A Chromosomal Region 7p11.2 Transcript Map: Its Development and Application to the Study of EGFR Amplicons in Glioblastoma

Neuro-oncology. Apr, 2002  |  Pubmed ID: 11916499

Cumulative information available about the organization of amplified chromosomal regions in human tumors suggests that the amplification repeat units, or amplicons, can be of a simple or complex nature. For the former, amplified regions generally retain their native chromosomal configuration and involve a single amplification target sequence. For complex amplicons, amplified DNAs usually undergo substantial reorganization relative to the normal chromosomal regions from which they evolve, and the regions subject to amplification may contain multiple target sequences. Previous efforts to characterize the 7p11.2 epidermal growth factor receptor ) amplicon in glioblastoma have relied primarily on the use of markers positioned by linkage analysis and/or radiation hybrid mapping, both of which are known to have the potential for being inaccurate when attempting to order loci over relatively short (<1 Mb) chromosomal regions. Due to the limited resolution of genetic maps that have been established through the use of these approaches, we have constructed a 2-Mb bacterial and P1-derived artificial chromosome (BAC-PAC) contig for the EGFR region and have applied markers positioned on its associated physical map to the analysis of 7p11.2 amplifications in a series of glioblastomas. Our data indicate that EGFR is the sole amplification target within the mapped region, although there are several additional 7p11.2 genes that can be coamplified and overexpressed with EGFR. Furthermore, these results are consistent with EGFR amplicons retaining the same organization as the native chromosome 7p11.2 region from which they are derived.

Mucosa-associated Lymphoid Tissue Lymphomas with T(11;18)(q21;q21) and Mucosa-associated Lymphoid Tissue Lymphomas with Aneuploidy Develop Along Different Pathogenetic Pathways

The American Journal of Pathology. Jul, 2002  |  Pubmed ID: 12107090

t(11;18)(q21;q21) and aneuploidy are recurrent chromosomal aberrations in mucosa-associated lymphoid tissue (MALT) lymphomas. To investigate their relationship and clinical significance, we developed a two-color fluorescence in situ hybridization (FISH) technique to detect t(11;18) and aneuploidy in nuclei isolated from paraffin-embedded tissue. Thirty-seven MALT lymphomas (all previously evaluated for t(11;18) by reverse transcriptase-polymerase chain reaction), 1 large cell lymphoma (LCL) arising subsequent to MALT lymphoma, and 16 controls were tested by FISH using the t(11;18) probe set and multiple centromeric probes. t(11;18)(q21;q21) was present by FISH in 11 of 12 polymerase chain reaction-positive MALT lymphomas (92%). The LCL and its clonally identical antecedent MALT lymphoma both showed t(11;18). The LCL had trisomy 12, and a small subset of MALT lymphoma cells had trisomy 3 and/or 12. Only one other MALT lymphoma with t(11;18) showed aneuploidy (trisomy 3) in a small clone, whereas 15 of 25 t(11;18)-negative MALT lymphomas (60%) showed trisomy of chromosomes 18 (n = 12), 3 (n = 8), 7 (n = 2), and/or 11 (n = 1). t(11;18) and aneuploidy are primarily mutually exclusive events, suggesting different pathogenetic pathways in the development of MALT lymphomas. Both t(11;18) and aneuploidy were seen disproportionately in lung, and both were associated with recurrent disease.

Neuropathology of Genetically Engineered Mice: Consensus Report and Recommendations from an International Forum

Oncogene. Oct, 2002  |  Pubmed ID: 12386807

The Mouse Models of Cancer Consortium of the NCI sponsored a meeting of neuropathologists and veterinary pathologists in New York City in November of 2000. A rapidly growing number of genetically engineered mice (GEM) predisposed to tumors of the nervous system have led to a concomitant need for neuropathological evaluation and validation of these models. A panel of 13 pathologists reviewed material representing most of the available published and unpublished GEM models of medulloblastoma, primitive neuroectodermal tumor, astrocytoma, oligodendroglioma, mixed glioma, and tumors of the peripheral nerve. The GEM tumors were found to have many similarities and some distinct differences with respect to human disease. After review of the biology and pathology for all models presented, participants were split into groups reflective of clinical expertise in human pathology, tumor biology, neuroimaging, or treatment/intervention. Recommendations were made detailing an extensive and complete neuropathological characterization of animals. Importance was placed on including information on strains, tumor clonality, and examination for genetic mutation or altered gene expression characteristics of the corresponding human malignancy. Specific proposals were made to incorporate GEM models in emerging neuroradiological modalities. Recommendations were also made for preclinical validation of these models in cancer therapeutics, and for incorporation of surrogate markers of tumor burden to facilitate preclinical evaluation of new therapies.

