Membrane Cholesterol Decreased Chemotherapy Sensitivity In HER2-positive Trastuzumab Resistant Gastric Cancer Via Upregulation Of ABCB1

BackgroundIncidence of gastric cancer is particularly high in East Asia.Nearly half of the world's gastric cancer patients are located in China.As the initial symptoms of gastric cancer are hidden,80%of gastric cancer patients in China are in the advanced stage when firstly diagnosed.For unresectable gastric cancer,chemotherapy is the basis of systemic treatment,but limited efficacy and obvious adverse reactions of chemotherapy impair patients' survival benefits.The median survival time is less than ? year and the 5-year survival rate is less than 30%.With the development of tumor molecular biology,targeted therapy has become one of the treatment methods for malignant tumor.In 2010,the ToGA study showed for the first time that trastuzumab that targets HER2 combined with chemotherapy(cisplatin combined with fluorouracil or modified protocol)compared to chemotherapy alone could prolong the overall survival time by nearly 6 months(NCT01041404),which made gastric cancer enter an era of targeted treatment.However,no suitable drug targeting other targets such as VEGF,MET,and EGFR has succeeded in the first-line clinical research of unresectable gastric cancer:trastuzumab occupied a unique position in the first-line treatment of gastric cancer.Unfortunately,acquired resistance of trastuzumab that occurs in about one year after first-line treatment is difficult to avoid.Moreover,trastuzumab,which is approved for cross-line use in HER2-positive breast cancer,has failed in cross-line treatment studies for HER2-positive gastric cancer.Thus,patients with HER2-positive gastric cancer in the post-ToGA era urgently need new therapeutic schemes.However,the clinical trials that have been conducted on patients with HER2-positive gastric cancer progressing after the first-line treatment have failed.Lapatinib and Trastuzumab emtansine(T-DM1),which have been approved for HER2-positive breast cancer,have not shown efficacy in second-line treatment of HER2-positive gastric cancer.In addition,the treatment option for second-line chemotherapy does not differ based on HER2 expression in NCCN(National Comprehensive Cancer Network)guidelines for gastric cancer.In summary,there is currently no available targeted treatment strategy for patients who have progressed after the treatment of first-line trastuzumab combined with chemotherapy.Clinically,this part of patients is treated with second-line chemotherapy for gastric cancer as well as HER2-negative patients.The two most recommended options are:Single-agent taxanes and single-agent irinotecan,which have similar efficacy in patients undergoing second-line treatments of gastric cancer.No research has investigated whether the sensitivity of second-line chemotherapy drugs to HER2-positive gastric cancer has changed after acquiring trastuzumab-resistant phenotype.Therefore,there is still no breakthrough in the choice of chemotherapy drugs and targeted drugs for HER2-positive gastric cancer patients after the progress of first-line treatment.On the one hand,if trastuzumab resistance has an effect on the sensitivity of chemotherapeutic drugs for gastric cancer,it will be of great value for the development of second-line treatment plans for HER2-positive gastric cancer patients.On the other hand,in the above-mentioned exploration of second-line treatment plans for HER2-positive gastric cancer in clinical trials,taxanes were selected as the basic medication or a baseline for comparison.The effect of trastuzumab resistance on the sensitivity of taxanes will directly affect the results of the clinical trials mentioned above.More importantly,if trastuzumab resistance changes the sensitivity of HER2-positive gastric cancer to chemotherapeutic drugs,it will indicate that there may be a correlation between the sensitivity of targeted drugs and chemotherapeutic drugs,and it will be worth exploring the different compatibility or order of the use of targeted drugs and chemotherapy drugs in different cancer.Previous study on estrogen-positive breast cancer has found that resistance to the endocrine therapy tamoxifen can lead to resistance to DNA-damaging chemotherapy,which suggests that resistance to other types of cancer therapy drugs may lead to resistance to chemotherapy.Therefore,in this study,we explored the difference of sensitivity of varies of chemotherapeutic drugs,especially taxanes,between trastuzumab resistant and wild-type HER2-positive gastric cancer.This will pave the way for the exploration