Pilot study of frozen platelet extracellular vesicles as a therapeutic agent in hemorrhagic shock in rats.

BACKGROUND: Hemorrhage accounts for the most preventable deaths after trauma. Resuscitation is guided by studies that demonstrate improved outcomes in patients receiving whole blood or balanced administration of blood products. Platelets present a logistical challenge due to short shelf life and need for refrigeration. Platelet-derived extracellular vesicles (PEVs) are a possible platelet alternative. Platelet-derived extracellular vesicles are secreted from platelets, have hemostatic effects and mitigate inflammation and vascular injury, similar to platelets. This pilot study aimed to elucidate the therapeutic effects of PEVs in a rat model of uncontrolled hemorrhage. METHODS: Male rats were anesthetized and femoral vessels cannulated. Vital signs (MAP, HR, and RR) were monitored. Electrolytes, lactate and ABG were obtained at baseline, 1-hour and 3-hours post injury. Laparotomy was performed, 50% of the middle hepatic lobe excised and the abdomen packed with gauze. Rats received 2 mL PEVs or lactated Ringers (LR) over 6 minutes immediately after injury. Peritoneal blood loss was quantified using preweighed gauze at 5 minutes, 15 minutes, 30 minutes, 45 minutes, and 60 minutes. Laparotomy was closed 1-hour postinjury. Animals were monitored for 3 hours postinjury then euthanized. Generalized Linear Mixed Effects models were performed to assess effects of treatment and time on lactate and MAP. RESULTS: Twenty-one rats were included (11 LR, 10 PEV). Overall blood loss was between 6 mL and 10 mL and not significantly different between groups. There was a 36% mortality rate in the LR group and 0% mortality in the PEV group ( p = 0.03). The LR group had significantly higher lactates at 1 hour ( p = 0.025). At 15 minutes, 45 minutes, 60 minutes, and 180 minutes, the MAP of the PEV group was significantly higher than the LR group. CONCLUSION: Early studies are encouraging regarding the potential use of PEVs in uncontrolled hemorrhagic shock based on improved survival and hemodynamics.

A multi-omic analysis of MCF10A cells provides a resource for integrative assessment of ligand-mediated molecular and phenotypic responses.

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix proteins. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods ( synapse.org/LINCS_MCF10A ). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes. Beyond these analyses, this dataset will serve as a resource for the broader scientific community to mine for biological insights, to compare signals carried across distinct molecular modalities, and to develop new computational methods for integrative data analysis.

INHBA is a mediator of aggressive tumor behavior in HER2+ basal breast cancer.

BACKGROUND: Resistance to HER2-targeted therapeutics remains a significant clinical problem in HER2+ breast cancer patients with advanced disease. This may be particularly true for HER2+ patients with basal subtype disease, as recent evidence suggests they receive limited benefit from standard of care HER2-targeted therapies. Identification of drivers of resistance and aggressive disease that can be targeted clinically has the potential to impact patient outcomes. METHODS: We performed siRNA knockdown screens of genes differentially expressed between lapatinib-responsive and -resistant HER2+ breast cancer cells, which corresponded largely to luminal versus basal subtypes. We then validated hits in 2-d and 3-d cell culture systems. RESULTS: Knockdown of one of the genes, INHBA, significantly slowed growth and increased sensitivity to lapatinib in multiple basal HER2+ cell lines in both 2-d and 3-d cultures, but had no effect in luminal HER2+ cells. Loss of INHBA altered metabolism, eliciting a shift from glycolytic to oxidative phosphorylative metabolism, which was also associated with a decrease in tumor invasiveness. Analysis of breast cancer datasets showed that patients with HER2+ breast cancer and high levels of INHBA expression had worse outcomes than patients with low levels of INHBA expression. CONCLUSIONS: Our data suggest that INHBA is associated with aggressiveness of the basal subtype of HER2+ tumors, resulting in poor response to HER2-targeted therapy and an invasive phenotype. We hypothesize that targeting this pathway could be an effective therapeutic strategy to reduce invasiveness of tumor cells and to improve therapeutic response.

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.

Enzalutamide response in a panel of prostate cancer cell lines reveals a role for glucocorticoid receptor in enzalutamide resistant disease.

