Cancer Letters

Cancer Letters

Volume 433, 1 October 2018, Pages 186-198
Cancer Letters

Original Articles
USP22 promotes resistance to EGFR-TKIs by preventing ubiquitination-mediated EGFR degradation in EGFR-mutant lung adenocarcinoma

https://doi.org/10.1016/j.canlet.2018.07.002Get rights and content

Highlights

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    USP22 promotes cell malignant biological functions, and contributes to resistance to EGFR-TKIs in EGFR mutant lung ADC cells.

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    USP22 prevents ubiquitination mediated EGFR degradation and enhances recycling of EGFR after EGF stimulation.

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    USP22 sustains the activation of multiple EGFR downstream signaling pathways in lung ADC cell lines.

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    USP22 stabilizes EGFR protein, positively correlated with USP22 expression in EGFR-mutant lung ADC tissues of patients.

Abstract

As a newly discovered deubiquitinating enzyme, ubiquitin-specific protease 22 (USP22) is predictive of therapeutic outcomes in individual cancer patients. However, its clinical effects on malignancy and its roles in conferring resistance to EGFR-TKIs (epidermal growth factor receptor-tyrosine kinase inhibitors) in lung adenocarcinoma (ADC) remain largely unknown. Here, we showed that USP22 promotes cell proliferation, migration and invasion, and contributes to resistance to EGFR-TKIs in EGFR mutant lung ADC cells. Mechanistically, USP22 deubiquitinates EGFR localized on late endosomes, prevents ubiquitination mediated EGFR degradation and enhances recycling of EGFR after EGF stimulation. Additionally, USP22 sustained the activation of multiple EGFR downstream signaling pathways, including STAT3, AKT/mTOR and MEK/ERK pathways, in lung ADC cell lines H1975 and PC9. Furthermore, USP22 stabilizes EGFR protein expression, which correlates with USP22 expression in EGFR-mutant lung ADC patient samples. We are the first to demonstrate that silencing USP22 counteracts EGFR-TKIs resistance both in vitro and in vivo. We propose USP22 as a potential therapeutic target for EGFR-TKIs-resistant lung ADC.

Introduction

Activation of epidermal growth factor receptor (EGFR)-receptor tyrosine kinases (RTKs) is a key factor in the progression of non-small cell lung cancer (NSCLC). EGFR-RTKs cause proliferation, invasion, metastasis, and angiogenesis, as well as suppress apoptosis, in lung adenocarcinoma (ADC). Mutations in EGFR are detected in about 10%ā€“17% of human ADCs in the United States and Europe and in about 30%ā€“65% of lung cancers in Asia [1,2]. Pharmacologic inhibitors of EGFR-TK, including gefitinib and erlotinib, have notable therapeutic effects in patients with NSCLCs [3,4]. Strategies targeting EGFR-TKIs have been the first therapeutic choice for patients with advanced lung ADC with EGFR mutations. Unfortunately, drug resistance is inevitable in almost all of these patients after a median treatment period of 10ā€“16 months [5]. Furthermore, patients with EGFR gene T790M-mutations are resistant to erlotinib and gefitinib. Although another second-generation EGFR-TKI, afatinib, can circumvent drug resistance to some degree, it has a very narrow therapeutic window due to intolerability. More recently, the third generation anti-EGFR agent osimertinib has been approved to treat NSCLC with the EGFR T790M mutation. However, acquired resistance to osimertinib has already been reported [6]. Therefore, developing new therapeutic strategies to overcome resistance to conventional EGFR inhibitors is urgently required.

Deubiquitinating enzymes (DUBs) deconjugate ubiquitin from substrates and negatively regulate ubiquitination. As a newly discovered member of DUBs and a key subunit of the human Spt-Ada-Gcn5-acetyltransferase (hSAGA) transcriptional coactivator complex, ubiquitin-specific protease 22 (USP22) interacts with the hSAGA complex to hydrolyze ubiquitins conjugated to histones H2A and H2B, thus activating target gene transcription by altering histone ubiquitination levels [7]. Increasing evidence suggests that USP22 has clinicopathological significance in oncology. Identified in many kinds of tumors, USP22 plays a key role in cell cycle regulation, embryo development, and telomere homeostasis [[7], [8], [9]]. For example, knockdown of USP22 in bladder cancer cells led to cell cycle arrest in the G0/G1 phase and resulted in decreased tumor growth in vivo [7,10,11]. Moreover, USP22 was shown to be required for the transcriptional activity of c-MYC, a key player driving tumor growth [7].

