Candace A. Gilbert

Cancer stem cells: Cell culture, markers, and targets for new therapies

A cancer stem cell (CSC) is defined as an undifferentiated cell with the ability to self‐renew, differentiate to multiple lineages and initiate tumors that mimic the parent tumor. In this review, we focus on glioblastomas, describing recent progress and problems in characterizing these cells. There have been advances in CSC culture, but tumor cell heterogeneity has made purification of CSCs difficult. Indeed, it may be that CSCs significantly vary from tumor to tumor. We also discuss the proposal that CSCs are resistant to radiotherapy and chemotherapy and play a major role in repopulating tumors following treatment. To overcome their resistance to conventional therapies, we may be able to use our extensive knowledge of the signaling pathways essential for stem cells during development. These pathways have potential as targets for new glioblastoma therapies. Hence, although there is an ongoing debate on the nature of CSCs, the theory continues to suggest new ideas for both the lab and the clinic. J. Cell. Biochem. 108: 1031–1038, 2009.

γ-Secretase Inhibitors Enhance Temozolomide Treatment of Human Gliomas by Inhibiting Neurosphere Repopulation and Xenograft Recurrence

Malignant gliomas are treated with a combination of surgery, radiation, and temozolomide (TMZ), but these therapies ultimately fail due to tumor recurrence. In glioma cultures, TMZ treatment significantly decreases neurosphere formation; however, a small percentage of cells survive and repopulate the culture. A promising target for glioma therapy is the Notch signaling pathway. Notch activity is upregulated in many gliomas and can be suppressed using gamma-secretase inhibitors (GSI). Using a neurosphere recovery assay and xenograft experiments, we analyzed if the addition of GSIs with TMZ treatment could inhibit repopulation and tumor recurrence. We show that TMZ + GSI treatment decreased neurosphere formation and inhibited neurosphere recovery. This enhancement of TMZ treatment occurred through inhibition of the Notch pathway and depended on the sequence of drug administration. In addition, ex vivo TMZ + GSI treatment of glioma xenografts in immunocompromised mice extended tumor latency and survival, and in vivo TMZ + GSI treatment blocked tumor progression in 50% of mice with preexisting tumors. These data show the importance of the Notch pathway in chemoprotection and repopulation of TMZ-treated gliomas. The addition of GSIs to current treatments is a promising approach to decrease brain tumor recurrence.

Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells

Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors.

Sorafenib exerts anti-glioma activity in vitro and in vivo.

Despite conventional treatment strategies glioblastoma, the most common malignant primary brain tumor, has a bad prognosis with median survival times of 12-15 months. In this study, the efficacy of sorafenib (Nexavar, BAY43-9006), a multikinase inhibitor, on glioblastoma cells was evaluated both in vitro and in vivo. Treatment of established or patient-derived glioblastoma cells with low concentrations of sorafenib caused a dramatic dose dependent inhibition of proliferation (IC(50), 1.5 microM) and induction of apoptosis and autophagy. Sorafenib inhibited phosphorylation of signal transducer and activator of transcription 3 (Stat3) and expression of cyclins, D and E. In contrast, AKT was not modulated by sorafenib. Most important, systemic delivery of sorafenib was well tolerated, and significantly suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis and autophagy, and reduction of angiogenesis. Furthermore, intracranial growth inhibition by sorafenib was accompanied by a significant reduction in ph-Stat3 (Tyr 705) levels. In summary, sorafenib has potent anti-glioma activity in vitro and in vivo.

Global targeting of subcellular heat shock protein-90 networks for therapy of glioblastoma.

Drug discovery for complex and heterogeneous tumors now aims at dismantling global networks of disease maintenance, but the subcellular requirements of this approach are not understood. Here, we simultaneously targeted the multiple subcellular compartments of the molecular chaperone heat shock protein-90 (Hsp90) in a model of glioblastoma, a highly lethal human malignancy in urgent need of fresh therapeutic strategies. Treatment of cultured or patient-derived glioblastoma cells with Shepherdin, a dual peptidomimetic inhibitor of mitochondrial and cytosolic Hsp90, caused irreversible collapse of mitochondria, degradation of Hsp90 client proteins in the cytosol, and tumor cell killing by apoptosis and autophagy. Stereotactic or systemic delivery of Shepherdin was well tolerated and suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis, and reduction of angiogenesis in vivo. These data show that disabling Hsp90 cancer networks in their multiple subcellular compartments improves strategies for drug discovery and may provide novel molecular therapy for highly recalcitrant human tumors. Mol Cancer Ther; 9(6); 1638–46. ©2010 AACR.

Clinically relevant doses of chemotherapy agents reversibly block formation of glioblastoma neurospheres.

Glioblastoma patients have a poor prognosis, even after surgery, radiotherapy, and chemotherapy with temozolomide or 1,3-bis(2-chloroethy)-1-nitrosourea. We developed an in vitro recovery model using neurosphere cultures to analyze the efficacy of chemotherapy treatments, and tested whether glioblastoma neurosphere-initiating cells are resistant. Concentrations of chemotherapy drugs that inhibit neurosphere formation are similar to clinically relevant doses. Some lines underwent a transient cell cycle arrest and a robust recovery of neurosphere formation. These results indicate that glioblastoma neurospheres can regrow after treatment with chemotherapy drugs. This neurosphere recovery assay will facilitate studies of chemo-resistant subpopulations and methods to enhance glioblastoma therapy.

