N4-acetylcytidine modifies primary microRNAs for processing in cancer cells.

N4 acetylcytidine (ac4C) modification mainly occurs on tRNA, rRNA, and mRNA, playing an important role in the expression of genetic information. However, it is still unclear whether microRNAs have undergone ac4C modification and their potential physiological and pathological functions. In this study, we identified that NAT10/THUMPD1 acetylates primary microRNAs (pri-miRNAs) with ac4C modification. Knockdown of NAT10 suppresses and augments the expression levels of mature miRNAs and pri-miRNAs, respectively. Molecular mechanism studies found that pri-miRNA ac4C promotes the processing of pri-miRNA into precursor miRNA (pre-miRNA) by enhancing the interaction of pri-miRNA and DGCR8, thereby increasing the biogenesis of mature miRNA. Knockdown of NAT10 attenuates the oncogenic characters of lung cancer cells by regulating miRNA production in cancers. Moreover, NAT10 is highly expressed in various clinical cancers and negatively correlated with poor prognosis. Thus, our results reveal that NAT10 plays a crucial role in cancer initiation and progression by modulating pri-miRNA ac4C to affect miRNA production, which would provide an attractive therapeutic strategy for cancers.

SUMOylation of YTHDF2 promotes mRNA degradation and cancer progression by increasing its binding affinity with m6A-modified mRNAs.

N 6-Methyladenosine (m6A) is the most abundant modification within diverse RNAs including mRNAs and lncRNAs and is regulated by a reversible process with important biological functions. Human YTH domain family 2 (YTHDF2) selectively recognized m6A-RNAs to regulate degradation. However, the possible regulation of YTHDF2 by protein post-translational modification remains unknown. Here, we show that YTHDF2 is SUMOylated in vivo and in vitro at the major site of K571, which can be induced by hypoxia while reduced by oxidative stress and SUMOylation inhibitors. SUMOylation of YTHDF2 has little impact on its ubiquitination and localization, but significantly increases its binding affinity of m6A-modified mRNAs and subsequently results in deregulated gene expressions which accounts for cancer progression. Moreover, Disease-free survival analysis of patients with lung adenocarcinoma derived from TCGA dataset reveals that higher expression of YTHDF2 together with higher expression of SUMO1 predicts poor prognosis. Our works uncover a new regulatory mechanism for YTHDF2 recognition of m6A-RNAs and highlight the importance of YTHDF2 SUMOylation in post-transcriptional gene expression regulation and cancer progression.

PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

USP7 reduces the level of nuclear DICER, impairing DNA damage response and promoting cancer progression.

Endoribonuclease DICER is an RNase III enzyme that mainly processes microRNAs in the cytoplasm but also participates in nuclear functions such as chromatin remodelling, epigenetic modification and DNA damage repair. The expression of nuclear DICER is low in most human cancers, suggesting a tight regulation mechanism that is not well understood. Here, we found that ubiquitin carboxyl-terminal hydrolase 7 (USP7), a deubiquitinase, bounded to DICER and reduced its nuclear protein level by promoting its ubiquitination and degradation through MDM2, a newly identified E3 ubiquitin-protein ligase for DICER. This USP7-MDM2-DICER axis impaired histone γ-H2AX signalling and the recruitment of DNA damage response (DDR) factors, possibly by influencing the processing of small DDR noncoding RNAs. We also showed that this negative regulation of DICER by USP7 via MDM2 was relevant to human tumours using cellular and clinical data. Our findings revealed a new way to understand the role of DICER in malignant tumour development and may offer new insights into the diagnosis, treatment and prognosis of cancers.

Phosphorylation of AGO2 by TBK1 Promotes the Formation of Oncogenic miRISC in NSCLC.

Non-small-cell lung cancer (NSCLC) is a highly lethal tumor that often develops resistance to targeted therapy. It is shown that Tank-binding kinase 1 (TBK1) phosphorylates AGO2 at S417 (pS417-AGO2), which promotes NSCLC progression by increasing the formation of microRNA-induced silencing complex (miRISC). High levels of pS417-AGO2 in clinical NSCLC specimens are positively associated with poor prognosis. Interestingly, the treatment with EGFR inhibitor Gefitinib can significantly induce pS417-AGO2, thereby increasing the formation and activity of oncogenic miRISC, which may contribute to NSCLC resistance to Gefitinib. Based on these, two therapeutic strategies is developed. One is jointly to antagonize multiple oncogenic miRNAs highly expressed in NSCLC and use TBK1 inhibitor Amlexanox reducing the formation of oncogenic miRISC. Another approach is to combine Gefitinib with Amlexanox to inhibit the progression of Gefitinib-resistant NSCLC. This findings reveal a novel mechanism of oncogenic miRISC regulation by TBK1-mediated pS417-AGO2 and suggest potential therapeutic approaches for NSCLC.