Combination of Epidermal Growth Factor Receptor Targeted Therapy with Radiation Therapy for Malignant Gliomas

Frontiers in Bioscience : a Journal and Virtual Library. Jan, 2003  |  Pubmed ID: 12456322

Glioblastoma multiforme (GBM) are extremely aggressive brain tumors characterized by resistance to standard treatment modalities including surgery, radiation therapy and chemotherapy. While radiation therapy is the standard treatment after surgical resection, these tumors invariably recur and are associated with a uniformly dismal prognosis. Cytotoxic chemotherapy has failed to improve on the modest gains conferred by radiation therapy. Our understanding of the molecular events driving gliomagenesis has led to the recognition of frequent alterations in the epidermal growth factor receptor (EGFR) pathway, leading to increased aggressiveness and a poorer prognosis. Based on the importance of EGFR in the development of malignancy in multiple tumor types, several classes of novel therapeutic agents have been developed that specifically target EGFR. This review outlines the relevance of normal and aberrant EGFR signaling in the biology of gliomas, the strategies for inhibiting EGFR activity and the rationale for combining EGFR inhibitors with radiation therapy in the treatment of GBM.

Lanthionine Synthetase Components C-like 2 Increases Cellular Sensitivity to Adriamycin by Decreasing the Expression of P-glycoprotein Through a Transcription-mediated Mechanism

Cancer Research. Feb, 2003  |  Pubmed ID: 12566319

Although the coincidental amplification and accompanying overexpression of bystander genes that neighbor oncogene targets occur frequently during the development of human tumors, little has been done to investigate the functional or biological consequences of amplified bystander gene overexpression. LANCL2 (LANC-like 2) is a bystander gene that is coamplified and overexpressed with epidermal growth factor receptor in approximately 20% of all glioblastomas. This gene has also been designated as Testis Adriamycin Sensitivity Protein because it is most highly expressed in testis and its expression has been noted to increase cellular sensitivity to Adriamycin. Because of the latter association, we have examined potential relationships between LANCL2 and the expression of multidrug-resistance (MDR)1, as well as its cognate protein, P-glycoprotein (P-gp), because elevated expression of P-gp is known to increase cell resistance to many cytotoxic drugs, including Adriamycin. Using the Dx5 derivative of MES-SA cells in which P-gp is overexpressed, we show that the level of endogenous P-gp decreases with increased expression of exogenous LanCl-2 and that cells with reduced P-gp show increased sensitivity to Adriamycin. Results from reverse transcription-PCR and MDR1 promoter activity analyses suggest that LanCl-2 transcriptionally suppresses MDR1, and this interpretation of LanCl-2 function is consistent with results from immunofluorescence analysis, which shows that LanCl-2 resides in the nucleus, as well as at the plasma membrane. With respect to this study, our data indicate that LanCl-2 increases cellular sensitivity to Adriamycin by decreasing the expression of P-gp, but more generally, these results indicate that the identification of bystander gene amplification in human tumors can have clinical implications.

Genetic and Signaling Pathway Alterations in Glioblastoma: Relevance to Novel Targeted Therapies

Frontiers in Bioscience : a Journal and Virtual Library. May, 2003  |  Pubmed ID: 12700121

Glioblastomas multiforme (GBM) is the most common malignant primary brain tumor in adults. GBM patients have a dismal prognosis, with a median survival of less than 1 year. During the past decade, significant advances have been made in our understanding of the molecular pathogenesis of these tumors. Specific genetic defects have been identified that appear to be important for the development, as well as maintenance of the malignant characteristics that are associated with GBM. Some of these genetic aberrations appear to have prognostic significance. However, even more exciting in this era of molecularly targeted therapy are the clues these gene alterations provide for identifying signaling mechanisms responsible for carcinogenesis, and for identifying potential therapeutic targets. Cancer drug therapy is currently undergoing a major transition with an attempt to move from the use of cytotoxic drugs towards the use of tumor mechanism-based drugs. Advances such as the decoding of the human genome, combinatorial chemistry, and gene expression profiling have led to an increase in the rate at which new drugs are being developed. In this review, we will describe the most common genetic and signaling pathway alterations that have relevance to new drug development for the treatment of GBM.

Contrasting in Vivo and in Vitro Fates of Glioblastoma Cell Subpopulations with Amplified EGFR

Genes, Chromosomes & Cancer. Jan, 2004  |  Pubmed ID: 14603439

Despite the high incidence of EGFR amplification in patient glioblastoma multiforme (GBM) tissues, only a single GBM cell line, of the many described in the literature, is known to contain and maintain amplified EGFR. Because EGFR mutations in GBM manifest primarily, if not exclusively, in amplified form, it follows that the availability of cell lines with mutation of endogenous EGFR would also be in short supply. In fact, there are no GBM cell lines harboring the common EGFR mutants described in patient GBMs. These observations suggest that in vivo environments select for EGFR amplification, whereas in vitro environments, specifically cell cultures, select against this gene alteration. To contrast directly the fates of EGFR amplification in vivo and in vitro, as well as to examine potential relationships between EGFR amplification and mutation, we have established and maintained GBM explants as xenografts by serial passaging in nude mice. Analysis of EGFR copy number and EGFR mutation status in 11 patient tumors and their corresponding xenografts, as well as the monitoring of EGFR copy number during the establishment of a GBM cell line from a xenograft with amplified EGFR, indicated that selection for EGFR amplification is an in vivo phenomenon. Furthermore, our data indicated that EGFR mutation occurs only in tumors with EGFR amplification and showed that the selection of amplified mutant EGFR over amplified wild-type EGFR as a xenograft occurred rapidly and completely during tumor propagation.

