Study On The Regulatory Mechanism Of Zinc Transporter ZnT7 On Tumor Development

Zinc,an essential nutrient,is closely related to human health.Zinc dyshomeostasis in the body can cause many diseases,including cancers.Clinical studies have found that deficiency of zinc and abnormal expression of multiple zinc transporters have been observed in human cancers.Dietary zinc supplementation can inhibit tumor growth,but the underlying mechanisms remain unclear.The Cancer Genome Atlas(TCGA)showed that the decreased expression ofZnT7 appeared in various human cancers,but the relevant mechanisms remain incompletely understood.Hence,we tend to explore regulatory mechanisms of nutrient zinc on tumorigenesis and tumor development,aiming to provide a certain theoretical basis for delaying tumorigenesis and developing functional foods.Drosophila melanogaster is a classical genetic model organism.It has been estimated that nearly 75%of disease-related genes in humans have functional orthologs in Drosophila.In recent years,Drosophila is increasingly recognized as a model organism in identifying tumor-related genes and researching food and nutrition.Zn T7,located on the Golgi apparatus,is responsible for transferring zinc from cytoplasm to Golgi apparatus.In this study,we found that silencingZnT7 gene(dZnT7 RNAi)can aggravate the phenotypes of benign Ras^V12 and malignant Raf^GOFscrib^-/-tumors.The regulatory mechanism of intracellular zinc on tumorigenesis was investigated by using biochemistry and molecular biology techniques.We found that zinc dyshomeostasis affected tumor progression by regulating JNK-dependent autophagy pathway.Based on this discovery,we screened several bioactive compounds from natural products that can regulate JNK pathway and influence tumor progression.We found that black bean skin coat extract(BBSCE),as well as its anthocyanin active monomer cyanidin-3-O-glucoside(C3G)exert their antitumor effect by regulating tumor growth and the microenvironment via JNK signaling pathway.The main research contents and results are as follows:(1)Silencing dZnT7 promotes tumor growth and invasion.The results showed that overexpression of Ras^V12 caused tumor overgrowth,the phenotype that can be enhanced by dZnT7 RNAi.In addition,knockdown of dZnT7 in Raf^GOFscrib^-/-tumor cells can significantly promote tumor growth,invasion and distant metastasis,and upregulate the expression of metastasis-associated protein MMP1(a onefold increase,P<0.001).(2)Zinc homeostasis in vivo is closely related to tumor growth.Compared with wild type flies,the zinc level was decreased by 35%in Raf^GOFscrib^-/-tumor flies.Compared with Raf^GOFscrib^-/-tumor model,dZnT7 RNAi resulted in a 15%reduction in zinc levels.This indicates that tumorigenesis and development is strongly linked to zinc deficiency in vivo.Further studies found that dietary zinc supplement can inhibit tumor growth and delay tumor progression.(3)dZnT7 RNAi promoted tumor growth by activating JNK signaling pathway.Silencing dZnT7 in tumors significantly upregulated phosphorylated JNK(p JNK)(?90%,P<0.001)and MMP1 expression(?175%,P<0.01),which can be suppressed by ectopic expression of a dominant negative form of the Jun kinase Basket(Bsk^DN).These data suggested that enhancement of dZnT7 RNAi on tumor phenotypes could be rescued by inhibiting the JNK signaling.(4)Zinc dyshomeostasis in Golgi caused by dZnT7 RNAi can activate the JNK signaling pathway.We found that dZnT7 RNAi upregulated puc expression(?43%,P<0.01),indicating that silencing dZnT7 can activate JNK signaling.Further,we found that zinc deficiency in Golgi caused by dZnT7 RNAi was able to activate JNK signaling,which can be rescued by regulating zinc homeostasis in Golgi with genetic means.These data suggested that zinc dyshomeostasis in Golgi by dZnT7 RNAi resulted in JNK signaling activation,which could be responsible for aggravating tumor phenotypes.(5)dZnT7 RNAi promoted tumor growth and invasion by activating JNK-dependent autophagy pathway.We found that dZnT7 RNAi activated autonomous and non-autonomous autophagy and increased autophagy level in tumors(?25%,P<0.05),which can be rescued by blocking JNK signaling.(6)BBSCE and its anthocyanin monomer C3G inhibit tumor growth by blocking JNK-dependent autophagy pathways.Cell experiments in vitro have shown that C3G could regulate JNK pathway,but its effect on tumor microenvironment in vivo remains unclear.We found that BBSCE and C3G can improve survival of tumor flies and inhibit tumor growth,invasion and distant migration.Further study indicated that C3G may inhibit tumor progression by repressing JNK-dependent autonomous and non-autonomous autophagy.Based on our findings,we hope to promote the possible application of C3G in tumor prevention and treatment.Intriguingly,we found that a combination of C3G and anti-tumor drug chloroquine(CQ)showed a greater antitumor effect than C3G or CQ treatment alone,indicating that C3G synergistically enhanced the antitumor effect of CQ.In summary,here using a Drosophila model of a malignant tumor,we found that dZnT7 RNAi resulted in zinc disorder in Golgi which activated JNK-dependent autonomous and non-autonomous autophagy,this process that accelerated tumor progression.In addition,BBSCE and C3G can remarkably suppress tumor growth.Moreover,C3G can assist clinical anti-tumor drug CQ and enhance its antitumor effect.This work provides in vivo evidence that the intake of dietary nutritional factor zinc and BBSCE can alleviate the tumor phenotypes,which can not only be used as a guide for dietary and nutritional administration of tumor patients,but also provide a certain theoretical support for the development of functional foods.