EGFR/MEK/ERK/HIF-1α Loop Regulates Glycolysis In Pancreatic Cancer

Objective:The total survival rate of the fatal disease,pancreatic cancer is less than 5% in5 years.Due to earlier local or metastasis recurrence of the cancer,the 5-year survival rate of the cured patients is merely 18%,the reason of which the disease is called “the King of Cancers”.Therefore,it has been the urgent clinical task to be solved to better know the potential mechanism of attack and recurrence of pancreatic cancer and search new therapeutic target.The glycometabolism in cancer cells is abnormal and the excessive growth of cancer cells leaves scarce oxygen for the cells.Therefore,cancer cells tend to stop aerobic oxidation requiring mitochondria and use anaerobic glycolysis of more glucose to provide energy.On the basis of specificity of pancreatic histology,many studies in recent years proved that diabetes was closely related to attack and development of pancreatic cancer.Abnormal glucose metabolism has been an important clue for early diagnosis and prognosis of pancreatic cancer.Meanwhile,abnormal glucose metabolism will cause resistance to conventional treatment method.All these factors facilitate exploration of new mechanism and therapeutic plan for regulation of abnormal glucose metabolism.Hypoxia-inducible factors1α(HIF-1α)and its upstream factor are the important regulatory factors for glycolysis of cancers.When oxygen partial pressure of cells is reduced or stimulated,HIF-1α will be accumulated and activated in cytoplasm,transferred to cell nucleus and combined with corresponding target sequences to regulate genetic expression of transcription factors related to glycolysis,such as PGK,GLUT and LDH etc to regulate glycometabolism eventually.In vitro experiment proved that extracellular signal-regulated kinase(ERK)/MAPK signal transduction pathway phosphorylation are of great importance for regulation of activity of HIF-1α.Studies showed that EGFR affected glycometabolism of cancers by regulating PKM2 through ERK1/2 pathway and NF-KB pathway,the latter of which was mediated by HIF-1α.In the earlier study,it had been proved that EGFR affected capacity of invasion and metastasis of pancreatic cancer cells by regulating the dissociated state of primarily cultivated cancer cells through MAPK/ERK pathway,which indicated that EGFR/MEK/ERK/HIF-1α could be the possible pathway to regulate glycolysis of pancreatic cancer.However,there is no report about how EGFR affects metastasis of pancreatic cancer cells by participating in cancer cells glycolysis mediated by HIF-1α through MAPK/ERK pathway.This study will discuss whether and how activation of EGFR/MEK/ERK pathway participates in regulation of pancreatic cancer glycolysis mediated by HIF-1α and its possible mechanism,whether individual or combined regulation of EGFR and HIF-1α will affect glycometabolism level of pancreatic cancer cells on the background of different glycometabolism levels and further affect characteristics of pancreatic cancer,and whether EGFR and HIF-1α is the potential therapeutic target rectifying abnormal glycometabolism.Methods:I.Object of study:1.Hamsters’ pancreatic cancer cell line PC1(low dissociation and metastasis)and PC1.0(high dissociation and metastasis)primarily cultured by this study team.2.6 cases of clinical inpatient patients with pancreatic cancer and PETCT result of hypermetabolism and 6 cases with hypometabolism;content of blood glucose in serum,C-peptide,insulin,glucagon,GHb A1 c,GA and GSP.II.Method of study: 1.Normoxic and hypoxic cell culture: primarily culture two kinds of pancreatic cancer cell lines;establish hypoxic model by putting cells into hypoxic incubator with temperature of 37 ℃、0.5% O2,94.5% N2,5% CO2.2.Cell treatment and grouping: Transfect expression of HIF-1α in Si RNA silencing PC1.0 cells;after transfection,pave the cells again;after treated by 10μM AG1478,the cells are grouped as follows: A Control group;NC transfection group;HIF-1α si RNA transfection group;AG1478 group;NC+AG1478 group;HIF-1α si RNA+AG1478 group.There are six groups in total.3.Test of glycometabolism level(glucose uptake test;lactic acid detection experiment).2-NBDG fluorescence microplate(excitation wavelength is 488 nm and emission wavelength is 520nm)is used to detect fluorescence intensity of cells in each group.Detect lactic acid secretion level of cells in each group according to instruction of lactic acid detection kit.4.Protein expression(western and immunohistochemistry)Detect protein expression level of EGFR,p-EGFR,p-MEK1/2,p-ERK1/2,HIF-1α,GLUT1 of cells in each group.5.Oxidative stress test(ROS)Analyze ROS output through DCF-DA fluorescence staining