The Mechanism Of GDF-15 Facilitate Pancreatic Ductal Adenocarcinoma Proliferation And Metastasis Through Orphan Receptor GFRAL

Background:Pancreatic ductal adenocarcinoma(PDAC)is one of the most malignant tumor with very poor curative effect and survival prognosis in the digestive system.Epidemiological studies have shown that more than 330,000 people worldwide die from pancreatic ductal adenocarcinoma each year,and the overall 5-year survival rate is about 6%(2%-9%).Due to the early metastasis and rapid progress of pancreatic cancer,most patients are already in the middle and advanced stages when they are diagnosed and have local invasion and distant metastasis.Therefore,only about 10-15%of pancreatic cancer patients are eligible for initialresection.Currently,most patients can only receive palliative surgery,chemotherapy,radiotherapy,immunotherapy and so on.However,the clinical effect is still poor.Therefore,it is a critical and urgent need to study the molecular mechanism of pancreatic cancer cell proliferation,invasion and metastasis.Furthermore,it is of significance for pancreatic cancer patients to identify new early diagnostic markers,and improve the survival rate and prognosis.Transforming growth factor-?superfamily(TGF-?s)is a kind of peptide cytokine with multiple biological functions.The current studies have found that there are about 33 subtypes,TGF-?1,TGF-?2,TGF-?3,Inhibin-?,Inhibin-?A,Inhibin-?B,Inhibin-?C,Inhibin-?E,Nodal,Myostatin,BMP-2,BMP-3,BMP-4,BMP-5,BMP-6,BMP-7,BMP-8A,BMP-8B,BMP-9,BMP-10,GDF-1,GDF-3,GDF-5,GDF-6,GDF-7,GDF-9,GDF-9B,GDF-10,GDF-11,GDF-15,MIS,Lefty A,Lefty B.All of them can play important roles in cell morphology,differentiation,proliferation,apoptosis,aging,migration and other biological processes.Growth and differentiation factor 15(GDF-15)also known as macrophage inhibitory cytokine-1(MIC-1),nonsteroidal anti-inflammatory drug-activated gene-1(NAG-1),prostate-derived growth factor(PDF),placental transforming growth factor beta(PTGF?),placental bone morphogenetic protein(PLAB)and PL74.It is a member of the growth differentiation factor subfamily in the transforming growth factor-?superfamily.GDF-15 is firstly isolated from a c DNA library of human bone marrow monocytes cell line U937 by Australian scholars Bootcov MR in 1997.As a member of the TGF-?family,the amino acid similarity between GDF-15 and other members is only 15%-29%.The gene of GDF-15 is7007bp DNA long and it is located on human chromosome 19 GRCh38.p13 which is contained two exons and one intron.The GDF-15 precursor protein contains 308 amino acids which is cut and processed by Furin-like protease in the cell.The molecular weight of GDF-15 is about 30k Da,and it is secreted by cells toplay its biological function.In physiological conditions,GDF-15 is slightly expressed in normal tissues excepted placental tissues.However,in pathological conditions,such as inflammation,stress,injury,ischemia reperfusion,heart failure,type II diabetes,etc.,the expression of GDF-15 is upregulated significantly,and the corresponding expression of GDF-15 in plasma also increased.Studies have reported that GDF-15 is abnormally over-expression in many types of tumors,such as malignant glioma,testicular cancer,ovarian cancer,oral squamous cell carcinoma,uveal melanoma,lung cancer,prostate cancer,breast cancer,colorectal cancer,gastric cancer and hepatocellular carcinoma,which can be used as a potential biomarker for detecting and judging tumor prognosis.GDF-15 plays an important role in the development of tumors,which is mainly secreted by activated tumor-associated macrophages and tumor cells.In pancreatic cancer,GDF-15 gene is continuously expressed under the regulation of Twist1,which enhances the proliferation,invasion and metastasis of pancreatic cancer cells,and induces cell resistance to chemotherapy drugs,but its specific molecular mechanism needs to be further elucidated.Glial-derived neurotrophic factor receptor alpha-like(GFRAL)is an orphan receptor of the glial cell-derived neurotrophic factor(GDNF)receptor alpha family.Studies have reported that the GDNF protein family has four members,namely GDNF family receptor-?1(GFR-?1),GDNF family receptor-?2(GFR-?2),GDNF family receptor-?3(GFR-?3)and GDNF family receptor-?4(GFR-?4).The GFRAL gene is located on human chromosome 6,and contains 9exons with alternative splicing bodies.GFRAL protein consists of 395 amino acid residues,with 20-30 stretchable hydrophobic amino acids at the C-terminus and a signal peptide at the N-terminus.The GFRAL protein is anchored to the cell membrane via glycosylphosphatidylinositol(GPI).As a single-pass transmembrane protein,GFRAL has a short structural domain in the cell and lacks the ability to transmit signals into the cell.Therefore,when it plays its biological role,GFRAL requires tyrosine kinase co-receptor Ret to assist the related signaling pathways conduction.From four