Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Structural Analysis of Human Fascin-1: Essential Protein for Actin Filaments Bundling.

Fascin, a major actin cross-linking protein, is expressed in most vertebrate epithelial tissues. It organizes actin filaments into well-ordered bundles that are responsible for the extension of dynamic membrane protrusions, including microspikes, filopodia, and invadopodia from cell surfaces, which are involved in cell migration and invasion as critical components of cancer metastasis. However, it is not well-understood how fascin-1 induces actin binding/bundling and where fascin-1 localizes along the actin filaments, thus facilitating actin bundle formation. In the present study, we attempted to clarify these problems by using biochemical and electron microscopic analyses using various fascin-1 constructs. Three dimensional structures of actin/fascin-1 complex were obtained by electron microscopy (EM) with iterative helical real-space reconstruction (IHRSR) and tomography. We revealed that the N-terminal region containing the Actin-Binding Site 2 (ABS2) of fascin-1 is responsible for actin bundling and the C-terminal region is important for the dimerization of fascin-1. We also found that the dimerization of fascin-1 through intermolecular interactions of the C-terminal region is essential for actin bundling. Since fascin is an important factor in cancer development, it is expected that the findings of present study will provide useful information for development of therapeutic strategies for cancer.

Myo5b Transports Fibronectin-Containing Vesicles and Facilitates FN1 Secretion from Human Pleural Mesothelial Cells.

Pleural mesothelial cells (PMCs) play a central role in the progression of pleural fibrosis. As pleural injury progresses to fibrosis, PMCs transition to mesenchymal myofibroblast via mesothelial mesenchymal transition (MesoMT), and produce extracellular matrix (ECM) proteins including collagen and fibronectin (FN1). FN1 plays an important role in ECM maturation and facilitates ECM-myofibroblast interaction, thus facilitating fibrosis. However, the mechanism of FN1 secretion is poorly understood. We report here that myosin 5b (Myo5b) plays a critical role in the transportation and secretion of FN1 from human pleural mesothelial cells (HPMCs). TGF-ß significantly increased the expression and secretion of FN1 from HPMCs and facilitates the close association of Myo5B with FN1 and Rab11b. Moreover, Myo5b directly binds to GTP bound Rab11b (Rab11b-GTP) but not GDP bound Rab11b. Myo5b or Rab11b knockdown via siRNA significantly attenuated the secretion of FN1 without changing FN1 expression. TGF-ß also induced Rab11b-GTP formation, and Rab11b-GTP but not Rab11b-GDP significantly activated the actin-activated ATPase activity of Myo5B. Live cell imaging revealed that Myo5b- and FN1-containing vesicles continuously moved together in a single direction. These results support that Myo5b and Rab11b play an important role in FN1 transportation and secretion from HPMCs, and consequently may contribute to the development of pleural fibrosis.

Mitochondria-associated myosin 19 processively transports mitochondria on actin tracks in living cells.

Mitochondria are fundamentally important in cell function, and their malfunction can cause the development of cancer, cardiovascular disease, and neuronal disorders. Myosin 19 (Myo19) shows discrete localization with mitochondria and is thought to play an important role in mitochondrial dynamics and function; however, the function of Myo19 in mitochondrial dynamics at the cellular and molecular levels is poorly understood. Critical missing information is whether Myo19 is a processive motor that is suitable for transportation of mitochondria. Here, we show for the first time that single Myo19 molecules processively move on actin filaments and can transport mitochondria in cells. We demonstrate that Myo19 dimers having a leucine zipper processively moved on cellular actin tracks in demembraned cells with a velocity of 50 to 60 nm/s and a run length of ∼0.4 µm, similar to the movement of isolated mitochondria from Myo19 dimer-transfected cells on actin tracks, suggesting that the Myo19 dimer can transport mitochondria. Furthermore, we show single molecules of Myo19 dimers processively moved on single actin filaments with a large step size of ∼34 nm. Importantly, WT Myo19 single molecules without the leucine zipper processively move in filopodia in living cells similar to Myo19 dimers, whereas deletion of the tail domain abolished such active movement. These results suggest that Myo19 can processively move on actin filaments when two Myo19 monomers form a dimer, presumably as a result of tail-tail association. In conclusion, Myo19 molecules can directly transport mitochondria on actin tracks within living cells.

