The Molecular Mechanism Of Protein Deubiquitinase USP9X In Breast Carcinogenesis

Objective:Ubiquitination is among the most widely used protein modifications involved in regulating diverse cellular signaling and homeostasis.Ubiquitination is constantly opposed by deubiquitination,exemplified by the existence of a large family of deubiquitinating enzymes(DUBs).The human genome encodes approximately 95 putative DUBs.Among these DUBs,the ubiquitin specific peptidase 9,X-linked(USP9X,also known as FAM for Drosophila fat facets in mouse),has been reported to regulate multiple cellular activities including protein trafficking/endocytosis,polarity,apoptosis and death,neurogenesis,and autophagy.In addition,USP9 X has been implicated in several pathological states including X-linked intellectual disability,seizures,and various types of malignancies.However,mechanistic insights into the role of USP9 X in cancer development and progression remain to be investigated.Similar to the chromosomes,the centrosome is precisely reproduced once and only once during each cell cycle.However,it remains unclear how this protein-based structure undergoes accurate duplication in a semiconservative manner.Acquisition of more than two centrosomes(centrosome amplification),severely disturbs mitotic process and cytokinesis via formation of more than two spindle poles,resulting in an increased frequency of chromosome segregation errors(chromosome instability).Destabilization of chromosomes by centrosome amplification aids gain of further malignant phenotypes,hence promoting tumor development/progression.Given the serious consequences of centrosome amplification,it is clearly important to understand how centrosome biogenesis/duplication is regulated and centrosome amplification is introduced.In this study,we employed multiple experimental methods to investigate whether/how dysregulation of USP9 X is linked to centrosome amplification and breast carcinogenesis.Results:A.USP9 X is physically associated with centrosome satellite protein CEP131.B.USP9 X was co-localized with the centrosomal markers Centrin,g-tubulin,PCM1 and CEP131,and the centrosomal localization of USP9 X is dependent on CEP131.C.USP9 X controls the stabilization of CEP131 and USP9 X depletion-associated loss of CEP131 from centrosome is not a consequence of a general loss of satellite proteins..D.USP9 X deubiquitinates CEP131 and K48-linked ubiquitin species are major forms of linkages opposed by USP9 X.E.USP9X-promoted centrosome biogenesis requires CEP131-regulated CDK2 localization and overexpression of USP9 X promotes centrosome amplification and mitotic defects in a CEP131-dependent manner.F.USP9 X and CEP131 are highly expressed in breast carcinoma samples,and the levels of their expression were strongly correlated with each other and with the progression of breast cancer.G.USP9 X is required for breast cancer cell survival in vitro and promotes breast carcinogenesis in vivo,and USP9 X promotes breast carcinogenesis through stabilizing CEP131.H.We also identified several other substrates of USP9 X with SILAC-based ubiquitome analysis.Conclusion:We demonstrate that USP9 X is an integral component of centrosome and required for centrosome biogenesis.Loss-of-function of USP9 X impairs centrosome duplication and gain-of-function of USP9 X promotes centrosome amplification and chromosome instability.Significantly,USP9 X,through regulation of CEP131 abundance,promotes breast carcinogenesis.Next,we utilized SILAC based ubiquitome analysis and identified several other substrates of USP9 X.Collectively,our experiments identified USP9 X as an important regulator of centrosome biogenesis,and our study uncovered a critical role for USP9X/CEP131 in breast carcinogenesis,supporting the pursuit of USP9X/CEP131 as potential targets for breast cancer intervention.