Publications by authors named "Dario C Altieri"

153 Publications

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

Small Extracellular Vesicle Regulation of Mitochondrial Dynamics Reprograms a Hypoxic Tumor Microenvironment.

Dev Cell 2020 10 10;55(2):163-177.e6. Epub 2020 Aug 10.

Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, PA 19104, USA; Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that breast cancer cells maintained in hypoxia release small extracellular vesicles (sEVs) that activate mitochondrial dynamics, stimulate mitochondrial movements, and promote organelle accumulation at the cortical cytoskeleton in normal mammary epithelial cells. This results in AKT serine/threonine kinase (Akt) activation, membrane focal adhesion turnover, and increased epithelial cell migration. RNA sequencing profiling identified integrin-linked kinase (ILK) as the most upregulated pathway in sEV-treated epithelial cells, and genetic or pharmacologic targeting of ILK reversed mitochondrial reprogramming and suppressed sEV-induced cell movements. In a three-dimensional (3D) model of mammary gland morphogenesis, sEV treatment induced hallmarks of malignant transformation, with deregulated cell death and/or cell proliferation, loss of apical-basal polarity, and appearance of epithelial-to-mesenchymal transition (EMT) markers. Therefore, sEVs released by hypoxic breast cancer cells reprogram mitochondrial dynamics and induce oncogenic changes in a normal mammary epithelium.
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http://dx.doi.org/10.1016/j.devcel.2020.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606608PMC
October 2020

Interplay Between V-ATPase G1 and Small EV-miRNAs Modulates ERK1/2 Activation in GBM Stem Cells and Nonneoplastic Milieu.

Mol Cancer Res 2020 11 4;18(11):1744-1754. Epub 2020 Aug 4.

Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem cells (GSC) maintenance and tumorigenicity. Moreover, V-ATPase reprograms the tumor microenvironment through acidification and release of extracellular vesicles (EV). Therefore, we investigated the role of V1G1 in GSC small EVs and their effects on primary brain cultures. To this end, small EVs were isolated from patients-derived GSCs grown as neurospheres (NS) with high (V1G1-NS) or low (V1G1-NS) V1G1 expression and analyzed for V-ATPase subunits presence, miRNA contents, and cellular responses in recipient cultures. Our results show that NS-derived small EVs stimulate proliferation and motility of recipient cells, with small EV derived from V1G1-NS showing the most pronounced activity. This involved activation of ERK1/2 signaling, in a response reversed by V-ATPase inhibition in NS-producing small EV. The miRNA profile of V1G1-NS-derived small EVs differed significantly from that of V1G1-NS, which included miRNAs predicted to target MAPK/ERK signaling. Mechanistically, forced expression of a MAPK-targeting pool of miRNAs in recipient cells suppressed MAPK/ERK pathway activation and blunted the prooncogenic effects of V1G1 small EV. These findings propose that the GSC influences the brain milieu through a V1G1-coordinated EVs release of MAPK/ERK-targeting miRNAs. Interfering with V-ATPase activity could prevent ERK-dependent oncogenic reprogramming of the microenvironment, potentially hampering local GBM infiltration. IMPLICATIONS: Our data identify a novel molecular mechanism of gliomagenesis specific of the GBM stem cell niche, which coordinates a V-ATPase-dependent reprogramming of the brain microenvironment through the release of specialized EVs.
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http://dx.doi.org/10.1158/1541-7786.MCR-20-0078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642188PMC
November 2020

The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells.

Sci Signal 2020 07 28;13(642). Epub 2020 Jul 28.

Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, PA 19104, USA.

Mitochondria are signaling hubs in eukaryotic cells. Here, we showed that the mitochondrial FUN14 domain-containing protein-1 (FUNDC1), an effector of Parkin-independent mitophagy, also participates in cellular plasticity by sustaining oxidative bioenergetics, buffering ROS production, and supporting cell proliferation. Targeting this pathway in cancer cells suppressed tumor growth but rendered transformed cells more motile and invasive in a manner dependent on ROS-mediated mitochondrial dynamics and mitochondrial repositioning to the cortical cytoskeleton. Global metabolomics and proteomics profiling identified a FUNDC1 interactome at the mitochondrial inner membrane, comprising the AAA+ protease, LonP1, and subunits of oxidative phosphorylation, complex V (ATP synthase). Independently of its previously identified role in mitophagy, FUNDC1 enabled LonP1 proteostasis, which in turn preserved complex V function and decreased ROS generation. Therefore, mitochondrial reprogramming by a FUNDC1-LonP1 axis controls tumor cell plasticity by switching between proliferative and invasive states in cancer.
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http://dx.doi.org/10.1126/scisignal.aaz8240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484983PMC
July 2020

The αvβ6 integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis.