and flowcytometry.6.Cell proliferation,invasion and metastasis capacity test(MTT,wound scratch assay and transwell)MTT experiment: carry out the test every 24 h from first day to fifth day;measure and calculate light absorption value of 570 nm wavelength of each hole by microplate reader;carry out statistical treatment to make growth curve for observation of cell proliferation of each group.Cell wound scratch assay: make cell wound model after cellular fusion;culture it in serum-free 1640 culture medium for 3h,6h,12 h and 30h;take out the cells and take photos under microscope to observe cell migration.Transwell experiment: Transwell chamber is coated by Matrigel glue;add cell suspension of each group into upper chamber;after culture for 48 h under corresponding culture conditions,it is fixed by 4% paraformaldehyde and stained by 0.5% crystal violet solution;it is counted by inverted microscope and migrates to cells of lower layer of microporous membrane.5 views of each sample are selected to count cells and obtain the mean numbers.Observe migration capacity of cells.III.Statistical treatment Data is expressed by mean number ± standard deviation or median and quartile;t test is adopted for mean comparison between two groups of data obeying normal distribution;one-way analysis of variance(ANOVA)is used for mean comparison among multiple groups;Dunnett’s test is carried out for comparison among multiple groups and statistical software is Graph Pad Prism 5.0(Graph Pad Software).P<0.05 means the difference is of statistical significance.Results:I.There are differences in glycometabolism levels of pancreatic cancer patients’ samples with different cancer cell lines and 18F-FDG concentration level.In PC1.0 cells under high dissociation,the intake of 2-NBDG is increased and lactic acid secretion level is increased;GLUT1 expression in pancreatic cancer patients’ samples with high concentration level of 18F-FDG is increased.II.Difference in glycometabolism level is related to pancreatic cancer cell proliferation and staging,invasion and metastasis of tumor.Proliferation of pancreatic cancer cell line PC1.0 with high glycometabolism is rapid with strong migration capacity;IHC result of pancreatic cancer patients with high glycometabolism indicated expression of proliferation marker ki-67 is increased.In two groups of samples with high glycometabolism and lower glycometabolism,the difference in staging,grading and metastasis degree of tumor is of statistical significance.III.There is difference in expression level of factor related to EGFR/ERK/HIF-1α pathway among cell lines with different glycometabolism levels and different patients’ samples.In pancreatic cancer cells and tissues with high glycometabolism,EGFR/MEK/ERK/ HIF-1α pathway participates in regulation of cancer cell glycometabolism.In cells with high metastasis,the phosphorylation levels of related factors such as EGFR,MEK and ERK etc are simultaneously increased with HIF-1αand are positively related to glycometabolism level and lactic acid formation level.IV.Oxidative stress mechanism participates in regulation of EGFR/MEK/ERK/HIF-1αfor glycometabolism.ROS level of pancreatic cancer cell lines with high glycometabolism activated by EGFR/MEK/ERK/HIF-1αpathway is low and EGFR expression level is further promoted by ROS inhibitor.V.Inhibiting EGFR and HIF-1αwill weaken regulatory function of EGFR/MEK/ERK/ HIF-1α pathway for glycometabolism,which will regulate pancreatic cancer cell proliferation,invasion and metastasis.Reduce EGFR or HIF-1αand jointly reduce both in cell lines with high metastasis:1.Glycometabolism level of cancer cells will be inhibited,2-NBDG intake and lactic acid secretion level are reduced in different extent.2.Invasion and metastasis of cancer cells are inhibited in different extent.Conclusion:1.Expression of EGFR/MEK/ERK/HIF-1αpathway is increased in cells and tissue samples with high glycometabolism.2.The activity of EGFR/MEK/ERK/ HIF-1αpathway is related to increase of glycometabolism level,invasion and metastasis capacity of cells and patients’ prognosis.3.Oxidative stress participates in regulation of EGFR/MEK/ERK/HIF-1αpathway for glycometabolism.Under hypoxic condition,increase of HIF-1α impedes excessive increase of ROS and the lowered ROS level will further activate EGFR to form a feedback loop.4.Reducing EGFR or HIF-1α and jointly reducing both in cell lines with high metastasis will inhibit glycometabolism level of cancer cells in different extent to further inhibit invasion and metastasis of cancer cells.Further studies can be carried out on EGFR and HIF-1α as the therapeutic target rectifying abnormal glycometabolism of cancer cells.