W. K.A. Yung

Se-methylselenocysteine inhibits phosphatidylinositol 3-kinase activity of mouse mammary epithelial tumor cells in vitro

IntroductionSe-methylselenocysteine (MSC), a naturally occurring selenium compound, is a promising chemopreventive agent against in vivo and in vitro models of carcinogen-induced mouse and rat mammary tumorigenesis. We have demonstrated previously that MSC induces apoptosis after a cell growth arrest in S phase in a mouse mammary epithelial tumor cell model (TM6 cells) in vitro. The present study was designed to examine the involvement of the phosphatidylinositol 3-kinase (PI3-K) pathway in TM6 tumor model in vitro after treatment with MSC.MethodsSynchronized TM6 cells treated with MSC and collected at different time points were examined for PI3-K activity and Akt phosphorylation along with phosphorylations of Raf, MAP kinase/ERK kinase (MEK), extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The growth inhibition was determined with a [3H]thymidine incorporation assay. Immunoblotting and a kinase assay were used to examine the molecules of the survival pathway.ResultsPI3-K activity was inhibited by MSC followed by dephosphorylation of Akt. The phosphorylation of p38 MAPK was also downregulated after these cells were treated with MSC. In parallel experiments MSC inhibited the Raf–MEK–ERK signaling pathway.ConclusionThese studies suggest that MSC blocks multiple signaling pathways in mouse mammary tumor cells. MSC inhibits cell growth by inhibiting the activity of PI3-K and its downstream effector molecules in mouse mammary tumor cells in vitro.

Suppression of human glioma growth by adenovirus-mediated Rb gene transfer

Objective: This study was conducted to obtain evidence that restoration of the retinoblastoma protein function may have therapeutic application for gliomas. Background: The development of glioblastoma multiforme involves progressive inactivation of several tumor suppressor genes. Abnormalities of the retinoblastoma tumor suppressor gene are found in the majority of cancers, including at least 30% of malignant gliomas. No final evidence has been produced about the role of Rb in suppressing glioma growth. Methods: To address this question, the Ad5CMV-Rb adenovirus carrying a 3.2-kb cDNA of the Rb gene was constructed. Expression of the exogenous protein was assessed by immunoblot and immunohistochemistry analyses. Growth curve assays were used to evaluate the effect of the Rb protein on glioma cell growth. Flow-cytometry analyses were used to analyze the phenotype of the cell cycle after the transfer of Rb. Human glioma xenografts implanted subcutaneously in nude mice were used for the tumorigenicity assay. Results: After the transfer of Rb, 80% of the treated cells expressed high levels of the retinoblastoma protein for at least 7 days. Within 5 days of treatment, the cells lost the neoplastic morphology and showed marked growth suppression. The majority of the Rb-expressing cells were arrested in the G1 phase of the cell cycle. In addition, the restoration of the retinoblastoma activity rendered the human glioma cells unable to form tumors in nude mice. Conclusions: These findings provide direct evidence that inactivation of the retinoblastoma protein is a critical event in gliomas, and suggest that the restoration of wild-type retinoblastoma activity in these tumors may have therapeutic utility.

Soluble Tie2 overrides the heightened invasion induced by anti-angiogenesis therapies in gliomas

Glioblastoma recurrence after treatment with the anti–vascular endothelial growth factor (VEGF) agent bevacizumab is characterized by a highly infiltrative and malignant behavior that renders surgical excision and chemotherapy ineffective. Our group has previously reported that Tie2-expressing monocytes (TEMs) are aberrantly present at the tumor/normal brain interface after anti-VEGF therapies and their significant role in the invasive outgrowth of these tumors. Here, we aimed to further understand the mechanisms leading to this pro-invasive tumor microenvironment. Examination of a U87MG xenogeneic glioma model and a GL261 murine syngeneic model showed increased tumor expression of angiopoietin 2 (Ang2), a natural ligand of Tie2, after anti-angiogenesis therapies targeting VEGF or VEGF receptor (VEGFR), as assessed by immunohistochemical analysis, immunofluorescence analysis, and enzyme-linked immunosorbent assays of tumor lysates. Migration and gelatinolytic assays showed that Ang2 acts as both a chemoattractant of TEMs and an enhancing signal for their tumor-remodeling properties. Accordingly, in vivo transduction of Ang2 into intracranial gliomas increased recruitment of TEMs into the tumor. To reduce invasive tumor outgrowth after anti-angiogenesis therapy, we targeted the Ang-Tie2 axis using a Tie2 decoy receptor. Using syngeneic models, we observed that overexpression of soluble Tie2 within the tumor prevented the recruitment of TEMs to the tumor and the development of invasion after anti-angiogenesis treatment. Taken together, these data indicate an active role for the Ang2-Tie2 pathway in invasive glioma recurrence after anti-angiogenesis treatment and provide a rationale for testing the combined targeting of VEGF and Ang-Tie2 pathways in patients with glioblastoma.

PAF promotes stemness and radioresistance of glioma stem cells.

