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Small cell lung cancer (SCLC) is an extremely aggressive neuroendocrine tumor, accounting for approximated 13% of all lung cancer cases. SCLC is characterized by rapid growth and early metastasis. Despite marked improvements in the number and efficacy of targeted, therapeutic options and overall survival rates in SCLC have remained nearly unchanged for almost three decades. The lack of significant progress can be attributed to our poor understanding of the biology of SCLC. Although immune checkpoint inhibitors were recently approved as front-line therapies for SCLC, we still need to better understand the mechanisms responsible for the selective vulnerability of some SCLCs to these inhibitors. Recent work utilizing sequencing data and single cell analyses identified four distinct subsets of SCLC, based on the expression levels of the transcription factors ASCL1, NEUROD1, POU2F3 and YAP1. Each subset was found to have its own distinct biology and therapeutic vulnerabilities. However, these subsets appear to be phenotypically unstable, representing snapshots in the gradual evolution of a tumor that exhibits significant plasticity. Tumor evolution, a product of this plasticity, results in the emergence of significant intratumoral heterogeneity which plays an important role in multiple aspects of SCLC development and progression, including cell survival and proliferation, metastasis and angiogenesis. The recent paradigm shifting discoveries in the biology of SCLC are now beginning to inform the design of new therapeutic strategies for the management of this intractable disease.

Protein arginine methyltransferase 5 (PRMT5) is the major methyltransferase (MT) catalyzing symmetric dimethylation (SDM). PRMT5 regulates developmental, homeostatic and disease processes in vertebrates and invertebrates, and a carcinogenic role has been observed in mammals. Recently, tools generated for PRMT5 loss of function have allowed researchers to demonstrate essential roles for PRMT5 in mouse and human lymphocyte biology. PRMT5 modulates CD4+ and CD8+ T cell development in the thymus, peripheral homeostasis, and differentiation into CD4+ helper T lymphocyte (Th)17 cell phenotypes. Here, we provide a timely review of the milestones leading to our current understanding of PRMT5 in T cell biology, discuss current tools to modify PRMT5 expression/activity, and highlight mechanistic pathways.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-β expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-β/δ. Treatment with the selective PI3K-β/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.

Soft tissue sarcomas (STS) are a biologically diverse group of mesenchymal tumors that predominantly exhibit a poor prognosis. Surgical resection is considered the mainstay of treatment and provides the only chance for long-term survival. However, some patients present with locally advanced, unresectable disease, and for those who are able to undergo resection, tumor recurrence occurs in over half of patients. In addition, the efficacy of conventional systemic therapies remains dismal. The serine/threonine kinase AKT pathway is one of the most frequently aberrantly activated signaling pathways that has been verified in many types of human cancer. Dysregulation of the AKT cascade is known to result in tumorigenesis and aggressive clinical behavior for many tumor types, including STS. EGFRs, with its downstream effectors, PI3K and protein kinase B (AKT)/mTOR, have been investigated for decades as promising targets for the treatment of STS, but significant challenges remain and the prognosis of patients with advanced STS has not improved in over two decades. In this review, we will first describe the AKT pathway and its role in STS tumor biology and then discuss the current challenges in targeting the AKT pathway to treat patients with advanced sarcoma.

Despite the unprecedented growth in our understanding of cell biology, it still remains challenging to connect it to experimental data obtained with cells and tissues’ physiopathological status under precise circumstances. This knowledge gap often results in difficulties in designing validation experiments, which are usually labor-intensive, expensive to perform, and hard to interpret. Here we propose PHENSIM, a computational tool using a systems biology approach to simulate how cell phenotypes are affected by the activation/inhibition of one or multiple biomolecules, and it does so by exploiting signaling pathways. Our tool’s applications include predicting the outcome of drug administration, knockdown experiments, gene transduction, and exposure to exosomal cargo. Importantly, PHENSIM enables the user to make inferences on well-defined cell lines and includes pathway maps from three different model organisms. To assess our approach’s reliability, we built a benchmark from transcriptomics data gathered from NCBI GEO and performed four case studies on known biological experiments. Our results show high prediction accuracy, thus highlighting the capabilities of this methodology. PHENSIM standalone Java application is available at https://github.com/alaimos/phensim, along with all data and source codes for benchmarking. A web-based user interface is accessible at https://phensim.tech/., Author summary Despite the unprecedented growth in our understanding of cell biology, it still remains challenging to connect it to experimental data obtained with cells and tissues’ physiopathological status under precise circumstances. This knowledge gap often results in difficulties in designing validation experiments, which are usually labor-intensive, expensive to perform, and hard to interpret. In this context, ’in silico’ simulations can be extensively applied in massive scales, testing thousands of hypotheses under various conditions, which is usually experimentally infeasible. At present, many simulation models have become available. However, complex biological networks might pose challenges to their performance. We propose PHENSIM, a computational tool using a systems biology approach to simulate how cell phenotypes are affected by the activation/inhibition of one or multiple biomolecules, and it does so by exploiting signaling pathways. We implemented our tool as a freely accessible web application, hoping to allow ’in silico’ simulations to play a more central role in the modeling and understanding of biological phenomena.

Metformin activates AMP-related pathways leading to inactivation of mammalian target of rapamycin (mTOR) and suppression of its downstream effectors, crucial for cancer growth. Epidemiologic studies showed a reduced incidence and improved survival in cancer patients. We conducted a prospective phase I study to assess the safety of metformin in combination with chemotherapy in patients with solid tumors. We conducted a delayed-start randomized trial of non-diabetic patients in two stages. In Stage 1, we randomized patients to two arms: concurrent arm (metformin with chemo) vs. delayed arm (chemo alone). In Stage 2, patients in delayed arm were crossed over to receive metformin. Patients received metformin 500 mg twice daily with chemotherapy to define dose-limiting toxicities (DLTs) in both stages. Secondary endpoints assessed adverse events (AEs) and response rates. Translational correlates included effects of metformin on expression and phosphorylation of 5′ adenosine monophosphate-activated protein kinase (AMPK) by western blot in PBMCs. A total of 100 patients were enrolled (51 in delayed arm vs. 49 concurrent arm). Rate of DLTs in patients receiving metformin with chemotherapy was 6.1% vs. 7.8% in patients receiving chemotherapy alone. DLTs seen with addition of metformin included those associated with established chemo adverse events. No lactic acidosis or hypoglycemia occurred. Restaging showed stable disease in 46% at cessation of metformin. 28% of patients with measurable tumor markers showed improvement. AMPK phosphorylation showed a four- to sixfold increase in AMPK phosphorylation after metformin. This is the largest phase I study of metformin combined with chemotherapy, which suggests that metformin can be given safely with chemotherapy, and offers a platform for future studies. Post-metformin increase in AMPK phosphorylation may potentially explain lack of disease progression in nearly half of our patients. UL1 TR001064. NCT01442870.

