Suppression of G-protein-coupled receptor kinase 3 expression is a feature of classical GBM that is required for maximal growth

G-protein-coupled receptor kinases (GRK) regulate the function of G-protein-coupled receptors (GPCR). Previously, we found that GPCR (CXCR4)-mediated astrocytoma growth was dependent upon abnormally sustained CXCR4 signaling and was correlated with decreased GRK-mediated receptor phosphorylation. As CXCR4 has also been implicated in the stimulation of high-grade glioma growth, we sought to determine whether dysregulation of GRK expression and/or function might also be present in high-grade gliomas. In an analysis of data from The Cancer Genome Atlas, we found that GRK3 expression is frequently decreased in glioblastoma (GBM) of the classical subtype, which possesses signature amplification or mutational activation of the epidermal growth factor (EGF) receptor. We tested the correlation between GRK3 expression and GBM subtypes, as well as the relationship between the activation of the EGF and other growth factor receptor pathways and GRK expression. In analyses of primary GBM tissue and RNA specimens, we found that GRK3 expression is correlated with established criteria for GBM subtyping including expression of EGF receptor, platelet-derived growth factor receptor (PDGFR)α, NF1, PTEN, CDKN2A, and neurofilament. We also found that established drivers of gliomagenesis, the EGF, PDGF, and TGF-β pathways, all regulate GRK expression. Coculture experiments, designed to mimic critical interactions between tumor and brain microvascular endothelial cells, showed that specifically increasing GRK3 expression reduced the trophic effect of endothelial cells on tumor cells. Together, these experiments show that GRK3 is a negative regulator of cell growth whose expression is preferentially reduced in GBM of the classical subtype as a consequence of activity in primary gliomagenic pathways.     

Identification of Tumor Precursor Cells in the Brains of Primates with Radiation-Induced de novo Glioblastoma Multiforme

The pathogenesis of de novo glioblastoma multiforme (GBM) is poorly understood and precursor cells are not known. To gain insight into the pathogenesis of GBM we analyzed brains from primates that developed de novo tumors 10 years after whole brain radiation. Four animals had clinical and radiological evidence of GBM, and two animals had no evidence of GBM at the time of euthanization. Tumor precursor cells were identified diffusely scattered in the grossly normal white matter of all animals including two monkeys without evidence of GBM by MR-imaging or on autopsy examination. Tumor precursors demonstrated cellular atypia and mitoses, and were negative for tumor-associated markers GFAP, EGFR and p53. The cells were positive for Ki67 and N-CoR, the nuclear co-repressor of astroglial differentiation. These results suggest that radiation-induced nuclear damage to neural stem cells or early astrocytic precursor cells can prevent normal differentiation and lead to tumor development. The findings provide insight into the tumorigenesis of de novo GBMs and suggest a new strategy for treatment of these lethal tumors by targeting both inactivation of N-CoR and inhibition of EGFR.     

Cancer stem cells and human malignant melanoma

Cancer stem cells (CSC) have been identified in hematological malignancies and several solid cancers. Similar to physiological stem cells, CSC are capable of self-renewal and differentiation and have the potential for indefinite proliferation, a function through which they may cause tumor growth. Although conventional anti-cancer treatments might eradicate most malignant cells in a tumor, they are potentially ineffective against chemoresistant CSC, which may ultimately be responsible for recurrence and progression. Human malignant melanoma is a highly aggressive and drug-resistant cancer. Detection of tumor heterogeneity, undifferentiated molecular signatures, and increased tumorigenicity of melanoma subsets with embryonic-like differentiation plasticity strongly suggest the presence and involvement of malignant melanoma stem cells (MMSC) in the initiation and propagation of this malignancy. Here, we review these findings in the context of functional properties ascribed to melanocyte stem cells and CSC in other cancers. We discuss the association of deregulated signaling pathways, genomic instability, and vasculogenic mimicry phenomena observed in melanoma subpopulations in light of the CSC concept. We propose that a subset of MMSC may be responsible for melanoma therapy-resistance, tumor invasiveness, and neoplastic progression and that targeted abrogation of a MMSC compartment could therefore ultimately lead to stable remissions and perhaps cures of metastatic melanoma.     

Elevated invasive potential of glioblastoma stem cells

Glioblastomas (GBMs) are the most lethal and common types of primary brain tumors. The hallmark of GBMs is their highly infiltrative nature. The cellular and molecular mechanisms underlying the aggressive cancer invasion in GBMs are poorly understood. GBM displays remarkable cellular heterogeneity and hierarchy containing self-renewing glioblastoma stem cells (GSCs). Whether GSCs are more invasive than non-stem tumor cells and contribute to the invasive phenotype in GBMs has not been determined. Here we provide experimental evidence supporting that GSCs derived from GBM surgical specimens or xenografts display greater invasive potential in vitro and in vivo than matched non-stem tumor cells. Furthermore, we identified several invasion-associated proteins that were differentially expressed in GSCs relative to non-stem tumor cells. One of such proteins is L1CAM, a cell surface molecule shown to be critical to maintain GSC tumorigenic potential in our previous study. Immunohistochemical staining showed that L1CAM is highly expressed in a population of cancer cells in the invasive fronts of primary GBMs. Collectively, these data demonstrate the invasive nature of GSCs, suggesting that disrupting GSCs through a specific target such as L1CAM may reduce GBM cancer invasion and tumor recurrence.     

