Phosphorylation and Stabilization of PIN1 by JNK Promote Intrahepatic Cholangiocarcinoma Growth.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of the c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and therefore the key downstream effectors of this pathway, remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signaling in ICC that could open up therapeutic opportunities. APPROACH AND RESULTS: Using loss-of-function and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumors, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 through all-trans retinoic acid, a Food and Drug Administration-approved drug, impairs the growth of both cultured and xenografted ICC cells. CONCLUSIONS: Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation through PIN1 inhibition.

Targeting myosin 1c inhibits murine hepatic fibrogenesis.

Myosin 1c (Myo1c) is an unconventional myosin that modulates signaling pathways involved in tissue injury and repair. In this study, we observed that Myo1c expression is significantly upregulated in human chronic liver disease such as nonalcoholic steatohepatitis (NASH) and in animal models of liver fibrosis. High throughput data from the GEO-database identified similar Myo1c upregulation in mice and human liver fibrosis. Notably, transforming growth factor-ß1 (TGF-ß1) stimulation to hepatic stellate cells (HSCs), the liver pericyte and key cell type responsible for the deposition of extracellular matrix, upregulates Myo1c expression, whereas genetic depletion or pharmacological inhibition of Myo1c blunted TGF-ß-induced fibrogenic responses, resulting in repression of α-smooth muscle actin (α-SMA) and collagen type I α 1 chain (Col1α1) mRNA. Myo1c deletion also decreased fibrogenic processes such as cell proliferation, wound healing response, and contractility when compared with vehicle-treated HSCs. Importantly, phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) and mothers against decapentaplegic homolog 3 (SMAD3) were significantly blunted upon Myo1c inhibition in GRX cells as well as Myo1c knockout (Myo1c-KO) mouse embryonic fibroblasts (MEFs) upon TGF-ß stimulation. Using the genetic Myo1c-KO mice, we confirmed that Myo1c is critical for fibrogenesis, as Myo1c-KO mice were resistant to carbon tetrachloride (CCl4)-induced liver fibrosis. Histological and immunostaining analysis of liver sections showed that deposition of collagen fibers and α-SMA expression were significantly reduced in Myo1c-KO mice upon liver injury. Collectively, these results demonstrate that Myo1c mediates hepatic fibrogenesis by modulating TGF-ß signaling and suggest that inhibiting this process may have clinical application in treating liver fibrosis.NEW & NOTEWORTHY The incidences of liver fibrosis are growing at a rapid pace and have become one of the leading causes of end-stage liver disease. Although TGF-ß1 is known to play a prominent role in transforming cells to produce excessive extracellular matrix that lead to hepatic fibrosis, the therapies targeting TGF-ß1 have achieved very limited clinical impact. This study highlights motor protein myosin-1c-mediated mechanisms that serve as novel regulators of TGF-ß1 signaling and fibrosis.

Screening Kinase-Dependent Phosphorylation of Key Metabolic Reprogramming Regulators.

Aerobic glycolysis has been commonly linked to cell proliferation, especially in cancer cells where it serves to generate sufficient energy and biosynthesis of new cell constituents needed for cell growth and division. The M2 isoform of pyruvate kinase (PKM2) catalyzes the last reaction of the glycolytic process. PKM2 promotes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, generating ATP and releasing pyruvate. This rate-limiting reaction relies therefore on the enzymatic activity of PKM2. The switching between the high- and low-activity states of PKM2 is subjected to a combination of allosteric mechanisms and fine-tuned regulation by oncogenes and tumor suppressor genes. These regulatory mechanisms involve primarily post-translational modifications of PKM2. Recent findings suggest that phosphorylation contributes to the regulation of PKM2 activity.Here, we describe an in vitro kinase assay we used to assess PKM2 phosphorylation by c-Jun N-terminal kinase (JNK), a master regulator of apoptosis, cell proliferation, and differentiation. While the use of phospho-specific antibodies gives information in terms of measuring the effects of a given kinase on its substrate, specific antibodies for newly identified phospho-groups are not readily available. The in vitro kinase assay allows the immediate measuring of phosphorylation of any substrate of interest. Although there are several options that do not use radioactive materials, we continue to rely on this biochemical method for robust quantitation of results. More interestingly, this protocol can be easily adapted to measure the activity of other kinases by using their specific substrates.

An Integrated Methodology to Quantify the Glycolytic Stress in Plasma Cell Myeloma in Response to Cytotoxic Drugs.

