Development of a peptide-based delivery platform for targeting malignant brain tumors.

Despite extensive molecular characterization, human glioblastoma remains a fatal disease with survival rates measured in months. Little improvement is seen with standard surgery, radiotherapy and chemotherapy. Clinical progress is hampered by the inability to detect and target glioblastoma disease reservoirs based on a diffuse invasive pattern and the presence of molecular and phenotypic heterogeneity. The goal of this study was to target the invasive and stem-like glioblastoma cells that evade first-line treatments using agents capable of delivering imaging enhancers or biotherapeutic cargo. To accomplish this, a combinatorial phage display library was biopanned against glioblastoma cell model systems that accurately recapitulate the intra- and inter-tumor heterogeneity and infiltrative nature of the disease. Candidate peptides were screened for specificity and ability to target glioblastoma cells in vivo. Cargo-conjugated peptides delivered contrast-enhancing agents to highly infiltrative tumor populations in intracranial xenograft models without the obvious need for blood brain barrier disruption. Simultaneous use of five independent targeting peptides provided greater coverage of this complex tumor and selected peptides have the capacity to deliver a therapeutic cargo (oncolytic virus VSVΔM51) to the tumor cells in vivo. Herein, we have identified a series of peptides with utility as an innovative platform to assist in targeting glioblastoma for the purpose of diagnostic or prognostic imaging, image-guided surgery, and/or improved delivery of therapeutic agents to glioblastoma cells implicated in disease relapse.

Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Elesclomol restores mitochondrial function in genetic models of copper deficiency.

Copper is an essential cofactor of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain. Inherited loss-of-function mutations in several genes encoding proteins required for copper delivery to CcO result in diminished CcO activity and severe pathologic conditions in affected infants. Copper supplementation restores CcO function in patient cells with mutations in two of these genes, COA6 and SCO2, suggesting a potential therapeutic approach. However, direct copper supplementation has not been therapeutically effective in human patients, underscoring the need to identify highly efficient copper transporting pharmacological agents. By using a candidate-based approach, we identified an investigational anticancer drug, elesclomol (ES), that rescues respiratory defects of COA6-deficient yeast cells by increasing mitochondrial copper content and restoring CcO activity. ES also rescues respiratory defects in other yeast mutants of copper metabolism, suggesting a broader applicability. Low nanomolar concentrations of ES reinstate copper-containing subunits of CcO in a zebrafish model of copper deficiency and in a series of copper-deficient mammalian cells, including those derived from a patient with SCO2 mutations. These findings reveal that ES can restore intracellular copper homeostasis by mimicking the function of missing transporters and chaperones of copper, and may have potential in treating human disorders of copper metabolism.

Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth.

DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg(+/-) offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg(-/-) mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg(+/-) and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg(-/-) animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg(-/-) animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg(-/-) mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

Urinary mitochondrial DNA is a biomarker of mitochondrial disruption and renal dysfunction in acute kidney injury.

Recent studies show the importance of mitochondrial dysfunction in the initiation and progression of acute kidney injury (AKI). However, no biomarkers exist linking renal injury to mitochondrial function and integrity. To this end, we evaluated urinary mitochondrial DNA (UmtDNA) as a biomarker of renal injury and function in humans with AKI following cardiac surgery. mtDNA was isolated from the urine of patients following cardiac surgery and quantified by quantitative PCR. Patients were stratified into no AKI, stable AKI, and progressive AKI groups based on Acute Kidney Injury Network (AKIN) staging. UmtDNA was elevated in progressive AKI patients and was associated with progression of patients with AKI at collection to higher AKIN stages. To evaluate the relationship of UmtDNA to measures of renal mitochondrial integrity in AKI, mice were subjected to sham surgery or varying degrees of ischemia followed by 24 h of reperfusion. UmtDNA increased in mice after 10-15 min of ischemia and positively correlated with ischemia time. Furthermore, UmtDNA was predictive of AKI in the mouse model. Finally, UmtDNA levels were negatively correlated with renal cortical mtDNA and mitochondrial gene expression. These translational studies demonstrate that UmtDNA is associated with recovery from AKI following cardiac surgery by serving as an indicator of mitochondrial integrity. Thus UmtDNA may serve as valuable biomarker for the development of mitochondrial-targeted therapies in AKI.

