ADAM10 Regulation Of Gemcitabine Sensitivity Of Pancreatic Cancer And Its Immunological Mechanism

Pancreatic cancer is a malignant tumor of the digestive system with insidious onset,rapid development and poor prognosis.Due to its early diagnosis and limited treatment,mortality is almost as high as morbidity.In the United States,the mortality rate of pancreatic cancer has risen from the fifth to the fourth,and pancreatic cancer is expected to be the second leading cause of cancer death by 2030.The most recent Epidemiology database estimates an overall five-year survival rate of merely 8.2%(2007-2013)for all stages of pancreatic cancer,which is among the lowest of all solid cancer types.Almost 90%of all pancreatic malignancies are pancreatic ductal adenocarcinomas(PDAC).Despite decades of extensive work towards improvement in diagnostic techniques,surgical procedures and chemotherapy,the overall prognosis of PDAC patients remains extremely poor.Most common risk factors associated with pancreatic cancer include tobacco smoking,family history,chronic pancreatitis,obesity,diabetes and occupational hazards.Due to lack of effective screening methods for early detection and few specific early symptoms,the large majority of PDAC patients are diagnosed with advanced or metastatic disease.While surgery offers the only potential curative treatment,only 15-20 percent of patients have resectable disease at the time of diagnosis,and even in patients for whom surgical resections are performed,most patients develop disease recurrence within a year.Gemcitabine(also known as dFdC:2',2'-difluorodeoxycytidine),originally used for its antiviral effects,has been widely used as an anti-cancer chemotherapeutic agent for various solid tumors and currently in certain lymphomas.Since 1997,when Burris et al.showed that gemcitabine can significantly improve patient symptoms and prolong the median survival of patients,gemcitabine has become the standard treatment choice for locally advanced and metastatic pancreatic cancer.Moreover,gemcitabine is now widely used as a radiosensitizer in clinical practice.A variety of gemcitabine-based combination therapies are being widely developed.Gemcitabine was superior to fluorouracil(5-FU)with respect to performance status and pain control.The clinical benefit response(CBR)of gemcitabine was more profound,almost five-fold higher,compared to 5-FU(23.8%vs.4.8%).The failure of translating good clinical response into relative survival benefits could at least partly be attributed to development of gemcitabine chemoresistance in initially sensitive tumors within weeks of treatment.Therefore,molecular basis studies of gemcitabine resistance may be the development of new strategies to increase the efficiency of this chemotherapy.This may not only be beneficial for patients with PDAC,but also for other malignant tumor patients treated with gemcitabine.Part 1:Clinical study of ADAM10 expression in human pancreatic cancer and gemcitabine resistanceObjective:Recent studies have shown that A Disintegrin and metalloproteinase domain-containing protein 10(ADAM 10)not only participates in tumor proliferation,angiogenesis,migration and invasion,but also participates in tumor chemoresistance.This study aims to detect the different expression of ADAM 10 in pancreatic cancers and its relationship with gemcitabine sensitivity.Methods:The expression levels of ADAM 10,VEGF and CD31 were detected by immunohistochemistry in pancreatic cancer specimens,and the correlation between ADAM 10 expression and VEGF and microvessel density(MVD)was analyzed.Univariate Kaplan-Meier and multivariate Cox survival analysis were used to determine the correlation between ADAM 10 expression level and the efficacy of gemcitabine treatment in pancreatic cancer.Results:Immunohistochemistry showed a significant positive correlation between ADAM 10 protein expression and microvessel density(p<0.001)and VEGF expression(p<0.001).The MVD value of ADAM 10 high expression group was significantly higher than that of ADAM 10 low expression group.At the same time,ADAM 10 expression in pancreatic cancer was shown to be significantly associated with histological grade(p = 0.001),lymph node metastasis(p = 0.012),perineural infiltration(p = 0.022),and tumor,lymph node,and