Hypoxia-induced mixed-lineage leukemia 1 regulates glioma stem cell tumorigenic potential.

Normal stem cells reside in functional niches critical for self-renewal and maintenance. Neural and hematopoietic stem cell niches, in particular, are characterized by restricted availability of oxygen and the resulting regulation by hypoxia-inducible factors (HIFs). Glioblastoma multiforme (GBM) is the most common malignant brain tumor and also contains high degrees of hypoxia. Heterogeneity within the neoplastic compartment has been well characterized in GBM and may be derived from genetic and epigenetic sources that co-evolve during malignant progression. Recent experimental evidence has supported the importance of hypoxia in glioma stem cell (GSC) niches. We hypothesized that HIFs require epigenetic-modifying proteins to promote tumor malignancy in GBM. Here we demonstrate that in GBM the histone methyltransferase mixed-lineage leukemia 1 (MLL1) is induced by hypoxia and enhances hypoxic responses. Loss of MLL1 reduces the expression of HIF transcripts and HIF2α protein. Targeting MLL1 by RNA interference inhibited the expression of HIF2α and target genes, including vascular endothelial growth factor (VEGF). GSCs expressed higher levels of MLL1 than matched non-stem tumor cells and depletion of MLL1 reduced GSC self-renewal, growth, and tumorigenicity. These studies have uncovered a novel mechanism mediating tumor hypoxic responses linking microenvironmental regulation of epigenetic-modifying proteins to cellular heterogeneity and provide rationale for the design of more sophisticated clinical approaches targeting epigenetic regulation.

Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions.

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

Acidic stress promotes a glioma stem cell phenotype.

Malignant gliomas are lethal cancers that display cellular hierarchies with cancer stem cells at the apex. Glioma stem cells (GSCs) are not uniformly distributed, but rather located in specialized niches, suggesting that the cancer stem cell phenotype is regulated by the tumor microenvironment. Indeed, recent studies show that hypoxia and its molecular responses regulate cancer stem cell maintenance. We now demonstrate that acidic conditions, independent of restricted oxygen, promote the expression of GSC markers, self-renewal and tumor growth. GSCs exert paracrine effects on tumor growth through elaboration of angiogenic factors, and low pH conditions augment this expression associated with induction of hypoxia inducible factor 2α (HIF2α), a GSC-specific regulator. Induction of HIF2α and other GSC markers by acidic stress can be reverted by elevating pH in vitro, suggesting that raising intratumoral pH may be beneficial for targeting the GSC phenotype. Together, our results suggest that exposure to low pH promotes malignancy through the induction of a cancer stem cell phenotype, and that culturing cancer cells at lower pH reflective of endogenous tumor conditions may better retain the cellular heterogeneity found in tumors.

Hypoxia inducible factors in cancer stem cells.

Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). Under hypoxia, HIFs regulate a variety of pro-angiogenic and pro-glycolysis pathways. In solid cancers, regions of hypoxia are commonly present throughout the tissue because of the chaotic vascular architecture and regions of necrosis. In these regions, the hypoxic state fluctuates in a spatial and temporal manner. Transient hypoxic cycling causes an increase in the activity of the HIF proteins above what is typical for non-pathologic tissue. The extent of hypoxia strongly correlates to poor patient survival, therapeutic resistance and an aggressive tumour phenotype, but the full contribution of hypoxia and the HIFs to tumour biology is an area of active investigation. Recent reports link resistance to conventional therapies and the metastatic potential to a stem-like tumour population, termed cancer stem cells (CSCs). We and others have shown that within brain tumours CSCs reside in two niches, a perivascular location and the surrounding necrotic tissue. Restricted oxygen conditions increase the CSC fraction and promote acquisition of a stem-like state. Cancer stem cells are critically dependant on the HIFs for survival, self-renewal, and tumour growth. These observations and those from normal stem cell biology provide a new mechanistic explanation for the contribution of hypoxia to malignancy. Further, the presence of hypoxia in tumours may present challenges for therapy because of the promotion of CSC phenotypes even upon successful killing of CSCs. The current experimental evidence suggests that CSCs are plastic cell states governed by microenvironmental conditions, such as hypoxia, that may be critical for the development of new therapies targeted to disrupt the microenvironment.