Recently, three mammalian DUBs, AMSH, UBPY and USP2a, have been implicated in the endocytic down-regulation of EGFR-RTKs [[12], [13], [14], [15]]. These studies suggested that resistance to EGFR-TKIs is closely related to endocytosis-mediated EGFR activation via ubiquitination. Our previous studies revealed that USP22 promotes tumor progression and induces epithelial-mesenchymal transition (EMT) in lung ADC [16]. Considering the critical roles of USP22 in the pathogenesis of lung ADCs, we sought to understand if USP22 had a non-redundant and independent role in endocytosis-mediated regulation of EGFR-RTKs.

In this study, we performed global gene expression microarray profiling of H1975ā€Æcells transfected with either scrambled (Scr)-shRNA or USP22-shRNA. Our data revealed that USP22 is associated with specific gene expression profiles regulated by endocytosis. Our study suggests that USP22 antagonizes EGFR degradation and amplifies EGFR signaling activity to promote EGFR-TKIs resistance in EGFR-mutated lung ADCs. Our findings suggest a new mechanism of EGFR-TKIs resistance from the novel perspective of deubiquitination modification, and we propose USP22 as a potential therapeutic target for EGFR-TKIs resistance in EGFR-mutant lung ADCs.

Section snippets

Cell lines and tissue specimens

Human NSCLC cell lines H1975 (mutant EGFR of L858R and T790M; erlotinib resistant) and PC9 (mutant EGFR with deletion of E746_A750; erlotinib sensitized) were maintained in our laboratory as previously reported [16]. H1975 and PC9 were maintained in RPMI-1640 and DMEM, respectively. Both media were supplemented with 5% heat-inactivated fetal bovine serum (Hyclone), 100 units/mL of penicillin, and 100ā€ÆĪ¼g/mL of streptomycin (Gibco). Cells were grown in a humidified atmosphere with 5% CO2 in air

Evaluation of USP22 expression in lung ADC tissues and EGFR-mutant lung ADC cell lines

Our previous study revealed that the frequency of USP22 positive staining was 76% in lung tissues of patients with lung ADC. Kaplan-Meier analysis also demonstrated that patients with USP22 positive staining had significantly poorer overall survival (OS) and disease-free survival (DFS) compared to patients with USP22 negative staining [16]. We evaluated publicly available microarray datasets and found that USP22 mRNA expression was significantly elevated in lung ADC tissues compared to normal

Discussion

Aberrant USP22 expression can promote tumor progression and induce EMT in lung ADC [16,19]. Our previous study also suggests that USP22 expression is significantly increased in NSCLC tissues. However, the underlying mechanisms and the potential regulatory roles of USP22 in the pathogenesis of lung ADC and sensitization of EGFR-mutant targeted drugs have not been fully elucidated. Our current study demonstrates that USP22 promotes EGFR-TKIs resistance by preventing EGFR degradation in

Disclosure of conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

We thank Dr. Liangde Xu from Department of Bioinformatics of Harbin Medical University for assistance with the microarray data analysis.

This work was supported in part by the National Natural Science Fund of China grant (No. 81572276 to LC, No. 81573001 to XC, No. 81673024 to YZ, and No. 81672931 to QM); the National Natural Science Fund Youth of Fund of China grant (No. 81501960 to JH and No. 81602717 to HL); the 60th Postdoctoral Natural Science Fund of China grant (No. 2016M601442 to JH);

Authors' contributions

HZ and BH performed experiments, analyzed data, and wrote the paper. JH and LC initiated and organized the study. JH designed the experiments. JH and HL critically revised the manuscript. YZ performed some experiments. XC provided samples. QM and MC analyzed the data. All authors read and approved the final version of the manuscript.

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