The hypoxia-inducible factor 1 transcriptional activator complex (HIF-1) is involved in the activation of the erythropoietin and several other hypoxia-responsive genes. The HIF-1 complex is composed of two protein subunits: HIF-1␤/ARNT (aryl hydrocarbon receptor nuclear translocator), which is constitutively expressed, and HIF-1␣, which is not present in normal cells but induced under hypoxic conditions. The HIF-1␣ subunit is continuously synthesized and degraded under normoxic conditions, while it accumulates rapidly following exposure to low oxygen tensions. The involvement of the ubiquitin-proteasome system in the proteolytic destruction of HIF-1 in normoxia was studied by the use of specific inhibitors of the proteasome system. Lactacystin and MG-132 were found to protect the degradation of the HIF-1 complex in cells transferred from hypoxia to normoxia. The same inhibitors were able to induce HIF-1 complex formation when added to normoxic cells.

One of the key mediators of the hypoxic response in animal cells is the hypoxia-inducible transcription factor-1 (HIF-1) complex, in which the ␣-subunit is highly susceptible to oxygen-dependent degradation. The hypoxic response is manifested in many pathophysiological processes such as tumor growth and metastasis. During hypoxia, cells shift to a primarily glycolytic metabolic mode for their energetic needs. This is also manifested in the HIF-1-dependent up-regulation of many glycolytic genes. Paradoxically, tumor cells growing under conditions of normal oxygen tension also show elevated glycolytic rates that correlate with the increased expression of glycolytic enzymes and glucose transporters (the Warburg effect). A key regulator of glycolytic flux is the relatively recently discovered fructose-2,6-bisphosphate (F-2,6-P2), an allosteric activator of 6-phosphofructo-1-kinase (PFK-1). Steady state levels of F-2,6-P2 are maintained by the bifunctional enzyme PFK-2/F2,6-Bpase, which has both kinase and phosphatase activities. Herein, we show that one isozyme, PFKFB3, is highly induced by hypoxia and the hypoxia mimics cobalt and desferrioxamine. This induction could be replicated by the use of an inhibitor of the prolyl hydroxylase enzymes responsible for the von Hippel Lindau (VHL)-dependent destabilization and tagging of HIF-1␣. The absolute dependence of the PFKFB3 gene on HIF-1 was confirmed by its overexpression in VHL-deficient cells and by the lack of hypoxic induction in mouse embryonic fibroblasts conditionally nullizygous for HIF-1␣.

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Adaptation to hypoxic microenvironment is critical for tumor survival and metastatic spread. Hypoxiainducible factor 1␣ (HIF-1␣) plays a key role in this adaptation by stimulating the production of proangiogenic factors and inducing enzymes necessary for anaerobic metabolism. Histone deacetylase inhibitors (HDACIs) produce a marked inhibition of HIF-1␣ expression and are currently in clinical trials partly based on their potent antiangiogenic effects. Although it has been postulated that HDACIs affect HIF-1␣ expression by enhancing its interactions with VHL (von Hippel Lindau), thus promoting its ubiquitination and degradation, the actual mechanisms by which HDACIs decrease HIF-1␣ levels are not clear. Here, we present data indicating that HDACIs induce the proteasomal degradation of HIF-1␣ by a mechanism that is independent of VHL and p53 and does not require the ubiquitin system. This degradation pathway involves the enhanced interaction of HIF-1␣ with HSP70 and is secondary to a disruption of the HSP70/HSP90 axis function that appears mediated by the activity of HDAC-6.

RepoRtRecently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancerassociated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, AtG16L, BNIp3, BNIp3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSo, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. thus, it appears that oxidative stress mediated induction of HIF1-and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α-and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of tIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIp3L. taken together, our results provide direct support for the "autophagic tumor stroma model of cancer metabolism", and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. our results imply that cancer patients lacking stromal Cav-1 should benefit...