Hemolysin BL (HBL) is a tripartite α‑pore‑forming toxin and a major Bacillus cereus virulence factor. Although LITAF and its homolog CDIP1 were previously identified as host receptors that promote HBL‑induced cytolysis, mice lacking both proteins remain only partially protected, indicating the existence of additional determinants of susceptibility. Using genome‑wide CRISPR knockout screens in Litaf-/-/Cdip1-/- mouse embryonic fibroblasts and human HT1080 cells, we identify caveolin‑1 (CAV1), a caveola‑associated membrane protein, as a third conserved host factor required for HBL cytolytic activity. Confocal imaging and co‑immunoprecipitation analyses show that CAV1 colocalizes with HBL and physically associates with the toxin at the plasma membrane. Notably, mice lacking all three factors (Litaf/Cdip1/Cav1 triple knockouts) are completely resistant to HBL toxin challenge, establishing CAV1 as an additional host factor mediating HBL toxin cytotoxicity. These findings define a tripartite host factor system required for HBL toxin activity and position CAV1 as an additional host determinant to B. cereus virulence.

Anthrax lethal toxin (LT) and edema toxin (ET) are two of the major virulence factors of Bacillus anthracis, the causative pathogen of anthrax disease. While the roles of LT in anthrax pathogenesis have been extensively studied, the pathogenic mechanism of ET remains poorly understood. ET is a calmodulin-dependent adenylate cyclase that elevates intracellular cAMP by converting ATP to cAMP. Thus, it was postulated that the ET-induced in vivo toxicity is mediated by certain cAMP-dependent events. However, mechanisms linking cAMP elevation and ET-induced damage have not been established. Cholera toxin is another bacterial toxin that increases cAMP. This toxin is known to cause severe intestinal fluid secretion and dehydration by cAMP-mediated activation of protein kinase A (PKA), which in turn activates cystic fibrosis transmembrane conductance regulator (CFTR). The cAMP-activated PKA phosphorylation of CFTR on the surface of intestinal epithelial cells leads to an efflux of chloride ions accompanied by secretion of H2O into the intestinal lumen, causing rapid fluid loss, severe diarrhea and dehydration. Due to similar in vivo effects, it was generally believed that ET and cholera toxin would exhibit a similar pathogenic mechanism. Surprisingly, in this work, we found that cAMP-mediated PKA/CFTR activation is not essential for ET to exert its in vivo toxicity. Instead, our data suggest that ET-induced ATP depletion may play an important role in the toxin's pathogenesis.

Lethal toxin (LT), the major virulence factor of Bacillus anthracis, proteolytically inactivates MEKs and disables downstream ERK, p38 and JNK pathway signalling leading to tissue damage and mortality. Therapies for LT-induced damage after host cell internalization of the toxin are lacking. Here we constructed MEK variants in which the LT proteolytic site was modified: MEK2(P10V/A11D), MEK3(I27D) and MEK6(I15D). These variants were resistant to proteolysis by LT. Expression in cells enabled sustained activation of ERK and p38 pathways and promoted cell survival upon LT treatment. Survival of LT- or B. anthracis-challenged MEK variant transgenic mice also increased compared with controls. We found that LT-mediated disruption of both ERK and p38 pathway is essential for anthrax pathogenesis. We show that engagement of upstream receptor tyrosine kinases reactivated the LT-disrupted ERK pathway, as did administering a cocktail of EGF, GM-CSF and FGF2 growth factors, which significantly increased survival of LT- or B. anthracis-challenged mice. These findings offer potential towards developing damage-limiting therapeutic strategies for anthrax.

