HIF1a-mediated STAT3 activation promotes self-renewal and malignancy of stem-like cells derived from spontaneous murine glioma

Hypoxia occurs commonly within solid tumors and is associated with a poor prognosis. Hypoxia-inducible factor (HIF)-1alpha is critical for the molecular responses to hypoxia. Recently tumor-derived cells exhibiting the characteristics of stem cells have been identified in many cancers including brain tumors and have been shown to play a key role in tumor development. Brain tumor-derived sphere cells cultured under conditions optimized for maintaining neural stem cells are shown to be enriched for cells with stem-cell properties and more accurately reflect the biology of primary tumors than do conventionally cultured adherent cells. To examine the effect of hypoxia on brain tumor-derived stem cells, we isolated sphere forming cells from oligodendroglioma arising in S100beta-nu-erbB/p53-/- mice. These tumor sphere cells were capable of self-renewal, differentiation and potent tumor initiation. When exposed to hypoxic conditions (1% oxygen), their anchorage-independent growth in soft agar and self-renewal were enhanced compared to the levels in normoxia (21% oxygen). HIF-1alpha protein was elevated in nu- erbB/p53-/- tumor sphere cells compared to normal neural stem cells. Constitutive activation of the PI3K-AKT pathway in nu- erbB/p53-/- tumor sphere cells contributed to the high HIF-1alpha level. HIF-1alpha expression was further induced by hypoxia. Inhibition of HIF-1alpha expression reduced anchorage-independent growth, self-renewal, proliferation and survival of the tumor sphere cells in both normoxia and hypoxia. Signal transducer and activator of transcription 3 (STAT3), a transcriptional factor essential for self-renewal of normal stem cells, was expressed at an elevated level in nu-erbB/p53 -/- oligodendroglioma and was aberrantly activated in the sphere cells derived from these tumors. STAT3 activation was required for anchorage-independent growth and self-renewal of nu-erbB/p53-/- tumor sphere cells. In response to hypoxia, activation of STAT3 was enhanced, and the inhibition of HIF-1alpha expression reduced STAT3 activation. Finally, the expression of a constitutively active STAT3 rescued the anchorage-independent growth and self-renewal that was inhibited by the reduction of HIF-1alpha. Our results suggest that HIF-1alpha-mediated STAT3 activation may constitute a critical stem cell-regulatory pathway, and hypoxia, an environmental factor prevalent in solid tumors, may contribute to the malignant phenotype of tumors by enhancing the function of tumor-derived stem cells through a stem cell pathway.