TrkA expression directs the anti-neoplastic activity of MLK3 inhibitors in triple-negative breast cancer.

Mixed Lineage Kinase 3 (MLK3) is a viable target for neoplastic diseases; however, it is unclear whether its activators or inhibitors can act as anti-neoplastic agents. We reported that the MLK3 kinase activity was higher in triple-negative (TNBC) than in hormone receptor-positive human breast tumors, where estrogen inhibited MLK3 kinase activity and provided a survival advantage to ER+ breast cancer cells. Herein, we show that in TNBC, the higher MLK3 kinase activity paradoxically promotes cancer cell survival. Knockdown of MLK3 or MLK3 inhibitors, CEP-1347 and URMC-099, attenuated tumorigenesis of TNBC cell line and Patient-Derived (PDX) xenografts. The MLK3 kinase inhibitors decreased both the expression and activation of MLK3, PAK1, and NF-kB protein and caused cell death in TNBC breast xenografts. RNA-seq analysis identified several genes downregulated by MLK3 inhibition, and the NGF/TrkA MAPK pathway was significantly enriched in tumors sensitive to growth inhibition by MLK3 inhibitors. The TNBC cell line unresponsive to kinase inhibitor had substantially lower TrkA, and overexpression of TrkA restored the sensitivity to MLK3 inhibition. These results suggest that the functions of MLK3 in breast cancer cells depend on downstream targets in TNBC tumors expressing TrkA, and MLK3 kinase inhibition may provide a novel targeted therapy.

Mixed lineage kinase 3 and CD70 cooperation sensitize trastuzumab-resistant HER2+ breast cancer by ceramide-loaded nanoparticles.

Trastuzumab is the first-line therapy for human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but often patients develop acquired resistance. Although other agents are in clinical use to treat trastuzumab-resistant (TR) breast cancer; still, the patients develop recurrent metastatic disease. One of the primary mechanisms of acquired resistance is the shedding/loss of the HER2 extracellular domain, where trastuzumab binds. We envisioned any new agent acting downstream of the HER2 should overcome trastuzumab resistance. The mixed lineage kinase 3 (MLK3) activation by trastuzumab is necessary for promoting cell death in HER2+ breast cancer. We designed nanoparticles loaded with MLK3 agonist ceramide (PPP-CNP) and tested their efficacy in sensitizing TR cell lines, patient-derived organoids, and patient-derived xenograft (PDX). The PPP-CNP activated MLK3, its downstream JNK kinase activity, and down-regulated AKT pathway signaling in TR cell lines and PDX. The activation of MLK3 and down-regulation of AKT signaling by PPP-CNP induced cell death and inhibited cellular proliferation in TR cells and PDX. The apoptosis in TR cells was dependent on increased CD70 protein expression and caspase-9 and caspase-3 activities by PPP-CNP. The PPP-CNP treatment alike increased the expression of CD70, CD27, cleaved caspase-9, and caspase-3 with a concurrent tumor burden reduction of TR PDX. Moreover, the expressions of CD70 and ceramide levels were lower in TR than sensitive HER2+ human breast tumors. Our in vitro and preclinical animal models suggest that activating the MLK3-CD70 axis by the PPP-CNP could sensitize/overcome trastuzumab resistance in HER2+ breast cancer.

MAP4K4 promotes pancreatic tumorigenesis via phosphorylation and activation of mixed lineage kinase 3.

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients.