Bacillus anthracis causes anthrax through a combination of bacterial infection and toxemia. As a major virulence factor of B. anthracis, anthrax lethal toxin (LT) is a zinc-dependent metalloproteinase, exerting its cytotoxicity through proteolytic cleavage of the mitogen-activated protein kinase kinases, thereby shutting down the MAPK pathways. Anthrax lethal toxin induces host lethality mostly by targeting the cardiovascular system. Although the enzymatic activity and the molecular targets of LT have long been known, the detailed mechanisms underlying cellular/tissue/organ toxicity are still poorly understood. In this work, we sought to investigate the mechanism of LT-induced cellular damage in the cardiovascular system. We demonstrate for the first time that anthrax lethal toxin has potent inhibitory effects on the central metabolism of cardiomyocytes and endothelial cells. This is likely due to the observed downregulating of c-Myc expression through the toxin-induced inhibition of the ERK pathway. Since c-Myc is a master transcription factor controlling the expression of many rate-limiting metabolic enzymes in glycolysis and the tricarboxylic acid cycle, LT's downregulation of c-Myc may lead to the observed bioenergetic collapse, particularly, in cardiomyocytes. Since cardiac cell contraction requires continuous production of large amounts of ATP, potent inhibition of the bioenergetics of cardiomyocytes would be incompatible with life. Thus, LT-induced lethality through targeting cardiomyocytes and endothelial cells appears to be a consequence of a bioenergetic collapse, likely due to the toxin's potent inhibitory activity on the MEK-ERK-c-Myc-metabolic/bioenergetic axis within these target cells of cardiovascular system.IMPORTANCEAnthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis, the causative pathogen of anthrax disease. Anthrax lethal toxin is a metalloproteinase that cleaves and inactivates MEKs, thereby shutting down MAPK pathways, leading to host mortality primarily through targeting of the cardiovascular system. However, the detailed mechanisms underlying the toxin's cellular and tissue toxicity are still poorly understood. Here, we found that anthrax lethal toxin has potent inhibitory activity on glycolysis and oxidative phosphorylation of cardiomyocytes and endothelial cells. These effects appear to be the consequence of downregulation of c-Myc, a master transcription factor that controls many rate-limiting enzymes of glycolysis and the tricarboxylic acid cycle. With the high demand on energy for cardiac contraction, the potent inhibition of cardiomyocyte metabolism by LT would be incompatible with life. This work provides critical insights into why the cardiovascular system is the major in vivo target of LT-induced lethality.

Cancer driver mutations in the RAS-RAF-MEK-ERK pathway occur in 46% of all human cancers. This has inspired the successful development of many small molecule inhibitors of BRAF and MEK. However, development of RAS small molecule inhibitors has not been successful in large part due to the lack of a sufficiently large and deep hydrophobic pocket in RAS proteins to allow for small molecule binding. Notably, in this regard, many bacterial pathogens have evolved potent protein toxins to disrupt the RAS-RAF-MEK-ERK pathway. Fortunately, these naturally designed potent toxins, such as anthrax toxin, Fortunately, these potent, naturally designed toxins, such as anthrax toxin and DUF5, can be structurally modified to achieve high tumor specificity. DUF5 (amino acids Glu3581-Gln4089) is a toxin effector domain of MARTX (a multifunctional-autoprocessing repeats-in-toxin) from Vibrio vulnificus. DUF5 was recently identified to be a specific and potent endopeptidase that cleaves and inactivates RAS. To address the above critical unmet medical needs, in this work, we have generated an anthrax toxin/DUF5-based, highly tumor-selective RAS inactivator and evaluated its anti-tumor activity in a variety of tumor models. This tumor-selective RAS inactivator can specifically bind to the major anthrax toxin receptor CMG2 on tumor cells and tumor stromal cells, and strictly relies on the simultaneous presence of two distinct tumor-associated proteases, MMPs and urokinase, to gain entry into tumor cells and tumor stromal cells to proteolytically inactivate RAS signaling, achieving potent selective targeting. Our data show that our tumor-selective RAS inactivator displays potent cytotoxicity to cancer cells whose survival depends on oncogenic RAS signaling. Our engineered toxin displayed potent in vivo antitumor activity to a range of tumors with oncogenic RAS mutations in syngeneic and human xenograft mouse tumor models. Therefore, we have developed a tumor-selective, safe, and highly potent RAS inactivator that has high potential for targeting tumors with oncogenic RAS mutations. Citation Format: Shihui Liu, Zehua Zuo, Jie Liu. A potent and highly tumor-selective KRAS inactivator for tumor targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2717.

FDA-approved BRAF and MEK small molecule inhibitors have demonstrated some level of efficacy in patients with metastatic melanomas. However, these "targeted" therapeutics have a very low therapeutic index, since these agents affect normal cells, causing undesirable, even fatal, side effects. To address these significant drawbacks, here, we have reengineered the anthrax toxin-based protein delivery system to develop a potent, tumor-selective MEK inactivator. This toxin-based MEK inactivator exhibits potent activity against a wide range of solid tumors, with the highest activity seen when directed toward tumors containing the BRAFV600E mutation. We demonstrate that this reengineered MEK inactivator also exhibits an extremely high therapeutic index (>15), due to its in vitro and in vivo activity being strictly dependent on the expression of multiple tumor-associated factors including tumor-associated proteases matrix metalloproteinase, urokinase plasminogen activator, and anthrax toxin receptor capillary morphogenesis protein-2. Furthermore, we have improved the specificity of this MEK inactivator, restricting its enzymatic activity to only target the ERK pathway, thereby greatly diminishing off-target toxicity. Together, these data suggest that engineered bacterial toxins can be modified to have significant in vitro and in vivo therapeutic effects with high therapeutic index.