J Extracell Vesicles 2020 20;9(1):1763594. Epub 2020 May 20.

Prostate Cancer Discovery and Development Program, Thomas Jefferson University, Philadelphia, USA.

Prostate cancer (PrCa) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sEVs). sEVs, as well as large extracellular vesicles (LEVs), isolated via iodixanol density gradients from PrCa cell culture media, express the epithelial-specific αvβ6 integrin, which is known to be induced in cancer. In this study, we show sEV-mediated protein transfer of αvβ6 integrin to microvascular endothelial cells (human microvascular endothelial cells 1 - HMEC1) and demonstrate that αvβ6 integrin expression is not caused by increased mRNA levels. Incubation of HMEC1 with sEVs isolated from PrCa PC3 cells that express the αvβ6 integrin results in a highly significant increase in the number of nodes, junctions and tubules. In contrast, incubation of HMEC1 with sEVs isolated from β6 negative PC3 cells, generated by shRNA against β6, results in a reduction in the number of nodes, junctions and tubules, a decrease in survivin levels and an increase in a negative regulator of angiogenesis, pSTAT1. Furthermore, treatment of HMEC1 with sEVs generated by CRISPR/Cas9-mediated down-regulation of β6, causes up-regulation of pSTAT1. Overall, our findings suggest that αvβ6 integrin in cancer sEVs regulates angiogenesis during PrCa progression.
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http://dx.doi.org/10.1080/20013078.2020.1763594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301698PMC
May 2020

MFF Regulation of Mitochondrial Cell Death Is a Therapeutic Target in Cancer.

Cancer Res 2019 Dec 3;79(24):6215-6226. Epub 2019 Oct 3.

Prostate Cancer Discovery and Development Program.

The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non-small cell lung cancer and formed homo- and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 d-enantiomeric peptidomimetic disrupted the MFF-VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patient-derived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF-VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer. SIGNIFICANCE: These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF-VDAC complex and displays anticancer activity in multiple tumor models..
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http://dx.doi.org/10.1158/0008-5472.CAN-19-1982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911621PMC
December 2019

Mitochondrial fission factor is a novel Myc-dependent regulator of mitochondrial permeability in cancer.

EBioMedicine 2019 Oct 18;48:353-363. Epub 2019 Sep 18.

Prostate Cancer Discovery and Development Program, USA; Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

Background: Mitochondrial functions are exploited in cancer and provide a validated therapeutic target. However, how this process is regulated has remained mostly elusive and the identification of new pathways that control mitochondrial integrity in cancer is an urgent priority.

Methods: We studied clinically-annotated patient series of primary and metastatic prostate cancer, representative cases of multiple myeloma (MM) and publicly available genetic databases. Gene regulation studies involved chromatin immunoprecipitation, PCR amplification and Western blotting of conditional Myc-expressing cell lines. Transient or stable gene silencing was used to quantify mitochondrial functions in bioenergetics, outer membrane permeability, Ca homeostasis, redox balance and cell death. Tumorigenicity was assessed by cell proliferation, colony formation and xenograft tumour growth.

Findings: We identified Mitochondrial Fission Factor (MFF) as a novel transcriptional target of oncogenic Myc overexpressed in primary and metastatic cancer, compared to normal tissues. Biochemically, MFF isoforms, MFF1 and MFF2 associate with the Voltage-Dependent Anion Channel-1 (VDAC1) at the mitochondrial outer membrane, in vivo. Disruption of this complex by MFF silencing induces general collapse of mitochondrial functions with increased outer membrane permeability, loss of inner membrane potential, Ca unbalance, bioenergetics defects and activation of cell death pathways. In turn, this inhibits tumour cell proliferation, suppresses colony formation and reduces xenograft tumour growth in mice.

Interpretation: An MFF-VDAC1 complex is a novel regulator of mitochondrial integrity and actionable therapeutic target in cancer.
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http://dx.doi.org/10.1016/j.ebiom.2019.09.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838406PMC
October 2019

IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1αregulated pseudohypoxic state.

FASEB J 2019 12 17;33(12):13398-13411. Epub 2019 Sep 17.

The Wistar Institute, Philadelphia, Pennsylvania, USA.