Representative in vitro model systems that accurately model response to therapy and allow the identification of new targets are important for improving our treatment of prostate cancer. Here we describe molecular characterization and drug testing in a panel of 20 prostate cancer cell lines. The cell lines cluster into distinct subsets based on RNA expression, which is largely driven by functional Androgen Receptor (AR) expression. KLK3, the AR-responsive gene that encodes prostate specific antigen, shows the greatest variability in expression across the cell line panel. Other common prostate cancer associated genes such as TMPRSS2 and ERG show similar expression patterns. Copy number analysis demonstrates that many of the most commonly gained (including regions containing TERC and MYC) and lost regions (including regions containing TP53 and PTEN) that were identified in patient samples by the TCGA are mirrored in the prostate cancer cell lines. Assessment of response to the anti-androgen enzalutamide shows a distinct separation of responders and non-responders, predominantly related to status of wild-type AR. Surprisingly, several AR-null lines responded to enzalutamide. These AR-null, enzalutamide-responsive cells were characterized by high levels of expression of glucocorticoid receptor (GR) encoded by NR3C1. Treatment of these cells with the anti-GR agent mifepristone showed that they were more sensitive to this drug than enzalutamide, as were several of the enzalutamide non-responsive lines. This is consistent with several recent reports that suggest that GR expression is an alternative signaling mechanism that can bypass AR blockade. This study reinforces the utility of large cell line panels for the study of cancer and identifies several cell lines that represent ideal models to study AR-null cells that have upregulated GR to sustain growth.

Microenvironment-Mediated Mechanisms of Resistance to HER2 Inhibitors Differ between HER2+ Breast Cancer Subtypes.

Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ∼2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-ß1 (NRG1ß), a ligand for HER3, induced resistance that could be reversed with pertuzumab, an inhibitor of HER2-HER3 heterodimerization. The subtype-specific responses were also observed in 3D cultures and murine xenografts. These results, along with bioinformatic pathway analysis and siRNA knockdown experiments, suggest different mechanisms of resistance specific to each HER2+ subtype: MET signaling for HER2E and HER2-HER3 heterodimerization for L-HER2+ cells.

Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution.

Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.

Two ABC transporter systems participate in siderophore transport in the marine pathogen Vibrio anguillarum 775 (pJM1).

ORF40 (named fatE) in the Vibrio anguillarum pJM1 plasmid-encoding anguibactin iron transport systems is a homolog of ATPase genes involved in ferric-siderophore transport. Mutation of fatE did not affect ferric-anguibactin transport, indicating that there must be other ATPase gene(s) in addition to fatE. By searching the genomic sequence of V. anguillarum 775(pJM1), we identified a homolog of fatE named fvtE on chromosome 2. It is of interest that in this locus, we also identified homologs of fatB, fatC, and fatD that we named fvtB, fvtC and fvtD, respectively. The fvtE mutant still showed ferric-anguibactin transport, while the double fatE and fvtE mutation completely abolished the ferric-anguibactin transport indicating that fatE and fvtE are functional ATPase homologs for ferric-anguibactin transport. Furthermore, we demonstrate that fvtB, fvtC, fvtD, and fvtE are essential for ferric-vanchrobactin and ferric-enterobactin transport.

The regulator HlyU, the repeat-in-toxin gene rtxA1, and their roles in the pathogenesis of Vibrio vulnificus infections.

HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.

Homodimerization and binding of specific domains to the target DNA are essential requirements for HlyU to regulate expression of the virulence gene rtxA1, encoding the repeat-in-toxin protein in the human pathogen Vibrio vulnificus.

The virulence gene rtxA1, encoding the repeat-in-toxin protein, plays an essential role in the pathogenesis of Vibrio vulnificus infections. Expression of this gene is controlled by the HlyU regulator by direct contact of the DNA upstream of the rtxA1 toxin operon acting as a derepressor of the H-NS protein. The crystal structure suggests that HlyU forms a homodimer in vitro. However, knowledge of the biological implications of these findings in vivo is limited. In this work, we endeavored to dissect, using genetic and biochemical approaches, the domains of this protein that are essential for homodimer formation and the interaction of HlyU with the target DNA. We identified that residues L18, N22, R25, S54, Q55, L57, W59, R61, K70, and Y77 are essential for the HlyU protein binding to the DNA and that amino acids L17 and L91 are important for HlyU dimerization. We also determined that HlyU homodimer formation is an essential requirement for binding to the upstream region of the rtxA1 operon and is the key feature in relieving the H-NS repression of rtxA1 transcription.

HlyU acts as an H-NS antirepressor in the regulation of the RTX toxin gene essential for the virulence of the human pathogen Vibrio vulnificus CMCP6.