Caveolin-1-Derived Peptide Reduces ER Stress and Enhances Gelatinolytic Activity in IPF Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease characterized by an excess deposition of extracellular matrix in the pulmonary interstitium. Caveolin-1 scaffolding domain peptide (CSP) has been found to mitigate pulmonary fibrosis in several animal models. However, its pathophysiological role in IPF is obscure, and it remains critical to understand the mechanism by which CSP protects against pulmonary fibrosis. We first studied the delivery of CSP into cells and found that it is internalized and accumulated in the Endoplasmic Reticulum (ER). Furthermore, CSP reduced ER stress via suppression of inositol requiring enzyme1α (IRE1α) in transforming growth factor ß (TGFß)-treated human IPF lung fibroblasts (hIPF-Lfs). Moreover, we found that CSP enhanced the gelatinolytic activity of TGFß-treated hIPF-Lfs. The IRE1α inhibitor; 4µ8C also augmented the gelatinolytic activity of TGFß-treated hIPF-Lfs, supporting the concept that CSP induced inhibition of the IRE1α pathway. Furthermore, CSP significantly elevated expression of MMPs in TGFß-treated hIPF-Lfs, but conversely decreased the secretion of collagen 1. Similar results were observed in two preclinical murine models of PF, bleomycin (BLM)- and adenovirus expressing constitutively active TGFß (Ad-TGFß)-induced PF. Our findings provide new insights into the mechanism by which lung fibroblasts contribute to CSP dependent protection against lung fibrosis.

PD-L1 mediates lung fibroblast to myofibroblast transition through Smad3 and ß-catenin signaling pathways.

Programmed death ligand-1 (PD-L1) is an immune checkpoint protein that has been linked with idiopathic pulmonary fibrosis (IPF) and fibroblast to myofibroblast transition (FMT). However, it remains largely unclear how PD-L1 mediates this process. We found significantly increased PD-L1 in the lungs of idiopathic pulmonary fibrosis patients and mice with pulmonary fibrosis induced by bleomycin and TGF-ß. In primary human lung fibroblasts (HLFs), TGF-ß induced PD-L1 expression that is dependent on both Smad3 and p38 pathways. PD-L1 knockdown using siRNA significantly attenuated TGF-ß-induced expression of myofibroblast markers α-SMA, collagen-1, and fibronectin in normal and IPF HLFs. Further, we found that PD-L1 interacts with Smad3, and TGF-ß induces their interaction. Interestingly, PD-L1 knockdown reduced α-SMA reporter activity induced by TGF-ß in HLFs, suggesting that PD-L1 might act as a co-factor of Smad3 to promote target gene expression. TGF-ß treatment also phosphorylates GSK3ß and upregulates ß-catenin protein levels. Inhibiting ß-catenin signaling with the pharmaceutical inhibitor ICG001 significantly attenuated TGF-ß-induced FMT. PD-L1 knockdown also attenuated TGF-ß-induced GSK3ß phosphorylation/inhibition and ß-catenin upregulation, implicating GSK3ß/ß-catenin signaling in PD-L1-mediated FMT. Collectively, our findings demonstrate that fibroblast PD-L1 may promote pulmonary fibrosis through both Smad3 and ß-catenin signaling and may represent a novel interventional target for IPF.

DOCK2 Promotes Pleural Fibrosis by Modulating Mesothelial to Mesenchymal Transition.

Mesothelial to mesenchymal transition (MesoMT) is one of the crucial mechanisms underlying pleural fibrosis, which results in restrictive lung disease. DOCK2 (dedicator of cytokinesis 2) plays important roles in immune functions; however, its role in pleural fibrosis, particularly MesoMT, remains unknown. We found that amounts of DOCK2 and the MesoMT marker α-SMA (α-smooth muscle actin) were significantly elevated and colocalized in the thickened pleura of patients with nonspecific pleuritis, suggesting the involvement of DOCK2 in the pathogenesis of MesoMT and pleural fibrosis. Likewise, data from three different pleural fibrosis models (TGF-ß [transforming growth factor-ß], carbon black/bleomycin, and streptococcal empyema) consistently demonstrated DOCK2 upregulation and its colocalization with α-SMA in the pleura. In addition, induced DOCK2 colocalized with the mesothelial marker calretinin, implicating DOCK2 in the regulation of MesoMT. Our in vivo data also showed that DOCK2-knockout mice were protected from Streptococcus pneumoniae-induced pleural fibrosis, impaired lung compliance, and collagen deposition. To determine the involvement of DOCK2 in MesoMT, we treated primary human pleural mesothelial cells with the potent MesoMT inducer TGF-ß. TGF-ß significantly induced DOCK2 expression in a time-dependent manner, together with α-SMA, collagen 1, and fibronectin. Furthermore, DOCK2 knockdown significantly attenuated TGF-ß-induced α-SMA, collagen 1, and fibronectin expression, suggesting the importance of DOCK2 in TGF-ß-induced MesoMT. DOCK2 knockdown also inhibited TGF-ß-induced Snail upregulation, which may account for its role in regulating MesoMT. Taken together, the current study provides evidence that DOCK2 contributes to the pathogenesis of pleural fibrosis by mediating MesoMT and deposition of neomatrix and may represent a novel target for its prevention or treatment.

