Somatic MAP3K3 mutation defines a subclass of cerebral cavernous malformation.

Cerebral cavernous malformations (CCMs) are vascular disorders that affect up to 0.5% of the total population. About 20% of CCMs are inherited because of familial mutations in CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10, whereas the etiology of a majority of simplex CCM-affected individuals remains unclear. Here, we report somatic mutations of MAP3K3, PIK3CA, MAP2K7, and CCM genes in CCM lesions. In particular, somatic hotspot mutations of PIK3CA are found in 11 of 38 individuals with CCMs, and a MAP3K3 somatic mutation (c.1323C>G [p.Ile441Met]) is detected in 37.0% (34 of 92) of the simplex CCM-affected individuals. Strikingly, the MAP3K3 c.1323C>G mutation presents in 95.7% (22 of 23) of the popcorn-like lesions but only 2.5% (1 of 40) of the subacute-bleeding or multifocal lesions that are predominantly attributed to mutations in the CCM1/2/3 signaling complex. Leveraging mini-bulk sequencing, we demonstrate the enrichment of MAP3K3 c.1323C>G mutation in CCM endothelium. Mechanistically, beyond the activation of CCM1/2/3-inhibited ERK5 signaling, MEKK3 p.Ile441Met (MAP3K3 encodes MEKK3) also activates ERK1/2, JNK, and p38 pathways because of mutation-induced MEKK3 kinase activity enhancement. Collectively, we identified several somatic activating mutations in CCM endothelium, and the MAP3K3 c.1323C>G mutation defines a primary CCM subtype with distinct characteristics in signaling activation and magnetic resonance imaging appearance.

Somatic variants of MAP3K3 are sufficient to cause cerebral and spinal cord cavernous malformations.

Cerebral cavernous malformations (CCMs) and spinal cord cavernous malformations (SCCMs) are common vascular abnormalities of the CNS that can lead to seizure, haemorrhage and other neurological deficits. Approximately 85% of patients present with sporadic (versus congenital) CCMs. Somatic mutations in MAP3K3 and PIK3CA were recently reported in patients with sporadic CCM, yet it remains unknown whether MAP3K3 mutation is sufficient to induce CCMs. Here we analysed whole-exome sequencing data for patients with CCM and found that ∼40% of them have a single, specific MAP3K3 mutation [c.1323C>G (p.Ile441Met)] but not any other known mutations in CCM-related genes. We developed a mouse model of CCM with MAP3K3I441M uniquely expressed in the endothelium of the CNS. We detected pathological phenotypes similar to those found in patients with MAP3K3I441M. The combination of in vivo imaging and genetic labelling revealed that CCMs were initiated with endothelial expansion followed by disruption of the blood-brain barrier. Experiments with our MAP3K3I441M mouse model demonstrated that CCM can be alleviated by treatment with rapamycin, the mTOR inhibitor. CCM pathogenesis has usually been attributed to acquisition of two or three distinct genetic mutations involving the genes CCM1/2/3 and/or PIK3CA. However, our results demonstrate that a single genetic hit is sufficient to cause CCMs.

Endothelial hyperactivation of mutant MAP3K3 induces cerebral cavernous malformation enhanced by PIK3CA GOF mutation.

Cerebral cavernous malformations (CCMs) refer to a common vascular abnormality that affects up to 0.5% of the population. A somatic gain-of-function mutation in MAP3K3 (p.I441M) was recently reported in sporadic CCMs, frequently accompanied by somatic activating PIK3CA mutations in diseased endothelium. However, the molecular mechanisms of these driver genes remain elusive. In this study, we performed whole-exome sequencing and droplet digital polymerase chain reaction to analyze CCM lesions and the matched blood from sporadic patients. 44 of 94 cases harbored mutations in KRIT1/CCM2 or MAP3K3, of which 75% were accompanied by PIK3CA mutations (P = 0.006). AAV-BR1-mediated brain endothelial-specific MAP3K3I441M overexpression induced CCM-like lesions throughout the brain and spinal cord in adolescent mice. Interestingly, over half of lesions disappeared at adulthood. Single-cell RNA sequencing found significant enrichment of the apoptosis pathway in a subset of brain endothelial cells in MAP3K3I441M mice compared to controls. We then demonstrated that MAP3K3I441M overexpression activated p38 signaling that is associated with the apoptosis of endothelial cells in vitro and in vivo. In contrast, the mice simultaneously overexpressing PIK3CA and MAP3K3 mutations had an increased number of CCM-like lesions and maintained these lesions for a longer time compared to those with only MAP3K3I441M. Further in vitro and in vivo experiments showed that activating PI3K signaling increased proliferation and alleviated apoptosis of endothelial cells. By using AAV-BR1, we found that MAP3K3I441M mutation can provoke CCM-like lesions in mice and the activation of PI3K signaling significantly enhances and maintains these lesions, providing a preclinical model for the further mechanistic and therapeutic study of CCMs.