Glioma Stem Cells: Cell Culture, Markers and Targets for New Combination Therapies

Gliomas are brain tumors with glial cell characteristics, and are composed of a heterogeneous mix of cells, which includes glioma stem cells. Gliomas include astrocytomas, oligodendrogliomas, ependymoma, and mixed gliomas. Gliomas account for 32% of all brain and central nervous system tumors (CNS) and 80% of all malignant brain and CNS tumors (CBTRUS, 2010). The WHO grade III anaplastic astrocytomas (AAs) and grade IV glioblastoma multiforme (GBMs) are highly invasive tumors and make up approximately three-quarters of all gliomas (CBTRUS, 2010). GBM is the most common and malignant form of brain tumor. GBMs make up 17% of all primary brain tumors in the United States, with an incidence of 3.17 cases per 100,000 persons per year (CBTRUS, 2010). Although both the knowledge of glioma biology and the available resources for treatment have greatly increased over the past decade, the expected survival of malignant glioma patients remains dismal. For AA patients, the current five-year and ten-year survival rates are 27.4% and 21.3%, respectively (CBTRUS, 2010). GBM patients have a much lower survival. The current five-year and ten-year survival rates for GBM patients are 4.5% and 2.7%, respectively (CBTRUS, 2010). Clinical treatment for gliomas consists of a combination of surgical resection, radiotherapy and chemotherapy. Due to the infiltrative nature of GBMs, complete removal of the tumor by surgery is not possible. Following surgery, the conventional radiation dosage of up to 60 Gy is given daily in 2 Gy fractions (Buatti et al 2008). The commonly used chemotherapy drug, temozolomide (Temodar®), is an alkylating agent that is taken orally and readily penetrates the blood-brain barrier (Ostermann et al., 2004). 1,3bis(2-chloroethyl)-1-nitrosourea (BCNU) is an older drug that surgeons deposit in the tumor bed as dissolvable wafers (Grossman et al., 1992). Both of these drugs alkylate DNA at multiple sites, including the O6 position of guanine, which can result in futile cycles of DNA repair and, ultimately, cell death (Sarkaria et al., 2008). These alkylating agents can also induce senescence (Gunther et al., 2003). Temozolomide is administered as both concomitant and adjuvant treatments to radiotherapy. This aggressive treatment increases the two-year survival rate for GBM patients from 10.4%, with radiotherapy alone, to 26.5% (Stupp et al., 2005). Cells that escape radiotherapyand chemotherapy-induced cell death eventually reenter the cell cycle and contribute to local tumor recurrence. Despite advances in chemotherapy regimens, the median progression free survival in AA and GBM patients is, 15.2 months (Chamberlain et al., 2008) and 6.9 months (Stupp et al., 2005), respectively. The median overall survival time for GBMs is 14.6 months (Stupp et al., 2005).

Abstract 963: Glioma treatment with temozolomide and Notch inhibition blocks tumor recovery through the induction of senescence and tumor clearance

Current glioma therapy relies on induction of cytotoxicity after removal of the bulk tumor through the combination of surgery, radiation and temozolomide (TMZ); however, these therapies do not result in a long-term cure. Our lab previously demonstrated that some glioma cells undergo a transient cell cycle arrest in response to chemotherapy. Treatment with TMZ decreases sphere formation; however, after a short recovery period, a small number of cells resume sphere formation and self-renewal, measured by secondary sphere formation. Blocking the Notch pathway in neurosphere cultures with gamma-secretase inhibitors (GSIs) after TMZ treatment targeted the cells capable of recovery. TMZ + GSI treated cells do not recover and are no longer capable of self-renewal. TMZ + GSI synergy is dependent on the sequence of the drug treatments. Recovery was inhibited when GSI was administered 24 hrs after TMZ treatment. TMZ + GSI treatment also decreases tumorigenicity. When glioma cell lines were treated in vitro and implanted in immunodeficient mice, TMZ + GSI treatment extended latency and greatly increased survival. In addition, in vivo TMZ + GSI treatment completely blocked tumor progression and resulted in the loss of a palpable tumor in 50% of mice, while none of the TMZ-only treated mice survived. TMZ + GSI treated cultures and xenografts display a senescent phenotype. We observed an increase in the number of cells expressing senescence-associated beta-galactosidase and a decrease in Ki67 positive cells. Gene expression was also analyzed after drug treatments to confirm the induction of senescence. p21 is upregulated in cells that have undergone either a transient cell cycle arrest or senescence. We found that upregulation of p21 occurred initially in both TMZ-only and TMZ + GSI treatments, but only remained upregulated in the TMZ + GSI samples. This demonstrates that the addition of GSIs shifts TMZ-treated cells from a transient arrested state to a permanent senescent state. New therapy combinations, such as TMZ + GSI, are arising in a promising new field of cytostatic therapy and therapy-induced senescence (TIS). A key feature of TIS is the secretory profile of senescent cells. It was previously demonstrated that senescent tumor cells secrete inflammatory cytokines and activate the innate immune system for tumor clearance. We found that TMZ + GSI treatment resulted in upregulation of secreted IL-6 and IL-8 cytokines. We are currently the effect of senescent glioma cells on the innate immune system and tumor clearance. Overall, this data demonstrates the importance of the Notch pathway in chemoprotection and maintenance of TMZ-treated gliomas. The addition of GSIs to current treatments is promising target-directed therapy to decrease the rate of brain tumor recurrence inducing senescence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 963. doi:10.1158/1538-7445.AM2011-963