Gemin6 promotes c‐Myc stabilisation and non‐small cell lung cancer progression via accelerating AURKB mRNA maturation

Abstract

Lung cancer is the leading cause of cancer death worldwide, and around 85% of patients are grouped into non-small cell lung cancer (NSCLC) based on the histological characteristics.1 The survival of motor neurons (SMN) complex has been demonstrated to play critical roles in the biogenesis of ribonucleoprotein complexes (RNPs), and the reduced expression of the causative gene SMN leads to spinal muscular atrophy (SMA).2, 3 The SMN complex mainly consists of SMN, Gemin2, 3, 4, 5, 6 and 7. SMN interacts with Gemin2, 3, 5 and 7 directly, whereas Gemin4 and 6 binding to SMN relies on Gemin3 and 7, respectively. In this study, we revealed the oncogenic role of Gemin6/AURKB/c-Myc axis in lung adenocarcinoma, and the promising anticancer potential of sertindole treatment with probable clinical application in the future. As the functional roles of SMN complex in NSCLC remain elusive, we examined the expressions of individual components of SMN complex, and found that Gemin6 is unanimously upregulated in multiple types of human cancer, including NSCLC (Figure 1A,B; Figure S1A–C). Gemin6 high expression was identified to correlate with worse clinical outcome (Figure 1C–H; Figure S1D,E; Table S1). Furthermore, the ROC curve analysis of the SMN complex factors showed that Gemin6 exhibits the highest AUC value of 0.936 (Figure 1I). As expected, real-time RT-PCR and immunoblot assays validated that Gemin6 was highly expressed in NSCLC tissues and cell lines (Figure 1J–M; Figure S1F,G). We also identified the mutation pattern of Gemin6 in pan-cancers including NSCLC (Figure S1H; Table S2). Hypomethylation in Gemin6 promoter region was uncovered to be positively associated with its transcript expression level in multiple tumours (Figure 1N; Figure S1I,J). In addition, the decreased expression of Gemin6 in normal control cell line BEAS-2B and tumour cells could be reversed by DNA methylases inhibitor 5-azacytidine (5-Aza) treatment (Figure 1O). Gemin6 transcript was then inhibited with two lenti-viral shRNAs in A549 and H1975 (Figure 2A; Figure S2A). We found that Gemin6 knockdown repressed tumour cell proliferation examined by growth curve, BrdU incorporation and colony formation assays (Figure 2B–G; Figure S2B–D). Furthermore, we uncovered that the cell cycle was arrested at G0/G1 phase after Gemin6 knockdown, and the key regulators for G0/G1 cell cycle transition, including CDK2, CDK4 and CDK6 were also markedly reduced upon Gemin6 knockdown (Figure 2H–J; Figure S2E–G). The cell migration ability was also reduced upon Gemin6 knockdown both in vitro and in vivo (Figure 2K–M; Figure S2H–P). In line with the findings in vitro, the xenograft tumour masses, tumour weights and volumes in Gemin6 inhibition groups were markedly impeded compared to the control group (Figure 2N–Q; Figure S2N). To decipher the underlying mechanism by which Gemin6 regulates NSCLC progression, we found that Gemin6 was involved in c-Myc, but not E2F, related signaling pathway (Figure 3A,B; Figure S3A). However, the reduced c-Myc proteins, but not the transcripts, in Gemin6 knockdown groups were detected compared to control (Figure 3C–F; Figure S3B–E). As expected, Gemin6 knockdown-reduced cell proliferation ability could be overcome by c-Myc overexpression (Figure 3G). These findings prompted us to hypothesise that Gemin6 regulates the post-translational modification of c-Myc proteins. As documented by other studies, c-Myc proteins could be stabilised by deubiquitinases USP36/USP28,4, 5 or degraded by E3 ligase FBXW7/SKP2.6, 7 To our surprise, the above c-Myc regulators’ mRNA expressions were not markedly deregulated upon Gemin6 inhibition (Figure 3H; Figure S3F,G). However, recent findings identified that phosphorylation of c-Myc Serine 