The Regulation And Mechanism Of E3 Ubiquitin Ligase NEDD4L On Stemness In Hepatocellular Carcinoma

Background&AimsThe mortality rate of hepatocellular carcinoma（HCC）is in the second place among China’s malignant tumor and the survival rate of liver cancer for five years is only 12.1%.There is no definite single carcinogen mutation in liver cancer and it is one of the most heterogeneous tumors,which can cause patients with liver cancer to suffer from routine treatment quite differently and the clinical treatment is not good.Liver cancer stem cells（LCSCs）are not only one of the factors that are involved in the heterogeneous of liver cancer,but also the main reason for drug resistance and the recurrence of liver cancer.The theory of tumor stem cells points out the important direction of the tumor treatment,which is not only the frontiers and difficulties of tumor research,but also the research trend and future of liver cancer treatment.In the past,the study of tumor stem cells in LCSCs is limited to the search for genetic markers and source of tumor stem cells,and little is known about how LCSCs regulate the development of tumor.Therefore,it is of significance to research the regulation mechanism of LCSCs and develop combination treatment to improve clinical treatment effect,which is very important to clinical diagnose and drug development in liver cancer.NEDD4L（Neural precursor cell expressed,developmentally 4-like）is a member of NEDD4 E3 ubiquitin ligase family,and the early studies focused on the role of NEDD4L in the pathogenesis of sodium ion pathways in the renal small tube epithelial cells and hypertension.The recent studies have found that NEDD4L also has an important regulatory role in inflammation and tumor.NEDD4L has various expressions in different tumors and promotes or inhibits tumor cell proliferation,apoptosis,metastasis and chemotherapy resistance in tumors such as colorectal cancer,melanoma,lung cancer and pancreatic cancer by regulating the signal pathways of WNT/β-catenin,MAPK/ERK,and other signal molecules such as STK35 and TGF-β.The research shows that NEDD4 and NEDD4L can target the degradation of the LGR5 receptor and inhibit WNT signaling pathway,which inhibit the stemness of colorectal cancer and slow the proliferation of colorectal cancer cells.TRIB3 can inhibit the degradation of FOXO1 through NEDD4L,promote the transcription of SOX2,which promote the maintenance of stemness in breast cancer cells.Thess two studies demonstrate that NEDD4L can be involved in regulating the characteristics of tumor stem cells.However,it has not yet been found that NEDD4L plays the role in the regulation of LCSCs.We previously found that NEDD4L promoted antiviral immunity through K29-linked cysteine ubiquitination of TRAF3,enhanced the production of IFN and cytokines in macrophages induced by TLR and RLR,and positively regulated the antiviral innate immunity（Nature Communications,2021;12:1194）.In the later study,we further studied whether NEDD4L was involved in the regulation of LCSCs and tumor immunity.In the present study,we intend to verify the regulatory role of NEDD4L on the stemness of HCC both in vitro and in vivo and we will clarify the molecular mechanism of NEDD4L in regulating HCC stemness.This study is expected to provide a new theoretical basis for the regulation mechanism of LCSCs and provide new targets for the diagnosis and treatment of HCC.Methods1.The expression of NEDD4L,the correlation with the prognosis of patients,and with HCC stemness score were determined by bioinformatics and immunohistochemistry according to the analysis of database and clinical cohort.2.The regulatory role of NEDD4L in HCC stemness was determined by knocking down and over-expressing NEDD4L in HCC cells.The effects of NEDD4L on HCC sphere formation efficiency,stemness,cell viability,and drug resistance were determined by sphere formation,real-time quantitative PCR,western blot,CCK-8,clone formation,and flow cytometry analyses.3.To investigate the role of NEDD4L during HCC development in vivo,we administered chemical carcinogen DEN plus CCl₄ to NEDD4L-deficient mice and utilized xenograft transplantation mouse model by real-time quantitative PCR and western blot.4.SHP2 was determined as the interaction protein of NEDD4L through mass spectrometry analysis,as well as immunoprecipitation,western blot and GST-pulldown analyses.5.The effects of NEDD4L on the expression and ubiquitination of SHP2 were determined by real-time quantitative PCR,western blot,immunohistochemistry and immunoprecipitation analyses.6.The effects of NEDD4L on signaling pathway dependent on SHP2 were determined by western blot,flow cytometry,ELISA and immunohistochemistry analyses.7.SHP2 inhibitor SHP099 was used to detect whether the regulation of NEDD4L in HCC stemness depends on SHP2 by real-time quantitative PCR,western blot,CCK-8,clone formation,and flow cytometry analyses.Results1.NEDD4L was highly expressed in HCC patients and negatively correlated with the prognosis of the patients.In addition,NEDD4L was positively correlated with HCC stemness score.2.NEDD4L promoted HCC spheroid formation capacity,stemness gene expression,cell proliferation,and drug resistance.3.NEDD4L directly combined with SHP2 and the combination site was the WW domain of NEDD4L and the PTP domain of SHP2.4.NEDD4L inhibited the combination of TRIM52 and SHP2,preventing the degradation of k48-linked SHP2 ubiquitination by TRIM52,resulting in the enrichment of SHP2,which stabilized and promoted the expression of SHP2.5.The K244,K358 and T357 residues were the ubiquitination sites to which NEDD4L-catalyzed k63-linked ubiquitin chains were attached.6.NEDD4L promoted the signaling pathway dependent on SHP2 in HCC,including the activation of ERK and AKT.What’s more,NEDD4L promoted the expression of PD-L1 in HCC cells,suppressed the secretion of the related anti-tumor cytokines,and inhibited the infiltration of antitumor immune cells,and therefore promoted the progress of tumor immune evasion.7.The inhibition of SHP2 restricted the influence of HCC sphere formation efficiency,expression of stemness markers,drug resistance,cell malignancy,signal activation of NEDD4L,demonstrating that the regulation of NEDD4L on HCC stemness was dependent on SHP2.ConclusionsOur data identified NEDD4L functions as a previously unknown modulator of K63-linked ubiquitination of SHP2,and it upregulates SHP2 protein expression thus augmenting HCC stemness and immune evasion.The present study will provide a new theoretical basis for the regulation mechanism of LCSCs and provide new targets for the diagnosis and treatment of HCC.