Myocardin Is Involved in Mesothelial-Mesenchymal Transition of Human Pleural Mesothelial Cells.

Pleural fibrosis is characterized by severe inflammation of the pleural space and pleural reorganization. Subsequent thickening of the visceral pleura contributes to lung stiffness and impaired lung function. Pleural mesothelial cells (PMCs) can become myofibroblasts via mesothelial-mesenchymal transition (MesoMT) and contribute to pleural organization, fibrosis, and rind formation. However, the mechanisms that underlie MesoMT remain unclear. Here, we investigated the role of myocardin in the induction of MesoMT. Transforming growth factor ß (TGF-ß) and thrombin induced MesoMT and markedly upregulated the expression of myocardin, but not myocardin-related transcription factor A (MRTF-A) or MRTF-B, in human PMCs (HPMCs). TGF-ß stimulation notably induced the nuclear translocation of myocardin in HPMCs, whereas nuclear translocation of MRTF-A and MRTF-B was not observed. Several genes under the control of myocardin were upregulated in cells undergoing MesoMT, an effect that was accompanied by a dramatic cytoskeletal reorganization of HPMCs consistent with a migratory phenotype. Myocardin gene silencing blocked TGF-ß- and thrombin-induced MesoMT. Although myocardin upregulation was blocked, MRTF-A and MRTF-B were unchanged. Myocardin, α-SMA, calponin, and smooth muscle myosin were notably upregulated in the thickened pleura of carbon black/bleomycin and empyema mouse models of fibrosing pleural injury. Similar results were observed in human nonspecific pleuritis. In a TGF-ß mouse model of pleural fibrosis, PMC-specific knockout of myocardin protected against decrements in lung function. Further, TGF-ß-induced pleural thickening was abolished by PMC-specific myocardin knockout, which was accompanied by a marked reduction of myocardin, calponin, and α-SMA expression compared with floxed-myocardin controls. These novel results show that myocardin participates in the development of MesoMT in HPMCs and contributes to the pathogenesis of pleural organization and fibrosis.

The Antiparallel Dimerization of Myosin X Imparts Bundle Selectivity for Processive Motility.

Myosin X is an unconventional actin-based molecular motor involved in filopodial formation, microtubule-actin filament interaction, and cell migration. Myosin X is an important component of filopodia regulation, localizing to tips of growing filopodia by an unclear targeting mechanism. The native α-helical dimerization domain of myosin X is thought to associate with antiparallel polarity of the two amino acid chains, making myosin X the only myosin that is currently considered to form antiparallel dimers. This study aims to determine if antiparallel dimerization of myosin X imparts selectivity toward actin bundles by comparing the motility of parallel and antiparallel dimers of myosin X on single and fascin-bundled actin filaments. Antiparallel myosin X dimers exhibit selective processivity on fascin-bundled actin and are only weakly processive on single actin filaments below saturating [ATP]. Artificial forced parallel dimers of myosin X are robustly processive on both single and bundled actin, exhibiting no selectivity. To determine the relationship between gating of the reaction steps and observed differences in motility, a mathematical model was developed to correlate the parameters of motility with the biochemical and mechanical kinetics of the dimer. Results from the model, constrained by experimental data, suggest that the probability of binding forward, toward the barbed end of the actin filament, is lower in antiparallel myosin X on single actin filaments compared to fascin-actin bundles and compared to constructs of myosin X with parallel dimerization.

Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca2+ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.

Myosin X is recruited to nascent focal adhesions at the leading edge and induces multi-cycle filopodial elongation.

