SIGNIFICANCE:B cell lymphoma-2 (Bcl-2) was discovered over three decades ago and is the prototype antiapoptotic member of the Bcl-2 family that comprises proteins with contrasting effects on cell fate. First identified as a consequence of chromosomal translocation (t 14:18) in human lymphoma, subsequent studies have revealed mutations and/or gene copy number alterations as well as posttranslational modifications of Bcl-2 in a variety of human cancers. The canonical function of Bcl-2 is linked to its ability to inhibit mitochondrial membrane permeabilization, thereby regulating apoptosome assembly and activation by blocking the cytosolic translocation of death amplification factors. Of note, the identification of specific domains within the Bcl-2 family of proteins (Bcl-2 homology domains; BH domains) has not only provided a mechanistic insight into the various interactions between the member proteins but has also been the impetus behind the design and development of small molecule inhibitors and BH3 mimetics for clinical use. Recent Advances: Aside from its role in maintaining mitochondrial integrity, recent evidence provides testimony to a novel facet in the biology of Bcl-2 that involves an intricate cross talk with cellular redox state. Bcl-2 overexpression modulates mitochondrial redox metabolism to create a "pro-oxidant" milieu, conducive for cell survival. However, under states of oxidative stress, overexpression of Bcl-2 functions as a redox sink to prevent excessive buildup of reactive oxygen species, thereby inhibiting execution signals. Emerging evidence indicates various redox-dependent transcriptional changes and posttranslational modifications with different functional outcomes.
CRITICAL ISSUES:Understanding the complex interplay between Bcl-2 and the cellular redox milieu from the standpoint of cell fate signaling remains vital for a better understanding of pathological states associated with altered redox metabolism and/or aberrant Bcl-2 expression.
FUTURE DIRECTIONS:Based on its canonical functions, Bcl-2 has emerged as a potential druggable target. Small molecule inhibitors of Bcl-2 and/or other family members with similar function, as well as BH3 mimetics, are showing promise in the clinic. The emerging evidence for the noncanonical activity linked to cellular redox metabolism provides a novel avenue for the design and development of diagnostic and therapeutic strategies against cancers refractory to conventional chemotherapy by the overexpression of this prosurvival protein.
SIGNIFICANCE: here is evidence to implicate reactive oxygen species (ROS) in tumorigenesis and its progression. This has been associated with the interplay between ROS and oncoproteins, resulting in enhanced cellular proliferation and survival. Recent Advances: To date, studies have investigated specific contributions of the crosstalk between ROS and signaling networks in cancer initiation and progression. These investigations have challenged the established dogma of ROS as agents of cell death by demonstrating a secondary function that fuels cell proliferation and survival. Studies have thus identified (onco)proteins (Bcl-2, STAT3/5, RAS, Rac1, and Myc) in manipulating ROS level as well as exploiting an altered redox environment to create a milieu conducive for cancer formation and progression.
CRITICAL ISSUES: Despite these advances, drug resistance and its association with an altered redox metabolism continue to pose a challenge at the mechanistic and clinical levels. Therefore, identifying specific signatures, altered protein expressions, and modifications as well as protein-protein interplay/function could not only enhance our understanding of the redox networks during cancer initiation and progression but will also provide novel targets for designing specific therapeutic strategies.
FUTURE DIRECTIONS: Not only a heightened realization is required to unravel various gene/protein networks associated with cancer formation and progression, particularly from the redox standpoint, but there is also a need for developing more sensitive tools for assessing cancer redox metabolism in clinical settings. This review attempts to summarize our current knowledge of the crosstalk between oncoproteins and ROS in promoting cancer cell survival and proliferation and treatment strategies employed against these oncoproteins.
SIGNIFICANCE: Breast cancer is a unique disease characterized by heterogeneous cell populations causing roadblocks in therapeutic medicine, owing to its complex etiology and primeval understanding of the biology behind its genesis, progression, and sustenance. Globocan statistics indicate over 1.7 million new breast cancer diagnoses in 2012, accounting for 25% of all cancer morbidities.
RECENT ADVANCES: Despite these dismal statistics, the introduction of molecular gene signature platforms, progressive therapeutic approaches in diagnosis, and management of breast cancer has led to more effective treatment strategies and control measures concurrent with an equally reassuring decline in the mortality rate.
CRITICAL ISSUES: However, an enormous body of research in this area is requisite as high mortality associated with metastatic and/or drug refractory tumors continues to present a therapeutic challenge. Despite advances in systemic chemotherapy, the median survival of patients harboring metastatic breast cancers continues to be below 2 years.
FUTURE DIRECTIONS: Hence, a massive effort to scrutinize and evaluate chemotherapeutics on the basis of the molecular classification of these cancers is undertaken with the objective to devise more attractive and feasible approaches to treat breast cancers and improve patients' quality of life. This review aims to summarize the current understanding of the biology of breast cancer as well as challenges faced in combating breast cancer, with special emphasis on the current battery of treatment strategies. We will also try and gain perspective from recent encounters on novel findings responsible for the progression and metastatic transformation of breast cancer cells in an endeavor to develop more targeted treatment options.
Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1-Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson's disease.
Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is conventionally considered to be solely dependent on local protein synthesis. Given the impact of epigenetics on memory, the intriguing question is whether epigenetic regulation influences mGluR-LTD as well. G9a/GLP histone lysine methyltransferase complex is crucial for brain development and goal-directed learning as well as for drug-addiction. In this study, we analyzed whether the epigenetic regulation by G9a/GLP complex affects mGluR-LTD in CA1 hippocampal pyramidal neurons of 5-7 weeks old male Wistar rats. In hippocampal slices with intact CA1 dendritic regions, inhibition of G9a/GLP activity abolished mGluR-LTD. The inhibition of this complex upregulated the expression of plasticity proteins like PKMζ, which mediated the prevention of mGluR-LTD expression by regulating the NSF-GluA2-mediated trafficking of AMPA receptors towards the postsynaptic site. G9a/GLP inhibition during the induction of mGluR-LTD also downregulated the protein levels of phosphorylated-GluA2 and Arc. Interestingly, G9a/GLP inhibition could not impede the mGluR-LTD when the cell-body was severed. Our study highlights the role of G9a/GLP complex in intact neuronal network as a bidirectional switch; when turned on, it facilitates the expression of mGluR-LTD, and when turned off, it promotes the expression of long-term potentiation.
The tuberal nucleus (TN) is a surprisingly understudied brain region. We found that somatostatin (SST) neurons in the TN, which is known to exhibit pathological or cytological changes in human neurodegenerative diseases, play a crucial role in regulating feeding in mice. GABAergic tuberal SST (TNSST) neurons were activated by hunger and by the hunger hormone, ghrelin. Activation of TNSST neurons promoted feeding, whereas inhibition reduced it via projections to the paraventricular nucleus and bed nucleus of the stria terminalis. Ablation of TNSST neurons reduced body weight gain and food intake. These findings reveal a previously unknown mechanism of feeding regulation that operates through orexigenic TNSST neurons, providing a new perspective for understanding appetite changes.
The regulation of host factors is a key strategy employed by viruses to suppress host defense systems and enhance their propagation; however, the mechanisms that underlie this regulation is still unclear. Formyl peptide receptor 2 (FPR2) recognizes numerous proinflammatory and anti-inflammatory stimuli, and emerging reports indicate elevated levels of FPR2 in several disease conditions. Although studies have implicated FPR2 in a myriad of inflammatory conditions, how viruses exploit this cell-surface receptor to facilitate disease progression remains unknown. In this study, we show that the activation of TLR3 and TLR7 induces the up-regulation of FPR2. We provide evidence that signal transducer and activator of transcription 3 (STAT3) phosphorylation is critical for the induction of FPR2 by double-stranded RNA, but not single-stranded RNA viral mimetics. Use of bone marrow-derived macrophages (BMDMs) from IFN-αβ receptor-deficient mice revealed that signaling via the type I IFN-STAT3 pathway is essential for FPR2 induction. We demonstrate that virus infection with enterovirus 71 and H1N1 PR8 influenza virus results in increased FPR2 expression. Inhibition of STAT3 phosphorylation in virus-infected cells repressed the induction of FPR2, which led to a reduction in viral loads. Finally, the absence of FPR2 in murine BMDMs resulted in lower viral loads, which suggests that FPR2 may be important for virus replication. Altogether, our study provides novel insights into how RNA viruses may hijack the immune system to facilitate their replication and survival. Identification of these regulatory elements may be useful in designing therapeutics for inflammatory disease conditions that are associated with elevated levels of FPR2.-Ampomah, P. B., Moraes, L. A., Lukman, H. M., Lim, L. H. K. Formyl peptide receptor 2 is regulated by RNA mimics and viruses through an IFN-β-STAT3-dependent pathway.
Evolutionarily conserved across eukaryotic cells, macroautophagy (herein autophagy) is an intracellular catabolic degradative process targeting damaged and superfluous cellular proteins, organelles, and other cytoplasmic components. Mechanistically, it involves formation of double-membrane vesicles called autophagosomes that capture cytosolic cargo and deliver it to lysosomes, wherein the breakdown products are eventually recycled back to the cytoplasm. Dysregulation of autophagy often results in various disease manifestations, including neurodegeneration, microbial infections, and cancer. In the case of cancer, extensive attention has been devoted to understanding the paradoxical roles of autophagy in tumor suppression and tumor promotion. In this review, while we summarize how this self-eating process is implicated at various stages of tumorigenesis, most importantly, we address the link between autophagy and hallmarks of cancer. This would eventually provide a better understanding of tumor dependence on autophagy. We also discuss how therapeutics targeting autophagy can counter various transformations involved in tumorigenesis. Finally, this review will provide a novel insight into the mutational landscapes of autophagy-related genes in several human cancers, using genetic information collected from an array of cancers.
Macrophages are potent immune cells with well-established roles in the response to stress, injury, infection and inflammation. The classically activated macrophages (M1) are induced by lipopolysaccharide (LPS) and express a wide range of pro-inflammatory genes. M2 macrophages are induced by T helper type 2 cytokines such as interleukin-4 (IL4) and express high levels of anti-inflammatory and tissue repair genes. The strong association between macrophages and tumour cells as well as the high incidences of leukocyte infiltration in solid tumours have contributed to the discovery that tumour-associated macrophages (TAMs) are key to tumour progression. Here, we investigated the role of Annexin A1 (ANXA1), a well characterized immunomodulatory protein on macrophage polarization and the interaction between macrophages and breast cancer cells. Our results demonstrate that ANXA1 regulates macrophage polarization and activation. ANXA1 can act dually as an endogenous signalling molecule or as a secreted mediator which acts via its receptor, FPR2, to promote macrophage polarization. Furthermore, ANXA1 deficient mice exhibit reduced tumour growth and enhanced survival in vivo, possibly due to increased M1 macrophages within the tumor microenvironment. These results provide new insights into the molecular mechanisms of macrophage polarization with therapeutic potential to suppress breast cancer growth and metastasis.
While tremendous improvement has been made for the treatment of breast cancers, the treatment of triple negative breast cancer (TNBC) still remains a challenge due to its aggressive characteristics and limited treatment options. Most of the studies on TNBC were conducted in Western population and TNBC is reported to be more frequent in the African women. This review encapsulates the studies conducted on TNBC patients in Asian population and elucidates the similarities and differences between these two regions. The current treatment of TNBC includes surgery, radiotherapy and chemotherapy. In addition to the current chemotherapies, which mainly include cytotoxic agents, such as taxanes and anthracyclines, many clinical trials are investigating the potential use of other chemotherapy drugs, targeted therapeutics and combinational therapies to treat TNBC. Moreover, this review also integrates the studies involving novel markers, which will help us to dissect the pathologic process of TNBC and in turn facilitate the development of better treatment strategies to combat TNBC.
Tumor recurrence remains the main reason for breast cancer–associated mortality, and there are unmet clinical demands for the discovery of new biomarkers and development of treatment solutions to benefit patients with breast cancer at high risk of recurrence. Here we report the identification of chromosomal copy-number amplification at 1q21.3 that is enriched in subpopulations of breast cancer cells bearing characteristics of tumor-initiating cells (TICs) and that strongly associates with breast cancer recurrence. Amplification is present in ~10–30% of primary tumors but in more than 70% of recurrent tumors, regardless of breast cancer subtype. Detection of amplification in cell-free DNA (cfDNA) from blood is strongly associated with early relapse in patients with breast cancer and could also be used to track the emergence of tumor resistance to chemotherapy. We further show that 1q21.3-encoded S100 calcium-binding protein (S100A) family members, mainly S100A7, S100A8, and S100A9 (S100A7/8/9), and IL-1 receptor–associated kinase 1 (IRAK1) establish a reciprocal feedback loop driving tumorsphere growth. Notably, this functional circuitry can be disrupted by the small-molecule kinase inhibitor pacritinib, leading to preferential impairment of the growth of 1q21.3-amplified breast tumors. Our study uncovers the 1q21.3-directed S100A7/8/9–IRAK1 feedback loop as a crucial component of breast cancer recurrence, serving as both a trackable biomarker and an actionable therapeutic target for breast cancer.
Yes-associated protein (YAP) is regulated by mechanical cues via the interaction of the Hippo pathway with cytoskeleton. Previous studies showed that YAP plays a role in regulating the actomyosin network by suppressing Rho GTPase in medaka fish. Here, we identify Rho GTPase activating protein 29 (ARHGAP29) as a transcriptional target of YAP in a human gastric cancer cell line. YAP promotes the expression of ARHGAP29 to suppress the RhoA-LIMK-cofilin pathway, destabilizing F-actin. The overexpression of YAP causes cytoskeletal rearrangement by altering the dynamics of F-actin/G-actin turnover, thus promoting migration. In a mouse model, circulating tumor cells (CTCs) exhibit an increased ARHGAP29 RNA level compared with cells at primary tumor sites, and the metastatic potential of CTCs is positively correlated with ARHGAP29 expression. Moreover, increased ARHGAP29 expression is correlated with shortened survival of human gastric cancer patients. Our study provides a model to understand YAP’s contribution to cancer metastasis via regulation of actin dynamics.
