br Materials and methods br Results br

Materials and methods
Results
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 EGFR-mutant lung ADC. Our functional characterization of USP22 is critical for understanding the biological significance in lung ADC tumorigenesis and may reveal novel therapeutic targets to advance the development of more effective clinical treatment strategies. To uncover the mechanisms underlying USP22-mediated transformation in lung cancer, we performed microarray analysis using publicly available datasets of human lung ADC patients. We identified 2171 differentially expressed genes in our profiling analysis. Subsequent functional pathway analysis based on the gene signature indicated that multiple pathways involved in cancer progression are affected in lung ADCs (Fig. 1), which we further validated. For example, USP22 positively correlated with cell proliferation and cell invasion in H1975 and PC9 Omaveloxolone in vitro (Figs. 2 and 3). Previous studies showed that USP22 modulates the multidrug resistance of hepatocellular carcinoma [26,27]. Here, we substantiated this finding by showing that higher USP22 expression facilitated resistance to EGFR-TKIs in vitro and in vivo in erlotinib-resistant H1975 cells that harbor a T790M mutation in EGFR (Figs. 3C and 7). Our study and other reports underscore the profound impact of USP22 in impacting on numerous downstream pathways. However, we did not observe morphological changes in cancer cell lines following USP22 knockdown in culture. The attachment of ubiquitin to RTKs, such as EGFR, mediates efficient delivery through the endosome-lysosome system for proteolysis. In this way, RTK degradation enables cessation of signal transduction. A recent siRNA screen identified a number of USPs that can significantly modulate EGFR levels and degradation kinetics [28]. Our data strongly indicate that USP22 plays complex roles in conferring drug resistance in lung ADCs. However, our finding on prevention of endocytosis-mediated EGFR degradation by USP22 reveals one vital aspect of USP22\'s diverse functions. Although more intensive investigation is necessary, our study provides a plausible explanation for how USP22 promotes EGFR-TKIs resistance in EGFR-mutant lung adenocarcinoma. Down-regulation of EGFR-dependent signaling is achieved by internalization of activated EGFR and its degradation in lysosome [29]. Endosomal sorting triggered by ubiquitination of EGFR can also be attenuated by DUBs. There are multiple ubiquitin protein ligases (E3s) and accompanying DUBs that function in the endocytic pathway as regulators of membrane protein trafficking and degradation [30]. Three mammalian DUBs, AMSH, UBPY and USP2a, have been assessed in terms of their effect on EGFR ubiquitination and degradation. While both AMSH [31] and UBPY [32,33] show deubiquitinating activity on Ubn-EGFR, the effects of these DUBs on EGFR down-regulation have been controversial. Some studies showed that AMSH [31] and UBPY [32,33] prevent EGFR down-regulation by deubiquitinating EGFR. USP2a antagonizes endocytosis-mediated EGFR degradation and thus amplifies signaling activity from the receptor [15]. Nevertheless, both AMSH [31,34,35] and UBPY [32,33,[36], [37], [38]] have been reported to increase EGFR down-regulation. In the present study, we showed that USP22 localized to late endosomes, decreased Ubn-EGFR, and promoted degradation of EGFR in a proteasome-dependent pathway, which eventually increased plasma membrane-localized EGFR. Taken together, we propose a working model that helps explain the potential roles of USP22 in stabilizing EGFR by reducing endocytosis-mediated degradation (Fig. 9). In this model, late endosome-localized USP22 deubiquitinates EGFR and impedes sorting of EGFR to the lysosome, thus sustaining the trafficking of EGFR to the plasma membrane (Figs. 6 and 9).