| RAS is the first identified human proto-oncogene,and the RAS protein encoded by it is converted between the activated and inactivated states through the guanosine exchange cycle.The guanosine exchange factors(GEFs)promoted the activation process of RAS protein,and GAPs promoted the inactivation process of RAS protein.When Ras is mutated,its internal ATP hydrolase activity is impaired or its binding with GAP is blocked,which leads to its inactivation process being blocked.Then RAS protein is overactivated and continuously activates downstream MAPK,PI3K signaling pathways to lead to carcinogenesis.RAS is mutated in approximately 30%of human solid tumors.The most common mutant subtype,KRAS,exists in 97.7%of pancreatic ductal adenocarcinoma.RAS was known as an "undruggable target" for nearly 40 years,because there were no targeted drugs on the market since discovered.In the past two years,small molecule compounds represented by AMG 510 specifically targeted KRASG12C mutated tumors and successively entered the clinical trials,breaking the impasse of no way out.On May 29,2021,the AMG 510 received FDA approval for marketing,officially ending the"undruggable" history of RAS and marking a landmark breakthrough.SOS1 protein was first discovered in the studies of eye development in Drosophila,and its main function is to promote the activation of RAS and RAC proteins as one of the most important guanosine exchange factors(GEFs).The CDC25 domain of SOS 1 binds to RAS and promotes its transformation from an inactive state bound with GDP to an active state bound with GTP.The REM domain promotes the function of CDC25 domain by reducing steric hindrance by rotating an alpha helix.Rasfonin,an α-pyranone derivative extracted by fermentation from a fungus,has been reported to induce autophagy and apoptosis in RAS-dependent cancer cell lines.However,the development of extraction and chemical synthesis is hindered by the problems of complex steps,high cost and low yield.Until 2014,Professor Che Yongsheng’s team found Doratomyces sp in soil samples from the Qinghai-Tibet Plateau.Rasfonin was extracted by fermentation with hundreds of milligrams in high purity(over 98%),providing material support for systematic pharmacological studies.Previous studies of our laboratory have shown that Rasfonin inhibited the proliferation,invasion and migration in varieties of cancer cells with high incidence of RAS mutation,including G12C.Rasfonin could also inhibit the growth of xenograft tumors constructed from human pancreatic carcinoma Panc-1 cells in nude mice.Further studies of its anti-tumor mechanism have showed that Rasfonin could down-regulate the expression of SOS1 protein,and inhibit the activities of RAS protein,and then block the phosphorylation of the related proteins in MAPK signaling pathway.The inhibition of Rasfonin on the expression of SOS1 may be an important part of its tumor suppressive mechanism.But the relationship between this inhibition and KRAS are still unclear,which is worthy of further study.Based on these backgrounds and previous results,this study intends to investigate the inhibition mechanism of Rasfonin on the expression of SOS 1 protein,and to explore the role of KRAS protein in the regulation of SOS1 expression by Rasfonin.The aims are to explore the anti-tumor mechanism of Rasfonin further,and to provide experimental basis for Rasfonin as a potential drug targeted RAS mutated cancer.1.The effects of Rasfonin on SOS1 expressionIn order to study the effects of Rasfonin on the expression level of SOS1 in various KRAS mutant cancer cell lines,Quantitative real-time PCR was used to determine the expression level of SOS1 mRNA.Western Blot was used to determine the expression level of SOS1 protein.To investigate the effects of Rasfonin and its negative control A321 on the mRNA and protein expression of SOS1,we selected human pancreatic cancer cells Panc-1,human non-small cell lung cancer cells CACU-1,and human bladder epithelial transitional cell carcinoma cells UM-UC-3 as our study objects.And we selected wild-type human breast cancer cells MCF-7 as control.The results showed that the wild-type RAS cancer cell line were insensitive to Rasfonin,while the mutant RAS cancer cell lines were sensitive to Rasfonin.Compared with the control group,Rasfonin incubated for 12 h at 10 t,M dose did not significantly reduce the expression level of SOS1 mRNA in wild-type KRAS breast cancer cell line MCF-7,and only after treatment for 24 h could the protein expression level be down regulated.But within 6 h,in KRASG12C and KRASG12D mutant tumor cell lines,SOS1 mRNA levels were significantly down-regulated,and protein levels were significantly down-regulated within 12 h after treatment.In Panc-1 and Cacu-1 cells,Rasfonin could significantly down-regulate mRNA levels at 3 h and protein levels at 6 h after treatment.The negative control of Rasfonin(A321)had no effect on the expression of SOS 1 protein in various cells.2.Regulation mechanism of Rasfonin on SOS1 expression2.1 Effects of Rasfonin on the SOS1 promoterThrough the study in the previous part,we found that Rasfonin significantly inhibited the mRNA and protein expression level of SOS1,so in this part we will further investigate its inhibition mechanism.Firstly,we focused on the promoter activity of SOS1.293T cells are selected as our research object.The luciferase reporter gene system was used to study it.Luciferase reporter gene system is an effective method to study the interaction between DNA sequence and transcription factor in promoter region of target