MDA-7 Regulates Cell Growth and Radiosensitivity in Vitro of Primary (non-established) Human Glioma Cells

Cancer Biology & Therapy. Aug, 2004  |  Pubmed ID: 15197348

We examined the impact of purified bacterially synthesized GST-MDA-7 (IL-24) and ionizing radiation on the proliferation and survival of nonestablished human glioblastoma multiforme (GBM) cells. Glioma cell types expressing mutated PTEN and p53 molecules, activated ERBB1VIII, overexpressing wild type ERBB1 or without receptor overexpression were selected. In MTT assays, GST-MDA-7 caused a dose-dependent reduction in the proliferation of nonestablished glioma cells; however only at higher concentrations did GST-MDA-7 reduce cell viability. The anti-proliferative and cytotoxic effects of GST-MDA-7 were enhanced by radiation in a greater than additive fashion that correlated with JNK1/2/3 activation. The reduction in cell growth and enhancement in cell killing by the combination of GST-MDA-7 and radiation were blocked by an ROS scavenger, N-acetyl cysteine (NAC), a JNK1/2/3 inhibitor SP600125, a pan-caspase inhibitor (zVAD) and by an inhibitor of caspase 9 (LEHD), but not by an inhibitor of caspase 8 (IETD). Low concentrations of either GST-MDA-7 or radiation reduced clonogenic survival, however colony formation ability was significantly further decreased when the two treatments were combined, which was also blocked by inhibition of caspase 9 function. In general agreement with activation of the intrinsic caspase pathway, cell death correlated with reduced BCL-XL expression and with increased levels of the pro-apoptotic proteins BAD and BAX. Inhibition of caspase 9 after combination treatment blunted neither JNK1/2/3 activation nor the enhanced expression of BAD and BAX, but did block caspase 3 cleavage, reduced expression of BCL-XL and inhibition of ERK1/2 activity. In contrast, incubation with NAC blocked JNK1/2/3 activation and cell killing, but not the increases in BAD and BAX expression. These findings argue that after combination treatment JNK1/2/3 activation is a primary pro-apoptotic event and loss of BCL-XL expression and ERK1/2 activity are secondary caspase-dependent processes. This data also argues that GST- MDA-7 induces two parallel pro-apoptotic pathways via ROS-dependent and -independent mechanisms. Infection of primary human astrocytes with a recombinant adenovirus to express MDA-7, Ad.mda-7, but not infection with either Ad.cmv or Ad.mda-7SP- lacking MDA-7 secretion, resulted in the suppression of GBM cell colony formation in soft agar overlay assays, an effect that was enhanced in a greater than additive fashion by radiation. Collectively, our findings demonstrate that MDA-7 reduces proliferation and enhances the radiosensitivity of nonestablished human GBM cells in vitro, and when grown in 3 dimensions, and that sensitization occurs independently of basal EGFR/ERK1/2/AKT activity or the functions of PTEN and p53.

Correlation of the Response of Recurrent Malignant Gliomas Treated with Interferon Alpha with Tumor Interferon Alpha Gene Content

International Journal of Oncology. Aug, 2004  |  Pubmed ID: 15254740

Malignant gliomas are treated by combining surgery and radiation with chemotherapy. Cure is rare and utilizing information arising from our improved understanding of brain tumor biology may be of value. Interferon alpha (IFNalpha) treatment as restorative immunotherapy has been utilized in malignant gliomas in the past. Interferon alpha/beta gene presence is variable in these tumors. The relationship between response to IFNalpha therapy and gene status has not been assessed prospectively. Patients with recurrent malignant gliomas were treated with 8-week courses of IFNalpha. Clinical and laboratory toxicity was assessed and response determined by MRI scans. Tumor interferon alpha/beta gene content was measured. Toxicities included fourteen grade 3/4 neuro-motor events, and eleven grade 3 neuro-cortical events. Rapid tolerance developed and with dose reductions few doses were missed. Three individuals with glioblastoma multiforme demonstrated a partial response. Median time to progression was 24.6 (+/-17.6) weeks for all glioblastomas. The correlation between longer time to progression and lower tumor IFNalpha gene content as measured here was significant. A minority of patients with recurrent malignant gliomas will respond to IFNalpha therapy at starting doses of 20 Mu/m(2) and above. These doses are associated with significant toxicity. A relationship between the tumor IFNalpha gene status and tumor response to therapy may be present. With current improved understanding of IFNalpha toxicities and ability to measure tumor IFNalpha function, this therapy warrants further evaluation for identifying patients whose tumors are likely to be responsive to IFNalpha therapy.