Filopodia protrude from the leading edge of cells and play important roles in cell motility. Here we report the mechanism of myosin X (encoded by Myo10)-induced multi-cycle filopodia extension. We found that actin, Arp2/3, vinculin and integrin-ß first accumulated at the cell's leading edge. Myosin X was then gathered at these sites, gradually clustered by lateral movement, and subsequently initiated filopodia formation. During filopodia extension, we found the translocation of Arp2/3 and integrin-ß along filopodia. Arp2/3 and integrin-ß then became localized at the tip of filopodia, from where myosin X initiated the second extension of filopodia with a change in extension direction, thus producing long filopodia. Elimination of integrin-ß, Arp2/3 and vinculin by siRNA significantly attenuated the myosin-X-induced long filopodia formation. We propose the following mechanism. Myosin X accumulates at nascent focal adhesions at the cell's leading edge, where myosin X promotes actin convergence to create the base of filopodia. Then myosin X moves to the filopodia tip and attracts integrin-ß and Arp2/3 for further actin nucleation. The tip-located myosin X then initiates the second cycle of filopodia elongation to produce the long filopodia.

Inhibition of Glycogen Synthase Kinase 3ß Blocks Mesomesenchymal Transition and Attenuates Streptococcus pneumonia-Mediated Pleural Injury in Mice.

Pleural loculation affects about 30,000 patients annually in the United States and in severe cases can resolve with restrictive lung disease and pleural fibrosis. Pleural mesothelial cells contribute to pleural rind formation by undergoing mesothelial mesenchymal transition (MesoMT), whereby they acquire a profibrotic phenotype characterized by increased expression of α-smooth muscle actin and collagen 1. Components of the fibrinolytic pathway (urokinase plasminogen activator and plasmin) are elaborated in pleural injury and strongly induce MesoMT in vitro. These same stimuli enhance glycogen synthase kinase (GSK)-3ß activity through increased phosphorylation of Tyr-216 in pleural mesothelial cells and GSK-3ß mobilization from the cytoplasm to the nucleus. GSK-3ß down-regulation blocked induction of MesoMT. Likewise, GSK-3ß inhibitor 9ING41 blocked induction of MesoMT and reversed established MesoMT. Similar results were demonstrated in a mouse model of Streptococcus pneumoniae-induced empyema. Intraperitoneal administration of 9ING41, after the induction of pleural injury, attenuated injury progression and improved lung function (lung volume and compliance; P < 0.05 compared with untreated and vehicle controls). MesoMT marker α-smooth muscle actin was reduced in 9ING41-treated mice. Pleural thickening was also notably reduced in 9ING41-treated mice (P < 0.05). Collectively, these studies identify GSK-3ß as a newly identified target for amelioration of empyema-related pleural fibrosis and provide a strong rationale for further investigation of GSK-3ß signaling in the control of MesoMT and pleural injury.

Human myosin VIIa is a very slow processive motor protein on various cellular actin structures.

Human myosin VIIa (MYO7A) is an actin-linked motor protein associated with human Usher syndrome (USH) type 1B, which causes human congenital hearing and visual loss. Although it has been thought that the role of human myosin VIIa is critical for USH1 protein tethering with actin and transportation along actin bundles in inner-ear hair cells, myosin VIIa's motor function remains unclear. Here, we studied the motor function of the tail-truncated human myosin VIIa dimer (HM7AΔTail/LZ) at the single-molecule level. We found that the HM7AΔTail/LZ moves processively on single actin filaments with a step size of 35 nm. Dwell-time distribution analysis indicated an average waiting time of 3.4 s, yielding ∼0.3 s-1 for the mechanical turnover rate; hence, the velocity of HM7AΔTail/LZ was extremely slow, at 11 nm·s-1 We also examined HM7AΔTail/LZ movement on various actin structures in demembranated cells. HM7AΔTail/LZ showed unidirectional movement on actin structures at cell edges, such as lamellipodia and filopodia. However, HM7AΔTail/LZ frequently missed steps on actin tracks and exhibited bidirectional movement at stress fibers, which was not observed with tail-truncated myosin Va. These results suggest that the movement of the human myosin VIIa motor protein is more efficient on lamellipodial and filopodial actin tracks than on stress fibers, which are composed of actin filaments with different polarity, and that the actin structures influence the characteristics of cargo transportation by human myosin VIIa. In conclusion, myosin VIIa movement appears to be suitable for translocating USH1 proteins on stereocilia actin bundles in inner-ear hair cells.

