Jianxiu Yu

lncRNA H19/miR-675 axis represses prostate cancer metastasis by targeting TGFBI

Prostate cancer is a leading cause of cancer‐related mortality in men worldwide and there is a lack of effective treatment options for advanced (metastatic) prostate cancer. Currently, limited knowledge is available concerning the role of long non‐coding RNAs in prostate cancer metastasis. In this study, we found that long non‐coding RNA H19 (H19) and H19‐derived microRNA‐675 (miR‐675) were significantly downregulated in the metastatic prostate cancer cell line M12 compared with the non‐metastatic prostate epithelial cell line P69. Upregulation of H19 in P69 and PC3 cells significantly increased the level of miR‐675 and repressed cell migration; however, ectopic expression of H19 in M12 cells could not increase the level of miR‐675 and therefore had no effect on cell migration. Furthermore, we found that the expression level of either H19 or miR‐675 in P69 cells was negatively associated with the expression of transforming growth factor β induced protein (TGFBI), an extracellular matrix protein involved in cancer metastasis. Dual luciferase reporter assays showed that miR‐675 directly bound with 3′UTR of TGFBI mRNA to repress its translation. Taken together, we show for the first time that the H19–miR‐675 axis acts as a suppressor of prostate cancer metastasis, which may have possible diagnostic and therapeutic potential for advanced prostate cancer.

Suppression of MIM by microRNA-182 activates RhoA and promotes breast cancer metastasis

Breast cancer is the most common type of cancer among women worldwide, and metastasis represents the most devastating stage of the disease. Recent studies have revealed that microRNAs (miRNA) have critical roles to regulate cancer cell invasion and metastasis. Here we present evidence to show the role of miR-182 in breast cancer metastasis. miR-182 is upregulated in the malignant cell line variants of both human MCF10 and mouse 4T1 series. Ectopic expression of miR-182 enhanced breast cancer cell motility and invasiveness, whereas miR-182 inhibition resulted in opposite changes. In nude mice, miR-182 led to increased pulmonary colonization of cancer cells. We further demonstrated that miR-182 directly targets MIM (Missing in Metastasis), which suppresses metastasis by inhibiting ras homolog family member A (RhoA) activity and stress fiber formation in breast cancer cells. Restoring MIM expression completely blocked the pro-metastasis function of miR-182, while RhoA inhibition reversed the phenotypes of both miR-182 overexpression and MIM knockdown. In breast tumor samples, miR-182 induction is linked to downregulation of MIM, RhoA activation and poor prognosis. Hence, our data delineates the molecular pathway by which miR-182 promotes breast cancer invasion and metastasis, and may have important implication for the treatment of metastatic cancers.

miR-146a functions as a tumor suppressor in prostate cancer by targeting Rac1

miR‐146a (miR‐146a‐5p) has been reported to be aberrantly expressed in different types of cancers, the current knowledge about the role of miR‐146a in prostate cancer is still limited.

MiR-130b suppresses prostate cancer metastasis through down-regulation of MMP2

Prostate cancer (PCa) is the most prevalent malignant carcinoma among males in western countries. Currently no treatments can cure advanced prostate cancers, so new diagnostic and therapeutic strategies are in urgent need. At present limited knowledge is available concerning the roles of dysregulated microRNAs in prostate cancer metastasis. In this study, we found that the expression of miR‐130b was significantly down‐regulated in prostate cancer cell lines and clinical prostate cancer tissues. Enforced over‐expression of miR‐130b in prostate cancer cells suppressed whereas knock‐down of miR‐130b increased cell migration and invasion. Using mouse model, we revealed that miR‐130b‐expressed prostate cancer cells displayed significant reduction in tumor metastasis. Furthermore, we identified and validated matrix metalloproteinase‐2 (MMP2) as a direct target of miR‐130b. Ectopic expression of MMP2 rescued miR‐130b‐suppressed cell migration and invasion, and knock‐down of MMP2 antagonized the effect of silencing miR‐130b.Taken together, our data reveal for the first time that miR‐130b exerts a suppressive effect in prostate cancer metastasis through down‐regulation of MMP2.

