title: Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma.
abstract: The fast proliferation of tumor cells develops faster than the vasculature, resulting, in most malignant tumors, in generation of hypoxic regions. Hypoxia renders solid tumors resistant to radiation and chemotherapeutics while providing opportunities for tumor-selective therapies targeting tumor hypoxia. Here we exploit two properties of tumors: propagation of tumor cells and ongoing generation of hypoxic regions to construct a system that preferentially leads to the death of tumor cells and thus hinders tumor growth. We constructed murine leukemia virus replication-competent (RCR) viruses that infect only propagating cells. These viruses express small hairpin RNAs (shRNAs) targeting cyclic AMP-response-element binding protein (CREB), hypoxia-inducible factors 1 (HIF)-1 or HIF-2 individually or all three together (X3). These viruses efficiently infected in vitro human hepatocellular carcinoma (HepG2 and FLC4) cells and established persistence of the virus and knocked down the expression of the regulators of the hypoxia-responding genes. Knockdown of either HIF-1 or CREB or both in hypoxia reduced the expression of hypoxia-response elements- and CRE-mediated gene expression, diminished cell proliferation and increased caspase-3 activity. We did not detect any significant effect of the efficiently knocked down HIF-2 on any of the functions tested in vitro. Moreover, severe combined immunodeficiency mice implanted subcutaneously with HepG2 stably infected with recombinant RCRs showed reduction of tumor growth and vascular endothelial growth factor expression, and no hypoxia-guided neovascularization. Combined treatment (RCRs+doxorubicin) improved efficacy in the context of in vitro hypoxia and in vivo (with either vACE-CREB or vACE-X3). This synergistic effect may lead to an improved efficacy and safety profile of the treatment that may result in fewer side effects.
authors.affiliation: Department of Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.|Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Shneor
authors.affiliation: Departments of Pathology, Ophthalmology and Biomedical Sciences, Oakland University William Beaumont School of Medicine, Rochester, MI, USA.
authors.forename: R
authors.initials: R
authors.lastname: Folberg
authors.affiliation: Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: J
authors.initials: J
authors.lastname: Pe'er
authors.affiliation: Department of Biochemistry and Molecular Biology, IMRIC, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.|Department of Biotechnology, Hadassah Academic College, Jerusalem, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Honigman
authors.affiliation: Department of Ophthalmology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.
authors.forename: S
authors.identifier: http://orcid.org/0000-0002-3688-8676
authors.initials: S
authors.lastname: Frenkel
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000378:metabolism; D015415:Biomarkers; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics* / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009389:Neovascularization, Pathologic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D047368:Tumor Burden / Q000235:genetics; D023041:Xenograft Model Antitumor Assays; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D015415:Biomarkers; D017362:Cyclic AMP Response Element-Binding Protein; D051793:Hypoxia-Inducible Factor 1; D034741:RNA, Small Interfering; D000099162:Endothelial PAS Domain-Containing Protein 1