The hippocampal area Cornu Ammonis (CA) CA2 is important for social interaction and is innervated by Substance P (SP)-expressing supramammillary (SuM) nucleus neurons. SP exerts neuromodulatory effects on pain processing and central synaptic transmission. Here we provide evidence that SP can induce a slowly developing NMDA receptor- and protein synthesis-dependent potentiation of synaptic transmission that can be induced not only at entorhinal cortical (EC)-CA2 synapses but also at long-term potentiation (LTP)-resistant Schaffer collateral (SC)-CA2 synapses. In addition, SP-induced potentiation of SC-CA2 synapses transforms a short-term potentiation of EC-CA2 synaptic transmission into LTP, consistent with the synaptic tagging and capture hypothesis. Interestingly, this SP-induced potentiation and associative interaction between the EC and SC inputs of CA2 neurons is independent of the GABAergic system. In addition, CaMKIV and PKMζ play a critical role in the SP-induced effects on SC-CA2 and EC-CA2 synapses. Thus, afferents from SuM neurons are ideally situated to prime CA2 synapses for the formation of long-lasting plasticity and associativity.
Metastatic breast cancer is still remain incurable so far, new specifically targeted and more effective therapies for triple negative breast cancer (TNBC) are required in the clinic. In this study, our clinical data has established that basal and claudin-low subtypes of breast cancer (TNBC types) express significantly higher levels of Annexin A1 (ANXA1) with poor survival outcomes. Using human cancer cell lines which model the TNBC subtype, we observed a strong positive correlation between expression of ANXA1 and Peroxisome Proliferator-Activated Receptor gamma (PPARγ). A similar correlation between these two markers was also established in our clinical breast cancer patients' specimens. To establish a link between these two markers in TNBC, we show de novo expression of ANXA1 is induced by activation of PPARγ both in vitro and invivo and it has a predictive value in determining chemo-sensitivity to PPARγ ligands. Mechanistically, we show for the first time PPARγ-induced ANXA1 protein directly interacts with Receptor Interacting Protein-1 (RIP1), promoting its deubiquitination and thereby activating the caspase 8-dependent death pathway. We further identified this underlying mechanism also involved a PPARγ-induced ANXA1-dependent autoubiquitination of cIAP1, the direct E3 ligase of RIP1, shifting cIAP1 towards proteosomal degradation. Collectively, our study provides first insight for the suitability of using drug-induced expression of ANXA1 as a new player in RIP1-induced death machinery in TNBCs. Hitherto, presenting itself both as an inclusion criterion for patient selection and surrogate marker for drug response in future PPARγ chemotherapy trials.
Recent discoveries in the non-coding genome have challenged the original central dogma of molecular biology; non-coding RNAs and related processes have been found to be important in regulating gene expression. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are amongst some that have gained attention recently in human diseases, including cancer, with the involvement of many more ncRNAs waiting to be discovered. Non-coding RNAs (ncRNAs) are a group of ribonucleic acids transcribed from regions of the human genome, which do not become translated into proteins, despite having essential roles in cellular physiology. Deregulation of ncRNA expression and function has been observed in cancer pathogenesis. Recently, the roles of a group of ncRNA known as lncRNA have gained attention in cancer, with increasing reported literature on their oncogenic involvement. Female reproductive cancers remain a leading cause of death in the female population, accounting for almost a third of all female cancer deaths in 2016. The Wnt signaling pathway is one of the most important oncogenic signaling pathways found hyperactivated in cancers, including female reproductive cancers. The advent of ncRNA research into their mechanistic roles in human cancers has also led to novel reported roles of ncRNAs in the Wnt pathway and Wnt-mediated oncogenesis. This review aims to provide a critical summary of the respective roles and cellular functions of Wnt-associated lncRNAs in female reproductive cancers and explores the potential of circulating cell-free lncRNAs as diagnostic markers and lncRNAs as therapeutic targets.
Altered epigenetic mechanisms are implicated in the cognitive decline associated with neurodegenerative diseases such as in Alzheimer's disease (AD). AD is the most prevalent form of dementia worldwide; amyloid plaques and neurofibrillary tangles are the histopathological hallmarks of AD. We have recently reported that the inhibition of G9a/GLP complex promotes long-term potentiation (LTP) and its associative mechanisms such as synaptic tagging and capture (STC). However, the role of this complex in plasticity impairments remains elusive. Here, we investigated the involvement of G9a/GLP complex in alleviating the effects of soluble Amyloid-β 1-42 oligomers (oAβ) on neuronal plasticity and associativity in the CA1 region of acute hippocampal slices from 5- to 7-week-old male Wistar rats. Our findings demonstrate that the regulation of G9a/GLP complex by inhibiting its catalytic activity reverses the amyloid-β oligomer-induced deficits in late-LTP and STC. This is achieved by releasing the transcription repression of the brain-derived neurotrophic factor (Bdnf) gene. The catalytic inhibition of G9a/GLP complex leads to the upregulation of Bdnf expression in the slices treated with oAβ. This further ensures the availability of BDNF that subsequently binds its receptor tyrosine kinase B (TrkB) and maintains the late-LTP. Furthermore, the capture of BDNF by weakly activated synapses re-establishes STC. Our findings regarding the reinstatement of functional plasticity and associativity in AD-like conditions provide the first evidence for the role of G9a/GLP complex in AD. We propose G9a/GLP complex as the possible target for preventing oAβ-induced plasticity deficits in hippocampal neurons.
Alternative splicing changes the CaV1.2 calcium channel electrophysiological property, but the in vivo significance of such altered channel function is lacking. Structure–function studies of heterologously expressed CaV1.2 channels could not recapitulate channel function in the native milieu of the cardiomyocyte. To address this gap in knowledge, we investigated the role of alternative exon 33 of the CaV1.2 calcium channel in heart function. Exclusion of exon 33 in CaV1.2 channels has been reported to shift the activation potential −10.4 mV to the hyperpolarized direction, and increased expression of CaV1.2Δ33 channels was observed in rat myocardial infarcted hearts. However, how a change in CaV1.2 channel electrophysiological property, due to alternative splicing, might affect cardiac function in vivo is unknown. To address these questions, we generated mCacna1c exon 33−/−-null mice. These mice contained CaV1.2Δ33 channels with a gain-of-function that included conduction of larger currents that reflects a shift in voltage dependence and a modest increase in single-channel open probability. This altered channel property underscored the development of ventricular arrhythmia, which is reflected in significantly more deaths of exon 33−/− mice from β-adrenergic stimulation. In vivo telemetric recordings also confirmed increased frequencies in premature ventricular contractions, tachycardia, and lengthened QT interval. Taken together, the significant decrease or absence of exon 33-containing CaV1.2 channels is potentially proarrhythmic in the heart. Of clinical relevance, human ischemic and dilated cardiomyopathy hearts showed increased inclusion of exon 33. However, the possible role that inclusion of exon 33 in CaV1.2 channels may play in the pathogenesis of human heart failure remains unclear.
Dynamic regulation of plasticity thresholds in a neuronal population is critical for the formation of long-term plasticity and memory and is achieved by mechanisms such as metaplasticity. Metaplasticity tunes the synapses to undergo changes that are necessary prerequisites for memory storage under physiological and pathological conditions. Here we discovered that, in amyloid precursor protein (APP)/presenilin-1 (PS1) mice (age 3-4 mo), a prominent mouse model of Alzheimer's disease (AD), late long-term potentiation (LTP; L-LTP) and its associative plasticity mechanisms such as synaptic tagging and capture (STC) were impaired already in presymptomatic mice. Interestingly, late long-term depression (LTD; L-LTD) was not compromised, but the positive associative interaction of LTP and LTD, cross-capture, was altered in these mice. Metaplastic activation of ryanodine receptors (RyRs) in these neurons reestablished L-LTP and STC. We propose that RyR-mediated metaplastic mechanisms can be considered as a possible therapeutic target for counteracting synaptic impairments in the neuronal networks during the early progression of AD.
Aging is accompanied by a general decline in the physiological functions of the body with the deteriorating organ systems. Brain is no exception to this and deficits in cognitive functions are quite common in advanced aging. Though a variety of age-related alterations are observed in the structure and function throughout the brain, certain regions show selective vulnerability. Medial temporal lobe, especially the hippocampus, is one such preferentially vulnerable region and is a crucial structure involved in the learning and long-term memory functions. Hippocampal synaptic plasticity, such as long-term potentiation (LTP) and depression (LTD), are candidate cellular correlates of learning and memory and alterations in these properties have been well documented in aging. A related phenomenon called synaptic tagging and capture (STC) has been proposed as a mechanism for cellular memory consolidation and to account for temporal association of memories. Mounting evidences from behavioral settings suggest that STC could be a physiological phenomenon. In this article, we review the recent data concerning STC and provide a framework for how alterations in STC-related mechanisms could contribute to the age-associated memory impairments. The enormity of impairment in learning and memory functions demands an understanding of age-associated memory deficits at the fundamental level given its impact in the everyday tasks, thereby in the quality of life. Such an understanding is also crucial for designing interventions and preventive measures for successful brain aging.
Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.
Cancer is one of the most studied areas of human biology over the past century. Despite having attractedmuch attention, hype, and investments, the search to find a cure for cancer remains an uphill battle. Recent discoveries that challenged the central dogma ofmolecular biology not only further increase the complexity but also demonstrate how various types of noncoding RNAs such as microRNA and long noncoding RNA, as well as their related processes such as RNA editing, are important in regulating gene expression. Parallel to this aspect, an increasing number of reports have focused on a family of proteins known as DEAD/H-box helicases involved in RNA metabolism, regulation of long and short noncoding RNAs, and novel roles as “editing helicases” and their association with cancers. This review summarizes recent findings on the roles of RNA helicases in various cancers, which are broadly classified into adult solid tumors, childhood solid tumors, leukemia, and cancer stem cells. The potential small molecule inhibitors of helicases and their therapeutic value are also discussed. In addition, analysing next generation sequencing data obtained from public portals and reviewing existing literature, we provide new insights on the potential of DEAD/H-box helicases to act as pharmacological drug targets in cancers.
Epithelial-mesenchymal transition (EMT) is characterized by the acquisition of invasive fibroblast-like morphology by epithelial cells that are highly polarized. EMT is recognized as a crucial mechanism in cancer progression and metastasis. In this study, we sought to assess the involvement of manganese superoxide dismutase (MnSOD) during the switch between epithelial-like and mesenchymal-like phenotypes in breast carcinoma.
RESULTS: Analysis of breast carcinomas from The Cancer Genome Atlas database revealed strong positive correlation between tumors' EMT score and the expression of MnSOD. This positive correlation between MnSOD and EMT score was significant and consistent across all breast cancer subtypes. Similarly, a positive correlation of EMT score and MnSOD expression was observed in established cell lines derived from breast cancers exhibiting phenotypes ranging from the most epithelial to the most mesenchymal. Interestingly, using phenotypically distinct breast cancer cell lines, we provide evidence that constitutively high or induced expression of MnSOD promotes the EMT-like phenotype by way of a redox milieu predominantly driven by hydrogen peroxide (H2O2). Conversely, gene knockdown of MnSOD results in the reversal of EMT to a mesenchymal-epithelial transition (MET)-like program, which appears to be a function of superoxide (O2(-•))-directed signaling.
INNOVATION AND CONCLUSION: These data underscore the involvement of MnSOD in regulating the switch between the EMT and MET-associated phenotype by influencing cellular redox environment via its effect on the intracellular ratio between O2(-•) and H2O2. Strategies to manipulate MnSOD expression and/or the cellular redox milieu vis-a-vis O2(-•):H2O2 could have potential therapeutic implications. Antioxid. Redox Signal. 25, 283-299.
Aging is associated with decline in cognitive functions, prominently in the memory consolidation and association capabilities. Hippocampus plays a crucial role in the formation and maintenance of long-term associative memories, and a significant body of evidence shows that impairments in hippocampal function correlate with aging-related memory loss. A number of studies have implicated alterations in hippocampal synaptic plasticity, such as long-term potentiation (LTP), in age-related cognitive decline although exact mechanisms underlying are not completely clear. Zinc deficiency and the resultant adverse effects on cognition have been well studied. However, the role of excess of zinc in synaptic plasticity, especially in aging, is not addressed well. Here, we have investigated the hippocampal zinc levels and the impairments in synaptic plasticity, such as LTP and synaptic tagging and capture (STC), in the CA1 region of acute hippocampal slices from 82- to 84-week-old male Wistar rats. We report increased zinc levels in the hippocampus of aged rats and also deficits in the tetani-induced and dopaminergic agonist-induced late-LTP and STC. The observed deficits in synaptic plasticity were restored upon chelation of zinc using a cell-permeable chelator. These data suggest that functional plasticity and associativity can be successfully established in aged neural networks by chelating zinc with cell-permeable chelating agents.