Activated full-length myosin-X moves processively on filopodia with large steps toward diverse two-dimensional directions.

Myosin-X, (Myo 10), is an unconventional myosin that transports the specific cargos to filopodial tips, and is associated with the mechanism underlying filopodia formation and extension. To clarify the innate motor characteristic, we studied the single molecule movement of a full-length myosin-X construct with leucine zipper at the C-terminal end of the tail (M10FullLZ) and the tail-truncated myosin-X without artificial dimerization motif (BAP-M101-979HMM). M10FullLZ localizes at the tip of filopodia like myosin-X full-length (M10Full). M10FullLZ moves on actin filaments in the presence of PI(3,4,5)P3, an activator of myosin-X. Single molecule motility analysis revealed that the step sizes of both M10FullLZ and BAP-M101-979HMM are widely distributed on single actin filaments that is consistent with electron microscopy observation. M10FullLZ moves on filopodial actin bundles of cells with a mean step size (~36 nm), similar to the step size on single actin filaments (~38 nm). Cartesian plot analysis revealed that M10FullLZ meandered on filopodial actin bundles to both x- and y- directions. These results suggest that the lever-arm of full-length myosin-X is flexible enough to processively steps on different actin filaments within the actin bundles of filopodia. This characteristic of myosin-X may facilitate actin filament convergence for filopodia production.

NK-CD11c+ Cell Crosstalk in Diabetes Enhances IL-6-Mediated Inflammation during Mycobacterium tuberculosis Infection.

In this study, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and identified factors that increase susceptibility of T2DM mice to infection by Mycobacterium tuberculosis (Mtb). All Mtb-infected T2DM mice and 40% of uninfected T2DM mice died within 10 months, whereas all control mice survived. In Mtb-infected mice, T2DM increased the bacterial burden and pro- and anti-inflammatory cytokine and chemokine production in the lungs relative to those in uninfected T2DM mice and infected control mice. Levels of IL-6 also increased. Anti-IL-6 monoclonal antibody treatment of Mtb-infected acute- and chronic-T2DM mice increased survival (to 100%) and reduced pro- and anti-inflammatory cytokine expression. CD11c+ cells were the major source of IL-6 in Mtb-infected T2DM mice. Pulmonary natural killer (NK) cells in Mtb-infected T2DM mice further increased IL-6 production by autologous CD11c+ cells through their activating receptors. Anti-NK1.1 antibody treatment of Mtb-infected acute-T2DM mice increased survival and reduced pro- and anti-inflammatory cytokine expression. Furthermore, IL-6 increased inflammatory cytokine production by T lymphocytes in pulmonary tuberculosis patients with T2DM. Overall, the results suggest that NK-CD11c+ cell interactions increase IL-6 production, which in turn drives the pathological immune response and mortality associated with Mtb infection in diabetic mice.

Exosomes in Human Breast Milk Promote EMT.

PURPOSE: Pregnancy increases breast cancer risk for all women for at least 5 years after parturition. During weaning and involution, the breast microenvironment becomes tumor promotional. Exosomes provide cell-cell communication during physiologic processes such as lactation, but also in breast cancer. We determined whether molecules in milk exosomes from healthy lactating women modulate the development and progression of breast cancer. EXPERIMENTAL DESIGN: Thirteen nursing women provided three (transitional, mature, and wean) milk samples. Exosomes were extracted and MCF7 and MCF10A breast cells labeled. The expression of six proteins linked to breast cancer was measured. On the basis of the findings, TGFß2 concentration in exosome samples was measured, breast cells incubated with the exosomes and effect (epithelial-mesenchymal transition, EMT) on EMT-related proteins [E-cadherin, α-smooth muscle actin (α-SMA), filamentous (F)-actin and vimentin] measured. RESULTS: Human milk exosomes entered benign and malignant breast cells. The greatest change in wean milk protein was in TGFß2 (P = 0.01). Exosomes with a high (but not low) level of TGFß2 led to EMT in both cancer and benign cells, based on (i) change in cell morphology, actin cytoskeleton, and loss of cell-cell junction structure and (ii) increased α-SMA and vimentin and decreased E-cadherin. CONCLUSIONS: TGFß2 is significantly upregulated in breast milk exosomes during weaning/early involution. Breast milk exosomes containing high levels of TGFß2 induce changes in both benign and malignant breast epithelial cells, consistent with the development and progression of breast cancer, suggesting a role for high TGFß2-expressing breast milk exosomes in influencing breast cancer risk. Clin Cancer Res; 22(17); 4517-24. ©2016 AACR.

