KCNJ15 Deficiency Promotes Drug Resistance Via Affecting The Function Of Lysosomes

Objective: Drug resistance-induced cancer progression represents as a main challenge for cancer treatment.Patients who developed multidrug resistance after receiving treatments typically pose significantly shortened disease-free survival(DFS).In 2022,more than600,000 cancer deaths were projected to occur due to primary or acquired therapeutic resistance in the United States.Therefore,the investigation on the mechanisms of drug resistance could improve the therapeutic effectiveness and patient prognosis.Lysosome,an organelle filled with various acidic hydrolases,is mainly responsible for the degradation of proteins,nucleic acids,polysaccharides and other biological macromolecules.Compared to the neutral microenvironment of cytoplasm(p H is about7.2),lysosomes exhibit their maximal enzymatic activity at a relatively low p H(p H?≤?5).On the surfaces of lysosomes,V-ATPase,a multi-subunit complex and proton pump,transports protons into the lysosomal lumen to maintain the low p H.V-ATPase contains 8V1 domains(A–H)for ATP hydrolysis and 5 membrane-spanning V0 domains(a,c,c’,d,e,and ATP6AP1).Growing attention has been drawn into the effects of V-ATPase in increasing the metastatic potential and survival of cancer cells in acidic tumor environment.However,the underlying mechanisms of how lysosomes regulate drug metabolism in tumor cells remain unclear.Potassium voltage-gated channel subfamily J member 15(KCNJ15)protein,a type of inward rectifying potassium ion channel,is widely distributed among various tissues to maintain the resting potential of cell membrane.KCNJ15 protein has been shown to participate in the tumor proliferation,senescence,invasion,and metastasis.However,the relationship between KCNJ15 protein and lysosomes,as well as their interactions in tumor drug resistance remains unclear.Here,we first report that KCNJ15 was essential to maintain the functions as well as stabilities of lysosomes in breast cancer cells.Low KCNJ15 expression in breast tumors were observed to interact with the subunits of V-ATPase which served as an ATP-driven proton pump positioned at lysosomal surfaces,thereby leading to the dysfunction of lysosomes and drug resistance.In addition,upregulation of KCNJ15 protein could subsequently decrease lysosome quantity,inhibit the cell proliferation,induce the apoptosis of tumor cells,and enhance the cell sensitivity to therapeutic drugs.To explore the clinical significance of this study,breast cancer tissues were collected from patients and analyzed.Our results indicated that KCNJ15 expression was lower in neoadjuvant chemoresistant tumors compared with chemosensitive tumors,which was significantly related to poorer DFS of patients.These findings suggested that the expression status of KCNJ15 might act as an underlying indicator for the drug resistance and chemotherapy efficacy of breast cancer,which might provide insights to the choice of strategies for cancer treatment.Methods: 1.KCNJ15 overexpression and control plasmid were purchased from Gene Pharma(China),MDA-MB-231 cells were transfected using Lipo3000(Invitrogen),and q RT-PCR was used to quantitatively detect transfection efficiency.CCK8,scratch experiment,Transwell invasion experiment and Annexin V-APC/7-AAD apoptosis assay were used to detect changes in cell proliferation,migration,invasion,drug resistance and apoptosis.Eighteen NOD/SCID mice were randomly divided into three groups,injected with tumor cells(KCNJ15-OE,KCNJ15-NC and control).Six mice were in each group,and injected docetaxel with tail veins.Western blot method was used to detect and compare the changes of KCNJ15 protein expression in tumor tissues of each group.Mouse body weight,tumor size,and final tumor weight were recorded.2.Potential downstream pathways affected by KCNJ15 were sought by m RNA sequencing and KEGG pathway enrichment analysis of KCNJ15-OE cells and KCNJ15-NC cells.Coimmunoprecipitation and scattered mass spectrometry were used to search for potential downstream interacting proteins of KCNJ15.Through these high-throughput methods,the key downstream interaction protein of KCNJ15 was discovered.Western blot,coimmunoprecipitation,immunofluorescence staining,and immunohistochemical staining were used to explore the regulation of KCNJ15 to its downstream proteins and verify the regulatory effects of KCNJ15 on downstream signaling pathways.3.Molecular dynamics simulation,Western blot,and co-immunoprecipitation experiments were used to verify the blocking effect of small molecule compounds on KCNJ15 and its interacting proteins as well as their effects on downstream signaling pathways.Furthermore,the inhibitory effect of V-ATPase inhibitors on drug-resistant cells in triplenegative breast cancer was verified by chemosensitivity test,immunofluorescence staining,and tumor chemosensitivity test in mice.Results: 1.Triple-negative breast cancer(TNBC)cells,including MDA-MB-231,MDAMB-468,and BT549,showed significantly lower levels of KCNJ15 than normal breast cells(MCF-10A).Overexpression of KCNJ15 could significantly inhibit cell proliferation and migration,induce apoptosis of tumor cells,and increase cell sensitivity to drugs including docetaxel,doxorubicin,and cyclophosphamide.After 4 weeks of inoculation of the cells,the tumors in the OE KCNJ15 group were much smaller than those in the 231-WT and 231-NC groups,and at the same time,the survival time of mice in the OE KCNJ15 group was longer than in other groups,indicating that KCNJ15 overexpression could improve the drug sensitivity of tumor cells and inhibit tumor growth.2.The results of co-immunoprecipitation(Co-IP)assay showed that KCNJ15 bound to ATP6V0A1 and ATP6V1B2.ATP6V0A1 is the main regulator of V-ATPase structure,and ATP6V1B2 is responsible for V-ATPase assembly.KCNJ15 could reduce the interaction of ATP6V0A1-ATP6V1B2 in 231-WT cells.In contrast,KCNJ15 depletion enhanced ATP6V0A1-ATP6V1B2 binding in 231-WT cells.The confocal results showed that KCNJ15 was clearly colocalized with lysosomes and ATP6V1B2.At the same time,overexpression of KCNJ15 reduced the number of lysosomes in cells and the distribution of ATP6V1B2.3.BAF and CMA could weaken or even block the binding of KCNJ15 to ATP6V0A1 and ATP6V1B2,respectively.The results of computer simulation showed that CMA had specific interaction sites with KCNJ15 and V-ATPase subunit ATP6V0A1/ATP6V1B2,which might inhibit the interaction between KCNJ15 and V-ATPase.After tumor formation in NOD/SCID mice,treated with PBS or CMA combined with docetaxel,the CMA group showed reduced tumor volume and ATP6V0A1 expression compared to the PBS group,and mice treated with CMA showed a decrease in the number of lysosomes in the cytoplasm.Conclusion: The study found that KCNJ15 had low specific expression in triple-negative breast cancer,especially in paclitaxel-resistant triple-negative breast cancer cells,and clarified the regulatory effect of KCNJ15 on triple-negative breast cancer.It was found that KCNJ15 could regulate the assembly of V-ATPase by binding to ATP6V0A1,thereby affecting the mechanism of lysosomal acidity-mediated drug resistance.Based on blocking the binding plane of KCNJ15 and ATP6V0A1,it was found that the V-ATPase inhibitor CMA could effectively reverse chemotherapy resistance,which provided a new therapeutic target and opened up a new path for the treatment of triple-negative breast cancer.