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Cited by 16Virus-based gene therapy researchCancer, Hypoxia, and MetabolismAngiogenesis and VEGF in Cancer

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Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma

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Shneor, D, Folberg, R, Pe'er, J, Honigman, A, Frenkel, S·2017·Cancer Gene Therapy
DOI 10.1038/cgt.2016.68PMC PMC5339434
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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.

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Figure 1 A schematic presentation of the various replication-competent viruses (RCRs). (a) A proviral construct of pACE-GFP. (b–f) The IRES-GFP sequences were replaced with an H1 promoter driving the transcription of the various small hairpin RNAs (shRNA) targeting cyclic AMP-response-element binding protein (CREB) (b), hypoxia-inducible factor (HIF)-1 (c), HIF-2 (d), all three genes (e) and a non-target sequence (f). The DNA sequence of the various shRNAs is marked.
Figure 1 A schematic presentation of the various replication-competent viruses (RCRs). (a) A proviral construct of pACE-GFP. (b–f) The IRES-GFP sequences were replaced with an H1 promoter driving the transcription of the various small hairpin RNAs (shRNA) targeting cyclic AMP-response-element binding protein (CREB) (b), hypoxia-inducible factor (HIF)-1 (c), HIF-2 (d), all three genes (e) and a non-target sequence (f). The DNA sequence of the various shRNAs is marked.CC BY-NC-ND · PubMed Central
Figure 2 Quantification of the efficiency of the knockdown in infected cell lines and the effect on the expression of regulated genes. Cells, HepG2 and FLC4 were infected with each of the recombinant replication-competent retroviruses (RCRs) as specified in the figure and cultured in hypoxic conditions for 24 h. Purified mRNA was quantified by real-time quantitative PCR. Levels of messenger RNA (mRNA) were normalized to mRNAs in cells infected with vACE-NT (set as 100%) (a–c). Proteins were quantified following western blot analysis (g), and band intensities were compared with those of α-tubulin and to band intensity of cells infected with vACE-NT (set as 100%) (d–f). Levels of all measured mRNAs (a–c) and proteins (d–f) were statistically significantly lower when compared with vACE-NT-infected cells in both cell lines (P<0.05). HepG2 cells infected with the various RCRs were transiently transfected with either pCRE-LUC or pHRE-LUC. Luciferase activity was determined after 48 h of exposure to hypoxia relative to cells infected with vACE-NT (presented as 100%) for each transfection. CRE (dark columns) and hypoxia-response elements (HRE) (light columns) mediated LUC activity are presented in relative light units (P<0.001) (h). The levels of vascular endothelial growth factor (VEGF) secreted by HepG2 cells were determined by enzyme-linked immunosorbent assays as described in the Materials and methods section. The VEGF secretion levels in hypoxia relative to normoxia were normalized to the ratio in vACE-NT-infected cells (P<0.01) (i). α-Tub=α-tubulin. Bars represent mean+s.d. for each measurement.
Figure 2 Quantification of the efficiency of the knockdown in infected cell lines and the effect on the expression of regulated genes. Cells, HepG2 and FLC4 were infected with each of the recombinant replication-competent retroviruses (RCRs) as specified in the figure and cultured in hypoxic conditions for 24 h. Purified mRNA was quantified by real-time quantitative PCR. Levels of messenger RNA (mRNA) were normalized to mRNAs in cells infected with vACE-NT (set as 100%) (a–c). Proteins were quantified following western blot analysis (g), and band intensities were compared with those of α-tubulin and to band intensity of cells infected with vACE-NT (set as 100%) (d–f). Levels of all measured mRNAs (a–c) and proteins (d–f) were statistically significantly lower when compared with vACE-NT-infected cells in both cell lines (P<0.05). HepG2 cells infected with the various RCRs were transiently transfected with either pCRE-LUC or pHRE-LUC. Luciferase activity was determined after 48 h of exposure to hypoxia relative to cells infected with vACE-NT (presented as 100%) for each transfection. CRE (dark columns) and hypoxia-response elements (HRE) (light columns) mediated LUC activity are presented in relative light units (P<0.001) (h). The levels of vascular endothelial growth factor (VEGF) secreted by HepG2 cells were determined by enzyme-linked immunosorbent assays as described in the Materials and methods section. The VEGF secretion levels in hypoxia relative to normoxia were normalized to the ratio in vACE-NT-infected cells (P<0.01) (i). α-Tub=α-tubulin. Bars represent mean+s.d. for each measurement.CC BY-NC-ND · PubMed Central
Figure 3 The role of cyclic AMP-response-element binding protein (CREB), hypoxia-inducible factor (HIF)-1 and HIF-2 in protecting HepG2 and FLC4 cells from hypoxia-induced apoptosis and on the response to treatment with doxorubicin (DOX). HepG2 (a, c) and FLC4 (b, d) infected with each of the viruses specified in the figure were cultured for 72 h. Viability (a, b) and activation of caspase-3 (Cas3) (c, d) were determined every 24 h. The relative viability and activation of caspase-3 after 72 h of hypoxia (light gray columns), and those after treatment with 1 mm of DOX in normoxia (black columns) and hypoxia (dark gray columns) were normalized to cells infected with each respective replication competent retroviruses in normoxia (P<0.01 for all comparisons). Bars represent mean+s.d. for each measurement.