Multiple myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). Myeloma plasma cells, like many other cancer cells, change their metabolism in response to internal and external stimuli. The main metabolic alterations of MM cells include deregulated glycolysis (commonly associated with enhanced uptake and utilization of glucose), lipid metabolism dysregulation, as well as deregulated mitochondrial respiration (commonly associated with the deregulated formation of reactive oxygen species). Over the past decade, the discovery of novel methodologies and the commercialization of sophisticated instrumentation and reagents have facilitated the detection of real-time changes in cellular bioenergetics. Of those, the Seahorse™ extracellular flux (XF) analyzer has been widely used to evaluate the glycolytic flux and mitochondrial respiration in many cell types. While adherent cell lines are easy to use with this technology, non-adherent suspension cells are more difficult to handle especially when their metabolic activities are being investigated in response to drug treatment. Here, we provide an integrated protocol that allows the detection of extracellular acidification rate (ECAR) of live myeloma plasma cells in response to chemotherapeutic drugs. Our optimized protocol consists of treating myeloma cells with cytotoxic drug of interest in a standard culture plate prior to the real-time analysis in the XF analyzer. Furthermore, we provide results of experiments in which the metabolic activities of myeloma cells in response to cytotoxic treatment were compared between the manufacturer's basic procedure and our optimized protocol. Our observations suggest that our integrated protocol can be used to achieve consistent, well-standardized results and thus it may have broad applications in studies focusing on the characterization of metabolic events in non-adherent suspension cells.

Gadd45beta promotes hepatocyte survival during liver regeneration in mice by modulating JNK signaling.

In the liver, the JNK cascade is induced downstream of TNF receptors (TNFRs) in response to inflammatory, microbial, and toxic challenges. Sustained activation of JNK triggers programmed cell death (PCD), and hepatocyte survival during these challenges requires induction of the NF-kappaB pathway, which antagonizes this activation by upregulating target genes. Thus, modulation of JNK activity is crucial to the liver response to TNFR-mediated challenge. The basis for this modulation, however, is unknown. Here, we investigated the role of the NF-kappaB target Gadd45b in the regulation of hepatocyte fate during liver regeneration after partial hepatectomy. We generated Gadd45b(-/-) mice and found that they exhibited decreased hepatocyte proliferation and increased PCD during liver regeneration. Notably, JNK activity was markedly increased and sustained in livers of Gadd45b(-/-) mice compared with control animals after partial hepatectomy. Furthermore, imposition of a Jnk2-null mutation, attenuating JNK activity, completely rescued the regenerative response in Gadd45b(-/-) mice. Interestingly, Gadd45beta ablation did not affect hepatotoxic JNK signaling after a TNFR-mediated immune challenge, suggesting specificity in the inducible hepatic program for JNK restraint activated during distinct TNFR-mediated challenges. These data provide a basis for JNK suppression during liver regeneration and identify Gadd45beta as a potential therapeutic target in liver diseases.

Gadd45 beta mediates the NF-kappa B suppression of JNK signalling by targeting MKK7/JNKK2.

NF-kappa B/Rel transcription factors control apoptosis, also known as programmed cell death. This control is crucial for oncogenesis, cancer chemo-resistance and for antagonizing tumour necrosis factor alpha (TNFalpha)-induced killing. With regard to TNFalpha, the anti-apoptotic activity of NF-kappa B involves suppression of the c-Jun N-terminal kinase (JNK) cascade. Using an unbiased screen, we have previously identified Gadd45 beta/Myd118, a member of the Gadd45 family of inducible factors, as a pivotal mediator of this suppressive activity of NF-kappa B. However, the mechanisms by which Gadd45 beta inhibits JNK signalling are not understood. Here, we identify MKK7/JNKK2--a specific and essential activator of JNK--as a target of Gadd45 beta, and in fact, of NF-kappa B itself. Gadd45 beta binds to MKK7 directly and blocks its catalytic activity, thereby providing a molecular link between the NF-kappa B and JNK pathways. Importantly, Gadd45 beta is required to antagonize TNFalpha-induced cytotoxicity, and peptides disrupting the Gadd45 beta/MKK7 interaction hinder the ability of Gadd45 beta, as well as of NF-kappa B, to suppress this cytotoxicity. These findings establish a basis for the NF-kappa B control of JNK activation and identify MKK7 as a potential target for anti-inflammatory and anti-cancer therapy.

Oxygen JNKies: phosphatases overdose on ROS.