Significance Glioblastoma multiforme (GBM) is uniformly lethal and shows resistance to all forms of therapy. Glioma stem cells (GSCs) have been shown to support GBM maintenance and exhibit enhanced resistance to ionizing radiation, a cornerstone of GBM therapy. This study establishes that proliferating cell nuclear antigen-associated factor (PAF) depletion profoundly reduces GSC frequency and tumorigenicity, in part, by down-regulating DNA replication and pyrimidine metabolism. Moreover, PAF depletion impairs error-prone DNA translesion synthesis (TLS) and enhances sensitivity of GSCs to radiation treatment. Pharmacological impairment of DNA replication and TLS diminished GSC maintenance and radioresistance, illuminating a potential GBM treatment strategy of combined TLS inhibition and radiation therapy. An integrated genomic and functional analysis to elucidate DNA damage signaling factors promoting self-renewal of glioma stem cells (GSCs) identified proliferating cell nuclear antigen (PCNA)-associated factor (PAF) up-regulation in glioblastoma. PAF is preferentially overexpressed in GSCs. Its depletion impairs maintenance of self-renewal without promoting differentiation and reduces tumor-initiating cell frequency. Combined transcriptomic and metabolomic analyses revealed that PAF supports GSC maintenance, in part, by influencing DNA replication and pyrimidine metabolism pathways. PAF interacts with PCNA and regulates PCNA-associated DNA translesion synthesis (TLS); consequently, PAF depletion in combination with radiation generated fewer tumorspheres compared with radiation alone. Correspondingly, pharmacological impairment of DNA replication and TLS phenocopied the effect of PAF depletion in compromising GSC self-renewal and radioresistance, providing preclinical proof of principle that combined TLS inhibition and radiation therapy may be a viable therapeutic option in the treatment of glioblastoma multiforme (GBM).

Abstract 1720: MSK1 mediates PI3K inhibition resistance by induction of β-catenin phosphorylation in glioblastoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Glioblastoma (GBM), the most common malignant brain tumor in adults, represents a compelling disease for kinase inhibitor therapy because majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. PI3K/mTOR-pathway is dysregulated in over 50% of human glioblastomas (GBM) and represents an attractive target for therapy. One major challenge for single pathway PI3K inhibition targeting therapy is intrinsic resistance. To study the mechanism of intrinsic resistance to PI3K inhibition, we analyzed the pre- and post-treatment reverse phase protein arrays (RPPA) data of both giloma cells and giloma initiating cells (GIC). Mitogen- and stress-activated protein kinase 1 (MSK1) was markedly induced after PI3K inhibitor treatment and it was linked with increase expression of β-catenin. We further found that MSK1 directly interacts with β-catenin and stabilizes β-catenin protein level. MSK1 also regulates β-catenin nuclear translocation and β-catenin transcriptional activity. More interestingly, using in vitro kinase assay, we identified MSK1 is a kinase that phosphorylates β-catenin. Finally, we showed that depletion of β-catenin potentiates PI3K inhibitors induced cytotoxity in a series of GICs. Taken together, our results suggest that MSK1/β-catenin signaling may serve as an escape survival signaling upon PI3K inhibition, and combination of PI3K inhibition and MSK1/β-catenin inhibition is required to induce lethal growth inhibition in human glioblastoma cells. Citation Format: Shaofang Wu, Jun Fu, Siyuan Zheng, Roel G. Verhaak, W. K Yung, Dimpy Koul. MSK1 mediates PI3K inhibition resistance by induction of β-catenin phosphorylation in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1720. doi:10.1158/1538-7445.AM2014-1720

Abstract 4170: Oncogenic activation of LMO2 by EGFR signaling regulates STAT3 phosphorylation and transcriptional activity in human glioma

Glioblastoma multiforme (GBM) is a highly heterogeneous disease characterized by multiple genetic abnormalities. Amplification and mutation of the epidermal growth factor receptor (EGFR) gene represent signature genetic abnormalities occurred in over 60% of GBM. The successful application of EGFR-targeted therapy for the treatment of GBM is thus dependent on a thorough understanding of the intricate signal pathways cross-linkage that underlies the pathobiology of this disease. The LIM-domain only factor 2 (LMO2) gene is a crucial oncogene involved in hematopoietic development and leukemogenesis. Herein, we showed, from the TCGA GBM data, that LMO2 is highly expressed in EGFR-aberrant GBM samples and might serve as a downstream effector of EGFR signaling in GBM. Depletion of LMO2 expression impairs the maintenance of glioma cell proliferation in vitro and in vivo. In addition, EGFR signaling induces LMO2 nuclear translocation and enables LMO2 to form a triplex with EGFR and Signal transducer and activator of transcription 3 (STAT3). More importantly, LMO2 serves as a bridging molecule to promote the formation of EGFR/STAT3 complex and to enhance EGFR-mediated STAT3 Y705 phosphorylation upon EGF stimulation, which in turn up-regulates the expression of STAT3-responsive genes involved in the regulation of cell proliferation and tumor development. In conclusion, these findings reveal that LMO2 might serve as a critical effector of EGFR signaling in human glioma and highlight the essential function of LMO2 in EGF-stimulated STAT3 activation and tumorigenesis. Citation Format: Jun Fu, Dimpy Koul, Shaofang Wu, Siyuan Zheng, Roel G. Verhaak, W.K Yung. Oncogenic activation of LMO2 by EGFR signaling regulates STAT3 phosphorylation and transcriptional activity in human glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4170. doi:10.1158/1538-7445.AM2014-4170

