Background: The importance of mRNA methylation erased by ALKBH5 in mRNA biogenesis, decay, and translation control is an emerging research focus. Ectopically activated YAP is associated with the development of many human cancers. However, the mechanism whereby ALKBH5 regulates YAP expression and activity to inhibit NSCLC tumor growth and metastasis is not clear. Methods: Protein and transcript interactions were analyzed in normal lung cell and NSCLC cells. Gene expression was evaluated by qPCR and reporter assays. Protein levels were determined by immunochemical approaches. Nucleic acid interactions and status were analyzed by immunoprecipitation. Cell behavior was analyzed by standard biochemical tests. The m⁶A modification was analyzed by MeRIP. Results: Our results show that YAP expression is negatively correlated with ALKBH5 expression and plays an opposite role in the regulation of cellular proliferation, invasion, migration, and EMT of NSCLC cells. ALKBH5 reduced m⁶A modification of YAP. YTHDF3 combined YAP pre-mRNA depending on m⁶A modification. YTHDF1 and YTHDF2 competitively interacted with YTHDF3 in an m⁶A-independent manner to regulate YAP expression. YTHDF2 facilitated YAP mRNA decay via the AGO2 system, whereas YTHDF1 promoted YAP mRNA translation by interacting with eIF3a; both these activities are regulated by m⁶A modification. Furthermore, ALKBH5 decreased YAP activity by regulating miR-107/LATS2 axis in an HuR-dependent manner. Further, ALKBH5 inhibited tumor growth and metastasis in vivo by reducing the expression and activity of YAP. Conclusions: The presented findings suggest m⁶A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2–mediated YAP activity in NSCLC. Moreover, effective inhibition of m⁶A modification of ALKBH5 might constitute a potential treatment strategy for lung cancer.

The roles of aberrantly regulated autophagy in human malignancy and the mechanisms that initiate and sustain the repression of autophagy in carcinogenesis are less well defined. Activation of the oncogene UBE2C and repression of autophagy are concurrently underlying the initiation, progression, and metastasis of lung cancer and exploration of essential association of UBE2C with autophagy will confer more options in searching novel molecular therapeutic targets in lung cancer. Here we report that aberrant activation of UBE2C in lung tumors from patients associates with adverse prognosis and enhances cell proliferation, clonogenicity, and invasive growth of NSCLC. UBE2C selectively represses autophagy in NSCLC and disruption of UBE2C-mediated autophagy repression attenuates cell proliferation, clonogenicity, and invasive growth of NSCLC. Autophagy repression is essentially involved in UBE2C-induced cell proliferation, clonogenicity, and invasive growth of NSCLC. Interference of UBE2C-autophagy repression axis by Norcantharidin arrests NSCLC progression. UBE2C is repressed post-transcriptionally via tumor suppressor miR-381 and epitranscriptionally stabilized with maintenance of lower m⁶A level within its mature RNAs due to the upregulation of m⁶A demethylase ALKBH5 in NSCLC. Collectively, our results indicated that deregulated UBE2C-autophagy repression axis drives NSCLC progression which renders varieties of potential molecular targets in cancer therapy of NSCLC.

Cisplatin (DDP) resistance has become the leading cause of mortality in non-small cell lung cancer (NSCLC). miRNA dysregulation significantly contributes to tumor progression. In this study, we found that miR-495 was significantly downregulated in lung cancer tissue specimens. This study aimed to elucidate the functions, direct target genes, and molecular mechanisms of miR-495 in lung cancer. miR-495 downregulated its substrate UBE2C through direct interaction with UBE2C 3′- untranslated region. UBE2C is a proto-oncogene activated in lung cancer; however, its role in chemotherapeutic resistance is unclear. Herein, UBE2C expression levels were higher in DDP-resistant NSCLC cells; this was associated with the proliferation, invasion, and DDP resistance in induced cisplatin-resistant NSCLC cells. Furthermore, epithelial–mesenchymal transitions (EMT) contributed to DDP resistance. Moreover, UBE2C knockdown downregulated vimentin. In contrast, E-cadherin was upregulated. Importantly, miR-495 and UBE2C were associated with cisplatin resistance. We attempted to evaluate their effects on cell proliferation and cisplatin resistance. We also performed EMT, cell migration, and invasion assays in DDP-resistant NSCLC cells overexpressing miR-495 and under-expressing UBE2C. Furthermore, in silico assays coupled with western blotting and luciferase assays revealed that UBE2C directly binds to the 5′-UTR of the drug-resistance genes ABCG2 and ERCC1. Furthermore, miR-495 downregulated ABCG2 and ERCC1 via regulation of UBE2C. Together, the present results indicate that the miR495-UBE2C-ABCG2/ERCC1 axis reverses DDP resistance via downregulation of anti-drug genes and reducing EMT in DDP-resistant NSCLC cells.

Background: Recent evidence indicates that metformin inhibits mammalian cancer growth and metastasis through the regulation of microRNAs. Metformin regulates miR-381 stability, which plays a vital role in tumor progression. Moreover, increased YAP expression and activity induce non-small cell lung cancer (NSCLC) tumor growth and metastasis. However, the molecular mechanism underpinning how metformin-induced upregulation of miR-381 directly targets YAP or its interactions with the epithelial-mesenchymal transition (EMT) marker protein Snail in NSCLC is still unknown. Methods: Levels of RNA and protein were analyzed using qPCR, western blotting and immunofluorescence staining. Cellular proliferation was detected using a CCK8 assay. Cell migration and invasion were analyzed using wound healing and transwell assays. Promoter activity and transcription were investigated using the luciferase reporter assay. Chromatin immunoprecipitation was used to detect the binding of YAP to the promoter of Snail. The interaction between miR-381 and the 3′UTR of YAP mRNA was analyzed using the MS2 expression system and co-immunoprecipitation with biotin. Results: We observed that miR-381 expression is negatively correlated with YAP expression and plays an opposite role to YAP in the regulation of cellular proliferation, invasion, migration, and EMT of NSCLC cells. The miR-381 function as a tumor suppressor was significantly downregulated in lung cancer tissue specimens and cell lines, which decreased the expression of its direct target YAP. In addition, metformin decreased cell growth, migration, invasion, and EMT via up-regulation of miR-381. Moreover, YAP, which functions as a co-transcription factor, enhanced NSCLC progression and metastasis by upregulation of Snail. Snail knockdown downregulated the mesenchymal marker vimentin and upregulated the epithelial marker E-cadherin in lung cancer cells. Furthermore, miR-381, YAP, and Snail constitute the miR-381-YAP-Snail signal axis, which is repressed by metformin, and enhances cancer cell invasiveness by directly regulating EMT. Conclusions: Metformin-induced repression of miR-381-YAP-Snail axis activity disrupts NSCLC growth and metastasis. Thus, we believe that the miR-381-YAP-Snail signal axis may be a suitable diagnostic marker and a potential therapeutic target for lung cancer.