LAMP2A Upregulates ING5 Protein Stability Though NF-?B Pathway To Support Lipid Droplet Production And Facilitate Chemo-Resistance In Colorectal Cancer Cells

Objective: Colorectal cancer(CRC)is one of the most frequent causes of cancer-related deaths worldwide.It is the fourth most common cancer among adults in the United States and the second leading cause of death from cancer.More than50,000 people die of colorectal cancer each year.Although lots of improvements have been made in early diagnosis and systemic therapies,only approximately 50% of patients with CRC survive longer than 5 years.Drug resistance remains a major failure of chemotherapy regimens and in charge of low 5-year survival in CRC patients,so the related research is still a hot spot at home and abroad.A number of studies have confirmed that lipid metabolic reprogramming has profound effects on chemo-resistance,tumorigenesis,and cellular differentiation.Also affects the prognosis of CRC patients.Chaperone-mediated autophagy(CMA)is a highly selective form of autophagy first found.Certain proteins are recognized by specific chaperones and transported into the lysosome for degradation though a lysosomal membrane receptor/translocation complex.CMA plays an important role in maintaining cell protein homeostasis,and also participates in tumorigenesis and development as well as energy metabolism.Lysosome-associated membrane protein2A(LAMP2A)is a highly glycosylated transmembrane protein on the lysosomal membrane.It acts as a receptor for the substrate protein and regulates the rate-limiting step of CMA.As it is directly related to CMA activity,make it the best criterion to measure the effect of CMA.Inhibitor of growth family 5(ING5)is a member of the ING protein family and functions as a type-II tumor suppressor gene.Our previous research found that the expression of ING5 protein and mRNA in colon cancer increased.Nuclear ING5 expression was negatively correlated with tumor size,depth of invasion,dedifferentiation,clinicopathological staging and poor prognosis,while plasma ING5 expression was positively correlated with tumor invasion depth,lymph tube invasion and clinicopathological staging.Therefore,we believe that the abnormal expression of ING5 and nucleocytoplasmic translocation play an important role in the occurrence and progression of malignant epithelial tumors.Interestingly,a positive co-relationship between ING5 expression and lipid metabolic reprogramming,chemo-resistance in gastric cancer was found too.Based on domestic and abroad literatures and the basis of previous work,we propose that LAMP2 A upregulates ING5 protein stability to support lipid droplet production and facilitate chemo-resistance in colorectal cancer cells.Methods:1.LAMP2 A upregulated the protein stability of ING5 in CRC cell lines by activating NF-?B pathway.Firstly,Western blot was performed on 95 clinical specimens of CRC patients' pathological tissues.And Immunohistochemical staining was performed on 425 cases of colorectal tissues.All to analyze the expression of LAMP2 A and ING5 protein and the relationship between them.Secondly,we transfected LAMP2 A short hairpinRNA(shRNA)or LAMP2 A overexpression plasmid to downregulate or upregulate LAMP2 A expression in CRC cell lines(HCT116 and DLD-1).We used Western blot to detect ING5 protein expression.PDTC(ammonium pyrrolidine dithiocarbamate)or CQ(chloroquine)was used to inhibit the NF-?B signal transduction pathway or lysosome pathway,CHX(cycloheximide)was used to inhibit protein synthesis,then ING5 protein expression was detected by Western blot.Finally,the results were verified by immunoprecipitation.SPSS24.0 was used to analyze data and P<0.05 was considered statistically different.2.LAMP2 A and ING5 promoted LD production in CRC cell lines HCT116 and DLD-1.Firstly,we detected the effect of LAMP2 A on the malignant phenotype of CRC cell lines HCT116 and DLD-1:Western blot was used to detect related protein expression;MTT was used to detect cell proliferation;APC and PI double staining was used to detect apoptosis;transwell assays was used to detect cell migration and invasion.Then ING5 short hairpinRNA(shRNA)or ING5 overexpression plasmid was transfected to CRC cell lines HCT116 and DLD-1 to downregulate or upregulate ING5 expression.Continue transfected with plasmid to downregulate or upregulate the expression of ING5 in sh LAMP2A-modified or LAMP2A-modified CRC cells.Western blot was used to detect related protein expression;Nile