Cancer stem cells in glioblastoma — molecular signaling and therapeutic targeting

Glioblastomas (GBMs) are highly lethal primary brain tumors. Despite current therapeutic advances in other solid cancers, the treatment of these malignant gliomas remains essentially palliative. GBMs are extremely resistant to conventional radiation and chemotherapies. We and others have demonstrated that a highly tumorigenic subpopulation of cancer cells called GBM stem cells (GSCs) promotes therapeutic resistance. We also found that GSCs stimulate tumor angiogenesis by expressing elevated levels of VEGF and contribute to tumor growth, which has been translated into a useful therapeutic strategy in the treatment of recurrent or progressive GBMs. Furthermore, stem cell-like cancer cells (cancer stem cells) have been shown to promote metastasis. Although GBMs rarely metastasize beyond the central nervous system, these highly infiltrative cancers often invade into normal brain tissues preventing surgical resection, and GSCs display an aggressive invasive phenotype. These studies suggest that targeting GSCs may effectively reduce tumor recurrence and significantly improve GBM treatment. Recent studies indicate that cancer stem cells share core signaling pathways with normal somatic or embryonic stem cells, but also display critical distinctions that provide important clues into useful therapeutic targets. In this review, we summarize the current understanding and advances in glioma stem cell research, and discuss potential targeting strategies for future development of anti-GSC therapies.     

Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.     

Glioblastoma Formation from Cell Population Depleted of Prominin1-Expressing Cells

Prominin1 (Prom1, also known as CD133 in human) has been widely used as a marker for cancer stem cells (CSCs), which self-renew and are tumorigenic, in malignant tumors including glioblastoma multiforme (GBM). However, there is other evidence showing that Prom1-negative cancer cells also form tumors in vivo. Thus it remains controversial whether Prom1 is a bona fide marker for CSCs. To verify if Prom1-expressing cells are essential for tumorigenesis, we established a mouse line, whose Prom1-expressing cells can be eliminated conditionally by a Cre-inducible DTA gene on the Prom1 locus together with a tamoxifen-inducible CreER^TM, and generated glioma-initiating cells (GICs-LD) by overexpressing both the SV40 Large T antigen and an oncogenic H-Ras^L61 in neural stem cells of the mouse line. We show here that the tamoxifen-treated GICs-LD (GICs-DTA) form tumor-spheres in culture and transplantable GBM in vivo. Thus, our studies demonstrate that Prom1-expressing cells are dispensable for gliomagenesis in this mouse model.     

Gene therapy of rat malignant gliomas using neural stem cells expressing IL-12

Primary malignant brain tumors have a poor prognosis. This report investigates the potential for gene therapy of experimental brain tumors using neural stem cells (NSCs) expressing IL-12. In this study NSCs were isolated from the hippocampi of 3-5-month human embryos and used for lipofectamine mediated transfer of the IL-12 gene. Positive clones of anti-G418 were obtained and were proliferated in culture and expression of IL-12 was demonstrated by RT-PCR. For the in vivo studies three groups of rats were used and stereotactic injections were made into the striatum. In the first group C6 tumor cells were injected, in the second C6 cells and hNSCs. IL-12, and in the third C6 cells on Day 0 followed by hNSCs.IL-12 on day 5. The growth of the resulting tumors was monitored by magnetic resonance imaging (MRI) and after sacrifice by immunohistochemistry. Rats injected with C6 cells and hNSCs.IL-12 had a significantly prolonged survival. Injections of hNSCs.IL-12 were also made into established gliomas. The survival time was also significantly prolonged compared to controls. MR imaging demonstrated that there was initial growth of tumor followed by shrinkage and then disappearance. After sacrifice, tumor areas were studied by histochemistry. NSCs were often seen intermingled with tumor cells, particularly when they had been injected into established tumors; they were also present at the boundaries of the tumor mass. The immunohistochemical analysis showed that these infiltrates were mostly constituted by CD4(+) and CD8(+) T-lymphocytes, the CD8(+) being more numerous than the CD4(+). These findings indicated that NSCs engineered to release IL-12 could have a strong antitumor effect. Neural stem/precursor cells could be useful vectors in genetic approaches to brain tumors.     

Role and mechanism of neural stem cells of the subventricular zone in glioblastoma

Glioblastoma multiforme (GBM), the most frequently occurring malignant brain tumor in adults, remains mostly untreatable. Because of the heterogeneity of invasive gliomas and drug resistance associated with the tumor microenvironment, the prognosis is poor, and the survival rate of patients is low. Communication between GBMs and non-glioma cells in the tumor microenvironment plays a vital role in tumor growth and recurrence. Emerging data have suggested that neural stem cells (NSCs) in the subventricular zone (SVZ) are the cells-of-origin of gliomas, and SVZ NSC involvement is associated with the progression and recurrence of GBM. This review highlights the interaction between SVZ NSCs and gliomas, summarizes current findings on the crosstalk between gliomas and other non-glioma cells, and describes the links between SVZ NSCs and gliomas. We also discuss the role and mechanism of SVZ NSCs in glioblastoma, as well as the interventions targeting the SVZ and their therapeutic implications in glioblastoma. Taken together, understanding the biological mechanism of glioma-NSC interactions can lead to new therapeutic strategies for GBM.