gene.Usually,purpose gene promoter fragment and luciferase gene are constructed in the same plasmid which are transfected into the tool cells.When transcription factors can combine with the promoter fragment of purpose gene,luciferase gene will express.And then when the luciferase substrate was added after about 48h for expression,the fluorescence which are reflecting the purpose gene promoter activity can be detected.At present,the dual luciferase reporter gene system is wildly used,that is,in addition to the above luciferase gene,another luciferase gene is selected as a statically expressed reference gene to eliminate the error of different transfection efficiency.The luciferase gene of Firefly and Renilla is commonly used.In this study,Firefly luciferase gene and Renilla were used,and Renilla was used as an internal reference gene.2.1.1 Influence of KRAS on SOS1 promoterIn order to investigate the effect of KRAS on SOS 1 promoter’s activity,dual luciferase reporter assay was used to detect SOS1 promoter activity.293T cells were transfected with SOS 1-Luc plasmid(constructed from the-2000bp to+38bp fragment of SOS1 promoter and firefly luciferase gene),KRAS plasmid(negative control NC,wild-type WT,mutant G12C,mutant G12D)and internal reference Renilla plasmid.The transfection ratio was 25:25:1.24 h later,luciferase substrate was added,and the fluorescence value was measured.We found that compared with KRAS-NC group,KRASWT and KRASG12D significantly increased SOS1 promoter activity,and KRASG12C tended to increase SOS1 promoter activity.2.1.2 Effect of Rasfonin on SOS1 promoterIn order to further investigate the effect of Rasfonin on SOS1 promoter activity,dual luciferase reporter assay was used.Compared with model group,Rasfonin at 10 μM dose treated for 12 h had no significant effect on SOS1 promoter activity in SOS1 mono-expression group.However,it significantly down-regulated SOS1 promoter activity in the co-expression group of KRAS and SOS1,suggesting that Rasfonin may rely on KRAS to inhibit SOS1 promoter activity.2.2 Correlationship between the inhibition of Rasfonin on SOS1 expression and KRASThese results suggest that Rasfonin may rely on KRAS to inhibit SOS1 promoter activity.To further determine whether the inhibition of Rasfonin on SOS1 expression is KRAS dependent,we transfected 293T cells with SOS1 and KRAS overexpressing plasmids to construct their mono-expression and co-expression systems,excluding the interference of many other factors.The role of KRAS protein in the inhibition of SOS1 by Rasfonin was studied in a relatively simple environment.2.2.1 Influence of overexpressing KRAS on SOS1 expressionEGFR has been reported to regulate the expression of SOS1 protein,EGFR is located in the upstream of SOS1 signaling pathway,so whether the downstream KRAS protein has a regulatory effect on SOS 1 expression is not clear,which is worthy of further study.Therefore,we first constructed the co-expression system of SOS1 and KRAS by plasmid transfection.In our experiment,it was found that the time periods for the high expression of KRAS and SOS1 proteins to reach and maintain high expression levels were different.After the simultaneous transfection of KRAS and SOS1 plasmids in 293T cells at 24 h,48 h,72 h and 96 h,no time points for the high expression of both proteins were detected.It may also be that the large SOS1 plasmid is much larger than KRAS plasmid,and the two plasmids compete during cotransfection,making it difficult for the large SOS 1 plasmid to be transfected into 293T cells.Therefore,the strategy of transfecting two plasmids was adopted in this study.The amount of protein expressed in the first place can be in a stable state during the expression of protein expressed in the second place.Therefore,groups with different expression sequences can be used as controls.Compared with the group transfected with SOS1 alone,the expression level of SOS 1 protein was significantly up-regulated in the group co-transfected with KRASWT or KRASG12D before transfection,and the expression level of SOS 1 protein was up-regulated in the group co-transfected with KRASG12C.However,these changes were not observed in the first transfection of SOS1 plasmid.When 293T cells first expressed KRAS,the SOS1 co-expression group was significantly upregulated compared with the mono-expression group,while there was no significant change when the SOS1 was first expressed,suggesting that KRAS could positively regulate the expression of SOS1 when its own expression level was stable.When KRAS plasmid was transfected first,the expression of KRAS protein in SOS1 and KRAS co-transfected group was almost unchanged compared with KRAS alone transfected group.However,when the SOS1 plasmid was transfected first,there was no significant change in the expression level of KRAS in the group co-transfected with SOS1 and KRASWT compared with the group transfected with KRASWT alone.Compared with the group transfected with KRASG12C alone,the expression level of KRAS was significantly up-regulated in the group co-transfected with SOS1 and KRASG12C,Compared with the group transfected with KRASG12D alone,the expression level of KRAS in the group transfected with SOS1 and KRASG12D together tended to be up-regulated.When 293T cells first expressed SOS1,KRAS co-expression group was significantly upregulated compared with the single expression group,while no significant change was found when KRAS was first expressed,suggesting that SOS1 could positively regulate the expression of KRAS when its own expression was stable.2.2.2 Effects of KRAS in the