CPT1A-mediated fatty acid oxidation confers cancer cell resistance to immune-mediated cytolytic killing.

Although tumor-intrinsic fatty acid ß-oxidation (FAO) is implicated in multiple aspects of tumorigenesis and progression, the impact of this metabolic pathway on cancer cell susceptibility to immunotherapy remains unknown. Here, we report that cytotoxicity of killer T cells induces activation of FAO and upregulation of carnitine palmitoyltransferase 1A (CPT1A), the rate-limiting enzyme of FAO in cancer cells. The repression of CPT1A activity or expression renders cancer cells more susceptible to destruction by cytotoxic T lymphocytes. Our mechanistic studies reveal that FAO deficiency abrogates the prosurvival signaling in cancer cells under immune cytolytic stress. Furthermore, we identify T cell-derived IFN-γ as a major factor responsible for induction of CPT1A and FAO in an AMPK-dependent manner, indicating a dynamic interplay between immune effector cells and tumor targets. While cancer growth in the absence of CPT1A remains largely unaffected, established tumors upon FAO inhibition become significantly more responsive to cellular immunotherapies including chimeric antigen receptor-engineered human T cells. Together, these findings uncover a mode of cancer resistance and immune editing that can facilitate immune escape and limit the benefits of immunotherapies.

Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

Lysophosphatidic acid induces tumor necrosis factor-alpha to regulate a pro-inflammatory cytokine network in ovarian cancer.

Epithelial ovarian carcinoma tissues express high levels of tumor necrosis factor-alpha (TNF-α) and other inflammatory cytokines. The underlying mechanism leading to the abnormal TNF-α expression in ovarian cancer remains poorly understood. In the current study, we demonstrated that lysophosphatidic acid (LPA), a lipid mediator present in ascites of ovarian cancer patients, induced expression of TNF-α mRNA and release of TNF-α protein in ovarian cancer cells. LPA also induced expression of interleukin-1ß (IL-1ß) mRNA but no significant increase in IL-1ß protein was detected. LPA enhanced TNF-α mRNA through NF-κB-mediated transcriptional activation. Inactivation of ADAM17, a disintegrin and metalloproteinase, with a specific inhibitor TMI-1 or by shRNA knockdown prevented ovarian cancer cells from releasing TNF-α protein in response to LPA, indicating that LPA-mediated TNF-α production relies on both transcriptional upregulations of the TNF-α gene and the activity of ADAM17, the membrane-associated TNF-α-converting enzyme. Like many other biological responses to LPA, induction of TNF-α by LPA also depended on the transactivation of the epidermal growth factor receptor (EGFR). Interestingly, our results revealed that ADAM17 was also the shedding protease responsible for the transactivation of EGFR by LPA in ovarian cancer cells. To explore the biological outcomes of LPA-induced TNF-α, we examined the effects of a TNF-α neutralizing antibody and recombinant TNF-α soluble receptor on LPA-stimulated expression of pro-tumorigenic cytokines and chemokines overexpressed in ovarian cancer. Blockade of TNF-α signaling significantly reduced the production of IL-8, IL-6, and CXCL1, suggesting a hierarchy of mechanisms contributing to the robust expression of the inflammatory mediators in response to LPA in ovarian cancer cells. In contrast, TNF-α inhibition did not affect LPA-dependent cell proliferation. Taken together, our results establish that the bioactive lipid LPA drives the expression of TNF-α to regulate an inflammatory network in ovarian cancer.

The lignan manassantin is a potent and specific inhibitor of mitochondrial complex I and bioenergetic activity in mammals.

Dineolignans manassantin A and B from the plant Saururus cernuus are used in traditional medicine to manage a wide range of ailments such as edema, jaundice, and gonorrhea. Cell-based studies have identified several molecular target candidates of manassantin including NF-κB, MAPK, STAT3, and hypoxia-inducible factor 1α (HIF-1α). It is unclear whether or how these structurally diverse proteins or pathways mediate any of the medical benefits of manassantin in vivo Moreover, it has recently been reported that manassantin causes developmental arrest in zebrafish by inhibiting the mitochondrial complex I, but it is unknown whether manassantin inhibits mitochondrial respiration in intact mammalian cells and live animals. Here, we present direct evidence that manassantin potently and specifically inhibits the mitochondrial complex I and bioenergetic activity in mammalian systems. Manassantin had no effect on complex II- or complex IV-mediated respiration. Of note, it decreased NADH-ubiquinone reductase activity but not the activity of NADH-ferricyanide reductase. Treatment with manassantin reduced cellular ATP levels and concomitantly stimulated AMP-activated protein kinase in vitro and in vivo As an adaptive response to manassantin-induced bioenergetic deficiency, mammalian cells up-regulated aerobic glycolysis, a process mediated by AMP-activated protein kinase (AMPK) independently of HIF-1α. Together these results demonstrate a biologically important activity of manassantin in the control of complex I-mediated respiration and its profound effects on oxygen utilization, energy homeostasis, and glucose metabolism in mammalian cells.

