The Mechanism Of Downregulation Of Pancreatic FGF21 By Kras^G12D In Initiation Of Pancreatic Cancer

Part 1.Test the expression of Fibroblast growth factor 21（FGF21）and its receptors in pancreasObjective: to test the expression of FGF21,it receptor FGFR1 and co-receptor KLB n pancreas and compare FGF21 level in normal pancreas and pancreatic cancerMethods: To understand the role of FGF21 in the pan Bacas,we measured pancreatic FGF21 gene expression in normal 70 days old Bac mice.To further compare FGF21 level,we performed Immunohistologic chemistry（IHC）analysis and western blotting analysis.To better understand the functional role of pancreatic derived FGF21,we analyzed its receptor FGFR1 in the pancreas and liver tissue from 70-day-old Bac mice by using semi-quantitative RT-PCR and qRT-PCR.We compared gene expression of its co-receptor,Beta Klotho（KLB）,in liver and pancreas by using qRT-PCR analysis.To further confirm KLB expression in the pancreas,we analyzed KLB protein level in normal Bac pancreas by western blot and using recombinant KLB as a positive control.Further analysis was conducted with immunohistochemistry.We also compared FGF21,FGFR1,and KLB gene expression in liver and pancreas tissues in 7-month-old mice to see if it maintains the similar pattern in 70 day-old-mice.To test if FGF21 plays a role in pancreatic cancer,we performed human pancreatic tissue array to detect FGF21 level.We further analyzed FGF21 gene expressing in human pancreatic cancer cell lines carrying oncogenic Kras mutations with normal human pancreatic tissue samples.Results: In 70-day-old Bac mice,we observed that in the normal feeding condition,pancreatic FGF21 gene expressing is highly expressed in pancreas and significantly higher than its expression in liver by using qRT-PCR、Western Blot and IHC staining（p<0.05）.qRT-PCR showed that pancreatic FGF21 gene expressing is approximately 80 folds higher than that in liver tissue.Immunohistochemistry（IHC）analysis also confirmed that FGF21 is highly expressed in the pancreas.We found that pancreatic FGFR1 gene expression in the same mice was substantially higher than that in liver by using semi-quantitative RT-PCR.Further study confirmed that pancreatic FGFR1 gene expression was about 12 folds higher than that of liver by qRT-PCR（p<0.05）.We found that pancreatic KLB gene expression is comparable to that in liver by using qRT-PCR analysis（p>0.05）.We analyzed KLB protein level in normal Bac pancreas by western blot and using recombinant KLB as a positive control.urther analysis with immunohistochemistry,we observed that KLB is mainly expressed in acinar cells of the pancreas,with lighter staining in islet cells.In 7-month-old mice,q-RT-PCR showed that FGF21 gene expression is about 60 folds than that in liver（p<0.05）;pancreatic FGFR1 is about 12 folds higher than that in liver（p<0.05）;Pancreatic KLB gene expression is comparable to that in liver（p>0.05）.By human pancreatic tissue array we found that comparing to the normal human pancreatic tissues,patients with PDAC express have substantially lower level of FGF21 by IHC of FGF21.In a panel of 59 human pancreatic cancer specimens,the mean level of FGF21 protein was significantly lower compared with normal pancreatic samples（n=45）.Statistical analysis further confirmed that PDAC patients have significant lower FGF21 protein level as compared with control group with normal pancreas（n=45,****p<0.0001）（p<0.05）.QRT-PCR found that FGF21 gene expression was significantly decreased（****P<0.0001）in human pancreatic cancer cell lines carrying oncogenic Kras mutation samples as compared with that of normal human pancreatic tissue samples（p<0.05）。Conclusion:.FGF21 and its functional components FGFR1 and KLB are expressed in pancreas in normal control mice in the normal feed condition.Pancreatic FGF21 and FGFR1 expression is significantly higher than that of in liver while KLB is mainly expressed in the acini that in the islet suggesting its crucial role in pancreatic exocrine process in mice.Older mouse maintains the similar expression pattern shown in 70 day-old-mice.Analysis on human pancreatic tissue and cell lines confirmed that FGF21 expression is significantly decreased in human pancreatic cancer。