Yiwen Jiang

The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes

Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.

On the origin of glioma

Abstract Glioma is the most frequent primary brain tumor of adults that has a presumably glial origin. Although our knowledge regarding molecular mechanisms and signaling pathways involved in gliomagenesis has increased immensely during the past decade, high-grade glioma remains a lethal disease with dismal prognosis. The failure of current therapies has to a large extent been ascribed the functional heterogeneity of glioma cells. One reason for this heterogeneity is most certainly the large number of variations in genetic alterations that can be found in high-grade gliomas. Another factor that may influence glioma heterogeneity could be the cell type from which the glioma is initiated. The cell of origin for glioma is still undefined, and additional knowledge about this issue may prove critical for a more complete understanding of glioma biology. Based on information from patients, developmental biology, and experimental glioma models, the most putative target cells include astrocytes, neural stem cells, and oligodendrocyte precursor cells, which are all discussed in more detail in this article. Animal modeling of glioma suggests that these three cell types have the capability to be the origin of glioma, and we have reason to believe that, depending on the initiating cell type, prognosis and response to therapy may be significantly different. Thus, it is essential to explore further the role of cellular origin in glioma.

Oncogenic Signaling Is Dominant to Cell of Origin and Dictates Astrocytic or Oligodendroglial Tumor Development from Oligodendrocyte Precursor Cells

Stem cells, believed to be the cellular origin of glioma, are able to generate gliomas, according to experimental studies. Here we investigated the potential and circumstances of more differentiated cells to generate glioma development. We and others have shown that oligodendrocyte precursor cells (OPCs) can also be the cell of origin for experimental oligodendroglial tumors. However, the question of whether OPCs have the capacity to initiate astrocytic gliomas remains unanswered. Astrocytic and oligodendroglial tumors represent the two most common groups of glioma and have been considered as distinct disease groups with putatively different origins. Here we show that mouse OPCs can give rise to both types of glioma given the right circumstances. We analyzed tumors induced by K-RAS and AKT and compared them to oligodendroglial platelet-derived growth factor B-induced tumors in Ctv-a mice with targeted deletions of Cdkn2a (p16Ink4a−/−, p19Arf−/−, Cdkn2a−/−). Our results showed that glioma can originate from OPCs through overexpression of K-RAS and AKT when combined with p19Arf loss, and these tumors displayed an astrocytic histology and high expression of astrocytic markers. We argue that OPCs have the potential to develop both astrocytic and oligodendroglial tumors given loss of p19Arf, and that oncogenic signaling is dominant to cell of origin in determining glioma phenotype. Our mouse data are supported by the fact that human astrocytoma and oligodendroglioma display a high degree of overlap in global gene expression with no clear distinctions between the two diagnoses.

miRNA-21 is developmentally regulated in mouse brain and is co-expressed with SOX2 in glioma

BackgroundMicroRNAs (miRNAs) and their role during tumor development have been studied in great detail during the last decade, albeit their expression pattern and regulation during normal development are however not so well established. Previous studies have shown that miRNAs are differentially expressed in solid human tumors. Platelet-derived growth factor (PDGF) signaling is known to be involved in normal development of the brain as well as in malignant primary brain tumors, gliomas, but the complete mechanism is still lacking. We decided to investigate the expression of the oncogenic miR-21 during normal mouse development and glioma, focusing on PDGF signaling as a potential regulator of miR-21.MethodsWe generated mouse glioma using the RCAS/tv-a system for driving PDGF-BB expression in a cell-specific manner. Expression of miR-21 in mouse cell cultures and mouse brain were assessed using Northern blot analysis and in situ hybridization. Immunohistochemistry and Western blot analysis were used to investigate SOX2 expression. LNA-modified siRNA was used for irreversible depletion of miR-21. For inhibition of PDGF signaling Gleevec (imatinib mesylate), Rapamycin and U0126, as well as siRNA were used. Statistical significance was calculated using double-sided unpaired Student´s t-test.ResultsWe identified miR-21 to be highly expressed during embryonic and newborn brain development followed by a gradual decrease until undetectable at postnatal day 7 (P7), this pattern correlated with SOX2 expression. Furthermore, miR-21 and SOX2 showed up-regulation and overlapping expression pattern in RCAS/tv-a generated mouse brain tumor specimens. Upon irreversible depletion of miR-21 the expression of SOX2 was strongly diminished in both mouse primary glioma cultures and human glioma cell lines. Interestingly, in normal fibroblasts the expression of miR-21 was induced by PDGF-BB, and inhibition of PDGF signaling in mouse glioma primary cultures resulted in suppression of miR-21 suggesting that miR-21 is indeed regulated by PDGF signaling.ConclusionsOur data show that miR-21 and SOX2 are tightly regulated already during embryogenesis and define a distinct population with putative tumor cell of origin characteristics. Furthermore, we believe that miR-21 is a mediator of PDGF-driven brain tumors, which suggests miR-21 as a promising target for treatment of glioma.

