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 (p75NTR) as a critical regulator of glioma invasion. Through a series of functional, biochemical, and clinical studies, we found that p75NTR dramatically enhanced migration and invasion of genetically distinct glioma and frequently exhibited robust expression in highly invasive glioblastoma patient specimens. Moreover, we found that p75NTR-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 p75NTR as a major contributor to the highly invasive nature of malignant gliomas and identify a novel therapeutic target.

Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx₉CxnCx₁₀C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx₉CxnCx₁₀C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient.

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.

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.

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 qPCR. 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 hours of reperfusion. UmtDNA increased in mice after 10-15 minutes 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.

Highlights d Dipeptidase 1 (DPEP1) serves as a vascular adhesion molecule in lungs and liver d DPEP1 acts as a physical adhesion receptor independent of its dipeptidase activity d Targeting DPEP1 reduces mortality in murine models of sepsis

The multifunctional signaling protein p75 neurotrophin receptor (p75NTR) 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 p75NTR is required for p75NTR-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75NTR or treatment of animals bearing p75NTR-positive intracranial tumors with clinically applicable γ-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75NTR was observed in p75NTR-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75NTR as a therapeutic target, suggesting that γ-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.

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 mitogenactivated 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.The MUC1 mucin has been well established as a tumor marker in breast cancer and is implicated in metastatic spread (1). Phosphorylation of the MUC1 cytoplasmic tail (2-4) allows MUC1 to associate with potential oncogenes (5-8). MUC1 can also be indirectly stimulated to initiate signaling cascades, through epidermal growth factor receptor (EGFR) 1 (9, 10) or antibodies directed against CD8-MUC1 chimeras (11, 12), resulting in activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) pathway. Our laboratory has previously identified intercellular adhesion molecule-1 (ICAM-1) as a natural, endogenous ligand for MUC1 (13,14). ICAM-1 is currently the only reported direct ligand for the MUC1 extracellular domain, and binding promotes adhesion of tumor cells to a simulated vessel wall construct under fluid flow conditions (15). In this report, we demonstrate a novel signaling paradigm, in which MUC1 participates in initiating intracellular calcium oscillations after direct stimulation by ICAM-1. As calcium signaling has previously been implicated in cytoskeletal remodeling and motility (16), we hypothesize that this signal is involved in tumor cell migration. Thus, the MUC1/ICAM-1 interaction may facilitate extravasation of tumor cells after mediating binding to the blood vessel wall. EXPERIMENTAL PROCEDURESReagents-The CT2 antibody against the MUC1 cytoplasmic domain and the pC1Neo TRϩ FLAG plasmid carrying the MUC1 gene were generously provided by Dr. Sandra Gendler, Mayo Clinic, Scottsdale, AZ. The MUC1 gene was PCR amplified from the plasmid and inserted into the Clontech pEYFP-N1 plasmid at the BsrGI/NotI cut sites. A synthetic MUC1-specific signal sequence, TCGACTAG...