| Currently, Lung cancer has been one of common malignant tumors in the world,and NSCLC accounts for75%to85%of lung cancer. The treatment of advancedNSCLC has changed from traditional chemotherapy drugs therapy to moleculartargeted therapy stage. Gefitinib is characterized by high specificity, rapid onset andless adverse reaction. Although gefitinib showed good therapeutic effect in NSCLCpatients, but resistance has greatly limited the application of gefitinib.STAT3is an important member of signal transducer and activator of transcription(STAT) family, and widely expressed in various kinds of cells and tissues. STAT3canthansfer signals from cytokine in the plasma membrane to the nucleus, where it canmodulate the transcription of multiple genes. It is reported that, STAT3siRNA orinhibitors such as JSI-124, AG490, Stattic and so on can enhance gefitinib sensitivityto NSCLC cells and improve the effect of gefitinib in NSCLC cells by inhibitingSTAT3activation. However, the mechanism of STAT3-mediated gefitinib resistance isnot very clear and required to further study. Here, we studied STAT3-mediatedgefitinib-resistance and its related mechanism.Part one: Constitutively active STAT3resists the effects of gefitinib in NSCLCcellsWe first evaluated the levels of STAT3activation in various NSCLC cells, andanalyzed the relation of EGFR phosphorylation and STAT3activation. we found thatPC14, HCC827and H1975cells harboring mutations of EGFR possessed higherlevels of constitutively activate STAT3compared with A549cells with wild-typeEGFR. And, in H1975, A549and HCC827cells, we found that gefitinib inhibitedEGFR tyrosine kinase, but it had no effect on STAT3activation at the same concentration and time. In addtion, stattic in combination with gefitinib significantlyenhanced the anti-cell growth effects of gefitinib in H1975cells, which furtherdemonstrates constitutively active STAT3affects the effect of gefitinib in H1975cells.Part two: The mechanism of constitutively active STAT3-mediated gefitinibresistance1. The influence of EGFR mutation on constitutively active STAT3p-Babe-EGFR-ins retroviral vector was stable transferred into NIH-3T3cells byretroviral infection. Followed by Western blot analysis, stable transferred NIH3T3cells were found expressed excessive exon20insertion (3T3/EGFR ins) mutation,which displayed elevated EGFR and STAT3tyrosine phosphorylation compared withNIH3T3cells. The activation of STAT3can be completely inhibited by gefitinib.Those results suggest that excessive EGFR directly leading to enhanced Stat3tyrosinephosphorylation in NIH3T3cells, and constitutively active STAT3mediatedgefitinib-resistance seems to be cell line dependent.Futhermore, we used EGFR siRNA to examine the effect of EGFR expression onSTAT3activation. H1975cells were transfected with EGFR shRNA to inhibit theexpression of EGFR, correspondingly, STAT3activity were down-regulated,indicating sustained activation of STAT3mediated-gefitinib resistance was related toEGFR molecule expression in H1975cells.2. The effect of GP130/JAK signaling activation on constitutively active STAT3GP130/JAK signaling pathway is a main signaling that can activate STAT3. Inthis part, we first compared the activity of JAK1and JAK2in A549and H1975cells,no significant differences in the activity of JAK1and JAK2were found in both cells.However, We found higher levels of JAK1/JAK2heterodimerization in H1975cellscompared with A549cells, which may be responsible for excessive activation ofSTAT3in H1975cells. We next evaluated whether gp130recepetor, upstream ofJAK1/JAK2affected STAT3activation. We found that inhibition of gp130by RNAinterference resulted in decrease of STAT3tyrosine phosphorylation in H1975cells,indicating sustained activation of STAT3-mediated gefitinib resistance is related togp130recepetor expression in H1975cells. Gp130is a membrane receptor of cytokine, it can mediate signaling transductionof gp130/JAK/STAT3pathway, and promote the activation of STAT3. Therefore, weused a cytokine array to analyze the secretion of cytokines in H1975cells conditionedmedia. We found that among the cytokines in H1975cells supernatant media, onlyIL-6was closely related to STAT3. To determine whether any other cytokinesinvolved in the activation of STAT3, we pretreated H1975cells with cycloheximide toblock newly protein synthesis and secretion, and found conditioned medium from thecells pretreated with cycloheximide didn’t block the activation of STAT3, indicatingother than IL-6, H1975cells did not secret any other cytokines that can mediateSTAT3activation. However, both of the FBS (containing IL-6) and the recombinantIL-6can not affect the activation of STAT3in H1975cells. The above experimentssuggest the activation of STAT3cells don’t associate with autocrine cytokines inH1975.Above studies showed that STAT3activation in H1975cells is related to theexpression of EGFR and gp130molecule, suggestting that abnormal activation ofSTAT3within H1975cell line have occured. Considering EGFR/HER2heterodimerformation is much higher in H1975cells than in A549cells and EGFR/HER2heterodimers can mediate STAT3activation through HER2binding to STAT3, wefurther used gp130siRNA to study its effect on EGFR/HER2heterodimer formationin H1975cells. Results showed that the knockout of gp130resulted in greatlydecrease of EGFR/HER2heterodimer formation, indicating that gp130mediatedformation of EGFR/HER2heterodimer. Altogether, our results demonstratesformation of gp130/EGFR/HER2complex in H1975cells is at least partly accountfor sustained activation of STAT3.3. The effect of gefitinib on socs-3expression in H1975cellsSTAT3activation is negatively regulated by a variety of endogenous proteinregulators in cells. Therefore, after treatment of gefitinib, we further used Westernblot method to detect socs-3expression, which is a gp130/JAK/stat3pathwayendogenous negative regulate molecule. Resultes showed that gefitinib was able toinhibit level of scos3protein in a manner of time-dependency in H1975cells, thereby H1975cells maintain sustained activation of STAT3. This effect was related togefitinib inhibiting ERK1/2activation.This research showed that the formation of gp130/EGFR/HER2complexes inH1975cells, which promoted interactive dialogue of gp130/JAK and EGFR/HER2signaling pathways, induced an increase of JAK1/JAK2heterodimer formation,eventually led to constitutive activation of STAT3. At the same time, gefitinibinhibiting socs-3expression impacted the endogenous negative regulation ofgp130/JAK/STAT3signal. In summary, due to formation of gp130/EGFR/HER2complexes, H1975cells have primary resistant to gefitinib, while gefitinib inhibitedthe expression of socs-3reducing the endogenous negative regulation of STAT3results in H1975cells acquired resistance to gefitinib. Two aspects work together tomaintain the sustained activation of STAT3, which mediates gefitinib resistance. Thispaper clarified the mechanisms of constitutively active STAT3-mediated gefitinibresistance, suggesting that inhibition of the formation of gp130/EGFR/HER2complexis an important way to reduce STAT3activation, which provides a potential newtarget for drugs of antagonizing gefitinib resistance, therefore has importanttheoretical significance and application value for drug development. |
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