Part 2.Oncogenic KrasG12 D expression led to significant decrease of FGF21 in the pancreasObjective: Understand the role of pancreatic FGF21 and its relation to oncogenic Kras in pancreatic cancerMethods: we chose to utilize LSL-Kras^G12D/+ mice to allow endogenous levels of pancreatic oncogenic Kras^G12D/+ expression upon tamoxifen induced activation of Elastase-Cre recombinase.We induced oncogenic Kras^G12D/+ expression in the adult mice（>60 old）and analyzed pancreatic FGF21 expression in 7-month-old mice by qRT-PCR and immunohistochemistry.To test whether decreased expression of pancreatic FGF21 is an early event of oncogenic Kras expression,we induced oncogenic Kras^G12D/+ expression in young adult mice for one week and assessed FGF21 m RNA levels.To investigate if FGFR1 and KLB have the same response after oncogenic Kras^G12D/+ expression,we evaluated FGFR1 and KLB gene expression in the same mice.To test if MIST1 is a mediator that is responsible for oncogenic Kras mediated decrease of pancreatic FGF21 gene expression,we evaluated MIST1 gene expression after 7 days,one month,or 5 months of oncogenic Kras expression by qRT-PCR and confirmed with western blot.Analysis of Ezh2 expression in WT mice and mice harboring the LSL-KRASG12 D was done by qRT-PCR.Further we include a floxed Ezh2 homozygous allele into Kras G12 D mice（allows inducible loss of Ezh2 when oncogenic KRAS is expressed;）to see if Ezh2 silencing can alleviated Fgf21 repression one after tamoxifen treatment.Treatment of Panc1 cells with DZNep,an EZH2 inhibitor,with different dosages was done to see FGF21 expression by semi-quantitative RT-PCRResults: qRT-PCR showed a striking more than 100 fold decrease of FGF21 expression in 7 month-old mice with oncogenic Kras expression when compared with Cre mice of the same age.IHC for these mouse tissues found that pancreatic acinar cells have substantially lower level of FGF21 staining when compared with that in Cre control mice,which have rich stain of FGF21 Kras^G12D+.We observed a 7-fold decrease in pancreatic m RNA expression of FGF21 in the pancreas harboring oncogenic Kras^G12D/+ mutation when compared with Bac control mice of the same age（*p<0.05）.while FGFR1 and KLB gene expression and found that none of them respond to early stage of oncogenic Kras^G12D/+ expression.MIST1 gene expression was significantly decrease in a time dependent manner.MIST1 protein level was also decreased,correlating to MIST gene alteration.Analysis of Ezh2 expression showed elevated levels of Ezh2 expression in mice harboring the LSL-KRASG12 D compared with Cre mice.However,the inclusion of a floxed Ezh2 homozygous allele into KM mice significantly alleviated Fgf21 repression.Treatment of Panc1 cells with DZNep,an EZH2 inhibitor,lead to decreased increased FGF21 expression in a dose dependent fashion.Conclusion: FGF21 is abundant in normal metabolic conditions and its expressing was lost in pancreas of the mice with oncogenic Kras expression.FGF21 is a potential player responsible for oncogenic Kras mediated disease initiation.pancreatic FGF21 decrease and eventually silence may relate to oncogenic Kras mediated MIST1 decrease.EZH2 is the mediator by which KrasG12 D leads to FGF21 decrease.This repression of FGF21 also occurs in human PDAC.These data suggest that pancreatic FGF21 decrease is through MIST1 decrease caused by Kras mediated EZH2 increase.Part 3.Recombinant human FGF21（rh FGF21）treatment protected pancreatic injury induced by HFDObjective: To test if the vulnerability of oncogenic Kras to HFD mediated PDAC development is due to the loss of FGF21 and if supply with rh FGF21 could alleviate high-fat-diet effect on promoting PDAC formation.Methods: To test this,we chose to use a traditional mouse model with endogenous level of oncogenic Kras G12D/+ expression.We induced oncogenic Kras expression with tamoxifen in adult mice,including both male and female.These mice were randomly grouped and treated with normal diet,high-fat-diet,or high-fat-diet + rh FGF21 for a duration of 10 weeks.Cre mice fed with normal diet were also recruit as control group.After 10 weeks of treatment,we dissected mice and examined the gross anatomy and HE staining of their pancreas and compare the incidence of PDAC of each group.we analyzed triglyceride level of pancreas.We used Western blot to analyze