SUMOylation modulates the LIN28A-let-7 signaling pathway in response to cellular stresses in cancer cells.

LIN28A is a conserved RNA-binding protein that inhibits the biogenesis of let-7 microRNAs, thus promoting cancer progression. However, mechanisms underlying the activation of the LIN28A-let-7 signaling pathway remain poorly understood. Here, we show that LIN28A is SUMOylated in vivo and in vitro at K15, which is increased by hypoxia but reduced by chemotherapy drugs such as Cisplatin and Paclitaxel. SUMOylation of LIN28A aggravates its inhibition of let-7 maturation, resulting in a stark reduction in let-7, which promotes cancer cell proliferation, migration, invasion, and tumor growth in vivo. Mechanistically, SUMOylation of LIN28A increases its binding affinity with the precursor let-7 (pre-let-7), which subsequently enhances LIN28A-mediated recruitment of terminal uridylyltransferase TUT4 and simultaneously blocks DICER processing of pre-let-7, thereby reducing mature let-7 production. These effects are abolished in SUMOylation-deficient mutant LIN28A-K15R. In summary, these findings shed light on a novel mechanism by which SUMOylation could regulate the LIN28A-let-7 pathway in response to cellular stress in cancer cells.

Correction: Atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 controls the core epithelial-to-mesenchymal transition-inducing transcription factors.

[This corrects the article DOI: 10.18632/oncotarget.2825.].

SUMOylation of Csk Negatively Modulates its Tumor Suppressor Function.

Csk, a non-receptor tyrosine kinase, serves as an indispensable negative regulator of the Src family kinases (SFKs). However, little is known about regulation of Csk expression so far. SUMOylation, a reversible post-translational modification, has been shown to regulate many biological processes especially in tumor progression. Here we report that Csk is covalently modified by SUMO1 at lysine 53 (K53) both in vitro and in vivo. Treatment with hydrogen peroxide inhibited this modification to a certain extent, but PIAS3, identified as the main specific SUMO E3 ligase for Csk, could significantly enhance SUMO1-Csk level. In addition, phosphorylation at Ser364, the active site in Csk, had no effect on this modification. Ectopic expression of SUMO-defective mutant, Csk K53R, inhibited tumor cell growth more potentially than Csk wild-type. Consistent with the biological phenotype, the SUMO modification of Csk impaired its activity to interact with Cbp (Csk binding protein) leading to decreased c-Src phosphorylation at Y527. Our results suggest that SUMOylation of Csk mainly at lysine 53 negatively modulates its tumor suppressor function by reducing its binding with Cbp and consequently, inducing c-Src activation.

Acetylation of AGO2 promotes cancer progression by increasing oncogenic miR-19b biogenesis.

Argonaute2 (AGO2) is an effector of small RNA mediated gene silencing. Increasing evidence show that post-translational modifications of AGO2 can change miRNA activity at specific or global levels. Among the six mature miRNAs that are encoded by miR-17-92, miR-19b1 is the most powerful to exert the oncogenic properties of the entire cluster. Here we identify that AGO2 can be acetylated by P300/CBP and deacetylated by HDAC7, and that acetylation occurs at three sites K720, K493, and K355. Mutation of K493R/K720R, but not K355R at AGO2, inhibits miR-19b biogenesis. We demonstrate that acetylation of AGO2 specifically increases its recruiting pre-miR-19b1 to form the miPDC (miRNA precursor deposit complex), thereby to enhance miR-19b maturation. The motif UGUGUG in the terminal-loop of pre-miR-19b1, as a specific processing feature that is recognized and bound by acetylated AGO2, is essential for the assembly of miRISC (miRNA-induced silencing complex) loading complex. Analyses on public clinical data, xenograft mouse models, and IHC and ISH staining of lung cancer tissues, further confirm that the high levels of both AGO2 acetylation and miR-19b correlate with poor prognosis in lung cancer patients. Our finding reveals a novel function of AGO2 acetylation in increasing oncogenic miR-19b biogenesis and suggests that modulation of AGO2 acetylation has potential clinical implications.

Sumoylation of EphB1 Suppresses Neuroblastoma Tumorigenesis via Inhibiting PKCγ Activation.