Immunohistochemical Detection of EGFRvIII in High Malignancy Grade Astrocytomas and Evaluation of Prognostic Significance

Journal of Neuropathology and Experimental Neurology. Jul, 2004  |  Pubmed ID: 15290895

The purpose of this study was to establish an accurate and accessible immunohistochemical (IHC) method for detecting vIII Egf receptor and to assess the prognostic significance of the method as applied to the detection of vIII in malignant astrocytomas. The accuracy of the method was determined by comparing vIII immunoreactivity in formalin-fixed and paraffin-embedded tumor sections versus RT-PCR results from the analysis of RNA extracted from corresponding frozen specimens. RT-PCR revealed vIII transcript in 18 of 44 cases in this series, and IHC analysis of matched formalin-fixed and paraffin-embedded sections showed EGFRvIII reactivity in each of these 18 tumors, as well as 1 additional tumor that was negative for vIII transcript. EGFR amplification was evident in all tumors expressing vIII; none of the 15 tumors lacking amplified EGFR were positive for vIII transcript or vIII protein. IHC analysis for vill expression was next applied to a large series of anaplastic astrocytomas (AAs) and glioblastoma multiforme (GBMs) from clinical trial patients with complete follow-up and that had been previously examined by FISH for amplified EGFR. Among the GBMs, vIII detection by IHC was determined in 19 of 46 cases (41.3%) with EGFR amplification, and in only 3 of 59 tumors lacking amplified EGFR (5.1%). Among the AAs, vIII expression was observed in 3 of 14 cases with amplified EGFR (21.4%) and in 6 of 49 cases without EGFR amplification (12.2%). GBM and AA patient survival analysis as a function of vIII expression showed contrasting results, with vIII positivity having no association with survival among GBM patients (p = 0.84), but being highly associated with reduced survival among AA patients (p = 0.0016). This latter finding, though quite possibly a result of vIII's association with increasing AA patient age, suggests that vIII IHC will be useful for identifying and/or confirming the identity of malignant astrocytomas whose clinical behavior is consistent with that of GBM.

Altered Molecular Pathways in Gliomas: an Overview of Clinically Relevant Issues

Seminars in Oncology. Oct, 2004  |  Pubmed ID: 15497113

Primary central nervous system (CNS) tumors constitute a small fraction of the overall incidence of human cancer, but they represent a major source of cancer-related morbidity and mortality. The most common CNS tumor subtype in adults, high-grade astrocytoma, confers a dismal prognosis with a median survival of only 1 to 2 years. Other common adult CNS tumors, ie, low-grade astrocytomas and oligodendrogliomas, carry a less ominous, yet still poor prognosis. Unfortunately, there has been little progress in extending the survival or quality of life for glioma patients, despite nearly four decades of extensive research. This research has, however, greatly increased our understanding of the underlying molecular biology of these tumors, examples of which include the determination of elevated epidermal growth factor receptor (EGFR) as well as platelet-derived growth factor receptor (PDGF) signaling, and the inactivation of p53 , p16 , and PTEN tumor-suppressor genes (TSGs) that negatively regulate specific enzymatic activities in normal glial cells. Such observations have greatly improved our understanding of the pathogenesis of these tumors and have potential diagnostic as well as therapeutic relevance. With respect to the latter of these two issues, the identification of aberrant enzymatic activities in gliomas has promoted the development of novel therapeutic agents that target specific signaling effectors, and whose inhibition should, in theory, prove to be cytostatic, if not cytotoxic, to tumor cells. Several clinical trials are currently underway for testing these therapeutic agents in patients with primary brain tumors, and it is hoped that the targeting of pro-tumorigenic enzymatic activities will lead to better patient outcomes. In this review, we will describe the most pertinent genetic and signaling pathway alterations that are clinically relevant to the management of glial tumors.

Patient Tumor EGFR and PDGFRA Gene Amplifications Retained in an Invasive Intracranial Xenograft Model of Glioblastoma Multiforme

Neuro-oncology. Apr, 2005  |  Pubmed ID: 15831234

We have previously described a panel of serially transplantable glioblastoma multiforme xenograft lines established by direct subcutaneous injection of patient tumor tissue in the flanks of nude mice. Here we report the characterization of four of these lines with respect to their histopathologic, genetic, and growth properties following heterotopic-to-orthotopic (flank-to-intracranial) transfer. Cells from short-term cultures, established from excised flank xenografts, were harvested and injected into the brains of nude mice (10(6) cells per injection). The intracranial tumors generated from these injections were all highly mitotic as well as highly invasive, but they lacked necrotic features in most instances and failed to show endothelial cell proliferation in all instances. For mice receiving injections from a common explant culture, tumor intracranial growth rate was consistent, as indicated by relatively narrow ranges in survival time. In contrast to the loss of epidermal growth factor receptor gene (EGFR) amplification in cell culture, high-level amplification and overexpression of EGFR were retained in intracranial tumors established from two EGFR-amplified flank tumors. A third intracranial tumor retained patient tumor amplification and high-level expression of platelet-derived growth factor receptor alpha gene. Because the heterotopic-to-orthotopic transfer and propagation of glioblastoma multiforme preserves the receptor tyrosine kinase (RTK) gene amplification of patient tumors, this approach should facilitate investigations for determining the extent to which RTK amplification status influences tumor response to RTK-directed therapies. The fact that such studies were carried out by using an invasive tumor model in an anatomically appropriate context should ensure a rigorous preclinical assessment of agent efficacy.