different medical research teams,Emmerson PJ,Yang L,Mullican SE and Hsu JY,report in the magazine of Nature Medicine and Nature,GFRAL is confinedly expressed in the mouse brainstem area postrema(AP)and Nucleus tractus solitarius(NTS)neurons.GDF-15 can specifically bind to GFRAL,and its binding rate is proportional to the concentration of GDF-15 positively correlated.The GDF-15/GFRAL/Ret complex is formed on the nerve cell membrane to activate the intracellular PI3K-Akt,Erk1/2,MAPK and phospholipase C(PLC)?signaling pathways,thereby inhibiting the feeding behavior of mice,regulating metabolism,reducing weight,and improving blood sugar.So,GDF-15 and GFRAL can be used as potential target for the treatment of severe obesity,type 2 diabete and anorexia/cachexia.However,there is no relevant research report on GFRAL in tumors.In order to further study the molecular mechanism of GDF-15 in pancreatic cancer and explore its clinical significance,we propose the following research assumptions based on the previous research work that pancreatic ductal adenocarcinoma cells autocrine differentiation growth factor GDF-15,specifically binding with the receptor GFRAL on the cancer cell membrane,and promoting the proliferation,invasion and metastasis of pancreatic cancer cells through the GDF-15/GFRAL signaling pathway.Objectives:1.Collect clinical pancreatic cancer cases,detect the expression of GDF-15 and GFRAL in pancreatic cancer tissues and plasma samples,analyze the correlation between GDF-15expression and GFRAL expression,and clarify the influence of their abnormal expression on the clinicopathological factors and survival prognosis of PDAC patients.Provide basis for the follow-up mechanism research.2.Detect the expression of GDF-15 and GFRAL in pancreatic cancer cell lines,and analyze the correlation between the two molecules expression at the cellular level.3.Clarify that pancreatic cancer autocrine GDF-15 can directly bind to the cell membrane receptor GFRAL,and promote the proliferation,invasion and metastasis of pancreatic cancer cells through the GDF-15/GFRAL signaling pathway.4.Construct a nude mouse subcutaneous tumor model,and explore the effects of GDF-15 and GFRAL on the growth of pancreatic cancer at the animal level,and provide new ideas for the development of pancreatic cancer diagnosis and treatment strategies.Methods:1.Enzyme-linked immunosorbent assays(ELISAs)are used to detect the expression of GDF-15 in the plasma of normal people and patients with pancreatic cancer,and the expression level of GDF-15 in the plasma of normal people and patients with pancreatic cancer is analyzed with Graph Pad Prism 5 software.At the same time,immunohistochemistry(IHC)experiments are used to verify the expression of GDF-15 in normal people and pancreatic cancer tissues.According to the level of GDF-15 expression in the plasma of pancreatic cancer patients,PDAC cases are divided into high expression groups and low expression groups(the mean value of GDF-15 expression in all pancreatic cancer plasma cases is used to distinguish between high and low expression groups).We analyse respectively of the differences among clinical data of PDAC patients'age,gender,tumor clinical staging,tumor size,and postoperative survival prognosis between the above two groups of cases.2.Through ELISA experiments,the expression of GDF-15 in normal pancreatic ductal epithelial cell line HPDE and four pancreatic cancer cell lines(As PC-1,Bx PC-3,Panc-1 and Hs766t)are detected.The expression level of GDF-15 in normal and pancreatic cancer cell lines is analyzed via Graph Pad Prism 5 software.Thereby,we verify the over-expression of GDF-15 at the level of cell lines in vitro,which is also upregualted in clinical tissues and plasma samples.3.Immunohistochemistry(IHC)experiments are used to detect the expression of GFRAL in normal people and pancreatic cancer tissues.According to the level of immunohistochemical score,PDAC cases are divided into high expression group and low expression group,and the differences in clinical data such as survival and prognosis of PDAC patients between the above two groups were respectively counted.At the same time,the Western Blot(WB)experiments are used to detect the expression of GFRAL in normal pancreatic duct epithelial cell line HPDE and six pancreatic cancer cell lines,As PC-1,Bx PC-3,CFPAC-1,Panc-1,SW1990 and Hs766t.4.Using Graph Pad Prism 5 software,we analyse the correlation between GDF-15 and GFRAL expression respectively in pancreatic cancer clinical samples and pancreatic cancer cell lines.5.Through immunofluorescence(IF),laser scanning confocal(CLSM)experimental technology,we detect the expression of GDF-15 and GFRAL in paraffin sections of pancreatic cancer tissues and pancreatic cancer cell lineswith double-labeled immunofluorescence respectively.Subsequently,co-immunoprecipitation(Co-IP)experiments are used to verify the direct