The role of mitochondria in cancer continues to be debated and paradoxically implicated in opposing functions in tumor growth and tumor suppression. To understand this dichotomy, we explored the function of mitochondrial isocitrate dehydrogenase (IDH)2, a tricarboxylic acid cycle enzyme mutated in subsets of acute leukemias and gliomas, in cancer. Silencing of IDH2 in prostate cancer cells impaired oxidative bioenergetics, elevated reactive oxygen species (ROS) production, and promoted exaggerated mitochondrial dynamics. This was associated with increased subcellular mitochondrial trafficking, turnover of membrane focal adhesion complexes, and enhanced tumor cell migration and invasion, without changes in cell cycle progression. Mechanistically, loss of IDH2 caused ROS-dependent stabilization of hypoxia-inducible factor-1α in normoxia, which was required for increased mitochondrial trafficking and tumor cell movements. Therefore, IDH2 is a dual regulator of cancer bioenergetics and tumor cell motility. This pathway may reprogram mitochondrial dynamics to differentially adjust energy production or promote tumor cell invasion in response to microenvironment conditions.-Wang, Y., Agarwal, E., Bertolini, I., Ghosh, J. C., Seo, J. H., Altieri, D. C. IDH2 reprograms mitochondrial dynamics in cancer through a HIF-1α-regulated pseudohypoxic state.
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http://dx.doi.org/10.1096/fj.201901366RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894043PMC
December 2019

Akt phosphorylation of mitochondrial Lonp1 protease enables oxidative metabolism and advanced tumor traits.

Oncogene 2019 10 12;38(43):6926-6939. Epub 2019 Aug 12.

Prostate Cancer Discovery and Development Program, Philadelphia, PA, USA.

Tumor mitochondria have heightened protein folding quality control, but the regulators of this process and how they impact cancer traits are not completely understood. Here we show that the ATP-directed mitochondrial protease, LonP1 is upregulated by stress conditions, including hypoxia, in tumor, but not normal cells. In mitochondria, LonP1 is phosphorylated by Akt on Ser173 and Ser181, enhancing its protease activity. Interference with this pathway induces accumulation of misfolded subunits of electron transport chain complex II and complex V, resulting in impaired oxidative bioenergetics and heightened ROS production. Functionally, this suppresses mitochondrial trafficking to the cortical cytoskeleton, shuts off tumor cell migration and invasion, and inhibits primary and metastatic tumor growth, in vivo. These data identify LonP1 as a key effector of mitochondrial reprogramming in cancer and potential therapeutic target.
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http://dx.doi.org/10.1038/s41388-019-0939-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814529PMC
October 2019

Myc-mediated transcriptional regulation of the mitochondrial chaperone TRAP1 controls primary and metastatic tumor growth.

J Biol Chem 2019 07 16;294(27):10407-10414. Epub 2019 May 16.

From the Prostate Cancer Discovery and Development Program,

The role of mitochondria in cancer continues to be debated, and whether exploitation of mitochondrial functions is a general hallmark of malignancy or a tumor- or context-specific response is still unknown. Using a variety of cancer cell lines and several technical approaches, including siRNA-mediated gene silencing, ChIP assays, global metabolomics and focused metabolite analyses, bioenergetics, and cell viability assays, we show that two oncogenic Myc proteins, c-Myc and N-Myc, transcriptionally control the expression of the mitochondrial chaperone TNFR-associated protein-1 (TRAP1) in cancer. In turn, this Myc-mediated regulation preserved the folding and function of mitochondrial oxidative phosphorylation (OXPHOS) complex II and IV subunits, dampened reactive oxygen species production, and enabled oxidative bioenergetics in tumor cells. Of note, we found that genetic or pharmacological targeting of this pathway shuts off tumor cell motility and invasion, kills Myc-expressing cells in a TRAP1-dependent manner, and suppresses primary and metastatic tumor growth We conclude that exploitation of mitochondrial functions is a general trait of tumorigenesis and that this reliance of cancer cells on mitochondrial OXPHOS pathways could offer an actionable therapeutic target in the clinic.
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http://dx.doi.org/10.1074/jbc.AC119.008656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615691PMC
July 2019

Syntaphilin Is a Novel Biphasic Biomarker of Aggressive Prostate Cancer and a Metastasis Predictor.

Am J Pathol 2019 06 9;189(6):1180-1189. Epub 2019 May 9.

Department of Pathology, Yale School of Medicine, New Haven, Connecticut. Electronic address:

Easily accessible biomarkers that may inform on the metastatic potential of localized prostate cancer are urgently needed. Herein, we show that syntaphilin (SNPH), a molecule originally identified as a negative regulator of mitochondrial dynamics in neurons, is abundantly expressed in prostate cancer. SNPH distribution in prostate cancer is spatially biphasic, with high expression at the invasive front, correlating with increased proliferative rates, as determined by Ki-67 labeling, and reduced levels in the central tumor bulk, which are further decreased in patients with distant metastases. Higher levels of SNPH are observed with increasing Gleason grade. Prostate tumors predominantly express a novel, extraneuronal isoform of SNPH that accumulates in mitochondria and maintains oxidative metabolism and tumor cell proliferation. These data suggest that SNPH is a novel marker of high Gleason grade prostate cancer, differentially expressed at the invasive front compared with the central tumor bulk, and is potentially down-regulated in metastatic disease. This biphasic pattern of expression may reflect a dual function of SNPH in controlling the balance between cell proliferation and invasion in tumors.
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http://dx.doi.org/10.1016/j.ajpath.2019.02.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560381PMC
June 2019

Myc Regulation of a Mitochondrial Trafficking Network Mediates Tumor Cell Invasion and Metastasis.

