Publications by authors named "Lucia R Languino"

92 Publications

Hitting the Bullseye: Are extracellular vesicles on target?

J Extracell Vesicles 2020 Nov 29;10(1):e12032. Epub 2020 Nov 29.

Department of Cancer Biology Sidney Kimmel Cancer Center Jefferson University Philadelphia Pennsylvania USA.

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http://dx.doi.org/10.1002/jev2.12032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890543PMC
November 2020

Differential expression of αVβ3 and αVβ6 integrins in prostate cancer progression.

PLoS One 2021 22;16(1):e0244985. Epub 2021 Jan 22.

Prostate Cancer Discovery and Development Program, Thomas Jefferson University, Philadelphia, PA, United States of America.

Neuroendocrine prostate cancer (NEPrCa) arises de novo or after accumulation of genomic alterations in pre-existing adenocarcinoma tumors in response to androgen deprivation therapies. We have provided evidence that small extracellular vesicles released by PrCa cells and containing the αVβ3 integrin promote neuroendocrine differentiation of PrCa in vivo and in vitro. Here, we examined αVβ3 integrin expression in three murine models carrying a deletion of PTEN (SKO), PTEN and RB1 (DKO), or PTEN, RB1 and TRP53 (TKO) genes in the prostatic epithelium; of these three models, the DKO and TKO tumors develop NEPrCa with a gene signature comparable to those of human NEPrCa. Immunostaining analysis of SKO, DKO and TKO tumors shows that αVβ3 integrin expression is increased in DKO and TKO primary tumors and metastatic lesions, but absent in SKO primary tumors. On the other hand, SKO tumors show higher levels of a different αV integrin, αVβ6, as compared to DKO and TKO tumors. These results are confirmed by RNA-sequencing analysis. Moreover, TRAMP mice, which carry NEPrCa and adenocarcinoma of the prostate, also have increased levels of αVβ3 in their NEPrCa primary tumors. In contrast, the αVβ6 integrin is only detectable in the adenocarcinoma areas. Finally, analysis of 42 LuCaP patient-derived xenografts and primary adenocarcinoma samples shows a positive correlation between αVβ3, but not αVβ6, and the neuronal marker synaptophysin; it also demonstrates that αVβ3 is absent in prostatic adenocarcinomas. In summary, we demonstrate that αVβ3 integrin is upregulated in NEPrCa primary and metastatic lesions; in contrast, the αVβ6 integrin is confined to adenocarcinoma of the prostate. Our findings suggest that the αVβ3 integrin, but not αVβ6, may promote a shift in lineage plasticity towards a NE phenotype and might serve as an informative biomarker for the early detection of NE differentiation in prostate cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0244985PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822502PMC
January 2021

Announcing the ISEV2020 special achievement award recipients: Andrew Hill and Edit Buzás; and the recipient of the ISEV2020 special education award: Carolina Soekmadji.

J Extracell Vesicles 2020 Oct 5;10(1):e12021. Epub 2020 Nov 5.

Department of Biochemistry and Cell Biology Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands.

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http://dx.doi.org/10.1002/jev2.12021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710126PMC
October 2020

Methods for extracellular vesicle isolation from cancer cells.

Cancer Drug Resist 2020 25;3:371-384. Epub 2020 Apr 25.

Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.
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http://dx.doi.org/10.20517/cdr.2019.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556721PMC
April 2020

Small extracellular vesicles modulated by αVβ3 integrin induce neuroendocrine differentiation in recipient cancer cells.

J Extracell Vesicles 2020 May 24;9(1):1761072. Epub 2020 May 24.

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

The ability of small extracellular vesicles (sEVs) to reprogram cancer cells is well established. However, the specific sEV components able to mediate aberrant effects in cancer cells have not been characterized. Integrins are major players in mediating sEV functions. We have previously reported that the αVβ3 integrin is detected in sEVs of prostate cancer (PrCa) cells and transferred into recipient cells. Here, we investigate whether sEVs from αVβ3-expressing cells affect tumour growth differently than sEVs from control cells that do not express αVβ3. We compared the ability of sEVs to stimulate tumour growth, using sEVs isolated from PrCa C4-2B cells by iodixanol density gradient and characterized with immunoblotting, nanoparticle tracking analysis, immunocapturing and single vesicle analysis. We incubated PrCa cells with sEVs and injected them subcutaneously into nude mice to measure tumour growth or analysed their anchorage-independent growth. Our results demonstrate that a single treatment with sEVs shed from C4-2B cells that express αVβ3, but not from control cells, stimulates tumour growth and induces differentiation of PrCa cells towards a neuroendocrine phenotype, as quantified by increased levels of neuroendocrine markers. In conclusion, the expression of αVβ3 integrin generates sEVs capable of reprogramming cells towards an aggressive phenotype.
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http://dx.doi.org/10.1080/20013078.2020.1761072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448905PMC
May 2020

