Tumor cell differentiation and hypoxia
Authors: Sven Pahlman
Journal: Cancer Research (2008)

SY34-02   Impaired oxygenation of solid tumors is associated with aggressive tumor behavior. Oxygen shortage, hypoxia, is a major driving force behind not only tumor vascularization, but also in the development of other hallmarks of aggressive tumors, e.g. high mutational rate, metastatic spread, lowered pH and resistance to treatment by radiation or cytotoxic drugs. It is well established that growth of a solid tumor over a size of a few mm requires vascularization of the tumor. Tumor hypoxia and expression of vascular endothelial growth factor (VEGF) via stabilization and activation of the hypoxia inducible transcription factors (HIFs) are major players in the formation of new blood vessels. Consequently hypoxia, VEGF signaling and HIF activity are targets for novel treatment strategies aiming at blocking the angiogenic process, and thus tumor growth. In addition, we recently discovered in neuroblastoma and breast cancer that low tumor oxygen levels lead to loss of expression of differentiation lineage specific genes and the development of stem cell-like phenotypes. We conclude that this effect of hypoxia has a direct bearing on tumor aggressiveness, as immature tumor features are generally associated with aggressive behavior and adverse outcome, which is particularly true in neuroblastoma and breast cancer.  Hypoxia-induced stabilization and activation of the transcription factors HIF-1a and HIF-2a are as far as we know the major mechanisms behind the phenotypic changes of hypoxic cells. Gene elimination studies have demonstrated that HIFs are essential for normal development. For instance, with regards to the sympathetic nervous system (SNS), HIF-2a appears to be important and is expressed in developing mouse and human SNS structures during discrete embryonal and fetal time periods. In neuroblastoma, an SNS-derived tumor, we recently demonstrated high HIF-2a protein expression in well-vascularized tumor areas. To mimic these conditions in vitro, neuroblastoma cells were maintained at 5% oxygen (an approximation of the oxygen level in end-capillaries) and the phenotype of such cells were compared to that of cells grown at 21% (normoxia) or 1% (hypoxia) oxygen. HIF-1a protein was virtually absent at 5 or 21% oxygen and showed a transient, fast increase at 1%, while HIF-2a protein was abundant at 1% oxygen, remained high over time, and, importantly was also stabilized at 5% oxygen. We further showed that HIF-2a is active and drives VEGF expression at 5% oxygen (reporter assays, VEGF-HRE binding shown by ChIP, inhibited VEGF expression by HIF-2a-siRNA). Other known hypoxia-driven genes were also transcribed at 5% oxygen, presumably driven by activated HIF-2a (verified for VEGF, NDRG1 and DEC1). Analyzing neuroblastoma patient tumor specimens for HIF-2a and VEGF protein, we further showed a correlation between the expression of these two proteins and that high HIF-2a protein levels associate with adverse outcome. Similar results were also obtained when a large cohort of specimens of invasive breast cancer was analyzed. In neuroblastoma, we further found a subset of perivascularly located strongly HIF-2 positive tumor cells (MYCN amplified) that lack expression of differentiation markers, but expressed neural crest and early sympathetic progenitor marker genes such as Notch-1, HES-1, c-Kit, dHAND and vimentin. We speculate that these HIF-2a positve cells have tumor initiating/stem cells features and that HIF-2a acts oncogenically at non-hypoxic conditions in neuroblastoma by activating classical hypoxia-driven genes and thus is directly involved in tumor vascularization by transcribing VEGF.