Mol Cell Biol 2019 07 27;39(14). Epub 2019 Jun 27.

Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, Pennsylvania, USA

The Myc gene is a universal oncogene that promotes aggressive cancer, but its role in metastasis has remained elusive. Here, we show that Myc transcriptionally controls a gene network of subcellular mitochondrial trafficking that includes the atypical mitochondrial GTPases RHOT1 and RHOT2, the adapter protein TRAK2, the anterograde motor Kif5B, and an effector of mitochondrial fission, Drp1. Interference with this pathway deregulates mitochondrial dynamics, shuts off subcellular organelle movements, and prevents the recruitment of mitochondria to the cortical cytoskeleton of tumor cells. In turn, this inhibits tumor chemotaxis, blocks cell invasion, and prevents metastatic spreading in preclinical models. Therefore, Myc regulation of mitochondrial trafficking enables tumor cell motility and metastasis.
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http://dx.doi.org/10.1128/MCB.00109-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597883PMC
July 2019

Survivin at a glance.

J Cell Sci 2019 04 4;132(7). Epub 2019 Apr 4.

The Wistar Institute Cancer Center, Philadelphia, PA 19104, USA.

Survivin (also known as BIRC5) is an evolutionarily conserved eukaryotic protein that is essential for cell division and can inhibit cell death. Normally it is only expressed in actively proliferating cells, but is upregulated in most, if not all cancers; consequently, it has received significant attention as a potential oncotherapeutic target. In this Cell Science at a Glance article and accompanying poster, we summarise our knowledge of survivin 21 years on from its initial discovery. We describe the structure, expression and function of survivin, highlight its interactome and conclude by describing anti-survivin strategies being trialled.
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http://dx.doi.org/10.1242/jcs.223826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467487PMC
April 2019

Mitochondrial dynamics and metastasis.

Authors:
Dario C Altieri

Cell Mol Life Sci 2019 Mar 10;76(5):827-835. Epub 2018 Nov 10.

Prostate Cancer Discovery and Development Program and Immunology, Microenvironment and Metastasis Program, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA.

Changes in cellular metabolism are now a recognized hallmark of cancer. Although this process is ripe with therapeutic potential in the clinic, its complexity and extraordinary plasticity have systematically defied dogmas and oversimplifications. Perhaps, best exemplifying this intricacy is the role of mitochondria in cancer, which in just a few years has gone from largely unnoticed to pivotal disease driver. The underlying mechanisms are only beginning to emerge. However, there is now clear evidence linking the dynamic nature of mitochondria to the machinery of tumor cell motility and metastatic spreading. These studies may open fresh therapeutic options for patients with disseminated cancer, currently an incurable and mostly lethal condition.
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http://dx.doi.org/10.1007/s00018-018-2961-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559795PMC
March 2019

Unique pattern of neutrophil migration and function during tumor progression.

Nat Immunol 2018 11 15;19(11):1236-1247. Epub 2018 Oct 15.

Immunology, Microenvironment, and Metastasis, Wistar Institute, Philadelphia, PA, USA.

Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils' contribution to early tumor dissemination.
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http://dx.doi.org/10.1038/s41590-018-0229-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195445PMC
November 2018

αβ Integrin Mediates Radioresistance of Prostate Cancer Cells through Regulation of Survivin.

Mol Cancer Res 2019 02 28;17(2):398-408. Epub 2018 Sep 28.

Department of Radiation Oncology, University of Massachusetts Medical School, Worcester, Massachusetts.