The histone demethylase KDM2B is an epigenetic factor with oncogenic properties that is regulated by the basic fibroblasts growth factor (FGF-2). It has recently been shown that KDM2B co-operates with Polycomb Group proteins to promote cell migration and angiogenesis in tumors. In the present study we addressed the role of KDM2B in regulating actin cytoskeleton signaling, cell-cell adhesion and migration of prostate tumor cells. We report here that KDM2B is functionally expressed in DU-145 prostate cancer cells, activated by FGF-2 and regulates EZH2. KDM2B knockdown induced potent up-regulation of gene transcription and protein expression of the epithelial markers E-cadherin and ZO-1, while KDM2B overexpression down-regulated the levels of both markers, suggesting control of cell adhesion by KDM2B. RhoA and RhoB protein expression and activity were diminished upon KDM2B-knockdown and upregulated in KDM2B-overexpressing cell clones. In accordance, actin reorganization with formation of stress fibers became evident in KDM2B-overexpressing cells and abolished in the presence of the Rho inhibitor C3 transferase. DU-145 cell migration was significantly enhanced in KDM2B overexpressing cells and abolished in C3-pretreated cells. Conversely, the retardation of cell migration observed in KDM2B knockdown cells was enhanced in C3-pretreated cells. These results establish a clear functional link between the epigenetic factor KDM2B and the regulation of cell adhesion and Rho-GTPases signaling that controls actin reorganization and cell migration.

Most normal and tumor cells are protected from tumor necrosis factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 kinase tumor progression locus-2 (TPL2) as a player contributing to the protection of a subset of tumor cell lines. The combination of TPL2 knockdown and TNFα gives rise to a synthetic lethality phenotype via receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent and -independent mechanisms. Whereas wild-type TPL2 rescues the phenotype, its kinase-dead mutant does not. Comparison of the molecular events initiated by small interfering RNA for TPL2 (siTPL2) ± TNFα in treatment-sensitive and -resistant lines revealed that the activation of caspase-8, downstream of miR-21-5p and cFLIP, is the dominant TPL2-dependent event. More important, comparison of the gene expression profiles of all of the tested cell lines results in the clustering of sensitive and resistant lines into distinct groups, providing proof of principle for the feasibility of generating a predictive tool for treatment sensitivity.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the World Health Organization Prioritized Pathogens, is an emerging phlebovirus with a high fatality(1–4). Owing to the lack of therapies and vaccines(5,6), there is a pressing need to understand SFTSV pathogenesis. SFSTV non-structural protein (NSs) has been shown to block type I interferon induction(7–11) and facilitate disease progression(12,13). Here, we report that SFTSV-NSs targets the tumour progression locus 2 (TPL2)-A20-binding inhibitor of NF-κB activation 2 (ABIN2)-p105 complex to induce the expression of interleukin-10 (lL-10) for viral pathogenesis. Using a combination of reverse genetics, a TPL2 kinase inhibitor and Tpl2(−/−) mice showed that NSs interacted with ABIN2 and promoted TPL2 complex formation and signalling activity, resulting in the marked upregulation of ll10 expression. Whereas SFTSV infection of wild-type mice led to rapid weight loss and death, Tpl2(−/−) mice or ll10(−/−) mice survived an infection. Furthermore, SFTSV-NSs P(102)A and SFTSV-NSs K(211)R that lost the ability to induce TPL2 signalling and IL-10 production showed drastically reduced pathogenesis. Remarkably, the exogenous administration of recombinant IL-10 effectively rescued the attenuated pathogenic activity of SFTSV-NSs P(102)A, resulting in a lethal infection. Our study demonstrates that SFTSV-NSs targets the TPL2 signalling pathway to induce immune-sup-pressive IL-10 cytokine production as a means to dampen the host defence and promote viral pathogenesis.

Background/Aims: The epigenetic factor KDM2B is a histone demethylase expressed in various tumors. Recently, we have shown that KDM2B regulates actin cytoskeleton organization, small Rho GTPases signaling, cell-cell adhesion and migration of prostate tumor cells. In the present study, we addressed its role in regulating EMT and small GTPases expression in colon tumor cells. Methods: We used RT-PCR for the transcriptional analysis of various genes, Western blotting for the assessment of protein expression and immunofluorescence microscopy for visualization of fluorescently labeled proteins. Results: We report here that KDM2B regulates EZH2 and BMI1 in HCT116 colon tumor cells. Knockdown of this epigenetic factor induced potent up-regulation of the protein levels of the epithelial markers E-cadherin and ZO-1, while the mesenchymal marker N-cadherin was downregulated. On the other hand, KDM2B overexpression downregulated the levels of both epithelial markers and upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B-knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing HCT116 cell clones. Interestingly, Rac1 GTPase level increased upon KDM2B-knockdown and diminished in KDM2B-overexpressing HCT116 colon tumor- and DU-145 prostate cancer cells. Conclusions: These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT and small-GTPases expression in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors.

Progressive multifocal leukoencephalopathy (PML) is an often-fatal demyelinating disease of the central nervous system. PML results when oligodendrocytes within immunocompromised individuals are infected with the human JC virus (JCV). We have identified an oligodendrocyte precursor cell line, termed G144, that supports robust levels of JCV DNA replication, a central part of the JCV life cycle. In addition, we have determined that JC virus readily infects G144 cells. Furthermore, we have determined that JCV DNA replication in G144 cells is stimulated by myristoylated (i.e., constitutively active) Akt and reduced by the Akt-specific inhibitor MK2206. Thus, this oligodendrocyte-based model system will be useful for a number of purposes, such as studies of JCV infection, establishing key pathways needed for the regulation of JCV DNA replication, and identifying inhibitors of this process. IMPORTANCE The disease progressive multifocal leukoencephalopathy (PML) is caused by the infection of particular brain cells, termed oligodendrocytes, by the JC virus. Studies of PML, however, have been hampered by the lack of an immortalized human cell line derived from oligodendrocytes. Here, we report that the G144 oligodendrocyte cell line supports both infection by JC virus and robust levels of JCV DNA replication. Moreover, we have established that the Akt pathway regulates JCV DNA replication and that JCV DNA replication can be inhibited by MK2206, a compound that is specific for Akt. These and related findings suggest that we have established a powerful oligodendrocyte-based model system for studies of JCV-dependent PML.

