Clinical and experimental data indicate that increased HER2 activity is an important step in breast cancer progression that impacts negatively on patient survival (36
). HER2 signaling provides increased resistance against apoptosis (induced by adverse conditions in the tumor microenvironment or chemotherapy) that is mediated by the PI3K-AKT pathway (31
). Another important consequence of HER2 signaling is increased VEGF expression (38
). We have demonstrated that HER2 signaling in nonhypoxic cells induces transcriptional activation of the VEGF
gene by HIF-1 that is dependent upon PI3K and AKT activity (Fig. ). Furthermore, activity of the downstream kinase FRAP is also required for HIF-1α expression under nonhypoxic conditions. The clinical relevance of these results is underscored by the recent demonstration that HIF-1α overexpression is significantly associated with HER2 and VEGF expression and with microvascular density in human ductal carcinoma in situ and invasive breast cancer (5
FIG. 9 Dual mechanisms for induction of HIF-1α protein and VEGF mRNA expression. Signal transduction from HER2 (and possibly other tyrosine kinases such as EGFR and V-SRC) to PI3K, AKT, and FRAP increases the rate of HIF-1α synthesis, whereas (more ...)
The most surprising result of the present study is the novel finding that activation of the PI3K-AKT-FRAP pathway by heregulin stimulation of MCF-7 human breast cancer cells does not affect HIF-1α stability but instead dramatically increases the rate of HIF-1α protein synthesis, as determined by three independent experimental approaches involving cycloheximide addition, pulse-chase labeling, and reporter gene transfection assays. The effect of heregulin-HER2 signaling is therefore similar to the forced expression of recombinant HIF-1α in transient transfection experiments (12
) in which VHL becomes limiting (54
), resulting in failure to degrade all of the HIF-1α that is expressed under nonhypoxic conditions. In contrast, previous studies have demonstrated that hypoxia and loss of p53 or VHL activity affect HIF-1α protein stability via altered ubiquitination (Fig. ). Whereas hypoxia increases both the stability of HIF-1α protein and its specific transcriptional activity (25
), heregulin-HER2 signaling induces HIF-1α protein synthesis, such that the combination of HER2 overexpression and hypoxia has a synergistic effect on VEGF
gene expression (Fig. B).
Data from cycloheximide-addition experiments suggest that activation of the PI3K-AKT-FRAP pathway by other receptor and nonreceptor tyrosine kinases, including EGFR and V-SRC, also induces HIF-1α protein synthesis (K. Chiles, E. Laughner, P. Taghavi, and G. L. Semenza, unpublished data), although this conclusion will need to be confirmed by pulse-chase analyses. The PI3K-AKT-FRAP pathway may also be activated by physiological stimulation of normal cells, such as the induction of HIF-1α expression in vascular smooth muscle cells exposed to angiotensin II, platelet-derived growth factor BB, or thrombin (42
). Thus, stimulation of HIF-1α synthesis by the PI3K-AKT-FRAP pathway is likely to represent a major mechanism for induction of HIF-1 and its downstream target genes in a variety of physiological and pathophysiological conditions.
The pulse-chase studies demonstrate a requirement for FRAP activity, as rapamycin markedly inhibited heregulin-induced HIF-1α protein synthesis (Fig. A). How does FRAP regulate the rate of HIF-1α synthesis? One possible mechanism involves the phosphorylation of 4E-BP1 by FRAP (14
). The eukaryotic translation initiation factor 4F (eIF-4F) performs the rate-limiting function of recruiting the 40S ribosomal subunit to mRNA, with the eIF-4E subunit binding directly to the 5′ cap structure. 4E-BP1 binds eIF-4E and inhibits its activity. Phosphorylation of 4E-BP1 by FRAP decreases its ability to bind eIF-4E. Thus, FRAP activity positively regulates translation. The other major downstream targets of FRAP are the p70 and p85 kinases, which phosphorylate the S6 protein of the 40S ribosomal subunit. S6 kinases have been shown to control the translation of mRNAs that containing polypyrimidine tracts within their 5′-UTR (8
). The HIF-1α 5′-UTR contains tracts of 8, 9, and 17 pyrimidines downstream of nucleotide +32 (23
). However, 4E-BP1, p70s6k
, and p85s6k
were highly phosphorylated in MCF-7 cells exposed to either serum or heregulin, whereas only heregulin markedly induced HIF-1α expression (Fig. ). Thus, further studies are required to determine whether phosphorylation of 4E-BP1 or S6 kinases is necessary for HIF-1α induction. Phosphorylation of the translation initiation factor eIF-2α has recently been shown to control stress-induced protein synthesis (18
), but the PI3K-AKT-FRAP pathway has not been implicated in this process.
Recent studies have revealed that a consequence of dysregulated expression of multiple tumor suppressor proteins and signal transduction pathways is an increase in HIF-1 transcriptional activity that occurs via three different molecular mechanisms. First, loss of p53 or VHL increases HIF-1α protein expression by interfering with its ubiquitination and proteasomal degradation (7
). Second, RAF/MEK/extracellular signal-regulated kinase signaling stimulates transcription of HIF-1-dependent target genes but does not increase HIF-1α expression, suggesting a direct effect on transactivation (41
). Third, PI3K-AKT-FRAP signaling increases the rate of HIF-1α synthesis, as demonstrated in this study. The consequences of activating signal transduction pathways may be cell type specific, since treatment of mouse embryo fibroblasts with the organomercurial compound mersalyl induces HIF-1α protein expression via a signaling pathway that requires MAP kinase activity (2
). In addition to genetic alterations involving oncogene and tumor suppressor gene products, HIF-1α protein stability and transcriptional activity are also induced by intratumoral hypoxia which, as in the case of HER2 overexpression, is associated with poor clinical outcome (reviewed in reference 49
). The molecular data indicating that multiple genetic and physiological stimuli induce HIF-1 in human cancers are consistent with immunohistochemical data indicating that HIF-1α overexpression occurs frequently in breast and other common human cancers (5
) and correlates with tumor grade and vascularization (5
) and patient survival (1
). Thus, HER2 overexpression does not activate HIF-1-dependent gene transcription in isolation but rather in combination with other tumor-specific genetic and physiological alterations. Taken together, the clinical and molecular studies suggest that increased HIF-1α expression may contribute to tumor progression by mediating angiogenesis, metabolic adaptation, and other aspects of invasion and metastasis that define the lethal cancer phenotype.