Designing reduced-calorie foods and beverages without compromising their satiating effect could benefit weight management, assuming that consumers do not compensate for the missing calories at other meals. Though research has demonstrated that compensation for overfeeding is relatively limited, the extent to which energy reductions trigger adjustments in later food intake is less clear. The current study tested satiety responses (characterised by changes in appetite and later food intake) to both a covert 200 kcal reduction and an addition of maltodextrin to a soymilk test beverage. Twenty-nine healthy male participants were recruited to consume three sensory-matched soymilk beverages across four non-consecutive study days: a medium energy control (ME: 300 kcal) and a lower energy (LE: 100 kcal) and higher energy (HE: 500 kcal) version. The ME control was consumed twice to assess individual consistency in responses to this beverage. Participants were unaware of the energy differences across the soymilks. Lunch intake 60 min later increased in response to the LE soymilk, but was unchanged after consuming the HE version. These adjustments accounted for 40% of the energy removed from the soymilk and 13% of the energy added in. Rated appetite was relatively unaffected by the soymilk energy content. No further adjustments were noted for the rest of the day. These data suggest that adult men tested were more sensitive to calorie dilution than calorie addition to a familiar beverage.
Parents and caregivers play a powerful role in shaping the food environment in which their children learn about appropriate portion sizes. Parents often report just ‘knowing’ the amount of food to serve to their children, but evidence indicates that these decisions are driven by the portions parents pick for themselves. This is potentially problematic because, like adults, young children are susceptible to overeating when served large portions, which raises questions about how and when children develop their own portion knowledge. Understanding the social, emotional and cultural factors that shape children's learning about appropriate portion sizes and self-regulation will lead to a better understanding of the calories that end up on children's plates and in their mouths, and the eating behaviours that define them as adults.
The sensory experience of eating is an important determinant of food intake control, often attributed to the positive hedonic response associated with certain sensory cues. However, palatability is just one aspect of the sensory experience. Sensory cues based on a food's sight, smell, taste and texture are operational before, during and after an eating event. The focus of this review is to look beyond palatability and highlight recent advances in our understanding of how certain sensory characteristics can be used to promote better energy intake control. We consider the role of visual and odour cues in identifying food in the near environment, guiding food choice and memory for eating, and highlight the ways in which tastes and textures influence meal size and the development of satiety after consumption. Considering sensory characteristics as a functional feature of the foods and beverages we consume provides the opportunity for research to identify how sensory enhancements might be combined with energy reduction in otherwise palatable foods to optimize short-term energy intake regulation in the current food environment. Moving forward, the challenge for sensory nutritional science will be to assess the longer-term impact of these principles on weight management.
Numerous studies have examined energy compensation following overfeeding regimes whereas much less is known about the impact of acute underfeeding on energy compensation and fewer still have compared energy reduction and addition in the same group of individuals. This study compared the effects of consuming lunches with varying energy content (7.2-fold difference) on subsequent energy intake. A total of 27 healthy males took part in this randomized, crossover study with five treatments: 163kcal (very low energy meal, VLEM), 302kcal (low energy meal, LEM), 605kcal (control), 889kcal (high energy meal, HEM), and 1176kcal (very high energy meal, VHEM) served as a noodle soup. Participants were instructed to consume a standardized breakfast in the morning and they were provided with one of the five treatments for lunch on non-consecutive test day. Test lunches were matched for palatability, sensory properties, and volume. Participants were provided with an afternoon snack and ad libitum dinner on each test day and recorded food intake for the rest of the day. Appetite ratings were measured at regular intervals. As the energy content of treatments increased, participants' hunger, desire to eat, and prospective consumption decreased significantly whereas fullness increased significantly. However, no significant difference in subsequent meal intake was found between the treatments (P=0.458): 1003kcal VLEM, 1010kcal LEM, 1011kcal control, 940kcal HEM, and 919kcal VHEM. Total daily energy intake was statistically significantly different between the treatments (P<0.001) and was varied directly with the energy content of the lunchtime meal. Despite the large difference in energy content between the treatments, participants did not compensate for the "missing calories" or "additional calories" at subsequent meals. These results suggest that covertly manipulated, equally palatable, sensory and volume matched meals have the potential to promote either positive or negative energy balance if the effects seen in this single meal study are sustained.
Alveolar rhabdomyosarcoma (ARMS) is an aggressive paediatric cancer of skeletal muscle with poor prognosis. A PAX3-FOXO1 fusion protein acts as a driver of malignancy in ARMS by disrupting tightly coupled but mutually exclusive pathways of proliferation and differentiation. While PAX3-FOXO1 is an attractive therapeutic target, no current treatments are designed to block its oncogenic activity. The present work shows that the histone acetyltransferase P/CAF (KAT2B) is overexpressed in primary tumours from ARMS patients. Interestingly, in fusion-positive ARMS cell lines, P/CAF acetylates and stabilizes PAX3-FOXO1 rather than MyoD, a master regulator of muscle differentiation. Silencing P/CAF, or pharmacological inhibition of its acetyltransferase activity down-regulates PAX3-FOXO1 levels concomitant with reduced proliferation and tumour burden in xenograft mouse models. Our studies identify a P/CAF-PAX3-FOXO1 signalling node that promotes oncogenesis and may contribute to MyoD dysfunction in ARMS. This work exemplifies the therapeutic potential of targeting chromatin-modifying enzymes to inhibit fusion-oncoproteins that are a frequent event in sarcomas.
The actin-binding protein, gelsolin, is a well known regulator of cancer cell invasion. However, the mechanisms by which gelsolin promotes invasion are not well established. As reactive oxygen species (ROS) have been shown to promote cancer cell invasion, we investigated on the hypothesis that gelsolin-induced changes in ROS levels may mediate the invasive capacity of colon cancer cells. Herein, we show that increased gelsolin enhances the invasive capacity of colon cancer cells, and this is mediated via gelsolin's effects in elevating intracellular superoxide (O2.-) levels. We also provide evidence for a novel physical interaction between gelsolin and Cu/ZnSOD, that inhibits the enzymatic activity of Cu/ZnSOD, thereby resulting in a sustained elevation of intracellular O2.-. Using microarray data of human colorectal cancer tissues from Gene Omnibus, we found that gelsolin gene expression positively correlates with urokinase plasminogen activator (uPA), an important matrix-degrading protease invovled in cancer invasion. Consistent with the in vivo evidence, we show that increased levels of O2.- induced by gelsolin overexpression triggers the secretion of uPA. We further observed reduction in invasion and intracellular O2.- levels in colon cancer cells, as a consequence of gelsolin knockdown using two different siRNAs. In these cells, concurrent repression of Cu/ZnSOD restored intracellular O2.- levels and rescued invasive capacity. Our study therefore identified gelsolin as a novel regulator of intracellular O2.- in cancer cells via interacting with Cu/ZnSOD and inhibiting its enzymatic activity. Taken together, these findings provide insight into a novel function of gelsolin in promoting tumor invasion by directly impacting the cellular redox milieu.
In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.
AIM: Epithelial Mesenchymal Transition (EMT) is characterized by the acquisition of invasive fibroblast-like morphology by epithelial cells that are highly polarized. EMT is recognized as a crucial mechanism in cancer progression and metastasis. Here we sought to assess the involvement of Manganese Superoxide Dismutase (MnSOD) during the switch between epithelial-like and mesenchymal-like phenotypes in breast carcinoma.
RESULTS: Analysis of breast carcinomas from The Cancer Genome Atlas (TCGA) database revealed strong positive correlation between tumors' EMT score and the expression of MnSOD. This positive correlation between MnSOD and EMT score was significant and consistent across all breast cancer subtypes. Similarly, a positive correlation of EMT score and MnSOD expression was observed in established cell lines derived from breast cancers exhibiting phenotypes ranging from the most epithelial to the most mesenchymal. Interestingly, using phenotypically distinct breast cancer cell lines, we provide evidence that constitutively high or induced expression of MnSOD promotes the EMT-like phenotype by way of a redox milieu predominantly driven by hydrogen peroxide (H2O2). Conversely, gene knockdown of MnSOD results in the reversal of EMT to a MET-like program, which appears to be a function of superoxide (O2-.)-directed signaling.
INNOVATION AND CONCLUSION: These data underscore the involvement of MnSOD in regulating the switch between the EMT and MET-associated phenotype by influencing cellular redox environment via its effect on the intracellular ratio between O2-. and H2O2. Strategies to manipulate MnSOD expression and/or the cellular redox milieu vis a vis O2-.:H2O2 could have potential therapeutic implications.
Epigenetic regulations play an important role in regulating the learning and memory processes. G9a/G9a-like protein (GLP) lysine dimethyltransferase complex controls a prominent histone H3 lysine9 dimethylation (H3K9me2) that results in transcriptional silencing of the chromatin. Here, we report that the inhibition of G9a/GLP complex by either of the substrate competitive inhibitors UNC 0638 or BIX 01294 reinforces protein synthesis-independent long-term potentiation (early-LTP) to protein synthesis-dependent long-term potentiation (late-LTP). The reinforcement effect was observed if the inhibitors were present during the induction of early-LTP and in addition when G9a/GLP complex inhibition was carried out by priming of synapses within an interval of 30 min before or after the induction of early-LTP. Surprisingly, the reinforced LTP by G9a/GLP complex inhibition was able to associate with a weak plasticity event from nearby independent synaptic populations, resulting in synaptic tagging/capture (STC). We have identified brain-derived neurotrophic factor (BDNF) as a critical plasticity protein that maintains G9a/GLP complex inhibition-mediated LTP facilitation and its STC. Our study reveals an epigenetic mechanism for promoting plasticity and associativity by G9a/GLP complex inhibition, and it may engender a promising epigenetic target for enhancing memory in neural networks.
Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.
Neurodegenerative diseases such as Alzheimer’s disease (AD) are associated with alterations in epigenetic factors leading to cognitive decline. Histone deacetylase 3 (HDAC3) is a known critical epigenetic negative regulator of learning and memory. In this study, attenuation of long-term potentiation by amyloid-β oligomer, and its reversal by specific HDAC3 inhibitor RGFP966, was performed in rat CA1 pyramidal neurons using whole cell voltage-clamp and field recording techniques. Our findings provide the first evidence that amyloid-β oligomer-induced synaptic plasticity impairment can be prevented by inhibition of HDAC3 enzyme both at the single neuron as well as in a population of neurons, thus identifying HDAC3 as a potential target for ameliorating AD related plasticity impairments.
KIF1Bβ is a candidate 1p36 tumor suppressor that regulates apoptosis in the developing sympathetic nervous system. We found that KIF1Bβ activates the Ca2+-dependent phosphatase calcineurin (CN) by stabilizing the CN-calmodulin complex, relieving enzymatic autoinhibition and enabling CN substrate recognition. CN is the key mediator of cellular responses to Ca2+ signals and its deregulation is implicated in cancer, cardiac, neurodegenerative, and immune disease. We show that KIF1Bβ affects mitochondrial dynamics through CN-dependent dephosphorylation of Dynamin-related protein 1 (DRP1), causing mitochondrial fission and apoptosis. Furthermore, KIF1Bβ actuates recognition of all known CN substrates, implying a general mechanism for KIF1Bβ in Ca2+ signaling and how Ca2+-dependent signaling is executed by CN. Pathogenic KIF1Bβ mutations previously identified in neuroblastomas and pheochromocytomas all fail to activate CN or stimulate DRP1 dephosphorylation. Importantly, KIF1Bβ and DRP1 are silenced in 1p36 hemizygous-deleted neuroblastomas, indicating that deregulation of calcineurin and mitochondrial dynamics contributes to high-risk and poor-prognosis neuroblastoma.
The mitochondrial uniporter (MCU) is an ion channel that mediates Ca(2+) uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca(2+) signaling. Matrix Ca(2+) concentration is similar to that in cytoplasm, despite an enormous driving force for entry, but the mechanisms that prevent mitochondrial Ca(2+) overload are unclear. Here, we show that MCU channel activity is governed by matrix Ca(2+) concentration through EMRE. Deletion or charge neutralization of its matrix-localized acidic C terminus abolishes matrix Ca(2+) inhibition of MCU Ca(2+) currents, resulting in MCU channel activation, enhanced mitochondrial Ca(2+) uptake, and constitutively elevated matrix Ca(2+) concentration. EMRE-dependent regulation of MCU channel activity requires intermembrane space-localized MICU1, MICU2, and cytoplasmic Ca(2+). Thus, mitochondria are protected from Ca(2+) depletion and Ca(2+) overload by a unique molecular complex that involves Ca(2+) sensors on both sides of the inner mitochondrial membrane, coupled through EMRE.
Aging is associated with impaired plasticity and memory. Altered epigenetic mechanisms are implicated in the impairment of memory with advanced aging. Histone deacetylase 3 (HDAC3) is an important negative regulator of memory. However, the role of HDAC3 in aged neural networks is not well established. Late long-term potentiation (late-LTP), a cellular correlate of memory and its associative mechanisms such as synaptic tagging and capture (STC) were studied in the CA1 area of hippocampal slices from 82–84 week old rats. Our findings demonstrate that aging is associated with deficits in the magnitude of LTP and impaired STC. Inhibition of HDAC3 augments the late-LTP and re-establishes STC. The augmentation of late-LTP and restoration of STC is mediated by the activation of nuclear factor kappa B (NFκB) pathway. We provide evidence for the promotion of associative plasticity in aged neural networks by HDAC3 inhibition and hence propose HDAC3 and NFκB as the possible therapeutic targets for treating age -related cognitive decline.
Metastatic tumour recurrence due to failed treatments remains a major challenge of breast cancer clinical management. Here we report that interleukin-1 receptor-associated kinase 1 (IRAK1) is overexpressed in a subset of breast cancers, in particular triple-negative breast cancer (TNBC), where it acts to drive aggressive growth, metastasis and acquired resistance to paclitaxel treatment. We show that IRAK1 overexpression confers TNBC growth advantage through NF-κB-related cytokine secretion and metastatic TNBC cells exhibit gain of IRAK1 dependency, resulting in high susceptibility to genetic and pharmacologic inhibition of IRAK1. Importantly, paclitaxel treatment induces strong IRAK1 phosphorylation, an increase in inflammatory cytokine expression, enrichment of cancer stem cells and acquired resistance to paclitaxel treatment. Pharmacologic inhibition of IRAK1 is able to reverse paclitaxel resistance by triggering massive apoptosis at least in part through inhibiting p38-MCL1 pro-survival pathway. Our study thusdemonstrates IRAK1 as a promising therapeutic target for TNBC metastasis and paclitaxel resistance.