The αβ integrin is involved in various physiologic and pathologic processes such as wound healing, angiogenesis, tumor growth, and metastasis. The impact of αβ integrin on the radiosensitivity of prostate cancer cells and the molecular mechanism controlling cell survival in response to ionizing radiation (IR) was investigated. Both LNCaP cells stably transfected with αβ integrin and PC-3 cells that contain endogenous β integrin were used. This study demonstrated that αβ integrin increases survival of αβ-LNCaP cells upon IR while small hairpin RNA (shRNA)-mediated knockdown of αβ integrin in PC-3 cells sensitizes to radiation. Expression of αβ integrin in LNCaP cells also enhances anchorage-independent cell growth while knockdown of αβ integrin in PC-3 cells inhibits anchorage-independent cell growth. The αβ antagonist, cRGD, significantly increases radiosensitivity in both αβ-LNCaP and PC-3 cells. Moreover, αβ integrin prevents radiation-induced downregulation of survivin. Inhibition of survivin expression by siRNA or shRNA enhances IR-induced inhibition of anchorage-independent cell growth. Overexpression of wild-type survivin in PC-3 cells treated with αβ integrin shRNA increases survival of cells upon IR. These findings reveal that αβ integrin promotes radioresistance and regulates survivin levels in response to IR. IMPLICATIONS: Future translational research on targeting αβ integrin and survivin may reveal novel approaches as an adjunct to radiotherapy for patients with prostate cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359981PMC
February 2019

Syntaphilin Ubiquitination Regulates Mitochondrial Dynamics and Tumor Cell Movements.

Cancer Res 2018 08 13;78(15):4215-4228. Epub 2018 Jun 13.

Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, Pennsylvania.

Syntaphilin (SNPH) inhibits the movement of mitochondria in tumor cells, preventing their accumulation at the cortical cytoskeleton and limiting the bioenergetics of cell motility and invasion. Although this may suppress metastasis, the regulation of the SNPH pathway is not well understood. Using a global proteomics screen, we show that SNPH associates with multiple regulators of ubiquitin-dependent responses and is ubiquitinated by the E3 ligase CHIP (or STUB1) on Lys111 and Lys153 in the microtubule-binding domain. SNPH ubiquitination did not result in protein degradation, but instead anchored SNPH on tubulin to inhibit mitochondrial motility and cycles of organelle fusion and fission, that is dynamics. Expression of ubiquitination-defective SNPH mutant Lys111→Arg or Lys153→Arg increased the speed and distance traveled by mitochondria, repositioned mitochondria to the cortical cytoskeleton, and supported heightened tumor chemotaxis, invasion, and metastasis Interference with SNPH ubiquitination activated mitochondrial dynamics, resulting in increased recruitment of the fission regulator dynamin-related protein-1 (Drp1) to mitochondria and Drp1-dependent tumor cell motility. These data uncover nondegradative ubiquitination of SNPH as a key regulator of mitochondrial trafficking and tumor cell motility and invasion. In this way, SNPH may function as a unique, ubiquitination-regulated suppressor of metastasis. These findings reveal a new mechanism of metastasis suppression by establishing the role of SNPH ubiquitination in inhibiting mitochondrial dynamics, chemotaxis, and metastasis. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072605PMC
August 2018

Exploiting TERT dependency as a therapeutic strategy for NRAS-mutant melanoma.

Oncogene 2018 07 26;37(30):4058-4072. Epub 2018 Apr 26.

Molecular & Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.

Targeting RAS is one of the greatest challenges in cancer therapy. Oncogenic mutations in NRAS are present in over 25% of melanomas and patients whose tumors harbor NRAS mutations have limited therapeutic options and poor prognosis. Thus far, there are no clinical agents available to effectively target NRAS or any other RAS oncogene. An alternative approach is to identify and target critical tumor vulnerabilities or non-oncogene addictions that are essential for tumor survival. We investigated the consequences of NRAS blockade in NRAS-mutant melanoma and show that decreased expression of the telomerase catalytic subunit, TERT, is a major consequence. TERT silencing or treatment of NRAS-mutant melanoma with the telomerase-dependent telomere uncapping agent, 6-thio-2'-deoxyguanosine (6-thio-dG), led to rapid cell death, along with evidence of both telomeric and non-telomeric DNA damage, increased ROS levels, and upregulation of a mitochondrial antioxidant adaptive response. Combining 6-thio-dG with the mitochondrial inhibitor Gamitrinib attenuated this adaptive response and more effectively suppressed NRAS-mutant melanoma. Our study uncovers a robust dependency of NRAS-mutant melanoma on TERT, and provides proof-of-principle for a new combination strategy to combat this class of tumors, which could be expanded to other tumor types.
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http://dx.doi.org/10.1038/s41388-018-0247-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062502PMC
July 2018

Exosomal αvβ6 integrin is required for monocyte M2 polarization in prostate cancer.

Matrix Biol 2018 09 9;70:20-35. Epub 2018 Mar 9.