HAI-1 is an independent predictor of lung cancer mortality and is required for M1 macrophage polarization.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Though immune checkpoint inhibitors (ICIs) have revolutionized lung cancer therapy in recent years, there are several factors limiting the therapeutic efficacy of ICI-based immunotherapy in lung cancer. Recent evidence suggests that one such mechanism is the phenotypic shift of tumor-infiltrating macrophages away from an anti-tumor M1 phenotype and towards an anti-inflammatory and tumor-permissive M2 phenotype. Though this phenomenon is well documented, the means through which the lung tumor microenvironment (TME) usurps macrophage function are poorly described. Hepatocyte growth factor (HGF) is a known driver of both lung cancer pathobiology as well as M2 polarization, and its signaling is antagonized by the tumor suppressor gene HAI-1 (SPINT1). Using a combination of genomic databases, primary NSCLC specimens, and in vitro models, we determined that patients with loss of HAI-1 have a particularly poor prognosis, hallmarked by increased HGF expression and an M2-dominant immune infiltrate. Similarly, conditioned media from HAI-1-deficient tumor cells led to a loss of M1 and increased M2 polarization in vitro, and patient NSCLC tissues with loss of HAI-1 showed a similar loss of M1 macrophages. Combined, these results suggest that loss of HAI-1 is a potential means through which tumors acquire an immunosuppressive, M2-dominated TME, potentially through impaired M1 macrophage polarization. Hence, HAI-1 status may be informative when stratifying patients that may benefit from therapies targeting the HGF pathway, particularly as an adjuvant to ICI-based immunotherapy.

Mixed Lineage Kinase 3 phosphorylates prolyl-isomerase PIN1 and potentiates GLI1 signaling in pancreatic cancer development.

The transcription factor Glioma-Associated Oncogene Homolog 1 (GLI1) is activated by sonic hedgehog (SHH) cascade and is an established driver of pancreatic ductal adenocarcinoma (PDAC). However, therapies targeting upstream hedgehog signaling have shown little to no efficacy in clinical trials. Here, we identify Mixed Lineage Kinase 3 (MLK3) as a druggable regulator of oncogenic GLI1. Earlier, we reported that MLK3 phosphorylated a peptidyl-prolyl isomerase PIN1 on the S138 site, and the PIN1-pS138 translocated to the nucleus. In this report, we identify GLI1 as one of the targets of PIN1-pS138 and demonstrate that PIN1-pS138 is upregulated in human PDAC and strongly associates with the upregulation of GLI1 and MLK3 expression. Moreover, we also identified two new phosphorylation sites on GLI1, T394, and S1089, which are directly phosphorylated by MLK3 to promote GLI1 nuclear translocation, transcriptional activity, and cell proliferation. Additionally, pharmacological inhibition of MLK3 by CEP-1347 promoted apoptosis in PDAC cell lines, reduced tumor burden, extended survival, and reduced GLI1 expression in the Pdx1-Cre x LSL-KRASG12D x LSL-TP53R172H (KPC) mouse model of PDAC. These findings collectively suggest that MLK3 is an important regulator of oncogenic GLI1 and that therapies targeting MLK3 warrant consideration in the management of PDAC patients.

Rationalized inhibition of mixed lineage kinase 3 and CD70 enhances life span and antitumor efficacy of CD8+ T cells.

BACKGROUND: The mitogen-activated protein kinases (MAPKs) are important for T cell survival and their effector function. Mixed lineage kinase 3 (MLK3) (MAP3K11) is an upstream regulator of MAP kinases and emerging as a potential candidate for targeted cancer therapy; yet, its role in T cell survival and effector function is not known. METHODS: T cell phenotypes, apoptosis and intracellular cytokine expressions were analyzed by flow cytometry. The apoptosis-associated gene expressions in CD8+CD38+ T cells were measured using RT2 PCR array. In vivo effect of combined blockade of MLK3 and CD70 was analyzed in 4T1 tumor model in immunocompetent mice. The serum level of tumor necrosis factor-α (TNFα) was quantified by enzyme-linked immunosorbent assay. RESULTS: We report that genetic loss or pharmacological inhibition of MLK3 induces CD70-TNFα-TNFRSF1a axis-mediated apoptosis in CD8+ T cells. The genetic loss of MLK3 decreases CD8+ T cell population, whereas CD4+ T cells are partially increased under basal condition. Moreover, the loss of MLK3 induces CD70-mediated apoptosis in CD8+ T cells but not in CD4+ T cells. Among the activated CD8+ T cell phenotypes, CD8+CD38+ T cell population shows more than five fold increase in apoptosis due to loss of MLK3, and the expression of TNFRSF1a is significantly higher in CD8+CD38+ T cells. In addition, we observed that CD70 is an upstream regulator of TNFα-TNFRSF1a axis and necessary for induction of apoptosis in CD8+ T cells. Importantly, blockade of CD70 attenuates apoptosis and enhances effector function of CD8+ T cells from MLK3-/- mice. In immune-competent breast cancer mouse model, pharmacological inhibition of MLK3 along with CD70 increased tumor infiltration of cytotoxic CD8+ T cells, leading to reduction in tumor burden largely via mitochondrial apoptosis. CONCLUSION: Together, these results demonstrate that MLK3 plays an important role in CD8+ T cell survival and effector function and MLK3-CD70 axis could serve as a potential target in cancer.