Angioarchitecture and genetic variants of spinal cord cavernous malformations and associated developmental venous anomalies: a case report.

Cavernous malformations (CM) have long been considered congenital of central nervous system, while the mechanism of CMs detailed development process associated with genetic factors remains unclear. We reported an uncommon case which suffered spinal cord cavernous malformations. In this work, representative samples were obtained, and the sequenced results were described for the first time. A 9-year-old boy was found oblique shoulder with slightly weakness of left limbs; MRI indicated spinal cord cavernous malformations (CMs) located at the C4-C6 vertebral level. On genetic analysis, a shared mutation of PIK3CA (p.H1047R) in CMs and associated developmental venous anomalies (DVAs) was detected, with a different abundance (2% and 7%, respectively), and a somatic mutation of MAP3K3 (p.I441M) was detected in the CM tissue samples. This case provides better knowledge of the formation history and genetic triggers of the DVA-associated CMs. This evidence allows us to speculate the developmental history of the CM lesion: The DVA with PIK3CA mutation might be genetic precursor, and then the associated CM could be derived from terminal cell population of the DVA by acquiring a somatic mutation in MAP3K3.

Somatic MAP3K3 and PIK3CA mutations in sporadic cerebral and spinal cord cavernous malformations.

Cavernous malformations affecting the CNS occur in ∼0.16-0.4% of the general population. The majority (85%) of cavernous malformations are in a sporadic form, but the genetic background of sporadic cavernous malformations remains enigmatic. Of the 81 patients, 73 (90.1%) patients were detected carrying somatic missense variants in two genes: MAP3K3 and PIK3CA by whole-exome sequencing. The mutation spectrum correlated with lesion size (P = 0.001), anatomical distribution (P < 0.001), MRI appearance (P = 0.004) and haemorrhage events (P = 0.006). PIK3CA mutation was a significant predictor of overt haemorrhage events (P = 0.003, odds ratio = 11.252, 95% confidence interval = 2.275-55.648). Enrichment of endothelial cell population was associated with a higher fractional abundance of the somatic mutations. Overexpression of the MAP3K3 mutation perturbed angiogenesis of endothelial cell models in vitro and zebrafish embryos in vivo. Distinct transcriptional signatures between different genetic subgroups of sporadic cavernous malformations were identified by single cell RNA sequencing and verified by pathological staining. Significant apoptosis in MAP3K3 mutation carriers and overexpression of GDF15 and SERPINA5 in PIK3CA mutation carriers contributed to their phenotype. We identified activating MAP3K3 and PIK3CA somatic mutations in the majority (90.1%) of sporadic cavernous malformations and PIK3CA mutations could confer a higher risk for overt haemorrhage. Our data provide insights into genomic landscapes, propose a mechanistic explanation and underscore the possibility of a molecular classification for sporadic cavernous malformations.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

MiroRNA-188 Acts as Tumor Suppressor in Non-Small-Cell Lung Cancer by Targeting MAP3K3.

Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer. MicroRNAs have been increasingly implicated in NSCLC and may serve as novel therapeutic targets to combat cancer. Here we investigated the functional implication of miR-188 in NSCLC. We first analyzed miR-188 expression in both NSCLC clinical samples and cancer cell lines. Next we investigated its role in A549 and H2126 cells with cell proliferation, migration, and apoptosis assays. To extend the in vitro study, we employed both xenograft model and LSL- K-ras G12D lung cancer model to examine the role of miR-188 in tumorigenesis. Last we tested MAP3K3 as miR-188 target in NSCLC model. MiR-188 expression was significantly downregulated at the NSCLC tumor sites and lung cancer cells. In vitro transfection of miR-188 reduced cell proliferation and migration potential and promoted cell apoptosis. In xenograft model, miR-188 inhibited tumor growth derived from cancer cells. Intranasal miR-188 administration reduced tumor formation in NSCLC animal model. MAP3K3 was validated as direct target of miR-188. Knocking down MAP3K3 in mice also inhibited tumorigenesis in LSL- K-ras G12D model. Our results demonstrate that miR-188 and its downstream target MAP3K3 could be a potential therapeutic target for NSCLC.

microRNA-122 regulates hypoxia-inducible factor-1 and vimentin in hepatocytes and correlates with fibrosis in diet-induced steatohepatitis.