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

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

Identification of monocyte-like precursors of granulocytes in cancer as a mechanism for accumulation of PMN-MDSCs.

J Exp Med 2019 09 25;216(9):2150-2169. Epub 2019 Jun 25.

Immunology, Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA

We have identified a precursor that differentiates into granulocytes in vitro and in vivo yet belongs to the monocytic lineage. We have termed these cells monocyte-like precursors of granulocytes (MLPGs). Under steady state conditions, MLPGs were absent in the spleen and barely detectable in the bone marrow (BM). In contrast, these cells significantly expanded in tumor-bearing mice and differentiated to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Selective depletion of monocytic cells had no effect on the number of granulocytes in naive mice but decreased the population of PMN-MDSCs in tumor-bearing mice by 50%. The expansion of MLPGs was found to be controlled by the down-regulation of Rb1, but not IRF8, which is known to regulate the expansion of PMN-MDSCs from classic granulocyte precursors. In cancer patients, putative MLPGs were found within the population of CXCR1CD15CD14HLA-DR monocytic cells. These findings describe a mechanism of abnormal myelopoiesis in cancer and suggest potential new approaches for selective targeting of MDSCs.
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http://dx.doi.org/10.1084/jem.20181952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719429PMC
September 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

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

Tumor-Derived Extracellular Vesicles Require β1 Integrins to Promote Anchorage-Independent Growth.

iScience 2019 Apr 27;14:199-209. Epub 2019 Mar 27.

Prostate Cancer Discovery and Development Program, Thomas Jefferson University, Philadelphia, PA, USA; Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia PA 19107, USA; Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, USA. Electronic address:

The β1 integrins, known to promote cancer progression, are abundant in extracellular vesicles (EVs). We investigated whether prostate cancer (PrCa) EVs affect anchorage-independent growth and whether β1 integrins are required for this effect. Specifically using a cell-line-based genetic rescue and an in vivo PrCa model, we show that gradient-purified small EVs (sEVs) from either cancer cells or blood from tumor-bearing TRAMP (transgenic adenocarcinoma of the mouse prostate) mice promote anchorage-independent growth of PrCa cells. In contrast, sEVs from cultured PrCa cells harboring a short hairpin RNA to β1, from wild-type mice or from TRAMP mice carrying a β1 conditional ablation in the prostatic epithelium (β1), do not. We find that sEVs, from cancer cells or TRAMP blood, are functional and co-express β1 and sEV markers; in contrast, sEVs from β1/TRAMP or wild-type mice lack β1 and sEV markers. Our results demonstrate that β1 integrins in tumor-cell-derived sEVs are required for stimulation of anchorage-independent growth.
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http://dx.doi.org/10.1016/j.isci.2019.03.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461598PMC
April 2019

Activated Extracellular Vesicles as New Therapeutic Targets?

Trends Cell Biol 2019 04 27;29(4):276-278. Epub 2019 Feb 27.

Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

A recent study by Genschmer et al. (Cell 2019;176:113-126) presents provocative new evidence that extracellular vesicles/exosomes released by activated neutrophils can degrade extracellular proteins. Elastase that is produced by neutrophils both coats the exosomes and is protected by the exosomes from inactivation by α1 antitrypsin, ultimately causing the pathological degradation of extracellular protein fibrils in lung alveoli.
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http://dx.doi.org/10.1016/j.tcb.2019.02.003DOI Listing
April 2019

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

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

Prostate cancer sheds the αvβ3 integrin in vivo through exosomes.

Matrix Biol 2019 04 8;77:41-57. Epub 2018 Aug 8.

