Regulation of hematopoietic stem cell fate by the ubiquitin proteasome system.

Hematopoietic stem cells (HSCs) residing in the bone marrow generate mature blood cells throughout the life of the organism. This is accomplished by careful regulation of HSC activity to balance quiescence, self-renewal and differentiation. Studies of the molecular mechanisms governing HSC maintenance have mostly focused on the role of signaling and transcriptional processes. However, it has recently been demonstrated that protein regulation via the ubiquitin proteasome system (UPS) is crucial for normal HSC function; the loss of which can lead to transformation and leukemogenesis. The effective use of a general and reversible inhibitor of the UPS, bortezomib, in treating mantle cell lymphoma and multiple myeloma has demonstrated that targeting the UPS has therapeutic potential. Thus, understanding the emerging field of how the UPS regulates HSC activity may lead to novel targets for therapy of leukemia.

The phosphorylation of ephrin-B2 ligand promotes glioma cell migration and invasion.

To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan-Meier analysis demonstrated ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin-B2 were high in GBM. Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin-B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin-B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a strong predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.

Glioma cells on the run - the migratory transcriptome of 10 human glioma cell lines.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. RESULTS: Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. CONCLUSION: Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

Loss of the mismatch repair protein MSH6 in human glioblastomas is associated with tumor progression during temozolomide treatment.

PURPOSE: Glioblastomas are treated by surgical resection followed by radiotherapy [X-ray therapy (XRT)] and the alkylating chemotherapeutic agent temozolomide. Recently, inactivating mutations in the mismatch repair gene MSH6 were identified in two glioblastomas recurrent post-temozolomide. Because mismatch repair pathway inactivation is a known mediator of alkylator resistance in vitro, these findings suggested that MSH6 inactivation was causally linked to these two recurrences. However, the extent of involvement of MSH6 in glioblastoma is unknown. We sought to determine the overall frequency and clinical relevance of MSH6 alterations in glioblastomas. EXPERIMENTAL DESIGN: The MSH6 gene was sequenced in 54 glioblastomas. MSH6 and O(6)-methylguanine methyltransferase (MGMT) immunohistochemistry was systematically scored in a panel of 46 clinically well-characterized glioblastomas, and the corresponding patient response to treatment evaluated. RESULTS: MSH6 mutation was not observed in any pretreatment glioblastoma (0 of 40), whereas 3 of 14 recurrent cases had somatic mutations (P = 0.015). MSH6 protein expression was detected in all pretreatment (17 of 17) cases examined but, notably, expression was lost in 7 of 17 (41%) recurrences from matched post-XRT + temozolomide cases (P = 0.016). Loss of MSH6 was not associated with O(6)-methylguanine methyltransferase status. Measurements of in vivo tumor growth using three-dimensional reconstructed magnetic resonance imaging showed that MSH6-negative glioblastomas had a markedly increased rate of growth while under temozolomide treatment (3.17 versus 0.04 cc/mo for MSH6-positive tumors; P = 0.020). CONCLUSIONS: Loss of MSH6 occurs in a subset of post-XRT + temozolomide glioblastoma recurrences and is associated with tumor progression during temozolomide treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro. MSH6 deficiency may therefore contribute to the emergence of recurrent glioblastomas during temozolomide treatment.

MAP-ing glioma invasion: mitogen-activated protein kinase kinase 3 and p38 drive glioma invasion and progression and predict patient survival.

Although astrocytic brain tumors do not metastasize systemically, during tumorigenesis glioma cells adopt an invasive phenotype that is poorly targeted by conventional therapies; hence, glioma patients die of recurrence from the locally invasive tumor population. Our work is aimed at identifying and validating novel therapeutic targets and biomarkers in invasive human gliomas. Transcriptomes of invasive glioma cells relative to stationary cognates were produced from a three-dimensional spheroid in vitro invasion assay by laser capture microdissection and whole human genome expression microarrays. Qualitative differential expression of candidate invasion genes was confirmed by quantitative reverse transcription-PCR, clinically by immunohistochemistry on tissue microarray, by immunoblotting on surgical specimens, and on two independent gene expression data sets of glial tumors. Cell-based assays and ex vivo brain slice invasion studies were used for functional validation. We identify mitogen-activated protein kinase (MAPK) kinase 3 (MKK3) as a key activator of p38 MAPK in glioma; MKK3 activation is strongly correlated with p38 activation in vitro and in vivo. We further report that these members of the MAPK family are strong promoters of tumor invasion, progression, and poor patient survival. Inhibition of either candidate leads to significantly reduced glioma invasiveness in vitro. Consistent with the concept of synthetic lethality, we show that inhibition of invasion by interference with these genes greatly sensitizes arrested glioma cells to cytotoxic therapies. Our findings therefore argue that interference with MKK3 signaling through a novel treatment combination of p38 inhibitor plus temozolomide heightens the vulnerability of glioma to chemotherapy.

Regulation of self-renewal in normal and cancer stem cells.

Mutations can confer a selective advantage on specific cells, enabling them to go through the multistep process that leads to malignant transformation. The cancer stem cell hypothesis postulates that only a small pool of low-cycling stem-like cells is necessary and sufficient to originate and develop the disease. Normal and cancer stem cells share important functional similarities such as 'self-renewal' and differentiation potential. However, normal and cancer stem cells have different biological behaviours, mainly because of a profound deregulation of self-renewal capability in cancer stem cells. Differences in mode of division, cell-cycle properties, replicative potential and handling of DNA damage, in addition to the activation/inactivation of cancer-specific molecular pathways confer on cancer stem cells a malignant phenotype. In the last decade, much effort has been devoted to unravel the complex dynamics underlying cancer stem cell-specific characteristics. However, further studies are required to identify cancer stem cell-specific markers and targets that can help to confirm the cancer stem cell hypothesis and develop novel cancer stem cell-based therapeutic approaches.

Autotaxin: a secreted autocrine/paracrine factor that promotes glioma invasion.

Glioblastoma multiforme (GBM) is inherently invasive, and it is from the invasive cell population that the tumor recurs. The GBM invasion transcriptome reveals over-expression of various autocrine factors that could act as motility drivers, such as autotaxin (ATX). Some of these factors could also have paracrine roles, modulating the behavior of cells in the peri-tumoral brain parenchyma. ATX generates lysophosphatidic acid (LPA), which signals through LPA receptors expressed by GBM as well as in astrocytes, oligodendrocytes (ODC) and microglia; their activation manifest cell specific effects. ATX stimulates invasion of GBM cells in vitro and ex vivo invasion assays. ATX activity enhances GBM adhesion in cells expressing the LPA1 receptor, as well as stimulating rac activation. GBM secreted ATX can also have paracrine effects: ATX activity results in reduced ODC adhesion. ODC monolayer invasion showed that U87 and U251 GBM cells expressing ATX invaded through an ODC monolayer significantly more than cells depleted of ATX or cells expressing inactive ATX, suggesting that GBM cells secreting ATX find ODCs less of a barrier than cells that do not express ATX. Secreted factors that drive GBM invasion can have autocrine and paracrine roles; one stimulates GBM motility and the other results in ODC dis-adhesion.