Regulation of SUMOylation on RNA metabolism in cancers.

Post-translational modifications of proteins play very important roles in regulating RNA metabolism and affect many biological pathways. Here we mainly summarize the crucial functions of small ubiquitin-like modifier (SUMO) modification in RNA metabolism including transcription, splicing, tailing, stability and modification, as well as its impact on the biogenesis and function of microRNA (miRNA) in particular. This review also highlights the current knowledge about SUMOylation regulation in RNA metabolism involved in many cellular processes such as cell proliferation and apoptosis, which is closely related to tumorigenesis and cancer progression.

Glucose-mediated N-glycosylation of RPTPα affects its subcellular localization and Src activation.

Receptor-type protein tyrosine phosphatase α (RPTPα) is one of the typical PTPs that play indispensable roles in many cellular processes associated with cancers. It has been considered as the most powerful regulatory oncogene for Src activation, however it is unclear how its biological function is regulated by post-translational modifications. Here, we show that the extracellular segment of RPTPα is highly N-glycosylated precisely at N21, N36, N68, N80, N86, N104 and N124 sites. Such N-glycosylation modifications mediated by glucose concentration alter the subcellular localization of RPTPα from Golgi apparatus to plasma membrane, enhance the interaction of RPTPα with Src, which in turn enhances the activation of Src and ultimately promotes tumor development. Our results identified the N-glycosylation modifications of RPTPα, and linked it to glucose starvation and Src activation for promoting tumor development, which provides new evidence for the potential antitumor therapy.

Linear ubiquitination of PTEN impairs its function to promote prostate cancer progression.

PTEN is frequently mutated in human cancers, which leads to the excessive activation of PI3K/AKT signaling and thus promotes tumorigenesis and drug resistance. Met1-linked ubiquitination (M1-Ubi) is also involved in cancer progression, but the mechanism is poorly defined. Here we find that HOIP, one important component of linear ubiquitin chain assembly complex (LUBAC), promotes prostate cancer (PCa) progression by enhancing AKT signaling in a PTEN-dependent manner. Mechanistically, PTEN is modified by M1-Ubi at two sites K144 and K197, which significantly inhibits PTEN phosphatase activity and thus accelerates PCa progression. More importantly, we identify that the high-frequency mutants PTENR173H and PTENR173C in PCa patients showed the enhanced level of M1-Ubi, which impairs PTEN function in inhibition of AKT phosphorylation and cell growth. We also find that HOIP depletion sensitizes PCa cells to therapeutic agents BKM120 and Enzalutamide. Furthermore, the clinical data analyses confirm that HOIP is upregulated and positively correlated with AKT activation in PCa patient specimen, which may promote PCa progression and increase the risk of PCa biochemical relapse. Together, our study reveals a key role of PTEN M1-Ubi in regulation of AKT activation and PCa progression, which may propose a new strategy for PCa therapy.

Hypoxia regulates overall mRNA homeostasis by inducing Met1-linked linear ubiquitination of AGO2 in cancer cells.

Hypoxia is the most prominent feature in human solid tumors and induces activation of hypoxia-inducible factors and their downstream genes to promote cancer progression. However, whether and how hypoxia regulates overall mRNA homeostasis is unclear. Here we show that hypoxia inhibits global-mRNA decay in cancer cells. Mechanistically, hypoxia induces the interaction of AGO2 with LUBAC, the linear ubiquitin chain assembly complex, which co-localizes with miRNA-induced silencing complex and in turn catalyzes AGO2 occurring Met1-linked linear ubiquitination (M1-Ubi). A series of biochemical experiments reveal that M1-Ubi of AGO2 restrains miRNA-mediated gene silencing. Moreover, combination analyses of the AGO2-associated mRNA transcriptome by RIP-Seq and the mRNA transcriptome by RNA-Seq confirm that AGO2 M1-Ubi interferes miRNA-targeted mRNA recruiting to AGO2, and thereby facilitates accumulation of global mRNAs. By this mechanism, short-term hypoxia may protect overall mRNAs and enhances stress tolerance, whereas long-term hypoxia in tumor cells results in seriously changing the entire gene expression profile to drive cell malignant evolution.