metastatic stage(p = 0.029).In univariate and multivariate Cox regression analysis,ADAM 10 was an independent prognostic factor for poor overall survival and low disease-free survival in patients with pancreatic cancer treated with gemcitabine-assisted chemotherapy.Conclusions:High expression of ADAM 10 protein in pancreatic cancer may be significantly associated with the low efficiency of patients receiving gemcitabine adjuvant chemotherapy.Part 2:Establishment and in vitro study of gemcitabine-resistant pancreatic cancer cell linesObjective:This study aimed to establish an in vitro model of gemcitabine resistance in pancreatic cancer as a tool to study the mechanism of ADAM 10 involved in gemcitabine resistance in pancreatic cancer.Methods:Gemcitabine-resistant human pancreatic cancer cell line PANC-1-GR was established by PANC-1 culture with increasing concentration of gemcitabine.Cell viability was measured by MTT assay,while cell growth curve was detected by real-time label-free cell analyze.MDR1 and ADAM10 expression were detected by Western blot.The level of soluble NKG2D ligand(sMICA/B,sULBP1-3)secreted by PANC-1-GR and its parental cell line was detected by ELISA.The killing efficiency of NK cells against PANC-1-GR and its parental cell lines was determined by MTT assay.After RNA silencing of ADAM 10,the soluble proteins of NKG2D ligand and NK cell cytotoxicity were determined.Results:Compared with the PANC-1 parental cell line,PANC-1-GR cells were resistant to gemcitabine,and the half-inhibitory concentration(IC50)of PANC-1-GR cells to gemcitabine was 56.2 ± 2.16?g/ml,while the IC50 of PANC-1 is 0.06 ±0.003 ?g/ml.The expression of the drug resistance-related gene MDR1(multidrug resistance 1)was significantly increased in PANC-1-GR cells.The proliferation rate of PANC-1-GR cells was decreased compared with the parental strain.PANC-1-GR cells expressed higher ADAM 10 than PANC-1 cells,and more soluble MICA and ULBP2 can be detected in the cell supernatant of PANC-1-GR cells.PANC-1-GR cells have reduced sensitivity to NK cell killing.RNA interference of ADAM 10 and down-regulation of its expression can restore the sensitivity of PANC-1-GR cells to NK cell killing.Conclusions:The established PANC-1-GR cell line has gemcitabine resistance and high expression of MDR1 and ADAM10.ADAM10 secretes MICA and ULBP2 soluble proteins by cleavage,resulting in the escape of NK cell killing by PANC-1-GR cells.Part 3:Upstream mechanism study of ADAM10 regulating the sensitivity of gemcitabine in pancreatic cancerObjective:This study aimed to explore the upstream mechanism of ADAM 10 regulation of gemcitabine sensitivity in pancreatic cancer,especially the circular RNA-miRNA mechanism,in order to find plasma markers that predicts gemcitabine sensitivity.Methods:Differential circRNA profiles of pancreatic cancer cell PANC-1 and its gemcitabine resistant strain PANC-1-GR were analyzed by circular RNA sequencing.Q-PCR was used to validate two circular RNAs that are closely related to gemcitabine sensitivity.A circRNA-miRNA binding network map was established to screen for miRNA-145 that may be involved in the regulation of gemcitabine sensitivity in pancreatic cancer.After overexpression of miRNA-145 in PANC-1 cells,the expression of metalloproteinase was detected by quantitative PCR.Results:Circular RNA sequencing revealed that 126 differentially expressed circRNA,of which 68 circRNAs were up-regulated in PANC-1-GR cells and 58 circRNAs were down-regulated.Quantitative PCR confirmed that two distinctly distinct circRNAs were up-regulated in PANC-1-GR cells and that the circRNA was inversely correlated with gemcitabine sensitivity in patients.Bioinformatics analysis revealed that miR145 has binding ability to both circRNAs.Overexpression of miR145 inhibited the expression of ADAM 10 and ADAM 17 in pancreatic cancer cells,and the expression of miR145 in plasma of PDAC patients was positively correlated with its response to gemcitabine.Conclusions:Circular RNAs,including chr14:101402109-101464448+ and chr4:52729603-52780244+,regulate the expression of the metalloproteinases ADAM 10 and ADAM 17 through the ceRNA mechanism of miR145-5p,thereby participating in the immunological regulation of gemcitabine sensitivity in pancreatic cancer.