Prostate Cancer Discovery and Development Program, Thomas Jefferson University, Philadelphia, Pennsylvania, USA; Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. Electronic address:

Therapeutic approaches aimed at curing prostate cancer are only partially successful given the occurrence of highly metastatic resistant phenotypes that frequently develop in response to therapies. Recently, we have described αvβ6, a surface receptor of the integrin family as a novel therapeutic target for prostate cancer; this epithelial-specific molecule is an ideal target since, unlike other integrins, it is found in different types of cancer but not in normal tissues. We describe a novel αvβ6-mediated signaling pathway that has profound effects on the microenvironment. We show that αvβ6 is transferred from cancer cells to monocytes, including β6-null monocytes, by exosomes and that monocytes from prostate cancer patients, but not from healthy volunteers, express αvβ6. Cancer cell exosomes, purified via density gradients, promote M2 polarization, whereas αvβ6 down-regulation in exosomes inhibits M2 polarization in recipient monocytes. Also, as evaluated by our proteomic analysis, αvβ6 down-regulation causes a significant increase in donor cancer cells, and their exosomes, of two molecules that have a tumor suppressive role, STAT1 and MX1/2. Finally, using the Pten prostate cancer mouse model, which carries a prostate epithelial-specific Pten deletion, we demonstrate that αvβ6 inhibition in vivo causes up-regulation of STAT1 in cancer cells. Our results provide evidence of a novel mechanism that regulates M2 polarization and prostate cancer progression through transfer of αvβ6 from cancer cells to monocytes through exosomes.
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http://dx.doi.org/10.1016/j.matbio.2018.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6081240PMC
September 2018

Mitochondrial HSP90 Accumulation Promotes Vascular Remodeling in Pulmonary Arterial Hypertension.

Am J Respir Crit Care Med 2018 07;198(1):90-103

1 Pulmonary Hypertension Research Group, Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec, Université Laval, Québec City, Québec, Canada.

Rationale: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival.

Objectives: We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis.

Methods: Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats.

Measurements And Main Results: We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat).

Conclusions: Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.
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http://dx.doi.org/10.1164/rccm.201708-1751OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835039PMC
July 2018

A Mitochondrial-targeted purine-based HSP90 antagonist for leukemia therapy.

Oncotarget 2017 Dec 11;8(68):112184-112198. Epub 2017 Dec 11.

Prostate Cancer Discovery and Development Program, The Wistar Institute, Philadelphia, PA, USA.

Reprogramming of mitochondrial functions sustains tumor growth and may provide therapeutic opportunities. Here, we targeted the protein folding environment in mitochondria by coupling a purine-based inhibitor of the molecular chaperone Heat Shock Protein-90 (Hsp90), PU-H71 to the mitochondrial-targeting moiety, triphenylphosphonium (TPP). Binding of PU-H71-TPP to ADP-Hsp90, Hsp90 co-chaperone complex or mitochondrial Hsp90 homolog, TRAP1 involved hydrogen bonds, π-π stacking, cation-π contacts and hydrophobic interactions with the surrounding amino acids in the active site. PU-H71-TPP selectively accumulated in mitochondria of tumor cells (17-fold increase in mitochondria/cytosol ratio), whereas unmodified PU-H71 showed minimal mitochondrial localization. Treatment of tumor cells with PU-H71-TPP dissipated mitochondrial membrane potential, inhibited oxidative phosphorylation in sensitive cell types, and reduced ATP production, resulting in apoptosis and tumor cell killing. Unmodified PU-H71 had no effect. Bioinformatics analysis identified a "mitochondrial Hsp90" signature in Acute Myeloid Leukemia (AML), which correlates with worse disease outcome. Accordingly, inhibition of mitochondrial Hsp90s killed primary and cultured AML cells, with minimal effects on normal peripheral blood mononuclear cells. These data demonstrate that directing Hsp90 inhibitors with different chemical scaffolds to mitochondria is feasible and confers improved anticancer activity. A potential "addiction" to mitochondrial Hsp90s may provide a new therapeutic target in AML.
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http://dx.doi.org/10.18632/oncotarget.23097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762502PMC
December 2017

Evaluation of drug combination effect using a Bliss independence dose-response surface model.

Stat Biopharm Res 2018 13;10(2):112-122. Epub 2018 Feb 13.

Immunology, Microenvironment & Metastasis, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104.

To test the anticancer effect of combining two drugs targeting different biological pathways, the popular way to show synergistic effect of drug combination is a heat map or surface plot based on the percent excess the Bliss prediction using the average response measures at each combination dose. Such graphs, however, are inefficient in the drug screening process and it doesn't give a statistical inference on synergistic effect. To make a statistically rigorous and robust conclusion for drug combination effect, we present a two-stage Bliss independence response surface model to estimate an overall interaction index () with 95% confidence interval (CI). By taking into all data points account, the overall with 95% CI can be applied to determine if the drug combination effect is synergistic overall. Using some example data, the two-stage model was compared to a couple of classic models following Bliss rule. The data analysis results obtained from our model reflect the pattern shown from other models. The application of overall helps investigators to make decision easier and accelerate the preclinical drug screening.
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http://dx.doi.org/10.1080/19466315.2018.1437071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6415926PMC
February 2018

AML Therapy: Wake Up the Guardian and Cut Loose the Executioners.