In-depth LC-MS/MS analysis of the chicken ovarian cancer proteome reveals conserved and novel differentially regulated proteins in humans.

Ovarian cancer (OVC) remains the most lethal gynecological malignancy in the world due to the combined lack of early-stage diagnostics and effective therapeutic strategies. The development and application of advanced proteomics technology and new experimental models has created unique opportunities for translational studies. In this study, we investigated the ovarian cancer proteome of the chicken, an emerging experimental model of OVC that develops ovarian tumors spontaneously. Matched plasma, ovary, and oviduct tissue biospecimens derived from healthy, early-stage OVC, and late-stage OVC birds were quantitatively characterized by label-free proteomics. Over 2600 proteins were identified in this study, 348 of which were differentially expressed by more than twofold (p ≤ 0.05) in early- and late-stage ovarian tumor tissue specimens relative to healthy ovarian tissues. Several of the 348 proteins are known to be differentially regulated in human cancers including B2M, CLDN3, EPCAM, PIGR, S100A6, S100A9, S100A11, and TPD52. Of particular interest was ovostatin 2 (OVOS2), a novel 165-kDa protease inhibitor found to be strongly upregulated in chicken ovarian tumors (p = 0.0005) and matched plasma (p = 0.003). Indeed, RT-quantitative PCR and Western blot analysis demonstrated that OVOS2 mRNA and protein were also upregulated in multiple human OVC cell lines compared to normal ovarian epithelia (NOE) cells and immunohistochemical staining confirmed overexpression of OVOS2 in primary human ovarian cancers relative to non-cancerous tissues. Collectively, these data provide the first evidence for involvement of OVOS2 in the pathogenesis of both chicken and human ovarian cancer.

Inhibitor of streptokinase gene expression improves survival after group A streptococcus infection in mice.

The widespread occurrence of antibiotic resistance among human pathogens is a major public health problem. Conventional antibiotics typically target bacterial killing or growth inhibition, resulting in strong selection for the development of antibiotic resistance. Alternative therapeutic approaches targeting microbial pathogenicity without inhibiting growth might minimize selection for resistant organisms. Compounds inhibiting gene expression of streptokinase (SK), a critical group A streptococcal (GAS) virulence factor, were identified through a high-throughput, growth-based screen on a library of 55,000 small molecules. The lead compound [Center for Chemical Genomics 2979 (CCG-2979)] and an analog (CCG-102487) were confirmed to also inhibit the production of active SK protein. Microarray analysis of GAS grown in the presence of CCG-102487 showed down-regulation of a number of important virulence factors in addition to SK, suggesting disruption of a general virulence gene regulatory network. CCG-2979 and CCG-102487 both enhanced granulocyte phagocytosis and killing of GAS in an in vitro assay, and CCG-2979 also protected mice from GAS-induced mortality in vivo. These data suggest that the class of compounds represented by CCG-2979 may be of therapeutic value for the treatment of GAS and potentially other gram-positive infections in humans.

Inhibition of Staphylococcus epidermidis biofilm by trimethylsilane plasma coating.

Biofilm formation on implantable medical devices is a major impediment to the treatment of nosocomial infections and promotes local progressive tissue destruction. Staphylococcus epidermidis infections are the leading cause of biofilm formation on indwelling devices. Bacteria in biofilms are highly resistant to antibiotic treatment, which in combination with the increasing prevalence of antibiotic resistance among human pathogens further complicates treatment of biofilm-related device infections. We have developed a novel plasma coating technology. Trimethylsilane (TMS) was used as a monomer to coat the surfaces of 316L stainless steel and grade 5 titanium alloy, which are widely used in implantable medical devices. The results of biofilm assays demonstrated that this TMS coating markedly decreased S. epidermidis biofilm formation by inhibiting the attachment of bacterial cells to the TMS-coated surfaces during the early phase of biofilm development. We also discovered that bacterial cells on the TMS-coated surfaces were more susceptible to antibiotic treatment than their counterparts in biofilms on uncoated surfaces. These findings suggested that TMS coating could result in a surface that is resistant to biofilm development and also in a bacterial community that is more sensitive to antibiotic therapy than typical biofilms.