Mixed lineage kinase 3 inhibition induces T cell activation and cytotoxicity.

Mixed lineage kinase 3 (MLK3), also known as MAP3K11, was initially identified in a megakaryocytic cell line and is an emerging therapeutic target in cancer, yet its role in immune cells is not known. Here, we report that loss or pharmacological inhibition of MLK3 promotes activation and cytotoxicity of T cells. MLK3 is abundantly expressed in T cells, and its loss alters serum chemokines, cytokines, and CD28 protein expression on T cells and its subsets. MLK3 loss or pharmacological inhibition induces activation of T cells in in vitro, ex vivo, and in vivo conditions, irrespective of T cell activating agents. Conversely, overexpression of MLK3 decreases T cell activation. Mechanistically, loss or inhibition of MLK3 down-regulates expression of a prolyl-isomerase, Ppia, which is directly phosphorylated by MLK3 to increase its isomerase activity. Moreover, MLK3 also phosphorylates nuclear factor of activated T cells 1 (NFATc1) and regulates its nuclear translocation via interaction with Ppia, and this regulates T cell effector function. In an immune-competent mouse model of breast cancer, MLK3 inhibitor increases Granzyme B-positive CD8+ T cells and decreases MLK3 and Ppia gene expression in tumor-infiltrating T cells. Likewise, the MLK3 inhibitor in pan T cells, isolated from breast cancer patients, also increases cytotoxic CD8+ T cells. These results collectively demonstrate that MLK3 plays an important role in T cell biology, and targeting MLK3 could serve as a potential therapeutic intervention via increasing T cell cytotoxicity in cancer.

Mitogen-Activated Protein Kinase Inhibitors and T-Cell-Dependent Immunotherapy in Cancer.

Mitogen-activated protein kinase (MAPK) signaling networks serve to regulate a wide range of physiologic and cancer-associated cell processes. For instance, a variety of oncogenic mutations often lead to hyperactivation of MAPK signaling, thereby enhancing tumor cell proliferation and disease progression. As such, several components of the MAPK signaling network have been proposed as viable targets for cancer therapy. However, the contributions of MAPK signaling extend well beyond the tumor cells, and several MAPK effectors have been identified as key mediators of the tumor microenvironment (TME), particularly with respect to the local immune infiltrate. In fact, a blockade of various MAPK signals has been suggested to fundamentally alter the interaction between tumor cells and T lymphocytes and have been suggested a potential adjuvant to immune checkpoint inhibition in the clinic. Therefore, in this review article, we discuss the various mechanisms through which MAPK family members contribute to T-cell biology, as well as circumstances in which MAPK inhibition may potentiate or limit cancer immunotherapy.

Correction: Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

The original version of this Article did not acknowledge Pradeep Sathyanarayana as an author. His affiliation is Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, USA.

Mixed lineage kinase 3 promotes breast tumorigenesis via phosphorylation and activation of p21-activated kinase 1.