BACKGROUND & AIMS: miR-122 is the most abundant miRNA in the liver particularly in hepatocytes where it targets cholesterol metabolism. Steatosis, a key component of non-alcoholic fatty liver disease, is regulated by hypoxia-inducible factor-1α (HIF-1α). Here, we hypothesized that reduced miR-122 has a pathogenic role in steatohepatitis. METHODS: miR-122 and its target genes were evaluated in mouse livers and/or isolated hepatocytes after methionine-choline-deficient (MCD) or methionine-choline-supplemented (MCS) diet. RESULTS: Liver and hepatocyte miR-122 expression was significantly decreased in steatohepatitis. A maximum reduction in miR-122 occurred at the fibrosis stage (8 weeks of MCD diet). MAP3K3, a miR-122 target gene, was induced at all stages of non-alcoholic steatohepatitis (NASH; 3-8 weeks) only at the mRNA level. Increased NF-κB activation was found in MCD diet-fed mice and MAP3K3 regulated the NF-κB DNA binding in naive hepatocytes. HIF-1α mRNA and DNA binding and expression of the HIF-1α target gene, profibrotic lysyl oxidase, was increased in advanced steatohepatitis (8 weeks). In addition, increase in vimentin and Sirius red staining (liver fibrosis) was found at 8 weeks of MCD diet. Using miR-122 overexpression and inhibition approaches, we confirmed that HIF-1α, vimentin and MAP3K3 are novel miR-122 targets in hepatocytes. We report transcriptional repression of miR-122 in NASH. Decreased liver miR-122 was associated with elevated circulating miR-122 in both exosome-rich and protein-rich serum fractions. CONCLUSIONS: Our novel data suggest that decreased liver miR-122 contributes to upregulation of modulators of tissue remodelling (HIF-1α, vimentin and MAP3K3) and might play a role in NASH-induced liver fibrosis.

Amplification and over-expression of MAP3K3 gene in human breast cancer promotes formation and survival of breast cancer cells.

Gene amplifications in the 17q chromosomal region are observed frequently in breast cancers. An integrative bioinformatics analysis of this region nominated the MAP3K3 gene as a potential therapeutic target in breast cancer. This gene encodes mitogen-activated protein kinase kinase kinase 3 (MAP3K3/MEKK3), which has not yet been reported to be associated with cancer-causing genetic aberrations. We found that MAP3K3 was amplified in approximately 8-20% of breast cancers. Knockdown of MAP3K3 expression significantly inhibited cell proliferation and colony formation in MAP3K3-amplified breast cancer cell lines MCF-7 and MDA-MB-361 but not in MAP3K3 non-amplified breast cancer cells. Knockdown of MAP3K3 expression in MAP3K3-amplified breast cancer cells sensitized breast cancer cells to apoptotic induction by TNFα and TRAIL, as well as doxorubicin, VP-16 and fluorouracil, three commonly used chemotherapeutic drugs for treating breast cancer. In addition, ectopic expression of MAP3K3, in collaboration with Ras, induced colony formation in both primary mouse embryonic fibroblasts and immortalized human breast epithelial cells (MCF-10A). Combined, these results suggest that MAP3K3 contributes to breast carcinogenesis and may endow resistance of breast cancer cells to cytotoxic chemotherapy. Therefore, MAP3K3 may be a valuable therapeutic target in patients with MAP3K3-amplified breast cancers, and blocking MAP3K3 kinase activity with a small molecule inhibitor may sensitize MAP3K3-amplified breast cancer cells to chemotherapy.

Treadmill training improves lung function and inhibits alveolar cell apoptosis in spinal cord injured rats.