Abstract 5481: Therapy of human LN229 glioblastoma growing in the brain of nude mice by Macitentan, a dual endothelin receptor A and B antagonist, combined with Temozolomide.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Purpose: We determined whether the blockade of endothelin receptor pathways can reverse the chemoprotection mediated by astrocytes and produce therapy of Human LN 229 Glioblastoma cells growing in the brain of nude mice treated with the dual endothelin receptor antagonist, Macitentan and Temozolomide (TMZ). Materials and Methods: In the first set of experiments, murine astrocytes were cultured with tumor cells in the presence or absence of Macitentan and TMZ. For therapy studies the leuciferas labeled LN229 cells were implanted (stereotactically) into the brains of nude mice. The mice were ear tagged and imaged by IVIS spectrometer at multiple times. On day 24 the mice were randomized to receive vehicle, TMZ (p.o., 7.5mg/kg, one week on and two weeks off), Macitentan (p.o., 10mg/kg, daily) and Macitentan plus TMZ. Survival of mice was compared by Kaplan-Meyer curve (log-rank test) and immunohistochemical analyses were carried out for multiple markers. Results: Simultaneous blocking of endothelin receptor A and B pathways reversed the in culture protection induced by astrocytes against TMZ (p<0.05). Therapy with Macitentan and TMZ significantly prolonged the survival of mice (p<0.0001) .The blockade of endothelin receptor A and B significantly decreased the level of expression of phosphorylated AKT and MAPK, and down-regulated the expression of BCL2L1, TWIST1 and GSTA5 in LN 229 cells growing in the brain. Proliferation of tumor cells and tumor-associated endothelial cells was significantly decreased. Moreover the apoptosis of LN229 cells was significantly increased in the brain of the combination treatment group. Conclusion: Blockade of endothelin receptor A and B pathways by Macitentan combined with TMZ provides a new approach for the treatment of patients with Glioblastoma. Citation Format: Sun Jin Kim, Seung Wook Kim, Hyun Jin Choi, Ho Jeong Lee, Francois Lehembre, Junqin He, Urs Regenass, Charles A Conrad, W.K. Yung, Isaiah J. Fidler. Therapy of human LN229 glioblastoma growing in the brain of nude mice by Macitentan, a dual endothelin receptor A and B antagonist, combined with Temozolomide. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5481. doi:10.1158/1538-7445.AM2013-5481

Abstract LB-114: Molecular and lineage analysis of glioblastoma stem cells identifies clinically relevant models of glioblatoma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The Cancer Genome Atlas project described a robust gene expression-based molecular classification of GBM. However, the functional and biological significance of the molecular subclasses are being determined. In this study, we hypothesize that Glioma Stem Cells (GSCs) isolated from individual patient tumor samples will recapitulate the molecular characteristics of tumor samples and provide a relevant model for functional analysis of the molecular subclasses. Thus we conducted a comprehensive analysis of 26 GSC lines with expression array, RPPA, and a series of neural lineage markers. Analysis of the expression data classified the 26 GSC lines into four subtypes ( Classical, Proneural 1, Proneural 2 and Mesenchymal ,) closely similar to the TCGA subclasses with a distinct profile for each subtype (c-Myc, Cyclin D2 for Classical ; Olig2, NKX2–2, Notch-1, Notch-3 for Proneural 2 ; BMP4, DCX, p16INK4a, ID2 for Proneural 1 ; CD44, CAV1, TGFBR2 for Mesenchymal ). Further analysis showed that GSC subtypes exhibit divergent patterns of signaling pathway activation. The major pathways activated in 4 subtypes were Notch pathway in Proneural 2 , Wnt/β-Catenin in Proneural 2 and Classical , while FGF/VEGF and TGF-β in Mesenchymal. In vitro treatment with SB 431542, a TGF-βR inhibitor, showed that Mesenchymal subtype is more sensitive than other subtypes. More importantly, lineage analysis of GSCs subtypes show that Proneural and Classical GSCs differentially express lineage markers for neural stem/progenitor cells and were responsive to differentiating agent retinoic acid. In addition, GSC subtypes exhibit distinct biological behaviors in self-renewal capacity, proliferation, invasiveness, angiogenic potential, response to growth factor stimulus, and differentiation in vitro and in vivo with Classical subtype being more proliferative and Mesenchymal subtype being more angiogenic and invasive. In conclusion, our comprehensive analysis showed that GSCs reflect patient tumor subclass and these subtypes showed distinct regulatory pathway activation, lineage profile and biological behaviors. Thus GSC is biologically and molecularly a more relevant model system for preclinical studies of therapeutic intervention and to expand our molecular understanding of human GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2011-LB-114