Mixed lineage kinase 3 (MLK3), a MAP3K member has been envisioned as a viable drug target in cancer, yet its detailed function and signaling is not fully elucidated. We identified that MLK3 tightly associates with an oncogene, PAK1. Mammalian PAK1 being a Ste20 (MAP4K) member, we tested whether it is an upstream regulator of MLK3. In contrast to our hypothesis, MLK3 activated PAK1 kinase activity directly, as well as in the cells. Although, MLK3 can phosphorylate PAK1 on Ser133 and Ser204 sites, PAK1S133A mutant is constitutively active, whereas, PAK1S204A is not activated by MLK3. Stable overexpression of PAK1S204A in breast cancer cells, impedes migration, invasion, and NFĸB activity. In vivo breast cancer cell tumorigenesis is significantly reduced in tumors expressing PAK1S204A mutant. These results suggest that mammalian PAK1 does not act as a MAP4K and MLK3-induced direct activation of PAK1 plays a key role in breast cancer tumorigenesis.

Transcriptional regulation of mixed lineage kinase 3 by estrogen and its implication in ER-positive breast cancer pathogenesis.

Mixed Lineage Kinase 3 (MLK3), also called as MAP3K11 is a tightly regulated MAP3K member but its cellular function is still not fully understood. Earlier we reported post-translational regulation of MLK3 by estrogen (E2) that inhibited the kinase activity and favored survival of ER+ breast cancer cells. Here we report that MLK3 is also transcriptionally downregulated by E2 in ER+ breast cancer cells. Publicly available data and in situ hybridization of human breast tumors showed significant down regulation of MLK3 transcripts in ER+ tumors. The basal level of MLK3 transcripts and protein in ER+ breast cancer cell lines were significantly lower, and the protein expression was further down regulated by E2 in a time-dependent manner. Analysis of the promoter of MLK3 revealed two ERE sites which were regulated by E2 in ER+ but not in ER- breast cancer cell lines. Both ERα and ERß were able to bind to MLK3 promoter and recruit nuclear receptor co-repressors (NCoR, SMRT and LCoR), leading to down-regulation of MLK3 transcripts. Collectively these results suggest that recruitment of nuclear receptor co-repressor is a key feature of ligand-dependent transcriptional repression of MLK3 by ERs. Therefore coordinated transcriptional and post-translational repression of pro-apoptotic MLK3 probably is one of the mechanisms by which ER+ breast cancer cells proliferate and survive.

Human Epidermal Growth Factor Receptor 2 (HER2) Impedes MLK3 Kinase Activity to Support Breast Cancer Cell Survival.

Human epidermal growth factor receptor 2 (HER2) is amplified in ∼ 15-20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2-) breast cancer tissues, whereas both HER2+ and HER2- tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser(674) by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors.

Mixed Lineage Kinase-c-Jun N-Terminal Kinase Axis: A Potential Therapeutic Target in Cancer.

Mixed lineage kinases (MLKs) are members of the mitogen-activated protein kinase kinase kinase (MAP3K) family and are reported to activate MAP kinase pathways. There have been at least 9 members of the MLK family identified to date, although the physiological functions of all the family members are yet unknown. However, MLKs in general have been implicated in neurodegenerative diseases, including Parkinson and Alzheimer diseases. Recent reports suggest that some of the MLK members could play a role in cancer via modulating cell migration, invasion, cell cycle, and apoptosis. This review article will first describe the biology of MLK members and then discuss the current progress that relates to their functions in cancer.

The peptidyl-prolyl isomerase Pin1 up-regulation and proapoptotic function in dopaminergic neurons: relevance to the pathogenesis of Parkinson disease.

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117-4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP(+)) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP(+)-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP(+)-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.

Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function.

Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is an essential and novel regulatory mechanism for protein phosphorylation. Therefore, tight regulation of Pin1 localization and catalytic activity is crucial for its normal nuclear functions. Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; however, the mechanism(s) by which Pin1 catalytic activity and nuclear localization are increased is unknown. Here we demonstrate that mixed-lineage kinase 3 (MLK3), a MAP3K family member, phosphorylates Pin1 on a Ser138 site to increase its catalytic activity and nuclear translocation. This phosphorylation event drives the cell cycle and promotes cyclin D1 stability and centrosome amplification. Notably, Pin1 pSer138 is significantly up-regulated in breast tumors and is localized in the nucleus. These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role in regulating the cell cycle, centrosome numbers, and oncogenesis.

