When oxygen demand is greater than oxygen supply, cells need to rapidly adjust their metabolism in order for the tissue to survive. Oxygen sensing by an organism influences a host of processes including growth, development, metabolism, pH homeostasis, and angiogenesis. Hypoxia also contributes to a wide number of human diseases including vascular disease, inflammatory conditions and cancer. Recently, major advances have been made in understanding the response of cells and tissues to hypoxia with the goal of providing mechanistic insight and novel therapeutic targets. In this article we review both the normal biological effects of hypoxia as well as the alterations that occur in specific disease conditions with an emphasis on the cell signaling and gene transcription mechanisms that underlie the changes associated with chronic hypoxia. Comparisons of studies in the fields of cardiac ischemia and tumor angiogenesis reveal the complexities within the microenvironment that control responses to hypoxia. It is clear that more interaction between researchers in these fields will improve the development of therapies that either promote or prevent hypoxic responses.
Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCa, PKCi and PKCc. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.
Hypoxia inducible factor-1α (HIF-1α) stimulates expression of genes associated with angiogenesis and is associated with poor outcomes in ovarian and other cancers. In normoxia, HIF-1α is ubiquitinated and degraded through the E3 ubiquitin ligase, von Hippel-Lindau; however, little is known about the regulation of HIF-1α in hypoxic conditions. FBW7 is an E3 ubiquitin ligase that recognizes proteins phosphorylated by glycogen synthase kinase 3β (GSK3β) and targets them for destruction. This study used an ovarian cancer cell model to test the hypothesis that HIF-1α phosphorylation by GSK3β in hypoxia leads to interaction with FBW7 and ubiquitin-dependent degradation. Expression of constitutively active GSK3β reduced HIF-1α protein and transcriptional activity and increased ubiquitination of HIF-1α in hypoxia, whereas pharmacologic inhibition of GSK3 or expression of siGSK3β promoted HIF-1α stabilization and activity. A mechanism through FBW7 was supported by the observed decrease in HIF-1α stabilization when FBW7 was overexpressed and both the elevation of HIF-1α levels and decrease in ubiquitinated HIF-1α when FBW7 was suppressed. Furthermore, HIF-1α associated with FBW7γ by co-immunoprecipitation, and the interaction was weakened by inhibition of GSK3 or mutation of GSK3β phosphorylation sites. The relevance of this pathway to angiogenic signaling was supported by the finding that endothelial cell tube maturation was increased by conditioned media from hypoxic SK-OV-3 cell lines expressing suppressed GSK3β or FBW7. These data introduce a new mechanism for regulation of HIF-1α during hypoxia that utilizes phosphorylation to target HIF-1α for ubiquitin-dependent degradation through FBW7 and may identify new targets in the regulation of angiogenesis.
Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers (1, 2), however, the precise mechanistic basis for its role remains unknown. Here we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biological characteristics to full length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism.
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