| Gastric cancer is a deadly disease with poor overall survival statistics throughout the world,which is a serious threat to human health.At present,gastric cancer is the fifth leading cancer type and has been identified as one of the main causes of cancer-related deaths worldwide.It is the end result of a complex interplay between host genetics,environmental factors(smoking,alcohol,high salt),and microbial factors(Helicobacter pylori infection).These indicate that in-depth study of the molecular mechanisms of the tumorigenesis and development of gastric cancer is of great significance for the clinical improvement and prognosis of gastric cancer.Ubiquitination regulates numerous cellular functions,including proteasomal degradation,membrane trafficking,DNA repair,and signal transduction.As a newly discovered ubiquitin linkage,linear ubiquitin chain is in a dynamic equ ilibrium that is catalyzed by the linear ubiquitin chain assembly complex(LUBAC)and hydrolyzed by the specific deubiquitination enzyme OTULIN.LUBAC complex composed of the HOIP(HOIL1-interacting protein),SHARPIN(SHANK associated RH domain-interacting protein)and HOIL1L(haem-oxidized IRP2 ubiquitin ligase 1L),is the sole E3 that generates the N-terminal M1-linked linear polyubiquitin chain.HOIP is the enzymatic core of the LUBAC complex,and SHARPIN and HOIL1 play a key role in the activation of HOIP.Studies have shown that linear ubiquitination can occur in several substrates such as NEMO,TNFR1,and RIPK1,and thereby regulating signal pathways such as NF-κB and Wnt/β-catenin,which plays important roles in innate immunity,inflammation and angiogenesis.However,the effect of linear ubiquitination on the biological behavior of gastric cancer in tumorigenesis and development is still unclear.Therefore,it is intriguing to explore the regulatory mechanism of linear ubiquitination on the biological behavior of gastric cancer,which has important theoretical significance and potential clinical value.Therefore,our article is mainly divided into two parts,respectively studying the effects and mechanism of linear ubiquitination-related proteins OTULIN and SHARPIN on the malignant biological behavior of gastric cancer.The main results and conclusions of this research are as follows:Section 1.Effect and mechanism of the deubiquitination enzyme OTULIN on the biological behavior of gastric cancer1.OTULIN is highly expressed in gastric cancer tissues(1)The abundance of OTULIN was increased in gastric cancer in different public databases.(2)The expression of OTULIN in gastric cancer tissues was significantly higher than that in corresponding para-cancer tissues.2.Effect of deubiquitinating enzyme OTULIN on the proliferation,migration and invasion of gastric cancer cells(1)OTULIN promoted the proliferation and tumorigenesis of gastric cancer cells.(2)OTULIN promoted the invasion and migration of gastric cancer cells.(3)The role of OTULIN in promoting migration and invasion partly depended on its deubiquitinase activity.3.Identification of RACK1 as a new linear ubiquitination substrate(1)Pull-down assay combined with mass spectrometry analysis,screen out the possible substrate protein RACK1.(2)Immunoprecipitation proved that RACK1 protein was modified by linear ubiquitination.4.OTULIN interact with RACK1 and formed a complex with the adhesion spot kinase FAK(1)Immunofluorescence experiments confirmed the co-localization of OTULIN and RACK1.(2)Immunoprecipitation proved that OTULIN,RACK1 and FAK formed complexes.5.OTULIN regulated the recruitment of FAK by RACK1 and affected the migration movement of gastric cancer cells(1)Knockout OTULIN inhibit the phosphorylation of FAK.(2)OTLUIN could promote RACK1 to recruit FAK.(3)Knockout OTULIN decreased the adhesion ability of gastric cancer cells.Section 2.Effect and mechanism of SHARPIN on the biological behavior of gastric cancer1.SHARPIN promotes β-catenin expression and enhances its signaling activity in a linear ubiquitination-independent manner(1)LUBAC and OTULIN were involved in the regulation of Wnt/ β-catenin signaling pathway.(2)SHARPIN interacts with β-catenin to regulate the Wnt/β-catenin signaling pathway.2.SHARPIN promoted malignant biological behavior of gastric cancer(1)SHARPIN promoted proliferation,clonal formation,migration and invasion of gastric cancer cells.(2)SHARPIN promoted subcutaneous tumorigenic ability of gastric cancer cells.3.SHARPIN decreases β-catenin ubiquitination levels in part by competing with the E3 ligase β-Trcp1 for β-catenin binding(1)SHARPIN reduced the ubiquitination levels of β-catenin.(2)SHARPIN competed with β-Trcp1 for binding β-catenin.(3)SHARPIN competed with β-Trcp1 for β-catenin binding partly by occupying similar residues of β-catenin.4.The SHARPIN-β-catenin signaling axis was essential for tumorigenicity and invasiveness of GC cells(1)Ectopic expression of β-catenin rescued cell proliferation,and colony formation,and anchorage-independent colony formation capacity of SHARPIN-depleted gastric cancer cells.(2)Reintroducing β-catenin rescued tumor growth in mice subcutaneously injected with SHARPIN-depleted cells.5.SHARPIN expression is correlated with activation of β-catenin signaling in cancer tissues and associated with disease progression of the GC patients.(1)SHARPIN expression in gastric cancer tissues was significantly higher than that in para-cancer tissues.(2)High expression of SHARPIN in gastric cancer tissue was correlated with shorter overall survival time.(3)The expression of SHARPIN was positively correlated with β-catenin expression in gastric cancer tissues.In conclusion,the above data suggested that1.The deubiquitinating enzyme OTULIN can promote the interaction between RACK1 and FAK by removing the linear ubiquitination modification of RACK1,thereby promoting the proliferation and metastasis of gastric cancer.2.Although SHARPIN is a key component of LUBAC,it functions as a regulator of Wnt/β-catenin signaling,contributing to gastric tumorigenesis independent of linear ubiquitination.Here,we found that SHARPIN interact with and stabilized β-catenin by competing with the E3 ubiquitin ligase β-Trcp1 for β-catenin binding,thereby promotingβ-catenin signaling to fuel the development of GC.Our study reported for the first time the effect of linear ubiquitin-related proteins OTULIN and SHARPIN on the malignant biological behavior of gastric cancer.We have discovered a new substrate protein RACK1 modified by linear ubiquitination.Our research preliminarily revealed the molecular mechanism by which the deubiquitinating enzyme OTULIN affected the proliferation and metastasis of gastric cancer cells by regulating RACK1,providing a new basis for studying the role of linear ubiquitination in tumors.In addition,we also found that SHARPIN regulated the activity of Wnt/β-catenin signaling pathway in a way that independent of linear ubiquitination,thereb y promoting the tumorigenesis and development of gastric cancer.These studies provide new research directions for exploring the molecular mechanism of gastric cancer,and may become potential strategies to improve the treatment and prognosis of gastric cancer patients. |
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