interaction between GDF-15 and GFRAL protein at the molecular level of pancreatic cancer cell lines.6.Construct interference lentivirus Lv-GDF15-RNAi and synthetic recombinant human GDF-15 cytokines,correspondingly down-regulate and simulate up-regulate the expression of GDF-15 in pancreatic cancer cells,and adopt CCK-8,Transwell,Wound healing assay and other techniques to observe the effect of GDF-15 on the proliferation,invasion and metastasis of pancreatic cancer cells.7.Construct over-expressing lentivirus Lv-EGFP-GFRAL(+)and its negative control lentivirus Lv-EGFP-GFRAL(-),infect pancreatic cancer cells,screen out cells stably up-regulated expressing GFRAL protein by flow cytometry,and performed WB experiment to detect the efficiency of overexpression.Using human recombinant GDF-15 cytokine to stimulate Lv-EGFP-GFRAL(+)and Lv-EGFP-GFRAL(-)cells respectively,observe the changes in cell proliferation,invasion and metastasis ability.8.Using 4-week-old BALB/c female nude mice as the research objects,we construct subcutaneous tumor animal models of pancreatic cancer by inoculating As PC-1 cells(2×10~6cells/mouse)with interfering lentivirus Lv-GDF15-RNAi stably down-regulating the expression of GDF-15.After 30 days,we use the IVIS~?Spectrum in vivo imaging system to observe the effect of GDF-15 on pancreatic cancer cell tumor formation in nude mice.Results:1.Compared with 7 normal human pancreatic tissues and 20 normal plasma samples,GDF-15 is significantly higher expressed in 21 pancreatic cancer tumor tissues and 34pancreatic cancer plasma(P<0.0001).The 5-year survival rate of pancreatic cancer patients in the GDF-15 high expression group is significantly lower than that in the GDF-15 low expression group(P=0.0120).2.To verify the expression of GDF-15 at the cell lines level,the expression level in different pancreatic cancer cell lines are different,with the highest GDF-15 expression in As PC-1 and the lowest expression in Hs766t.3.Compared with 13 normal pancreatic tissues,GFRAL expression is significantly upregulated in 117 pancreatic cancer tissues;the 5-year survival rate of pancreatic cancer patients in the GFRAL high expression group is significantly lower than that in the GFRAL low expression group(P=0.0272).The expression of GFRAL in pancreatic cancer cell lines is consistent with clinical samples.4.In order to explore the correlation between expression of GDF-15 and GFRAL in pancreatic cancer samples,we perform a statistical analysis via Graph Pad Prism 5 software.The result show that the expression of GDF-15 and GFRAL is positively correlated(r~2=0.6501,P=0.0009).5.Laser scanning confocal experiments show that GDF-15 and GFRAL are co-expressed in pancreatic cancer tissues,and the co-immunoprecipitation experiments prove that GDF-15protein can directly bind with GFRAL to play its biological role.6.Human recombinant GDF-15 cytokine can promote proliferation,invasion and metastasis of pancreatic cancer cells;after down-regulated the expression of GDF-15,the proliferation,invasion and metastasis ability of pancreatic cancer cell lines is significantly weakened.After up-regulated the expression of GFRAL by lentivirus in pancreatic cancer cell lines,GDF-15 can significantly enhance tumor cells'proliferation,invasion and metastasis ability.7.Compared with the negative control group(Lv-GDF15-NC),the weight and volume of the subcutaneous tumors of nude mice are significantly smaller than the interfering group(Lv-GDF15-RNAi),P=0.0286.Conclusions:1.GDF-15 and GFRAL are significantly over-expressed in plasma and tissues of pancreatic cancer,and are closely related to the clinical prognosis of pancreatic cancer patients.These protein molecules can be used as tumor markers for clinical diagnosis and treatment for pancreatic cancer patients.2.In clinical samples and cell lines of pancreatic cancer,the expression of GDF-15 and GFRAL are positively correlated.3.GDF-15 and GFRAL are expressed and co-localized in pancreatic cancer.These protein molecules can directly combine with each other.4.Pancreatic cancer cells can autocrine GDF-15.5.GDF-15 promotes proliferation,invasion and metastasis of pancreatic cancer cells through orphan receptor GFRAL.Through in-depth explore,this study investigated the influence of GDF-15/GFRAL signaling pathway during the development of pancreatic cancer.It is the first time to propose and confirm the research hypothesis thatpancreatic cancer cells can highly express the orphan receptor GFRAL and autocrine GDF-15,which can directly bind to the PDAC cell membrane receptor GFRAL to promote proliferation,invasion and metastasis of pancreatic cancer cells.Our study further enrich the theoretical treasure house of the study on the mechanism of pancreatic cancer cells'ability of invasion and metastasis,and provid a new potential target for the prevention and treatment of PDAC invasion and metastasis in clinical practice.