The Role Of Ubiquitination Modification And Degradation Regulation Axis Of The Cancer Driving Factors(SIX1 And RPS16)in The Treatment Of Hepatocellular Carcinoma

Background & Aims:Hepatocellular carcinoma(HCC)remains challenging due to the lack of efficient therapy.Promoting degradation of certain cancer drivers has become an innovative therapy.The nuclear transcription factor sine oculis homeobox 1(sine oculis homeobox1,SIX1)is a key driver for the progression of HCC.In previous studies,we focused on the degradation mechanism of SIX1 and identified ubiquitin-specific peptidase 1(USP1)as a deubiquitinase of SIX1 that promotes cancer cell proliferation by maintaining high expression levels of SIX1 in prostate cancer cells,and targeted the USP1-SIX1 axis to identify a degradation inducer of SIX1: SNS-032.Not only that,we also found that ribosomal protein S16(RPS16)is an important substrate of USP1 in HCC.Analogously,we found that USP1 maintains its protein stability and promotes the invasion and metastasis of HCC.However,whether USP1 is still the dominant factor in inhibiting SIX1 ubiquitination and degradation in HCC remains unknown.The inhibitory effect and mechanism of SNS-032 on HCC need to be clarified.The upstream regulation path of the USP1-SIX1\ RPS16 axis is unknown.Through this study,we will clarify the degradation regulation mechanism of SIX1 in HCC,and clarify the inhibitory effect and mechanism of SNS-032 on HCC.Methods:Co-immunoprecipitation assay was used to detect ubiquitination levels and protein interactions,western blotting was used to detect changes in protein levels,and immunofluorescence was used to verify the changes of interacting proteins and protein levels.Cell viability detection,Ed U proliferation detection,colony formation assays were used to comprehensively evaluate cell proliferation ability.Flow cytometry was used to detect cell cycle distribution and apoptosis.Trans-well was used to assess cell invasion and metastasis;In vivo tumor models were constructed using nude mouse subcutaneous transplant tumors to evaluate anticancer effects in vivo.Results:Here,we detected the ubiquitination level of SIX1 in clinical HCC tissues and analyzed TCGA and GEPIA databases.We gradually found that USP1 is a main reason for SIX1 to maintain low ubiquitination and high protein levels in HCC tissues,which is related to the malignant progression of cancer.We demonstrated the saturated binding of USP1 to SIX1 and RPS16,suggesting that targeting the USP1-SIX1\ RPS16 axis may be an efficient strategy for inhibiting HCC.Further study found that the USP1-SIX1\ RPS16 axis is regulated by the EGFR-AKT signaling pathway.Besides,we demonstrated that SNS-032 can inactivate the EGFR-AKT-USP1 axis and induce the degradation of SIX1 and RPS16 in HCC,leading to cell cycle arrest,inhibition of cell proliferation and metastasis,and triggering apoptosis.Further,reactivation of the EGFR-AKT pathway or overexpression of USP1 reversed SNS-023-induced degradation of SIX1 and RPS16.In addition,in vitro and in vivo models have confirmed that sorafenib combined with SNS-032 or EGFR pathway inhibitor gefitinib can synergistically inhibit liver cancer.Conclusions & Significance:The decreased level of SIX1 ubiquitination in HCC is related to the high expression status of USP1;USP1 interacts with SIX1 and RPS16 through the Cterminal;EGFR signaling pathway promotes the expression of USP1 and its substrates;SNS-032 induces ubiquitination and degradation of SIX1 and RPS16 by inhibiting the EGFR-AKT-USP1 axis;SNS-032 inhibits HCC cell proliferation and invasion metastasis,triggers apoptosis,and enhances the sensitivity of HCC to sorafenib therapy.In summary,starting from exploring the molecular mechanism of SIX1 ubiquitination in HCC,combined with previous studies,we elaborated on the mechanism of the EGFR-AKT-USP1-SIX1\ RPS16 regulatory axis,and further targeted the regulatory axis to provide a potential strategy for the treatment of HCC.