BACKGROUND/AIMS: An increasing number of studies have linked erythropoietin-producing hepatocellular carcinoma (Eph) family receptor tyrosine kinases to cancer progression. However, little knowledge is available about the regulation of their functions in cancer. METHODS: SUMOylation was analyzed by performing Ni2+-NTA pull-down assay and immunoprecipitation. Cell proliferation, anchorage-independent growth, and tumorigenesis in vivo were examined by cell counting kit-8, soft agar colony formation assay, and a xenograft tumor mouse model, respectively. RESULTS: We found that EphB1 was post-translationally modified by the small ubiquitin-like modifier (SUMO) protein at lysine residue 785. Analysis of wild-type EphB1 and SUMOylation-deficient EphB1 K785R mutant revealed that SUMOylation of EphB1 suppressed cell proliferation, anchorage-independent cell growth, and xenograft tumor growth. Mechanistic study showed that SUMOylation of EphB1 repressed activation of its downstream signaling molecule PKCγ, and consequently inhibited tumorigenesis. A reciprocal regulatory loop between PKCγ and SUMOylation of EphB1 was also characterized. CONCLUSION: Our findings identify SUMO1 as a novel key regulator of EphB1-mediated tumorigenesis.

SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function.

The methyltransferase like 3 (METTL3) is a key component of the large N6-adenosine-methyltransferase complex in mammalian responsible for N6-methyladenosine (m6A) modification in diverse RNAs including mRNA, tRNA, rRNA, small nuclear RNA, microRNA precursor and long non-coding RNA. However, the characteristics of METTL3 in activation and post-translational modification (PTM) is seldom understood. Here we find that METTL3 is modified by SUMO1 mainly at lysine residues K177, K211, K212 and K215, which can be reduced by an SUMO1-specific protease SENP1. SUMOylation of METTL3 does not alter its stability, localization and interaction with METTL14 and WTAP, but significantly represses its m6A methytransferase activity resulting in the decrease of m6A levels in mRNAs. Consistently with this, the abundance of m6A in mRNAs is increased with re-expression of the mutant METTL3-4KR compared to that of wild-type METTL3 in human non-small cell lung carcinoma (NSCLC) cell line H1299-shMETTL3, in which endogenous METTL3 was knockdown. The alternation of m6A in mRNAs and subsequently change of gene expression profiles, which are mediated by SUMOylation of METTL3, may directly influence the soft-agar colony formation and xenografted tumor growth of H1299 cells. Our results uncover an important mechanism for SUMOylation of METTL3 regulating its m6A RNA methyltransferase activity.

Src SUMOylation Inhibits Tumor Growth Via Decreasing FAK Y925 Phosphorylation.

Src, a non-receptor tyrosine kinase protein, plays a critical role in cell proliferation and tumorigenesis. SUMOylation, a reversible ubiquitination-like post-translational modification, is vital for tumor progression. Here, we report that the Src protein can be SUMOylated at lysine 318 both in vitro and in vivo. Hypoxia can induce a decrease of Src SUMOylation along with an increase of Y419 phosphorylation, a phosphorylation event required for Src activation. On the other hand, treatment with hydrogen peroxide can enhance Src SUMOylation. Significantly, ectopic expression of SUMO-defective mutation, Src K318R, promotes tumor growth more potently than that of wild-type Src, as determined by migration assay, soft agar assay, and tumor xenograft experiments. Consistently, Src SUMOylation leads to a decrease of Y925 phosphorylation of focal adhesion kinase (FAK), an established regulatory event of cell migration. Our results suggest that SUMOylation of Src at lysine 318 negatively modulate its oncogenic function by, at least partially, inhibiting Src-FAK complex activity.

miR186 suppresses prostate cancer progression by targeting Twist1.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths in north American men, and most its related deaths are due to advanced and metastatic PCa. However, the molecular mechanisms underlying PCa progression are still unclear. Here we use a pair of prostate cell lines P69/M12, which have the same genetic background and the highly metastatic cell line M12 is a subline derived from P69, to identify the pathogenesis of PCa. We find that a key miRNA--miR186 is significantly reduced in M12 compared to that in P69. Further, we validate that miR186 is also downregulated in human PCa specimens, most significantly in the metastatic patient specimens. The low miR186 expression is correlated with poor patient survival. Through knockdown or overexpression of miR186 in PCa cell lines, we discover that miR186 strongly inhibits cell motility, invasive, soft-agar colony formation, 3D culture growth and vasculogenic mimicry (VM) formation capacity, as well as the epithelial-to-mesenchymal transition (EMT) process by downregulation of its target Twist1. Moreover, the inverse relationship between the expression levels of miR186 and Twist1 is confirmed in vivo tumor metastasis experiment and clinical specimens. Taken together, our findings demonstrate an important role of miR186/Twist1 axis in the regulation of PCa progression, suggesting a potential application of miR186/Twist1 in PCa treatment.