Since its discovery more than 25 years ago, the kinase AKT has become a central figure in cell signaling. We highlight some of the landmark findings in those 25 years that contributed to our understanding of the regulation and function of AKT in directing cellular processes and behavior. Future progress toward fully understanding the roles of AKT in cell, tissue, and organismal biology will depend on technological innovations and the combination of in-depth reductionist analyses with systems-based strategies.

Stat5 (signal transducer and activator of transcription 5) is an essential mediator of cytokine receptor signaling and plays important roles in the proliferation of alveolar progenitors and the survival of functionally differentiated epithelial cells in the mammary gland. A deregulated expression and activation of Stat5 leads to precocious alveolar development in the absence of pregnancy hormones, impaired mammary gland remodeling following the cessation of lactation, and mammary tumor formation. We reported previously that Stat5 induces the transcription of the Akt1 gene from a novel promoter. In this report, we provide experimental evidence that Akt1 is an essential mediator for the biological function of Stat5 as a survival factor. Additionally, Stat5 controls the expression of the regulatory and catalytic subunits of the phosphatidylinositol 3-kinase (PI3K) (p85α and p110α), thereby greatly augmenting signaling through the prosurvival PI3K/Akt pathway. In agreement with this model, we observed that the constitutive activation of Stat5 cooperates with the loss of function of the tumor suppressor PTEN by accelerating the formation of preneoplastic lesions and mammary tumors. The mammary gland-specific ablation of Stat5 is sufficient to prevent mammary carcinogenesis in a genuine mouse model for Cowden syndrome. Therefore, targeting the Jak2/Stat5 pathway might be a suitable strategy to prevent breast cancer in patients that carry a mutant PTEN allele.

Interferon-stimulated transcription is thought to occur mainly through the action of the JAK/STAT pathway. However, recent findings revealed an additional PI3K/Akt-dependent pathway, which contributes to the induction of a set of interferon-stimulated genes (ISGs) through the regulation of the transcriptional repressor EMSY.

The histone H3K27 methyltransferase EZH2 plays an important role in oncogenesis, by mechanisms that are incompletely understood. Here we show that the JmjC domain histone H3 demethylase NDY1 synergizes with EZH2 to silence the EZH2 inhibitor miR-101. NDY1 and EZH2 repress miR-101 by binding its promoter in concert, via a process triggered by upregulation of NDY1. Whereas EZH2 binding depends on NDY1, the latter binds independently of EZH2. However, both are required to repress transcription. NDY1 and EZH2 acting in concert, upregulate EZH2 and stabilize the repression of miR-101 and its outcome. NDY1 is induced by FGF-2 via CREB phosphorylation and activation, downstream of DYRK1A, and mediates the FGF-2 and EZH2 effects on cell proliferation, migration and angiogenesis. The FGF-2-NDY1/EZH2-miR-101-EZH2 axis described here, was found to be active in bladder cancer. These data delineate a novel oncogenic pathway that functionally links FGF-2 with EZH2 via NDY1 and miR-101.

The growth and survival of tumor cells in an unfavorable hypoxic environment depend upon their adaptability. Here, we show that both normal and tumor cells expressing the protein kinase Akt2 are more resistant to hypoxia than cells expressing Akt1 or Akt3. This is due to the differential regulation of microRNA (miR) 21, which is upregulated by hypoxia only in Akt2-expressing cells. By upregulating miR-21 upon oxygen deprivation, Akt2 downregulates PTEN and activates all three Akt isoforms. miR-21 also targets PDCD4 and Sprouty 1 (Spry1), and the combined downregulation of these proteins with PTEN is sufficient to confer resistance to hypoxia. Furthermore, the miR-21 induction by Akt2 during hypoxia depends upon the binding of NF-κB, cAMP responsive element–binding protein (CREB), and CBP/p300 to the miR-21 promoter, in addition to the regional acetylation of histone H3K9, all of which are under the control of Akt2. Analysis of the Akt2/miR-21 pathway in hypoxic MMTV-PyMT–induced mouse mammary adenocarcinomas and human ovarian carcinomas confirmed the activity of the pathway in vivo. Taken together, this study identifies a novel Akt2-dependent pathway that is activated by hypoxia and promotes tumor resistance via induction of miR-21. Cancer Res; 71(13); 4720–31. ©2011 AACR.

The cancer stem cell hypothesis suggests that, unlike most cancer cells within a tumor, cancer stem cells resist chemotherapeutic drugs and can regenerate the various cell types in the tumor, thereby causing relapse of the disease. Thus, drugs that selectively target cancer stem cells offer great promise for cancer treatment, particularly in combination with chemotherapy. Here, we show that low doses of metformin, a standard drug for diabetes, inhibits cellular transformation and selectively kills cancer stem cells in four genetically different types of breast cancer. The combination of metformin and a well-defined chemotherapeutic agent, doxorubicin, kills both cancer stem cells and non–stem cancer cells in culture. Furthermore, this combinatorial therapy reduces tumor mass and prevents relapse much more effectively than either drug alone in a xenograft mouse model. Mice seem to remain tumor-free for at least 2 months after combinatorial therapy with metformin and doxorubicin is ended. These results provide further evidence supporting the cancer stem cell hypothesis, and they provide a rationale and experimental basis for using the combination of metformin and chemotherapeutic drugs to improve treatment of patients with breast (and possibly other) cancers. [Cancer Res 2009;69(19):7507–11]

