The ISG15-specific protease USP18 regulates stability of PTEN

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.

Deubiquitinase USP18 Loss Mislocalizes and Destabilizes KRAS in Lung Cancer

“…Given this, it is not surprising that proteins downstream of KRAS like cyclin D1 (35) can rescue growth of USP18-repressed lung cancer cells (data not shown). It is interesting to note that many proteins regulated by ISG15 and USP18 are involved in the KRAS signaling pathway (8,46). Simultaneous destabilization of KRAS and other USP18-regulated proteins downstream of KRAS may play a functional role in reducing formation of Kras mutant lung cancers.…”

“…Simultaneous destabilization of KRAS and other USP18-regulated proteins downstream of KRAS may play a functional role in reducing formation of Kras mutant lung cancers. Recent work indicates that many growth-regulatory proteins are altered by USP18 loss (46). Future work will establish whether these or other growth-regulatory proteins are affected by USP18, if they are downstream of KRAS signaling and whether they are repressed in Kras LA2/+ /Usp18 −/− lung cancers.…”

The functions of tumor suppressor PTEN in innate and adaptive immunity

“…In addition, ubiquitin-specific peptidase 18 (USP18) could help to stabilize PTEN by acting on ISG-15. 80 …”

PI3‐kinase pathway biomarkers in oral cancer and tumor immune cells

“…[13][14][15] Expression of each biomarker was jointly quantified by 2 pathologists (MN and IW) in 3 cores per sample and the average was used for downstream statistical analysis. Immunohistochemical staining was performed on deparaffinized and hydrated sections.…”

“…These antibodies had been shown to be specific by Western blot analysis using nonsmall cell lung cancer cell lines and human bronchial epithelial cell line models and had been optimized for immunohistochemistry using cell line pellets fixed in formalin and embedded in paraffin, as previously reported by our group. [13][14][15] Expression of each biomarker was jointly quantified by 2 pathologists (MN and IW) in 3 cores per sample and the average was used for downstream statistical analysis. Scoring of 3 cores allowed us to account for and potentially minimize (but not completely eliminate) interpretation bias resulting from tumor heterogeneity that certainly exists when assessing protein expression levels in different regions of the tumor.…”