SUMOylation of Grb2 enhances the ERK activity by increasing its binding with Sos1

BackgroundGrb2 (Growth factor receptor-bound protein 2) is a key adaptor protein in maintaining the ERK activity via linking Sos1 (Son of sevenless homolog 1) or other proteins to activated RTKs, such as EGFR. Currently, little knowledge is available concerning the post-translational modification (PTM) of Grb2 except for its phosphorylation. Since emerging evidences have highlighted the importance of SUMOylation (Small ubiquitin-related modifier), a reversible PTM, in modulating protein functions, we wondered if Grb2 could be SUMOylated and thereby influences its functions especially involved in the Ras/MEK/ERK pathway.MethodsSUMOylation of Grb2 was analyzed with the in vivo SUMOylation assay using the Ni2+-NTA affinity pulldown and the in vitro E.coli-based SUMOylation assay. To test the ERK activity and cell transformation, the murine fibroblast cell line NIH/3T3 and the murine colon cancer cell line CMT-93 were used for the experiments including Grb2 knockdown, ectopic re-expression, cell transformation and migration. Immunoprecipitation (IP) was employed for seeking proteins that interact with SUMO modified Grb2. Xenograft tumor model in mice was conducted to verify that Grb2 SUMOylation regulated tumorigenesis in vivo.ResultsGrb2 can be SUMOylated by SUMO1 at lysine 56 (K56), which is located in the linker region between the N-terminal SH3 domain and the SH2 domain. Knockdown of Grb2 reduced the ERK activity and suppressed cell motility and tumorigenesis in vitro and in vivo, which were all rescued by stable ectopic re-expression of wild-type Grb2 but not the mutant Grb2K56R. Furthermore, Grb2 SUMOylation at K56 increased the formation of Grb2-Sos1 complex, which sequentially leads to the activation of Ras/MEK/MAPK pathway.ConclusionsOur results provide evidences that Grb2 is SUMOylated in vivo and this modification enhances ERK activities via increasing the formation of Grb2-Sos1 complex, and may consequently promote cell motility, transformation and tumorigenesis.

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 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.

SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function

Abstract 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.

Regulation of RPTPα–c‐Src signalling pathway by miR‐218

Receptor protein tyrosine phosphatase alpha (RPTPα), an activator of Src family kinases, is found significantly overexpressed in human cancer tissues. However, little is known about the regulation of RPTPα expression. miRNAs target multiple genes and play important roles in many cancer processes. Here, we identified a miRNA, miR‐218 that binds directly to the 3′‐UTR of RPTPα. Ectopic overexpression of miR‐218 decreased RPTPα protein leading to decreased dephosphorylation of c‐Src and decreased tumour growth in vitro and in vivo. A feedback loop between c‐Src and miR‐218 was revealed where c‐Src inhibits transcription of SLIT2, which intronically hosts miR‐218. These results show a novel regulatory pathway for RPTPα–c‐Src signalling.

Nuclear PTEN safeguards pre-mRNA splicing to link Golgi apparatus for its tumor suppressive role

Dysregulation of pre-mRNA alternative splicing (AS) is closely associated with cancers. However, the relationships between the AS and classic oncogenes/tumor suppressors are largely unknown. Here we show that the deletion of tumor suppressor PTEN alters pre-mRNA splicing in a phosphatase-independent manner, and identify 262 PTEN-regulated AS events in 293T cells by RNA sequencing, which are associated with significant worse outcome of cancer patients. Based on these findings, we report that nuclear PTEN interacts with the splicing machinery, spliceosome, to regulate its assembly and pre-mRNA splicing. We also identify a new exon 2b in GOLGA2 transcript and the exon exclusion contributes to PTEN knockdown-induced tumorigenesis by promoting dramatic Golgi extension and secretion, and PTEN depletion significantly sensitizes cancer cells to secretion inhibitors brefeldin A and golgicide A. Our results suggest that Golgi secretion inhibitors alone or in combination with PI3K/Akt kinase inhibitors may be therapeutically useful for PTEN-deficient cancers.Cytoplasmic PTEN is a tumor suppressor that antagonises PI3K signalling. Here, the authors show that nuclear PTEN can interact with the spliceosomal proteins and drive pre-mRNA splicing in a phosphatase-independent manner, in particular, PTEN depletion promotes Golgi extension and secretion through GOLGA2 exon skipping.