Figure 3 The role of cyclic AMP-response-element binding protein (CREB), hypoxia-inducible factor (HIF)-1 and HIF-2 in protecting HepG2 and FLC4 cells from hypoxia-induced apoptosis and on the response to treatment with doxorubicin (DOX). HepG2 (a, c) and FLC4 (b, d) infected with each of the viruses specified in the figure were cultured for 72 h. Viability (a, b) and activation of caspase-3 (Cas3) (c, d) were determined every 24 h. The relative viability and activation of caspase-3 after 72 h of hypoxia (light gray columns), and those after treatment with 1 mm of DOX in normoxia (black columns) and hypoxia (dark gray columns) were normalized to cells infected with each respective replication competent retroviruses in normoxia (P<0.01 for all comparisons). Bars represent mean+s.d. for each measurement.CC BY-NC-ND · PubMed Central
Figure 4 Testing the effect of cyclic AMP-response-element binding protein (CREB), HIF-1 and 2 on tumor growth in vivo in a mouse xenograft model. Severe combined immunodeficiency mice were implanted with 4 × 106 HepG2 cells as described in Materials and methods section. Tumor growth was monitored every 7 days with the IVIS InVivo System. Images of light emission from representative mice taken on days 7 and 35 are presented. The same color scale was used for all mice at both time points (a). Relative light units (RLU) measurements were normalized to the readings on day 7 post injection and plotted along the extent of the experiment. Each point represents the mean+s.e. (b). At the end of the experiment, tumors were harvested for histopathological analysis. Hematoxylin–eosin staining and the corresponding vascular endothelial growth factor immunohistochemistry of tumors are presented in (c). ( × 4 magnification, scale bar 100 μm).
Figure 4 Testing the effect of cyclic AMP-response-element binding protein (CREB), HIF-1 and 2 on tumor growth in vivo in a mouse xenograft model. Severe combined immunodeficiency mice were implanted with 4 × 106 HepG2 cells as described in Materials and methods section. Tumor growth was monitored every 7 days with the IVIS InVivo System. Images of light emission from representative mice taken on days 7 and 35 are presented. The same color scale was used for all mice at both time points (a). Relative light units (RLU) measurements were normalized to the readings on day 7 post injection and plotted along the extent of the experiment. Each point represents the mean+s.e. (b). At the end of the experiment, tumors were harvested for histopathological analysis. Hematoxylin–eosin staining and the corresponding vascular endothelial growth factor immunohistochemistry of tumors are presented in (c). ( × 4 magnification, scale bar 100 μm).CC BY-NC-ND · PubMed Central
Figure 5 Immunohistochemical analysis of hypoxia and blood vessels (CD34) of the tumors. Tumors infected with replication-competent retroviruses expressing small hairpin RNA (shRNA)-targeting genes specified on the left column were stained for hypoxia (left column) and for blood vessels (right column) as described in the Materials and methods section. It is clear that knockdown of CREB, HIF-1 and all three genes together ( × 3) abolished neovascularization within the tumors. In tumors infected with the non-target shRNA or with shRNA targeting HIF-2 blood vessel growth toward the hypoxic regions is noticed. Correlation between the amount of CD34 and hypoxia staining is presented in the top right column for each virus. ( × 10 magnification, scale bar 500 μm).