Tumor progression locus 2 (Tpl2), a mitogen-activated protein kinase kinase kinase (MAP3K) that is activated by provirus insertion in retrovirus-induced rodent lymphomas and mammary adenocarcinomas, is known to transduce Toll-like receptor, interleukin 1, tumor necrosis factor α, and CD40 signals and to play an important role in inflammation. Here we show that Tpl2 is also required for the transduction of cell migration and gene expression signals originating in the G-protein–coupled receptor proteinase-activated receptor 1 (PAR1). PAR1 signals transduced by Tpl2 activate Rac1 and focal adhesion kinase, and they are required for reorganization of the actin cytoskeleton and cell migration. PAR1 expressed in fibroblasts can be triggered by proteinases produced by tumor cells, and PAR1 expressed in tumor cells can be triggered by proteinases produced by fibroblasts. These data suggest that signals that regulate cell migration and gene expression flow between stromal and tumor cells in both directions and that Tpl2 plays a pivotal role in this process. [Cancer Res 2008;68(6):1851–61]

Tumor progression locus 2 (TPL-2) kinase is essential for Toll-like receptor 4 activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) and for upregulation of the inflammatory cytokine tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-stimulated macrophages. LPS activation of ERK requires TPL-2 release from associated NF-kappaB1 p105, which blocks TPL-2 access to its substrate, the ERK kinase MEK. Here we demonstrate that TPL-2 activity is also regulated independently of p105, since LPS stimulation was still needed for TPL-2-dependent activation of ERK in Nfkb1(-/-) macrophages. In wild-type macrophages, LPS induced the rapid phosphorylation of serine (S) 400 in the TPL-2 C-terminal tail. Mutation of this conserved residue to alanine (A) blocked the ability of retrovirally expressed TPL-2 to induce the activation of ERK in LPS-stimulated Nfkb1(-/-) macrophages. TPL-2(S400A) expression also failed to reconstitute LPS activation of ERK and induction of TNF in Map3k8(-/-) macrophages, which lack endogenous TPL-2. Consistently, the S400A mutation was found to block LPS stimulation of TPL-2 MEK kinase activity. Thus, induction of TPL-2 MEK kinase activity by LPS stimulation of macrophages requires TPL-2 phosphorylation on S400, in addition to its release from NF-kappaB1 p105. Oncogenic C-terminal truncations of TPL-2 that remove S400 could promote its transforming potential by eliminating this critical control step.

Advances in analytical techniques, specifically in mass spectrometry, have allowed for both facile protein identification and routine sequencing of proteins at increased sensitivity levels. Protein modifications present additional challenges because they occur at low stoichiometries and often change the analytical behavior of the molecule. For example, characterization of protein phosphorylation provides crucial information to signaling processes that are often associated with disease. Research into protein phosphorylation requires inter-disciplinary co-operation involving multiple investigators with expertise in diverse scientific fields. As such, techniques must be simple, effective, and incorporate multiple checkpoints that confirm the sample contains a phosphorylated protein in order to ensure resources are conserved. In this study, tumor progression locus 2 (Tpl2), which has been implicated in cell cycle regulation and has been shown to play a significant role in critical signal transduction pathways, was transfected into 293T cells, overexpressed and isolated from the cell lysate. Isolated proteins were separated via 1D gel electrophoresis, and their phosphorylation was confirmed using phosphospecific staining. The bands were excised and subjected to tryptic digestion and immobilized metal affinity chromatography (IMAC) prior to analysis by capillary-LC-MS/MS. Three phosphorylation sites were detected on Tpl2. One site had previously been reported in the literature but had not been characterized by mass spectrometric methods until this time; two additional novel sites of phosphorylation were detected.

© 2015 AGA Institute. Background & Aims Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). Methods We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn's disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). Results A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. Conclusions Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer.

Tumor Progression Locus 2 (TPL2) is widely recognized as a cytoplasmic mitogen-activated protein 3 kinase with a prominent role in the regulation of inflammatory and oncogenic signal transduction. Herein we report that TPL2 may also operate in the nucleus as a physical and functional partner of nucleophosmin (NPM/B23), a major nucleolar phosphoprotein with diverse cellular activities linked to malignancy. We demonstrate that TPL2 mediates the phosphorylation of a fraction of NPM at threonine 199, an event required for its proteasomal degradation and maintenance of steady-state NPM levels. Upon exposure to ultraviolet C, Tpl2 is required for the translocation of de-phosphorylated NPM from the nucleolus to the nucleoplasm. NPM is an endogenous inhibitor of HDM2:p53 interaction and knockdown of TPL2 was found to result in reduced binding of NPM to HDM2, with concomitant defects in p53 accumulation following genotoxic or ribosomal stress. These findings expand our understanding of the function of TPL2 as a negative regulator of carcinogenesis by defining a nuclear role for this kinase in the topological sequestration of NPM, linking p53 signaling to the generation of threonine 199-phosphorylated NPM.

Tpl2/Cot is a serine/threonine kinase that plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including tumor necrosis factor-alpha (TNF-alpha). TNF-alpha stimulates the JNK, ERK, and p38 mitogen-activated protein kinases and the NF-kappaB pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1. Here we showed that Tpl2 activation by TNF-alpha signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2, which are required for the engagement of the ERK mitogen-activated protein kinase pathway. However, neither RIP1 nor TRAF2 overexpression was sufficient to activate Tpl2 and ERK. We also showed that Tpl2 activation by TNF-alpha depends on a tyrosine kinase activity that is detected in TNF-alpha-stimulated cells. Based on both genetic and biochemical evidence, we concluded that in a variety of cell types, Syk is the tyrosine kinase that plays an important role in the activation of Tpl2 upstream of ERK. These data therefore dissect the TNF receptor 1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2, and Syk in this pathway.

During lung injury alveolar epithelial cells are directly exposed to changes in PO 2 and PCO 2 . Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O 2 –5% CO 2 ) or hypercapnia (21% O 2– 15% CO 2 ) and expression of surfactant protein C was measured and compared to normal conditions (21% O 2 - 5% CO 2 ). Cell cycle progression and apoptosis were measured by flow cytometric analysis. Results A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-α partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. Conclusions Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.