MCU is the pore-forming subunit of the mitochondrial inner membrane Ca2+ uniporter ion channel that mediates Ca2+ uptake into the matrix to regulate metabolism, cell death, and cytoplasmic Ca2+ signaling. We previously identified MCUR1 (Mitochondrial Calcium Uniporter Regulator 1) as an important regulator of MCU activity and showed that MCUR1 biochemically interacted with MCU (Mallilankaraman et al., 2012). MCUR1 regulated MCU-dependent mitochondrial Ca2+ uptake driven by the inner membrane voltage (ψm) generated by the electron transport chain, and MCUR1 knockdown abrogated Ca2+ uptake by mitochondria in intact and permeabilized cells, and disrupted oxidative phosphorylation (OXPHOS).
Despite recent advances in breast cancer therapeutics, mortality of metastatic triple negative breast cancer (TNBC) subtype remains high; due to their lack of hormone receptors expression for targeted therapy. Aberrant activation of Wnt/β-catenin signaling has been associated with breast cancers; where 40% of total breast cancers have elevated β-catenin levels with increased Wnt activity. Recently, we identified DEAD-box RNA helicase DP103 as a novel prognostic biomarker and metastasis-driving oncogene; highly expressed in TNBC subtype. Interestingly, we found high DP103 expression to be positively correlated with high β-catenin expression in clinical specimens (n=400). This led us to hypothesize a possible role of DP103 in modulating the Wnt/β-catenin pathway in TNBCs. Depletion of DP103 in metastatic TNBC cells decreases Wnt/β-catenin activity and expression of downstream Wnt target genes, while overexpression of DP103 increases Wnt activity. Depletion of DP103 also decreases phosphorylation of LRP6 and several important Wnt modulators required for downstream Wnt activation. Moreover, induction of Wnt/β-catenin signaling in Wnt responsive TNBC cells also significantly increased DP103 expression, indicating a possible positive feedback loop. Both canonical and non-canonical Wnt signaling is known to independently promote stem cell growth in mammospheres. Herein, we will also provide evidence on the role of DP103 in promoting breast cancer stem cell-like properties. Collectively, our data show a novel regulatory role of DP103 in the Wnt/β-catenin signaling pathway and in promoting breast cancer stem cell-like behavior, presenting itself as a potential drug target in TNBC patients.
Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.
Synaptic co-operation and competition are important components of synaptic plasticity that tune synapses for the formation of associative long-term plasticity, a cellular correlate of associative long-term memory. We have recently reported that coincidental activation of weak synapses within the vicinity of potentiated synapses will alter the co-operative state of synapses to a competitive state thus leading to the slow decay of long-term plasticity, but the molecular mechanism underlying this is still unknown. Here, using acute hippocampal slices of rats, we have examined how increasing extracellular dopamine concentrations interact and/or affect electrically induced long-term potentiation (LTP) in the neighboring synapses. We demonstrate that D1/D5-receptor-mediated potentiation at the CA1 Schaffer collateral synapses differentially regulates synaptic co-operation and competition. Further investigating the molecular players involved, we reveal an important role for extracellular signal-regulated kinases-1 and 2 (ERK1/2) as signal integrators and dose-sensors. Interestingly, a sustained activation of ERK1/2 pathway seems to be involved in the differential regulation of synaptic associativity. The concentration-dependent effects of the modulatory transmitter, as demonstrated for dopaminergic signaling in the present study, might offer additional computational power by fine tuning synaptic associativity processes for establishing long-term associative memory in neural networks.
A balance of protein synthesis and degradation is critical for the dynamic regulation and implementation of long-term memory storage. The role of the ubiquitin-proteasome system (UPS) in regulating the plasticity at potentiated synapses is well studied, but its roles in depressed synaptic populations remain elusive. In this study, we probed the possibility of regulating the UPS by inhibiting the proteasome function during the induction of protein synthesis-independent form of hippocampal long-term depression (early-LTD), an important component of synaptic plasticity. Here, we show that protein degradation is involved in early-LTD induction and interfering with this process facilitates early-LTD to late-LTD. We provide evidence here that under the circumstances of proteasome inhibition brain-derived neurotrophic factor is accumulated as plasticity-related protein and it drives the weakly depressed or potentiated synapses to associativity. Thus, UPS inhibition promotes LTD and establishes associativity between weakly depressed or potentiated synapses through the mechanisms of synaptic tagging/capture or cross-capture.
Ischemic stroke causes a high rate of deaths and permanent neurological damage in survivors. Ischemic stroke triggers the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1), which activate toll-like receptors (TLRs) and receptor for advanced glycation endproducts (RAGE) in the affected area, leading to an exaggerated inflammatory response and cell death. Both TLRs and RAGE are transmembrane pattern recognition receptors (PRRs) that have been shown to contribute to ischemic stroke-induced brain injury. Intravenous immunoglobulin (IVIg) preparations obtained by fractionating human blood plasma are increasingly being used as an effective therapeutic agent in the treatment of several inflammatory diseases. Its use as a potential therapeutic agent for treatment of stroke has been proposed, but little is known about the direct neuroprotective mechanisms of IVIg. We therefore investigate whether IVIg exerts its beneficial effects on the outcome of neuronal injury by modulating HMGB1-induced TLR and RAGE expressions and activations.
METHODS:Primary cortical neurons were subjected to glucose deprivation or oxygen and glucose deprivation conditions and treated with IVIg and recombinant HMGB1. C57/BL6J mice were subjected to middle cerebral artery occlusion, followed by reperfusion, and IVIg was administered intravenously 3 h after the start of reperfusion. Expression of TLRs, RAGE and downstream signalling proteins in neurons and brain tissues were evaluated by immunoblot.
RESULTS:Treatment of cultured neurons with IVIg reduced simulated ischemia-induced TLR2, TLR4, TLR8 and RAGE expressions, pro-apoptotic caspase-3 cleavage and phosphorylation of the cell death-associated kinases such as c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK) as well as the p65 subunit of nuclear factor kappa B (NF-κB). These results were recapitulated in an in vivo model of stroke. IVIg treatment also upregulated the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) in cortical neurons under ischemic conditions. Finally, IVIg protected neurons against HMGB1-induced neuronal cell death by modulating TLR and RAGE expressions and signalling pathways.
CONCLUSIONS:Taken together, these results provide a rationale for the potential use of IVIg to target inappropriately activated components of the innate immune system following ischemic stroke.
OBJECTIVE:Stroke is a leading cause of mortality and disability. The peptidyl-prolyl cis/trans isomerase Pin1 regulates factors involved in cell growth. Recent evidence has shown that Pin1 plays a major role in apoptosis. However, the role of Pin1 in ischemic stroke remains to be investigated.
METHODS:We used Pin1 overexpression and knockdown to manipulate Pin1 expression and explore the effects of Pin1 in cell death on ischemic stress in vitro and in a mouse stroke model. We also used Pin 1 inhibitor, γ-secretase inhibitor, Notch1 intracellular domain (NICD1)-deleted mutant cells, and Pin1 mutant cells to investigate the underlying mechanisms of Pin1-NICD1-mediated cell death.
RESULTS:Our findings indicate that Pin1 facilitates NICD1 stability and its proapoptotic function following ischemic stroke. Thus, overexpression of Pin1 increased NICD1 levels and enhanced its potentiation of neuronal death in simulated ischemia. By contrast, depletion or knockout of Pin1 reduced the NICD1 level, which in turn desensitized neurons to ischemic conditions. Pin1 interacted with NICD1 and increased its stability by inhibiting FBW7-induced polyubiquitination. We also demonstrate that Pin1 and NICD1 levels increase following stroke. Pin1 heterozygous (+/-) and knockout (-/-) mice, and also wild-type mice treated with an inhibitor of Pin1, each showed reduced brain damage and improved functional outcomes in a model of focal ischemic stroke.
INTERPRETATION:These results suggest that Pin1 contributes to the pathogenesis of ischemic stroke by promoting Notch signaling, and that inhibition of Pin1 is a novel approach for treating ischemic stroke.
Myasthenia gravis (MG), the most common autoimmune disease of neuromuscular junction (NMJ), is heterogeneous in terms of pathophysiology, which is determined by the pathogenic antigen of autoantibodies targeting to synaptic proteins at the NMJs. Currently, patients suspected with MG are routinely screened for the presence of autoantibodies against acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) using a cell-based assay (CBA) that involves the expression of target synaptic membrane protein in heterologous cell lines. However, some autoantibodies may only show reactivity for binding to densely clustered AChR in the physiological conformation, while AChR clustering is known to involve signaling events orchestrated by over a dozen of postsynaptic proteins. To improve the existing serological diagnosis of MG, this study explored the possibility of using the well-established Xenopus primary culture system as a novel CBA for MG. Here, by examining the pathogenic effects of four MG human plasma samples, we found that the samples from both seropositive and seronegative MG patients effectively induced the disassembly of aneural AChR clusters in cultured Xenopus muscle cells, as well as the nerve-induced AChR clusters in the nerve-muscle co-cultures. Importantly, the disassembly of AChR clusters was spatio-temporally correlated to the disappearance of actin depolymerizing factor (ADF)/cofilin, an actin regulator involved in AChR trafficking and clustering. Taken together, this study develops a reliable CBA using Xenopus primary cultures for screening the pathogenicity of human MG plasma samples, and providing a platform for investigating the pathogenic mechanisms underlying the endocytic trafficking and degradation of AChRs at NMJs in MG patients.
A large number of etiological factors and the complexity of breast cancers present challenges for prevention and treatment. Recently, the emergence of microRNAs (miRNAs) as cancer biomarkers has added an extra dimension to the 'molecular signatures' of breast cancer. Bioinformatic analyses indicate that each miRNA can regulate hundreds of target genes and could serve functionally as 'oncogenes' or 'tumour suppressor' genes, and co-ordinate multiple cellular processes relevant to cancer progression. A number of studies have shown that miRNAs play important roles in breast tumorigenesis, metastasis, proliferation and differentiation of breast cancer cells. This review provides a comprehensive overview of miRNAs with established functional relevance in breast cancer, their established target genes and resulting cellular phenotype. The role and application of circulating miRNAs in breast cancer is also discussed. Furthermore, we summarize the role of miRNAs in the hallmarks of breast cancer, as well as the possibility of using miRNAs as potential biomarkers for detection of breast cancer.
Inflammatory cells may contribute to secondary brain injury following cerebral ischemia. The C57Bl/6 mouse strain is known to exhibit a T helper 1-prone, pro-inflammatory type response to injury, whereas the FVB strain is relatively T helper 2-prone, or anti-inflammatory, in its immune response. We tested whether stroke outcome is more severe in C57Bl/6 than FVB mice. Male mice of each strain underwent sham surgery or 1 h occlusion of the middle cerebral artery followed by 23 h of reperfusion. Despite no difference in infarct size, C57Bl/6 mice displayed markedly greater functional deficits than FVB mice after stroke, as assessed by neurological scoring and hanging wire test. Total numbers of CD45(+) leukocytes tended to be larger in the brains of C57Bl/6 than FVB mice after stroke, but there were marked differences in leukocyte composition between the two mouse strains. The inflammatory response in C57Bl/6 mice primarily involved T and B lymphocytes, whereas neutrophils, monocytes and macrophages were more prominent in FVB mice. Our data are consistent with the concept that functional outcome after stroke is dependent on the immune cell composition which develops following ischemic brain injury.
γ-Secretase is a distinct proteolytic complex required for the activation of many transmembrane proteins. The cleavage of substrates by γ-secretase plays diverse biological roles in producing essential products for the organism. More than 90 transmembrane proteins have been reported to be substrates of γ-secretase. Two of the most widely known and studied of these substrates are the amyloid precursor protein (APP) and the Notch receptor, which are precursors for the generation of amyloid-β (Aβ) and the Notch intracellular domain (NICD), respectively. The wide spectrum of γ-secretase substrates has made analyses of the pathology of γ-secretase-related diseases and underlying mechanisms challenging. Inflammation is an important aspect of disease pathology that requires an in-depth analysis. γ-Secretase may contribute to disease development or progression by directly increasing and regulating production of pro-inflammatory cytokines. This review summarizes recent evidence for a role of γ-secretase in inflammatory diseases, and discusses the potential use of γ-secretase inhibitors as an effective future treatment option.
Patients with triple-negative breast cancer (TNBC) have a high incidence of early relapse and metastasis; however, the molecular basis for recurrence in these individuals remains poorly understood. Here, we demonstrate that RASAL2, which encodes a RAS-GTPase–activating protein (RAS-GAP), is a functional target of anti-invasive microRNA-203 and is overexpressed in a subset of triple-negative or estrogen receptor–negative (ER-negative) breast tumors. As opposed to luminal B ER-positive breast cancers, in which RASAL2 has been shown to act as a RAS-GAP tumor suppressor, we found that RASAL2 is oncogenic in TNBC and drives mesenchymal invasion and metastasis. Moreover, high RASAL2 expression was predictive of poor disease outcomes in patients with TNBC. RASAL2 acted independently of its RAS-GAP catalytic activity in TNBC; however, RASAL2 promoted small GTPase RAC1 signaling, which promotes mesenchymal invasion, through binding and antagonizing the RAC1-GAP protein ARHGAP24. Together, these results indicate that activation of a RASAL2/ARHGAP24/RAC1 module contributes to TNBC tumorigenesis and identify a context-dependent role of RASAL2 in breast cancer.