Authors:
Dario C Altieri

Cancer Cell 2017 12;32(6):719-720

Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA. Electronic address:

In this issue of Cancer Cell, Pan et al. show that a combination therapy designed to reactivate the p53 tumor suppressor while antagonizing the anti-apoptotic function of Bcl-2 is highly active in preclinical models of refractory acute myeloid leukemia (AML). The results may move the needle in this hard-to-treat malignancy.
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http://dx.doi.org/10.1016/j.ccell.2017.11.010DOI Listing
December 2017

Syntaphilin controls a mitochondrial rheostat for proliferation-motility decisions in cancer.

J Clin Invest 2017 Oct 11;127(10):3755-3769. Epub 2017 Sep 11.

Prostate Cancer Discovery and Development Program.

Tumors adapt to an unfavorable microenvironment by controlling the balance between cell proliferation and cell motility, but the regulators of this process are largely unknown. Here, we show that an alternatively spliced isoform of syntaphilin (SNPH), a cytoskeletal regulator of mitochondrial movements in neurons, is directed to mitochondria of tumor cells. Mitochondrial SNPH buffers oxidative stress and maintains complex II-dependent bioenergetics, sustaining local tumor growth while restricting mitochondrial redistribution to the cortical cytoskeleton and tumor cell motility. Conversely, introduction of stress stimuli to the microenvironment, including hypoxia, acutely lowered SNPH levels, resulting in bioenergetics defects and increased superoxide production. In turn, this suppressed tumor cell proliferation but increased tumor cell invasion via greater mitochondrial trafficking to the cortical cytoskeleton. Loss of SNPH or expression of an SNPH mutant lacking the mitochondrial localization sequence resulted in increased metastatic dissemination in xenograft or syngeneic tumor models in vivo. Accordingly, tumor cells that acquired the ability to metastasize in vivo constitutively downregulated SNPH and exhibited higher oxidative stress, reduced cell proliferation, and increased cell motility. Therefore, SNPH is a stress-regulated mitochondrial switch of the cell proliferation-motility balance in cancer, and its pathway may represent a therapeutic target.
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http://dx.doi.org/10.1172/JCI93172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617650PMC
October 2017

Mitochondria on the move: emerging paradigms of organelle trafficking in tumour plasticity and metastasis.

Authors:
Dario C Altieri

Br J Cancer 2017 Jul 4;117(3):301-305. Epub 2017 Jul 4.

Prostate Cancer Discovery and Development Program, Tumour Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA.

There is now a resurgent interest in the role of mitochondria in cancer. Long considered controversial or outright unimportant, mitochondrial biology is now increasingly recognised as an important tumour driver. The underlying mechanisms remain to be fully elucidated. But recent studies have uncovered a complex landscape where reprogramming of mitochondrial homoeostasis, including organelle dynamics, metabolic output, apoptosis control and redox status converge to promote tumour adaptation to an unfavourable microenvironment and inject new traits of aggressive disease. In particular, mechanisms of subcellular mitochondrial trafficking have unexpectedly emerged as central regulators of metastatic competence in disparate tumours. Some of these pathways are druggable, opening fresh therapeutic opportunities for advanced and disseminated disease.
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http://dx.doi.org/10.1038/bjc.2017.201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5537502PMC
July 2017

Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower Antitumor Therapeutic Responses.

Cancer Res 2017 07 18;77(13):3513-3526. Epub 2017 May 18.

Department of Pathology & Cell Biology, Columbia University Medical Center, New York, New York.

