The signal transduction events that modulate the expression of HIF-1α, as well as the subsequent expression of VEGF
and other HIF-1-regulated genes, are currently under intensive scrutiny. The results of the present study confirm and extend the earlier report that rapamycin inhibits both the stabilization of HIF-1α and the transcriptional activity of HIF-1 in hypoxic cancer cells (50
). Furthermore, we provide genetic evidence to support the conclusion that the rapamycin target protein, mTOR, functions as a positive regulator of HIF-1 activation by hypoxia or the hypoxia-mimetic agent, CoCl2
A synthesis of the available data indicates that at least two integrated signaling pathways promote the accumulation of HIF-1α in mammalian cells. The first pathway is triggered by hypoxia or CoCl2
and involves the inhibition of a family of PHDs that modify Pro-564 and Pro-402 of HIF-1α (5
). The second pathway is triggered by polypeptide growth factors or oncogenic mutations (e.g., PTEN
gene loss) and leads to the activation of PI 3-kinase and its downstream targets, AKT and the Rac GTPases (17
). Although it seems clear that hypoxia regulates HIF-1α turnover at the level of degradation rather than synthesis (26
), PI 3-kinase-dependent signals appear to regulate both the synthesis and stabilization of HIF-1α in hypoxic tumor cells (26
The findings presented here substantially strengthen previous observations that mTOR-dependent signals stimulate HIF-1α accumulation and HIF-1-mediated transcription in cells exposed to hypoxia or hypoxia-mimetic agents (50
). As appears to be the case for PI 3-kinase, our results indicate that the rapamycin-sensitive functions of mTOR are not essential for the accumulation of HIF-1α but are needed for maximal expression of this protein, as well as for optimal HIF-1-dependent gene expression under hypoxic conditions. The notion that PI 3-kinase and mTOR both serve as amplifiers rather than essential triggers of HIF-1 activation is consistent with the model that these two signaling kinases reside in the same signaling pathway (33
). However, the evidence that supports a functional linkage between PI 3-kinase and mTOR is not definitive and, at this stage, we cannot exclude the alternative possibility that PI 3-kinase and mTOR converge on the HIF-1 regulatory machinery through parallel pathways. The proposed function of mTOR as a nutrient sensor (16
) may be particularly relevant to HIF-1 function, since decreased oxygen tensions are almost inevitably accompanied by limited supplies of glucose and amino acids in mammalian tissues.
Our initial studies demonstrated that pretreatment of PC-3 cells with rapamycin strongly inhibited the increase in HIF-1-dependent reporter gene expression provoked by hypoxia and CoCl2
. These results are in general agreement with those reported by Zhong et al., who observed that rapamycin exposure inhibited epidermal growth factor and phorbol myristate acetate-induced secretion of VEGF, a HIF-1-regulated gene product, from TSU prostate cancer cells (50
). On the other hand, our finding that rapamycin suppresses HIF-1-mediated gene transcription in PC-3 cells seems at odds with an earlier study, which demonstrated that rapamycin exposure had no effect on hypoxia-induced VEGF promoter activity in Ha-Ras
-transformed NIH 3T3 cells (29
). The latter findings suggest that mTOR is not an obligate intermediate in the transmission of activating signals to HIF-1 and that the host cell type, together with the particular oncogenic background, play determinant roles in the cellular response to mTOR inhibition by rapamycin. Further understanding of the signaling inputs that govern HIF-1α turnover will be critical in the event that rapamycin or other PI 3-kinase/mTOR inhibitors are approved for clinical use as cytostatic and/or cytotoxic agents in patients with solid tumors. This information may facilitate the selection of patients who are most likely to benefit from therapy with rapamycin or related drugs.
As previously reported, we observed that hypoxia- or CoCl2
-induced HIF-1 activation was strongly suppressed by cellular treatment with the PI 3-kinase inhibitor, LY294002 (50
). The inhibitory effects of LY294002 on both HIF-1α accumulation (50
) and HIF-1-dependent transcription (Fig. in the present study) are consistent with the idea that PI 3-kinase is involved in hypoxia-induced signaling to HIF-1 (10
). However, LY294002 is not a specific inhibitor of PI 3-kinase; indeed, this drug suppresses mTOR kinase activity at concentrations similar to those required for PI 3-kinase inhibition (9
). Therefore, although PI 3-kinase activity is strongly implicated in the stimulation of HIF-1α by growth factors (26
), its role in hypoxia-induced HIF-1α stabilization requires further investigation.
The inhibitory effect of rapamycin on HIF-1α accumulation indicated that this drug either decreased the rate of HIF-1α synthesis or increased the rate of HIF-1α degradation in hypoxic cells. By using a proteasome inhibitor to block the major pathway of HIF-1α degradation, we found that rapamycin treatment had little effect on the accumulation of HIF-1α in PC-3 cells cultured under reduced serum (either 2 or 0.1% FBS) conditions. On the other hand, rapamycin exposure significantly decreased the stability of HIF-1α in hypoxic PC-3 cells, when ongoing synthesis of HIF-1α protein was blocked with CHX. Collectively, these results suggest that rapamycin decreases the steady-state level of HIF-1α in PC-3 cells primarily through interference with the mechanism that promotes the stabilization of this protein under hypoxic conditions.
The negative effect of rapamycin on HIF-1α stability was somewhat unexpected, based on the recent report by Semenza and coworkers (26
). These investigators demonstrated that stimulation of HER2 receptors in normoxic cells increased the expression of HIF-1α via a pathway that involved PI 3-kinase, AKT, and mTOR. However, in contrast to the present findings in hypoxic PC-3 cells, HER2 receptor-mediated HIF-1α accumulation occurred primarily through the upregulation of HIF-1α synthesis, due largely to the stimulation of HIF-1α mRNA translation. These authors further demonstrated that the 5′-untranslated region of the HIF-1α mRNA contains a translational control element that is upregulated by HER2 receptor occupancy and is sensitive to inhibition by rapamycin. The latter result strongly implicates mTOR as a positive regulator of HIF-1α translation, a scenario that is highly reminiscent of the stimulatory roles of mTOR in the synthesis of ribosomal and other polypyrimidine tract-containing proteins in mitogen-stimulated cells (16
). Because hypoxia affects the turnover of HIF-1α at the level of degradation rather than synthesis (26
), the translational effect of mTOR might support, but should not drive, the increase in HIF-1α expression in hypoxic cells, particularly under conditions of limiting growth factor and nutrient availability. Our finding that rapamycin interferes with the stabilization of HIF-1α under hypoxic conditions is therefore of particular importance and strongly suggests that mTOR-dependent signals promote HIF-1α expression at the levels of both synthesis and proteolytic degradation. The nature of the stimulus (i.e., polypeptide hormone stimulation versus hypoxia), together with the cellular background, likely determine which of these signaling inputs from mTOR exerts dominant control over the increase in HIF-1α protein in different tumors.
The importance of mTOR in the transduction of hypoxia/CoCl2-initiated signals to HIF-1α was underscored by the results of genetic experiments involving the expression of wild-type or mutated mTOR constructs in PC-3 cells. Overexpression of wild-type mTOR significantly increased the level of HIF-1-dependent reporter gene expression provoked by exposure of the engineered cell lines to the hypoxia-mimetic agent, CoCl2 (Fig. ), as well as hypoxia itself (unpublished results). These results suggest that the endogenous level of mTOR activity in PC-3 cells limits the magnitude of the HIF-1-dependent transcriptional response to these stimuli. Furthermore, expression of the rapamycin-resistant AmTOR-SI mutant in the same cells abrogates the inhibitory effects of rapamycin on CoCl2-stimulated HIF-1α accumulation and HIF-1-dependent transcription. Thus, the results obtained with AmTOR-SI-transfected PC-3 sublines provide genetic evidence to support the conclusion that rapamycin suppresses hypoxia/CoCl2-induced HIF-1 function through inhibition of a single target protein, mTOR.
Transfection experiments with a panel of G4-HIF-1α fusion proteins pinpointed the ODD domain as an important target for the mTOR signaling pathway that amplifies HIF-1α stabilization at low oxygen tensions. In normoxic PC-3 cells, expression of the G4-HIF-1α (residues 498 to 603) construct was relatively low due to proteasome-mediated degradation of the fusion protein. This construct contains the carboxyl-terminal half of the ODD domain and includes the critical Pro-564 residue, which undergoes PHD-dependent hydroxylation in hypoxic cells (41
). Exposure of the cells to hypoxia or to the PHD inhibitor CoCl2
(unpublished results) led to significant stabilization of HIF-1α, which is consistent with the model that the ODD domain, and specifically the region surrounding Pro-564, confers instability on the HIF-1α protein at normal oxygen tensions (5
). The hypoxia-induced increase in G4-HIF-1α (residues 498 to 603) expression was strongly suppressed by rapamycin, whereas the expression of G4 fusion proteins containing amino- or carboxyl-terminal fragments of HIF-1α was neither hypoxia-inducible nor sensitive to rapamycin (see Fig. and unpublished results). We conclude from these findings that the mTOR-dependent signals that promote HIF-1α stabilization under hypoxic conditions impinge largely, if not entirely, on the ODD domain. Moreover, the suppressive effect of rapamycin on the expression of the chimeric G4-HIF-1α (residues 498 to 603) protein substantiates the conclusion that, in the setting of hypoxia/CoCl2
stimulation, the drug is not simply acting as a 5′-untranslated-region-dependent inhibitor of HIF-1α mRNA translation.
The exact mechanism whereby mTOR contributes to the stabilization of HIF-1α in hypoxic cells remains unclear. Given the complexity of the oxygen-responsive machinery that governs the stability of HIF-1α, the list of potential mTOR substrates in this pathway is extensive. Based on the results described above, we considered the ODD domain a potential site of intersection for both mTOR- and oxygen-dependent signals that govern HIF-1α stability. The ODD domain is serine- and threonine-rich and contains numerous Ser/Thr-Pro sites that represent potential targets for the mTOR kinase domain (7
). Consequently, we examined the ability of a series of glutathione S
-transferase-HIF-1α fusion proteins to serve as substrates for mTOR in immune complex kinase assays. The results revealed that the ODD domain was the only region of HIF-1α that was detectably phosphorylated by mTOR in vitro (unpublished results). However, the stoichiometry of ODD phosphorylation by mTOR was quite low, and additional studies will be required to determine the significance of this finding. We also cannot rule out the possibility that mTOR modulates the activities of the PHDs that modify HIF-1α or interferes with the recognition of proline-hydroxylated HIF-1α by the VHL-ubiquitin E3 ligase complex.
In summary, this study identifies mTOR as a positive regulator of HIF-1-dependent gene transcription in cells exposed to hypoxia or hypoxia-mimetic agents. Our results suggest that mTOR plays a key role in the transmission of signals that lead to the adaptation of mammalian cells to oxygen- and nutrient-poor environmental conditions. It is noteworthy that studies in budding yeast have established the mTOR orthologs, TOR1p and TOR2p, as central regulators of the pathway that coordinates nutrient availability with yeast growth (37
). The particular demands placed on nutrient-deprived cells sequestered in bodily tissues may have provided the evolutionary driving force for the insertion of mTOR into the hypoxia response pathway in metazoan organisms. Finally, our findings add further support to the idea that mTOR is an intriguing target for anticancer therapy (30
). If rapamycin or other mTOR inhibitors prove to be effective inhibitors of hypoxic adaptation in developing tumors, these drugs could have dramatic effects on tumor growth, invasiveness, and metastatic potential in human cancer patients.