Histopathological Diagnosis of Nonalcoholic Steatohepatitis (NASH).

Fatty acid beta oxidation (FAO) is a predominant bioenergetic pathway in mammals. Substantial investigations have demonstrated that FAO activity is dysregulated in many pathophysiological conditions including nonalcoholic steatohepatitis (NASH). Convenient and quantitative assays of FAO activities are important for studies of cell metabolism and the biological relevance of FAO to health and diseases. However, most current FAO assays are based on non-physiological culture conditions, measure FAO activity indirectly or lack adequate quantification. We herein describe details of practical protocols for measurement of basal and genetically or pharmacologically regulated FAO activities in the mammalian system. We also discuss the advantages and disadvantages of these assays in the context of experimental purposes.

Molecular diversity of toxic components from the scorpion Heterometrus petersii venom revealed by proteomic and transcriptome analysis.

Scorpion venoms contain a vast untapped reservoir of natural products, which have the potential for medicinal value in drug discovery. In this study, toxin components from the scorpion Heterometrus petersii venom were evaluated by transcriptome and proteome analysis.Ten known families of venom peptides and proteins were identified, which include: two families of potassium channel toxins, four families of antimicrobial and cytolytic peptides,and one family from each of the calcium channel toxins, La1-like peptides, phospholipase A2,and the serine proteases. In addition, we also identified 12 atypical families, which include the acid phosphatases, diuretic peptides, and ten orphan families. From the data presented here, the extreme diversity and convergence of toxic components in scorpion venom was uncovered. Our work demonstrates the power of combining transcriptomic and proteomic approaches in the study of animal venoms.

Functional analysis of molecular and pharmacological modulators of mitochondrial fatty acid oxidation.

Fatty acid oxidation (FAO) is a key bioenergetic pathway often dysregulated in diseases. The current knowledge on FAO regulators in mammalian cells is limited and sometimes controversial. Previous FAO analyses involve nonphysiological culture conditions or lack adequate quantification. We herein described a convenient and quantitative assay to monitor dynamic FAO activities of mammalian cells in physiologically relevant settings. The method enabled us to assess various molecular and pharmacological modulators of the FAO pathway in established cell lines, primary cells and mice. Surprisingly, many previously proposed FAO inhibitors such as ranolazine and trimetazidine lacked FAO-interfering activity. In comparison, etomoxir at low micromolar concentrations was sufficient to saturate its target proteins and to block cellular FAO function. Oxfenicine, on the other hand, acted as a partial inhibitor of FAO. As another class of FAO inhibitors that transcriptionally repress FAO genes, antagonists of peroxisome proliferator-activated receptors (PPARs), particularly that of PPARα, significantly decreased cellular FAO activity. Our assay also had sufficient sensitivity to monitor upregulation of FAO in response to environmental glucose depletion and other energy-demanding cues. Altogether this study provided a reliable FAO assay and a clear picture of biological properties of potential FAO modulators in the mammalian system.

Transcriptome analysis of the venom gland of the scorpion Scorpiops jendeki: implication for the evolution of the scorpion venom arsenal.

BACKGROUND: The family Euscorpiidae, which covers Europe, Asia, Africa, and America, is one of the most widely distributed scorpion groups. However, no studies have been conducted on the venom of a Euscorpiidae species yet. In this work, we performed a transcriptomic approach for characterizing the venom components from a Euscorpiidae scorpion, Scorpiops jendeki. RESULTS: There are ten known types of venom peptides and proteins obtained from Scorpiops jendeki. Great diversity is observed in primary sequences of most highly expressed types. The most highly expressed types are cytolytic peptides and serine proteases. Neurotoxins specific for sodium channels, which are major groups of venom components from Buthidae scorpions, are not detected in this study. In addition to those known types of venom peptides and proteins, we also obtain nine atypical types of venom molecules which haven't been observed in any other scorpion species studied to date. CONCLUSION: This work provides the first set of cDNAs from Scorpiops jendeki, and one of the few transcriptomic analyses from a scorpion. This allows the characterization of a large number of venom molecules, belonging to either known or atypical types of scorpion venom peptides and proteins. Besides, our work could provide some clues to the evolution of the scorpion venom arsenal by comparison with venom data from other scorpion lineages.

Imcroporin, a new cationic antimicrobial peptide from the venom of the scorpion Isometrus maculates.

