HER2-driven breast cancer suppression by the JNK signaling pathway.

The HER2+ subtype of human breast cancer is associated with the malignant transformation of luminal ductal cells of the mammary epithelium. The sequence analysis of tumor DNA identifies loss of function mutations and deletions of the MAP2K4 and MAP2K7 genes that encode direct activators of the JUN NH2-terminal kinase (JNK). We report that in vitro studies of human mammary epithelial cells with CRISPR-induced mutations in the MAPK and MAP2K components of the JNK pathway caused no change in growth in 2D culture, but these mutations promoted epithelial cell proliferation in 3D culture. Analysis of gene expression signatures in 3D culture demonstrated similar changes caused by HER2 activation and JNK pathway loss. The mechanism of signal transduction cross-talk may be mediated, in part, by JNK-suppressed expression of integrin α6ß4 that binds HER2 and amplifies HER2 signaling. These data suggest that HER2 activation and JNK pathway loss may synergize to promote breast cancer. To test this hypothesis, we performed in vivo studies using a mouse model of HER2+ breast cancer with Cre/loxP-mediated ablation of genes encoding JNK (Mapk8 and Mapk9) and the MAP2K (Map2k4 and Map2k7) that activate JNK in mammary epithelial cells. Kaplan-Meier analysis of tumor development demonstrated that JNK pathway deficiency promotes HER2+-driven breast cancer. Collectively, these data identify JNK pathway genes as potential suppressors for HER2+ breast cancer.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

A tumor-associated splice-isoform of MAP2K7 drives dedifferentiation in MBNL1-low cancers via JNK activation.

Master splicing regulator MBNL1 shapes large transcriptomic changes that drive cellular differentiation during development. Here we demonstrate that MBNL1 is a suppressor of tumor dedifferentiation. We surveyed MBNL1 expression in matched tumor/normal pairs across The Cancer Genome Atlas and found that MBNL1 was down-regulated in several common cancers. Down-regulation of MBNL1 predicted poor overall survival in breast, lung, and stomach adenocarcinomas and increased relapse and distant metastasis in triple-negative breast cancer. Down-regulation of MBNL1 led to increased tumorigenic and stem/progenitor-like properties in vitro and in vivo. A discrete set of alternative splicing events (ASEs) are shared between MBNL1-low cancers and embryonic stem cells including a MAP2K7∆exon2 splice variant that leads to increased stem/progenitor-like properties via JNK activation. Accordingly, JNK inhibition is capable of reversing MAP2K7∆exon2-driven tumor dedifferentiation in MBNL1-low cancer cells. Our work elucidates an alternative-splicing mechanism that drives tumor dedifferentiation and identifies biomarkers that predict enhanced susceptibility to JNK inhibition.

Dual inhibition of MEK1/2 and MEK5 suppresses the EMT/migration axis in triple-negative breast cancer through FRA-1 regulation.

Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.

Structural basis for producing selective MAP2K7 inhibitors.

Mitogen-activated protein kinase kinase 7 (MAP2K7) in the c-Jun N-terminal kinase signal cascade is an attractive drug target for a variety of diseases. The selectivity of MAP2K7 inhibitors against off-target kinases is a major barrier in drug development. We report a crystal structure of MAP2K7 complexed with a potent covalent inhibitor bearing an acrylamide moiety as an electrophile, which discloses a structural basis for producing selective and potent MAP2K7 inhibitors.

MiR-125b suppresses the carcinogenesis of osteosarcoma cells via the MAPK-STAT3 pathway.

The microRNA (miRNA) miR-125b is abnormally expressed in many different types of tumors, including osteosarcoma (OS). How aberrantly expressed miR-125b participates in regulating the initiation and progression of OS is still poorly understood. In the current study, we found that in OS, miR-125b can suppress the expression of MAP kinase kinase 7 (MKK7), which can dephosphorylate and inactivate signal transducer and activator of transcription 3 (STAT3). We also identified an elevated expression level of MKK7 in OS and an association between MKK7 expression and poor prognosis. Further, miR-125b inhibited OS cell proliferation and invasion by targeting and downregulating MKK7 in vitro and suppressed tumor formation in vivo. Moreover, using Western blot analysis, we preliminarily proved that the activation (phosphorylation) of STAT3 was regulated by MKK7 at the epigenetic level. MKK7 was overexpressed in OS and associated with poor clinical results. The miR-125b-MAPK-STAT3 axis may be one of the mechanisms of OS oncogenesis and a potential target for the treatment of OS.

High-resolution structure discloses the potential for allosteric regulation of mitogen-activated protein kinase kinase 7.