Unique Aspects of Mda-7/IL-24 Antitumor Bystander Activity: Establishing a Role for Secretion of MDA-7/IL-24 Protein by Normal Cells

Oncogene. Nov, 2005  |  Pubmed ID: 16044151

Melanoma differentiation associated gene-7 (mda-7) was cloned using subtraction hybridization from terminally differentiated human melanoma cells. Based on structural and functional properties, mda-7 is now recognized as interleukin-24 (IL-24), a new member of the expanding IL-10 gene family. Unique properties of mda-7/IL-24 include its ability to selectively induce growth suppression, apoptosis and radiosensitization in diverse human cancer cells, without causing similar effects in normal cells. The utility of mda-7/IL-24, administered by means of a replication-incompetent adenovirus, as a gene therapy for cancer has recently received validation in patients, highlighting an important phenomenon initially observed in pancreatic tumor cells, namely a 'potent bystander apoptosis-inducing effect' in adjacent tumor cells not initially receiving this gene product. We presently investigated the contribution of mda-7/IL-24 secreted by normal cells in mediating this 'bystander effect', and document that normal cells induced to produce mda-7/IL-24 following infection with recombinant adenoviruses expressing this cytokine secrete mda-7/IL-24, which modifies the anchorage-independent growth, invasiveness, survival and sensitivity to radiation of cancer cells that contain functional IL-20/IL-22 receptors, but not in cancer cells that lack a complete set of receptors. Moreover, the combination of secreted mda-7/IL-24 and radiation engenders a 'bystander antitumor effect' not only in inherently mda-7/IL-24 or radiation-sensitive cancer cells, but also in tumor cells overexpressing the antiapoptotic proteins bcl-2 or bcl-x(L) and displaying resistance to either treatment alone. The present studies provide definitive evidence that secreted mda-7/IL-24 from normal cells can induce direct antitumor and radiation-enhancing effects that are dependent on the presence of canonical receptors for this cytokine on tumor cells. Moreover, we now describe a novel means of enhancing mda-7/IL-24's therapeutic potential by targeting normal cells to produce and release this cancer-specific apoptosis-inducing cytokine, a strategy that could be employed as an innovative way of using this unique gene product for treating metastatic disease.

MTOR Controls FLIPS Translation and TRAIL Sensitivity in Glioblastoma Multiforme Cells

Molecular and Cellular Biology. Oct, 2005  |  Pubmed ID: 16199861

The tumor-selective, proapoptotic, death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a mediator of antitumor drug activity and in itself is a promising agent for the treatment of human malignancies. Like many tumors, however, glioblastoma multiforme (GBM), the most fatal form of glioma, exhibits a range of TRAIL sensitivity, and only a small percentage of GBM tumors undergo TRAIL-induced apoptosis. We here show that TRAIL resistance in GBM is a consequence of overexpression of the short isoform of the caspase-8 inhibitor, c-FLICE inhibitory protein (FLIP(S)), and that FLIP(S) expression is in turn translationally enhanced by activation of the Akt-mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (S6K1) pathway. Conversely, pharmacologic or genetic inhibition of mTOR, or the mTOR target S6K1, suppresses polyribosomal accumulation of FLIP(S) mRNA, FLIP(S) protein expression, and TRAIL resistance. In archived material from 12 human GBM tumors, PTEN status was a predictor of activation of the Akt-mTOR-S6K1 pathway and of FLIP(S) levels, while in xenografted human GBM, activation status of the PTEN-Akt-mTOR pathway distinguished the tumors inherently sensitive to TRAIL from those which could be sensitized by the mTOR inhibitor rapamycin. These results define the mTOR pathway as a key limiter of tumor elimination by TRAIL-mediated mechanisms, provide a means by which the TRAIL-sensitive subset of GBM can be identified, and provide rationale for the combined use of TRAIL with mTOR inhibitors in the treatment of human cancers.

Disruption of Parallel and Converging Signaling Pathways Contributes to the Synergistic Antitumor Effects of Simultaneous MTOR and EGFR Inhibition in GBM Cells

Neoplasia (New York, N.Y.). Oct, 2005  |  Pubmed ID: 16242075

Elevated epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) signaling are known to contribute to the malignant properties of glioblastoma multiforme (GBM), which include uncontrolled cell proliferation and evasion of apoptosis. Small molecule inhibitors that target these protein kinases have been evaluated in multiple clinical trials for cancer patients, including those with GBM. Here we have examined the cellular and molecular effects of a combined kinase inhibition of mTOR (rapamycin) and EGFR (EKI-785) in U87 and U251 GBM cells. Simultaneous treatment with rapamycin and EKI-785 results in synergistic antiproliferative as well as proapoptotic effects. At a molecular level, rapamycin alone significantly decreases S6 phosphorylation, whereas EKI-785 alone promotes substantially reduced signal transducer and activator of transcription (STAT3) phosphorylation. Treatment with rapamycin alone also increases Akt phosphorylation on Ser-473, but this effect is blocked by a simultaneous administration of EKI-785. Individually, EKI-785 diminishes while rapamycin promotes the binding of the translation inhibitor eukaryotic initiation factor 4E binding protein (4EBP1) to the eukaryotic translation initiation factor 4E (eIF4E). In spite of these opposing effects, the highest level of 4EBP1-eIF4E binding occurs with the combination of the two inhibitors. These results indicate that the inhibition of EGFR and mTOR has distinct as well as common signaling consequences and provides a molecular rationale for the synergistic antitumor effects of EKI-785 and rapamycin administration.

