| Background: Early onset pancreatic cancer(EOPC)is a subgroup of pancreatic cancer with distinctive clinical and molecular characteristics.However,the clinical and molecular characteristics of EOPC are still sparse and controversial.Transcriptome-based analysis has identified a significantly increased transcription level of FOXC2 in EOPC,and FOXC2 expression shows a significant correlation with epithelial-mesenchymal transition(EMT)-related pathways.FOXC2 can promote tumor metastasis through EMT and induce cancer-associated fibroblast(CAF)to shape an immunosuppressive tumor microenvironment.Therefore,there are potential interactions between FOXC2 and EMT,CAF,and tumor immune cell infiltration in pancreatic cancer.However,the underlying molecular alterations and potential regulatory mechanisms within EMT,CAF and immune cell infiltration interactions induced by increased expression of FOXC2 in EOPC compared to late onset pancreatic cancer(LOPC)remain unclear.Objective: To explore the clinical characteristics of EOPC patients and the underlying molecular alterations and possible regulatory mechanism involved in the interactions among EMT,CAF,and immune cell infiltration induced by increased FOXC2 expression in EOPC compared to LOPC.Methods: In our study,EOPC was defined as pancreatic cancer diagnosed before the age of 45 years,while LOPC was defined as diagnosed at or after the age of 45 years.(1)Clinical data from 119 patients with EOPC and 38 patients with LOPC were retrospectively analyzed to explore the clinical characteristics and prognosis of EOPC patients.(2)The expression of markers for FOXC2,EMT,CAF,and immune cell infiltration in EOPC,was evaluated by immunohistochemistry(IHC,21 EOPC tissues vs.38 LOPC paraffin section tissues).(3)Immune cell infiltration in EOPC was further explored by RNA sequencing(10 EOPC vs.10 LOPC frozen tissues).(4)The expression of FOXC2,EMT and CAF markers at the m RNA and protein levels was further verified by quantitative real time-polymerase chain reaction(q RT-PCR,12 EOPC vs.8 LOPC frozen tissues)and Western blot(WB,10 EOPC vs.7 LOPC frozen tissues),respectively.Results: In terms of clinical characteristics,(1)based on a single EOPC cohort(n = 119),the analysis found that patients aged 35-45 years accounted for 86.5%,47.1% of patients had a later stage of T(T3 + T4),74.8% of patients exhibited lymph node metastasis(N1 + N2),16.8% of patients presented metastasis(M1),and 65.5% of patients were already at an advanced stage(stage III + IV)when diagnosed;survival of resected EOPC patients were significantly prolonged,especially in patients with stage 0-II(the median survival was 52 months),followed by patients with stage IV;the median survival of EOPC patients was 21 months,and the 5-year survival rate was only 8.4%.(2)The proportion of advanced stage appeared to be higher in EOPC,EOPC patients had a higher proportion of T4 stage,R0 resection rate,and received more adjuvant chemotherapy compared to LOPC;median survival was not significantly different between the two groups.In terms of molecular characteristics,compared to LOPC,(3)IHC results showed that the expression of FOXC2,Ncadherin and Vimentin was significantly upregulated,and the expression of E-cadherin was significantly downregulated in EOPC.Moreover,the expression of FOXC2 was significantly correlated with the expression of E-cadherin and Vimentin in EOPC.(4)IHC results showed that alphasmooth muscle actin(α-SMA),fibroblast activation protein(FAP)and fibroblast-specific protein 1(FSP1/S100A4)were significantly upregulated in EOPC;myofibroblastic CAF(my CAF)were characterized by high expression of α-SMA and FAP induced by the transforming growth factor-β(TGF-β)signaling pathway.(5)IHC showed that the expression of CD3(T cells)and CD20(B cells)in EOPC was significantly downregulated,and the expression of CD68(macrophages)and CD138(plasma cells)in EOPC was significantly upregulated.(6)The expression of the CD56(natural killer cell,NK cell)protein evaluated by IHC did not differ between LOPC and EOPC.However,NK cells decreased significantly in EOPC according to transcript levels.(7)q RT-PCR results showed that FOXC2,N-cadherin,and Vimentin expression increased significantly and E-cadherin decreased significantly in EOPC;FAP and S100A4 increased significantly in EOPC,and α-SMA had a trend of upregulation in EOPC.(8)Compared to LOPC,the WB results showed that FOXC2 and N-cadherin increased significantly and E-cadherin decreased significantly in EOPC;Vimentin had a trend of upregulation in EOPC;S100A4 was significantly overexpressed and α-SMA had a trend of upregulation in EOPC;and no significant differences in FAP were observed between the two groups.(9)Infiltration of CD138-positive plasma cells were associated with longer median overall survival(OS)and relapse free survival(RFS)of patients with EOPC.Conclusions:(1)Aggressive surgical treatment can significantly improve the survival of early stage of patients with EOPC.(2)EOPC is a subgroup with a more biologically aggressive phenotype and presents a poor prognosis.(2)The activities of FOXC2,EMT,and CAF in EOPC are significantly enhanced,and EOPC is in a relatively stronger tumor immunosuppressive microenvironment.(3)FOXC2 may promote the activation of EMT and CAF through enhanced TGF-β signaling pathway,affecting immune cell infiltration to promote the relatively strong tumor immunosuppressive microenvironment in EOPC.(4)CD138 can act as an effective biomarker of prognosis in EOPC.25 figures,24 tables,276 references... |
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