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Oncolytic Adenovirus Therapy of Neuroendocrine Tumors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neuroendocrine tumors (NETs), originally described as carcinoids, represent a rare and heterogeneous group of neoplasms associated with intensive secretion of hormones, bioactive peptides and amines. Most of the patients are diagnosed at a late stage of disease, often with liver metastases. Surgery remains the main treatment to control metastatic disease, but is not curative. Oncolytic virotherapy represents a promising approach to treat cancer and different strategies have been exploited to restrict viral replication to tumor cells. We developed an oncolytic adenovirus based on serotype 5, Ad5[CgA-E1A], where the chromogranin A (CgA) promoter controls expression of the E1A gene and thereby virus replication. We found that Ad5[CgA-E1A], selectively replicates in NET cells and it is able to suppress fast-growing human BON carcinoid tumors in nude mice. The activity of Ad5[CgA-E1A] was not completely blocked in liver cells. We further repressed virus replication in hepatocytes by targeting E1A with miR122, an miRNA specifically expressed in the liver. miRNAs bind to mRNA and induce its cleavage or translational blockage. By insertion of tandem repeats of miR122 target sequences in 3’UTR of E1A gene, we observed reduced E1A protein expression and replication arrest in miR122 expressing liver cells. The oncolytic potency of the miR122-targeted virus was not affected in NET cells. Since some NET and neuroblastoma cells express high levels of somatostatin receptors (SSTRs), we introduced in the virus fiber knob cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site of somatostatin for SSTRs. The FWKT-modified Ad5 transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to a greater extent than Ad5, indicating that the fiber knob modification may prolong the systemic circulation time. NETs represent a huge therapeutic challenge and novel diagnostic markers are needed for early detection and effective treatment of NETs. We have profiled primary tumors and liver metastases of ileocaceal NETs, using Affymetrix microarrays and advanced bioinformatics. We have identified six novel marker genes and show high similarity between primary lesions and liver metastases transcriptome by hierarchical clustering analysis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 653
Keyword [en]
adenovirus, virotherapy, oncolytic virus, neuroendocrine tumors, chromogranin A, somatostatin receptors, microRNA, novel biomarkers
National Category
Cell and Molecular Biology Cancer and Oncology Microbiology in the medical area Cell and Molecular Biology
Research subject
Medical Science; Oncology; Medical Virology; Molecular Cellbiology
Identifiers
URN: urn:nbn:se:uu:diva-146966ISBN: 978-91-554-8022-6OAI: oai:DiVA.org:uu-146966DiVA: diva2:399527
Public defence
2011-04-15, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-03-25 Created: 2011-02-22 Last updated: 2011-05-04Bibliographically approved
List of papers
1.
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2. Double-detargeted oncolytic adenovirus shows replication arrest in liver cells and retains neuroendocrine cell killing ability
Open this publication in new window or tab >>Double-detargeted oncolytic adenovirus shows replication arrest in liver cells and retains neuroendocrine cell killing ability
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2010 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 5, no 1, e8916- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

We have previously developed an oncolytic serotype 5 adenovirus (Ad5) with chromogranin-A (CgA) promoter-controlled E1A expression, Ad[CgA-E1A], with the intention to treat neuroendocrine tumors, including carcinoids. Since carcinoids tend to metastasize to the liver it is important to fully repress viral replication in hepatocytes to avoid adenovirus-related liver toxicity. Herein, we explore miRNA-based regulation of E1A expression as a complementary mechanism to promoter-based transcriptional control.

METHODOLOGY/PRINCIPAL FINDINGS:

Ad[CgA-E1A-miR122], where E1A expression is further controlled by six tandem repeats of the target sequence for the liver-specific miR122, was constructed and compared to Ad[CgA-E1A]. We observed E1A suppression and replication arrest of the miR122-detargeted adenovirus in normal hepatocytes, while the two viruses killed carcinoid cells to the same degree. Repeated intravenous injections of Ad[CgA-E1A] induced liver toxicity in mice while Ad[CgA-E1A-miR122] injections did not. Furthermore, a miR122-detargeted adenovirus with the wild-type E1A promoter showed reduced replication in hepatic cells compared to wild-type Ad5 but not to the same extent as the miR122-detargeted adenovirus with the neuroendocrine-selective CgA promoter.