Eukaryotic initiation factor 2 (eIF2) is a central regulator of translational initiation in times of growth and times of stress. Here we discovered three new conserved regulators of eIF2 in Saccharomyces cerevisiae. cdc123, homolog of mammalian D123, is a new cell division cycle mutant with a G2 delay at permissive temperature and a terminal, mating-proficient G1 arrest point. Cdc123 protein is regulated by nutrient availability. CHF1 and CHF2, homologs of mammalian checkpoint forkhead associated with RING genes, are required for G2 delay and G1 arrest of cdc123-4 and promote G1 delay when over-expressed. Cell cycle delaying activity and the natural instability of Chf1 and Chf2 depend on the integrity of both domains and association with Cdc123. Genetic analysis maps the Chf1 forkhead associated domain-binding site to the conserved Thr-274 of Cdc123, suggesting that mammalian D123 is a key target of Chfr. Gcd11, the gamma subunit of eIF2, is an additional Cdc123-interacting protein that is an essential target of the Cdc123 cell cycle promoting and Chf cell cycle arresting activity whose abundance is regulated by Cdc123, Chf1, and Chf2. Loss of cdc123 activity promotes Chf1 and Chf2 accumulation and Gcd11 depletion, accounting for the essentiality of Cdc123. The data establish the Cdc123-Chf-Gcd11 axis as an essential pathway for nutritional control of START that runs parallel to the Tor-Gcn2-Sui2 system of translational control.

Growth factor independence-1 (GFI1) and GFI1B are closely related, yet differentially expressed transcriptional repressors with nearly identical DNA binding domains. GFI1 is upregulated in the earliest thymocyte precursors, while GFI1B expression is restricted to T lymphopoiesis stages coincident with activation. Transgenic expression of GFI1 potentiates T-cell activation, while forced GFI1B expression decreases activation. Both mice and humans with mutant Gfi1 display lymphoid abnormalities. Here we describe autoregulation of Gfi1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human Gfi1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of Gfi1 can also be mediated by GFI1B. These data provide the first example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of Gfi1 by GFI1 and GFI1B in maintaining the normal expression patterns of Gfi1, and suggest that GFI1B may indirectly affect T-cell activation through repression of Gfi1.

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.

The Epstein-Barr virus-encoded latent infection membrane protein 1 (LMP1) is a pleiotropic protein, the activities of which include effects on cell transformation and phenotype, growth, and survival. The ability of LMP1 to mediate at least some of these phenomena could be attributed to the activation of the transcription factor NF-κB. LMP1 promotes NF-κB activation through the recruitment of the adapter protein TRAF2 and the formation of a dynamic multiprotein complex that includes the NF-κB kinase, the IκB kinases, and their downstream targets, IκBs and p105. In this study, we have identified the oncogenic kinase Tpl-2/Cot as a novel component of LMP1-induced NF-κB signaling. We show that Tpl-2 is expressed in primary biopsies from patients with nasopharyngeal carcinoma and Hodgkin's disease, where LMP1 is also found. Inducible expression of LMP1 promotes the activation of Tpl-2, and a catalytically inactive Tpl-2 mutant suppresses LMP1-induced NF-κB signaling. In colocalization and coimmunoprecipitation experiments, Tpl-2 and TRAF2 were found to interact with Tpl-2 functioning downstream of TRAF2. Consistent with this observation, catalytically inactive Tpl-2 also blocked CD40-mediated NF-κB activation, which largely depends on TRAF2. The ability of Tpl-2 to influence LMP1-induced NF-κB occurs through modulation of both IκBα and p105 functions. Furthermore, Tpl-2 was found to influence the expression of angiogenic mediators, such as COX-2 in LMP1-transfected cells. These data identify Tpl-2 as a component of LMP1 signaling downstream of TRAF2 and as a modulator of LMP1-mediated effects.

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (TnfΔAREmouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon γ and requires the function of CD8+ T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell–derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor–deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow–derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase–deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

The JmjC domain histone H3K36me2/me1 demethylase NDY1/KDM2B is overexpressed in various types of cancer. Here we show that knocking down NDY1 in a set of 10 cell lines derived from a broad range of human tumors inhibited their anchorage-dependent and anchorage-independent growth by inducing senescence and/or apoptosis in some and by inhibiting G1 progression in all. We further show that the knockdown of NDY1 in mammary adenocarcinoma cell lines decreased the number, size, and replating efficiency of mammospheres and downregulated the stem cell markers ALDH and CD44, while upregulating CD24. Together, these findings suggest that NDY1 is required for the self-renewal of cancer stem cells and are in agreement with additional findings showing that tumor cells in which NDY1 was knocked down undergo differentiation and a higher number of them is required to induce mammary adenocarcinomas, upon orthotopic injection in animals. Mechanistically, NDY1 functions as a master regulator of a set of miRNAs that target several members of the polycomb complexes PRC1 and PRC2, and its knockdown results in the de-repression of these miRNAs and the downregulation of their polycomb targets. Consistent with these observations, NDY1/KDM2B is expressed at higher levels in basal-like triple-negative breast cancers, and its overexpression is associated with higher rates of relapse after treatment. In addition, NDY1-regulated miRNAs are downregulated in both normal and cancer mammary stem cells. Finally, in primary human breast cancer, NDY1/KDM2B expression correlates negatively with the expression of the NDY1-regulated miRNAs and positively with the expression of their PRC targets. Cancer Res; 74(14); 3935–46. ©2014 AACR.

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer. Keywords: PI3K; MEK; KRAS; Colorectal cancer; Mouse model of cancer

The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. Twenty-one of the twenty-four non-small-cell-lung carcinomas we analyzed express IWS1. More importantly, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer.

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

Multiple biological functions have been ascribed to the Ras-related G protein R-Ras. These include the ability to transform NIH 3T3 fibroblasts, the promotion of cell adhesion, and the regulation of apoptotic responses in hematopoietic cells. To investigate the signaling mechanisms responsible for these biological phenotypes, we compared three R-Ras effector loop mutants (S61, G63, and C66) for their relative biological and biochemical properties. While the S61 mutant retained the ability to cause transformation, both the G63 and the C66 mutants were defective in this biological activity. On the other hand, while both the S61 and the C66 mutants failed to promote cell adhesion and survival in 32D cells, the G63 mutant retained the ability to induce these biological activities. Thus, the ability of R-Ras to transform cells could be dissociated from its propensity to promote cell adhesion and survival. Although the transformation-competent S61 mutant bound preferentially to c-Raf, it only weakly stimulated the mitogen-activated protein kinase (MAPK) activity, and a dominant negative mutant of MEK did not significantly perturb R-Ras oncogenicity. Instead, a dominant negative mutant of phosphatidylinositol 3-kinase (PI3-K) drastically inhibited the oncogenic potential of R-Ras. Interestingly, the ability of the G63 mutant to induce cell adhesion and survival was closely associated with the PI3-K-dependent signaling cascades. To further delineate R-Ras downstream signaling events, we observed that while a dominant negative mutant of Akt/protein kinase inhibited the ability of R-Ras to promote cell survival, both dominant negative mutants of Rac and Ral suppressed cell adhesion stimulated by R-Ras. Thus, the biological actions of R-Ras are mediated by multiple effectors, with PI3-K-dependent signaling cascades being critical to its functions.