It is known that cells within the inflammatory background in classical Hodgkin lymphoma (cHL) provide signals essential for the continual survival of the neoplastic Hodgkin and Reed-Sternberg (HRS) cells. However, the mechanisms underlying the recruitment of this inflammatory infiltrate into the involved lymph nodes are less well understood. In this study, we show in vitro that HRS cells secrete lymphotoxin-α (LTα) which acts on endothelial cells to upregulate the expression of adhesion molecules that are important for T cell recruitment. LTα also enhances the expression of hyaluronan which preferentially contributes to the recruitment of CD4(+) CD45RA(+) naïve T cells under in vitro defined flow conditions. Enhanced expression of LTα in HRS cells and tissue stroma; and hyaluronan on endothelial cells are readily detected in involved lymph nodes from cHL patients. Our study also shows that although NF-κB and AP-1 are involved, the cyclooxygenase (COX) pathway is the dominant regulator of LTα production in HRS cells. Using pharmacological inhibitors, our data suggest that activity of COX1, but not of COX2, directly regulates the expression of nuclear c-Fos in HRS cells. Our findings suggest that HRS cell-derived LTα is an important mediator that contributes to T cell recruitment into lesional lymph nodes in cHL.
Although small-molecule targeting of EZH2 appears to be effective in lymphomas carrying EZH2 activating mutations, finding similar approaches to target EZH2-overexpressing epithelial tumors remains challenging. In MYC-driven, but not PI3K-driven prostate cancer, we show that interferon-γ receptor 1 (IFNGR1) is directly repressed by EZH2 in a MYC-dependent manner and is downregulated in a subset of metastatic prostate cancers. EZH2 knockdown restored the expression of IFNGR1 and, when combined with IFN-γ treatment, led to strong activation of IFN-JAK-STAT1 tumor-suppressor signaling and robust apoptosis. Pharmacologic depletion of EZH2 by the histone-methylation inhibitor DZNep mimicked the effects of EZH2 knockdown on IFNGR1 induction and delivered a remarkable synergistic antitumor effect with IFN-γ. In contrast, although they efficiently depleted histone Lysine 27 trimethylation, EZH2 catalytic inhibitors failed to mimic EZH2 depletion. Thus, EZH2-inactivated IFN signaling may represent a therapeutic target, and patients with advanced prostate cancer driven by MYC may benefit from the combination of EZH2 and IFN-γ-targeted therapy.
Bcl-2 is frequently overexpressed in haematopoietic malignancies, and selective phosphorylation at ser70 enhances its anti-apoptotic activity. Phospho-ser70 is dephosphorylated by specific heterotrimers of protein phosphatase 2A (PP2A). We report here that a mild pro-oxidant intracellular milieu induced by either pharmacological inhibition or genetic knockdown of superoxide dismutase 1 (SOD1) inhibits the functional holoenzyme assembly of PP2A and prevents Bcl-2 ser70 dephosphorylation. This redox-dependent regulation of Bcl-2 phosphorylation is due to nitrosative modification of B56δ, which we identify as the regulatory subunit mediating PP2A-dependent Bcl-2 dephosphorylation. Redox inhibition of PP2A results from peroxynitrite-mediated nitration of a conserved tyrosine residue within B56δ (B56δY289). While nitrated-B56δY289 binds efficiently to ser70-phosphorylated Bcl-2, this specific modification inhibits the recruitment of the PP2A catalytic core (A and C subunits). Furthermore, inhibition of B56δY289 nitration restores PP2A holoenzyme assembly, thereby permitting S70 dephosphorylation of Bcl-2 and inhibiting its anti-apoptotic activity. More importantly, in primary cells derived from clinical lymphomas Bcl-2 phosphorylation at S70 directly correlates with B56δ nitration and repression of SOD1, but inversely correlates with B56δ interaction with PP2A-C catalytic subunit. These data underscore the role of a pro-oxidant milieu in chemoresistance of haematopoeitic and other cancers via selective targeting of tumor suppressors such as PP2A.
Canonical models suggest that mechanisms of long-term memory consist of a synapse-specific, protein synthesis-independent induction phase (changes in synaptic weights/temporary tagging of such synapses) and, within adjacent dendritic compartments, a protein synthesis-dependent distribution phase that may accompany or immediately precede induction and whose protein products enable consolidation through synaptic capture.We now report that this distribution phase is competitive in a “winner-take-all” fashion when synapses potentiated at induction compete with each other for plasticity-related proteins. This finding highlights the importance of synaptic competition in creating stable long-lasting memory in neural networks without disruption.
Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.
Two voltage-gated calcium channel subtypes—CaV1.2 and CaV1.3—underlie the major L-type Ca2+ currents in the mammalian central nervous system. Owing to their high sequence homology, the two channel subtypes share similar pharmacological properties, and at high doses classic calcium channel blockers, such as dihydropyridines, phenylalkylamines and benzothiazepines, do not discriminate between the two channel subtypes. Recent progress in treating Parkinson’s disease (PD) was marked by the discovery of synthetic compound 8, which was reported to be a highly selective inhibitor of the CaV1.3 L-type calcium channels (LTCC). However, despite a previously reported IC50 of ~24 μM, in our hands inhibition of the full-length CaV1.342 by compound 8 at 50 μM reaches a maximum of 45%. Moreover, we find that the selectivity of compound 8 towards CaV1.3 relative to CaV1.2B15 channels is greatly influenced by the β-subunit type and its splice isoform variants.
Triple-negative breast cancer (TNBC) is characterized by unique aggressive behavior and lack of targeted therapies. Among the various molecular subtypes of breast cancer, it was observed that TNBCs express elevated levels of sphingosine kinase 1 (SPHK1) compared to other breast tumor subtypes. High levels of SPHK1 gene expression correlated with poor overall and progression- free survival, as well as poor response to Doxorubicin-based treatment. Inhibition of SPHK1 was found to attenuate ERK1/2 and AKT signaling and reduce growth of TNBC cells in vitro and in a xenograft SCID mouse model. Moreover, SPHK1 inhibition by siRNA knockdown or treatment with SKI-5C sensitizes TNBCs to chemotherapeutic drugs. Our findings suggest that SPHK1 inhibition, which effectively counteracts oncogenic signaling through ERK1/2 and AKT pathways, is a potentially important anti-tumor strategy in TNBC. A combination of SPHK1 inhibitors with chemotherapeutic agents may be effective against this aggressive subtype of breast cancer.
Objectives:Sphingosine kinase 1 (SphK1) phosphorylates the membrane sphingolipid, sphingosine, to sphingosine-1-phosphate (S1P), an oncogenic mediator, which drives tumor cell growth and survival. Although SphK1 has gained increasing prominence as an oncogenic determinant in several cancers, its potential as a therapeutic target in colon cancer remains uncertain. We investigated the clinical relevance of SphK1 expression in colon cancer as well as its inhibitory effects in vitro. Methods:SphK1 expression in human colon tumor tissues was determined by immunohistochemistry and its clinicopathological significance was ascertained in 303 colon cancer cases. The effects of SphK1 inhibition on colon cancer cell viability and the phosphoinositide 3-kinase (PI3K)/Akt cell survival pathway were investigated using a SphK1-selective inhibitor-compound 5c (5c). The cytotoxicity of a novel combination using SphK1 inhibition with the chemotherapeutic drug, 5-fluorouracil (5-FU), was also determined. Results:High SphK1 expression correlated with advanced tumor stages (AJCC classification). Using a competing risk analysis model to take into account disease recurrence, we found that SphK1 is a significant independent predictor for mortality in colon cancer patients. In vitro, the inhibition of SphK1 induced cell death in colon cancer cell lines and attenuated the serum-dependent PI3K/Akt signaling. Inhibition of SphK1 also enhanced the sensitivity of colon cancer cells to 5-FU. Conclusion:Our findings highlight the impact of SphK1 in colon cancer progression and patient survival, and provide evidence supportive of further development in combination strategies that incorporate SphK1 inhibition with current chemotherapeutic agents to improve colon cancer outcomes.
Inherited KIF1Bβ loss-of-function mutations in neuroblastomas and pheochromocytomas implicate the kinesin KIF1Bβ as a 1p36.2 tumor suppressor. However, the mechanism of tumor suppression is unknown. We found that KIF1Bβ interacts with RNA helicase A (DHX9) causing nuclear accumulation of DHX9, followed by subsequent induction of the pro-apoptotic XIAP-associated Factor 1 (XAF1) and, consequently, apoptosis. Pheochromocytoma and neuroblastoma arise from neural crest progenitors that compete for growth factors such as nerve growth factor NGF during development. KIF1Bβ is required for developmental apoptosis induced by competition for NGF. We show that DHX9 is induced by and required for apoptosis stimulated by NGF deprivation. Moreover, neuroblastomas with chromosomal deletion of 1p36 exhibit loss of KIF1Bβ expression and impaired DHX9 nuclear localization, implicating the loss of DHX9 nuclear activity in neuroblastoma pathogenesis.
Although statins are known to inhibit proliferation and induce death in a number of cancer cell types, the mechanisms through which downregulation of the mevalonate (MVA) pathway activates death signaling remain poorly understood. Here we set out to unravel the signaling networks downstream of the MVA pathway that mediate the death-inducing activity of simvastatin. Consistent with previous reports, exogenously added geranylgeranylpyrophosphate, but not farnesylpyrophosphate, prevented simvastatin's growth-inhibitory effect, thereby suggesting the involvement of geranylgeranylated proteins such as Rho GTPases in the anticancer activity of simvastatin. Indeed, simvastatin treatment led to increased levels of unprenylated Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42). Intriguingly, instead of inhibiting the functions of Rho GTPases as was expected with loss of prenylation, simvastatin caused a paradoxical increase in the GTP-bound forms of RhoA, Rac1 and Cdc42. Furthermore, simvastatin disrupted the binding of Rho GTPases with the cytosolic inhibitor Rho GDIα, which provides a potential mechanism for GTP loading of the cytosolic Rho GTPases. We also show that the unprenylated RhoA- and Rac1-GTP retained at least part of their functional activities, as evidenced by the increase in intracellular superoxide production and JNK activation in response to simvastatin. Notably, blocking superoxide production attenuated JNK activation as well as cell death induced by simvastatin. Finally, we provide evidence for the involvement of the B-cell lymphoma protein 2 family, Bcl-2-interacting mediator (Bim), in a JNK-dependent manner, in the apoptosis-inducing activity of simvastatin. Taken together, our data highlight the critical role of non-canonical regulation of Rho GTPases and involvement of downstream superoxide-mediated activation of JNK pathway in the anticancer activity of simvastatin, which would have potential clinical implications.
Engagement of the mitochondrial-death amplification pathway is an essential component in chemotherapeutic execution of cancer cells. Therefore, identification of mitochondria-targeting agents has become an attractive avenue for novel drug discovery. Here, we report the anticancer activity of a novel Osmium-based organometallic compound (hereafter named Os) on different colorectal carcinoma cell lines. HCT116 cell line was highly sensitive to Os and displayed characteristic features of autophagy and apoptosis; however, inhibition of autophagy did not rescue cell death unlike the pan-caspase inhibitor z-VAD-fmk. Furthermore, Os significantly altered mitochondrial morphology, disrupted electron transport flux, decreased mitochondrial transmembrane potential and ATP levels, and triggered a significant increase in reactive oxygen species (ROS) production. Interestingly, the sensitivity of cell lines to Os was linked to its ability to induce mitochondrial ROS production (HCT116 and RKO) as HT29 and SW620 cell lines that failed to show an increase in ROS were resistant to the death-inducing activity of Os. Finally, intra-peritoneal injections of Os significantly inhibited tumor formation in a murine model of HCT116 carcinogenesis, and pretreatment with Os significantly enhanced tumor cell sensitivity to cisplatin and doxorubicin. These data highlight the mitochondria-targeting activity of this novel compound with potent anticancer effect in vitro and in vivo, which could have potential implications for strategic therapeutic drug design.
The small chaperone protein Hsp27 confers resistance to apoptosis, and therefore is an attractive anticancer drug target. We report here a novel mechanism underlying the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitizing activity of the small molecule LY303511, an inactive analog of the phosphoinositide 3-kinase inhibitor inhibitor LY294002, in HeLa cells that are refractory to TRAIL-induced apoptosis. On the basis of the fact that LY303511 is derived from LY294002, itself derived from quercetin, and earlier findings indicating that quercetin and LY294002 affected Hsp27 expression, we investigated whether LY303511 sensitized cancer cells to TRAIL via a conserved inhibitory effect on Hsp27. We provide evidence that upon treatment with LY303511, Hsp27 is progressively sequestered in the nucleus, thus reducing its protective effect in the cytosol during the apoptotic process. LY303511-induced nuclear translocation of Hsp27 is linked to its sustained phosphorylation via activation of p38 kinase and MAPKAP kinase 2 and the inhibition of PP2A. Furthermore, Hsp27 phosphorylation leads to the subsequent dissociation of its large oligomers and a decrease in its chaperone activity, thereby further compromising the death inhibitory activity of Hsp27. Furthermore, genetic manipulation of Hsp27 expression significantly affected the TRAIL sensitizing activity of LY303511, which corroborated the Hsp27 targeting activity of LY303511. Taken together, these data indicate a novel mechanism of small molecule sensitization to TRAIL through targeting of Hsp27 functions, rather than its overall expression, leading to decreased cellular protection, which could have therapeutic implications for overcoming chemotherapy resistance in tumor cells.
AIMS: Although earlier reports highlighted a tumor suppressor role for manganese superoxide dismutase (MnSOD), recent evidence indicates increased expression in a variety of human cancers including aggressive breast carcinoma. In the present article, we hypothesized that MnSOD expression is significantly amplified in the aggressive breast carcinoma basal subtype, and targeting MnSOD could be an attractive strategy for enhancing chemosensitivity of this highly aggressive breast cancer subtype.