Mitogen-activated protein kinase kinase 7 (MAP2K7) regulates stress and inflammatory responses, and is an attractive drug discovery target for several diseases including arthritis and cardiac hypertrophy. Intracellular proteins such as MAP2K7 are prone to aggregation due to cysteine-driven oxidation in in vitro experiments. MAP2K7 instability due to the four free cysteine residues on the molecular surface abrogated the crystal growth and led to a low-resolution structure with large residual errors. To acquire a higher resolution structure for promoting rational drug discovery, we explored stable mutants of MAP2K7 by replacing the surface cysteine residues, Cys147, Cys218, Cys276 and Cys296. Single-site mutations, except for Cys147, maintained the specific activity and increased the protein yield, while all the multi-site mutations massively reduced the activity. The C218S mutation drastically augmented the protein production and crystallographic resolution. Furthermore, the C218S crystals grown under microgravity in a space environment yielded a 1.3 Å resolution structure, providing novel insights for drug discovery: the precisely assigned water molecules in the active site, the double conformations in the flexible region and the C-terminal extension bound to the N-terminal region of the adjacent molecules. The latter insight is likely to promote the production of allosteric MAP2K7 inhibitors.

A crucial role of Cys218 in configuring an unprecedented auto-inhibition form of MAP2K7.

Mitogen-activated protein kinase kinase 7 (MAP2K7) is an indispensable kinase of the c-Jun N-terminal kinase signal cascade and is rigorously regulated via phosphorylation. To investigate the regulatory mechanism of the inactive non-phosphorylated state of MAP2K7, the crystal structures of the wild-type and C218S mutant were solved. The wild-type apo-structure revealed an unprecedented auto-inhibition form that occluded the ATP site. This closed form was configured by the n-σ* interaction of Cys218, a non-conserved residue among the MAP2K family kinases, with Gly145 in the glycine-rich loop. The interaction was unaltered in the presence of an ATP analog, whereas the C218S mutation precluded the closed configuration. These structural insights are potentially valuable for drug discovery of highly selective MAP2K7 inhibitors.

LncRNA PCED1B-AS1 mediates miR-3681-3p/MAP2K7 axis to promote metastasis, invasion and EMT in gastric cancer.

BACKGROUND: LncRNA PCED1B-AS1 is abnormally expressed in multiple cancers and has been confirmed as an oncogene. Our study aimed to investigate the regulatory mechanism of lncRNA PCED1B-AS1 in gastric cancer. METHODS: TCGA database was used to analyze the abnormal expression of lncRNA PCED1B-AS1 in gastric cancer. By database prediction and mass spectrometric analysis, miR-3681-3p and MAP2K7 are potential downstream target molecules of lncRNA PCED1B-AS1 and verified by dual-luciferase report assay. RT-qPCR analysis and western blot were performed to detect the expressions of PCED1B-AS1 and MAP2K7 in gastric cancer cell lines and tissues. CCK-8 kit was applied to measure the cell viability. Wound healing and Transwell experiment were used to detect the migration and invasion. Western blot and immunohistochemical staining were performed to detect the expressions of EMT-related proteins in tissues. The changes of tumor proliferation were detected by xenograft experiment in nude mice. RESULTS: PCED1B-AS1 expression was higher but miR-3681-3 expression was lower in gastric cancer cell lines or tissues, compared to normal group. Function analysis verified PCED1B-AS1 promoted cell proliferation and inhibited cell apoptosis in gastric cancer cells in vitro and in vivo. LncRNA PCED1B-AS1 could bind directly to miR-3681-3p, and MAP2K7 was found to be a downstream target of miR-3681-3p. MiR-3681-3p mimics or si-MAP2K7 could partly reverse the effect of PCED1B-AS1 on gastric cancer cells. CONCLUSION: PCED1B-AS1 accelerated cell proliferation and inhibited cell apoptosis through sponging miR-3681-3p to upregulate MAP2K7 expression in gastric cancer, which indicated PCED1B-AS1/miR-3681-3p/MAP2K7 axis may serve as a potential therapeutic target for gastric cancer.

Cross-species analysis of transcriptome emphasizes a critical role of TNF-α in mediating MAP2K7/AKT2 signaling in zearalenone-induced apoptosis.

Zearalenone (ZEN) is a widespread and transgenerational toxicant that can cause serious reproductive health risks, which poses a potential threat to global agricultural production and human health; its estrogenic activity can lead to reproductive toxicity through the induction of granulosa cell apoptosis. Herein, comparative transcriptome analysis, single-cell transcriptome analysis, and weighted gene co-expression network analysis (WGCNA) combined with gene knockout in vivo and RNA interference in vitro were used to comprehensively describe the damage caused by ZEN exposure on ovarian granulosa cells. Comparative transcriptome analysis and WGCNA suggested that the tumor necrosis factor (TNF)-α-mediated mitogen-activated protein kinase 7 (MAP2K7)/ AKT serine/threonine kinase 2 (AKT2) axis was disordered after ZEN exposure in porcine granulosa cells (pGCs) and mouse granulosa cells (mGCs). In vivo gene knockout and in vitro RNA interference verified that TNF-α-mediated MAP2K7/AKT2 was the guiding signal in ZEN-induced apoptosis in pGCs and mGCs. Moreover, single-cell transcriptome analysis showed that ZEN exposure could induce changes in the TNF signaling pathway in offspring. Overall, we concluded that the TNF-α-mediated MAP2K7/AKT2 axis was the main signaling pathway of ZEN-induced apoptosis in pGCs and mGCs. This work provides new insights into the mechanism of ZEN toxicity and provides new potential therapeutic targets for the loss of livestock and human reproductive health caused by ZEN.