Secondary lung injury after SCI is a major cause of patient mortality, with apoptosis playing a key role. This study aimed to explore the impact of treadmill training and miR145-5p on the MAPK/Erk signaling pathway and apoptosis in rats with complete SCI. SD rats were used to establish T10 segmental complete SCI models and underwent treadmill training 3, 7, or 14 days postinjury. Various techniques including arterial blood gas analysis, lung wet/dry weight ratio, HE staining, immunofluorescence staining, immunohistochemical staining, qRT-PCR, and Western blotting were employed to assess alterations in lung function and the expression levels of crucial apoptosis-related factors. In order to elucidate the specific mechanism, the impact of miR145-5p on the MAPK/Erk pathway and its role in apoptosis in lung cells were confirmed through miR145-5p overexpression and knockdown experiments. Following spinal cord injury (SCI), an increase in apoptosis, activation of the MAPK/Erk pathway, and impairment of lung function were observed in SCI rats. Conversely, treadmill training resulted in a reduction in alveolar cell apoptosis, suppression of the MAPK/Erk pathway, and enhancement of lung function. The gene MAP3K3 was identified as a target of miR145-5p. The influence of miR145-5p on the MAPK/Erk pathway and its impact on apoptosis in alveolar cells were confirmed through the manipulation of miR145-5p expression levels. The upregulation of miR145-5p in spinal cord injury (SCI) rats led to a reduction in MAP3K3 protein expression within lung tissues, thereby inhibiting the MAPK/Erk signaling pathway and decreasing apoptosis. Contrarily, rats with miR145-5p knockdown undergoing treadmill training exhibited an increase in miR145-5p expression levels, resulting in the inhibition of MAP3K3 protein expression in lung tissues, suppression of the MAPK/Erk pathway, and mitigation of lung cell apoptosis. Ultimately, the findings suggest that treadmill training may attenuate apoptosis in lung cells post-spinal cord injury by modulating the MAP3K3 protein through miR145-5p to regulate the MAPK/Erk signaling pathway.

XJ-8, a natural compound isolated from Sanguis draxonis, inhibits platelet function and thrombosis by targeting MAP3K3.

BACKGROUND: Vascular injury initiates rapid platelet activation, which is critical for haemostasis, while it also causes fatal thrombotic diseases, such as myocardial infarction or ischemic stroke. OBJECTIVES: To study the inhibitory effects and underlying mechanisms of XJ-8, a natural compound isolated from Sanguis draxonis, on platelet activation and thrombosis. METHODS: The regulatory effects of XJ-8 on the dense granule release, thromboxane A2 (TxA2 ) synthesis, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by multiple agonists were investigated in in vitro experiments. The effects of XJ-8 on bleeding time and FeCl3 -induced carotid artery thrombosis were also evaluated in in vivo experiments. Furthermore, we investigated the underlying mechanisms by which XJ-8 exerted its pharmacological effects. RESULTS: XJ-8 not only significantly inhibited the dense granule release, TxA2 synthesis, and aggregation of platelets induced by multiple agonists, but also exerted extending effects on bleeding time and therapeutic effects on thrombotic disease. In addition, XJ-8 selectively and moderately inhibited the activity of mitogen-activated protein kinase kinase kinase 3 (MAP3K3) and the activation of signalling pathways downstream MAP3K3, which play important roles in platelet activation. CONCLUSION: XJ-8 can inhibit platelet function and thrombosis by targeting MAP3K3 and has potential to be developed into a novel therapeutic agent for the treatment of thrombotic diseases.

LncRNA OGFRP1 promotes cell proliferation and suppresses cell radiosensitivity in gastric cancer by targeting the miR-149-5p/MAP3K3 axis.

Gastric cancer (GC) is an aggressive malignancy with high incidence and mortality. Radiotherapy is a common treatment for patients with advanced GC. Many long noncoding RNAs (lncRNAs) have been verified to affect the radiosensitivity of multiple cancers in previous studies. Nevertheless, whether lncRNA opioid growth factor receptor pseudogene 1 (OGFRP1) affects the radiosensitivity of GC has not been determined. We hypothesized that OGFRP1 might affect cellular processes in GC development. The present study aims to explore the role of OGFRP1 in GC development. First, high expression of OGFRP1 in GC tissues and cells was determined through RT-qPCR. Subsequently, functional assays including colony formation assays, 5-Ethynyl-2'-deoxyuridine assays and flow cytometry analyses were performed to probe the biological functions of OGFRP1 in GC. Specifically, the effect of OGFRP1 on the radiosensitivity of GC cells was detected. Subsequently, with the help of the starBase tool, we found that miR-149-5p might bind to OGFRP1, which was confirmed through a luciferase reporter assay. Furthermore, we identified that MAP3K3 was targeted by miR-149-5p in GC cells. Finally, the results from rescue experiments validated that enhanced MAP3K3 expression counteracted the effect of OGFRP1 silencing on GC cell proliferation, apoptosis and radiosensitivity. Overall, OGFRP1 was determined to promote GC cell proliferation while suppressing cell apoptosis and radiosensitivity by regulating the miR-149-5p/MAP3K3 axis.