SUMOylation of TARBP2 regulates miRNA/siRNA efficiency.

Small RNA-induced gene silencing is essential for post-transcriptional regulation of gene expression; however, it remains unclear how miRNA/siRNA efficiency is regulated. Here we show that TARBP2 is SUMOylated at K52, which can be enhanced by its phosphorylation. This modification can stabilize TARBP2 via repressing its K(48)-linked ubiquitination. We find that TARBP2 SUMOylation does not influence the overall production of mature miRNAs, but it regulates miRNA/siRNA efficiency. SUMOylated TARBP2 recruits Ago2 to constitute the RNA-induced silencing complex (RISC)-loading complex (RLC), and simultaneously promotes more pre-miRNAs to load into the RLC. Consequently, Ago2 is stabilized and miRNAs/siRNAs bound by TARBP2/Dicer is effectively transferred to Ago2. Thus, these processes lead to the formation of the effective RISC for RNA interference (RNAi). Collectively, our data suggest that SUMOylation of TARBP2 is required for regulating miRNA/siRNA efficiency, which is a general mechanism of miRNA/siRNA regulation.

SUMOylation at K707 of DGCR8 controls direct function of primary microRNA.

DGCR8 (DiGeorge syndrome critical region gene 8) is essential for primary microRNA (pri-miRNA) processing in the cell nucleus. It specifically combines with Drosha, a nuclear RNase III enzyme, to form the Microprocessor complex (MC) that cleaves pri-miRNA to precursor miRNA (pre-miRNA), which is further processed to mature miRNA by Dicer, a cytoplasmic RNase III enzyme. Increasing evidences suggest that pri-/pre-miRNAs have direct functions in regulation of gene expression, however the underlying mechanism how it is fine-tuned remains unclear. Here we find that DGCR8 is modified by SUMO1 at the major site K(707), which can be promoted by its ERK-activated phosphorylation. SUMOylation of DGCR8 enhances the protein stability by preventing the degradation via the ubiquitin proteasome pathway. More importantly, SUMOylation of DGCR8 does not alter its association with Drosha, the MC activity and miRNA biogenesis, but rather influences its affinity with pri-miRNAs. This altered affinity of DGCR8 with pri-miRNAs seems to control the direct functions of pri-miRNAs in recognition and repression of the target mRNAs, which is evidently linked to the DGCR8 function in regulation of tumorigenesis and cell migration. Collectively, our data suggest a novel mechanism that SUMOylation of DGCR8 controls direct functions of pri-miRNAs in gene silencing.

Atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 controls the core epithelial-to-mesenchymal transition-inducing transcription factors.

Epithelial-mesenchymal transition (EMT) plays a critical role in the development of tumor metastases by enhancing migration/invasion. One of the hallmarks of EMT is loss of E-cadherin and gain of N-cadherin expression, which are regulated by the core EMT-inducing transcription factors (EMT-TFs), such as Zeb1/2, Snai1/2 and Twist1. Here, we find that EMT-TFs can be dynamically degraded by an atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 (SPFFbxo45) through the ubiquitin proteasome system (UPS). The key step is recognition of EMT-TFs by Fbxo45 through its SPRY domain for Zeb2, or F-box domain for the other three EMT-TFs Snai1, Snai2 and Twist1, respectively. The K48-linkaged ubiquitination capability on Zeb2 relies on its functional SBD domain. In addition, miR-27a* can directly down-regulate the expression of Fbxo45, preventing degradation of EMT-TFs and thus ensuring EMT phenotype. We suggest that Fbxo45 is a key node of the miR-27a*/Fbxo45/EMT-TFs signaling axis.