Figure 5 Immunohistochemical analysis of hypoxia and blood vessels (CD34) of the tumors. Tumors infected with replication-competent retroviruses expressing small hairpin RNA (shRNA)-targeting genes specified on the left column were stained for hypoxia (left column) and for blood vessels (right column) as described in the Materials and methods section. It is clear that knockdown of CREB, HIF-1 and all three genes together ( × 3) abolished neovascularization within the tumors. In tumors infected with the non-target shRNA or with shRNA targeting HIF-2 blood vessel growth toward the hypoxic regions is noticed. Correlation between the amount of CD34 and hypoxia staining is presented in the top right column for each virus. ( × 10 magnification, scale bar 500 μm).CC BY-NC-ND · PubMed Central
Figure 6 Effect of combined treatment on tumor growth monitored by light emission. HepG2 cells stably expressing the luc gene and infected with the viruses specified above were implanted subcutaneous in severe combined immunodeficiency (SCID) mice. After 3 weeks, the mice were injected twice a week with doxorubicin (DOX) in the concentrations specified in the picture. Tumor growth was monitored by light emission as described in Figure 4. (a) One mouse from each group at day 42 is depicted. A scale bar of light emission is localized on the right side of the picture. (b) The average (+s.d.) relative tumor growth rate between days 14 and 42 is presented on each bar.
Figure 6 Effect of combined treatment on tumor growth monitored by light emission. HepG2 cells stably expressing the luc gene and infected with the viruses specified above were implanted subcutaneous in severe combined immunodeficiency (SCID) mice. After 3 weeks, the mice were injected twice a week with doxorubicin (DOX) in the concentrations specified in the picture. Tumor growth was monitored by light emission as described in Figure 4. (a) One mouse from each group at day 42 is depicted. A scale bar of light emission is localized on the right side of the picture. (b) The average (+s.d.) relative tumor growth rate between days 14 and 42 is presented on each bar.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Shneor, D., Folberg, R., Pe'er, J., Honigman, A., & Frenkel, S.
Affiliations
  • Hebrew University of Jerusalem (IL)
  • Oakland University (US)
  • Hadassah Medical Center (IL)
Corresponding author
Frenkel, S
ORCID
Venue
Cancer Gene Therapy
Publisher
Springer Science and Business Media LLC
Published
2016-12-09
Volume
24
Issue
2
Pages
64-74
ISSN
  • 0929-1903 (ISSN-L)
  • 1476-5500
DOI
10.1038/cgt.2016.68
PMC
PMC5339434
OpenAlex
W2560657110
Cited by
16
References
4
Citations by year
2026: 1 · 2024: 1 · 2023: 3 · 2021: 2 · 2020: 2 · 2019: 5 · 2018: 1 · 2017: 1
FWCI
1.89
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Gene Therapy (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Hebrew University of Jerusalem
  • Israel Cancer Association
  • Israel Science Foundation
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
Keywords
hepatocellular carcinoma, tumor hypoxia, CREB, HIF-1, HIF-2, murine leukemia virus, shRNA, gene knockdown, vascular endothelial growth factor, tumor angiogenesis, doxorubicin combination therapy, caspase-3, tumor growth inhibition
MeSH terms
Endothelial PAS Domain-Containing Protein 1, Animals, Hypoxia, Disease Models, Animal, Genetic Vectors, Carcinoma, Hepatocellular, Humans, Liver Neoplasms, Neovascularization, Pathologic, Retroviridae, Biomarkers, Gene Expression Regulation, Neoplastic, Cyclic AMP Response Element-Binding Protein, Xenograft Model Antitumor Assays, Gene Order, RNA, Small Interfering, Cell Line, Tumor, Tumor Burden, Cell Proliferation, Mice, Basic Helix-Loop-Helix Transcription Factors, Basic Helix-Loop-Helix Proteins, Hypoxia-Inducible Factor 1, Gene Knockdown Techniques

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CoChat. (2026). Citation check: Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma. https://app.cochat.ai/papers/10.1038/cgt.2016.68
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Shneor, D., Folberg, R., Pe'er, J., Honigman, A., & Frenkel, S. (2017). Stable knockdown of CREB, HIF-1 and HIF-2 by replication-competent retroviruses abrogates the responses to hypoxia in hepatocellular carcinoma. Cancer Gene Therapy. https://doi.org/10.1038/cgt.2016.68