Proinflammatory cytokine TNFalpha triggers cell death by inducing reactive oxygen species (ROS). These then inflict cytotoxicity through downstream activation of the JNK MAPK cascade. Yet the mechanisms by which ROS trigger JNK signaling have remained elusive. In a recent issue of Cell, Kamata et al. now provide one such mechanism.

T cell-derived lymphotoxin regulates liver regeneration.

BACKGROUND & AIMS: The ability of the liver to regenerate hepatic mass is essential to withstanding liver injury. The process of liver regeneration is tightly regulated by distinct signaling cascades involving components of the innate immune system, cytokines, and growth factors. However, the role of the adaptive immune system in regulation of liver regeneration is not well-defined. The role of adaptive immune system in liver regeneration was investigated in lymphocyte-deficient mice and in conditional lymphotoxin-deficient mice. METHODS: A model of liver regeneration after 70% partial hepatectomy was used, followed by examination of liver pathology, survival, DNA synthesis, and cytokine expression. RESULTS: We found that mice deficient in T cells show a reduced capacity for liver regeneration following partial hepatectomy. Furthermore, surface lymphotoxin, provided by T cells, is critical for liver regeneration. Mice specifically deficient in T-cell lymphotoxin had increased liver damage and a reduced capacity to initiate DNA synthesis after partial hepatectomy. Transfer of splenocytes from wild-type but not lymphotoxin-deficient mice improved liver regeneration in T cell-deficient mice. We found that an agonistic antibody against the lymphotoxin beta receptor was able to facilitate liver regeneration by reducing liver injury, increasing interleukin-6 production, hepatocyte DNA synthesis, and survival of lymphocyte-deficient (Rag) mice after partial hepatectomy. CONCLUSIONS: The adaptive immune system directly regulates liver regeneration via a T cell-derived lymphotoxin axis, and pharmacological stimulation of lymphotoxin beta receptor might represent a novel therapeutic approach to improve liver regeneration.

Upregulation of Twist-1 by NF-kappaB blocks cytotoxicity induced by chemotherapeutic drugs.

NF-kappaB/Rel transcription factors are central to controlling programmed cell death (PCD). Activation of NF-kappaB blocks PCD induced by numerous triggers, including ligand engagement of tumor necrosis factor receptor (TNF-R) family receptors. The protective activity of NF-kappaB is also crucial for oncogenesis and cancer chemoresistance. Downstream of TNF-Rs, this activity of NF-kappaB has been linked to the suppression of reactive oxygen species and the c-Jun-N-terminal-kinase (JNK) cascade. The mechanism by which NF-kappaB inhibits PCD triggered by chemotherapeutic drugs, however, remains poorly understood. To understand this mechanism, we sought to identify unrecognized protective genes that are regulated by NF-kappaB. Using an unbiased screen, we identified the basic-helix-loop-helix factor Twist-1 as a new mediator of the protective function of NF-kappaB. Twist-1 is an evolutionarily conserved target of NF-kappaB, blocks PCD induced by chemotherapeutic drugs and TNF-alpha in NF-kappaB-deficient cells, and is essential to counter this PCD in cancer cells. The protective activity of Twist-1 seemingly halts PCD independently of interference with cytotoxic JNK, p53, and p19(ARF) signaling, suggesting that it mediates a novel protective mechanism activated by NF-kappaB. Indeed, our data indicate that this activity involves a control of inhibitory Bcl-2 phosphorylation. The data also suggest that Twist-1 and -2 play an important role in NF-kappaB-dependent chemoresistance.

Growth arrest and DNA damage protein 45b (Gadd45b) protects retinal ganglion cells from injuries.

We hypothesize that neurons have protective mechanisms against adverse local conditions that improve the chances of cell survival. In the present study, we find that growth arrest and DNA damage protein 45b (Gadd45b), a previously unknown molecule in neurons of any type, is neuroprotective in retinal ganglion cells (RGCs) in the retina. Gadd45b is upregulated in RGCs in response to oxidative stress, aging and elevated intraocular pressure. Using Gadd45b siRNA, we show that Gadd45b protects RGCs from dying against different neuronal injuries including oxidative stress, TNFalpha cytotoxicity, and glutamate excitotoxicity in vitro. Using Gadd45b knockout mice, we find that Gadd45b protects RGCs from dying against oxidative stress in vivo. Our data suggest that Gadd45b is an important component of the intrinsic neuroprotective mechanisms of RGC neurons in the retina and, perhaps in the CNS as well.

Mechanisms of liver disease: cross-talk between the NF-kappaB and JNK pathways.

