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. 2017 Jan 3;8(1):3-14.
doi: 10.18632/onco_target.13914.

The ISG15-specific protease USP18 regulates stability of PTEN

Lisa Maria Mustachio  1 , Masanori Kawakami  2 , Yun Lu  1 , Jaime Rodriguez-Canales  3 , Barbara Mino  3 , Carmen Behrens  2 , Ignacio Wistuba  3 , Neus Bota-Rabassedas  2 , Jun Yu  4 , J Jack Lee  4 , Jason Roszik  5   6 , Lin Zheng  2 , Xi Liu  2 , Sarah J Freemantle  1 , Ethan Dmitrovsky  1   2   7
Affiliations

The ISG15-specific protease USP18 regulates stability of PTEN

Lisa Maria Mustachio et al. Onco_target. .

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.

Keywords: ISG15; PTEN; USP18; and lung cancer; protein stability.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interests exist.

Figures

Figure 1
Figure 1. RPPA-based protein profiling revealed USP18 regulated PTEN levels
A. Independent RPPAs are displayed of ED1 and HOP62 lung cancer cell lines stably transfected with a control vector or one of two individual shRNAs against USP18. Signal intensities were normalized and transformed to linear values. RPPA highlighted species showing substantial differences in expression profiles between vector control and USP18 shRNA transfected lung cancer cells are represented by heat-maps for both ED1 and HOP62 lung cancer arrays. Red indicated high and green indicated low protein expression. Cell line clusters are shown as dendrograms. PTEN (red box) was highlighted as one of the proteins showing similar trends in both arrays. B. PTEN expression in the displayed ED1 and HOP62 transfected lung cancer cell lines was quantified using normalized linear values. Cell lines stably transfected with shRNAs against USP18 were compared relative to vector control transfectants. C. Representative immunoblots independently probed with USP18 and PTEN recognizing antibodies confirmed that engineered repression of USP18 decreased PTEN protein levels in ED1 and HOP62 lung cancer cells. PTEN expression was normalized to actin expression and compared between vector control and USP18 shRNA-transfected lung cancer cell lines. D. Decreased PTEN protein levels caused by loss of USP18 expression were rescued by transiently overexpressing GFP-tagged USP18 in ED1 lung cancer cells. Immunoblots were individually probed with GFP and PTEN recognizing antibodies. PTEN was normalized to actin expression and compared between vector control and USP18 shRNA- as well as USP18 shRNA/USP18 vector-transfected lung cancer cell lines.
Figure 2
Figure 2. Modifying USP18 and ISG15 protein levels altered PTEN protein stability
A. Loss of USP18 expression decreased PTEN protein stability. ED1 murine lung cancer cells stably transfected with a vector control or with one of two individual shRNAs against USP18 were subjected to an 8 hour CHX treatment. PTEN expression was quantified relative to respective actin expression at indicated time points and normalized to the 0 hour (before CHX treatment) time point. B. Gain of USP18 expression increased PTEN levels. ED1 and HOP62 lung cancer cells were independently transfected with a USP18 expression plasmid or a control vector. PTEN expression was normalized to actin expression and compared between vector control and USP18 vector-transfected lung cancer cell lines. C. Engineered gain of ISG15 expression decreased PTEN protein levels. ED1 and HOP62 lung cancer cells were independently transfected with ISG15 or a vector control. PTEN was normalized to actin expression and compared between vector control and ISG15 vector-transfected lung cancer cell lines. D. Decreased PTEN levels conferred by overexpressed ISG15 were rescued by transiently overexpressing GFP-tagged USP18 in HOP62 lung cancer cells. Immunoblots were independently probed with GFP and PTEN recognizing antibodies. PTEN protein levels were normalized to actin expression and compared between vector control and ISG15 vector as well as ISG15 vector/USP18 vector-transfected lung cancer cell lines.
Figure 3
Figure 3. Loss of USP18 expression decreased cytoplasmic PTEN protein
A. ED1 and B. HOP62 lung cancer cell lines stably transfected with a vector control or shRNA against USP18 were fixed and stained for PTEN, sodium potassium ATPase, and DAPI. Cells were imaged by confocal microscopy and the magnification was 63X. Percent immunostaining of PTEN in the cytoplasm and nucleus was quantified and compared. Data are representative of 20 cells per group for each analyzed experiment.
Figure 4
Figure 4. PTEN undergoes ISGylation
ISG15 conjugates to PTEN in lung cancer cell lines. Immunoprecipitation (IP) with an anti-HA antibody followed by immunoblot (IB) with an anti-PTEN antibody revealed ISG15-PTEN complex formation between HA-tagged ISG15 and A. exogenous and B. endogenous PTEN at ~75 kDa in HOP62 lung cancer cells. C. The ISG15-PTEN conjugation observed by IP was attenuated with introduction of USP18 in HOP62 lung cancer cells. D. Deletions of PTEN at indicated domains were subjected to E. IP by HA-tagged ISG15 using an anti-HA antibody followed by IB with an anti-GFP antibody revealed differential conjugation between PTEN deletions in ED1 lung cancer cells. Arrows displayed ISG15-PTEN conjugation bands.
Figure 5
Figure 5. PTEN and USP18 expression profiles are associated in human lung cancers
A. Blocking peptide antagonized PTEN immunostaining in the NIH/3T3 (PTEN positive) cell line. B. Representative staining for human lung cancers with low PTEN and USP18 or high PTEN and USP18 expression profiles. All magnifications were 20X. C. PTEN and USP18 immunostaining are positively correlated in human lung cancer (n = 461) and D. This association was confirmed using a contingency table by grouping negative, low, medium, or high PTEN and USP18 immunostaining profiles in lung cancer cases.
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