RESULTS: Using MDA-MB-231 and BT549 as a model of basal breast cancer cell lines, we show that knockdown of MnSOD decreased the colony-forming ability and sensitized the cells to drug-induced cell death, while drug resistance was associated with increased MnSOD expression. In an attempt to develop a clinically relevant approach to down-regulate MnSOD expression in patients with basal breast carcinoma, we employed activation of the peroxisome proliferator-activated receptor gamma (PPARγ) to repress MnSOD expression; PPARγ activation significantly reduced MnSOD expression, increased chemosensitivity, and inhibited tumor growth. Moreover, as a proof of concept for the clinical use of PPARγ agonists to decrease MnSOD expression, biopsies derived from breast cancer patients who had received synthetic PPARγ ligands as anti-diabetic therapy had significantly reduced MnSOD expression. Finally, we provide evidence to implicate peroxynitrite as the mechanism involved in the increased sensitivity to chemotherapy induced by MnSOD repression.
INNOVATION AND CONCLUSION: These data provide evidence to link increased MnSOD expression with the aggressive basal breast cancer, and underscore the judicious use of PPARγ ligands for specifically down-regulating MnSOD to increase the chemosensitivity of this subtype of breast carcinoma.
Aims: We explore the role of an elevated O2−:H2O2 ratio as a prosurvival signal in glioma-propagating cells (GPCs). We hypothesize that depleting this ratio sensitizes GPCs to apoptotic triggers.
Results: We observed that an elevated O2−:H2O2 ratio conferred enhanced resistance in GPCs, and depletion of this ratio by pharmacological and genetic methods sensitized cells to apoptotic triggers. We established the reactive oxygen species (ROS) Index as a quantitative measure of a normalized O2−:H2O2 ratio and determined its utility in predicting chemosensitivity. Importantly, mice implanted with GPCs of a reduced ROS Index demonstrated extended survival. Analysis of tumor sections revealed effective targeting of complementarity determinant 133 (CD133)- and nestin-expressing neural precursors. Further, we established the Connectivity Map to interrogate a gene signature derived from a varied ROS Index for the patterns of association with individual patient gene expression in four clinical databases. We showed that patients with a reduced ROS Index demonstrate better survival. These data provide clinical evidence for the viability of our O2−:H2O2-mediated chemosensitivity profiles.
Innovation and Conclusion: Gliomas are notoriously recurrent and highly infiltrative, and have been shown to arise from stem-like cells. We implicate an elevated O2−:H2O2 ratio as a prosurvival signal in GPC self-renewal and proliferation. The ROS Index provides quantification of O2−:H2O2-mediated chemosensitivity, an advancement in a previously qualitative field. Intriguingly, glioma patients with a reduced ROS Index correlate with longer survival and the Proneural molecular classification, a feature frequently associated with tumors of better prognosis. These data emphasize the feasibility of manipulating the O2−:H2O2 ratio as a therapeutic strategy.
OBJECTIVE: To test the hypothesis that Notch signalling plays a role in the pathogenesis of rheumatoid arthritis (RA) and to determine whether pharmacological inhibition of Notch signalling with γ-secretase inhibitors can ameliorate the RA disease process in an animal model.
METHODS: Collagen-induced arthritis was induced in C57BL/6 or Notch antisense transgenic mice by immunisation with chicken type II collagen (CII). C57BL/6 mice were administered with different doses of inhibitors of γ-secretase, an enzyme required for Notch activation, at disease onset or after onset of symptoms. Severity of arthritis was monitored by clinical and histological scores, and in vivo non-invasive near-infrared fluorescence (NIRF) images. Micro-CT was used to confirm joint destruction. The levels of CII antibodies and cytokines in serum were determined by ELISA and bead-based cytokine assay. The expression levels of cytokines were studied by quantitative PCR in rheumatoid synovial fibroblasts.
RESULTS: The data show that Notch signalling stimulates synoviocytes and accelerates their production of proinflammatory cytokines and immune responses involving the upregulation of IgG1 and IgG2a. Pharmacological inhibition of γ-secretase and antisense-mediated knockdown of Notch attenuates the severity of inflammatory arthritis, including arthritis indices, paw thickness, tissue damage and neutrophil infiltration, and reduces the levels of active NF-κB, ICAM-1, proinflammatory cytokines and matrix metalloproteinase-3 activity in the mouse model of RA.
CONCLUSIONS: These results suggest that Notch is involved in the pathogenesis of RA and that inhibition of Notch signalling is a novel approach for treating RA.
Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors that initiate signals in response to diverse pathogen-associated molecular patterns. Several groups have recently reported a role for TLR2 and TLR4 in ischemic stroke-induced brain injury. However, relatively little is known about the role of TLR8 in ischemic stroke. Here we provide the first evidence that TLR8 activation plays a detrimental role in stroke outcome by promoting neuronal apoptosis and T cell-mediated post-stroke inflammation. TLR8 is expressed in cerebral cortical neurons, where its levels and downstream signaling via JNK are increased in response to oxygen glucose deprivation (OGD). Treatment with a TLR8 agonist activated pro-apoptotic JNK and increased neuronal cell death during OGD. Furthermore, selective knockdown of TLR8 using siRNA protected SH-SY5Y cells following OGD, and TLR8 agonist administration in vivo increased mortality, neurological deficit and T cell infiltration following stroke. Taken together, our findings indicate a detrimental role for neuronal TLR8 signaling in the triggering of post-stroke inflammation and neuronal death.
In this study, we provide evidence for the first time that membrane-bound endopeptidase neurolysin is up-regulated in different parts of mouse brain affected by focal ischemia-reperfusion in a middle cerebral artery occlusion model of stroke. Radioligand binding, enzymatic and immunoblotting experiments in membrane preparations of frontoparietal cortex, striatum, and hippocampus isolated from the ischemic hemisphere of mouse brain 24 h after reperfusion revealed statistically significant increase (≥ twofold) in quantity and activity of neurolysin compared with sham-operated controls. Cerebellar membranes isolated from the ischemic hemisphere served as negative control supporting the observations that up-regulation of neurolysin occurs in post-ischemic brain regions. This study also documents sustained functional up-regulation of neurolysin in frontoparietal cortical membranes for at least 7 days after stroke, which appears not to be transcriptionally or translationally regulated, but rather depends on translocation of cytosolic neurolysin to the membranes and mitochondria. Considering diversity of endogenous neurolysin substrates (neurotensin, bradykinin, angiotensins I/II, substance P, hemopressin, dynorphin A(1-8), metorphamide, somatostatin) and the well-documented role of these peptidergic systems in pathogenesis of stroke, resistance to ischemic injury and/or post-stroke brain recovery, our findings suggest that neurolysin may play a role in processes modulating the brain's response to stroke and its recovery after stroke. We provide evidence that peptidase neurolysin is up-regulated in the mouse brain membranes after stroke. Neurolysin metabolizes several neuropeptides in the brain (neurotensin, bradykinin, angiotensins I and II, substance P, hemopressin, dynorphin A(1-8), metorphamide, somatostatin), which have various functions in stroke pathophysiology. We hypothesize that neurolysin plays a key role in processes modulating the brain's response to stroke and its recovery after stroke.
Inflammation is an innate immune response to infection or tissue damage that is designed to limit harm to the host, but contributes significantly to ischemic brain injury following stroke. The inflammatory response is initiated by the detection of acute damage via extracellular and intracellular pattern recognition receptors, which respond to conserved microbial structures, termed pathogen-associated molecular patterns or host-derived danger signals termed damage-associated molecular patterns. Multi-protein complexes known as inflammasomes (e.g. containing NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, AIM2 and/or Pyrin), then process these signals to trigger an effector response. Briefly, signaling through NLRP1 and NLRP3 inflammasomes produces cleaved caspase-1, which cleaves both pro-IL-1β and pro-IL-18 into their biologically active mature pro-inflammatory cytokines that are released into the extracellular environment. This review will describe the molecular structure, cellular signaling pathways and current evidence for inflammasome activation following cerebral ischemia, and the potential for future treatments for stroke that may involve targeting inflammasome formation or its products in the ischemic brain.
Multi-protein complexes called inflammasomes have recently been identified and shown to contribute to cell death in tissue injury. Intravenous immunoglobulin (IVIg) is an FDA-approved therapeutic modality used for various inflammatory diseases. The objective of this study is to investigate dynamic responses of the NLRP1 and NLRP3 inflammasomes in stroke and to determine whether the NLRP1 and NLRP3 inflammasomes can be targeted with IVIg for therapeutic intervention. Primary cortical neurons were subjected to glucose deprivation (GD), oxygen-glucose deprivation (OGD) or simulated ischemia-reperfusion (I/R). Ischemic stroke was induced in C57BL/6J mice by middle cerebral artery occlusion, followed by reperfusion. Neurological assessment was performed, brain tissue damage was quantified, and NLRP1 and NLRP3 inflammasome protein levels were evaluated. NLRP1 and NLRP3 inflammasome components were also analyzed in postmortem brain tissue samples from stroke patients. Ischemia-like conditions increased the levels of NLRP1 and NLRP3 inflammasome proteins, and IL-1β and IL-18, in primary cortical neurons. Similarly, levels of NLRP1 and NLRP3 inflammasome proteins, IL-1β and IL-18 were elevated in ipsilateral brain tissues of cerebral I/R mice and stroke patients. Caspase-1 inhibitor treatment protected cultured cortical neurons and brain cells in vivo in experimental stroke models. IVIg treatment protected neurons in experimental stroke models by a mechanism involving suppression of NLRP1 and NLRP3 inflammasome activity. Our findings provide evidence that the NLRP1 and NLRP3 inflammasomes have a major role in neuronal cell death and behavioral deficits in stroke. We also identified NLRP1 and NLRP3 inflammasome inhibition as a novel mechanism by which IVIg can protect brain cells against ischemic damage, suggesting a potential clinical benefit of therapeutic interventions that target inflammasome assembly and activity.
CaV1.3 ion channels are dominant Ca(2+) portals into pacemaking neurons, residing at the epicenter of brain rhythmicity and neurodegeneration. Negative Ca(2+) feedback regulation of CaV1.3 channels (CDI) is therefore critical for Ca(2+) homeostasis. Intriguingly, nearly half the CaV1.3 transcripts in the brain are RNA edited to reduce CDI and influence oscillatory activity. It is then mechanistically remarkable that this editing occurs precisely within an IQ domain, whose interaction with Ca(2+)-bound calmodulin (Ca(2+)/CaM) is believed to induce CDI. Here, we sought the mechanism underlying the altered CDI of edited channels. Unexpectedly, editing failed to attenuate Ca(2+)/CaM binding. Instead, editing weakened the prebinding of Ca(2+)-free CaM (apoCaM) to channels, which proves essential for CDI. Thus, editing might render CDI continuously tunable by fluctuations in ambient CaM, a prominent effect we substantiate in substantia nigral neurons. This adjustability of Ca(2+) regulation by CaM now looms as a key element of CNS Ca(2+) homeostasis.
Persistent activation of NF-kB has been associated with the development of asthma. Receptor-interacting protein 2 (Rip2) is a transcriptional product of NF-kB activation. It is an adaptor protein with serine/threonine kinase activity and has been shown to positively regulate NF-kB activity. We investigated potential protective effects of Rip2 gene silencing using small interfering RNA (siRNA) in an OVA-induced mouse asthma model. Rip2 protein level was found to be upregulated in allergic airway inflammation. A potent and selective Rip2 siRNA given intratracheally knocked down Rip2 expression in OVA-challenged lungs and reduced OVA-induced increases in total and eosinophil counts, and IL-4, IL-5, IL-13, IL-1b, IL-33, and eotaxin levels in bronchoalveolar lavage fluid. Rip2 silencing blocked OVA-induced inflammatory cell infiltration and mucus hypersecretion as observed in lung sections, and mRNA expression of ICAM-1, VCAM-1, E-selectin, RANTES, IL-17, IL-33, thymic stromal lymphopoietin, inducible NO synthase, and MUC5ac in lung tissues. In addition, elevation of serum OVA-specific IgE level in mouse asthma model was markedly suppressed by Rip2 siRNA, together with reduced IL-4, IL-5, and IL-13 production in lymph node cultures. Furthermore, Rip2 siRNA-treated mice produced significantly less airway hyperresponsiveness induced by methacholine. Mechanistically, Rip2 siRNA was found to enhance cytosolic level of IkBa and block p65 nuclear translocation and DNA-binding activity in lung tissues from OVA-challenged mice. Taken together, our findings clearly show that knockdown of Rip2 by gene silencing ameliorates experimental allergic airway inflammation, probably via interruption of NF-kB activity, confirming Rip2 a novel therapeutic target for the treatment of allergic asthma.
Hodgkin lymphoma is caused by a minority population of malignant Hodgkin and Reed-Sternberg (HRS) cells that recruit an abundance of inflammatory cells. The long-term survival of HRS cells among the vast majority of immune cells indicates that they have developed potent immune escape mechanisms. We report that the TNF receptor family member CD137 (TNFRSF9) is expressed on HRS cells, while normal B cells, from which HRS cells are most often derived, do not express CD137. In 48 of 53 cases of classical Hodgkin lymphoma, CD137 was detected on HRS cells. Ectopically expressed CD137 transferred by trogocytosis from HRS cells to neighboring HRS and antigen-presenting cells, which constitutively express the CD137 ligand (CD137L and TNFSF9), became associated with CD137L and the CD137-CD137L complex was internalized. Disappearance of CD137L from the surface of HRS and antigen-presenting cells led to reduced costimulation of T cells through CD137, reducing IFN-γ release and proliferation. Our results reveal a new regulatory mechanism for CD137L expression that mediates immune escape by HRS cells, and they identify CD137 as a candidate target for immunotherapy of Hodgkin lymphoma.