The pace of resistance against antibiotics almost exceeds that of the development of new drugs. As many bacteria have become resistant to conventional antibiotics, new drugs or drug resources are badly needed to combat antibiotic-resistant pathogens, like methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial peptides, rich sources existing in nature, are able to effectively kill multidrug-resistant pathogens. Here, imcroporin, a new antimicrobial peptide, was screened and isolated from the cDNA library of the venomous gland of Isometrus maculates. The MIC of imcroporin against MRSA was 50 microg/ml, 8-fold lower than that of cefotaxime and 40-fold lower than that of penicillin. Imcroporin killed bacteria rapidly in vitro, inhibited bacterial growth, and cured infected mice. These results revealed that imcroporin could be considered a potential anti-infective drug or lead compound, especially for treating antibiotic-resistant pathogens.

Characterization of LmTxLP11 and LmVP1.1 transcripts and genomic organizations: alternative splicing contributing to the diversity of scorpion venom peptides.

Scorpion venoms are rich resources of bioactive peptides with extreme variability. Multiple molecular mechanisms are involved in the diversity of scorpion venom peptides. However, alternative splicing, which plays a major role in the generation of proteomic and functional diversity in metazoan organisms, hasn't been reported in genes coding for scorpion venom peptides. In the EST analysis of venom peptide transcripts from scorpion Lychas mucronatus, we reported an alternative splicing event. Transcripts of LmTxLP11 and LmVP1.1 share identical 5' region. LmVP1.1 is a novel type of scorpion venom peptides constrained by one disulfide bridge, whereas LmTxLP11 is an extended version of LmVP1.1. By transcript alignment with its genomic sequence, it is found that both transcripts are generated from a single gene by alternative poly A site and terminal exon. The gene encoding LmTxLP11 and LmVP1.1 is the first one harboring three introns ever reported from scorpion venoms. This work demonstrates for the first time that alternative splicing is involved in regulating the diversity of scorpion venom peptides.

A HindIII BAC library construction of Mesobuthus martensii Karsch (Scorpiones:Buthidae): an important genetic resource for comparative genomics and phylogenetic analysis.

Scorpions are "living but sophisticated fossils" that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of scorpion genome and the relationships between scorpions and other organisms, we first determined the genome size of the scorpion Mesobuthus martensii Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA intergenic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.

Immunometabolism: A new target for improving cancer immunotherapy.

Fundamental metabolic pathways are essential for mammalian cells to provide energy, precursors for biosynthesis of macromolecules, and reducing power for redox regulation. While dysregulated metabolism (e.g., aerobic glycolysis also known as the Warburg effect) has long been recognized as a hallmark of cancer, recent discoveries of metabolic reprogramming in immune cells during their activation and differentiation have led to an emerging concept of "immunometabolism." Considering the recent success of cancer immunotherapy in the treatment of several cancer types, increasing research efforts are being made to elucidate alterations in metabolic profiles of cancer and immune cells during their interplays in the setting of cancer progression and immunotherapy. In this review, we summarize recent advances in studies of metabolic reprogramming in cancer as well as differentiation and functionality of various immune cells. In particular, we will elaborate how distinct metabolic pathways in the tumor microenvironment cause functional impairment of immune cells and contribute to immune evasion by cancer. Lastly, we highlight the potential of metabolically reprogramming the tumor microenvironment to promote effective and long-lasting antitumor immunity for improved immunotherapeutic outcomes.

Mucroporin, the first cationic host defense peptide from the venom of Lychas mucronatus.

The misuse of antibiotics has led our age to a dangerous edge, as antibiotic-resistant pathogens appear to evolve more quickly than antibiotics are invented. Thus, new agents to treat bacterial infection are badly needed. Cationic host defense peptides are on the first line of a host defense system and are thought to be good candidates for treating bacterial infection. Here, a novel cationic host defense peptide, mucroporin, was cloned and characterized from the venom of Lychas mucronatus. The MIC for Staphylococcus aureus was 25 microg/ml, including antibiotic-resistant pathogens. Based on the molecular template of mucroporin, mucroporin-M1 was designed by amino acid substitution. The MIC for S. aureus was 5 microg/ml, including the antibiotic-resistant pathogens methicillin-resistant S. aureus, methicillin-resistant coagulase-negative Staphylococcus, penicillin-resistant S. aureus, and penicillin-resistant S. epidermidis. Moreover, mucroporin-M1 also inhibited gram-negative bacteria. The modes of action of mucroporin and mucroporin-M1 were both rapid killing by disrupting the cell membrane of bacteria, and the number of surviving bacteria was reduced by about 4 to 5 orders of magnitude immediately after peptide delivery. These results showed that mucroporin could be considered a potential anti-infective drug, especially for treating antibiotic-resistant pathogens.

Fatty acid oxidation: An emerging facet of metabolic transformation in cancer.

