Epiregulin Regulates The Transition Between Normal Fibroblasts And Cancer-associated Fibroblasts And Promotes Oral Squamous Cell Carcinoma Invasion

As the second largest "disease killer" of human being,malignancy is the major disease cause of death among the 45-64 age group.Oral squamous cell carcinoma(OSCC)ranks the eighth among all malignancies in the world,with a significantly higher incidence in China than in developed countries.Despite continuous progress in surgical treatment,radiotherapy,chemotherapy,and biological therapy,the five-year survival rate of OSCC patients has been hovering around 50%-60%.In order to improve the prognosis of OSCC patients,it is urgent to further reveal the mechanism underlying the development and progression of OSCC.In recent years,more and more studies have shown that the development of tumor depends on the interaction of tumor and tumor microenvironment.As a major cellular component in the tumor microenvironment,cancer-associated fibroblast(CAF)plays an important role in the development of OSCC.However,the mechanism underlying the abnormal activation from normal fibroblasts(NF)to CAF stays unclear.In this study,to unveil the key participant in NF phenotype transition,we separated NF from normal mucosa specimen and CAF from OSCC specimen.Through high-throughput sequencing and bioinformatics analysis,we identified epiregulin(EREG)as a most significantly changed gene between NF and CAF.We then further verified this result in other 5 pairs of NF-CAF through qPCR and WB,and found that the genetic and protein expression of EREG was significantly higher in CAF than in NF.We then performed a retrospective study on 104 OSCC patients to unveil whether EREG expression in CAF was related to prognosis of OSCC,and found that the high EREG level in CAF was related to poorer overall survival(p=0.0398),poorer metastasis-free survival(p=0.0359)and poor disease-free survival(p=0.0314),and was a independent prognostic factor for poorer overall survival(p=0.049,Exp(B)=2.758,95%CI:1.007-7.566)and poorer disease-free survival(p=0.04,Exp(B)=2.842,95%CI:1.048-7.71).In addition,the high expression of EREG in CAF was closely related to advanced TNM stage(?2=8.248,p=0.0041),deeper invasion depth(?2=22.29,p<0.0001)and inferior worst pattern of invasion(?2=20.97,p<0.0001),suggesting that CAF with high EREG expression might promote the invasion and metastasis of OSCC,which led to poor prognosis of OSCC patientsBased on these results,we hypothesized that EREG promoted the malignant transition of fibroblasts,thus resulted in inferior prognosis of OSCC patients.To verify this hypothesis,we successfully constructed EREG-overexpression plasmid and EREG interference siRNA.We found that after EREG over-expression in NF,the expression of CAF markers such as SMA,Vimentin and N-cadherin was up-regulated significantly(p<0.05),and cell viability also increased significantly(p<0.05).After EREG-interference in CAF,the expression of CAF markers such as SMA,Vimentin and N-cadherin decreased significantly(p<0.05),and the cell viability was weakened(p<0.05).In addition,we revealed that after overexpression of EREG in NF,the JAK2-STAT3 pathway was activated significantly.After JAK2-STAT3 pathway inhibition,EREG-mediated NF phenotype transition was inhibited,indicating that EREG mediated NF-CAF phenotype activation was dependent on JAK2-STAT3 pathway.In addition,it is generally believed that CAF is an irreversible malignant activation form of NF.However,EREG-interference in CAF could deprive CAF of its malignant phenotype,further indicating that EREG played a key role in NF-CAF transition.In addition to the up-regulation of CAF marker and cell viability,another important phenotypic difference between CAF and NF is the tumor-supportive nature of CAF.Distant metastasis is among the major causes of death in OSCC patients,and invasion is the precondition for distant metastasis,thus the effect of CAF on the invasion of OSCC is especially worthy of attention.Through a review of clinical cases,we have found that CAF with high EREG expression was associated with a stronger invasiveness and higher distant metastasis rate of OSCC.Through further in vitro observation including wound healing assay,Transwell migration and invasion assay,we revealed that the high expression of EREG could enhance the invasion-promoting ability of fibroblasts to promote OSCC migration and invasion.To further study the invasion-supportive role of CAF in OSCC,we also developed a three-dimensional in vitro invasion model,which was easy to operate and with good repeatability,and provided a more direct imitation of in vivo invasion.With this model,we could provide in-depth understanding of the phenotypic transition between NF and CAF,thus adding up to the theoretical foundation for OSCC treatment.This three-dimensional model showed that compared with NF,CAF had a significant promoting effect on OSCC invasion,with decreased intercellular adhesion(a worse way of invasion)and a deeper invasion depth.After interfering with EREG in CAF,the invasion-promoting ability decreased significantly.After EREG over-expression,NF gained tumor-promoting ability,showing deeper invasion depth and worse invasion pattern.In order to further reveal the mechanism of EREG-mediated invasion-promoting ability of CAF,we performed genetic co-expression analysis,and found 35 protein encoding mRNAs and 2 long-chain non-coding RNAs with close expression relation with EREG.Among them,TAC1,S100B,KLK7,FUT3,SPRR2A,TMPRSS were reported to be related to tumor invasion.Through qPCR and WB,we found that the genetic and protein expression level of KLK7 was significantly augmented in NF after EREG over-expression(p<0.05),and in OSCC cell line after its supernatant treatment(p<0.05).These results indicated that KLK7 might be involved in the process of EREG-mediated invasion-promoting ability of CAF.Finally,to verify the above results in vivo,we co-injected OSCC HSC3 cell line with conditional NF or CAF into nude mice to establish OSCC xenograft model.This model further proved that CAF had a significant tumor-supportive abilities compared with NF.The tumor-promoting ability of CAF significantly decreased after EREG interference.Moreover,the CAF marker SMA,and invasion-ability marker MMP2 also decreased in CAF.Meanwhile,after EREG overexpression,NF obtained the CAF-like tumor-promoting phenotype in vivo,and the expression of SMA and MMP2 increased significantly.The above results showed that EREG was the key to maintaining the malignant phenotype of CAF and promoting the OSCC invasion.Moreover,qPCR and immunohistochemical staining showed that after EREG overexpression,(p-)JAK2,(p-)STAT3 and KLK7 increased in NF,confirming that JAK2-STAT3 was involved in EREG mediated NF-CAF transition,and KLK7 was involved in EREG mediated invasion-promoting role of fibroblastsIn summary,our study found that the high expression of EREG in CAF could promote the invasion and metastasis of OSCC,and led to poor OSCC prognosis.We revealed the EREG promoted NF-CAF transition in a JAK2-STAT3 pathway dependent way,and increased the invasion-promoting ability of fibroblasts through KLK7.EREG knockdown in CAF resulted in the reversion of malignant phenotype,and inhibition of its tumor-supportive role.Therefore,CAF with high EREG expression could be a potential target for OSCC treatment.Our study provides theoretical basis for OSCC treatment.