The spindle checkpoint ensures the proper partition of the chromosomal content of dividing cells, by controlling the transition from metaphase to anaphase. In a recent issue of Cancer Cell , Vecchione and coworkers report that the protein product of the tumor suppressor gene Lzts1 (Leucine zipper tumor suppressor-1) binds the Cdk1 phosphatase Cdc25C and stabilizes it by protecting it from proteasomal degradation (Vecchione et al., 2007). Partial or complete loss of Lzts1 downregulates Cdc25C and inhibits Cdk1 activity during mitosis, leading to premature transition from metaphase to anaphase.

AKT/protein kinase B (PKB) is a cardinal node in diverse signaling cascades important in both normal cellular physiology and various disease states. AKT signaling regulates cell proliferation and survival, cell growth (size), glucose metabolism, cell motility and angiogenesis. Aberrant regulation of these processes result in cellular perturbations considered hallmarks of cancer, and numerous studies testify to the frequent hyperactivation of AKT signaling in many human cancers. Various oncoproteins and tumor suppressors intersect the AKT signal transduction pathway and are activated or inactivated, respectively, in cancer. This issue of Oncogene Reviews includes a collection of perspectives on the normal cellular functions of various components of the AKT pathway, as well as biological consequences of alterations of these proteins as related to tumorigenesis. Two reviews focus on AKT regulation, one of which addresses various aspects of phosphoinositide metabolism, while the other emphasizes the role of AKT-interacting proteins in AKT activation. Several reviews highlight the role of major AKT substrates involved in cellular metabolism, transcription and translation; another focuses on the role of AKT signaling in epithelial-mesenchymal transition. Also included are articles on the involvement of AKT pathway deregulation in human cancer and certain hereditary cancer syndromes, as well as in murine models of cancer based on AKT pathway activation. Additional articles discuss current approaches to identify selective inhibitors of the AKT pathway.

Both genetic mutations and UV irradiation (UVR) can predispose individuals to melanoma. Although BRAFV600E is the most prevalent oncogene in melanoma, the BRAFV600E mutant is not sufficient to induce tumors in vivo. Mutation at the CDKN2A locus is another melanoma-predisposing event that can disrupt the function of both p16INK4a and ARF. Numerous studies have focused on the role of p16INK4a in melanoma, but the involvement of ARF, a well-known p53 activator, is still controversial. Using a transgenic BRAFV600E mouse model previously generated in our laboratory, we report that loss of ARF is able to enhance spontaneous melanoma formation and cause profound sensitivity to neonatal UVB exposure. Mechanistically, BRAFV600E and ARF deletion synergize to inhibit nucleotide excision repair by epigenetically repressing XPC and inhibiting the E2F4/DP1 complex. We suggest that the deletion of ARF promotes melanomagenesis not by abrogating p53 activation but by acting in concert with BRAFV600E to increase the load of DNA damage caused by UVR. Cancer Res; 73(14); 4337–48. ©2013 AACR.

Lung cancer is a heterogeneous disease at both clinical and molecular levels, posing conceptual and practical bottlenecks in defining key pathways affecting its initiation and progression. Molecules with a central role in lung carcinogenesis are likely to be targeted by multiple deregulated pathways and may have prognostic, predictive, and/or therapeutic value. Here, we report that Tumor Progression Locus 2 (TPL2), a kinase implicated in the regulation of innate and adaptive immune responses, fulfils a role as a suppressor of lung carcinogenesis and is subject to diverse genetic and epigenetic aberrations in lung cancer patients. We show that allelic imbalance at the TPL2 locus, up-regulation of microRNA-370, which targets TPL2 transcripts, and activated RAS (rat sarcoma) signaling may result in down-regulation of TPL2 expression. Low TPL2 levels correlate with reduced lung cancer patient survival and accelerated onset and multiplicity of urethane-induced lung tumors in mice. Mechanistically, TPL2 was found to antagonize oncogene-induced cell transformation and survival through a pathway involving p53 downstream of cJun N-terminal kinase (JNK) and be required for optimal p53 response to genotoxic stress. These results identify multiple oncogenic pathways leading to TPL2 deregulation and highlight its major tumor-suppressing function in the lung.

The mitogen-activated protein kinase kinase kinase (MAPKKK or MAP3K) tumor progression locus 2 (Tpl2) is required for the transduction of signals initiated by the thrombin-activated G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1), which promote reorganization of the actin cytoskeleton and cell migration. Here, we show that Tpl2 is activated through Gα(i2)-transduced GPCR signals. Activated Tpl2 promoted the phosphorylation and activation of phospholipase C-β3 (PLCβ(3)); consequently, Tpl2 was required for thrombin-dependent production of inositol 1,4,5-trisphosphate (IP(3)), IP(3)-mediated cytoplasmic calcium ion (Ca(2+)) signals, and the activation of classical and novel members of the protein kinase C (PKC) family. A PKC-mediated feedback loop facilitated extracellular signal-regulated kinase (ERK) activation in response to Tpl2 and contributed to the coordinate regulation of the ERK and Ca(2+) signaling pathways. Pharmacological and genetic studies revealed that stimulation of cell migration by Tpl2 depends on both of these pathways. Tpl2 also promoted Ca(2+) signals and cell migration from sphingosine 1-phosphate-responsive GPCRs, which also couple to Gα(i); from Wnt5a; and from the interleukin-1β (IL-1β) receptor, a member of the Toll-IL-1R (TIR) domain family. Our data provide new insights into the role of Tpl2 in GPCR-mediated Ca(2+) signaling and cell migration.

The role of specific members of the NF-κB family of transcription factors in CD8 T-cell selection and development is largely unknown. Here, we show that mice lacking NF-κB1 develop a unique population of conventional CD8 single-positive (SP) thymocytes with memory T cell-like properties that populate peripheral immune organs. Development of this memory-like population is not due to PLZF(+) thymocytes and instead coincides with changes in CD8 T-cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF-κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8(+) thymocytes with memory cell characteristics.