Tbc1d10c is a selective, constitutive suppressor of the CD8 T-cell anti-tumor response.

Cancer immunotherapy approaches target signaling pathways that are highly synonymous between CD4 and CD8 T-cell subsets and, therefore, often stimulate nonspecific lymphocyte activation, resulting in cytotoxicity to otherwise healthy tissue. The goal of our study was to identify intrinsic modulators of basic T lymphocyte activation pathways that could discriminately bolster CD8 anti-tumor effector responses. Using a Tbc1d10c null mouse, we observed marked resistance to a range of tumor types conferred by Tbc1d10c deficiency. Moreover, tumor-bearing Tbc1d10c null mice receiving PD-1 or CTLA-4 monotherapy exhibited a 33% or 90% cure rate, respectively. While Tbc1d10c was not expressed in solid tumor cells, Tbc1d10c disruption selectively augmented CD8 T-cell activation and cytotoxic effector responses and adoptive transfer of CD8 T cells alone was sufficient to recapitulate Tbc1d10c null tumor resistance. Mechanistically, Tbc1d10c suppressed CD8 T-cell activation and anti-tumor function by intersecting canonical NF-κB pathway activation via regulation of Map3k3-mediated IKKß phosphorylation. Strikingly, none of these cellular or molecular perturbations in the NF-κB pathway were featured in Tbc1d10c null CD4 T cells. Our findings identify a Tbc1d10c-Map3k3-NF-κB signaling axis as a viable therapeutic target to promote CD8 T-cell anti-tumor immunity while circumventing CD4 T cell-associated cytotoxicity and NF-κB activation in tumor cells.

Simplex cerebral cavernous malformations with MAP3K3 mutation have distinct clinical characteristics.

Objectives: To investigate the clinical characteristics of cerebral cavernous malformations (CCMs) with MAP3K3 somatic mutation. Methods: We performed a retrospective review of our CCMs database between May 2017 and December 2019. The patients with simplex CCMs identified to harbor a MAP3K3 or CCM gene somatic mutation were included. Clinical characteristics were recorded. Univariate and multivariate logistic analyses were used to assess the risk factors associated with hemorrhage events of CCMs. To explore the underlying mechanism, we transfected MEKK3-I441M-overexpressing and CCM2-knockdown lentiviruses into human umbilical vein endothelial cells (HUVECs) and investigated thrombomodulin (TM) and tight junctions (TJs) protein expression by western blotting and immunofluorescence. Finally, immunohistochemistry was used to validate TM and TJs protein expression in surgical samples. Results: Fifty simplex CCMs patients were included, comprising 38 MAP3K3 mutations and 12 CCM gene mutations. Nine (23.7%) patients with MAP3K3 mutations and 11(91.7%) patients with CCM gene mutations exhibited overt hemorrhage, respectively. Multivariate logistic analyses revealed that MAP3K3 mutation was associated with a lower risk of hemorrhage events. In the vitro experiments, ZO-1 expression was not reduced in MEKK3-I441M-overexpressing HUVECs compared with wild type, whereas it was significantly decreased in CCM2-knockdown HUVECs compared with control. In the MEKK3-I441M-overexpressing HUVECs, TM expression was increased, and the NF-κB pathway was significantly activated. After treatment with an NF-κB signaling inhibitor, TM expression was further upregulated. Meanwhile, TM expression was increased, but the NF-κB pathway was not activated in CCM2-knockdown HUVECs. Accordingly, immunohistochemistry showed that ZO-1 expression in the MAP3K3-mutant samples was significantly higher than that in the CCM-mutant samples. TM expression in the MAP3K3-mutant lesions was significantly lower than that in the CCM-mutant samples. Conclusion: Simplex CCMs with MAP3K3 mutation occasionally present with overt hemorrhage, which is associated with the biological function of MAP3K3 mutation.