Use of an Orthotopic Xenograft Model for Assessing the Effect of Epidermal Growth Factor Receptor Amplification on Glioblastoma Radiation Response

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Apr, 2006  |  Pubmed ID: 16609043

The influence of epidermal growth factor receptor (EGFR) amplification on glioblastoma patient prognosis following definitive radiotherapy has been extensively investigated in clinical studies, and yet the relationship between EGFR status and radiation response remains unclear. The intent of the current study was to address this relationship using several EGFR-amplified glioblastoma xenografts in an orthotopic athymic mouse model.

PIK3CA Gene Mutations in Pediatric and Adult Glioblastoma Multiforme

Molecular Cancer Research : MCR. Oct, 2006  |  Pubmed ID: 17050665

The phosphatidylinositol 3-kinases (PI3K) are a family of enzymes that relay important cellular growth control signals. Recently, a large-scale mutational analysis of eight PI3K and eight PI3K-like genes revealed somatic mutations in PIK3CA, which encodes the p110alpha catalytic subunit of class IA PI3K, in several types of cancer, including glioblastoma multiforme. In that report, 4 of 15 (27%) glioblastomas contained potentially oncogenic PIK3CA mutations. Subsequent studies, however, showed a significantly lower mutation rate ranging from 0% to 7%. Given this disparity and to address the relation of patient age to mutation frequency, we examined 10 exons of PIK3CA in 73 glioblastoma samples by PCR amplification followed by direct DNA sequencing. Overall, PIK3CA mutations were found in 11 (15%) samples, including several novel mutations. PIK3CA mutations were distributed in all sample types, with 18%, 9%, and 13% of primary tumors, xenografts, and cell lines containing mutations, respectively. Of the primary tumors, PIK3CA mutations were identified in 21% and 17% of pediatric and adult samples, respectively. No evidence of PIK3CA gene amplification was detected by quantitative real-time PCR in any of the samples. This study confirms that PIK3CA mutations occur in a significant number of human glioblastomas, further indicating that therapeutic targeting of this pathway in glioblastomas is of value. Moreover, this is the first study showing PIK3CA mutations in pediatric glioblastomas, thus providing a molecular target in this important pediatric malignancy.

Retargeted Oncolytic Measles Strains Entering Via the EGFRvIII Receptor Maintain Significant Antitumor Activity Against Gliomas with Increased Tumor Specificity

Cancer Research. Dec, 2006  |  Pubmed ID: 17178881

Among the best-characterized genetic alterations in gliomas is the amplification of the epidermal growth factor receptor (EGFR) gene, present in approximately 40% of glioblastoma multiforme, and frequently associated with the EGFRvIII gene rearrangement. We have previously shown that attenuated vaccine strains of measles virus have potent antitumor activity against gliomas, and identified H protein mutations, which ablate recognition of the natural measles virus receptors CD46 and SLAM. Retargeted recombinant viruses were generated from the measles Edmonston-NSe vaccine strain displaying a single-chain antibody against EGFRvIII at the COOH terminus of H and containing the marker green fluorescent protein (GFP) gene in position 1. Two different H mutants were employed: H(SNS) (V451S, Y481N, and A527S)-CD46 blind, and H(AA) (Y481A and R533A)-CD46 and SLAM blind. MV-GFP virus was used as a positive control. Both EGFRvIII-retargeted viruses had significant antitumor activity against EGFRvIII-expressing glioblastoma multiforme but no cytopathic effect against normal cells. In an orthotopic model of EGFRvIII-expressing GBM39 xenografts, there was comparable therapeutic efficacy between retargeted strains and unmodified MV-GFP and statistically significant prolongation of survival in treated animals compared with the control group (P = 0.001). Formation of syncytia was observed in tumors treated with retargeted viruses, with a surrounding infiltrate consisting of macrophages and natural killer cells. In summary, EGFRvIII-retargeted oncolytic measles virus strains have comparable therapeutic efficacy with the unmodified MV-GFP strain against EGFRvIII-expressing glioma lines and xenografts with improved therapeutic index, a finding with potential translational implications in glioma virotherapy.

Rarity of PTEN Deletions and EGFR Amplification in Malignant Gliomas of Childhood: Results from the Children's Cancer Group 945 Cohort

Journal of Neurosurgery. Nov, 2006  |  Pubmed ID: 17328268

In reporting on molecular studies involving malignant gliomas in adults, authors have noted that deletions of PTEN and amplification of EGFR are common and may contribute to tumor development, providing a rationale for a number of therapies aimed at these molecular targets. The frequency of comparable abnormalities has not been defined in a sizable pediatric cohort. To address this issue, we examined tumor samples from the Children's Cancer Group 945 study, a large randomized trial of treatment for childhood malignant gliomas.

