Mechanism And Role Of SUMO1 Targeted Small Molecular Degrader In Inhibiting The Progression Of Colorectal Cancer

Colorectal cancer(CRC)is one of the most common and deadliest cancers,with an increasing incidence in China and a trend of younger age of onset.The current treatment strategies for CRC are associated with low efficacy,significant toxicity,and dependence on specific gene mutation status.There is an urgent need in clinical practice to study the mechanisms underlying the progression of colorectal cancer and develop targeted small molecule drugs.SUMOylation,a post-translational modification process,is involved in the regulation of tumor signaling pathways,tumor stem cell renewal,and tumorigenesis.Small ubiquitin-like modifier 1(SUMO1)is a potential therapeutic target.By utilizing intracellular protein degradation systems,tumor-associated proteins can be targeted for drug intervention.Our study has discovered that a small molecule degrader of SUMO1(SMDS1)can induce the interaction between cytoplasmic activation/proliferation-associated protein 1(CAPRIN1)and F-box only protein 42(FBXO42),recruit SUMO1 to the E3 ligase complex,and facilitate the degradation of SUMO1.This finding provides a new option for the treatment of colorectal cancer.In order to explore the cellular changes induced by targeted degradation of SUMO1,we initially searched the Gene expression omnibus(GEO)public database and identified the GSE163884 dataset.The researchers uploaded this dataset used CRISPR/Cas9 technology to knock out the expression of SUMO1 and SUMO2 in human osteosarcoma U2 OS cells,respectively.They then analyzed the transcriptome of the SUMO1 and SUMO2 knockout cell lines using RNA-seq.Next,we selected the control group and the SUMO1 knockout group samples from the dataset and performed weighted gene co-expression network analysis(WGCNA),gene ontology(GO)functional enrichment,Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment,and gene set enrichment analysis(GSEA)on the differentially expressed genes that were identified.Through WGCNA analysis,we identified two co-expression modules that were closely associated with SUMO1 knockout.GO/KEGG enrichment analysis of the genes within these modules revealed a potential association between SUMO1 knockout and mitochondrial respiratory chain,oxidative phosphorylation pathways.Further GSEA analysis also indicated an upregulation of oxidative phosphorylation and respiratory chain electron transport pathways,as well as suppressed Wnt/ β-catenin pathway in the SUMO1 knockout samples.These findings suggest that SUMO1 knockout may affect mitochondrial respiratory function,with the Wnt/β-catenin pathway potentially playing a role.This provides us with a reference for subsequent experimental design and suggests that cellular metabolism indicators could be a research entry point.In terms of phenotype studies,we have demonstrated that SMDS1 exhibits potent tumor-inhibitory effects in colorectal cancer cell lines,mouse xenograft models,and patient-derived xenografts(PDX)models.Building upon the previous bioinformatics analysis,we were particularly interested in glucose-6-phosphate dehydrogenase(G6PD),a key enzyme in the pentose phosphate pathway(PPP),which emerged from the genome-wide CRISPR/Cas9 knockout screening results.Subsequent experiments revealed that SMDS1 significantly inhibits G6 PD enzyme activity without reducing G6 PD protein levels,leading to a significant decrease in the levels of the PPP’s main product,reduced form of nicotinamide adenine dinucleotide phosphate(NADPH).NADPH supply is crucial for maintaining normal lipid synthesis.Correspondingly,we observed that SMDS1 inhibits lipid synthesis in CRC cells.Furthermore,investigations showed that this small molecule leads to an accumulation of reactive oxygen species(ROS)in CRC cells,disrupting oxidative stress homeostasis and inducing endoplasmic reticulum stress.Additionally,through measurements of Caspase 8 activity and Annexin V signaling,combined with immunohistochemistry staining of the xenografts,we confirmed that SMDS1 does not induce apoptosis in CRC cells.In the GSEA analysis of differentially expressed genes from the GSE163884 dataset,we found that SUMO1 knockout resulted in downregulation of pathways related to DNA double-strand break repair,base excision