Circ_0006988 promotes the proliferation, metastasis and angiogenesis of non-small cell lung cancer cells by modulating miR-491-5p/MAP3K3 axis.

Circular RNAs (circRNAs) are related to the progression of non-small cell lung cancer (NSCLC). However, the roles and mechanism of circ_0006988 are largely unknown. The levels of circ_0006988, Low-Density Lipoprotein Receptor Class A Domain Containing 3 (LDLRAD3), microRNA-491-5p (miR-491-5p), Mitogen-Activated Protein Kinase Kinase Kinase 3 (MAP3K3) were measured using quantitative real-time polymerase-chain reaction (qRT-PCR) and western blot assay. The characteristic of circ_0006988 was analyzed by RNase R assay and Actinomycin D assay. Functional analyses were processed by Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, flow cytometry analysis, transwell assay, wound-healing assay and tube formation assay. The interactions between circ_0006988 and miR-491-5p as well as miR-491-5p and MAP3K3 were analyzed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Murine xenograft model assay was processed to verify the function of circ_0006988 in vivo. Immunohistochemistry (IHC) assay was conducted to examine the level of Ki67. Circ_0006988 abundance was increased in NSCLC tissues and cells. Circ_0006988 silencing restrained NSCLC cell proliferation, migration, invasion and angiogenesis, and induced apoptosis. Circ_0006988 sponged miR-491-5p, which directly targeted MAP3K3. MiR-491-5p overexpression repressed NSCLC cell malignant behaviors. MiR-491-5p downregulation or MAP3K3 overexpression reversed the effect of circ_0006988 silencing on NSCLC cell progression. In addition, circ_0006988 knockdown reduced xenograft tumor growth. ssCirc_0006988 contributed to the development of NSCLC by miR-491-5p/MAP3K3 axis.

CircSETDB1 knockdown inhibits the malignant progression of serous ovarian cancer through miR-129-3p-dependent regulation of MAP3K3.

BACKGROUND: Circular RNA (circRNA) is recently found to participate in the regulation of tumor progression, including ovarian cancer. However, the application of circRNA SET domain bifurcated histone lysine methyltransferase 1 (circSETDB1) as a therapeutic target in serous ovarian cancer (SOC) remains to be elucidated. Herein, circSETDB1 role in SOC malignant progression and underlying mechanism are revealed. METHODS: The expression of circSETDB1, microRNA-129-3p (miR-129-3p) and mitogen-activated protein kinase kinase kinase 3 (MAP3K3) messenger RNA (mRNA) was detected by quantitative real-time polymerase chain reaction. Protein abundance was determined by western blot analysis. Cell proliferation, apoptosis, invasion and migration were demonstrated by cell counting kit-8 and 5-Ethynyl-29-deoxyuridine assays, flow cytometry analysis, transwell invasion assay and wound-healing assay, respectively. The interaction between miR-129-3p and circSETDB1 or MAP3K3 was predicted by online database, and identified by mechanism assays. The effect of circSETDB1 knockdown on tumor formation in vivo was unveiled by mouse model experiment. RESULTS: CircSETDB1 and MAP3K3 expression were apparently upregulated, whereas miR-129-3p expression was downregulated in SOC tissues and cells in comparison with normal fallopian tube tissues or normal ovarian epithelial cells. CircSETDB1 knockdown inhibited cell proliferation, invasion and migration, but induced cell apoptosis in SOC cells. Additionally, miR-129-3p inhibitor impaired circSETDB1 silencing-mediated SOC malignant progression. MiR-129-3p repressed SOC cell processes via binding to MAP3K3. Furthermore, circSETDB1 knockdown suppressed tumor growth in vivo. CONCLUSION: CircSETDB1 silencing repressed SOC malignant progression through miR-129-3p/MAP3K3 pathway. This study supports circSETDB1 as a new therapeutic target for SOC.

1. CircSETDB1 expression was increased in SOC tissues and cells.2. CircSETDB1 silencing repressed the malignancy of SOC cells.3. CircSETDB1 mediated SOC malignant progression by interacting with miR-129-3p.4. MAP3K3 served as a target gene of miR-129-3p.5. CircSETDB1 knockdown inhibited tumor formation in vivo.

DNM3OS Facilitates Ovarian Cancer Progression by Regulating miR-193a-3p/MAP3K3 Axis.