acinar cell marker,amylase.To analyze the level of PSC activation,we measured α-SMA expression.We use CK19 and Sirius Red staining to evaluate the acinar-to-ductal metaplasia and fibrogenesis.we evaluated pancreatic inflammation and immune cell infiltration using COX2,F4/80,CD3 immunohistochemistry staining.we examined cell proliferation among each group using proliferative marker Ki67 and cyclin D1.To test how long-term treatment of FGF21 would prolong overall survival and delay tumor growth and metastasis,LSL/BAC mice were fed with ND,HFD,or HFD+rh FGF21 until ageing.Bac mice fed on ND were used as overall control.We recruited both male and female mice.We monitored all the mice until they die and make survival curve.To determine whether FGF21 treatment would have any effects on the food intake,we measured food intake weekly by normalizing the food consumed per mice.We also measured body weight.When the last mouse HFD treatment Kras G12D/+ mouse was sacrificed,we sacrificed the mice with various other treatment to visually compare the pancreas.Results: The mice fed on a high-fat-diet accumulated abdominal fat and presented pancreatic injury,In contrast,the Kras G12D/+ mice with HFD+rh FGF21 treatment substantially inhibited abdominal fat accumulation and pancreas injury,which are comparable to the Kras G12D/+ mice fed on a ND.Further histologically analysis using H&E staining revealed that mice fed on a high-fat-diet developed PDAC with 50% penetrance within 10 weeks.However,with FGF21 treatment,most of the mice are still in the Pan IN lesion I or II comparable to the mice fed on a normal diet and only one out of 8 mice developed PDAC（12.5% penetrance）.we analyzed triglyceride level of pancreas and found that oncogenic Kras expressing mice fed on a high-fat-diet contain significant higher level of TG than the same mice fed on normal diet.FGF21 administration significantly inhibit the buildup of TG in pancreas in the same mice fed on a HFD.Western blot found that HFD treatment substantially decreased amylase while FGF21 treatment preserved amylase in the same mice fed on a HFD.α-SMA expression was strongly increased in mice with HFD.No decrease of α-SMA expression were noted in mice with HFD+rh FGF21 treatment when compared with the mice on a ND.acinar cells.BAC mice on ND displayed normal collagen distribution which was primarily around the blood vessels in the pancreas as indicated by Sirius red.Kras G12D/+ mice on ND showed some peri-lobular collagen,and there was no difference between control Kras G12D/+ mice on the ND and Kras G12D/+ mice on HFD+rh FGF21 treatment.Consistent with tumor development,Kras G12D/+ mice on the HFD demonstrated extensive interlobular and Sirius red and CK19 staining.when mice expressing oncogenic Kras were treated with FGF21,the positive staining of Cox2,F4/80 and CD3 in pancreas was substantially decreased which is comparable to the oncogenic Kras expressing mice fed on ND.The Cox2,F4/80 and CD3 staining was abundant in mice fed on HFD.Supporting the presence of a neoplastic epithelium in mice fed on HFD,we observed increased proliferation using proliferative marker Ki67 and Cyclin D1.Despite feeding with HFD,rh FGF21 treatment inhibited Ki67 and Cyclin D1 ductal cell staining.For long-term mice,we did not observe any significant food consumption alteration in HFD+FGF21 group when compared with HFD only in the same agenda,however,we found that male consume more food than femal.We also found that mice with HFD treatment significantly increased body weight;however,FGF21 treatment significantly inhibited body weight increase,which is comparable to the mice fed on ND.Accompany with significant body weight increase,LSL/BAC mice on the HFD showed development tumor.LSL/BAC mice on the HFD had a significant decrease in median survival time compared to LSL/BAC mice on HFD+FGF21 remained healthyConclusion: The study showed confirmed that the vulnerability of oncogenic Kras to HFD mediated PDAC development is due to the loss of FGF21 and if supply with rh FGF21 could alleviate high-fat-diet effect on promoting PDAC formation.our data shed light on an unexpected anti-tumor action of HFD imposed before tumor onset and identify FGF21 as a putative therapeutic target to selectively hinder pancreatic cancer.