Justin D. Lathia

Hypoxia-Inducible Factors Regulate Tumorigenic Capacity of Glioma Stem Cells

Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). Because hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2alpha and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to non-stem tumor cells and normal neural progenitors. In tumor specimens, HIF2alpha colocalizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation, and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns, and HIF2alpha might represent a promising target for antiglioblastoma therapies.

Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits.

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We now report that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-γ stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death.

Notch Promotes Radioresistance of Glioma Stem Cells

Radiotherapy represents the most effective nonsurgical treatments for gliomas. However, gliomas are highly radioresistant and recurrence is nearly universal. Results from our laboratory and other groups suggest that cancer stem cells contribute to radioresistance in gliomas and breast cancers. The Notch pathway is critically implicated in stem cell fate determination and cancer. In this study, we show that inhibition of Notch pathway with γ‐secretase inhibitors (GSIs) renders the glioma stem cells more sensitive to radiation at clinically relevant doses. GSIs enhance radiation‐induced cell death and impair clonogenic survival of glioma stem cells but not non‐stem glioma cells. Expression of the constitutively active intracellular domains of Notch1 or Notch2 protect glioma stem cells against radiation. Notch inhibition with GSIs does not alter the DNA damage response of glioma stem cells after radiation but rather reduces Akt activity and Mcl‐1 levels. Finally, knockdown of Notch1 or Notch2 sensitizes glioma stem cells to radiation and impairs xenograft tumor formation. Taken together, our results suggest a critical role of Notch signaling to regulate radioresistance of glioma stem cells. Inhibition of Notch signaling holds promise to improve the efficiency of current radiotherapy in glioma treatment. STEM CELLS 2010;28:17–28

Integrin Alpha 6 Regulates Glioblastoma Stem Cells

Cancer stem cells (CSCs) are a subpopulation of tumor cells suggested to be critical for tumor maintenance, metastasis, and therapeutic resistance. Prospective identification and targeting of CSCs are therefore priorities for the development of novel therapeutic paradigms. Although CSC enrichment has been achieved with cell surface proteins including CD133 (Prominin-1), the roles of current CSC markers in tumor maintenance remain unclear. We examined the glioblastoma stem cell (GSC) perivascular microenvironment in patient specimens to identify enrichment markers with a functional significance and identified integrin alpha6 as a candidate. Integrin alpha6 is coexpressed with conventional GSC markers and enriches for GSCs. Targeting integrin alpha6 in GSCs inhibits self-renewal, proliferation, and tumor formation capacity. Our results provide evidence that GSCs express high levels of integrin alpha6, which can serve not only as an enrichment marker but also as a promising antiglioblastoma therapy.

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.

Cancer stem cells in glioblastoma

Tissues with defined cellular hierarchies in development and homeostasis give rise to tumors with cellular hierarchies, suggesting that tumors recapitulate specific tissues and mimic their origins. Glioblastoma (GBM) is the most prevalent and malignant primary brain tumor and contains self-renewing, tumorigenic cancer stem cells (CSCs) that contribute to tumor initiation and therapeutic resistance. As normal stem and progenitor cells participate in tissue development and repair, these developmental programs re-emerge in CSCs to support the development and progressive growth of tumors. Elucidation of the molecular mechanisms that govern CSCs has informed the development of novel targeted therapeutics for GBM and other brain cancers. CSCs are not self-autonomous units; rather, they function within an ecological system, both actively remodeling the microenvironment and receiving critical maintenance cues from their niches. To fulfill the future goal of developing novel therapies to collapse CSC dynamics, drawing parallels to other normal and pathological states that are highly interactive with their microenvironments and that use developmental signaling pathways will be beneficial.

Toll-Like Receptors in Neurodegeneration

The key roles of toll-like receptors (TLRs) as mediators of the detection and responses of immune cells to invading pathogens are well known. There are at least 13 mammalian TLRs which are integral membrane proteins with a leucine-rich extracellular domain and a cytoplasmic domain similar to that of the interleukin-1 receptor which initiates downstream signaling through kinases to activate transcription factors such as AP-1 and NFkappaB. TLRs are activated in glial cells (microglia, astrocytes and oligodendrocytes) and lymphocytes that infiltrate the nervous system in response to inflammation caused by infectious agents, tissue injury or autoimmune conditions. By inducing the production of pro-inflammatory cytokines and cell adhesion molecules in immune cells, TLRs may indirectly damage neurons in conditions such as ischemic stroke and multiple sclerosis. Recent findings suggest that neurons also express a subset of TLRs and that their activation promotes neuronal degeneration in experimental models of stroke and Alzheimers disease. TLRs may also play roles in regulating the processes of neurogenesis and neurite outgrowth, suggesting roles in neuronal plasticity. A better understanding of the molecular and cellular biology of TLRs in the normal and diseased nervous system, may lead to novel approaches for preventing neuronal degeneration and promoting recovery of function in an array of neurodegenerative conditions.

c-Myc Is Required for Maintenance of Glioma Cancer Stem Cells

Background Malignant gliomas rank among the most lethal cancers. Gliomas display a striking cellular heterogeneity with a hierarchy of differentiation states. Recent studies support the existence of cancer stem cells in gliomas that are functionally defined by their capacity for extensive self-renewal and formation of secondary tumors that phenocopy the original tumors. As the c-Myc oncoprotein has recognized roles in normal stem cell biology, we hypothesized that c-Myc may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Methodology/Principal Findings Based on previous methods that we and others have employed, tumor cell populations were enriched or depleted for cancer stem cells using the stem cell marker CD133 (Prominin-1). We characterized c-Myc expression in matched tumor cell populations using real time PCR, immunoblotting, immunofluorescence and flow cytometry. Here we report that c-Myc is highly expressed in glioma cancer stem cells relative to non-stem glioma cells. To interrogate the significance of c-Myc expression in glioma cancer stem cells, we targeted its expression using lentivirally transduced short hairpin RNA (shRNA). Knockdown of c-Myc in glioma cancer stem cells reduced proliferation with concomitant cell cycle arrest in the G0/G1 phase and increased apoptosis. Non-stem glioma cells displayed limited dependence on c-Myc expression for survival and proliferation. Further, glioma cancer stem cells with decreased c-Myc levels failed to form neurospheres in vitro or tumors when xenotransplanted into the brains of immunocompromised mice. Conclusions/Significance These findings support a central role of c-Myc in regulating proliferation and survival of glioma cancer stem cells. Targeting core stem cell pathways may offer improved therapeutic approaches for advanced cancers.

Targeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growth

Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6Rα) and glycoprotein 130 (gp130). Targeting IL6Rα or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6Rα or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti‐IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC‐derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients. STEM CELLS 2009;27:2393–2404

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.