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.     

Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; implications for prevention of stroke and AD

IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.     

Distinct types of tumor-initiating cells form human colon cancer tumors and metastases

Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.     

Identification of cancer stem cells from human glioblastomas: growth and differentiation capabilities and CD133/prominin-1 expression

CD133 can be a marker of tumorigenic CSCs (cancer stem cells) in human GBM (glioblastoma multiforme), although tumorigenic CD133-negative CSCs have been also isolated. Additional evidence indicates that CSCs from GBM exhibit different phenotypes, with increasing interest in the potential significance of the different CSCs with respect to diagnosis, prognosis and the development of novel targets for treatment. We have analysed the expression of CD133 in freshly isolated cells from 15 human GBM specimens. Only 4 of them contained cells positive for AC133 by FACS analysis, and all of them yielded distinct CSC lines, whereas only 6 CSC lines were obtained from the other 11 GBMs. Of these 10 CSCs lines, we further characterized 6 CSC lines. Three CSCs grew as fast-growing neurospheres with higher clonogenic ability, whereas the remaining 3 grew as slow-growing semi-adherent spheres of lower clonogenicity. In addition, the former CSC lines displayed better differentiation capabilities than the latter ones. PCR and Western blot analysis showed that all 6 GBM CSC lines expressed CD133/prominin-1, suggesting that cells negative by FACS analysis may actually represent cells expressing low levels of CD133 undetected by FACS. Nevertheless, all the 6 CSC lines were tumorigenic in nude mice. In conclusion, CSCs from human primary GBMs show different phenotypes and variable levels of CD133 expression, but these parameters did not directly correlate with the tumorigenic potential.     

Niche-Dependent Gene Expression Profile of Intratumoral Heterogeneous Ovarian Cancer Stem Cell Populations

Intratumoral heterogeneity challenges existing paradigms for anti-cancer therapy. We have previously demonstrated that the human embryonic stem cells (hESC)-derived cellular microenvironment in immunocompromised mice, enables functional distinction of heterogeneous tumor cells, including cells which do not grow into a tumor in a conventional direct tumor xenograft platform. We have identified and characterized six cancer cell subpopulations each clonally expanded from a single cell, derived from human ovarian clear cell carcinoma of a single tumor, to demonstrate striking intratumoral phenotypic heterogeneity that is dynamically dependent on the tumor growth microenvironment. These cancer cell subpopulations, characterized as cancer stem cell subpopulations, faithfully recapitulate the full spectrum of histological phenotypic heterogeneity known for human ovarian clear cell carcinoma. Each of the six subpopulations displays a different level of morphologic and tumorigenic differentiation wherein growth in the hESC-derived microenvironment favors growth of CD44+/aldehyde dehydrogenase positive pockets of self-renewing cells that sustain tumor growth through a process of tumorigenic differentiation into CD44-/aldehyde dehydrogenase negative derivatives. Strikingly, these derivative cells display microenvironment-dependent plasticity with the capacity to restore self-renewal markers and CD44 expression. In the current study, we delineate the distinct gene expression and epigenetic profiles of two such subpopulations, representing extremes of phenotypic heterogeneity in terms of niche-dependent self-renewal and tumorigenic differentiation. By combining Gene Set Enrichment, Gene Ontology and Pathway-focused array analyses with methylation status, we propose a suite of robust differences in tumor self-renewal and differentiation pathways that underlie the striking intratumoral phenotypic heterogeneity which characterize this and other solid tumor malignancies.     

REST regulates the pool size of the different neural lineages by restricting the generation of neurons and oligodendrocytes from neural stem/progenitor cells

REST is a master repressor of neuronal genes; however, whether it has any role during nervous system development remains largely unknown. Here, we analyzed systematically the role of REST in embryonic stem cells and multipotent neural stem/progenitor (NS/P) cells, including neurogenic and gliogenic NS/P cells derived from embryonic stem (ES) cells or developing mouse embryos. We showed that REST-null ES cells remained pluripotent and generated teratomas consisting of the three germ layers. By contrast, multipotent NS/P cells lacking REST displayed significantly reduced self-renewal capacity owing to reduced cell cycle kinetics and precocious neuronal differentiation. Importantly, although early-born neurogenic NS/P cells that lack REST were capable of differentiating to neurons and glia, the neuronal and oligodendrocytic pools were significantly enlarged and the astrocytic pool was shrunken. However, gliogenic NS/P cells lacking REST were able to generate a normal astrocytic pool size, suggesting that the shrinkage of the astrocytic pool generated from neurogenic NS/P cells lacking REST probably occurs by default. Microarray profiling of early-born NS/P cells lacking REST showed upregulation of neuronal as well as oligodendrocytic genes, specifically those involved in myelination. Furthermore, chromatin immunoprecipitation analyses showed that some of the upregulated oligodendrocytic genes contain an RE1 motif and are direct REST targets. Together, our data support a central role for REST during neural development in promoting NS/P cell self-renewal while restricting the generation and maturation of neurons and oligodendrocytes.     