The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos.

The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.

Bioenergetic profiling of zebrafish embryonic development.

Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility.

Etiology of MNU-induced melanomas in Xiphophorus hybrids.

Genetic hybrids of the genus Xiphophorus have historically been useful models for study of the genetic aspects of tumor formation. In the most studied Xiphophorus tumor model, two-gene loci, XMRK and DIFF, are implicated as critical both to UV-induced and spontaneous melanoma formation in BC(1) hybrids of crosses between X. maculatus and X. helleri, with X. helleri as the recurrent backcross parent. In addition to UV, the direct-acting carcinogen N-methyl-N-nitrosourea (MNU) has been used to induce tumors in Xiphophorus BC(1) hybrids from several cross types. In the present study, we address the hypothesis that excess melanomas in MNU-treated BC(1) hybrids may have been generated by direct mutation of CDKN2AB, a candidate gene for DIFF. MNU treatment of F(1) and BC(1) hybrid fish significantly increased tumor incidence at 6 months; however, no association was found between MNU-induced tumor formation and zygosity of the candidate tumor tumor-suppressor CDKN2AB in BC(1) hybrids, consistent with previously reported results. Sequence analysis of the X. maculatus CDKN2AB locus of heterozygous individuals (both BC(1) and F(1) hybrids) did not reveal any mutations caused by MNU, suggesting that the mechanism of MNU-induced melanoma formation in this Xiphophorus model does not involve direct mutation of CDKN2AB but may result from mutation of other critical genes.

Gamma-secretase represents a therapeutic target for the treatment of invasive glioma mediated by the p75 neurotrophin receptor.

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target, suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.

MUC1 initiates Src-CrkL-Rac1/Cdc42-mediated actin cytoskeletal protrusive motility after ligating intercellular adhesion molecule-1.

MUC1, a transmembrane glycoprotein of the mucin family, when aberrantly expressed on breast cancer cells is correlated with increased lymph node metastases. We have previously shown that MUC1 binds intercellular adhesion molecule-1 (ICAM-1) on surrounding accessory cells and facilitates transendothelial migration of MUC1-bearing cells. Nevertheless, the underlying molecular mechanism is still obscure. In the present study, we used a novel assay of actin cytoskeletal reorganization to show that by ligating ICAM-1, MUC1 triggers Rac1- and Cdc42-dependent actin cytoskeletal protrusive activity preferentially at the heterotypic cell-cell contact sites. Further, we show that these MUC1/ICAM-1 interaction-initiated lamellipodial and filopodial protrusions require Src family kinase and CT10 regulator of kinase like (CrkL) accompanied by the rapid formation of a Src-CrkL signaling complex at the MUC1 cytoplasmic domain. Through inhibition of Src kinase activity, we further revealed that Src is required for recruiting CrkL to the MUC1 cytoplasmic domain as well as mediating the observed actin cytoskeleton dynamics. These findings suggest a novel MUC1-Src-CrkL-Rac1/Cdc42 signaling cascade following ICAM-1 ligation, through which MUC1 regulates cytoskeletal reorganization and directed cell motility during cell migration.

The p75 neurotrophin receptor is a central regulator of glioma invasion.

The invasive nature of cancers in general, and malignant gliomas in particular, is a major clinical problem rendering tumors incurable by conventional therapies. Using a novel invasive glioma mouse model established by serial in vivo selection, we identified the p75 neurotrophin receptor (p75(NTR)) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75(NTR) dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75(NTR)-mediated invasion was neurotrophin dependent, resulting in the activation of downstream pathways and producing striking cytoskeletal changes of the invading cells. These results provide the first evidence for p75(NTR) as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.

MUC1 mediates transendothelial migration in vitro by ligating endothelial cell ICAM-1.