Cancer cells undergo metabolic reprogramming such as enhanced aerobic glycolysis, mutations in the tricarboxylic acid cycle enzymes, and upregulation of de novo lipid synthesis and glutaminolysis. These alterations are pivotal to the development and maintenance of the malignant phenotype of cancer cells in unfavorable tumor microenvironment or metastatic sites. Although mitochondrial fatty acid ß-oxidation (FAO) is a primary bioenergetic source, it has not been generally recognized as part of the metabolic landscape of cancer. The last few years, however, have seen a dramatic change in the view of cancer relevance of the FAO pathway. Many recent studies have provided significant evidence to support a "lipolytic phenotype" of cancer. FAO, like other well-defined metabolic pathways involved in cancer, is dysregulated in diverse human malignancies. Cancer cells rely on FAO for proliferation, survival, stemness, drug resistance, and metastatic progression. FAO is also reprogrammed in cancer-associated immune and other host cells, which may contribute to immune suppression and tumor-promoting microenvironment. This article reviews and puts into context our current understanding of multi-faceted roles of FAO in oncogenesis as well as anti-cancer therapeutic opportunities posed by the FAO pathway.

Ovarian Cancer Relies on Glucose Transporter 1 to Fuel Glycolysis and Growth: Anti-Tumor Activity of BAY-876.

The recent progresses in understanding of cancer glycolytic phenotype have offered new strategies to manage ovarian cancer and other malignancies. However, therapeutic targeting of glycolysis to treat cancer remains unsuccessful due to complex mechanisms of tumor glycolysis and the lack of selective, potent and safe glycolytic inhibitors. Recently, BAY-876 was identified as a new-generation inhibitor of glucose transporter 1 (GLUT1), a GLUT isoform commonly overexpressed but functionally poorly defined in ovarian cancer. Notably, BAY-876 has not been evaluated in any cell or preclinical animal models since its discovery. We herein took advantage of BAY-876 and molecular approaches to study GLUT1 regulation, targetability, and functional relevance to cancer glycolysis. The anti-tumor activity of BAY-876 was evaluated with ovarian cancer cell line- and patient-derived xenograft (PDX) models. Our results show that inhibition of GLUT1 is sufficient to block basal and stress-regulated glycolysis, and anchorage-dependent and independent growth of ovarian cancer cells. BAY-876 dramatically inhibits tumorigenicity of both cell line-derived xenografts and PDXs. These studies provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. BAY-876 is a potent blocker of GLUT1 activity, glycolytic metabolism and ovarian cancer growth, holding promise as a novel glycolysis-targeted anti-cancer agent.

Molecular cloning and electrophysiological studies on the first K(+) channel toxin (LmKTx8) derived from scorpion Lychas mucronatus.

LmKTx8, the first toxic gene isolated from the venom of scorpion Lychas mucronatus by constructing cDNA library method, was expressed and characterized physiologically. The mature peptide has 40 residues including six conserved cysteines, and is classified as one of alpha-KTx11 subfamily. Using patch-clamp recording, the recombinant LmKTx8 (rLmKTx8) was used to test the effect on voltage-gated K(+) channels (Kv1.3) stably expressed in COS7 cells and large conductance-Ca(2+)-activated K(+) (BK) channels expressed in HEK293. The results of electrophysiological experiments showed that the rLmKTx8 was a potent inhibitor of Kv1.3 channels with an IC(50)=26.40+/-1.62nM, but 100nM rLmKTx8 did not block the BK currents. LmKTx8 or its analogs might serve as a potential candidate for the development of new drugs for autoimmune diseases.

Adaptive evolution of insect selective excitatory ß-type sodium channel neurotoxins from scorpion venom.

Insect selective excitatory ß-type sodium channel neurotoxins from scorpion venom (ß-NaScTxs) are composed of about 70-76 amino acid residues and share a common scaffold stabilized by four unique disulfide bonds. The phylogenetic analysis of these toxins was hindered by limited sequence data. In our recent study, two new insect selective excitatory ß-NaScTxs, LmIT and ImIT, were isolated from Lychas mucronatus and Isometrus maculatus, respectively. With the sequences previously reported, we examined the adaptive molecular evolution of insect selective excitatory ß-NaScTxs by estimating the nonsynonymous-to-synonymous rate ratio (ω=dN/dS). The results revealed 12 positively selected sites in the genes of insect selective excitatory ß-NaScTxs. Moreover, these positively selected sites match well with the sites important for interacting with sodium channels, as demonstrated in previous mutagenesis study. These results reveal that adaptive evolution after gene duplication is one of the most important genetic mechanisms of scorpion neurotoxin diversification.