In Vibrio vulnificus, HlyU upregulates the expression of the large RTX toxin gene. In this work we identified the binding site of HlyU to -417 to -376 bp of the rtxA1 operon transcription start site. lacZ fusions for a series of progressive deletions from the rtxA1 operon promoter showed that transcriptional activity increased independently of HlyU when its binding site was absent. Thus HlyU must regulate the rtxA1 operon expression by antagonizing a negative regulator. Concomitantly we found that an hns mutant resulted in an increase in the expression of the rtxA1 operon genes. Multiple copies of HlyU can increase the promoter activity only in the presence of H-NS underscoring the hypothesis that HlyU must alleviate the repression by this protein. H-NS binds to a region that extends upstream and downstream of the rtxA1 operon promoter. In the upstream region it binds to five AT-rich sites of which two overlap the HlyU binding site. Competitive footprinting and gel shift data demonstrate HlyU's higher affinity as compared with H-NS resulting in the de-repression and a corresponding increased expression of the rtxA1 operon.

The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus.

Vibrio vulnificus is an opportunistic human pathogen that preferentially infects compromised iron-overloaded patients, causing a fatal primary septicemia with very rapid progress, resulting in a high mortality rate. In this study we determined that the HlyU protein, a virulence factor in V. vulnificus CMCP6, up-regulates the expression of VV20479, a homologue of the Vibrio cholerae RTX (repeats in toxin) toxin gene that we named rtxA1. This gene is part of an operon together with two other open reading frames, VV20481 and VV20480, that encode two predicted proteins, a peptide chain release factor 1 and a hemolysin acyltransferase, respectively. A mutation in rtxA1 not only contributes to the loss of cytotoxic activity but also results in a decrease in virulence, whereas a deletion of VV20481 and VV20480 causes a slight decrease in virulence but with no effect in cytotoxicity. Activation of the expression of the rtxA1 operon by HlyU occurs at the transcription initiation level by binding of the HlyU protein to a region upstream of this operon.

Construction, detection and microarray analysis on Shigella dysenteriae a1 IroN, ShuA single, double mutants.

In this study, we constructed single mutants MTS-1, MTS-2 of IroN and ShuA gene and double mutant MTS of them in Shigella dysenteriae A1 strain 51197 by insert and absence. The functional detection of every mutant was performed at the level of culture medium and cell experiment. The gene expression profiles of the mutants and the wild-type strains under iron-enriched and iron-limited conditions were analyzed by the SD51197 whole genomic microarray. The results showed that all the mutants grew obviously less well than the wild-type strains in L broth appending iron chelator DIP. The addition of iron to the cultures can stimulate the growth of mutants back to wild-type levels. In either the experiments on the ability of intracellular multiplication or the cell-to-cell spread in HeLa and U937 cell lines, mutants showed no obvious change in virulence compared with the parental strain SD51197. However when DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS-1, MTS-2, MTS has reduced about 23.4%, 25.2%, 43.6% respectively. The analysis of expression profiles under the iron-limited condition showed that the mutants were more sensitive for the changes of iron deficiency than the wild-type strains, many genes have been altered. Up-regulated genes mainly involved genes of transcription, coenzyme metabolism, amino acid transport and metabolism, and unknown functional genes, while down-regulated genes mainly involved genes of energy and carbohydrate metabolism and unknown function genes; the expression levels of known iron-transport associated genes generally showed up-regulated. The results demonstrated that iron-transport associated genes IroN, ShuA were likely to have some effects on the virulence and growth of S. dysenteriae.

Construction, detection and microarray analysis on the Shigella flexneri 2a sitC mutant.

In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442, Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study. With this improved knockout system, we inactivated sitC gene, which is associated with iron transport in Shigella flexneri 2a strain 301, to yield the mutant, MTS. The functional detection of the mutant was performed at the level of culture medium, cell and animal experiment, respectively. The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions. The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150 micromol/L iron chelator DIP (2,2'-dipyridyl). Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium. In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines, or the experiment on keratoconjunctivitis in guinea pigs, MTS showed no obvious changes in virulence compared with the parental strain Sf301. When 65 micromol/L DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS reduced about 51.6% as compared with that of Sf301. The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301. There are 106 more up-regulated genes in MTS than in wild-type strains, which are involved in membrane transportation, amino acid metabolism and uncategorized function genes, while down-regulated genes are mainly involved in energy and carbohydrate metabolism. Under low iron conditions, the expression levels of known iron-transport associated genes generally increased. Additionally, the number of these genes and their increase amplitude in MTS are more than those in Sf301. Together, these results confirmed that Sit iron-transport system is important for the growth of Shigella.