Snail depletes the tumorigenic potential of glioblastoma

Glioblastoma multiforme (GBM) is an aggressive brain malignancy characterized by high heterogeneity and invasiveness. It is increasingly accepted that the refractory feature of GBM to current therapies stems from the existence of few tumorigenic cells that sustain tumor growth and spreading, the so-called glioma-initiating cells (GICs). Previous studies showed that cytokines of the bone morphogenetic protein (BMP) family induce differentiation of the GICs, and thus act as tumor suppressors. Molecular pathways that explain this behavior of BMP cytokines remain largely elusive. Here, we show that BMP signaling induces Smad-dependent expression of the transcriptional regulator Snail in a rapid and sustained manner. Consistent with its already established promigratory function in other cell types, we report that Snail silencing decreases GBM cell migration. Consequently, overexpression of Snail increases GBM invasiveness in a mouse xenograft model. Surprisingly, we found that Snail depletes the GBM capacity to form gliomaspheres in vitro and to grow tumors in vivo, both of which are important features shared by GICs. Thus Snail, acting downstream of BMP signaling, dissociates the invasive capacity of GBM cells from their tumorigenic potential.

Forced expression of Sox21 inhibits Sox2 and induces apoptosis in human glioma cells

Numerous studies support a role for Sox2 to keep stem cells and progenitor cells in an immature and proliferative state. Coexpression of Sox2 and GFAP has been found in regions of the adult brain where neural stem cells are present and in human glioma cells. In our study, we have investigated the roles of Sox2 and its counteracting partner Sox21 in human glioma cells. We show for the first time that Sox21 is expressed in both primary glioblastoma and in human glioma cell lines. We found that coexpression of Sox2, GFAP and Sox21 was mutually exclusive with expression of fibronectin. Our result suggests that glioma consists of at least two different cell populations: Sox2+/GFAP+/Sox21+/FN− and Sox2−/GFAP−/Sox21−/FN+. Reduction of Sox2 expression by using siRNA against Sox2 or by overexpressing Sox21 using a tetracycline‐regulated expression system (Tet‐on) caused decreased GFAP expression and a reduction in cell number due to induction of apoptosis. We suggest that Sox21 can negatively regulate Sox2 in glioma. Our findings imply that Sox2 and Sox21 may be interesting targets for the development of novel glioma therapy.

Sox5 can suppress platelet-derived growth factor B-induced glioma development in Ink4a-deficient mice through induction of acute cellular senescence

SOX5 is a member of the high-mobility group superfamily of architectural non-histone proteins involved in gene regulation and maintenance of chromatin structure in a wide variety of developmental processes. Sox5 was identified as a brain tumor locus in a retroviral insertional mutagenesis screen of platelet-derived growth factor B (PDGFB)-induced mouse gliomas. Here we have investigated the role of Sox5 in PDGFB-induced gliomagenesis in mice. We show that Sox5 can suppress PDGFB-induced glioma development predominantly upon Ink4a-loss. In human glioma cell lines and tissues, we found very low levels of SOX5 compared with normal brain. Overexpression of Sox5 in human glioma cells led to a reduction in clone formation and inhibition of proliferation. Combined expression of Sox5 and PDGFB in primary brain cell cultures caused decreased proliferation and an increased number of senescent cells in the Ink4a−/− cells only. Protein analyses showed a reduction in the amount and activation of Akt and increased levels of p27Kip1 upon Sox5 expression that was dominant to PDGFB signaling and specific to Ink4a−/− cells. Upon inhibition of p27Kip1, the effects of Sox5 on proliferation and senescence could be reversed. Our data suggest a novel pathway, where Sox5 may suppress the oncogenic effects of PDGFB signaling during glioma development by regulating p27Kip1 in a p19Arf-dependent manner, leading to acute cellular senescence.

