Fig. 6.

Phd2 deletion leads to activation of the Akt–mTOR pathway. a RPPA analysis of tumors with homozygous deletion of Phd2. Tumor tissues from Tyr::CreER; BRaf^V600E; Phd2^−/− or Tyr::CreER; BRaf^V600E mice were processed and analyzed by RPPA assays. The analyses identified proteins that were significantly changed in mouse melanomas compared to nevi. b Activation of Akt–mTOR pathway after phd2 deletion. Tumor tissues were processed and western blots showed stabilization of HIF-1α and HIF-2α proteins after Phd2 depletion. Increased phosphorylation of Akt, 4EBP1 and S6K was observed in tumors from Tyr::CreER; BRaf^V600E; Phd2^−/− compared with those of Tyr::CreER; BRaf^V600E mice. c Re-expression of Phd2 inhibits the Akt–mTOR pathway. A BRaf^V600E; Phd2^−/− mouse melanoma cell line was established from melanomas in Tyr::CreER; BRaf^CA; Phd2^lox/lox mice. Phd2 was ectopically reintroduced in these tumor cells. Western blot analysis showed that degradation of HIF-1α and HIF-2α proteins with decreased expression of VEGFR2 decreased phosphorylation of Akt, 4EBP1 and S6K. d Pharmacological inhibition (FM19G11) of HIF pathway in BRaf^V600E; Phd2^−/− melanoma cells. A similar but more pronounced inhibition of the Akt–mTOR pathway was observed using the HIF inhibitor. β-Actin was used as a loading control. Results are representative of three independent experiments