MUC1 is a transmembrane glycoprotein expressed by normal breast epithelium and virtually all breast cancers. MUC1 is normally restricted to the apical surface of epithelia and loss of this polarized distribution in breast carcinomas is associated with lymph node metastasis. Our previous work found that MUC1 can bind intercellular adhesion molecule-1 (ICAM-1), mediating adhesion of breast cancer cells to a simulated blood vessel wall, and also triggering a calcium-based signal in the MUC1-bearing cells. It is possible that the depolarized membrane distribution of MUC1 in breast carcinomas may facilitate interactions with stromal/endothelial ICAM-1 leading to adhesion and subsequent migration through the vessel wall. In the current study, we provide evidence that ICAM-1 can influence the migration of cells that express endogenous or transfected MUC1. Migration across a gelatin-coated Transwell membrane could be increased in a step-wise manner by the sequential addition of ICAM-1-expressing cells (endothelial cells and fibroblasts), and ICAM-1-inducing inflammatory cytokines (tumour necrosis factor-alpha and interleukin-1 beta). Antibodies against MUC1 or ICAM-1, but not a control antibody, could abrogate migratory increases. Cells that did not express MUC1 were unresponsive to ICAM-1. Our current findings build on our earlier work, by suggesting that the end result of the MUC1/ICAM-1-mediated cell-cell adhesion and calcium-based signal is migration. This has implications for the exit of circulating tumour cells from the vasculature, as well as tumour cell migration through fibroblast-containing stroma underlying the endothelial wall.

Patterns of transcription of a virus-like agent in tumor and non-tumor tissues in bicolor damselfish.

Damselfish neurofibromatosis (DNF) is a transmissible disease characterized by peripheral nerve sheath and pigment cell tumors which occurs in bicolor damselfish (Stegastes partitus) on Florida reefs. The damselfish virus-like agent (DVLA) is associated with the development of DNF and contains a 2.4-kb DNA genome which was found at high levels in tumors and tumor-derived cell lines and at lower levels in non-tumor tissues of both spontaneously diseased fish (TF) and fish with experimentally induced tumors (EF). An analysis of transcription patterns revealed up to five DVLA derived RNAs ranging in size from 300 to 1400 bp in these cell types. DNA was the most commonly distributed DVLA component in TF and EF followed by RNA. Prevalence of transcripts varied by tissue type. The smallest transcripts were the most common in all cell types and the most complete patterns, which included the larger transcripts, were observed primarily in tumors. The presence of viral RNAs in addition to DNA in non-tumor tissues suggested these tissues were infected by DVLA and indicated a wide tissue tropism for this agent. The high levels of DVLA nucleic acids found in tumors suggest that replication is occurring there. However, the potential for DVLA replication in other tissues where only a limited range of transcripts were present is not known. The mechanism of tumorigenesis by this agent is unknown. However, the association of the larger transcripts with most tumor tissues and their absence in most non-tumor tissues suggests that these RNAs may be involved in tumor formation.

MUC1 initiates a calcium signal after ligation by intercellular adhesion molecule-1.

The MUC1 mucin is normally restricted to the apical surface of breast epithelial cells. In tumors, it is frequently overexpressed and underglycosylated. The MUC1 peptide core mediates firm adhesion of tumor cells to adjacent cells via binding to intercellular adhesion molecule-1 (ICAM-1). There is increasing evidence that MUC1 is involved in signaling, with current reports focusing on phosphorylation of the MUC1 cytoplasmic tail after indirect or artificial modes of stimulation. ICAM-1 is the only known direct ligand of the MUC1 extracellular domain. The data presented herein show that MUC1 expressed on the surface of breast cancer cell lines or transfected 293T cells can initiate a calcium-based oscillatory signal on contact with ICAM-1-transfected NIH 3T3 cells, and we present a novel method of quantifying and comparing calcium oscillations. The MUC1-induced signal appears to be distinct from those previously described, and may involve a Src family kinase, phosphoinositol 3-kinase, phospholipase C, and lipid rafts, but not mitogen-activated protein kinase. As calcium signaling has been associated with cytoskeletal change and motility, it is possible that the functions of MUC1 include heterotypic cell-cell adhesion followed by a calcium-based promigratory signal within tumor cells, thus facilitating metastasis.