of new solutions in the post-ToGA era.Next,we plan to explore the mechanism of trastuzumab resistance associated with decreased chemotherapy sensitivity in HER2-positive gastric cancer.As trastuzumab-resistant gastric cancer is simultaneously resistant to multiple chemotherapeutic drugs with different structures,we firstly consider the altered expression of ABC family(ATP-binding cassette transporters,ABC)mediating multidrug resistance(MDR)may be the reason for trastuzumab-resistant gastric cancer's decreased sensitivity to multiple chemotherapy drugs.Furthermore,wo examined the mechanism that concurrently mediating trastuzumab resistance and upregulation of ABCB1 expression.Membrane cholesterol is an important factor that regulates the transport activity of the ABC family.At the same time,the cholesterol homeostasis pathway is upregulated in HER2-positive trastuzumab-resistant gastric cancer cells.Previous studies have found that exhausting the membrane cholesterol of HER2-positive gastric cancer cells can increase its sensitivity to trastuzumab.Gene Ontology pathway enrichment analyses of GSE77346 chip showed that differential genes of trastuzumab resistant NCI-N87 cells compared with wild-type were enriched to membrane lipid metabolism and protein membrane localization-related pathways,which also suggested that the membrane protein localization in drug-resistant cells may be governed by the upregulation of membrane lipid metabolism.Therefore,we screened up-regulated ABC family members in trastuzumab-resistant cells,and tested the distribution characteristics of cholesterol.Moreover,we intervened with ABCB1 or membrane cholesterol to detect whether they could control the sensitive to chemotherapy drugs and whether there is a regulatory association between membrane cholesterol and ABCB1 expression.Finally,we further discovered the mechanism by which the enrichment of membrane cholesterol in trastuzumab-resistant cells up-regulated ABCB1 expression and reduced the sensitivity of taxanes.Firstly,membrane cholesterol could directly act as a ligand to activate the Hedgehog(Hh)pathway.When Hh pathway was activated,nuclear translocation of GLI1 initiated ABCB1 transcription.In addition,membrane cholesterol can also directly adjust the structure of lipid rafts on the membrane to affect membrane protein expression.Increased membrane cholesterol resulted in decreased membrane fluidity,and increased the expression of cavernous lipid raft and its marker CAV1 on the membrane.CAV1 is involved in the regulation of protein transport and degradation.For example,in NCI-N87 cells,CAV1 can reduce the stability of HER2 protein,and reducing membrane CAV1 can increase the sensitivity of trastuzumab in NCI-N87 cells.In NCI-N87 T-DM1 resistant strain,CAV1 mediates T-DM1 endocytosis to be degraded through lysosomal pathway.The combination of CAV1 and ABCB1 can inhibit the pumping activity of ABCB1,and CAV1 expression can inhibit the degradation of the ABC family via lysosomal pathway by inhibiting autophagy.Therefore,we tried to explain the reasons for the decrease in the sensitivity of taxanes through the transcriptional and posttranslational upregulation of ABCB1 in the membrane cholesterol of drug-resistant cells from the two perspectives of Hh pathway and CAV1 regulation.Methods and Results1.Trastuzumab-resistant gastric cancer decreased sensitivity to multiple chemotherapeutic drugsTwo human gastric cancer cell lines with HER2 gene amplification were screened:NCI-N87 and SNU-216.And the trastuzumab resistance cell model was constructed by continuous trastuzumab stimulation through 3D-2D conversion model.Then,we verified Trastuzumab resistance by the experiments of cell viability assay,clone formation and the flow cytometry assay.After confirming the successful construction of trastuzumab-resistant strains,we test the sensitivity of some common chemotherapeutic drugs in gastric cancer:Oxaliplatin,Cisplatin,5-fluorouracil,Paclitaxel and Docetaxel,between wild cell line and Trastuzumab-resistant cell line.We find that the Trastuzumab-resistant cell is resistant to OX,and its sensitivity to PTX and DTX is also decreased.After determining the changes in vitro,we selected wild and trastuzumab-resistant NCI-N87 to construct a subcutaneous tumor model in the left and right symmetrical parts of nude mice,and we treated these mice with saline,trastuzumab,FOLFOX(including 5-FU,folic acid(Leucovorin)and OX),PTX.Trastuzumab-resistant tumors are larger than wild-type tumors in the PTX group,which is consistent with the results of vitro studies.In summary,HER2-positive