Identification of Molecular Characteristics Correlated with Glioblastoma Sensitivity to EGFR Kinase Inhibition Through Use of an Intracranial Xenograft Test Panel

Molecular Cancer Therapeutics. Mar, 2007  |  Pubmed ID: 17363510

In the current study, we examined a panel of serially passaged glioblastoma xenografts, in the context of an intracranial tumor therapy response model, to identify associations between glioblastoma molecular characteristics and tumor sensitivity to the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. From an initial evaluation of 11 distinct glioblastoma xenografts, two erlotinib-sensitive tumors were identified, each having amplified EGFR and expressing wild-type PTEN. One of these tumors expressed truncated EGFRvIII, whereas the other expressed full-length EGFR. Subsequent cDNA sequence analysis revealed the latter tumor as expressing an EGFR sequence variant with arginine, rather than leucine, at amino acid position 62; this was the only EGFR sequence variant identified among the 11 xenografts, other than the aforementioned vIII sequence variant. EGFR cDNAs were then examined from 12 more xenografts to determine whether additional missense sequence alterations were evident, and this analysis revealed one such case, expressing threonine, rather than alanine, at amino acid position 289 of the extracellular domain. This glioblastoma was also amplified for EGFR, but did not display significant erlotinib sensitivity, presumably due to its lacking PTEN expression. In total, our study identified two erlotinib-sensitive glioblastoma xenografts, with the common molecular characteristics shared by each being the expression of wild-type PTEN in combination with the expression of amplified and aberrant EGFR.

Epidermal Growth Factor Receptor Variant III Status Defines Clinically Distinct Subtypes of Glioblastoma

Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. Jun, 2007  |  Pubmed ID: 17538175

The clinical significance of epidermal growth factor receptor variant III (EGFRvIII) expression in glioblastoma multiforme (GBM) and its relationship with other key molecular markers are not clear. We sought to evaluate the clinical significance of GBM subtypes as defined by EGFRvIII status.

Bioluminescence Monitoring of Intracranial Glioblastoma Xenograft: Response to Primary and Salvage Temozolomide Therapy

Journal of Neurosurgery. Sep, 2007  |  Pubmed ID: 17886562

Bioluminescence imaging (BLI) offers a rapid and accurate means for longitudinal study of tumor cell growth and response to therapy in rodent models. Because this technology has only recently come into use in the field of small animal imaging, applications in this area have been limited. In the current study we have applied BLI to the analysis of clinically relevant issues involving use of the DNA methylating agent temozolomide (TMZ) in a mouse model.

Epidermal Growth Factor Receptor (EGFR)-retargeted Measles Virus Strains Effectively Target EGFR- or EGFRvIII Expressing Gliomas

Molecular Therapy : the Journal of the American Society of Gene Therapy. Apr, 2007  |  Pubmed ID: 17299404

A retargeted measles virus strain MV-GFP-H(AA)-scEGFR was generated by engineering the MV-NSe Edmonston vaccine strain to incorporate both CD46 (Y481A) and signaling lymphocyte activation molecule (SLAM) (R533A) ablating mutations in the hemagglutinin protein in combination with the display of a single-chain antibody against epidermal growth factor receptor (EGFR) at the C terminus of hemagglutinin. The unmodified MV-GFP virus was used as a positive control. Specificity of the EGFR retargeted virus was demonstrated in non-permissive Chinese hamster ovary (CHO) cells stably transfected to express either the natural receptors CD46 or SLAM or the target receptors EGFR and EGFRvIII. In vitro, the retargeted virus had potent antitumor activity against EGFR- or EGFRvIII-overexpressing primary glioblastoma multi-forme (GBM) cell lines that was comparable to the activity of the unmodified MV-GFP virus. Intratumoral administration of MV-GFP-H(AA)-scEGFRvIII in orthotopic GBM12 xenografts resulted in tumor regression, as demonstrated by bioluminescence imaging and significant prolongation of survival, that was comparable to the effect of the unmodified strain. In contrast to MV-GFP, central nervous system administration of the targeted MV-GFP-H(AA)-scEGFR virus in measles replication-permissive Ifnar(ko) CD46 transgenic mice resulted in no neurotoxicity. In conclusion, EGFR-retargeted measles virus strains have comparable therapeutic efficacy to the unmodified virus in glioma cells overexpressing EGFR or EGFRvIII in vivo and in vitro, and improved therapeutic index, a finding with potential translational implications in glioma virotherapy.

Mapping the Future of Neuro-Oncology

Neuro-oncology. Jan, 2007  |  Pubmed ID: 17170381

Combination of Measles Virus Virotherapy and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Dec, 2007  |  Pubmed ID: 18056196

Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas.

Tumor Suppressor P16 Methylation in Multiple Myeloma: Biological and Clinical Implications

Blood. Feb, 2007  |  Pubmed ID: 16840723

The biological and clinical implications of p16 gene methylation in multiple myeloma (MM) are still unclear despite previous studies. In this comprehensive study, using methylation-specific PCR (MS-PCR), we show that p16 methylation is relatively common and occurs in monoclonal gammopathy of undetermined significance (MGUS; n=17), smoldering multiple myeloma (SMM; n=40), and MM (n=522) at a prevalence of 24%, 28%, and 34%, respectively. However, p16 methylation does not appear to affect gene expression level. In a large cohort of patients with long-term follow-up information (n=439), there was no difference in overall survival between patients with or without p16 methylation. We also found no association between p16 methylation and the main cytogenetic categories, although it was more common among patients with 17p13.1 deletions (p53 locus), a genetic progression event in MM. In addition, p16 methylation has no apparent effect on the cycle because there was also no difference in the plasma cell labeling index (a direct measurement of proliferation) between patients with and without p16 methylation. Our results question a major role for p16 methylation in the oncogenesis of the PC neoplasm, and we now believe p16 methylation may be a marker for overall epigenetic changes associated with disease progression, with no obvious direct biological or clinical consequences.

