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

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.

Figure 1. HIF2α Overrides Tumor Suppression by pVHL

(A) 786-O subclones that were transfected to produce wild-type pVHL (WT8) or with an empty plasmid (PRC3) cells, as well as WT8 cells infected with an empty retrovirus (Empty) or retroviruses encoding the indicated HIF2α variants [ (P→A)² = P405A;P531A and * = bHLH mutation] were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies. (B) In vitro proliferation of WT8 cells infected with the indicated retroviruses. (C) Tumor weights approximately 9 wk after subcutaneous implantation of WT8 cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.

Figure 2. Downregulation of HIF2α Is Sufficient to Suppress Tumor Growth by pVHL-Defective Renal Carcinoma Cells

(A) Parental 786-O cells (VHL[−/−]) and 786-O cells infected with an empty retrovirus (Empty) or retroviruses encoding HIF2α shRNAs (sequence #2 or #3) were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies. (B) In vitro proliferation of 786-O cells infected with the indicated retroviruses. (C) Tumor weights approximately 9 wk after subcutaneous implantation of 786-O 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 cells in left (upper) flank and 786-O cells infected with HIF2α shRNA (#3) retrovirus on right (lower) flank.

Figure 3. 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.

Figure 4. Tumor Suppression by HIF2α shRNA Is Not Restricted to a Single Cell Line

(A) Tumor weights approximately 8 wk after subcutaneous implantation of A498 cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error. (B) Representative histological sections after staining with hematoxylin and eosin of tumors formed by A498 cells infected with the indicated retroviruses.