repair,DNA replication,and cell cycle.Immunoblotting experiments also demonstrated that SMDS1 reduces the protein levels of cyclin-dependent kinases 4/6(CDK4/6),key regulators of the cell cycle.Therefore,we hypothesize that SMDS1 exerts its tumor growth-inhibitory effects by inducing cell cycle arrest.We further investigated the mechanisms underlying the phenotypic changes caused by SMDS1.Among the top hits from the genome-wide CRISPR/Cas9 knockout screen of SMDS1,steroidogenic acute regulatory protein(St AR)-related lipid transfer domain containing 7(StarD7)emerged as a key player in SMDS1 function.We validated that SMDS1 indeed reduces the protein levels of StarD7 in CRC cell lines,3D organoids,and PDX models.Furthermore,we demonstrated that StarD7 is closely associated with tumor proliferation.Compared to normal tissues,CRC exhibits an overexpression of StarD7.Knocking out StarD7 using CRISPR/Cas9-mediated gene editing significantly inhibited the growth of HCT116 cell line.The tumor formation rate was also significantly reduced in subcutaneous xenograft models established using StarD7 knockout cell lines,leading to prolonged survival in mice.Additionally,we observed that the knockout of StarD7 gene conferred resistance to the tumor growth inhibitory effect of SMDS1.In StarD7 knockout cell lines,the small molecule SMDS1 exhibited weakened inhibitory effects on G6 PD activity and significantly reduced its role in NADPH and lipid synthesis.Moreover,SMDS1 failed to induce oxidative stress and endoplasmic reticulum stress in StarD7 knockout cell lines.These findings indicate that the action of this small molecule is dependent on StarD7.The regulation of StarD7 by SMDS1 is dependent on SUMO1 degradation.We found that knocking out SUMO1,similar to SMDS1 treatment regarding cell behavior,leads to a decrease in StarD7 protein levels.However,on the basis of SUMO1 knockout,the small molecule SMDS1 is unable to further reduce StarD7 expression and induce oxidative stress nor endoplasmic reticulum stress.Through chromatin immunoprecipitation(ChIP)combined with polymerase chain reaction(PCR),we have discovered that the transcription factor T cell factor 4(TCF4)can bind to the promoter sequence of StarD7.Luciferase reporter gene experiments have shown that the small molecule SMDS1 greatly inhibits the transcriptional activity of TCF4.SMDS1 targets SUMO1 for degradation,leading to a corresponding decrease in SUMOylation of TCF4 by SUMO1.This directs TCF4 to the proteasome pathway for degradation,thereby inhibiting its transcriptional activity.This ultimately results in a decrease in the transcription level of the StarD7 gene,leading to a decrease in the protein level of StarD7.We observed that SMDS1 treatment significantly increased the ADP/ATP ratio in CRC cells,indicating that SMDS1 induces cellular energy stress.AMP-activated protein kinase(AMPK),as a "sensor" of intracellular energy stress,activates its catalytic α subunit,which inhibits G6 PD activity.The inhibition of G6 PD activity by SMDS1 can be reversed by using an AMPK inhibitor,but it does not alleviate the decrease in StarD7 protein caused by SMDS1.Knockout of StarD7 can alleviate the energy stress induced by SMDS1.We also demonstrated that the decrease in G6 PD activity is due to the inhibition of its active dimer formation by SMDS1.The mechanism underlying the phenotype of colorectal cancer caused by SMDS1 can be summarized as follows: 1.SMDS1 binds to CAPRIN1,promoting ubiquitination and degradation of SUMO1;2.Decreased levels of SUMO1 result in weakened SUMOylation of TCF4,leading to increased proteasomal degradation of TCF4 and inhibition of its transcriptional activity;3.Inhibition of TCF4 transcriptional activity leads to decreased transcription levels of the StarD7 gene,resulting in reduced levels of StarD7 protein;4.Decreased levels of StarD7 protein trigger endoplasmic reticulum stress and excessive accumulation of reactive oxygen species(ROS);5.SMDS1 induces cellular energy stress by reducing StarD7 protein levels and activates AMPK,which inhibits the activity of G6 PD,leading to decreased intracellular levels of NADPH and inhibition of fatty acid synthesis metabolism;6.The reduction of NADPH further exacerbates oxidative stress,causing cell cycle arrest and inhibition of colorectal cancer cell proliferation.