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Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth.

Kondo K, Kim WY, Lechpammer M, Kaelin WG - PLoS Biol. (2003)

Bottom Line: Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities.Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells.These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2alpha variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

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Effect of HIF2α shRNA Is Specifically Due to Downregulation of HIF2α(A) Parental 786-O cells (VHL[−/−]) and 786-O cells stably producing HIF2α shRNA #3 that were coinfected with an empty retrovirus (Empty) or a retrovirus encoding a HIF2α mRNA with three silent mutations in the #3 recognition site (MT*) were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of 786-O HIF2α shRNA #3 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of 786-O HIF2α shRNA cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.(D) Representative photograph of nude mouse 9 wk after subcutaneous injection of 786-O HIF2α shRNA #3 cells in left (upper) flank and 786-O HIF2α shRNA #3 cells infected with retrovirus encoding HIF2α MT* mRNA on right (lower) flank.
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pbio.0000083-g003: Effect of HIF2α shRNA Is Specifically Due to Downregulation of HIF2α(A) Parental 786-O cells (VHL[−/−]) and 786-O cells stably producing HIF2α shRNA #3 that were coinfected with an empty retrovirus (Empty) or a retrovirus encoding a HIF2α mRNA with three silent mutations in the #3 recognition site (MT*) were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of 786-O HIF2α shRNA #3 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of 786-O HIF2α shRNA cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.(D) Representative photograph of nude mouse 9 wk after subcutaneous injection of 786-O HIF2α shRNA #3 cells in left (upper) flank and 786-O HIF2α shRNA #3 cells infected with retrovirus encoding HIF2α MT* mRNA on right (lower) flank.

Mentions: To ask whether inhibition of HIF2α is likewise sufficient for tumor suppression by pVHL, we set out to inhibit HIF2α in VHL(−/−) renal carcinoma cells using short hairpin RNAs (shRNA). We tested five HIF2α shRNAs based on 19mer sequences that are unique to HIF2α according to GenBank. Two such shRNAs (#2 and #3) decreased HIF2α protein levels, as determined by anti-HIF2α immunoblot analysis and by diminished activity of a cotransfected HRE–luciferase reporter plasmid, when transiently introduced into 786-O cells (data not shown). Infection of 786-O cells with retroviruses encoding shRNA #2 or #3, but not the parental retrovirus, led to decreased steady-state levels of HIF2α protein as well as decreased levels of GLUT1, which is encoded by a HIF-responsive gene (Figure 2A). Downregulation of HIF2α did not affect cell growth in vitro, but was sufficient to impair tumor growth in vivo (Figure 2B–2D). The former observation is consistent with the finding that pVHL does not inhibit cell proliferation under standard cell culture conditions and argues against the idea that the latter was due to nonspecific toxicity. Moreover, these in vivo effects could be prevented by coadministration of a retrovirus encoding an HIF2α mRNA with silent third-base mutations within the shRNA recognition site (Figure 3) and were not observed with retroviruses encoding a scrambled HIF2α shRNA or luciferase shRNA (data not shown). Thus, tumor suppression by the HIF2α shRNA was unlikely to reflect a spurious interaction with an unintended target.


Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth.

Kondo K, Kim WY, Lechpammer M, Kaelin WG - PLoS Biol. (2003)

Effect of HIF2α shRNA Is Specifically Due to Downregulation of HIF2α(A) Parental 786-O cells (VHL[−/−]) and 786-O cells stably producing HIF2α shRNA #3 that were coinfected with an empty retrovirus (Empty) or a retrovirus encoding a HIF2α mRNA with three silent mutations in the #3 recognition site (MT*) were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of 786-O HIF2α shRNA #3 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of 786-O HIF2α shRNA cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.(D) Representative photograph of nude mouse 9 wk after subcutaneous injection of 786-O HIF2α shRNA #3 cells in left (upper) flank and 786-O HIF2α shRNA #3 cells infected with retrovirus encoding HIF2α MT* mRNA on right (lower) flank.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC300692&req=5

pbio.0000083-g003: Effect of HIF2α shRNA Is Specifically Due to Downregulation of HIF2α(A) Parental 786-O cells (VHL[−/−]) and 786-O cells stably producing HIF2α shRNA #3 that were coinfected with an empty retrovirus (Empty) or a retrovirus encoding a HIF2α mRNA with three silent mutations in the #3 recognition site (MT*) were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of 786-O HIF2α shRNA #3 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of 786-O HIF2α shRNA cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.(D) Representative photograph of nude mouse 9 wk after subcutaneous injection of 786-O HIF2α shRNA #3 cells in left (upper) flank and 786-O HIF2α shRNA #3 cells infected with retrovirus encoding HIF2α MT* mRNA on right (lower) flank.
Mentions: To ask whether inhibition of HIF2α is likewise sufficient for tumor suppression by pVHL, we set out to inhibit HIF2α in VHL(−/−) renal carcinoma cells using short hairpin RNAs (shRNA). We tested five HIF2α shRNAs based on 19mer sequences that are unique to HIF2α according to GenBank. Two such shRNAs (#2 and #3) decreased HIF2α protein levels, as determined by anti-HIF2α immunoblot analysis and by diminished activity of a cotransfected HRE–luciferase reporter plasmid, when transiently introduced into 786-O cells (data not shown). Infection of 786-O cells with retroviruses encoding shRNA #2 or #3, but not the parental retrovirus, led to decreased steady-state levels of HIF2α protein as well as decreased levels of GLUT1, which is encoded by a HIF-responsive gene (Figure 2A). Downregulation of HIF2α did not affect cell growth in vitro, but was sufficient to impair tumor growth in vivo (Figure 2B–2D). The former observation is consistent with the finding that pVHL does not inhibit cell proliferation under standard cell culture conditions and argues against the idea that the latter was due to nonspecific toxicity. Moreover, these in vivo effects could be prevented by coadministration of a retrovirus encoding an HIF2α mRNA with silent third-base mutations within the shRNA recognition site (Figure 3) and were not observed with retroviruses encoding a scrambled HIF2α shRNA or luciferase shRNA (data not shown). Thus, tumor suppression by the HIF2α shRNA was unlikely to reflect a spurious interaction with an unintended target.

Bottom Line: Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities.Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells.These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2alpha variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

Show MeSH
Related in: MedlinePlus