Emerging neurotoxic mechanisms in environmental factors-induced neurodegeneration.

Exposure to environmental neurotoxic metals, pesticides and other chemicals is increasingly recognized as a key risk factor in the pathogenesis of chronic neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Oxidative stress and apoptosis have been actively investigated as neurotoxic mechanisms over the past two decades, resulting in a greater understanding of neurotoxic processes. Nevertheless, emerging evidence indicates that epigenetic changes, protein aggregation and autophagy are important cellular and molecular correlates of neurodegenerative diseases resulting from chronic neurotoxic chemical exposure. During the Joint Conference of the 13th International Neurotoxicology Association and the 11th International Symposium on Neurobehavioral Methods and Effects in Occupational and Environmental Health, the recent progress made toward understanding epigenetic mechanisms, protein aggregation, autophagy, and deregulated kinase activation following neurotoxic chemical exposure and the relevance to neurodegenerative conditions were one of the themes of the symposium. Dr. Anumantha G. Kanthasamy described the role of acetylation of histones and non-histone proteins in neurotoxicant-induced neurodegenerative processes in the nigral dopaminergic neuronal system. Dr. Arthi Kanthasamy illustrated the role of autophagy as a key determinant in cell death events during neurotoxic insults. Dr. Ajay Rana provided evidence for posttranslational modification of α-synuclein protein by the Mixed Linage Kinase (MLK) group of kinases to initiate protein aggregation in cell culture and animal models of Parkinson's disease. These presentations outlined emerging cutting edge mechanisms that might set the stage for future mechanistic investigations into new frontiers of molecular neurotoxicology. This report summarizes the views of symposium participants, with emphasis on future directions for study of environmentally and occupationally linked chronic neurodegenerative diseases.

Mixed lineage kinase 3 modulates ß-catenin signaling in cancer cells.

Expression of ß-catenin is strictly regulated in normal cells via the glycogen synthase kinase 3ß (GSK3ß)- adenomatous polyposis coli-axin-mediated degradation pathway. Mechanisms leading to inactivation of this pathway (example: activation of Wnt/ß-catenin signaling or mutations of members of the degradation complex) can result in ß-catenin stabilization and activation of ß-catenin/T-cell factor (TCF) signaling. ß-Catenin-mediated cellular events are diverse and complex. A better understanding of the cellular signaling networks that control ß-catenin pathway is important for designing effective therapeutic strategies targeting this axis. To gain more insight, we focused on determining any possible cross-talk between ß-catenin and mixed lineage kinase 3 (MLK3), a MAPK kinase kinase member. Our studies indicated that MLK3 can induce ß-catenin expression via post-translational stabilization in various cancer cells, including prostate cancer. This function of MLK3 was dependent on its kinase activity. MLK3 can interact with ß-catenin and phosphorylate it in vitro. Overexpression of GSK3ß-WT or the S9A mutant was unable to antagonize MLK3-induced stabilization, suggesting this to be independent of GSK3ß pathway. Surprisingly, despite stabilizing ß-catenin, MLK3 inhibited TCF transcriptional activity in the presence of both WT and S37A ß-catenin. These resulted in reduced expression of ß-catenin/TCF downstream targets Survivin and myc. Immunoprecipitation studies indicated that MLK3 did not decrease ß-catenin/TCF interaction but promoted interaction between ß-catenin and KLF4, a known repressor of ß-catenin/TCF transcriptional activity. In addition, co-expression of MLK3 and ß-catenin resulted in significant G(2)/M arrest. These studies provide a novel insight toward the regulation of ß-catenin pathway, which can be targeted to control cancer cell proliferation, particularly those with aberrant activation of ß-catenin signaling.