Mitochondrial Reprogramming Regulates Breast Cancer Progression.

PURPOSE: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers. EXPERIMENTAL DESIGN: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development. RESULTS: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis-associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors. CONCLUSIONS: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348-60. ©2016 AACR.

p53- and PAI-1-mediated induction of C-X-C chemokines and CXCR2: importance in pulmonary inflammation due to cigarette smoke exposure.

We previously demonstrated that tumor suppressor protein p53 augments plasminogen activator inhibitor-1 (PAI-1) expression in alveolar epithelial cells (AECs) during chronic cigarette smoke (CS) exposure-induced lung injury. Chronic lung inflammation with elevated p53 and PAI-1 expression in AECs and increased susceptibility to and exacerbation of respiratory infections are all associated with chronic obstructive pulmonary disease (COPD). We recently demonstrated that preventing p53 from binding to the endogenous PAI-1 mRNA in AECs by either suppressing p53 expression or blockading p53 interactions with the PAI-1 mRNA mitigates apoptosis and lung injury. Within this context, we now show increased expression of the C-X-C chemokines (CXCL1 and CXCL2) and their receptor CXCR2, and the intercellular cellular adhesion molecule-1 (ICAM-1), in the lung tissues of patients with COPD. We also found a similar increase in lung tissues and AECs from wild-type (WT) mice exposed to passive CS for 20 wk and in primary AECs treated with CS extract in vitro. Interestingly, passive CS exposure of mice lacking either p53 or PAI-1 expression resisted an increase in CXCL1, CXCL2, CXCR2, and ICAM-1. Furthermore, inhibition of p53-mediated induction of PAI-1 expression by treatment of WT mice exposed to passive CS with caveolin-1 scaffolding domain peptide reduced CXCL1, CXCL2, and CXCR2 levels and lung inflammation. Our study reveals that p53-mediated induction of PAI-1 expression due to chronic CS exposure exacerbates lung inflammation through elaboration of CXCL1, CXCL2, and CXCR2. We further provide evidence that targeting this pathway mitigates lung injury associated with chronic CS exposure.

Complex-I Alteration and Enhanced Mitochondrial Fusion Are Associated With Prostate Cancer Progression.

Mitochondria (mt) encoded respiratory complex-I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi-myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA, and protein in sera exosomes was determined. A co-culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co-localization of exosomes and mt. We observed frequent RCI mutations in human and Hi-myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1, and decreased expression of MT-TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi-myc tumors. RCI-mtDNA, MFN2, and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co-localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development.

Structure and Regulation of the Movement of Human Myosin VIIA.

Human myosin VIIA (HM7A) is responsible for human Usher syndrome type 1B, which causes hearing and visual loss in humans. Here we studied the regulation of HM7A. The actin-activated ATPase activity of full-length HM7A (HM7AFull) was lower than that of tail-truncated HM7A (HM7AΔTail). Deletion of the C-terminal 40 amino acids and mutation of the basic residues in this region (R2176A or K2179A) abolished the inhibition. Electron microscopy revealed that HM7AFull is a monomer in which the tail domain bends back toward the head-neck domain to form a compact structure. This compact structure is extended at high ionic strength or in the presence of Ca(2+). Although myosin VIIA has five isoleucine-glutamine (IQ) motifs, the neck length seems to be shorter than the expected length of five bound calmodulins. Supporting this observation, the IQ domain bound only three calmodulins in Ca(2+), and the first IQ motif failed to bind calmodulin in EGTA. These results suggest that the unique IQ domain of HM7A is important for the tail-neck interaction and, therefore, regulation. Cellular studies revealed that dimer formation of HM7A is critical for its translocation to filopodial tips and that the tail domain (HM7ATail) markedly reduced the filopodial tip localization of the HM7AΔTail dimer, suggesting that the tail-inhibition mechanism is operating in vivo. The translocation of the HM7AFull dimer was significantly less than that of the HM7AΔTail dimer, and R2176A/R2179A mutation rescued the filopodial tip translocation. These results suggest that HM7A can transport its cargo molecules, such as USH1 proteins, upon release of the tail-dependent inhibition.