Promote Gli3 Processing To Inhibit Shh Signaling

Background and Objectives:The Shh signaling pathway plays a key role in embryonic development and adult tissue homeostasis.Abberant activation of the Shh signaling pathway is closely related to the development of several human malignancies,including the most common malignant brain tumor medulloblastoma（MB）in children.All Shh pathway inhibitors currently being studied in clinical or clinical trials target Smo,such as classic vismodegib from Genentech.However,more and more findings suggest that there is rapid emergence of drug resistance and severe pediatric developmental side effects when targeting Smo.Gli3 is an important transcription factor that regulates the Shh signaling pathway.The full-length form of Gli3FL can befurther processed into Gli3R by partial proteolysis（a process known as Gli3processing）.Gli3R mainly acts as a transcriptional repressor in the Shh signaling pathway,which plays a negative feedback regulatory role.However,Gli3 processing is inhibited in MB,the negative feedback regulation of Shh signaling pathway is unbalanced,and the overactivation of signaling pathway leads to tumorigenesis.This study attempts to find new drug targets from the downstream of Smo,specifically targeting Gli3 processing,which provides a new perspective for the treatment of medulloblastoma.It also helps to elucidate the molecular mechanism of Gli3 processing and deepen the understanding of the molecular mechanism regulating Shh signaling pathway.Methods:1.The Cre-GFP virus was used to infect MEF(Ptch1^fl/fl)cell lines,and the GFP-expressing cell lines were screened out by flow cytometry.The MEF(Ptch1^-/-)cell lines were constructed as Smoi sensitive cells lines.The MEF WT cell lines were infected by SmoA1-GFP（Smo mutation）lentivirus,and the successfully infected cell lines were screened by flow cytometry to form MEF（SmoA1）cell lines,which considered to be Smoi resistant cells lines.2.MEF(Ptch1^-/-)and MEF（SmoA1）cell lines were treated with vismodegib（Smoi）or transfected with Gli3R O/E plasmids respectively,and the activities of Shh signaling pathway of the treated cell lines were evaluated by Western Blot and quantitative fluorescence PCR.3.The recombinant plasmid of HA-Gli3FL-GFP was cloned,and the overexpressed Gli3FL protein was labeled with HA at the N-terminal domain and fused with GFP protein at the C-terminal domain.4.HEK293FT cell lines were transfected with the HA-Gli3FL-GFP plasmids to overexpress Gli3FL protein.The changes of Gli3 processing treated with cAMP or FSK were detected by WB.Meanwhile,the changes of GFP were detected by Microplate reader to observe the changes of fluorescence intensity when treated with cAMP or FSK.5.High-throughput drug screening model:the HEK293FT cell lines were transfected with the recombinant plasmids HA-Gli3FL-GFP.After expressing for 24 hours,the transfected cell lines were terated with compounds（10μM）for 24 hours,and the fluorescence intensity was detected by Microplate reader.6.A well-established high throughput drug screening platform was used to screen the compound that promote Gli3 processing based on FDA-approved drug libraries.7.The screened compounds were further tested on MEF(Ptch1^-/-)and MEF（SmoA1）cell lines by quantitative fluorescence PCR for pharmacodynamic tests.Results:1.After being treated with a series of concentrations of vismodegib in MEF(Ptch1^-/-)cell lines,real-time fluorescence quantitative PCR showed that transcription levels of target genes（Gli1,Ptch2）of Shh signaling pathway decreased.And the degree of decrease was dose-dependent,indicating that vismodegib could inhibit the Shh signaling pathway in sensitive strain.However,when MFE（SmoA1）cell lines were treated with vismodegib,no decrease of Gli1 transcription level was detected by real-time fluorescence quantitative PCR.These results indicate that vismodegib can inhibit the Shh signaling pathway in sensitive strains but not resistant strains,which also showed that we have successfully constructed sensitive and resistant strains.2.After overexpression of Gli3R in MEF(Ptch1^-/-)cell lines,the decrease of Gli1 was detected by Western blotting and real-time fluorescence quantitative PCR at protein and transcriptional levels,respectively.In addition,after the overexpression of Gli3R in the MFE（SmoA1）cell lines,the decrease of transcription levels of target genes of Shh signaling pathway（Gli1,Gli2,Ptch2,Sfrp1）was detected by real-time fluorescence quantitative PCR.Therefore,the overexpression of Gli3R can reverse the drug resistance induced by Smo mutation.3.After overexpression of Gli3FL protein in HEK293FT cell lines,Gli3 processing was promoted by cAMP.The expression of Gli3R increased with the increase of drug concentration,and the trend was dose-dependent.These results suggest that Gli3processing can be regulated by compounds.4.Establishment of drug screening system targeting Gli3 processing:After overexpression of Gli3FL protein in HEK293FT cell lines,the ratio of Gli3R/Gli3FL increased after treatment with cAMP/FSK,and the fluorescence intensity of cell lysates decreased at the same time.This result indicated that the process of Gli3 processing was consistent with the change of fluorescence intensity.Considering the cost of drug screening,this model uses the change in fluorescence intensity as the screening index to reflect the extent to which compounds promote Gli3 processing.5.The optimization of drug screening system:selecting the best transfection reagent,cell line,lysis buffer,lysis method,different expression and administration time to improvethe sensitivity,reliability and reproducibility of screening methods.6.Using the optimized high-throughput drug screening system,1700 compounds in the library were screened on a large scale.The results of screening showed that the relative fluorescence intensity of 10 compounds was about 0.5 or less.7.The results of pharmacodynamic tests of the top ten compounds showed that only compound 10^#E3 could inhibit the Shh signaling pathway in both MEF(Ptch1^-/-)and MFF（SmoA1）cell liness.Western blot analysis showed that compound 10^#E3 did increase the ratio of Gli3R/Gli3FL,which meaned promoting Gli3 processing.Conclusions:This study confirmed that the Shh signaling pathways both in MEF(Ptch1^-/-)sensitive cell lines and MEF（SmoA1）resistant cell lines could be inhibited by promoting Gli3processing.Drug development based on this new target was expected to solve the drug resistance problem of the Smo inhibitor vismodegib.Gli3 processing was proved to be controllable in vitro by further studies.According to the previous results,we established a high-throughput drug screening system targeting Gli3 processing:HEK293FT cell lines were transfected with the recombinant plasmid HA-Gli3FL-GFP.After treatment with the tested compound for 24 hours,the fluorescence intensity was detected by Microplate reader.The decrease of fluorescence intensity indicated that the compound could promote Gli3 processing.Based on this model,compound 10^#E3 was finally screened out.This compound significantly inhibited the Shh signaling pathway in both sensitive and resistant strains.The results of western blotting experiments also showed that the compound could promote Gli3 processing.The drug was expected to be used for the treatment of sensitive and drug-resistant medulloblastoma,and to overcome the resistance problem caused by Smoi.