Abstract
The ubiquitin-like modifier interferon-stimulated gene 15 (ISG15) is implicated in both oncogenic and tumor suppressive programs. Yet, few ISGylation substrates are known and functionally validated in cancer biology. We previously found specific oncoproteins were substrates of ISGylation and were stabilized by the ISG15-specific deubiquitinase (DUB) ubiquitin specific peptidase 18 (USP18). Using reverse-phase protein arrays (RPPAs), this study reports that engineered loss of the DUB USP18 destabilized the tumor suppressor protein phosphatase and tensin homologue (PTEN) in both murine and human lung cancer cell lines. In contrast, engineered gain of USP18 expression in these same lung cancer cell lines stabilized PTEN protein. Using the protein synthesis inhibitor cycloheximide (CHX), USP18 knockdown was shown to destabilize PTEN whereas USP18 overexpression stabilized PTEN protein. Interestingly, repression of USP18 decreased cytoplasmic PTEN relative to nuclear PTEN protein levels. We sought to identify mechanisms engaged in this PTEN protein destabilization using immunoprecipitation assays and found ISG15 directly conjugated with PTEN. To confirm translational relevance of this work, USP18 and PTEN immunohistochemical expression were compared in comprehensive lung cancer arrays. There was a significant (P < 0.0001) positive correlation and association between PTEN and USP18 protein expression profiles in human lung cancers. Taken together, this study identified PTEN as a previously unrecognized substrate of the ISGylation post-translational modification pathway. The deconjugase USP18 serves as a novel regulator of PTEN stability. This indicates inhibition of ISGylation is therapeutically relevant in cancers.
Highlights
Lung cancer is the most common cause of cancer death for men and women [1]
We previously identified specific ISGylation substrates involved in oncogenesis by manipulating expression of the interferon-stimulated gene 15 (ISG15)-specific protease ubiquitin specific peptidase 18 (USP18) [19,20]
Since the functions of ISGylation are under active study, we aimed to identify new target proteins of the pathway. Both ED1 murine and HOP62 human lung cancer cell lines engineered with stable knockdown of USP18 were used to monitor expression of 304 growth-regulatory proteins that contribute to tumorigenesis in an reverse-phase protein arrays (RPPAs) (Supplementary Table S1 and Supplementary Figure S1)
Summary
Lung cancer is the most common cause of cancer death for men and women [1]. Despite current treatments, lung cancer often becomes resistant to therapies [2], driving investigators to uncover alternative ways to target oncoproteins required for tumorigenesis. PTEN functions as a phosphatase for tyrosine and serine/threonine residues on phosphatidylinositol (3,4,5)-triphosphate (PIP3) This can inhibit downstream signaling to protein kinase B (AKT) and affect rapamycin (mTOR)-dependent pathways [5]. The ubiquitin specific peptidase 13 (USP13) was the only DUB yet uncovered to stabilize PTEN [10] This implicated that stability and activity of PTEN and other proteins involved in oncogenesis are altered by specific DUBs. This implicated that stability and activity of PTEN and other proteins involved in oncogenesis are altered by specific DUBs Understanding these processes will provide insights into PTEN biology, and advance our understanding of how this critical growth-regulatory protein can affect carcinogenesis
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