red staining to observe the LDs in cells.Finally,co-immunoprecipitation was used to detect the correlationship between ING5 and other proteins,which related to lipid metabolism.Statistical analysis was performed on the data by SPSS 24.0,P<0.05 was considered statistically different.3.Overexpression of LAMP2 A and ING5 increased LD production and promoted chemo-resistance in 5-FU-resistant CRC cells.To obtain 5-Fu–resistant cells,first we selected CRC cells(HCT116 and DLD-1)using increasing concentrations of 5-Fu on parental HCT116/DLD-1 cells.They were then intermittently treated with large doses of 5-Fu.MTT was used to detect cell proliferation and calculat IC50;Western blot was used to detect resistance-related proteins expression;observed the cells with microscope.Then the expression of related proteins were detected by Western blot.Next,we used plasmid transfection to downregulate the LAMP2 A or ING5 expression in 5-FU resistant CRC cells: Western blot was used to detect the related proteins expression,Nile red staining was used to observe the LDs in the cells.After treating the certain cells with chemotherapeutic drugs(5-FU,OXA,FOX)for a certain period of time,cell proliferation was detected by MTT assay.Finally,the immunoprecipitation experiment was used to detect the correlationship between ING5 and NF-?B with other drug-resistant related proteins.Statistical analysis was performed on the data by SPSS 24.0,P<0.05 was considered statistically different.Results:Part?:1.The expression status and correlationship of LAMP2 A and ING5 in clinicopathological tissues of CRC patients.(1)Western blot was performed on 95 pairs of clinical pathological tissue specimens: Compared with normal tissues adjacent to cancer,the expression levels of LAMP2 A and ING5 protein in CRC tissues were higher,P<0.05.(2)Immunohistochemical staining was performed on 425 cases of CRC tissues: Compared with the adjacent normal tissues and adenoma tissues,the expression of LAMP2 A in CRC tissues was increased,and the expression of ING5 was decreased,P<0.05.LAMP2 A expressed in cytoplasm only,while ING5 expressed both in the nucleus and cytoplasm.ING5 mainly expressed in the nucleus in normal and adenoma tissues,while in the cytoplasm in cancer tissues.In general,the expressions of LAMP2 A and ING5 were positively correlated(Pearson analysis,P=0.000);the expressions in the nucleus(Pearson analysis,P=0.000)and cytoplasm(Pearson analysis,P=0.009)were positively correlated respectively.2.LAMP2 A upragulated the protein stability of ING5 by activating NF-?B pathway.(1)Western blot was performed on several CRC cell lines HCT15,HCT116,HT29,DLD-1,RKO,SW480,SW620.We chose HCT116,which with higher expression of LAMP2 A and ING5,and DLD-1,which with both lower expression,to perform further experiments.(2)Knockdown and overexpression of LAMP2 A by transfection of LAMP2A-shRNA or LAMP2 A overexpression plasmid in HCT116 and DLD-1 cells were associated with decreased and increased expression of ING5 and NF-?B,respectively.We used PDTC to block the NF-?B pathway and CQ to inhibit the lysosomal pathway,and both resulted in a clear and significant reduction of ING5 compared to non-treated cells in the Western blot analysis.Then we used CHX to inhibiting protein synthesis,and found higher protein stability of ING5 in the cells overexpressing LAMP2 A.Co-immunoprecipitation experiments further confirmed the interaction of NF-?B with ING5,and HSC70 with I?B.Ik B was identified as a substrate of CMA.Therefore,when CMA was high in CRC cells,the I?B level in cytoplasm decreased,resulting in increased NF-?B activity.Thereafter,the combination forms of ING5 and NF-?B increased,which resulted in higher expression of ING5.Part?:1.The effect of LAMP2 A on the malignant phenotype of CRC cell lines HCT116 and DLD-1.Western blot analysis,MTT assay,APC and PI double-stained cell apoptosis detection,Transwell experiment were performed on HCT116 and DLD-1cell lines stably transfected with LAMP2A-shRNA or LAMP2 A overexpression plasmid.In sh LAMP2A-modified HCT116 and DLD-1 cells the expression of ACC/ACL and NF-?B decreased,cell proliferation slowed down,cell apoptosis increased,cell migration and invasion ability decreased;conversely,in LAMP2A-modified HCT116 and DLD-1 cells,the expression of ACC/ACL and NF-?B increased,cell proliferation accelerated,cell apoptosis decreased,cell migration and invasion ability increased.2.LAMP2 A and ING5 promoted LD production in CRC cell