Estrogen suppresses MLK3-mediated apoptosis sensitivity in ER+ breast cancer cells.

Little knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17beta-Estradiol (E(2)) hinders cytotoxic drug-induced cell death in estrogen receptor-positive (ER(+)) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor-negative (ER(-)) breast tumors, suggesting that E(2) might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH(2)-terminal kinase, were rapidly inhibited by E(2) in ER(+) but not in ER(-) cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E(2), indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E(2) involved phosphorylation of MLK3 Ser(674) by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E(2) accentuated this limitation. Taken together, our findings indicate that E(2) inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug-induced death of ER(+) breast cancer cells.

Mixed lineage kinase-3/JNK1 axis promotes migration of human gastric cancer cells following gastrin stimulation.

Gastrin is a gastrointestinal peptide hormone, secreted by the gastric G cells and can exist as a fully processed amidated form (G17) or as unprocessed forms. All forms of gastrin possess trophic properties towards the gastrointestinal mucosa. An understanding of the signaling pathways involved is important to design therapeutic approaches to target gastrin-mediated cellular events. The studies described here were designed to identify the signaling pathways by which amidated gastrin (G17) mediates cancer cell migration. These studies indicated a time- and dose-dependent increase in gastric cancer cell migration after G17 stimulation, involving cholecystokinin 2 receptor. G17-induced migration was preceded by activation of MAPK pathways and was antagonized after pretreatment with SP600125, a pharmacological inhibitor of c-Jun-NH(2)-terminal kinase (JNK) pathway. Knockdown of endogenous JNK1 expression via small interference RNA (JNK1-siRNA) inhibited G17-induced phosphorylation of c-Jun and migration, and overexpression of wild-type JNK1 or constitutive active JNK1 promoted G17-induced migration. Studies designed to identify the MAPK kinase kinase member mediating JNK activation indicated the involvement of mixed lineage kinase-3 (MLK3), which was transiently activated upon G17 treatment. Inhibition of MLK3 pathway via a pan-MLK inhibitor or knockdown of MLK3 expression by MLK3-siRNA antagonized G17-induced migration. Incubation with G17 also resulted in an induction of matrix metalloproteinase 7 promoter activity, which is known to mediate migration and invasion pathways in cancer cells. Modulation of MLK3, JNK1, and c-Jun pathways modulated G17-induced matrix metalloproteinase 7 promoter activation. These studies indicate that the MLK3/JNK1 axis mediates G17-induced gastric cancer cell migration, which can be targeted for designing novel therapeutic strategies for treating gastric malignancies.

TRAF2-MLK3 interaction is essential for TNF-alpha-induced MLK3 activation.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that is activated by tumor necrosis factor-alpha (TNF-alpha) and specifically activates c-Jun N-terminal kinase (JNK) on TNF-alpha stimulation. The mechanism by which TNF-alpha activates MLK3 is still not known. TNF receptor-associated factors (TRAFs) are adapter molecules that are recruited to cytoplasmic end of TNF receptor and mediate the downstream signaling, including activation of JNK. Here, we report that MLK3 associates with TRAF2, TRAF5 and TRAF6; however only TRAF2 can significantly induce the kinase activity of MLK3. The interaction domain of TRAF2 maps to the TRAF domain and for MLK3 to its C-terminal half (amino acids 511-847). Endogenous TRAF2 and MLK3 associate with each other in response to TNF-alpha treatment in a time-dependent manner. The association between MLK3 and TRAF2 mediates MLK3 activation and competition with the TRAF2 deletion mutant that binds to MLK3 attenuates MLK3 kinase activity in a dose-dependent manner, on TNF-alpha treatment. Furthermore the downstream target of MLK3, JNK was activated by TNF-alpha in a TRAF2-dependent manner. Hence, our data show that the direct interaction between TRAF2 and MLK3 is required for TNF-alpha-induced activation of MLK3 and its downstream target, JNK.