The limited efficacy of the current antitumor microenvironment strategies is due in part to the poor understanding of the roles and relative contributions of the various tumor stromal cells to tumor development. Here, we describe a versatile in vivo anthrax toxin protein delivery system allowing for the unambiguous genetic evaluation of individual tumor stromal elements in cancer. Our reengineered tumor-selective anthrax toxin exhibits potent antiproliferative activity by disrupting ERK signaling in sensitive cells. Since this activity requires the surface expression of the capillary morphogenesis protein-2 (CMG2) toxin receptor, genetic manipulation of CMG2 expression using our cell-type-specific CMG2 transgenic mice allows us to specifically define the role of individual tumor stromal cell types in tumor development. Here, we established mice with CMG2 only expressed in tumor endothelial cells (ECs) and determined the specific contribution of tumor stromal ECs to the toxin's antitumor activity. Our results demonstrate that disruption of ERK signaling only within tumor ECs is sufficient to halt tumor growth. We discovered that c-Myc is a downstream effector of ERK signaling and that the MEK-ERK-c-Myc central metabolic axis in tumor ECs is essential for tumor progression. As such, disruption of ERK-c-Myc signaling in host-derived tumor ECs by our tumor-selective anthrax toxins explains their high efficacy in solid tumor therapy.

Chromosome rearrangement is one of the hallmarks of human malignancies. Gene fusion is one of the consequences of chromosome rearrangements. In this report, we show that gene fusion between solute carrier family 45 member 2 (SLC45A2) and alpha‐methylacyl‐coenzyme A racemase (AMACR) occurs in eight different types of human malignancies, with frequencies ranging from 45% to 97%. The chimeric protein is translocated to the lysosomal membrane and activates the extracellular signal‐regulated kinase signaling cascade. The fusion protein promotes cell growth, accelerates migration, resists serum starvation‐induced cell death, and is essential for cancer growth in mouse xenograft cancer models. Introduction of SLC45A2‐AMACR into the mouse liver using a sleeping beauty transposon system and somatic knockout of phosphatase and TENsin homolog (Pten) generated spontaneous liver cancers within a short period. Conclusion: The gene fusion between SLC45A2 and AMACR may be a driving event for human liver cancer development.

Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.

Significant number of human cancers contains gene fusion between solute carrier family 45, member 2 (SLC45A2) and α‐methylacyl‐CoA racemase (AMACR). SLC45A2 is a transporter protein known to be overexpressed in melanoma, while AMACR is an enzyme involved in metabolism of branch fatty acid, and is known for its overexpression in several human malignancies. SLC45A2‐AMACR replaces 8 transmembrane and cytosolic domains of SLC45A2 with an intact racemase domain from AMACR. At least eight types of primary human malignancies and cell lines are positive for SLC45A2‐AMACR fusion. Our analysis showed that SLC45A2‐AMACR interacts and activates MAPK kinase pathway, and promote cell growth and cancer invasion both in vitro and in vivo. Hydrodynamic tail vein injection of “sleeping beauty” SLC45A2‐AMACR in conjunction with somatic Pten knockout produced spontaneous liver cancer in 4 months. Disruption of SLC45A2‐AMACR fusion in HUH7 and H1299 cells abrogated the transformation activity of these cancer cell lines, while over‐expression of the fusion gene in H1299 cells induced higher growth rate, mitgration and resistant to serum starvation induced cell death in vitro. As a result, we conclude that SLC45A2‐AMACR is a critical driver for human cancers.

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

Cellular stress response 1 (CSR1) is a tumor suppressor gene that plays an important role in regulating cell death. In this report, we show that the N-terminus of CSR1 interacts with splicing factor 3A, subunit 3 (SF3A3). The SF3A3 binding motif was identified in the region of amino acids 62-91 of CSR1 through cell-free binding analyses. The interaction between CSR1 and SF3A3 led to migration of SF3A3 from nucleus to cytoplasm. The cytoplasmic redistribution of SF3A3 significantly reduced the splicing efficiency of epidermal growth factor receptor and platelet-derived growth factor receptor. Induction of CSR1 or down-regulation of SF3A3 also significantly reduced the splicing activity of oxytocin reporter gene both in vivo and in vitro. Mutant CSR1 that lacks the SF3A3 binding motif contained no RNA splicing regulatory activity, while the peptide corresponding to the SF3A3 binding motif in CSR1 interfered with the wild-type CSR1 mediated inhibition of RNA splicing. Interaction of CSR1 and SF3A3 is essential for CSR1 mediated cell death. To our knowledge, this is the first report demonstrating that RNA splicing is negatively regulated by redistribution of a splicing factor. © 2016 Wiley Periodicals, Inc.

