Study On The Immune Molecular Classification Of Pancreatic Cancer And The Mechanism Of Pancreatic Stellate Cell FGF9 Regulating The Proliferation Of Pancreatic Cancer Cell

Part Ⅰ:Construction of immune molecular subtypes of pancreatic cancerPancreatic cancer remains treatment refractory.Immunotherapy has achieved success in the treatment of multiple malignancies.However,the efficacy of immunotherapy in pancreatic cancer is limited by a lack of promising biomarkers.In this research,we aimed to identify new classification based on immune components in pancreatic cancer microenvironment,to facilitate prognosis prediction and patient selection for immunotherapy.A training cohort of 149 pancreatic cancer samples from The Cancer Genome Atlas(TCGA)with mRNA expression data was analyzed.By means of non-negative matrix factorization(NMF),the immune-related signals were virtually dissected from bulk gene expression data.Detailed immunogenomic and survival analyses of the immune molecular subtypes were conducted to determine their biological and clinical relevance.Validation was performed in five independent datasets on a total of 615 samples.In this study,approximately 31%of pancreatic cancer samples(46/149)had higher immune cell infiltration,more active immune cytolytic activity,higher activation of the interferon pathway,a higher tumor mutational burden,and fewer copy number variations than the other samples(all P<0.001).This new molecular subtype was named Immune Class,which served as an independent favorable prognostic factor(Overall survival hazard ratio,0.56;95%confidence interval,0.33-0.97).Immune Class in cooperation with previously reported tumor and stroma classifications performed a better role in pancreatic cancer prognostic stratification.Moreover,programmed cell death-1(PD-1)inhibitors and other immune checkpoint inhibitors showed potential efficacy for Immune Class in SubMap analysis(P=0.04).The robustness of our immune molecular subtypes was further verified in the validation cohort.In conclusion,by capturing immune-related signals in the PDAC tumor microenvironment,we reveal a novel molecular subtype,Immune Class.Immune Class serves as an independent favorable prognostic factor in PDAC patients and provides potential implications for immunotherapy response prediction.Part Ⅱ:Molecular mechanisms of paracrine FGF9 from pancreatic stellate cells on regulating Myc protein stability and proliferation of pancreatic cancer cellsPancreatic stellate cells(PSCs)are the main stromal cells in the pancreatic cancer microenvironment.This study aimed to discover a novel mechanism of activated PSCs regulating pancreatic cancer development and progression.Firstly,transcriptome sequencing was performed on primary PSCs,and differential expression analysis was conducted between activated and deactivated PSCs.It was found that FGF9 was significantly highly expressed in activated PSCs.Paracrine FGF9 from PSCs can regulate the proliferation,colony formation,invasion and migration abilities of pancreatic cancer cells.Pancreatic cancer cells were treated with exogenous FGF9 protein and transcriptome sequencing was performed.Pathway enrichment analysis showed that cell cycle related pathways were significantly enriched.Transcription factor enrichment analysis showed that Myc was the core transcription factor downstream of FGF9.Subsequent mechanism studies have shown that FGF9 increases Myc phosphorylation levels at Ser62 site,and decreases Myc phosphorylation levels at Thr58 site.Through measuring Myc protein half-life,proteasome degradation and ubiquitination level,it was found that FGF9 inhibited degradation of Myc protein by ubiquitin-proteosome system.FGF9 activated downstream MAPK pathway and PI3K/Akt/GSKβ pathway,thus regulating Myc protein phosphorylation and ubiquitin-proteasome associated degradation.Finally,this study identified a non-coding RNA,LINC01963,involved in the regulation of Myc stability.LINC01963 is highly expressed in pancreatic cancer,promotes the proliferation of tumor cells and is associated with poor prognosis of pancreatic cancer.In conclusion,this study identified a novel molecular mechanism by which paracrine FGF9 from PSCs regulated Myc protein stability and proliferation in pancreatic cancer cells.The discovery of a specific secretory factor of PSCs and a novel non-coding RNA will contribute to the development of new Myc-targeted therapies.