Study On The Mechanism Of ZEB2/TWIST1/PRMT5/NuRD Complex Promoting EMT And Invasion And Metastasis Of Colorectal Cancer

Colorectal cancer is one of the most common malignant tumors of the digestive system,and its high incidence and metastasis rates make colorectal cancer a terrible threat to human health.The American Cancer Society’s "CA: A Cancer Journal for Clinicians" shows that colorectal cancer is the third most common cancer and the second most deadly cancer worldwide in 2020.In China,colorectal cancer incidence and mortality rates are also increasing,currently ranking the second in incidence and the fourth in mortality for all cancers.Clinical data show that 15-25%of patients have liver metastases at the time of initial diagnosis,while another 15-25%have liver metastases even after radical surgery of the primary site,and the 5-year survival rate of patients with metastatic colorectal cancer is less than 10% after diagnosis.Therefore,it is of great significance to explore the invasive metastasis mechanism of colorectal cancer cells and to develop specific targeted drugs for it,which is very positive for the prognosis of colorectal cancer patients.A large number of studies have shown that the epithelial-mesenchymal transition(EMT)process plays a crucial role in the invasive metastasis of colorectal cancer cells,and the elucidation of the regulatory mechanism of the EMT process has become a key scientific problem in the study of tumor metastasis.A variety of transcriptional repressors,including SNAIL,ZEB and TWIST,are involved in the regulation of the EMT process during tumor metastasis.In addition,a series of reports have demonstrated that transcriptional repressors involved in the EMT process can use different epigenetic mechanisms to regulate the expression of E-cadherin.Currently,the EMT process has been reported in a variety of epithelial-derived tumors,including breast cancer and prostate cancer,but compared with other tumor types such as breast cancer,experimental data on the role of the EMT process in colorectal cancer metastasis are very limited,and the main regulators and related molecular mechanisms are still not fully and deeply studied.In order to explore the transcriptional repressors and related mechanisms that regulate the development of invasive metastasis in colorectal cancer,we first screened for transcriptional factors which inhibit E-cadherin expression in colorectal cancer cells.The results showed that ZEB2 and TWIST1 were significantly overexpressed when TGF-β1 induced the EMT process in colorectal cancer cells.In contrast,the levels of other EMT transcriptional repressors,such as Snail1,Slug and ZEB1,increased less or remained unchanged.Also,the m RNA levels of both ZEB2 and TWIST1 were significantly higher in clinical tumor tissues of colorectal cancer patients compared to paracancerous tissues.In addition,the results of TCGA database analysis also indicated that there was a significant correlation between the expression of ZEB2 and TWIST1 in colorectal cancer patients,suggesting that ZEB2 and TWIST1 may be jointly involved in the invasive metastatic process of colorectal cancer.Next,the expression levels of E-cadherin were significantly suppressed in low metastatic potential colorectal cancer cell line SW480 when either ZEB2 or TWIST1 was overexpressed alone,and the suppression of E-cadherin expression was more pronounced after simultaneous expression of ZEB2 and TWIST1.The upregulation of E-cadherin was more dramatic after co-silencing ZEB2 and TWIST1 in the high metastatic potential colorectal cancer cell line SW620 than when ZEB2 or TWIST1 were silenced alone,indicating that the two can synergistically regulate the EMT process in colorectal cancer cells.Meanwhile,the results of Co-IP and GST-pull down assays indicated a direct interaction between ZEB2 and TWIST1,and the results of dual-luciferase reporter assay and chromatin immunoprecipitation further confirmed that ZEB2 could recruit TWIST1 to the E-cadherin promoter region,and the two synergistically inhibited the transcriptional process of E-cadherin.In addition,we confirmed that ZEB2 and TWIST1 can synergistically promote the migration and invasion ability of colorectal cancer cells in vitro by wound healing assay and transwell invasion assay.Through in vivo xenograft mouse model and mouse survival assay,we found that the combined inhibition of ZEB2 and TWIST1 significantly reduced the chance of metastasis and significantly prolonged the survival of colorectal cancer in mice,indicating that ZEB2 and TWIST1 can synergistically promote the invasive metastatic potential of colorectal cancer cells in vivo.To further analyze whether other cofactors are involved in the regulation of ZEB2/TWIST1 on the invasive metastasis of colorectal cancer cells,other components of the ZEB2/TWIST1 complex were next identified by affinity purification and mass spectrometry experiments.The results showed that ZEB2 and TWIST1 were able to interact with the protein arginine methyltransferase PRMT5 and the nucleosome remodeling complex NuRD protein members HDAC2 and MTA2 to form a novel inhibitory multi-protein complex.Ch IP and dual-luciferase reporter assays also showed that ZEB2 recruits PRMT5 to the promoter region of E-cadhrein,catalyzing the generation of the repressive epigenetic modification H4R3me2,and ultimately triggering transcriptional repression of E-cadherin.Meanwhile,ZEB2 was able to recruit more NuRD complexes to the E-cadherin promoter via TWIST1,removing more acetylation modifications at the H3K56 site and similarly leading to transcriptional repression of E-cadherin.In addition,we demonstrated that inhibition of PRMT5 or HDAC2 significantly reduced the migration and invasive potential of colorectal cancer cells and that treatment of tumor-bearing mice with PRMT5 or HDAC2 inhibitors also greatly prevented distal metastasis of colorectal cancer cells.In summary,this thesis shows for the first time that ZEB2 can form an inhibitory multiprotein complex with TWIST1,PRMT5 and NuRD,and demonstrates that ZEB2 and TWIST1 can catalyze the generation of H4R3me2 modification and the removal of H3K56 ac modification by synergistically recruiting PRMT5 and NuRD complexes to the promoter region of E-cadherin,thereby significantly inhibit the transcriptional level of E-cadherin,which ultimately induces the EMT process and invasive metastasis of colorectal cancer.Therefore,this thesis identifies transcriptional repressor complexes that regulate the development of EMT and invasive metastasis in colorectal cancer cells,reveals the regulatory role of epigenetic mechanisms in the invasive metastasis process of colorectal cancer,and confirms the therapeutic potential of PRMT5 and HDAC2 inhibitors for colorectal cancer,providing new ideas and new targets for clinical diagnosis and new drug development of metastatic colorectal cancer.