Rational therapeutic approaches based on synthetic lethality may improve cancer management. On the basis of a high-throughput drug screen, we provide preclinical proof of concept that targeting the mitochondrial Hsp90 chaperone network (mtHsp90) and inhibition of Bcl-2, Bcl-xL, and Mcl-1 is sufficient to elicit synthetic lethality in tumors recalcitrant to therapy. Our analyses focused on BH3 mimetics that are broad acting (ABT263 and obatoclax) or selective (ABT199, WEHI-539, and A1210477), along with the established mitochondrial matrix chaperone inhibitor gamitrinib-TPP. Drug combinations were tested in various therapy-resistant tumors and in murine model systems of melanoma, triple-negative breast cancer, and patient-derived orthotopic xenografts (PDX) of human glioblastoma. We found that combining BH3 mimetics and gamitrinib-TPP blunted cellular proliferation in a synergistic manner by massive activation of intrinsic apoptosis. In like manner, suppressing either Bcl-2, Bcl-xL, or Mcl-1 recapitulated the effects of BH3 mimetics and enhanced the effects of gamitrinib-TPP. Mechanistic investigations revealed that gamitrinib-TPP activated a PERK-dependent integrated stress response, which activated the proapoptotic BH3 protein Noxa and its downstream targets Usp9X and Mcl-1. Notably, in the PDX glioblastoma and BRAFi-resistant melanoma models, this drug combination safely and significantly extended host survival. Our results show how combining mitochondrial chaperone and Bcl-2 family inhibitors can synergize to safely degrade the growth of tumors recalcitrant to other treatments. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-3424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5503474PMC
July 2017

Mitochondrial matrix chaperone and c-myc inhibition causes enhanced lethality in glioblastoma.

Oncotarget 2017 Jun;8(23):37140-37153

Department of Pathology and Cell Biology, Columbia University Medical Center, New York, NY, USA.

Malignant gliomas display high levels of the transcription factor c-myc and organize a tumor specific chaperone network within mitochondria. Here, we show that c-myc along with mitochondrial chaperone inhibition displays massive tumor cell death. Inhibition of mitochondrial matrix chaperones and c-myc was established by utilizing genetic as well as pharmacological approaches. Bromodomain and extraterminal (BET) family protein inhibitors, JQ1 and OTX015, were used for c-myc inhibition. Gamitrinib was applied to interfere with mitochondrial matrix chaperones. A xenograft model was used to determine the in vivo efficacy. Combined inhibition of c-myc and mitochondrial matrix chaperones led to a synergistic reduction of cellular proliferation (CI values less than 1) in established glioblastoma, patient-derived xenograft and stem cell-like glioma cultures. The combinatorial treatment of BET inhibitors and Gamitrinib elicited massive apoptosis induction with dissipation of mitochondrial membrane potential and activation of caspases. Mechanistically, BET-inhibitors and Gamitrinib mediated a pronounced integrated stress response with a PERK-dependent up regulation of ATF4 and subsequent modulation of Bcl-2 family of proteins with down-regulation of Mcl-1 and its interacting partner, Usp9X, and an increase in pro-apoptotic Noxa. Blocking ATF4 by siRNA attenuated Gamitrinib/BET inhibitor mediated increase of Noxa. Knockdown of Noxa and Bak protected from the combinatorial treatment. Finally, the combination treatment of Gamitrinib and OTX015 led to a significantly stronger reduction of tumor growth as compared to single treatments in a xenograft model of human glioma without induction of toxicity. Thus, Gamitrinib in combination with BET-inhibitors should be considered for the development for clinical application.
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http://dx.doi.org/10.18632/oncotarget.16202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514897PMC
June 2017

Inhibition of Age-Related Therapy Resistance in Melanoma by Rosiglitazone-Mediated Induction of Klotho.

Clin Cancer Res 2017 Jun 23;23(12):3181-3190. Epub 2017 Feb 23.

The Wistar Institute, Philadelphia, Pennsylvania.

Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, and , and the implications of that for targeted therapy in young versus aged animals. We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor-sensitive and BRAF inhibitor-resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474161PMC
June 2017

Carcinoma-risk variant of EBNA1 deregulates Epstein-Barr Virus episomal latency.

Oncotarget 2017 Jan;8(5):7248-7264

The Wistar Institute, Philadelphia, PA USA.

Epstein-Barr Virus (EBV) latent infection is a causative co-factor for endemic Nasopharyngeal Carcinoma (NPC). NPC-associated variants have been identified in EBV-encoded nuclear antigen EBNA1. Here, we solve the X-ray crystal structure of an NPC-derived EBNA1 DNA binding domain (DBD) and show that variant amino acids are found on the surface away from the DNA binding interface. We show that NPC-derived EBNA1 is compromised for DNA replication and episome maintenance functions. Recombinant virus containing the NPC EBNA1 DBD are impaired in their ability to immortalize primary B-lymphocytes and suppress lytic transcription during early stages of B-cell infection. We identify Survivin as a host protein deficiently bound by the NPC variant of EBNA1 and show that Survivin depletion compromises EBV episome maintenance in multiple cell types. We propose that endemic variants of EBNA1 play a significant role in EBV-driven carcinogenesis by altering key regulatory interactions that destabilize latent infection.
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http://dx.doi.org/10.18632/oncotarget.14540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352318PMC
January 2017
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