERK expression levels were significantly reduced after 24 h of trastuzumab monotherapy compared with other groups(p<0.05),but pERK was not reduced in the trastuzumab combined with epirubicin group compared with the control group,suggesting that the MAPK signaling pathway did not play a significant role in the synergistic effect of trastuzumab and epirubicin.Part ?:Epirubicin accumulation in the nucleus did not change significantly after the addition of Herceptin in HER2-negative cell line MKN28,while epirubicin accumulation in the nucleus increased significantly after the addition of Herceptin in HER2-positive cell line NCI-N87,which was statistically significant(p<0.05),the fluorescence intensity of individual cell nuclei increased significantly,indicating an increase in intracellular accumulation of epirubicin,further suggesting a decrease in the activity of chemotherapeutic drug resistance-associated transporter proteins on the cell surface.The main members of the ABC superfamily resistance pump are ABCB1,ABCC1,ABCG2,but only ABCG2 was found correlated with HER2 expression significantly by bioinformatic analysis of TCGA database.Western blot showed that HER2-positive cell lines NCI-N87 and SGC7901 highly expressed ABCG2 protein,compared with HER2-negative cell line MKN28.which was statistically different(p<0.05).And the same results as the PPI subnet map of HER2-associated genes in the first part of the bioinformatic analysis,ABCG2 protein was a HER2-associated upregulated gene.In the drug treatment experiments,ABCG2 protein and pAKT were unchanged with epirubicin alone,significantly decreased simultaneously with the addition of Herceptin to inhibit PI3K/AKT pathway,and then increased simultaneously with the addition of PI3K activator 740Y-P,which reversed the effect of Herceptin,indicating that the ABCG2 protein expression was associated with PI3K/AKT pathway activity positively and was regulated by PI3K/AKT signaling pathway-related proteins.Conclusion:HER2 expression in gastric cancer was not significantly related to common clinicopathological data such as gender,age,tumor site,TNM stage,lymph node metastasis and depth of tumor infiltration.However,the addition of anti-HER2 drugs significantly improved the antitumor effect of chemotherapeutic drugs.HER2-positive gastric cancer showed resistance to epirubicin,and trastuzumab alone had no significant inhibitory effect on Her-2-positive N87 and SGC7901 cell lines,and the combination with epirubicin enhanced the effect of epirubicin.HER2-positive gastric cancer had a significant activation of MAPK and PI3K-AKT pathways,especially HER2 was found to be positively correlated with ABCG2 transporter protein expression by bioinformatic analysis.It was further found that Herceptin decreased the expression of ABCG2 transporter protein by inhibiting the phosphorylation level of PI3K/AKT pathway and decreased the efflux of epirubicin,which increased the aggregation of epirubicin in the nucleus and thus increased the rate of cell inhibition.Both epirubicin and Herceptin have cardiotoxicity,which is a concern for us with the combination of them.It has been noted that the safety and efficacy of sequential trastuzumab maintenance therapy after epirubicin-based chemotherapy in combination with trastuzumab is acceptable,and the decrease in EF is reversible and improved with carvedilol.And the cardiotoxicity of anthracyclines can be mitigated by modifying the chemical structural moiety.Significance:In recent years,it is significant to study the targeted therapy of HER2.Firstly,the abnormal expression of HER2 is widely present in a variety of solid tumors,and newly developed drugs can be considered for a variety of tumor indications.Secondly,due to the bystander effect of new ADC drugs,the scope of HER2 positivity is expanding,and HER2 immunohistochemistry 2+can be identified as HER2 positive without performing FISH,increasing the drug indications population.Therefore,more cancer types and more people are benefit from anti-HER2 therapy.The development of ADC drugs is still emerging,especially those targeting HER2,and we choose the advantageous combination of trastuzumab and chemotherapy drugs to provide candidates for ADC to achieve better therapeutic effects.The results of the study in gastric cancer can also suggest the potential clinical guidance on how to optimize the choice of targeted drugs in combination with chemotherapy in other solid tumors,and the exploration of the molecular mechanism of drug combination synergy will provide new clues for the design of future treatment regimens.This exploratory study is centered on clinical issues and has potential application and marketability.