CONCLUSIONS/SIGNIFICANCE:

A combination of transcriptional (promoter) and post-transcriptional (miRNA target) regulation to control virus replication may allow for the use of higher doses of adenovirus for efficient tumors treatment without liver toxicity.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-124295 (URN)10.1371/journal.pone.0008916 (DOI)000274114800019 ()20111709 (PubMedID)
Available from: 2010-05-03 Created: 2010-05-03 Last updated: 2015-06-16Bibliographically approved
3. Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells
Open this publication in new window or tab >>Oncolytic adenovirus modified with somatostatin motifs for selective infection of neuroendocrine tumor cells
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2011 (English)In: Gene Therapy, ISSN 0969-7128, E-ISSN 1476-5462, Vol. 18, no 11, 1052-1062 p.Article in journal (Refereed) Published
Abstract [en]

We have previously described the oncolytic adenovirus, Ad(CgA-E1A-miR122), herein denoted Ad5(CgA-E1A-miR122) that selectively replicates in and kills neuroendocrine cells, including freshly isolated midgut carcinoid cells from liver metastases. Ad5(CgA-E1A-miR122) is based on human adenovirus serotype 5 (Ad5) and infects target cells by binding to the coxsackie-adenovirus receptor (CAR) and integrins on the cell surface. Some neuroendocrine tumor (NET) and neuroblastoma cells express low levels of CAR and are therefore poorly transduced by Ad5. However, they often express high levels of somatostatin receptors (SSTRs). Therefore, we introduced cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site for SSTRs in the virus fiber knob. We show that FWKT-modified Ad5 binds to SSTR2 on NET cells and transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5 while it transduces normal hepatocytes at about 50% of Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to greater extent than Ad5, indicating that fiber knob modification may prolong the systematic circulation time. We conclude that modification of adenovirus with the FWKT motif may be beneficial for NET therapy.

Keyword
oncolytic adenovirus, somatostatin, neuroendocrine tumors, carcinoid, neuroblastoma, proximity ligation
National Category
Cell and Molecular Biology Cancer and Oncology Microbiology in the medical area
Research subject
Oncology; Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-146964 (URN)10.1038/gt.2011.54 (DOI)000296889500004 ()21490682 (PubMedID)
Available from: 2011-02-22 Created: 2011-02-22 Last updated: 2012-05-22Bibliographically approved
4. Novel markers for enterochromaffin cells and gastrointestinal neuroendocrine carcinomas
Open this publication in new window or tab >>Novel markers for enterochromaffin cells and gastrointestinal neuroendocrine carcinomas
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2009 (English)In: Modern Pathology, ISSN 0893-3952, E-ISSN 1530-0285, Vol. 22, no 2, 261-272 p.Article in journal (Refereed) Published
Abstract [en]

The gene expression profile of metastasizing serotonin-producing neuroendocrine carcinomas, which arise from enterochromaffin cells in the jejunum and ileum, is still largely unknown. The aim of this study was to identify genes and proteins, which are preferentially expressed by neuroendocrine carcinoma and enterochromaffin cells and therefore potential novel biomarkers and/or therapeutic targets. Six carcinoma specimens and six normal ileal mucosas were profiled by Affymetrix microarrays. Advanced bioinformatics identified differentially and specifically expressed genes, which were validated by quantitative real-time-PCR on tumor cells extracted by laser capture microdissection and normal enterochromaffin cells extracted by immunolaser capture microdissection. We identified six novel marker genes for neuroendocrine carcinoma cells: paraneoplastic antigen Ma2 (PNMA2), testican-1 precursor (SPOCK1), serpin A10 (SERPINA10), glutamate receptor ionotropic AMPA 2 (GRIA2), G protein-coupled receptor 112 (GPR112) and olfactory receptor family 51 subfamily E member 1 (OR51E1). GRIA2 is specifically expressed by neuroendocrine carcinoma cells whereas the others are also expressed by normal enterochromaffin cells. GPR112 and OR51E1 encode proteins associated with the plasma membrane and may therefore become targets for antibody-based diagnosis and therapy. Hierarchical clustering shows high similarity between primary lesions and liver metastases. However, chemokine C-X-C motif ligand 14 (CXCL14) and NK2 transcription factor related locus 3 Drosophila (NKX2-3) are expressed to a lower level in liver metastases than in primary tumors and normal enterochromaffin cells, which implies a role in neuroendocrine carcinoma differentiation. In conclusion, this study provides a list of genes, which possess relatively specific expression to enterochromaffin and neuroendocrine carcinoma cells and genes with differential expression between primary tumors and metastases. We verified six novel marker genes that may be developed as biomarkers and/or therapeutic targets.

Keyword
gastrointestinal neuroendocrine carcinoma, bioinformatics analysis, novel markers
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-102171 (URN)10.1038/modpathol.2008.174 (DOI)000263182500014 ()18953328 (PubMedID)
Available from: 2009-05-05 Created: 2009-05-05 Last updated: 2011-12-09Bibliographically approved

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