inhibition of Rasfonin on SOS1 expressionTo further clarify the role of KRAS in the downregulation of SOS1 expression by Rasfonin,293T cells were transfected with KRAS and SOS1 plasmids to construct a co-expression system of KRAS and SOS1.Previous laboratory studies have found that Rasfonin could inhibit the expression level of SOS 1 protein but had no significant effect on the expression of RAS protein,so the model constructed by first transfection of KRAS which has more obvious effect on the expression of SOS 1 was chosen for the study.The negative control of Rasfonin,A321,was selected as the negative control.AMG 510,the first specifically targeted drug for KRASG12C which was marketed on May 29,2021,was used as the control drug in the KRASG12C mutation group,and no control drug was used in the KRASG12D mutation group due to the lack of targeting drug.The results showed that,compared with the model group,Rasfonin treated for 12 h at 10 μM dose tended to down-regulate the expression level of SOS1 protein in the single SOS1 group,and significantly down-regulate the expression level of SOS1 protein in the mutant KRAS(G12C,G12D)and co-expression group.A321 had no significant effect on SOS1 protein expression.It suggested that KRAS might be involved in and promote the inhibition of SOS1 expression by Rasfonin.2.3 Effects of KRAS on SOS1 expressionThese results suggest that Rasfonin may inhibit the expression level of SOS1 through KRAS,suggesting that KRAS may have a regulatory effect on the expression of SOS 1.To further confirm the existence of this regulatory effect,we use RNAi technology to knockdown KRAS to study its influence on SOS1 expression and used GEPIA website to analyze the TCGA database.RNA interference(RNAi)can effectively silence the expression of target genes by introducing double-stranded RNA into cells to specifically degrade the mRNA of target genes and inhibit their protein expression.The double stranded RNAs commonly used include small interfering RNAs(siRNAs)and short hairpin RNAs(shRNA).The TCGA database is called The Cancer Genome Atlas.It is a collaborated research projects of cancer established by the National Cancer Institute(NCI)and the National Human Genome Research Institute(NHGRI)in 2006.TCGA provides a free,all-round,multi-dimensional and huge cancer database through collecting genome,transcriptome and epigenetic,protein group and other omics data from more than ten thousand of patients with more than 50 different types of cancer.Nearly 10 million genetic mutations have been identifiedGene Expression Profiling Interactive Analysis(GEPIA)platform is a simple and visual big data analysis website for cancer,which is designed by Tang Zefang and his companies come from Professor Zhang Zemin of Beijing University for researchers who do not have bioinformatics background.The platform provides interactive analysis such as pathological staging,tumor differential expression profile analysis,similar gene detection analysis and patient survival analysis.It provides convenience for cancer researchers that numbers of cancer patients data can be obtained by simple operation here.2.3.1 The influence of knocking down KRAS on SOS1 expressionTo study the regulation effects of KRAS on SOS1 expression,specifically shRNA for KRAS were used to knock down KRAS protein.A variety of KRAS mutant cancer cell lines including human pancreatic cancer cells Panc-1,human lung adenocarcinoma cells Cacu-1,human bladder cancer epithelial transitional cell carcinoma cells UM-UC-3 were selected as the research objects.Human breast cancer cells MCF-7 were used as control.The results showed that the expression of SOS1 was significantly reduced by knocking down KRAS in various KRAS mutant tumor cell lines,and the expression of KRAS was also significantly reduced by knocking down SOS1.These results suggest that SOS1 and KRAS can positively regulate the expression level of each other.2.3.2 The correlationship between the expression level of KRAS and SOS1 in cancer patientsTo further confirm the relationship between the expression levels of the two proteins,GEPIA website was used to analyze the TCGA database,and the three human cancers with the highest incidence of KRAS mutations were selected as the research objects.Transcriptome data from patients with pancreatic cancer,lung adenocarcinoma and colorectal cancer were analyzed,and the results showed that KRAS and SOS1 expression levels are positively correlated with each other.Based on the results of this study,we draw the following conclusions:1.There are mutual regulation effects between KRAS protein(wild type and mutant)and SOS 1 protein.KRAS protein can up-regulate the promoter activity of SOS 1.2.Rasfonin can inhibite the mRNA and protein expression of SOS1 in mutated KRASG12C and KRASG12D cancer cell lines.3.Rasfonin inhibited the activity of SOS1 promoter by mutant KRAS(G12C,G12D)protein and then reduce the expression of SOS1 protein.In conclusion,this study explored the anti-tumor mechanism of Rasfonin further.We found that one of its anti-tumor mechanisms might be that Rasfonin inhibit the activity of SOS1 promoter through KRAS,and down-regulate the expression levels of SOS1 mRNA and protein,then the RAS activation and downstream MAPK signaling pathway are inhibited to suppress tumor.Our findings enhance our understanding of the anti-tumor mechanism of Rasfonin and the regulation of SOS1 expression,which may provide new ideas for the treatment and disease prevention of RAS mutated cancer in the future. |
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