Increased airway smooth muscle (ASM) mass is believed to underlie the relatively fixed airway hyperresponsiveness (AHR) in asthma. Developments of therapeutic approaches to reverse airway remodeling are impeded by our lack of insight on the mechanisms behind the increase in mass of contractile ASM cells. Increased expression of laminin, an extracellular matrix protein, is associated with asthma. Our studies investigate the role of laminin-induced ASM survival signals in the development of increased ASM and AHR. Antagonizing laminin integrin binding using the laminin-selective competing peptide, YIGSR, and mimicking laminin with exogenous α2-chain laminin, we show that laminin is both necessary and sufficient to induce ASM cell survival, concomitant with the induction of ASM contractile phenotype. Using siRNA, we show that the laminin-binding integrin α7β1 mediates this process. Moreover, in laminin-211-deficient mice, allergen-induced AHR was not observed. Notably, ASM cells from asthmatic airways express a higher abundance of intracellular cell survival proteins, consistent with a role for reduced rates of cell apoptosis in development of ASM hyperplasia. Targeting the laminin-integrin α7β1 signaling pathway may offer new avenues for the development of therapies to reduce the increase in mass of contractile phenotype ASM cells that underlie AHR in asthma.
Air particulate matter (PM) samples were collected in Singapore from 21 to 29 October 2010. During this time period, a severe regional smoke haze episode lasted for a few days (21–23 October). Physicochemical and toxicological characteristics of both haze and non-haze aerosols were evaluated. The average mass concentration of PM2.5 (PM with aerodynamic diameter of ≤2.5 μm) increased by a factor of 4 during the smoke haze period (107.2 μg/m3) as compared to that during the non-smoke haze period (27.0 μg/m3). The PM2.5 samples were analyzed for 16 priority polycyclic aromatic hydrocarbons (PAHs) listed by the United States Environmental Protection Agency and 10 transition metals. Out of the seven PAHs known as potential or suspected carcinogens, five were found in significantly higher levels in smoke haze aerosols as compared to those in the background air. Metal concentrations were also found to be higher in haze aerosols. Additionally, the toxicological profile of the PM2.5 samples was evaluated using a human epithelial lung cell line (A549). Cell viability and death counts were measured after a direct exposure of PM2.5 samples to A459 cells for a period of 48 h. The percentage of metabolically active cells decreased significantly following a direct exposure to PM samples collected during the haze period. To provide further insights into the toxicological characteristics of the aerosol particles, glutathione levels, as an indirect measure of oxidative stress and caspase-3/7 levels as a measure of apoptotic death, were also evaluated.
In the Review article by Fyhrquist et al. (The roles of senescence and telomere shortening in cardiovascular disease. Nat. Rev. Cardiol. doi:10.1038/nrcardio.2013.30), the authors extensively appraise the role in cardiovascular diseases of telomere length homeostasis in response to senescence, oxidative stress, and various other factors. The measurement of leukocyte telomere length to determine telomere size might be an important biomarker to predict cardiac disease outcomes. We agree with most of the conclusions of the authors in this Review and wish to add a new dimension by proposing a possible mechanism by which chronic oxidative stress in the cardiac microenvironment, as a result of ion-channel defects, might cause telomere shortening, which supports the hypothesis of arrhythmia-induced cardiac dysfunction. We previously reported the direct correlation between telomere length attrition and sudden cardiac death (SCD) using the processes of leukocyte-telomere-length measurement and comparative genomic hybridization. This analysis revealed defects in key ion-channel genes that had a strong correlation with telomere attrition when compared with age-matched controls. Fyhrquist and colleagues reviewed some work on the role of the mitochondria in cardiovascular disease, but ion channels—which have a crucial role in maintaining cellular homeostasis, particularly in the heart—were not discussed. Fyhrquist et al. proposed that the hypothesis of telomere dysfunction might converge with that of mitochondrial dysfunction. They highlighted reports of the presence of increased numbers of senescent cardiomyocytes that express p16, p21, and p53, and which have short telomeres, in the ageing heart. In our study, the mean age of individuals who experienced SCD was 24.7 years (range 12–33 years), and they were compared with controls with a mean age of 23 years (range 21–28 years). Severe telomere shortening and ion-channel defects were present in both the cardiomyocytes and peripheral blood lymphocytes of individuals who experienced SCD when compared with age-matched and sex-matched controls. This phenomenon was observed independently of age. Post-mortem examinations did not reveal signs of atherosclerotic plaques or cardiomyopathy, which ruled out age-related cardiac dysfunction and degeneration. Therefore, given that in this population, age-induced telomere shortening is well controlled for, the SCD cohort can be used as a model to study telomere attrition. The association between SCD and arrhythmias is well established. In our study of SCD, we reported deletion of KCNA4 and amplification of RYR2 genes, which actively regulate potassium-channel homeostasis. These copy number changes in ion-channel genes correlate with telomere regulatory defects in regions such as 3q26 and 18q11.2. We have also reported that mouse cells that lack telomerase reverse transcriptase are hypersensitive to oxidative stress, and that these mice display rapid upregulation of inflammatory cytokines and increased mortality compared with wild-type mice. We speculate that ion-channel defects and electrical abnormalities of the heart might accelerate telomere shortening by increasing chronic oxidative stress and, thus, cause SCD.
Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8−/− mice, we have generated Rnf8−/−p53−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8−/− mice. Similarly, the senescence phenotype of Rnf8−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8−/−p53−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8−/−p53−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8−/− or p53−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.
Curcumin, a polyphenolic compound isolated from Curcuma longa (Turmeric) is widely used in traditional Ayurvedic medicine. Its potential therapeutic effects on a variety of diseases have long been known. Though anti-tumour effects of curcumin have been reported earlier, its mode of action and telomerase inhibitory effects are not clearly determined in brain tumour cells. In the present study, we demonstrate that curcumin binds to cell surface membrane and infiltrates into cytoplasm to initiate apoptotic events. Curcumin treatment has resulted in higher cytotoxicity in the cells that express telomerase enzyme, highlighting its potential as an anticancer agent. Curcumin induced growth inhibition and cell cycle arrest at G2/M phase in the glioblastoma and medulloblastoma cells used in the study. Gene and protein expression analyses revealed that curcumin down-regulated CCNE1, E2F1 and CDK2 and up-regulated the expression of PTEN genes resulting in growth arrest at G2/M phase. Curcumin-induced apoptosis is found to be associated with increased caspase-3/7 activity and overexpression of Bax. In addition, down-regulation of Bcl2 and survivin was observed in curcumin-treated cells. Besides these effects, we found curcumin to be inhibiting telomerase activity and down-regulating hTERT mRNA expression leading to telomere shortening. We conclude that telomerase inhibitory effects of curcumin underscore its use in adjuvant cancer therapy.
Calcium fluxes have been implicated in the specification of the vertebrate embryonic nervous system for some time, but how these fluxes are regulated and how they relate to the rest of the neural induction cascade is unknown. Here we describe Calfacilitin, a transmembrane calcium channel facilitator that increases calcium flux by generating a larger window current and slowing inactivation of the L-type CaV1.2 channel. Calfacilitin binds to this channel and is co-expressed with it in the embryo. Regulation of intracellular calcium by Calfacilitin is required for expression of the neural plate specifiers Geminin and Sox2 and for neural plate formation. Loss-of-function of Calfacilitin can be rescued by ionomycin, which increases intracellular calcium. Our results elucidate the role of calcium fluxes in early neural development and uncover a new factor in the modulation of calcium signalling.
Mutations in parkin and LRRK2 together account for the majority of familial Parkinson's disease (PD) cases. Interestingly, recent evidence implicates the involvement of parkin and LRRK2 in mitochondrial homeostasis. Supporting this, we show here by means of the Drosophila model system that, like parkin, LRRK2 mutations induce mitochondrial pathology in flies when expressed in their flight muscles, the toxic effects of which can be rescued by parkin coexpression. When expressed specifically in fly dopaminergic neurons, mutant LRRK2 results in the appearance of significantly enlarged mitochondria, a phenotype that can also be rescued by parkin coexpression. Importantly, we also identified in this study that epigallocatechin gallate (EGCG), a green tea-derived catechin, acts as a potent suppressor of dopaminergic and mitochondrial dysfunction in both mutant LRRK2 and parkin-null flies. Notably, the protective effects of EGCG are abolished when AMP-activated protein kinase (AMPK) is genetically inactivated, suggesting that EGCG-mediated neuroprotection requires AMPK. Consistent with this, direct pharmacological or genetic activation of AMPK reproduces EGCG's protective effects. Conversely, loss of AMPK activity exacerbates neuronal loss and associated phenotypes in parkin and LRRK mutant flies. Together, our results suggest the relevance of mitochondrial-associated pathway in LRRK2 and parkin-related pathogenesis, and that AMPK activation may represent a potential therapeutic strategy for these familial forms of PD.
Semaphorins are implicated in glioma progression, although little is known about the underlying mechanisms. We have reported plexin-B3 expression in human gliomas, which upon stimulation by Sema5A causes significant inhibition of cell migration and invasion. The concomitant inactivation of Rac1 is of mechanistic importance because forced expression of constitutively active Rac1 abolishes these inhibitory effects. Furthermore, Sema5A induces prominent cell collapse and ramification of processes reminiscent of astrocytic morphology, which temporally associate with extensive disassembly of actin stress fibers and disruption of focal adhesions, followed by accumulation of actin patches in protrusions. Mechanistically, Sema5A induces transient protein kinase C (PKC) phosphorylation of fascin-1, which can reduce its actin-binding/bundling activities and temporally parallels its translocation from cell body to extending processes. PKC inhibition or fascin-1 knockdown is sufficient to abrogate Sema5A-induced morphological differentiation, whereas the process is hastened by forced expression of fascin-1. Intriguingly, Sema5A induces re-expression of glial fibrillary acidic protein (GFAP), which when silenced restricts differentiation of glioma cells to bipolar instead of multipolar morphology. Therefore, we hypothesize complementary functions of fascin-1 and GFAP in the early and late phases of Sema5A-induced astrocytic differentiation of gliomas, respectively. In summary, Sema5A and plexin-B3 impede motility but promote differentiation of human gliomas. These effects are plausibly compromised in high-grade human astrocytomas in which Sema5A expression is markedly reduced, hence leading to infiltrative and anaplastic characteristics. This is evident by increased invasiveness of glioma cells when endogenous Sema5A is silenced. Therefore, Sema5A and plexin-B3 represent potential novel targets in counteracting glioma progression.
Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within CaV1.3 Ca2+ channels, renown for low-voltage Ca2+-influx and neuronal pacemaking. Significantly, editing occurs within the channel’s IQ domain, a calmodulin-binding site mediating inhibitory Ca2+-feedback CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of CaV1.3 editing seen across the brain. Edited CaV1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited CaV1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wild-type versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning CaV1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions.
Skeletal muscle cells have served as a paradigm for understanding mechanisms leading to cellular differentiation. The proliferation and differentiation of muscle precursor cells require the concerted activity of myogenic regulatory factors including MyoD. In addition, chromatin modifiers mediate dynamic modifications of histone tails that are vital to reprogramming cells toward terminal differentiation. Here, we provide evidence for a unique dimension to epigenetic regulation of skeletal myogenesis. We demonstrate that the lysine methyltransferase G9a is dynamically expressed in myoblasts and impedes differentiation in a methyltransferase activity-dependent manner. In addition to mediating histone H3 lysine-9 di-methylation (H3K9me2) on MyoD target promoters, endogenous G9a interacts with MyoD in precursor cells and directly methylates it at lysine 104 (K104) to constrain its transcriptional activity. Mutation of K104 renders MyoD refractory to inhibition by G9a and enhances its myogenic activity. Interestingly, MyoD methylation is critical for G9a-mediated inhibition of myogenesis. These findings provide evidence of an unanticipated role for methyltransferases in cellular differentiation states by direct posttranslational modification of a transcription factor.
Rationale: Ca(V)1.2 channels are essential for excitation-contraction coupling in the cardiovascular system, and alternative splicing optimizes its role. Galectin-1 (Gal-1) has been reported to regulate vascular smooth muscle cell (VSMC) function and play a role in pulmonary hypertension. We have identified Gal-1 multiple times in yeast 2-hybrid assays using the Ca(V)1.2 I-II loop as bait. Objective: Our hypothesis is that Gal-1 interacts directly with Ca(V)1.2 channel at the I-II loop to affect arterial constriction. Methods and Results: Unexpectedly, Gal-1 was found to selectively bind to the I-II loop only in the absence of alternatively spliced exon 9*. We found that the current densities of Ca(V)1.2(Δ9*) channels were significantly inhibited as a result of decreased functional surface expression due to the binding of Gal-1 at the export signal located on the C-terminus of exon 9. Moreover, the suppression of Gal-1 expression by siRNA in rat A7r5 and isolated VSMCs produced the opposite effect of increased I(Ca,L). The physiological significance of Gal-1 mediated splice variant-specific inhibition of Ca(V)1.2 channels was demonstrated in organ bath culture where rat MAs were reversibly permeabilized with Gal-1 siRNA and the arterial wall exhibited increased K(+)-induced constriction. Conclusion: The above data indicated that Gal-1 regulates I(Ca,L) via decreasing the functional surface expression of Ca(V)1.2 channels in a splice variant selective manner and such a mechanism may play a role in modulating vascular constriction.
