Kevin D. Woolard

SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma.

CD133+ populations of human glioblastoma multiforme (GBM) cells are reportedly enriched for tumor stem cells (TSCs) or tumor-initiating cells (TICs). Approximately 40% of freshly isolated GBM specimens, however, do not contain CD133+ tumor cells, raising the possibility that CD133 may not be a universal enrichment marker for GBM TSCs/TICs. Here we demonstrate that stage-specific embryonic antigen 1(SSEA-1/LeX)+ GBM cells fulfill the functional criteria for TSC/TIC, since (1) SSEA-1+ cells are highly tumorigenic in vivo, unlike SSEA-1- cells; (2) SSEA-1+ cells can give rise to both SSEA-1+ and SSEA-1- cells, thereby establishing a cellular hierarchy; and (3) SSEA-1+ cells have self-renewal and multilineage differentiation potentials. A distinct subpopulation of SSEA-1+ cells was present in all but one of the primary GBMs examined (n = 24), and most CD133+ tumor cells were also SSEA-1+, suggesting that SSEA-1 may be a general TSC/TIC enrichment marker in human GBMs.

Epigenetic-Mediated Dysfunction of the Bone Morphogenetic Protein Pathway Inhibits Differentiation of Glioblastoma-Initiating Cells

Despite similarities between tumor-initiating cells with stem-like properties (TICs) and normal neural stem cells, we hypothesized that there may be differences in their differentiation potentials. We now demonstrate that both bone morphogenetic protein (BMP)-mediated and ciliary neurotrophic factor (CNTF)-mediated Jak/STAT-dependent astroglial differentiation is impaired due to EZH2-dependent epigenetic silencing of BMP receptor 1B (BMPR1B) in a subset of glioblastoma TICs. Forced expression of BMPR1B either by transgene expression or demethylation of the promoter restores their differentiation capabilities and induces loss of their tumorigenicity. We propose that deregulation of the BMP developmental pathway in a subset of glioblastoma TICs contributes to their tumorigenicity both by desensitizing TICs to normal differentiation cues and by converting otherwise cytostatic signals to proproliferative signals.

Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.

No curative treatment exists for glioblastoma, with median survival times of less than 2 years from diagnosis. As an approach to develop immune-based therapies for glioblastoma, we sought to target antigens expressed in glioma stem cells (GSCs). GSCs have multiple properties that make them significantly more representative of glioma tumors than established glioma cell lines. Epidermal growth factor receptor variant III (EGFRvIII) is the result of a novel tumor-specific gene rearrangement that produces a unique protein expressed in approximately 30% of gliomas, and is an ideal target for immunotherapy. Using PCR primers spanning the EGFRvIII-specific deletion, we found that this tumor-specific gene is expressed in three of three GCS lines. Based on the sequence information of seven EGFRvIII-specific monoclonal antibodies (mAbs), we assembled chimeric antigen receptors (CARs) and evaluated the ability of CAR-engineered T cells to recognize EGFRvIII. Three of these anti-EGFRvIII CAR-engineered T cells produced the effector cytokine, interferon-γ, and lysed antigen-expressing target cells. We concentrated development on a CAR produced from human mAb 139, which specifically recognized GSC lines and glioma cell lines expressing mutant EGFRvIII, but not wild-type EGFR and did not recognize any normal human cell tested. Using the 139-based CAR, T cells from glioblastoma patients could be genetically engineered to recognize EGFRvIII-expressing tumors and could be expanded ex vivo to large numbers, and maintained their antitumor activity. Based on these observations, a γ-retroviral vector expressing this EGFRvIII CAR was produced for clinical application.

Effect of brain- and tumor-derived connective tissue growth factor on glioma invasion.

