LIN exhibits high expression in
LIN28 exhibits high expression in embryonic stem PSB 1115 (ESCs) and its expression level is reduced upon differentiation [6,7]. Accordingly, LIN28 is a stem cell core factor and participates in cellular reprogramming [, , ]. Moreover, LIN28-expressing cancers demonstrate a less-differentiated, more aggressive profile . Thus, LIN28-based control of cell proliferation is a pivotal factor in cancer aggressiveness [, , ]. Cancer stem cells (CSCs) are involved in tumor relapse and confer resistance to anticancer therapies [15,16]. A growing body of evidence indicates that LIN28 is a biomarker for CSCs . The elucidation of factors that contribute to CSC-mediated aggressive pathology is necessary for designing effective therapeutic measures against CSCs [4,18]. Upstream transcriptional factors or loss of transcriptional repressors triggers activation of LIN28 expression in tumor cells . For instance, depletion of c-Myc causes a reduction in LIN28 transcript and protein levels, while overexpressed c-Myc restores LIN28 expression level in breast carcinoma [19,20].
LIN28 undergoes post-translational modifications that affect stability and its protein level. A study demonstrated that ubiquitin ligase human TRIM71 polyubiquitinates and degrades LIN28B protein ; however, to the best of our knowledge, no study has reported on the ubiquitination and stability of LIN28A protein. Protein ubiquitination events are widely reported to be reversed by counteracting enzymes known as deubiquitinating enzymes (DUBs). Approximately 100 DUBs have been found in the human proteome, and they are divided into five major families. Ubiquitin-specific protease (USP) is one of the largest families, comprising approximately 55 DUB proteins . To date, no DUB(s) have been identified for LIN28A protein. Therefore, the impact and functional mechanisms of LIN28A protein regulation by DUBs remain unknown.
In this study, we demonstrated that the oncogenic protein LIN28A undergoes ubiquitination by the 26S proteasomal degradation pathway. We further report that ubiquitin-specific protease 28 (USP28) interacts with and deubiquitinates LIN28A. We found that USP28 stabilizes LIN28A protein and extends its half-life by circumventing LIN28A protein turnover. Additionally, our study demonstrates the role of USP28 in promoting LIN28A-mediated cancer cell viability, invasion and migration.
Materials and methods
Discussion A plethora of information is available on LIN28 transcriptional modulations [1,6,38]. However, knowledge about post-translational modifications on LIN28A such as acetylation, phosphorylation, methylation, and ubiquitination and their effects on activity, stability and protein-protein interactions are not fully characterized. P300/CBP-associated factor (PCAF) acetyltransferase directly interacts with and acetylates LIN28, thereby reducing LIN28 protein level. This process is reversible by the deacetylase Sirtuin 1 . Thus, the balance of post-translational modifications such as acetylation and deacetylation substantially affect regulation of LIN28 protein stability and activity. In pluripotent stem cells, LIN28 undergoes phosphorylation by MAPK/ERK, leading to protein stability . In this study, we found that the highly conserved RNA-binding protein LIN28A undergoes proteasomal degradation by the 26S proteasome pathway. Furthermore, we report that USP28 is a deubiquitinating enzyme for LIN28A that reverses LIN28A ubiquitination to stabilize LIN28A protein. LIN28A, a chief regulator of let-7 miRNA processing , is overexpressed in human malignancies and promotes proliferation of cancer cells and tumorigenesis [18,41,42]. LIN28 has emerged as a factor that can influence the stemness and self-renewal of mammalian embryonic stem cells . Although LIN28A is a widely known potential target for cancers, no studies have been conducted on the ubiquitin-mediated post-translational modification of LIN28A.