br Fig PPM D and SPOP attenuated the inhibition of
Fig. 5. PPM1D and SPOP attenuated the inhibition of cell proliferation induced by APPBP2 knockdown in NSCLC cells. (a) Ectopic expression of PPM1D or SPOP promoted colon formation on APPBP2 silenced A549 cells. Control was set as a blank group without protein overexpression. (b) The statistical results of colony formation indicate the impact of PPM1D and SPOP on NSCLC stable cells. A one-way ANOVA revealed a significant effect of group (F[4,10] = 324.5, p b 0.001 in A549 and F[4,10] = 63.45, p b 0.001 in H1299). (c) The proliferation effects of PPM1D or SPOP expression on A549 stable BYL 719 was measured by MTT assay. A one-way ANOVA revealed a significant effect of group (F[6,14] = 106.2, p b 0.001). (d) The proliferation effects of PPM1D or SPOP expression on H1299 stable cells was measured by MTT assay. A one-way ANOVA revealed a significant effect of group (F[6,14] = 393.9, p b .001). (e) PPM1D and SPOP had a significant impact on cell apoptosis of A549 stable cells by Annexin V and flow cytometric analysis. A one-way ANOVA revealed a significant effect of group (F[6,14] = 338.4, p b 0.001). (f) PPM1D and SPOP had a significant impact on cell apoptosis of H1299 stable cells by Annexin V and flow cytometric analysis. A one-way ANOVA revealed a significant effect of group (F[6,14] = 310.9, 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. 0.01 b *p b 0.05 and ***p b 0.001 for indicated comparison. n.s. refers as not significant.
Fig. 7. Graphical signalling pathway of the effect of APPBP2 on NSCLC via PPM1D and SPOP. The blue lines indicate the activation of the targets according to our experiments or published researches. The red lines indicate the possible inhibition of targets according to our reasonable speculation. AR: androgen receptor.
modulation of PPM1D by APPBP2 in NSCLC is probably through phos-phorylation and/or translocation.
Previous studies have proved the oncogenic roles of PPM1D in numerous types of cancers [5,6,8,32], mostly in a p38MAPK/p53/p16 pathway dependent manner [33,34]. Notably, H1299 cells have a homo-zygous partial deletion of the TP53 gene , suggesting the existence of a P53-independent manner of PPM1D affecting NSCLC cells. Consis-tently, a P53-independent manner of PPM1D action has been reported in clonal haematopoiesis and myelodysplastic syndrome [36,37].
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 , 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 . Interestingly, we found the elevated expression of SPOP in NSCLC (Fig. 4e,f and Fig. S3a,b). APPBP2 promotes a cytoplasmic retention of androgen receptor (AR) , leading to the dysfunction of AR upregulating the of CDK4 expression in prostatic can-cer . 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 .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.
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.