BACKGROUND Tumor cell invasion is the principal cause of treatment failure and death among patients with malignant gliomas. Connective tissue growth factor (CTGF) has been previously implicated in cancer metastasis and invasion in various tumors. We explored the mechanism of CTGF-mediated glioma cell infiltration and examined potential therapeutic targets. METHODS Highly infiltrative patient-derived glioma tumor-initiating or tumor stem cells (TIC/TSCs) were harvested and used to explore a CTGF-induced signal transduction pathway via luciferase reporter assays, chromatin immunoprecipitation (ChIP), real-time polymerase chain reaction, and immunoblotting. Treatment of TIC/TSCs with small-molecule inhibitors targeting integrin β1 (ITGB1) and the tyrosine kinase receptor type A (TrkA), and short hairpin RNAs targeting CTGF directly were used to reduce the levels of key protein components of CTGF-induced cancer infiltration. TIC/TSC infiltration was examined in real-time cell migration and invasion assays in vitro and by immunohistochemistry and in situ hybridization in TIC/TSC orthotopic xenograft mouse models (n = 30; six mice per group). All statistical tests were two-sided. RESULTS Treatment of TIC/TSCs with CTGF resulted in CTGF binding to ITGB1-TrkA receptor complexes and nuclear factor kappa B (NF-κB) transcriptional activation as measured by luciferase reporter assays (mean relative luciferase activity, untreated vs CTGF(200 ng/mL): 0.53 vs 1.87, difference = 1.34, 95% confidence interval [CI] = 0.69 to 2, P < .001). NF-κB activation resulted in binding of ZEB-1 to the E-cadherin promoter as demonstrated by ChIP analysis with subsequent E-cadherin suppression (fold increase in ZEB-1 binding to the E-cadherin promoter region: untreated + ZEB-1 antibody vs CTGF(200 ng/mL) + ZEB-1 antibody: 1.5 vs 6.4, difference = 4.9, 95% CI = 4.8 to 5.0, P < .001). Immunohistochemistry and in situ hybridization revealed that TrkA is selectively expressed in the most infiltrative glioma cells in situ and that the surrounding reactive astrocytes secrete CTGF. CONCLUSION A CTGF-rich microenvironment facilitates CTGF-ITGB1-TrkA complex activation in TIC/TSCs, thereby increasing the invasiveness of malignant gliomas.

Histone Demethylase Jumonji D3 (JMJD3) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

Histone methylation regulates normal stem cell fate decisions through a coordinated interplay between histone methyltransferases and demethylases at lineage specific genes. Malignant transformation is associated with aberrant accumulation of repressive histone modifications, such as polycomb mediated histone 3 lysine 27 (H3K27me3) resulting in a histone methylation mediated block to differentiation. The relevance, however, of histone demethylases in cancer remains less clear. We report that JMJD3, a H3K27me3 demethylase, is induced during differentiation of glioblastoma stem cells (GSCs), where it promotes a differentiation-like phenotype via chromatin dependent (INK4A/ARF locus activation) and chromatin independent (nuclear p53 protein stabilization) mechanisms. Our findings indicate that deregulation of JMJD3 may contribute to gliomagenesis via inhibition of the p53 pathway resulting in a block to terminal differentiation.

Glioma Stem Cells: Better Flat Than Round

Glioma-initiating cells/glioma stem cells (GIC/GSCs) are grown in vitro using a cumbersome and inefficient spheroid assay. In this issue of Cell Stem Cell, Pollard and coworkers present a protocol for the efficient derivation of GIC/GSC lines that may greatly improve the isolation and the potential clinical application of these cells.

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

Background Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. Methodology and Principal Findings Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. Conclusions S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.

Genetic evidence for the role of Erk activation in a lymphoproliferative disease of mice

Germline mutation of the linker for activation of T cells (LAT) gene at the phospholipase C-γ1 (PLC-γ1)-binding site leads to a fatal lymphoproliferative disease in mice. The hyperactivated T cells that develop in these mice have defective T-cell antigen receptor (TCR)-induced calcium flux but enhanced mitogen-activated protein kinase (MAPK) activation. We used genetic analysis to investigate genes whose products might suppress MAPK activation and lymphoproliferative disease in LAT mutant mice. B-lymphocyte adaptor molecule of 32 kDa (Bam32) is a known mediator of MAPK activation in B cells. We recently reported that in CD4+ T cells, Bam32 deficiency decreased MAPK activation and specifically extracellular-signal-regulated kinase (Erk) signaling, following TCR stimulation. By crossing the Bam32 null mutation onto the LAT knock-in background, we found that the Bam32 null mutation delayed the onset and decreased the severity of lymphoproliferative disease in LAT knock-in mice. The pulmonary lymphocyte infiltration seen in LAT knock-in mice was also markedly decreased in double-mutant mice. Additionally, Erk activation was diminished in LAT knock-in Bam32 knockout CD4+ T cells. To more accurately determine the role of Erk in this delay of lymphoproliferative disease, we also bred a transgenic, hypersensitive Erk allele (the Erk2 sevenmaker mutant) onto the LAT knock-in Bam32 knockout double-mutant background. These triple transgenic mice demonstrated a role for Erk activation in lymphoproliferative disease caused by the LAT knock-in mutation.

Identification of molecular pathways facilitating glioma cell invasion in situ.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3, a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.

The Raccoon Polyomavirus Genome and Tumor Antigen Transcription are Stable and Abundant in Neuroglial Tumors

ABSTRACT Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyVs association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. IMPORTANCE The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor tissue or in unaffected raccoons. Unlike other oncogenic polyomaviruses, RacPyV was episomal, not integrated, in these tumors. To determine the stability of the viral genome and sustained transcription of the oncogenic tumor antigen genes, we cultured primary raccoon tumor cells and passaged them in mice, confirming the nonintegrated state of the virus and the maintenance of viral gene transcription throughout. RacPyV provides a naturally occurring and tractable model for a novel mechanism of polyomavirus-mediated oncogenesis.