The liver plays a central role in the transformation and degradation of endogenous and exogenous chemicals, and in the removal of unwanted cells such as damaged, genetically mutated and virus-infected cells. Because of this function, the liver is susceptible to toxicity caused by the products generated during these natural occurrences. Hepatocyte death is the major feature of liver injury. In response to liver injury, specific intracellular processes are initiated to maintain liver integrity. Inflammatory cytokines including tumor necrosis factor (TNF)alpha and interleukin-6 (IL-6) are key mediators of these processes and activate different cellular response such as proliferation, survival and death. TNFalpha induces specific signaling pathways in hepatocytes that lead to activation of either pro-survival mediators or effectors of cell death. Whereas activation of transcription factor NF-kappaB promotes survival, c-Jun N-terminal kinases (JNKs) and caspases are strategic effectors of cell death in the TNFalpha-mediated signaling pathway. This review summarizes recent advances in the mechanisms of TNFalpha-induced hepatotoxicity and suggests that NF-kappaB plays a protective role against JNK-induced hepatocyte death. Identification of the mechanisms regulating interplay between the NF-kappaB and JNK pathways is required in the search for novel targets for the treatment of liver disease, including hepatitis and hepatocellular carcinoma.

The NF-kappaB transcription factor pathway as a therapeutic target in cancer: methods for detection of NF-kappaB activity.

NF-kappaB transcription factors marshal innate and adaptive immunity and inflammation. NF-kappaB also counters programmed cell death (PCD) induced by the proinflammatory cytokine tumor necrosis factor (TNF)alpha, and this activity of NF-kappaB is crucial for organismal physiology, chronic inflammation, and tumorigenesis. Indeed, whereas NF-kappaB contributes to many aspects of oncogenesis, it is now clear that its suppressive action on PCD is central to this process. Notably, recent studies indicate that NF-kappaB represents a crucial link in the well-established association between inflammation and carcinogenesis. In this link, NF-kappaB promotes synthesis of inflammatory mediators (e.g. TNFalpha) that stimulate growth of cancer cells, and upregulates genes that protect these cells against PCD induced by inflammatory signals. Elevated NF-kappaB activity also hampers tumor-cell killing inflicted by radiation and chemotherapeutic drugs, and in so doing, promotes resistance to anticancer therapy. Accordingly, NF-kappaB-targeting drugs are increasingly being used for treatment of human malignancies. Owing to the ubiquitous nature of the NF-kappaB pathway, however, these drugs have serious side effects, which limit their clinical use. Thus, a preferable approach would be to block, rather than NF-kappaB itself, its critical downstream targets that mediate discrete functions in cancer, such as prosurvival functions. Recent discoveries unraveling tissue specificity in the NF-kappaB-inducible mechanism(s) for control of PCD and identifying putative effectors of this control clearly validate this therapeutic approach. Given the emerging role of TNFkappa-induced signals of NF-kappaB activation in cancer and the potential of these signals for yielding new anticancer therapies, we focus herein on the methods most commonly used for analysis of the molecular steps leading from the triggering of TNF-Receptor (TNF-R)1 - the primary receptor of TNFalpha - to the induction of NF-kappaB. Specifically, we review the methods used for analysis of TNF-R1 trafficking, assembly of so-called TNF-R1 complex I, formation and activation of the IkappaB kinase (IKK) complex, phosphorylation and proteolysis of inhibitory IkappaB proteins, post-translational modifications and nuclear translocation of NF-kappaB dimers, induction of NF-kappaB transcriptional activity and binding to specific promoters, and upregulation of NF-kappaB target genes. The analysis of these events in cancerous cells may not only provide a better understanding of the basis for the role of NF-kappaB in carcinogenesis, but also potential new targets for selective anticancer therapy.

The ERK and JNK pathways in the regulation of metabolic reprogramming.

Most tumor cells reprogram their glucose metabolism as a result of mutations in oncogenes and tumor suppressors, leading to the constitutive activation of signaling pathways involved in cell growth. This metabolic reprogramming, known as aerobic glycolysis or the Warburg effect, allows tumor cells to sustain their fast proliferation and evade apoptosis. Interfering with oncogenic signaling pathways that regulate the Warburg effect in cancer cells has therefore become an attractive anticancer strategy. However, evidence for the occurrence of the Warburg effect in physiological processes has also been documented. As such, close consideration of which signaling pathways are beneficial targets and the effect of their inhibition on physiological processes are essential. The MAPK/ERK and MAPK/JNK pathways, crucial for normal cellular responses to extracellular stimuli, have recently emerged as key regulators of the Warburg effect during tumorigenesis and normal cellular functions. In this review, we summarize our current understanding of the roles of the ERK and JNK pathways in controlling the Warburg effect in cancer and discuss their implication in controlling this metabolic reprogramming in physiological processes and opportunities for targeting their downstream effectors for therapeutic purposes.