trastuzumab-resistant gastric cancer decreased sensitivity to multiple chemotherapeutic drugs.2.Enriched membrane cholesterol up-regulated ABCB1 expression owing to decreased sensitivity of trastuzumab-resistant gastric cancer cells to taxanesWe first performed fluorescent paclitaxel Flutax-2 uptake and efflux tests on trastuzumab-resistant and wild-type cells,and confirmed that Flutax-2 effluxed more in resistant cells.So we further screened the up-regulated ABC family in trastuzumab-resistant gastric cancer cells,and found that the expression levels of messenger RNA(Messenger ribonucleic acid,mRNA)and protein of ABCB1 increased significantl.And ABCB1 silencing increased sensitivity to taxanes in resistant cells.In addition,bioinformatics analysis showed that trastuzumab-resistant type versus wild-type differential genes in NCI-N87 cells can be enriched in cholesterol homeostasis related pathways.So we performed Filipin staining on the wild-type and drug-resistant types of the two cell lines to show the intracellular cholesterol distribution.The images showed that cholesterol was significantly enriched in the cell membrane in resistant cells.In the assay of cell viability and colony formation experiment,we use Methyl-?-Cyclodextrins(M?CD)to exhaust membrane cholesterol in resistant cells,then the sensitivity to PTX and DTX in resistant cells is significantly increased.After M?CD treatment,the expression of ABCB1 in trastuzumab-resistant cells decreased,with flutax-2 pumping reducing.Therefore,it was verified that plasma membrane cholesterol enrichment in trastuzumab-resistant gastric cancer cells up-regulated ABCB1,leading to a decrease in sensitivity to taxanes.3.Enriched membrane cholesterol up-regulated ABCB1 expression by promoting transcription and inhibiting protein degradation.Based on previous research,we explored the mechanisms by which membrane cholesterol regulated ABCB1 expression from Hh pathway activation and CAV1 regulation.We first verified the activation of the Hh pathway in trastuzumab-resistant cells versus wild-type cells by real-time quantitative polymerase chain reaction(qPCR)and Western blot(WB).The expression of the transcription factor-GLI1 target gene was up-regulated in drug-resistant cells,and significantly decreased when treated with Hh pathway inhibitor Vismodegib.Also the transcription of ABCB1 was significantly reduced after treatment with Vismodegib.When depleting membrane cholesterol with M?CD,the expression of GLI1 target gene is significantly reduced,and the transcriptional expression of ABCB1 is also reduced.Hh inhibitors can significantly increase the sensitivity of trastuzumab-resistant gastric cancer cells to taxanes.Further,in order to compare the protein stability of HER2 and ABCB1 in trastuzumab-resistant cells and wild cells,we applied Cycloheximide(CHX)time gradient treatment to inhibit protein synthesis and performed WB.WB proved that ABCB1 degradation rate is slower and protein stability is higher in resistant strains,while HER2 degradation was faster.IP experiments suggested that CAV1 reduced binding to ABCB1 and increased binding to HER2 in resistant strains.Inhibition of CAV1 by M?CD treatment in resistant cells showed that ABCB1 degradation increased,HER2 degradation slightly decreased,and trastuzumab sensitivity increased.The above experiments basically proved that the membrane cholesterol of trastuzumab-resistant cells promoted ABCB1 transcription by activating the Hh pathway,and increasing ABCB1 protein stability regulated by CAV1.CAV1 reduced ABCB1 protein degradation by dissociating ABCB1 and binding with HER2 promoting HER2 degradation through lysosomal pathway.All of these factors together linked trastuzumab resistance to decreased sensitivity of taxanes..ConclusionIn this study,we first investigated the change of sensitivity of HER2 positive trastuzumab-resistant gastric cancer to chemotherapeutic drugs,and found that its sensitivity to multiple drugs has decreased.Its mechanism is mainly related to the enrichment of membrane cholesterol in trastuzumab-resistant cells.On one hand,the Hh pathway promoted ABCB1 transcription,and on the other hand,it increased the stability of ABCB1 protein through CAV1,which eventually leads to the up-regulation of ABCB1 expression and the increasing pumping of taxane in resistant strains,therefore decreased sensitivity of taxanes.This study will provide ideas and theoretical support for the selection and improvement of second-line treatment options after the progressed first-line trastuzumab combined with chemotherapy treatment for HER2-positive gastric cancer.