The EEF1AKMT3/MAP2K7/TP53 axis suppresses tumor invasiveness and metastasis in gastric cancer.

The importance of methylation in the tumorigenic responses of nonhistone proteins, such as TP53, PTEN, RB1, AKT, and STAT3, has been emphasized in numerous studies. In parallel, the corresponding nonhistone protein methyltransferases have been acknowledged in the pathophysiology of cancer. Thus, this study aimed to explore the pathological role of a nonhistone methyltransferase in gastric cancer (GC), identify nonhistone substrate protein, and understand the underlying mechanism. Interestingly, among the 24 methyltransferases and methyltransferase family 16 (MTF16) proteins, EEF1AKMT3 (METTL21B) expression was prominently lower in GC tissues than in normal adjacent tissues and was associated with a worse prognosis. In addition, EEF1AKMT3-knockdown induced gastric tumor invasiveness and migration. Through gain and loss-of-function studies, mass spectrometry analysis, RNA-seq, and phospho-antibody array, we identified EEF1AKMT3 as a novel tumor-suppressive methyltransferase that catalyzes the monomethylation of MAP2K7 (MKK7) at K296, thereby decreasing the phosphorylation, ubiquitination, and degradation of TP53. Furthermore, EEF1AKMT3, p-MAP2K7, and TP53 protein levels were positively correlated in GC tissues. Collectively, our results delineate the tumor-suppressive function of the EEF1AKMT3/MAP2K7/TP53 signaling axis and suggest the dysregulation of the signaling axis as potential targeted therapy in GC.

Inhibition of the MAP2K7-JNK pathway with 5Z-7-oxozeaenol induces apoptosis in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric leukemia with a worse prognosis than most frequent B-cell ALL due to a high incidence of treatment failures and relapse. Our previous work showed that loss of the pioneer factor KLF4 in a NOTCH1-induced T-ALL mouse model accelerated the development of leukemia through expansion of leukemia-initiating cells and activation of the MAP2K7 pathway. Similarly, epigenetic silencing of the KLF4 gene in children with T-ALL was associated with MAP2K7 activation. Here, we showed the small molecule 5Z-7-oxozeaenol (5Z7O) induces dose-dependent cytotoxicity in a panel of T-ALL cell lines mainly through inhibition of the MAP2K7-JNK pathway, which further validates MAP2K7 as a therapeutic target. Mechanistically, 5Z7O-mediated apoptosis was caused by the downregulation of regulators of the G2/M checkpoint and the inhibition of survival pathways. The anti-leukemic capacity of 5Z7O was evaluated using leukemic cells from two mouse models of T-ALL and patient-derived xenograft cells generated using lymphoblasts from pediatric T-ALL patients. Finally, a combination of 5Z7O with dexamethasone, a drug used in frontline therapy, showed synergistic induction of cytotoxicity. In sum, we report here that MAP2K7 inhibition thwarts survival mechanisms in T-ALL cells and warrants future pre-clinical studies for high-risk and relapsed patients.

Tumorigenic de-differentiation: the alternative splicing way.

The mechanism of acquisition of tumorigenic properties by somatic cells at the onset of cancer and later during relapse is a question of paramount importance in cancer biology. We have recently discovered a Muscleblind like-1 (MBNL1)-driven alternative-splicing mediated mechanism of tumorigenic de-differentiation that is associated with poor prognosis, relapse and metastasis in common cancer types.

miR-125b inhibited epithelial-mesenchymal transition of triple-negative breast cancer by targeting MAP2K7.

MicroRNAs (miRNAs) play important roles in diverse biological processes and are emerging as key regulators of tumorigenesis and tumor progression. Among the differentially expressed miRNAs in breast cancer, miR-125b was revealed to be deregulated and associated with poor prognosis and chemoresistance in triple-negative breast cancer (TNBC), but the mechanism is still unknown. In our study, we showed downregulated expression of miR-125b in TNBC tissues and decreased migration and invasion in miR-125b-expressing Hs578T cells. MAP2K7 was then detected to be a novel target of miR-125b, and downregulation of MAP2K7 by miR-125b was similar to transient knockdown of MAP2K7 which hindered epithelial-mesenchymal transition (EMT) of Hs578T cells. Upregulation of MAP2K7 in miR-125b-overexpressing Hs578T cells partly rescued the migration and invasion suppression of miR-125b. Furthermore, MAP2K7 was overexpressed in TNBC samples compared with normal tissues and negatively correlated with miR-125b expression. In light of these findings, miR-125b emerged as a tumor suppressor in TNBC by targeting MAP2K7 to inhibit EMT.

The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells.

By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study selective macroautophagy in mammalian cells.