The miR 302-367 cluster drastically affects self-renewal and infiltration properties of glioma-initiating cells through CXCR4 repression and consequent disruption of the SHH-GLI-NANOG network

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in adults, often characterized by poor survival. Glioma-initiating cells (GiCs) are defined by their extensive self-renewal, differentiation, and tumor initiation properties. GiCs are known to be involved in tumor growth and recurrence, and in resistance to conventional treatments. One strategy to efficiently target GiCs in GBM consists in suppressing their stemness and consequently their tumorigenic properties. In this study, we show that the miR-302-367 cluster is strongly induced during serum-mediated stemness suppression. Stable miR-302-367 cluster expression is sufficient to suppress the stemness signature, self-renewal, and cell infiltration within a host brain tissue, through inhibition of the CXCR4 pathway. Furthermore, inhibition of CXCR4 leads to the disruption of the sonic hedgehog (SHH)-GLI-NANOG network, which is involved in self-renewal and expression of the embryonic stem cell-like signature. In conclusion, we demonstrated that the miR-302-367 cluster is able to efficiently trigger a cascade of inhibitory events leading to the disruption of GiCs stem-like and tumorigenic properties.     

The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype

Glioblastomas are highly lethal cancers that contain cellular hierarchies with self-renewing cancer stem cells that can propagate tumors in secondary transplant assays. The potential significance of cancer stem cells in cancer biology has been demonstrated by studies showing contributions to therapeutic resistance, angiogenesis, and tumor dispersal. We recently reported that physiologic oxygen levels differentially induce hypoxia inducible factor-2α (HIF2α) levels in cancer stem cells. HIF1α functioned in proliferation and survival of all cancer cells but also was activated in normal neural progenitors suggesting a potentially restricted therapeutic index while HIF2α was essential in only in cancer stem cells and was not expressed by normal neural progenitors demonstrating HIF2α is a cancer stem cell specific target. We now extend these studies to examine the role of hypoxia in regulating tumor cell plasticity. We find that hypoxia promotes the self-renewal capability of the stem and non-stem population as well as promoting a more stem-like phenotype in the non-stem population with increased neurosphere formation as well as upregulation of important stem cell factors, such as OCT4, NANOG, and c-MYC. The importance of HIF2α was further supported as forced expression of non-degradable HIF2α induced a cancer stem cell marker and augmented the tumorigenic potential of the non-stem population. This novel finding may indicate a specific role of HIF2α in promoting glioma tumorigenesis. The unexpected plasticity of the non-stem glioma population and the stem-like phenotype emphasizes the importance of developing therapeutic strategies targeting the microenvironmental influence on the tumor in addition to cancer stem cells.     

Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.     

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.     

Autotaxin is overexpressed in glioblastoma multiforme and contributes to cell motility of glioblastoma by converting lysophosphatidylcholine to lysophosphatidic acid

Autotaxin (ATX) is a multifunctional phosphodiesterase originally isolated from melanoma cells as a potent cell motility-stimulating factor. ATX is identical to lysophospholipase D, which produces a bioactive phospholipid, lysophosphatidic acid (LPA), from lysophosphatidylcholine (LPC). Although enhanced expression of ATX in various tumor tissues has been repeatedly demonstrated, and thus, ATX is implicated in progression of tumor, the precise role of ATX expressed by tumor cells was unclear. In this study, we found that ATX is highly expressed in glioblastoma multiforme (GBM), the most malignant glioma due to its high infiltration into the normal brain parenchyma, but not in tissues from other brain tumors. In addition, LPA1, an LPA receptor responsible for LPA-driven cell motility, is predominantly expressed in GBM. One of the glioblastomas that showed the highest ATX expression (SNB-78), as well as ATX-stable transfectants, showed LPA1-dependent cell migration in response to LPA in both Boyden chamber and wound healing assays. Interestingly these ATX-expressing cells also showed chemotactic response to LPC. In addition, knockdown of the ATX level using small interfering RNA technique in SNB-78 cells suppressed their migratory response to LPC. These results suggest that the autocrine production of LPA by cancer cell-derived ATX and exogenously supplied LPC contribute to the invasiveness of cancer cells and that LPA1, ATX, and LPC-producing enzymes are potential targets for cancer therapy, including GBM.