Thymidylate Synthase Prompts Metastatic Progression Through Epithelial-mesenchymal Transition In Pancreatic Ductal Adenocarcinoma

Background:Pancreatic ductal adenocarcinoma(PDA)is one of common malignant digestive diseases,and prognosis of this disease is very poor.Radical resection is the potentially curative treatment for PDA,but unrestrained growth(metastasis and locally advance)led to the low resectabilty and surgical failure.Tumor biology has developed rapidly in recent years,but the key molecule or pathway implicated in the metastasis of PDA is remaining to be elucidated.Thus,in-depth analysis of the pathologic features of pancreatic cancer development,and related molecular mechanism research to develop new diagnosis and treatment methods is the urgent task for the basic research and transformation of pancreatic cancer.As a fundamental metabolic enzyme,anti-TSstrategy has been shown to be an effective therapy for human cancers.However,the genuine effects of TS in PDA are still conflicting.A lot of research shown that TS high expression was associated with poor prognosis of cancer,such as colorectal cancer,lung cancer,pancreatic cancer.But there was another opposite view indicatied that TS high expression was associated better prognosis of pancreatic cancer patients.We systemically assessed the prognostic value and whether TS associated with malignant progression in PDA.In this study,the prognostic effect of TS expressed in cytoplasm or cytonuclear was determined separately in the first time.In addition,The impact of TS on tumor cell behaviors was assessed in in vitro assays,and the TS associated metastatic potential was further determined in two different PDA metastatic models.Methods:From January 2007 to January 2016,91 tumor specimens(71 paraffin-embedded tissue blocks and 20 freshly-frozen tissues)were collected and their corresponding normal tissues.The clinical data of tumor specimens were collected and q-PCR was used to analyze the expression difference of TS in PDA and normal pancreatic tissues.The IHC was used for evaluating the TS expression in tumor and adjacent tissues and combinded with clinical data for analyzing the clinical significance.The lentiviral vector encoding human TSshRNA was transfected into pancreatic cancer cell lines.PANC-1 cells with stably knock-down of TS was termed as PANC-1-sh-TS.The cell proliferation was quantified every 24h by using a cell counting kit-8.The viability of cell lines treated with varying chemodrugs was determined by the MTT assay.Besides,motility ability of PANC-1 cells was determined by scratch assay and transwell migration assay.The expression of key protein in EMT process was assessed by western blot and immunocytochemistry in PANC-1 cells.To further validate the important role of TS in the regulation of EMT process,we investigated the effect of TS depletion on TGF-β treated PDA cells.The dTMPconcentrationsinpancreaticcancer cell lysate were measured by HPLC assay.Two different animal models(orthotopic and pulmonary circulation metastatic PDA model)combined with MRI,CT and PET were used to determine whether TS could affect PDA metastasis in vivo.IHC analysis of the metastatic lesions in liver or lung was used to determine the tumor Ki-67 expression.Results:q-PCR analysis revealed a significant up-regulation of TS gene expression(>2 folds)in 45%(9/20)en bloc PDA tumor specimens with a highest 19 folds increase when compared to the matched normal tissue.Simultaneously,taking into account that TS protein expression was remarkably higher in cell lines known to be more chemoresistant and metastatic.The IHC analysis demonstrated that TS was extensively distributed in cytoplasm and nucleus in both PDA specimens and the matched adjacent tissues.However,no significant differences were observed in cytoplasmic TS expression ratio(p=0.637)or staining intensity(p=0.611)between tumor and the adjacent tissues.But it should be noted that tumor TS expression was correlated with perineural involvement and tumor TS staining intensity was associated with worse histological grade.Kaplan-Meier analysis observed that patients with strong tumor cytoplasmic TS staining showed a borderline significant lower overall survival(p=0.84),but the cytoplasmic TS staining ratio in both tumor andadjacent tissues were not noted to have statistically significant association with post operation survival.Both staining ratio and staining intensity of tumor cytoplasmic TS expression cannot be an effective predictive/prognostic factor for PDA patients.The cytonuclear TS positive staining ratio is significantly different between tumor and adjacent tissues.Furthermore,the tumor nuclear TS expression was obviously correlated with pathological stage(p=0.043)and lymphatic metastasis(p=0.016).Negative correlation between tumor cytonuclear TS staining and the OS was observed(p=0.015).So,tumor cytonuclear TS is a potential prognostic biomarker for PDA,and TS may be critical in malignant progression of PDA.TS was failure to affect the proliferation of PDA cell lines according to the CCK-8 assay.Following 48 h incubation with different concentrations of gemcitabine,the average inhibition rate is dramatically exaggerated in TS deficiency PANC-1 group.Surprisingly,less pronounced results were observed in 5-FU and Erlotinib treated cell.Transwell assays and scratch assays showed that TS depletion significantly decreased cell invasion and migration ability.The decreased protein levels of EMT transcription factors including Snail,Vimentin and ZEB1 were concomitantly observed with the elevated epithelial marker E-cadherin expression in TS depletion PANC-1 cells.The β-catenin nucleartranslocation is an important hallmarker of EMT process in PDA.The decreased β-catenin expression in both cytoplasma and cytonucleus was confirmed by western blot analysis.As expected,enhanced membranal E-cadherin and cytoplasmic re-distributed β-catenin were simultaneously detected by immunofluorescence staining.Correspondingly,reduced cytoplasmic Vimentin was also observed in PANC-1-sh-TS cells.In addition,TGF-β enhanced cytonucleusβ-catenin localization and Vimentin overexpression were alleviated in PANC-1-sh-TS cells according to western blot and immunocytochemistry.The similar level of dTMP in both WT(wild type)and control groups was observedand the TS knockdown naturally brought a conspicuously decreased concentration of dTMP.Our results clearly demonstrated that 24 h incubation with 100 mM dTMP abolished the E-cadherin expression in PANC-1 cells and even in the PANC-1-sh-TS cells.Concurrently,the increased expression of EMT markers(Snail,Vimentin and ZEB)was observed.As illustrated by the PET/CT scan,both the growth of the pancreas orthotopic tumor and thedevelopment of the liver metastasis lesions are significantly suppressed in the sh-TS injected mice.Next step,we further explored the impact of TS on metastasis in pulmonic circulation metastasis murine model.Without surprise,the parental and sh-control PANC-1 cells effectively metastasized to the normal lung region,but the situationwas markedly inhibited in sh-TS group.Meanwhile,survival analysis also demonstrated a significantly improved lifetime in sh-TS orthotopic models.IHC analysis of the metastatic lesions in liver or lung also revealed that the tumor Ki-67 expression was distinctly reduced in the sh-TS metastatic tumor regions.Conclusion:Tumor cytonuclear TS expression predicts lymphatic metastasis and indicates poorer prognosis in PDA.TS expression positively correlates with primary gemcitabine chemoresistance in PDA cell lines.TS depletion suppressed migratory and invasive capabilities in PDA cells.TS intimately related with the EMT markers expression and mesenchymal phenotype in PDA cells.TS influences EMT process through the regulation of dTMP biosynthesis in PDA cells.TS depletion simultaneously tamed distant metastasis in both orthotopic and pulmonary circulation metastatic PDA model and TS associated with proliferation marker expression and overall survival in orthotopically implanted PDA models.