Mechanism Of Deubiquitination Enzyme USP35 Promoting Cell Proliferation And Chemotherapeutic Resistance Through Stabilizing FUCA1 In Colorectal Cancer

Colorectal cancer(CRC)is a malignancy with worldwide significant morbidity and mortality,and its morbidity and mortality are the third most common malignant tumors in men and women.Although CRC progresses slowly and the 5-year survival rate of early-stage CRC patients is as high as 94%,nearly two-thirds of CRC patients in China are in the middle and late stages when first diagnosed,and the 5-year survival rate of patients is less than 10%.For mid to late-stage CRC,surgical resection of localized tumors combined with adjuvant chemotherapy,targeted therapy,immunotherapy,and radiotherapy is required.FOLFOX(Oxaliplatin[OXA],5-Fluorouracil[5-FU],and leucovorin[LV])chemotherapy regimens are still the first-line treatment regimens used in clinical practice,but patients inevitably develop drug resistance after a period of chemotherapy use,leading to treatment failure.Therefore,it is important to explore the mechanisms of CRC development and drug resistance to find more effective targets for CRC diagnosis and treatment,alleviate chemotherapy resistance,and prolong the survival of CRC patients.Ubiquitination is an important type of protein post-translational modification that plays an important regulatory role in protein stability,subcellular localization,and activity.Protein ubiquitination is a dynamic and reversible biological process,catalyzed by the ubiquitin ligase system and can be reversed by deubiquitination of deubiquitinating enzymes.,ubiquitinase and deubiquitinase abnormalities play an important function in tumor development,recurrence and metastasis,and chemotherapy resistance.Ubiquitin-specific-processing proteases 35(USP35),a newly discovered deubiquitinating enzyme,is involved in the regulation of various tumors such as breast,lung,ovarian,and prostate cancers,but the role of USP35 in CRC has not been reported in the literature.In the present study,we investigated the role and mechanism of action of USP35 in CRC for the first time.First,through raw letter analysis and clinical sample detection,we found that USP35 was overexpressed in CRC and was closely associated with the clinicopathological stage of CRC patients and the high recurrence rate of patients receiving postoperative chemotherapy.In vitro and in vivo functional assays showed that high USP35 expression promoted CRC cell proliferation and significantly decreased sensitivity to Oxaliplatin(OXA)and 5-Fluorouracil(5-FU)while silencing USP35 inhibited cell proliferation and increased cell sensitivity to OXA and 5-FU.Then,to investigate the possible mechanism of USP35-induced phenotypes,we applied co-immunoprecipitation(co-IP)combined with mass spectrometry(MS)analysis to screen for proteins that interact with USP35 and found that α-L-fucosidase(α-L-fucosidase 1,FU fucosidase 1(FUCA1)was found to interact with USP35.Further,we confirmed that USP35 could directly bind to FUCA1 and catalyze its deubiquitination to inhibit its proteasomal degradation and up-regulate its intracellular protein level by Proximity Ligation Assay(PLA),Immunofluorescence(IF),and protein stability assay.protein level.Meanwhile,the results of complementation experiments showed that USP35 promoted CRC cell proliferation by upregulating FUCA1,and FUCA1 mediated the regulation of chemotherapeutic drug sensitivity by USP35 in P53 mutant CRC cells.Finally,we conducted a preliminary investigation of the mechanism by which upregulation of FUCA1 by USP35 regulates drug sensitivity and found that USP35-induced upregulation of FUCA1 significantly increased the protein levels of nucleotide excision repair(NER)pathway key factors XPC,XPA,and ERCC1 in CRC cells while Silencing of XPA significantly reversed the drug resistance in CRC cells caused by upregulation of FUCA1 by USP35,suggesting that USP35-FUCA1 may mediate CRC chemoresistance through activation of the NER pathway,but the exact mechanism needs to be further elucidated.In conclusion,our study explored for the first time the role and mechanism of action of USP35 in CRC,which promotes CRC cell proliferation and chemoresistance through deubiquitination and stabilization of FUCA1.The current study not only identified FUCA1,a novel target protein of USP35 but also revealed a new mechanism of USP35 involvement in CRC development and chemoresistance,providing new targets and ideas for CRC diagnosis and treatment.