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  • br Our findings suggest that


    Our findings suggest that SPOP is also a key molecule responsible for the effects of APPBP2 on NSCLC. Although SPOP mutations were identi-fied in numerous types of tumours, especially in prostatic carcinoma [38], no SPOP mutations were observed in the analysis of human NSCLC samples from the TCGA cohort in the current study (Fig. 4c). Con-sistent with our findings, earlier study revealed that somatic mutation of SPOP is rare in lung cancer [39]. Interestingly, we found the elevated Etoposide of SPOP in NSCLC (Fig. 4e,f and Fig. S3a,b). APPBP2 promotes a cytoplasmic retention of androgen receptor (AR) [40], leading to the dysfunction of AR upregulating the of CDK4 expression in prostatic can-cer [41]. CDK4 induces the phosphorylation as well as the degradation of SPOP in multiple cancers [38,42]. In renal cell carcinoma, SPOP can be activated by HIF and SPOP has very important oncogenic role sup-pressing PTEN and other genes, as well as activating AKT and ERK [43].Consistently, researchers also found that SPOP, as a strong onco-genic protein, acted as a diagnostic biomarker in renal cell carcinoma [44–46]. Based on these reports, it is reasonable to hypothesize that APPBP2 enhances NSCLC proliferation and invasiveness partially through AR/CDK4/SPOP pathway (Fig. 7).
    Collectively, APPBP2 is strongly associated with NSCLC and pro-motes the initiation and progression of tumours, which is, at least partly, mediated by PPM1D as well as SPOP. Our work provides a novel molec-ular mechanism underlying the oncogenesis of NSCLC and supports APPBP2 as a potential valuable marker suitable for diagnosis and thera-peutic intervention in NSCLC.
    Funding sources
    the migration as well as the invasiveness of A549 and H1299 cells. Finally, the investigators provided evidence that APPBP2-knockdown reduces NSCLC xenograft growth. Taken together, the findings suggest that APPBP2 plays an oncogenic role in NSCLC.
    In the investigation of the molecular mechanism underlying APPBP2 biologic effects on NSCLC, the investigators discovered that the level of APPBP2 expression positively correlated with PPM1D and SPOP expres-sion as represented in Fig. 7. Notably, the overexpression of PPM1D and SPOP effectively attenuated APPBP2-silencing inhibition in NSCLC cell proliferation, migration, and invasiveness, indicating PPM1D and SPOP responsible for the oncogenic roles of APPBP2 in NSCLC. To explore the interaction of APPBP2 with PPM1D and SPOP, we performed co-IP assays and found that PPM1D (but not SPOP) could strongly bind to APPBP2 in both A549 and H1299 cell contexts, indicating the physiolog-ically relevant interactions of APPBP2 with PPM1D.
    APPBP2 contains a tetratricopeptide repeat (TPR) structural motif that frequently mediates protein-protein interactions [28,29], so the binding of APPBP2 with PPM1D is potentially mediated by the TPR structural motif. Since APPBP2 (PAT1) phosphorylates Mei2 and Ste11 in the controls of sex differentiation [30] and functions as a positive reg-ulator of Kinesin motility, resulting in cargo transport [31], the
    This work was supported by the Key Program of Natural Science Re-search of Higher Education of Anhui Province (grant no. KJ2017A241), the National Natural Science Foundation of China (grant no. 81772493) and the Science and Technology Program of Anhui Province (grant nos. 2017070503B037 and YDZX20183400002554). The funders had no role in the study design, data collection, data analysis, interpre-tation, or writing of the paper.
    Declarations of interest
    All co-authors agree with the content of this manuscript and report no conflicts of interest related to this study.
    Author contributions
    formed the experimental procedures: All authors; Analysed the data:
    Fig. 6. PPM1D and SPOP attenuated the inhibition of cell migration and invasiveness induced by APPBP2 knockdown in NSCLC cells. (a) Effects of PPM1D and SPOP on cell migration and invasion of A549 stable cells by transwell, n = 3. (b) Effects of PPM1D and SPOP on cell migration and invasion of H1299 stable cells by transwell, n = 3. (c) The statistical results of PPM1D and SPOP on cell migration and invasiveness of A549 stable cells by transwell. A one-way ANOVA revealed a significant effect of group (F[6,14] = 35.7, p b 0.001). (d) The statistical results of PPM1D and SPOP on cell migration and invasiveness of H1299 stable cells by transwell. A one-way ANOVA revealed a significant effect of group (F[6,14] = 72.58, p b 0.001). The addition or non-addition of plasmid or virus is present as “+” and “-”, respectively. Data are presented as mean ± SEM. The difference between means was compared by Tukey's Multiple Comparison test. 0.001 b **p b 0.01 and ***p b 0.001 for indicated comparison. n.s. refers as not significant. Scale bars: 50 μm.