High Expression of Glycolytic Genes in Cirrhosis Correlates With the Risk of Developing Liver Cancer.

A marked increase in the rate of glycolysis is a key event in the pathogenesis of hepatocellular carcinoma (HCC), the main type of primary liver cancer. Liver cirrhosis is considered to be a key player in HCC pathogenesis as it precedes HCC in up to 90% of patients. Intriguingly, the biochemical events that underlie the progression of cirrhosis to HCC are not well understood. In this study, we examined the expression profile of metabolic gene transcripts in liver samples from patients with HCC and patients with cirrhosis. We found that gene expression of glycolytic enzymes is up-regulated in precancerous cirrhotic livers and significantly associated with an elevated risk for developing HCC. Surprisingly, expression levels of genes involved in mitochondrial oxidative metabolism are markedly increased in HCC compared to normal livers but remain unchanged in cirrhosis. Our findings suggest that key glycolytic enzymes such as hexokinase 2 (HK2), aldolase A (ALDOA), and pyruvate kinase M2 (PKM2) may represent potential markers and molecular targets for early detection and chemoprevention of HCC.

GADD45ß Loss Ablates Innate Immunosuppression in Cancer.

T-cell exclusion from the tumor microenvironment (TME) is a major barrier to overcoming immune escape. Here, we identify a myeloid-intrinsic mechanism governed by the NF-κB effector molecule GADD45ß that restricts tumor-associated inflammation and T-cell trafficking into tumors. In various models of solid cancers refractory to immunotherapies, including hepatocellular carcinoma and ovarian adenocarcinoma, Gadd45b inhibition in myeloid cells restored activation of proinflammatory tumor-associated macrophages (TAM) and intratumoral immune infiltration, thereby diminishing oncogenesis. Our results provide a basis to interpret clinical evidence that elevated expression of GADD45B confers poor clinical outcomes in most human cancers. Furthermore, they suggest a therapeutic target in GADD45ß for reprogramming TAM to overcome immunosuppression and T-cell exclusion from the TME.Significance: These findings define a myeloid-based immune checkpoint that restricts T-cell trafficking into tumors, with potentially important therapeutic implications to generally improve the efficacy of cancer immunotherapy. Cancer Res; 78(5); 1275-92. ©2017 AACR.

A method for isolating prosurvival targets of NF-kappaB/Rel transcription factors.

NF-KappaB/Rel transcription factors are critical regulators of immunity, inflammation, development, and cell survival. Activation of NF-KB inhibits programmed cell death (PCD) triggered by tumor necrosis factor alpha (TNFalpha) and several other stimuli. The prosurvival activity of NF-KB is also crucial to lymphopoiesis, neuroprotection, tumorigenesis, and cancer chemoresistance. The characterization of the downstream targets that mediate the prosurvival activity of NF-KB is therefore a topic of intense investigation. Early screens aimed at identifying these genes were mainly based on expression criteria and so were poised to only isolate genes already known to have protective effects. Here, we describe a new method for the identification of these genes, whereby expression libraries are screened for their ability to halt PCD in NF-KB-deficient cells. This complementation approach provides substantial advantages over other approaches, as it enables functional assessment of isolated genes without any preconceived notion about their sequence or presumed role. Expression libraries are generated from cells that are resistant to TNFalpha-induced cytotoxicity and are then enriched in prosurvival genes upon selection with TNFa in NF-kappaB/RelA-null cells, which are highly susceptible instead to this cytotoxicity. Upon enrichment, libraries are screened through a randomized two-step approach, whereby cDNAs are first tested for cytoprotective function and then for differential expression in NF-kappaB-proficient and NF-KappaB-deficient cells.

Linking apoptosis to cancer metabolism: Another missing piece of JuNK.

Cancer cells become dependent on aerobic glycolysis to sustain rapid proliferation and escape apoptosis. How this metabolic change, also known as the Warburg effect, is linked to apoptosis remains largely unknown. Our new data place c-Jun N-terminal kinase in the center of a hub regulating apoptosis and cancer metabolism.