Pleiotrophin enhances PDGFB-induced gliomagenesis through increased proliferation of neural progenitor cells

Pleiotrophin (PTN) augments tumor growth by increasing proliferation of tumor cells and promoting vascular abnormalization, but its role in early gliomagenesis has not been evaluated. Through analysis of publically available datasets, we demonstrate that increased PTN mRNA expression is associated with amplification of chromosome 7, identified as one of the earliest steps in glioblastoma development. To elucidate the role of PTN in tumor initiation we employed the RCAS/tv-a model that allows glioma induction by RCAS-virus mediated expression of oncogenes in neural progenitor cells. Intracranial injection of RCAS-PTN did not induce glioma formation when administrated alone, but significantly enhanced RCAS-platelet derived growth factor (PDGF)B-induced gliomagenesis. PTN co-treatment augmented PDGFB-induced Akt activation in neural progenitor cells in vitro, and enhanced neural sphere size associated with increased proliferation. Our data indicates that PTN expression is associated with chromosome 7 gain, and that PTN enhances PDGFB-induced gliomagenesis by stimulating proliferation of neural progenitor cells.

Abstract 3343: Upregulation of SOX5 perturbs human glioma cell proliferation and is associated with proneural glioblastoma

Background: The proneural subtype of glioblastoma (GBM) is characterized by PDGFRα alterations. In a retroviral insertional mutagenesis screen (Johansson et al., PNAS, 2004) the transcription factor Sox5 was identified as a candidate brain tumor locus and the mode of insertions indicated an overexpression of the short version of Sox5 (s-Sox5). This led to an investigation of the role of s-Sox5 in glioma development, and we subsequently found that opposite to our hypothesis, s-Sox5 could inhibit glioma development in mice, reduce cell proliferation and clone formation ability of human glioma cell lines, and induce acute cellular senescence in primary mouse glial cells through regulation of AKT and p27Kip1 (Tchougounova et al., Oncogene, 2009). Aims: To investigate if s-SOX5 alterations are associated with a certain subtype of human GBM, and to identify the mechanism behind the anti-tumoral properties of s-SOX5 in newly established human glioma cell cultures (HGCCs). Methods: The TCGA dataset for GBM was analyzed via the cBio Cancer Genomics Portal (http://cbioportal.org) for alterations in SOX5 + other SOX genes and glioma genes. Endogenous SOX5 + expression of other SOX proteins were analyzed with immunostainings in six newly established HGCCs maintained under stem cell conditions. These HGCCs were also transfected with pcDNA-V5-s-Sox5, and analyzed by flow cytometry for EdU incorporation, cleaved Caspase-3 and V5. Results: We found that the majority of SOX5 alterations were confined to the proneural subtype (10/56 cases) with 90% of the alterations being upregulations. In fact, all upregulations of SOX5 were found in the proneural group. Only a few alterations (3/56; all downregulations) were found in the mesenchymal subtype, and none in the classical or neural subgroups. The upregulated SOX5 expression in the proneural subtype could be connected to a slight increase in patient survival. Co-occurring alterations in s-SOX5, PDGFRα, SOX6 and SOX10 were found in the proneural subtype. These findings are only suggestive and need to be validated in larger set of tumor samples. Immunostainings showed that all HGCCs expressed SOX2, SOX6, SOX9 and SOX10. For SOX5, two were completely negative while the remaining four showed varying expression. The transfection efficiency was very low (10%), but when specifically analyzing the V5-s-SOX5 positive cells, we found in all HGCCs a reduced number of EdU positive cells compared to the uninfected cell population of the same sample (statistically significant in three HGCCs). Conclusion: Our preliminary data suggest that upregulation of s-SOX5 could be a defining alteration in the proneural subgroup of GBM, and that s-SOX5 suppresses proliferation of human glioma cell cultures. Further studies are underway to solidify our findings and clarify the mechanism behind s-SOX5/Sox5 inhibition of glioma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3343. doi:1538-7445.AM2012-3343

LGR5 promotes tumorigenicity and invasion of glioblastoma stem-like cells and is a potential therapeutic target for a subset of glioblastoma patients: LGR5 regulates glioblastoma tumorigenicity and invasion

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine‐rich repeat‐containing G‐protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient‐derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5‐responding cultures could be identified by their significantly higher self‐renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high‐ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high‐ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5‐responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient‐derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright