ERK and c-Myc signaling in host-derived tumor endothelial cells is essential for solid tumor growth.

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.

A potent tumor-selective ERK pathway inactivator with high therapeutic index.

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.

Oncogenic Activity of Solute Carrier Family 45 Member 2 and Alpha-Methylacyl-Coenzyme A Racemase Gene Fusion Is Mediated by Mitogen-Activated Protein Kinase.

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.

Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen.

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.

Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

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.

Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene.

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.

Cellular stress response 1 down-regulates the expression of epidermal growth factor receptor and platelet-derived growth factor receptor through inactivation of splicing factor 3A3.

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.

Discovery and Classification of Fusion Transcripts in Prostate Cancer and Normal Prostate Tissue.

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.

Oncogenic Activity of miR-650 in Prostate Cancer Is Mediated by Suppression of CSR1 Expression.

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.

Metallothionein 1 h tumour suppressor activity in prostate cancer is mediated by euchromatin methyltransferase 1.

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.

Pivotal role of reduced let-7g expression in breast cancer invasion and metastasis.

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Signal transducer and activator of transcription (STAT)-5A and STAT5B differentially regulate human mammary carcinoma cell behavior.

Increased activation of signal transducer and activator of transcription (STAT)-5 has been reported in various malignancies including mammary carcinoma. However, it is only recently that potentially distinct roles of STAT5A and STAT5B in neoplasia have begun to emerge. Herein we systematically delineate the functions of STAT5A and STAT5B in human mammary carcinoma cell lines MCF-7 and T47D. Forced expression of constitutively active (CA) STAT5A enhanced both survival and anchorage-independent growth of human mammary carcinoma cells but concordantly suppressed cell motility as revealed in colony scattering, cell migration, and invasion assays. In contrast, forced expression of CA STAT5B exhibited lower potency than CA STAT5A in enhancing survival and anchorage-independent growth of mammary carcinoma cells and exerted no effects on cell motility. Differential expression of genes that regulate cellular survival and motility was concomitantly observed on forced expression of CA STAT5A or CA STAT5B. Small interfering RNA-mediated depletion of STAT5A significantly impaired anchorage-independent growth of human mammary carcinoma cells, whereas a smaller reduction was observed upon small interfering RNA-mediated depletion of STAT5B. Depletion of endogenous STAT5A also significantly enhanced cell motility, whereas depletion of endogenous STAT5B exhibited no effect. Xenograft studies provided data concordant with the in vitro effects of the two STAT5 isoforms. We therefore demonstrate that STAT5A and STAT5B differentially regulate behavior of human mammary carcinoma cells.

[Inhibitory effect of bladder cancer related protein gene on HeLa cell proliferation].

BACKGROUND & OBJECTIVE: Bladder cancer related protein (BLCAP) gene was found downregulated most in primary cervical cancer tissues using oncogene/tumor suppressor gene microarray screening in our previous study, therefore this study was to explore the possible correlation between BLCAP gene and cervical cancer. METHODS: BLCAP expression was investigated in 54 cervical cancer and 25 normal tissues by reverse transcription polymerase chain reaction (RT-PCR). Full length of BLCAP cDNA was cloned into pLXSN expression vector and stably transfected into cervical cancer cell line HeLa cells. Cell proliferation, colony formation ability and apoptosis were determined by cell number counting, colony formation and DNA ladder assay. Nude mice were used to study the anti-tumor effect of BLCAP gene in vivo. RESULTS: BLCAP gene expression was significantly down-regulated or even not observed in human cervical cancer tissues compared to the normal ones. The cell doubling time of HeLa cells transfected with BLCAP was significantly elevated to 69.4 hrs (P<0.05), compared to that of the parental cells (27.5 hrs) or cells transfected with empty vectors (30.2 hrs). Moreover, in comparison with control cells, the colony formation efficiency of cells transfected with BLCAP gene was significantly lower (t=5.98, P<0.01). BLCAP expression also sensitized Hela cells to apoptosis induced by serum deprivation. In vivo, smaller size of tumors were formed in mice after the injection of cells transfected with BLCAP for 30 days compared to those injected with parental cells or cells transfected with empty vector (tumor wet weights were 1.015 g, 1.612 g, and 1.530 g, respectively, P<0.05). Furthermore, under pathological examinations, tumor tissues formed by cells transfected with BLCAP gene displayed less invasive potential with integral vascular fibrous capsules; muscle or adipocyte tissue invasion was not observed. In comparison, necropsy revealed that the tumors formed from the control cells were attached to the underlying muscles; histologically, the neoplastic cells were locally invasive and associated with fibrous connective tissues. These cells exhibited severer cytoplasmic and nuclear pleomorphism compared to cells transfected with BLCAP. CONCLUSION: BLCAP gene is down-regulated in cervical carcinoma tissues and could suppress tumorigenic ability and growth of HeLa cells, thus it may be a potential suppressor candidate gene of cervical carcinoma.

Functional analysis of bladder cancer-related protein gene: a putative cervical cancer tumor suppressor gene in cervical carcinoma.

Our previous study has suggested thatthe bladder cancer-associated protein gene (BLCAP) was among the differentially expressed genes in cervical cancer. We confirm here that BLCAP is expressed in all noncancerous cervical tissues (10/10), but it is greatly lost in primary cervical cancer tissue (31/39). In order to further investigate the functional roles of BLCAP, we stably transfected BLCAP cDNA into HeLa cells. The HeLa cells expressing BLCAP show reduced cell growth and clone genicity compared to the vector-transfected cognate cells. BLCAP expression in HeLa cells leads to growth arrest and significantly enhanced apoptosis in vitro and reduced tumor formation in vivo. Thus, BLCAP might be a potential tumor suppressor gene in cervical carcinoma.