The Ox-44 (CD53) leukocyte antigen contributes to the transduction of CD2-generated signals in T cells and natural killer (NK) cells and has been suggested to play a role in growth regulation. The gene encoding this protein was assigned to the midportion of mouse chromosome 3 by interspecific backcross mapping and to human chromosome region 1p21→p13.3 and rat chromosome region 2q34→q41 by fluorescence in situ hybridization. Comparative mapping data presented in this report demonstrate conservation of synteny within the region encompassing this gene in mouse (Cd53), human (CD53), and rat (CD53).

Although Akt is known to play a role in human cancer, the relative contribution of its three isoforms to oncogenesis remains to be determined. We expressed each isoform individually in an Akt1(-/-)/Akt2(-/-)/Akt3(-/-) cell line. MicroRNA profiling of growth factor-stimulated cells revealed unique microRNA signatures for cells with each isoform. Among the differentially regulated microRNAs, the abundance of the miR-200 family was decreased in cells bearing Akt2. Knockdown of Akt1 in transforming growth factor-beta (TGFbeta)-treated MCF10A cells also decreased the abundance of miR-200; however, knockdown of Akt2, or of both Akt1 and Akt2, did not. Furthermore, Akt1 knockdown in MCF10A cells promoted TGFbeta-induced epithelial-mesenchymal transition (EMT) and a stem cell-like phenotype. Carcinomas developing in MMTV-cErbB2/Akt1(-/-) mice showed increased invasiveness because of miR-200 down-regulation. Finally, the ratio of Akt1 to Akt2 and the abundance of miR-200 and of the messenger RNA encoding E-cadherin in a set of primary and metastatic human breast cancers were consistent with the hypothesis that in many cases breast cancer metastasis may be under the control of the Akt-miR-200-E-cadherin axis. We conclude that induction of EMT is controlled by microRNAs whose abundance depends on the balance between Akt1 and Akt2 rather than on the overall activity of Akt.

Moloney murine leukemia virus (MoMuLV)-induced rat T-cell lymphomas express discrete 1.8-, 2.2-, and 4-kb mRNA transcripts hybridizing under conditions of reduced stringency to a probe derived from a region upstream of the first exon of the Tpl-1/Ets-1 gene. Screening a cDNA library from one rat T-cell lymphoma with this genomic probe yielded 15 cDNA clones which were derived from 10 different genes. One of these genes, defined by the cDNA clone pRcT7a, was expressed as a 1.8-kb mRNA transcript in spleen and thymus but not in other normal rat tissues. Expression of the gene defined by the pRcT7a cDNA clone in a series of MoMuLV-induced rat T-cell lymphomas showed a perfect correlation with the expression of the rat leukocyte antigen MRC OX-44. Because of this observation, the pRcT7a clone was sequenced and it was shown to identify a gene coding for a 219-amino-acid protein. The homology between pRcT7a and the Tpl-1 probe used for its detection mapped within the 3' untranslated region of the pRcT7a cDNA clone. The pRcT7a protein, which exhibits four putative transmembrane regions and three putative glycosylation sites, contains a region which is nearly identical in sequence to a peptide derived from the rat leukocyte antigen MRC OX-44. This finding suggested that the pRcT7a cDNA clone defines the gene coding for OX-44. To confirm this finding, a pRcT7a construct in the retrovirus vector pZipNeo was introduced into the OX-44- T-cell lymphoma line 2788. Immunostaining with the MRC OX-44 monoclonal antibody followed by flow cytometry revealed that following gene transfer, the 2788 cells became OX-44+. Sequence comparisons revealed that pRcT7a/MRC OX-44 is a member of a family of genes which includes the melanoma-specific antigen ME491; the human leukocyte antigen CD37; the protein TAPA-1, which is expressed on the surface of human T cells and appears to be involved in growth regulation; the human gastrointestinal tumor antigen CO-029; and the Schistosoma mansoni-associated antigen Sm23.

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor-alpha, Toll-like receptor, and G protein-coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-gamma production. Accordingly, Tpl2(-/-) mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-gamma production. Furthermore, reconstitution of Rag2(-/-) mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-gamma defect seen in the Tpl2-deficient mice, confirming a T cell-intrinsic defect. CD4(+) T cells isolated from Tpl2(-/-) mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses.

The growth factor, vascular endothelial growth factor (VEGF), induces angiogenesis and promotes endothelial cell (EC) proliferation. Affymetrix gene array analyses show that VEGF stimulates the expression of a cluster of nuclear-encoded mitochondrial genes, suggesting a role for VEGF in the regulation of mitochondrial biogenesis. We show that the serine threonine kinase Akt3 specifically links VEGF to mitochondrial biogenesis. A direct comparison of Akt1 vs. Akt3 gene silencing was performed in ECs and has uncovered a discrete role for Akt3 in the control of mitochondrial biogenesis. Silencing of Akt3, but not Akt1, results in a decrease in mitochondrial gene expression and mtDNA content. Nuclear-encoded mitochondrial gene transcripts are also found to decrease when Akt3 expression is silenced. Concurrent with these changes in mitochondrial gene expression, lower O2 consumption was observed. VEGF stimulation of the major mitochondrial import protein TOM70 is also blocked by Akt3 inhibition. In support of a role for Akt3 in the regulation of mitochondrial biogenesis, Akt3 silencing results in the cytoplasmic accumulation of the master regulator of mitochondrial biogenesis, PGC-1α, and a reduction in known PGC-1α target genes. Finally, a subtle but significant, abnormal mitochondrial phenotype is observed in the brain tissue of AKT3 knockout mice. These results suggest that Akt3 is important in coordinating mitochondrial biogenesis with growth factor-induced increases in cellular energy demands.—Wright, G. L., Maroulakou, I. G., Eldridge, J., Liby, T. L., Sridharan, V., Tsichlis, P. N., Muise-Helmericks, R. C. VEGF stimulation of mitochondrial biogenesis: requirement of AKT3 kinase.