Specifically targeting genomic rearrangements and mutations in tumor cells remains an elusive goal in cancer therapy. Here, we used Cas9-based genome editing to introduce the gene encoding the prodrug-converting enzyme herpes simplex virus type 1 thymidine kinase (HSV1-tk) into the genomes of cancer cells carrying unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135-CCDC67 and MAN2A1-FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9D10A and guide RNAs targeting the breakpoint sequences, and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed decreased tumor burden and no mortality during the study. Thus, Cas9-mediated suicide-gene insertion may be a viable genotype-specific cancer therapy.

目的:探讨真核细胞翻译起始因子4E(eIF4E)基因在卵巢癌细胞生长增殖、迁移侵袭以及对顺铂敏感性中的作用.方法:采用eIF4E基因的特异shRNA表达质粒转染卵巢癌A2780细胞,通过siRNA抑制eIF4E的表达,采用real time-PCR和Western印迹法检测siRNA对eIF4E mRNA和蛋白表达水平的抑制作用,优化转染作用条件.进而分析eIF4E敲降后,A2780细胞生物学行为的变化.结果:特定的siRNA对eIF4E基因的表达有显著的抑制作用;eIF4E敲降后,与对照组比较,A2780细胞的增殖明显受到抑制(P＜0.05),对顺铂的敏感性明显增强;细胞划痕实验和Transwell侵袭实验发现A2780细胞的运动侵袭能力显著下降(P＜0.05);细胞周期分析发现A2780细胞S期细胞比例略有下降,G1期细胞比例略有上升,但无统计学意义(P＞0.05).结论:抑制eIF4E基因的表达,可以显著抑制卵巢癌细胞增殖、抑制细胞的迁移侵袭能力,增强细胞对顺铂敏感性.上述结果为卵巢癌的分子机制及顺铂敏感性的研究提供了新依据;也为卵巢癌的临床治疗提供了新靶点基因.

Cellular stress response 1 (CSR1) is a tumor suppressor gene whose expression was frequently down-regulated in prostate cancer. The mechanism of its down-regulation, however, is not clear. Here, we show that the 3' untranslated region of CSR1 contains a target site of miR-650. High level of miR-650 was found in prostate cancer samples and cell lines. Degradation of miR-650 by specific inhibitor dramatically increased the expression levels of CSR1. Interaction between miR-650 and its target site in the 3' untranslated region was validated through luciferase reporter system. Mutation at the target site completely abrogated the activity of miR-650 on the 3' untranslated region of CSR1. Inhibition of miR-650 reversed the expression suppression of CSR1, suppressed colony formation, and blocked cell cycle entry to the S phase of both PC3 and DU145 cells. Animal model showed significant decrease of tumor volume, rate of metastasis, and mortality of severe combined immunodeficient mice xenografted with PC3 or DU145 cells transformed with inhibitor of miR-650. Our analyses demonstrate that suppression of CSR1 expression is a novel mechanism critical for the oncogenic activity of miR-650.

Fusion transcript formation is one of the fundamental mechanisms that drives the development of prostate cancer. Because of the advance of high-throughput parallel sequencing, many fusion transcripts have been discovered. However, the discovery rate of fusion transcripts specific for prostate cancer is Lagging behind the discoveries made on chromosome abnormalities of prostate cancer. Recent analyses suggest that many fusion transcripts are present in both benign and cancerous tissues. Some of these fusion transcripts likely represent important components of normal gene expression in cells. It is necessary to identify the criteria and features of fusion transcripts that are specific for cancer. In this review, we discuss optimization of RNA sequencing depth for fusion transcript discovery and the characteristics of fusion transcripts in normal prostate tissues and prostate cancer. We also propose a new classification of cancer-specific fusion transcripts on the basis of their tail gene fusion protein product and the roles that these fusions may play in cancer development.

Metallothioneins ( MTs ) are a group of metal binding proteins thought to play a role in the detoxification of heavy metals. Here we showed by microarray and validation analyses that MT1h , a member of MT , is down‐regulated in many human malignancies. Low expression of MT1h was associated with poor clinical outcomes in both prostate and liver cancer. We found that the promoter region of MT1h was hypermethylated in cancer and that demethylation of the MT1h promoter reversed the suppression of MT1h expression. Forced expression of MT1h induced cell growth arrest, suppressed colony formation, retarded migration, and reduced invasion. SCID mice with tumour xenografts with inducible MT1h expression had lower tumour volumes as well as fewer metastases and deaths than uninduced controls. MT1h was found to interact with euchromatin histone methyltransferase 1 ( EHMT1 ) and enhanced its methyltransferase activity on histone 3. Knocking down of EHMT1 or a mutation in MT1h that abrogates its interaction with EHMT1 abrogated MT1h tumour suppressor activity. This demonstrates tumour suppressor activity in a heavy metal binding protein that is dependent on activation of histone methylation. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.