The medial septum is anatomically and functionally linked to the hippocampus, a region implicated in nociception. However, the role of medial septum in nociception remains unclear. To investigate the role of the region in nociception in rats, muscimol, a GABA agonist, or zolpidem, a positive allosteric modulator of GABAA receptors, was microinjected into medial septum to attenuate the activity of neurons in the region. Electrophysiological studies in anesthetized rats indicated that muscimol evoked a stronger and longer-lasting suppression of medial septal-mediated activation of hippocampal theta field activity than zolpidem. Similarly, microinjection of muscimol (1 or 2 lg/0.5 ll) into the medial septum of awake rats suppressed both licking and flinching behaviors in the formalin test of inflammatory pain, whereas only the latter behavior was affected by zolpidem (8 or 12 lg/0.5 ll) administered into the medial septum. Interestingly, both drugs selectively attenuated nociceptive behaviors in the second phase of the formalin test that are partly driven by central plasticity. Indeed, muscimol reduced the second phase behaviors by 30% to 60%, which was comparable to the reduction seen with systemic administration of a moderate dose of the analgesic morphine. The reduction was accompanied by a decrease in formalin-induced expression of spinal c-Fos protein that serves as an index of spinal nociceptive processing. The drug effects on nociceptive behaviors were without overt sedation and were distinct from the effects observed after septal lateral microinjections. Taken together, these findings suggest that the activation of medial septum is pro-nociceptive and facilitates aspects of central neural processing underlying nociception.
The molecular mechanisms underlying constitutive nuclear factor-κB (NF-κB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-κB activity, suggesting that ANXA1 may be required for the constitutive activity of IκB kinase (IKK) and NF-κB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-κB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKγ or NEMO, but not IKKα or IKKβ. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-κB. Downstream targets of NF-κB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-κB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-κB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-κB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.
Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. Here, we identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. We isolated two ana2 alleles that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/ maintain the association of Mud with Pins at the apical cortex. Our findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division.
Both EZH2 and NF-kB contribute to aggressive breast cancer, yet whether the two oncogenic factors have functional crosstalk in breast cancer is unknown. Here, we uncover an unexpected role of EZH2 in conferring the constitutive activation of NF-kB target gene expression in ER-negative basal-like breast cancer cells. This function of EZH2 is independent of its histone methyltransferase activity but requires the physical interaction with RelA/RelB to promote the expression of NF-kB targets. Intriguingly, EZH2 acts oppositely in ER-positive luminal-like breast cancer cells and represses NF-kB target gene expression by interacting with ER and directing repressive histone methylation on their promoters. Thus, EZH2 functions as a double-facet molecule in breast cancers, either as a transcriptional activator or repressor of NF-B targets, depending on the cellular context. These findings reveal an additional mechanism by which EZH2 promotes breast cancer progression and underscore the need for developing context-specific strategy for therapeutic targeting of EZH2 in breast cancers.
The cAMP-metabolising enzyme, phosphodiesterase 4 (PDE4), has been implicated in a number of immune responses, including tumour necrosis factor α (TNFα) production. To date, few data have directly addressed whether synovial cytokine and chemokine production is modified by PDE4.
Using specific PDE4 inhibitors, roflumilast plus two novel inhibitors, INH 0061 and INH 0062, the authors studied the effect of PDE4 inhibition on proinflammatory cytokine and chemokine release from primary rheumatoid arthritis (RA) synovial digest suspensions and in a macrophage T cell co-culture assay system.
All PDE4 inhibitors dose-dependently reduced the release of TNFα from primary synovial membrane cultures (n=5), half maximal inhibitory concentration (IC(50)) 300-30 nM, p<0.05. Similarly, a significant suppression in the release the proinflammatory chemokines, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β (IC(50) 300-30 nM) and regulated upon activation normal T-cell expressed and secreted (RANTES) (IC(50) 3 nM) was also observed, p<0.05. While interleukin 1β was also reduced, it did not achieve an IC(50). These observations were further confirmed in a macrophage T cell co-culture system, demonstrating the importance of PDE4 pathways in regulating cytokine/chemokine release in a cellular interaction implicated in inflammatory synovitis. Subsequent studies using the human monocytic cell line U937 also demonstrated cytokine regulation with PDE4 knockdown utilising a small interfering RNA approach.
These data provide direct evidence of PDE4-dependent pathways in human RA synovial inflammatory cytokine and chemokine release and may provide a novel approach in treating chronic autoimmune conditions such as RA.
The small GTPase Rac1 is involved in the activation of the NADPH oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2-overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 blocks Bcl-2-mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and co-immunoprecipitation, as well as GST-fusion proteins, we provide evidence for a co-localization and physical interaction between the two proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2-mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B cell lymphoma overexpressing Bcl-2 but not in non-cancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anti-cancer drug design.
Fusion genes are chimeric genes formed in cancers through genomic aberrations such as translocations, amplifications, and rearrangements. To identify fusion genes in gastric cancer, we analyzed regions of chromosomal imbalance in a cohort of 106 primary gastric cancers and 27 cell lines derived from gastric cancers. Multiple samples exhibited genomic breakpoints in the 5′ region of SLC1A2/EAAT2, a gene encoding a glutamate transporter. Analysis of a breakpoint-positive SNU16 cell line revealed expression of a CD44-SLC1A2 fusion transcript caused by a paracentric chromosomal inversion, which was predicted to produce a truncated but functional SLC1A2 protein. In primary tumors, CD44-SLC1A2 gene fusions were detected in 1 to 2% of gastric cancers, but not in adjacent matched normal gastric tissues. When we specifically silenced CD44-SLC1A2, cellular proliferation, invasion, and anchorage-independent growth were significantly reduced. Conversely, CD44-SLC1A2 overexpression in gastric cells stimulated these pro-oncogenic traits. CD44-SLC1A2 silencing caused significant reductions in intracellular glutamate concentrations and sensitized SNU16 cells to cisplatin, a commonly used chemotherapeutic agent in gastric cancer. We conclude that fusion of the SLC1A2 gene coding region to CD44 regulatory elements likely causes SLC1A2 transcriptional dysregulation, because tumors expressing high SLC1A2 levels also tended to be CD44-SLC1A2–positive. CD44-SLC1A2 may represent a class of gene fusions in cancers that establish a pro-oncogenic metabolic milieu favoring tumor growth and survival.
The PP2A serine/threonine protein phosphatase serves as a critical cellular regulator of cell growth, proliferation, and survival. However, how this pathway is altered in human cancer to confer growth advantage is largely unknown. Here, we show that PPP2R2B, encoding the B55β regulatory subunit of the PP2A complex, is epigenetically inactivated by DNA hypermethylation in colorectal cancer. B55β-associated PP2A interacts with PDK1 and modulates its activity toward Myc phosphorylation. On loss of PPP2R2B, mTORC1 inhibitor rapamycin triggers a compensatory Myc phosphorylation in PDK1-dependent, but PI3K and AKT-independent manner, resulting in resistance. Reexpression of PPP2R2B, genetic ablation of PDK1 or pharmacologic inhibition of PDK1 abrogates the rapamycin-induced Myc phosphorylation, leading to rapamycin sensitization. Thus, PP2A-B55β antagonizes PDK1-Myc signaling and modulates rapamycin sensitivity.
Thyrotoxic hypokalemic periodic paralysis (TPP) is characterized by acute attacks of weakness, hypokalemia, and thyrotoxicosis of various etiologies. These transient attacks resemble those of patients with familial hypokalemic periodic paralysis (hypoKPP) and resolve with treatment of the underlying hyperthyroidism. Because of the phenotypic similarity of these conditions, we hypothesized that TPP might also be a channelopathy. While sequencing candidate genes, we identified a previously unreported gene (not present in human sequence databases) that encodes an inwardly rectifying potassium (Kir) channel, Kir2.6. This channel, nearly identical to Kir2.2, is expressed in skeletal muscle and is transcriptionally regulated by thyroid hormone. Expression of Kir2.6 in mammalian cells revealed normal Kir currents in whole-cell and single-channel recordings. Kir2.6 mutations were present in up to 33% of the unrelated TPP patients in our collection. Some of these mutations clearly alter a variety of Kir2.6 properties, all altering muscle membrane excitability leading to paralysis.
Biological membranes with cubic morphology are a hallmark of stressed or diseased cellular conditions; both protein-protein interactions and lipid alterations appear to contribute to their biogenesis, yet their specific cellular functions are unknown. The occurrence of cubic membranes strikingly correlates with viral infections; notably, virus entry, proliferation, and release are processes closely linked to cellular cholesterol metabolism, and dys-regulation of cholesterol synthesis at the level of HMG-CoA reductase also induces cubic membrane formation, in the absence of viral infection. We propose that virus-induced cubic membranes could result from viral interference of cellular cholesterol homeostasis, generating a protective membrane environment to facilitate virus assembly and proliferation. Preventing cubic membrane formation might thus disrupt the 'virus factory' and offer new avenues to combat viral infections.
Duchenne Muscular Dystrophy (DMD), caused by loss of dystrophin is characterized by progressive muscle cell necrosis. However, the mechanisms leading to muscle degeneration in DMD are poorly understood. Here, we demonstrate that Stra13 protects muscle cells from oxidative damage, and its absence leads to muscle necrosis in response to injury in Stra13-deficient mice. Interestingly, Stra13-/- mutants express elevated levels of TNFalpha, reduced levels of heme-oxygenase-1, and display apparent signs of oxidative stress prior to muscle death. Moreover, Stra13-/- muscle cells exhibit an increased sensitivity to pro-oxidants, and conversely, Stra13 overexpression provides resistance to oxidative damage. Consistently, treatment with anti-oxidant N-acetylcysteine ameliorates muscle necrosis in Stra13-/- mice. We also demonstrate that Stra13 expression is elevated in muscles from dystrophin-deficient (mdx) mice, and mdx/Stra13-/- double mutants exhibit an early onset of muscle degeneration. Our studies underscore the importance of oxidative stress-mediated muscle degeneration in muscular dystrophy, and reveal the contribution of Stra13 in maintenance of muscle integrity.
The corticotropin-releasing factor (CRF) peptides CRF and uro-cortins 1 to 3 are crucial regulators of mammalian stress and inflammatory responses, and they are also implicated in disorders such as anxiety, depression, and drug addiction. There is considerable interest in the physiological mechanisms by which CRF receptors mediate their widespread effects, and here we report that the native CRF receptor 1 (CRFR1) endogenous to the human embryonic kidney 293 cells can functionally couple to mammalian Ca(V)3.2 T-type calcium channels. Activation of CRFR1 by either CRF or urocortin (UCN) 1 reversibly inhibits Ca(V)3.2 currents (IC(50) of approximately 30 nM), but it does not affect Ca(V)3.1 or Ca(V)3.3 channels. Blockade of CRFR1 by the antagonist astressin abolished the inhibition of Ca(V)3.2 channels. The CRFR1-dependent inhibition of Ca(V)3.2 channels was independent of the activities of phospholipase C, tyrosine kinases, Ca(2+)/calmodulin-dependent protein kinase II, protein kinase C, and other kinase pathways, but it was dependent upon a cholera toxin-sensitive G protein-mediated mechanism relying upon G protein betagamma subunits (Gbetagamma). The inhibition of Ca(V)3.2 channels via the activation of CRFR1 was due to a hyperpolarized shift in their steady-state inactivation, and it was reversible upon washout of the agonists. Given that UCN affect multiple aspects of cardiac and neuronal physiology and that Ca(V)3.2 channels are widespread throughout the cardiovascular and nervous systems, the results point to a novel and functionally relevant CRFR1-Ca(V)3.2 T-type calcium channel signaling pathway.
SHARP1, a basic helix-loop-helix transcription factor, is expressed in many cell types; however, the mechanisms by which it regulates cellular differentiation remain largely unknown. Here, we show that SHARP1 negatively regulates adipogenesis. Although expression of the early marker CCAAT/enhancer binding protein beta (C/EBPbeta) is not altered, its crucial downstream targets C/EBPalpha and peroxisome proliferator-activated receptor gamma (PPARgamma) are downregulated by SHARP1. Protein interaction studies confirm that SHARP1 interacts with and inhibits the transcriptional activity of both C/EBPbeta and C/EBPalpha, and enhances the association of C/EBPbeta with histone deacetylase 1 (HDAC1). Consistently, in SHARP1-expressing cells, HDAC1 and the histone methyltransferase G9a are retained at the C/EBP regulatory sites on the C/EBPalpha and PPARgamma2 promoters during differentiation, resulting in inhibition of their expression. Interestingly, treatment with troglitazone results in displacement of HDAC1 and G9a, and rescues the differentiation defect of SHARP1-overexpressing cells. Our data indicate that SHARP1 inhibits adipogenesis through the regulation of C/EBP activity, which is essential for PPARgamma-ligand-dependent displacement of co-repressors from adipogenic promoters.
Mutations of human CaV1.2 channel gene were identified only recently. The gain-of-function mutations were found at two mutually exclusive exons in patients with Timothy syndrome (TS). These patients exhibit prolonged QT interval and lethal cardiac arrhythmias. In contrast, the loss-of-function mutations of CaV1.2 channel in patients with Brugada syndrome produce short QT interval that could result in sudden cardiac death. TS patients also suffer from multi-organ dysfunction that includes neurological disorder such as autism and mental retardation reflecting the wide tissue distribution of CaV1.2 channel. Mutations found on different mutually exclusive exons determine the severity of the disease. Unexpectedly, TS patients may develop recurrent infections and bronchitis that suggests a role of CaV1.2 channel in the immune system. Furthermore, recent reports revealed a linkage of CaV1.2 channel polymorphism with multiple central nervous system disorders including bipolar disorder, depression, and schizophrenia. Here, we will discuss how alternative splicing modulates CaV1.2 channelopathy and the role of CaV1.2 channel in both excitable and non-excitable tissues.