Energy depletion activates AMP-activated protein kinase (AMPK) and inhibits cell growth via TSC2-dependent suppression of mTORC1 signaling. Long term energy depletion also induces apoptosis by mechanisms that are not well understood to date. Here we show that AMPK, activated by energy depletion, inhibited cell survival by binding to and phosphorylating IRS-1 at Ser-794. Phosphorylation of IRS-1 at this site inhibited phosphatidylinositol 3-kinase/Akt signaling, suppressed the mitochondrial membrane potential, and promoted apoptosis. Of the treatments promoting energy depletion, glucose deprivation, hypoxia, and inhibition of ATP synthesis in the mitochondria stimulated phosphorylation of IRS-1 at Ser-794 via an LKB1/AMPK-dependent manner, whereas oxidative stress and 2-deoxyglucose stimulated phosphorylation at this site via a Ca2+/calmodulin-dependent protein kinase kinase beta/AMPK axis. These data define a novel pathway that cooperates with other adaptive mechanisms to formulate the cellular response to energy depletion.

Akt is a central regulator of cardiomyocyte survival after ischemic injury in vitro and in vivo, but the mechanisms regulating Akt activity in the postischemic cardiomyocyte are not known. Furthermore, although much is known about the detrimental role that the c-Jun N-terminal kinases (JNKs) play in promoting death of cells exposed to various stresses, little is known of the molecular mechanisms by which JNK activation can be protective. We report that JNKs are necessary for the reactivation of Akt after ischemic injury. We identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia, apparently by priming Akt for subsequent phosphorylation by 3-phosphoinositide–dependent protein kinase. The reduction in Akt activity that is induced by JNK inhibition may have significant biological consequences, as we find that JNKs, acting via Akt, are critical determinants of survival in posthypoxic cardiomyocytes in culture. Furthermore, in contrast to selective p38–mitogen-activated protein kinase inhibition, which was cardioprotective in vivo, concurrent inhibition of both JNKs and p38–mitogen-activated protein kinases increased ischemia/reperfusion injury in the heart of the intact rat. These studies demonstrate that reactivation of Akt after resolution of hypoxia and ischemia is regulated by JNKs and suggest that this is likely a central mechanism of the myocyte protective effect of JNKs.

Epithelial-mesenchymal transition (EMT) is an important process during development and oncogenesis by which epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. Squamous cell carcinoma lines engineered to express constitutively active Akt underwent EMT, characterized by down-regulation of the epithelial markers desmoplakin, E-cadherin, and beta-catenin and up-regulation of the mesenchymal marker vimentin. The cells lost epithelial cell morphology and acquired fibroblast-like properties. Additionally, E-cadherin was down-regulated transcriptionally. The cells expressing constitutively active Akt exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals. AKT is activated in many human carcinomas, and the AKT-driven EMT may confer the motility required for tissue invasion and metastasis. These findings suggest that future therapies based on AKT inhibition may complement conventional treatments by controlling tumor cell invasion and metastasis.

Earlier studies have shown that Tvl-1 gives rise to at least two differentially spliced mRNAs, one of which (Tvl-S) encodes a protein that lacks amino acids 91–112. DNA binding of RFX complexes assembled in the presence of Tvl-S is impaired. As a result, Tvl-S does not support the expression of Class II major histocompatibility complex (MHC) genes. Here, we show that the reason Tvl-S is inactive as a transcriptional regulator of Class II MHC genes is that the RFX complexes assembled in the presence of Tvl-S are unstable. Additionally, we show that interferon-γ, which induces Class II MHC gene expression in 293 cells, promotes a shift in the splicing pattern of RFXANK/Tvl-1 toward the transcriptionally active Tvl-L isoform, suggesting that differential splicing of Tvl-1 is a signal-regulated process. Finally, we show that Tvl-1 regulates the expression of non-MHC genes. One such gene encodes the ephrin receptor EphA3. Since both Tvl-L and Tvl-S are identical in their ability to induce the expression of EphA3, we conclude that Tvl-1 regulates the expression of non-MHC genes by RFX-independent mechanisms.

Earlier studies had suggested that epigenetic mechanisms play an important role in the control of human cytomegalovirus (HCMV) infection. Here we show that productive HCMV infection is indeed under the control of histone H3K27 trimethylation. The histone H3K27 methyltransferase EZH2, and its regulators JARID2 and NDY1/KDM2B repress GFI1, a transcriptional repressor of the major immediate-early promoter (MIEP) of HCMV. Knocking down EZH2, NDY1/KDM2B or JARID2 relieves the repression and results in the upregulation of GFI1. During infection, the incoming HCMV rapidly downregulates the GFI1 mRNA and protein in both wild-type cells and in cells in which EZH2, NDY1/KDM2B or JARID2 were knocked down. However, since the pre-infection levels of GFI1 in the latter cells are significantly higher, the virus fails to downregulate it to levels permissive for MIEP activation and viral infection. Following the EZH2-NDY1/KDM2B-JARID2-independent downregulation of GFI1 in the early stages of infection, the virus also initiates an EZH2-NDY1/ΚDM2Β-JARID2-dependent program that represses GFI1 throughout the infection cycle. The EZH2 knockdown also delays histone H3K27 trimethylation in the immediate early region of HCMV, which is accompanied by a drop in H3K4 trimethylation that may contribute to the shEZH2-mediated repression of the major immediate early HCMV promoter. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program., Author Summary Human cytomegalovirus (HCMV) is a significant pathogen that belongs to the herpesvirus family. Here we show that the histone H3K27 methyltransferase EZH2 and its regulators JARID2 and NDY1/KDM2B are required for the establishment of productive infection. Mechanistically, the EZH2-NDY1/KDM2B-JARID2 axis downregulates GFI1, a repressor of the HCMV major-immediate-early promoter (MIEP) and inhibition of this axis upregulates GFI1 and interferes with the activation of the MIEP and HCMV infection. GFI1 is rapidly downregulated during infection in both wild-type and EZH2, NDY1/KDM2B, JARID2 knockdown cells. However, since the starting levels of GFI1 in the latter are significantly higher, they remain high despite the virus-induced GFI1 downregulation, preventing the infection. Following the downregulation of GFI1 immediately after virus entry, HCMV initiates an EZH2-NDY1/KDM2B-JARID2-JMJD3-dependent program to maintain the low expression of GFI1 throughout the infection cycle. The knockdown of EZH2 also modulates the accumulation of histone H3K27me3 and H3K4me3 in the immediate-early region of HCMV, and by doing so, it may contribute directly to the MIEP repression induced by the knockdown of EZH2. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program.