lines HCT116 and DLD-1.Knockdown and overexpression of ING5 by transfection of ING5-shRNA or ING5 overexpression plasmid in HCT116 and DLD-1 cells,then continue transfected with plasmid to knockdown or overexpression the expression of ING5 in sh LAMP2A-modified or LAMP2A-modified CRC cells.The expression of ACC/ACL decreased or increased along with ING5 protein expression,and the expression of NF-?B was also the same in the Western blot analysis.In the double-transfected cells,ACC/ACL expression was also positively along with the expression of LAMP2 A and ING5.Nile red staining directly observes the LDs in the cells.Both LAMP2 A and ING5 boosted the synthesis of ACC/ACL and promote the production of lipid droplets in CRC cells.The results were further verified by co-immunoprecipitation.ING5 was related with ACC/ACL.Statistical analysis was performed on the data by SPSS 24.0,P<0.05 was considered statistically different.Part?:1.Establishment of 5-FU drug-resistant cell lines.HCT116 and DLD-1 were treated with increasing concentrations of 5-FU firstly,then intermittently treated with large doses of 5-Fu,until the cells were resistant,labeled as HCT116-R and DLD-1-R:(1)Western blot was used to detect the expression of drug-resistance related gene(CD147,LRP1,PGP,GST?,MLH1,MRP1 increased,and FBXW7 decreased).(2)We treated the cells with different concentrations of 5-FU(0,1.25,2.5,5,7.5,10,15,20,30ug/m L)for a certain period of time(48h),and detected cell proliferation by MTT assay.Calculated IC50: HCT116-R was 7.22;DLD-1-R was 6.7.(3)Then we treated the cells with different chemotherapy drugs,5-fluorouracil(5-FU 10 u M),oxaliplatin(OXA 10 u M),FOX regimen(5-FU 10 u M+OXA 10 u M))for a certain period of time(0h,24 h,48h,72 h,96h),and used MTT assay to detect cell proliferation.We also observed co-resistance to other chemotherapy drugs,such as OXA,in both cells.(4)Observed 5-FU-resistant CRC cells: Both cell types increased in size with irregular nuclei,and some cells had multiple,enlarged lysosomes when examined by Lyso Tracker staining.The proliferation rates of HCT116-R and DLD-1-R cells were similar to the parental cells,but the doubling time was prolonged in vitro.2.Overexpression of LAMP2 A and ING5 increased LD production and promoted drug resistance in 5-FU resistant CRC cell lines.(1)We found enhanced LAMP2 A and ING5 protein expression in HCT116-R and DLD-1-R.ACC/ACL and NF-?B protein expression was also increased in both cell lines.(2)Knockdown of LAMP2 A or ING5 in HCT116-R and DLD-1-R cells were associated with decreased expression of ACC/ACL.And LDs in cells were reduced when detected by Nile red staining.(3)After treating the certain cell lines(sh LAMP2A-modified HCT116-R cells and DLD-1-R cells;LAMP2A-modified HCT116 and DLD-1 cells;sh ING5-modified HCT116-R cells and DLD-1-R cells;ING5-modified HCT116 and DLD-1 cells and their parental ones)with chemotherapeutic drugs(5-FU,OXA,FOX)for a certain period of time,cell proliferation was detected by MTT assay.The cell proliferation rates of sh LAMP2A-modified or sh ING5-modified HCT116-R cells and DLD-1-R cells were significantly lower than that of the drug-resistant group;on the contrary,in the LAMP2A-modified or ING5-modified HCT116 and DLD-1 cells,the cell proliferation rates increased significantly,and the difference was statistically significant(P<0.05).It is suggested that,loss of function of LAMP2 A and ING5 in5-Fu-resistant CRC cells or gain of function in LAMP2A-deficient and ING5-deficient cells rendered them sensitive or resistant to 5-Fu.(4)We also found the interaction of NF-?B with some drug resistance genes,such as CD147,GST3,and MLH1,also of ING5 with CD147 and GST3,through co-immunoprecipitation experiments.This may be another important line of evidence that the NF-?B pathway and ING5 are associated with drug resistance in CRC.Statistical analysis was performed on the data by SPSS 24.0,P<0.05 was considered statistically different.Conclusions: 1.Compared with normal tissues adjacent to cancer,the protein expression of LAMP2 A and ING5 were higher in CRC tissues,and they were positively correlated.2.LAMP2 A upregulates ING5 protein stability to support lipid droplet production via NF-?B pathway in colorectal cancer cells.3.Loss of function of LAMP2 A or ING5 in 5-Fu-resistant CRC cells reduced lipid droplet production,increased chemotherapeutic cell death,decreased chemotherapeutic drug resistance,and rendered them sensitive to chemotherapeutic drug.