PURPOSE: Long non-coding RNAs (lncRNAs) are essential regulators in the development of ovarian cancer (OC). Nonetheless, the function of lncRNA DNM3 opposite strand/antisense RNA (DNM3OS) in OC remains unclear. This work aimed to investigate the biological roles and underlying mechanisms of DNM3OS in OC. MATERIALS AND METHODS: Quantitative real-time polymerase chain reaction was conducted to examine DNM3OS, microRNA (miR)-193a-3p, and mitogen-activated protein kinase 3 (MAP3K3) mRNA expression in OC tissues and cell lines. Kaplan-Meier survival analysis was employed to analyze the relationship between DNM3OS expression and the prognosis of OC patients. Cell counting kit-8, 5-ethynyl-2'-deoxyuridine, and transwell experiments were conducted to monitor cell proliferation, migration, and invasion, respectively. Western blot was applied to examine epithelial-mesenchymal transition associated protein (E-cadherin and N-cadherin) expression. Luciferase reporter gene and RNA immunoprecipitation experiments were performed to confirm the relationships among DNM3OS, miR-193a-3p, and MAP3K3. Pearson's correlation analysis was adopted to analyze the correlations among DNM3OS, miR-193a-3p, and MAP3K3 mRNA. RESULTS: DNM3OS expression was remarkably increased in OC tissues and cell lines, which was associated with the unfavorable prognosis of the patients. DNM3OS overexpression enhanced OC cell proliferation, migration, and invasion; suppressed E-cadherin protein expression; and facilitated N-cadherin protein expression, while the transfection of miR-193a-3p mimics had the opposite effects. DNM3OS directly interacted with miR-193a-3p, and miR-193a-3p targeted MAP3K3 by directly binding to 3'UTR. DNM3OS could up-regulate the expression of MAP3K3 via repressing miR-193a-3p expression. CONCLUSION: DNM3OS, as an oncogenic lncRNA, increases the malignancy of OC cells via regulation of an miR-193a-3p/MAP3K3 axis.

Concurrent targeting of MAP3K3 and BRD4 by miR-3140-3p overcomes acquired resistance to BET inhibitors in neuroblastoma cells.

Neuroblastoma (NB) harboring MYCN amplification is a refractory disease with a poor prognosis. As BRD4, an epigenetic reader belonging to the bromodomain and extra terminal domain (BET) family, drives transcription of MYCN in NB cells, BET inhibitors (BETis) are considered useful for NB therapy. However, clinical trials of BETis suggested that early acquired resistance to BETis limits their therapeutic benefit. MicroRNAs are small non-coding RNAs that mediate post-transcriptional silencing of target genes. We previously identified miR-3140-3p as a potent candidate for nucleic acid therapeutics for cancer, which directly targets BRD4. We demonstrated that miR-3140-3p suppresses tumor cell growth in MYCN-amplified NB by downregulating MYCN and MYC through BRD4 suppression. We established BETi-acquired resistant NB cells to evaluate the mechanism of resistance to BETi in NB cells. We revealed that activated ERK1/2 stabilizes MYCN protein by preventing ubiquitin-mediated proteolysis via phosphorylation of MYCN at Ser62 in BETi-acquired resistant NB cells, thereby attenuating the effects of BETi in these cells. miR-3140-3p efficiently downregulated MYCN expression by directly targeting the MAP3K3-ERK1/2 pathway in addition to BRD4 suppression, inhibiting tumor cell growth in BETi-acquired resistant NB cells. This study suggests that miR-3140-3p has the potential to overcome resistance to BETi in NB.

Late-onset multiple venous malformations confined to the upper limb: link to somatic MAP3K3 mutations.

Venous (cavernous) malformations are commonly seen in the upper limb. Almost all venous malformations are congenital. They may be sporadic, familial, or syndromic. Late-onset, multiple venous malformations confined to the upper limb are rare. Lesions present after puberty. All previously reported cases were located subcutaneously and were small in size. The condition is non-hereditary and non-syndromic. We present a unique series of eight patients with this rare condition. Unique features included the presence of large malformations (up to 20 cm in diameter) and the presence of subfascial lesions causing nerve compression. Surgical excision was curative. Mutational analysis in one patient identified a novel somatic MAP3K3 gene mutation (c.1723T > C, p.Tyr 575 His) in the affected veins. The encoded MAP3K3 protein is known to accelerate the RAS pathway of cellular proliferation.Level of evidence: IV.