Hunting for protein markers of hypoxia by combining plasma membrane enrichment with a new approach to membrane protein analysis.

Nontransient hypoxia is strongly associated with malignant lesions, resulting in aggressive behavior and resistance to treatment. We present an analysis of mRNA and protein expression changes in neuroblastoma cell lines occurring upon the transition from normoxia to hypoxia. The correlation between mRNA and protein level changes was poor, although some known hypoxia-driven genes and proteins correlated well. We present previously undescribed membrane proteins expressed under hypoxic conditions that are candidates for evaluation as biomarkers.

Transcriptional adaptation of neuroblastoma cells to hypoxia.

Low oxygen pressure (hypoxia) is a physiological condition that has been linked to tumor progression and increased malignancy in several cancer forms. Cells of the childhood neoplasm neuroblastoma respond to hypoxia by attaining a lower grade of differentiation, which clinically is associated with poor prognosis. Furthermore, expression of the hypoxia inducible factor-2alpha correlates to poor outcome in neuroblastoma patients. In this report we have by microarray analysis studied transcriptional changes in seven neuroblastoma cell lines subjected to long term hypoxia. We find the gene regulatory response to be highly dependent on cell line background, however, a set of genes was coherently regulated by hypoxia and these genes are correlated to known hypoxia-induced transcriptional profiles.

Hypoxia inducible factor-2alpha in cancer.

Poorly oxygenated (hypoxic) tumors are frequently more aggressive compared to corresponding tumors that are better oxygenated. Adaptation to hypoxia is primarily mediated by two closely related hypoxia inducible transcription factor complexes, HIF-1 and HIF-2, which become stabilized and activated at low oxygen levels. Whether HIF-1 and HIF-2 have different roles in tumorigenesis is an open question and an issue we discuss. With focus on HIF-2, we summarize reported phenotypical changes of HIF genetic models and HIF expression patterns during normal development, in adult non-malignant tissues and in tumors. We further address the much-discussed subject of target gene preferences between HIF-1 and HIF-2, given that both transcription factors bind to the same DNA motif. Finally, we also discuss the observations that the oxygen-sensitive HIF-2alpha subunit is accumulated and active under non-hypoxic conditions as exemplified by HIF-2alpha expressing tumor macrophages and neuroblastoma cells located in seemingly well-vascularized tumor regions and how this phenomenon is related to tumor aggressiveness.

Hypoxia-induced dedifferentiation of tumor cells--a mechanism behind heterogeneity and aggressiveness of solid tumors.

Histopathological examination of solid tumors frequently reveals pronounced tumor cell heterogeneity with regards to cell organization, cell morphology, cell size, nuclei morphology, etc. Analyses of gene expression patterns by immunohistochemistry or in situ hybridization techniques further strengthen the actual presence of phenotypic heterogeneity, often demonstrating substantial diversity within a given tumor. The molecular mechanisms underlying the phenotypic heterogeneity are very complex with genetic, epigenetic and environmental components. Hypoxia, shortage in oxygen, greatly influences cellular phenotypes by altering the expression of specific genes, and is an important contributor to intra- and inter-tumor cell diversity as revealed by the pronounced but non-uniform expression of hypoxia-driven genes in solid tumors (reviewed in [Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721-32; Harris AL. Hypoxia--a key regulatory factor in tumour growth. Nat Rev Cancer 2002;2:38-47.]). The oxygen pressure in solid tumors is generally lower than in the surrounding non-malignant tissues, and tumors exhibiting extensive hypoxia have been shown to be more aggressive than corresponding tumors that are better oxygenized [Vaupel P. Oxygen transport in tumors: characteristics and clinical implications. Adv Exp Med Biol 1996;388:341-51; Vaupel P, Thews O, Hoeckel M. Treatment resistance of solid tumors: role of hypoxia and anemia. Med Oncol 2001;18:243-59.]. We recently observed that hypoxic neuroblastoma cells and breast cancer cells lose their differentiated gene expression patterns and develop stem cell-like phenotypes [Jögi A, Øra I, Nilsson H, Lindeheim A, Makino Y, Poellinger L, et al. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci USA 2002;99:7021-6; Helczynska K, Kronblad A, Jögi A, Nilsson E, Beckman S, Landberg G, et al. Hypoxia promotes a dedifferentiated phenotype in ductal breast carcinoma in situ. Cancer Res 2003;63:1441-4.]. As low stage of differentiation in neuroblastoma and in breast cancer is linked to poor prognosis, hypoxia-induced dedifferentiation will not only contribute to tumor heterogeneity but could also be one mechanism behind increased aggressiveness of hypoxic tumors. The effect(s) of hypoxia on tumor cell differentiation status is the focus of this review.