67 site mediated by AURKB, prevents its degradation in a proteasome signaling pathway-dependent manner,8 which was uncovered to be significantly reduced upon Gemin6 knockdown (Figure 3I; Figure S3H). Furthermore, we found that Gemin6 expression positively correlates with AURKB, and the mRNA and protein expressions of AURKB were both decreased in Gemin6 knockdown cells (Figure 3J,K; Figure S3I). In addition, we showed that AURKB overexpression reversed Gemin6 knockdown-reduced cell proliferation and migration abilities (Figure 3L–Q). In line with the findings that SMN complex regulates the biogenesis of RNPs, we revealed that the SMN complex formation, the maturation process, but not the stability of AURKB mRNA, were decreased upon Gemin6 knockdown (Figure 3R–T; Figure S3J–L). As expected, we showed that AURKB serves as a prognostic biomarker and is highly expressed in NSCLC, which correlates with worse overall survival (OS) and disease-specific survival (DSS) rates in LUAD (Figure 3U,V; Figure S3M–P). To further explore the clinical value of Gemin6, we then examined the potential drug repurposing activities of 10 drugs, selectively targeting dopamine or serotonin receptors from FDA-Approved Drug Library Mini, in NSCLC by blocking Gemin6 expression (Table S3).9 Compound sertindole was identified as the only one with the activity decreasing Gemin6 proteins to less than 70% of control level (Figure 4A; Figure S4A). Sertindole has been previously identified as an antagonist targeting dopamine D2 receptors, serotonin 5HT2A receptors and α1-adrenoceptors, which is mainly produced in the central nervous system and gastrointestinal tract.10 The cell viability after sertindole treatment was examined, and more dramatic inhibitory effect on tumour cell (A549 and H1975) survival was detected (Figure 4B). Furthermore, we found that sertindole treatment decreased the mRNA expressions of Gemin6 and AURKB, but not c-Myc (Figure 4C,D; Figure S4B). The cell proliferation and cell migration abilities were also repressed after sertindole treatment, which were markedly reversed by Gemin6 or c-Myc forced expression, respectively (Figure 4E–I; Figure S4C–I). As expected, the xenograft tumour masses, tumour weights and volumes in sertindole treatment group were markedly inhibited compared to control group, evidenced by deceased Ki67, Gemin6 and c-Myc IHC-positive signals (Figure 4J–O). We observed that AURKB mRNA was reduced upon Gemin6 inhibition. However, how Gemin6 acts alone or cooperates with other SMN complex components to regulate AURKB mRNA splicing or transcription is still unclear. Therefore, it will be necessary to examine the integrity of RNPs or SMN complex upon Gemin6 knockdown in the future. We showed that sertindole, previous identified antagonist targeting dopamine D2 receptors, serotonin 5HT2A receptors and α1-adrenoceptors, inhibited NSCLC progression by inhibiting c-Myc, who lies at the crossroads of these signaling pathways, and reveals the promising anticancer potentials of sertindole against NSCLC even with lung-to-brain metastases in the future (Figure 4P). This study was supported by National Key Research and Development Program of China (2021YFF1000602), National Nature Science Foundation of China (U1902216, U2102206, 82173110, 82002439, 82060515, 81960423, 82160453, 82160593) and Yunnan Applied Basic Research Projects (2019FJ009, 202001AS070037, 2019HB076, 2019FE001-042, 202001AT070027, 202001AC070712). The specific phosphorylation antibody against Serine 67 site on c-Myc was kindly provided by Dr. Hudan Liu at Wuhan University, China. The NSCLC tissue microarray was provided by Dr. Songqing Fan at the Second Xiangya Hospital, Central South University, Changsha, Hunan, China. The authors declare that there is no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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