Prostate Cancer Discovery and Development Program, Thomas Jefferson University, Philadelphia, PA 19107, USA; Departments of Cancer Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Electronic address:

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.
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http://dx.doi.org/10.1016/j.matbio.2018.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6541230PMC
April 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

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

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

Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors.

Cancer Cell 2017 11;32(5):654-668.e5

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

Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.
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http://dx.doi.org/10.1016/j.ccell.2017.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827952PMC
November 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

A neuronal network of mitochondrial dynamics regulates metastasis.

Nat Commun 2016 12 19;7:13730. Epub 2016 Dec 19.

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

The role of mitochondria in cancer is controversial. Using a genome-wide shRNA screen, we now show that tumours reprogram a network of mitochondrial dynamics operative in neurons, including syntaphilin (SNPH), kinesin KIF5B and GTPase Miro1/2 to localize mitochondria to the cortical cytoskeleton and power the membrane machinery of cell movements. When expressed in tumours, SNPH inhibits the speed and distance travelled by individual mitochondria, suppresses organelle dynamics, and blocks chemotaxis and metastasis, in vivo. Tumour progression in humans is associated with downregulation or loss of SNPH, which correlates with shortened patient survival, increased mitochondrial trafficking to the cortical cytoskeleton, greater membrane dynamics and heightened cell invasion. Therefore, a SNPH network regulates metastatic competence and may provide a therapeutic target in cancer.
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http://dx.doi.org/10.1038/ncomms13730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5187409PMC
December 2016

v-Src Oncogene Induces Trop2 Proteolytic Activation via Cyclin D1.

Cancer Res 2016 11 15;76(22):6723-6734. Epub 2016 Sep 15.

Department of Cancer Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1-regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src-mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723-34. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330252PMC
November 2016

Transgenic Expression of the Mitochondrial Chaperone TNFR-associated Protein 1 (TRAP1) Accelerates Prostate Cancer Development.

J Biol Chem 2016 Nov 17;291(48):25247-25254. Epub 2016 Oct 17.

From the Prostate Cancer Discovery and Development Program,

Protein homeostasis, or proteostasis, is required for mitochondrial function, but its role in cancer is controversial. Here we show that transgenic mice expressing the mitochondrial chaperone TNFR-associated protein 1 (TRAP1) in the prostate develop epithelial hyperplasia and cellular atypia. When examined on a Pten background, a common alteration in human prostate cancer, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ Conversely, homozygous deletion of TRAP1 delays prostatic tumorigenesis in Pten mice without affecting hyperplasia or prostatic intraepithelial neoplasia. Global profiling of Pten-TRAP1 transgenic mice by RNA sequencing and reverse phase protein array reveals modulation of oncogenic networks of cell proliferation, apoptosis, cell motility, and DNA damage. Mechanistically, reconstitution of Pten prostatic epithelial cells with TRAP1 increases cell proliferation, reduces apoptosis, and promotes cell invasion without changes in mitochondrial bioenergetics. Therefore, TRAP1 is a driver of prostate cancer in vivo and an "actionable" therapeutic target.
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http://dx.doi.org/10.1074/jbc.M116.745950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5122790PMC
November 2016

A microRNA/Runx1/Runx2 network regulates prostate tumor progression from onset to adenocarcinoma in TRAMP mice.

Oncotarget 2016 Oct;7(43):70462-70474

Department of Biochemistry and University of Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA.

While decades of research have identified molecular pathways inducing and promoting stages of prostate cancer malignancy, studies addressing dynamic changes of cancer-related regulatory factors in a prostate tumor progression model are limited. Using the TRAMP mouse model of human prostate cancer, we address mechanisms of deregulation for the cancer-associated transcription factors, Runx1 and Runx2 by identifying microRNAs with reciprocal expression changes at six time points during 33 weeks of tumorigenesis. We molecularly define transition stages from PIN lesions to hyperplasia/neoplasia and progression to adenocarcinoma by temporal changes in expression of human prostate cancer markers, including the androgen receptor and tumor suppressors, Nkx3.1 and PTEN. Concomitant activation of PTEN, AR, and Runx factors occurs at early stages. At late stages, PTEN and AR are downregulated, while Runx1 and Runx2 remain elevated. Loss of Runx-targeting microRNAs, miR-23b-5p, miR-139-5p, miR-205-5p, miR-221-3p, miR-375-3p, miR-382-5p, and miR-384-5p, contribute to aberrant Runx expression in prostate tumors. Our studies reveal a Runx/miRNA interaction axis centered on PTEN-PI3K-AKT signaling. This regulatory network translates to mechanistic understanding of prostate tumorigenesis that can be developed for diagnosis and directed therapy.
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http://dx.doi.org/10.18632/oncotarget.11992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342565PMC
October 2016

Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

Cancer Cell 2016 08;30(2):257-272

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

Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer.
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http://dx.doi.org/10.1016/j.ccell.2016.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131882PMC
August 2016

αvβ6 Integrin Promotes Castrate-Resistant Prostate Cancer through JNK1-Mediated Activation of Androgen Receptor.

Cancer Res 2016 09 22;76(17):5163-74. Epub 2016 Jul 22.

Prostate Cancer Discovery and Development Program, Thomas Jefferson University, Philadelphia, Pennsylvania. Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania.

Androgen receptor signaling fuels prostate cancer and is a major therapeutic target. However, mechanisms of resistance to therapeutic androgen ablation are not well understood. Here, using a prostate cancer mouse model, Pten(pc-/-), carrying a prostate epithelial-specific Pten deletion, we show that the αvβ6 integrin is required for tumor growth in vivo of castrated as well as of noncastrated mice. We describe a novel signaling pathway that couples the αvβ6 integrin cell surface receptor to androgen receptor via activation of JNK1 and causes increased nuclear localization and activity of androgen receptor. This downstream kinase activation by αvβ6 is specific for JNK1, with no involvement of p38 or ERK kinase. In addition, differential phosphorylation of Akt is not observed under these conditions, nor is cell morphology affected by αvβ6 expression. This pathway, which is specific for αvβ6, because it is not regulated by a different αv-containing integrin, αvβ3, promotes upregulation of survivin, which in turn supports anchorage-independent growth of αvβ6-expressing cells. Consistently, both αvβ6 and survivin are significantly increased in prostatic adenocarcinoma, but are not detected in normal prostatic epithelium. Neither XIAP nor Bcl-2 is affected by αvβ6 expression. In conclusion, we show that αvβ6 expression is required for prostate cancer progression, including castrate-resistant prostate cancer; mechanistically, by promoting activation of JNK1, the αvβ6 integrin causes androgen receptor-increased activity in the absence of androgen and consequent upregulation of survivin. These preclinical results pave the way for further clinical development of αvβ6 antagonists for prostate cancer therapy. Cancer Res; 76(17); 5163-74. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012867PMC
September 2016

Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Nontumorigenic Cells Promotes a Migratory Phenotype.

Mol Cancer Res 2016 11 20;14(11):1136-1146. Epub 2016 Jul 20.

Prostate Cancer Discovery and Development Program, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania.

The αvβ3 integrin is known to be highly upregulated during cancer progression and promotes a migratory and metastatic phenotype in many types of tumors. We hypothesized that the αvβ3 integrin is transferred through exosomes and, upon transfer, has the ability to support functional aberrations in recipient cells. Here, for the first time, it is demonstrated that αvβ3 is present in exosomes released from metastatic PC3 and CWR22Pc prostate cancer cells. Exosomal β3 is transferred as a protein from donor to nontumorigenic and tumorigenic cells as β3 protein or mRNA levels remain unaffected upon transcription or translation inhibition in recipient cells. Furthermore, it is shown that upon exosome uptake, de novo expression of an αvβ3 increases adhesion and migration of recipient cells on an αvβ3 ligand, vitronectin. To evaluate the relevance of these findings, exosomes were purified from the blood of TRAMP mice carrying tumors where the expression of αvβ3 is found higher than in exosomes from wild-type mice. In addition, it is demonstrated that αvβ3 is coexpressed with synaptophysin, a biomarker for aggressive neuroendocrine prostate cancer.

Implications: Overall this study reveals that the αvβ3 integrin is transferred from tumorigenic to nontumorigenic cells via exosomes, and its de novo expression in recipient cells promotes cell migration on its ligand. The increased expression of αvβ3 in exosomes from mice bearing tumors points to its clinical relevance and potential use as a biomarker. Mol Cancer Res; 14(11); 1136-46. ©2016 AACR.
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http://dx.doi.org/10.1158/1541-7786.MCR-16-0058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5107121PMC
November 2016