An Orthotopic Skull Base Model of Malignant Meningioma

Brain Pathology (Zurich, Switzerland). Apr, 2008  |  Pubmed ID: 18093250

Meningioma tumor growth involves the subarachnoid space that contains the cerebrospinal fluid. Modeling tumor growth in this microenvironment has been associated with widespread leptomeningeal dissemination, which is uncharacteristic of human meningiomas. Consequently, survival times and tumor properties are varied, limiting their utility in testing experimental therapies. We report the development and characterization of a reproducible orthotopic skull-base meningioma model in athymic mice using the IOMM-Lee cell line. Localized tumor growth was obtained by using optimal cell densities and matrigel as the implantation medium. Survival times were within a narrow range of 17-21 days. The xenografts grew locally compressing surrounding brain tissue. These tumors had histopathologic characteristics of anaplastic meningiomas including high cellularity, nuclear pleomorphism, cellular pattern loss, necrosis and conspicuous mitosis. Similar to human meningiomas, considerable invasion of the dura and skull and some invasion of adjacent brain along perivascular tracts were observed. The pattern of hypoxia was also similar to human malignant meningiomas. We use bioluminescent imaging to non-invasively monitor the growth of the xenografts and determine the survival benefit from temozolomide treatment. Thus, we describe a malignant meningioma model system that will be useful for investigating the biology of meningiomas and for preclinical assessment of therapeutic agents.

Increased Expression of the Glioma-associated Antigen ARF4L After Loss of the Tumor Suppressor PTEN. Laboratory Investigation

Journal of Neurosurgery. Feb, 2008  |  Pubmed ID: 18240926

Despite recent advances in cancer immunotherapy, cellular mechanisms controlling expression of tumor-associated antigens are poorly understood. Mutations in cancer cells, such as loss of PTEN, may increase expression of tumor-associated antigens. The authors investigated the relationship between PTEN status and the expression of a glioma-associated antigen, adenosine diphosphate-ribosylation factor 4-like (ARF4L) protein.

Mechanisms of Chemoresistance to Alkylating Agents in Malignant Glioma

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. May, 2008  |  Pubmed ID: 18483356

Intrinsic or acquired chemoresistance to alkylating agents is a major cause of treatment failure in patients with malignant brain tumors. Alkylating agents, the mainstay of treatment for brain tumors, damage the DNA and induce apoptosis, but the cytotoxic activity of these agents is dependent on DNA repair pathways. For example, O6-methylguanine DNA adducts can cause double-strand breaks, but this is dependent on a functional mismatch repair pathway. Thus, tumor cell lines deficient in mismatch repair are resistant to alkylating agents. Perhaps the most important mechanism of resistance to alkylating agents is the DNA repair enzyme O6-methylguanine methyltransferase, which can eliminate the cytotoxic O6-methylguanine DNA adduct before it causes harm. Another mechanism of resistance to alkylating agents is the base excision repair (BER) pathway. Consequently, efforts are ongoing to develop effective inhibitors of BER. Poly(ADP-ribose)polymerase plays a pivotal role in BER and is an important therapeutic target. Developing effective strategies to overcome chemoresistance requires the identification of reliable preclinical models that recapitulate human disease and which can be used to facilitate drug development. This article describes the diverse mechanisms of chemoresistance operating in malignant glioma and efforts to develop reliable preclinical models and novel pharmacologic approaches to overcome resistance to alkylating agents.

PTEN Loss Does Not Predict for Response to RAD001 (Everolimus) in a Glioblastoma Orthotopic Xenograft Test Panel

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Jun, 2008  |  Pubmed ID: 18559622

Hyperactivation of the phosphatidylinositol 3-kinase/Akt signaling through disruption of PTEN function is common in glioblastoma multiforme, and these genetic changes are predicted to enhance sensitivity to mammalian target of rapamycin (mTOR) inhibitors such as RAD001 (everolimus).

MiR-124 and MiR-137 Inhibit Proliferation of Glioblastoma Multiforme Cells and Induce Differentiation of Brain Tumor Stem Cells

BMC Medicine. 2008  |  Pubmed ID: 18577219

Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.

Abnormal DNA Methylation of CD133 in Colorectal and Glioblastoma Tumors

Cancer Research. Oct, 2008  |  Pubmed ID: 18829568

Much recent effort has focused on identifying and characterizing cellular markers that distinguish tumor propagating cells (TPC) from more differentiated progeny. We report here an unusual promoter DNA methylation pattern for one such marker, the cell surface antigen CD133 (Prominin 1). This protein has been extensively used to enrich putative cancer propagating stem-like cell populations in epithelial tumors and, especially, glioblastomas. We find that, within individual cell lines of cultured colon cancers and glioblastomas, the promoter CpG island of CD133 is DNA methylated, primarily, in cells with absent or low expression of the marker protein, whereas lack of such methylation is evident in purely CD133+ cells. Differential histone modification marks of active versus repressed genes accompany these DNA methylation changes. This heterogeneous CpG island DNA methylation status in the tumors is unusual in that other DNA hypermethylated genes tested in such cultures preserve their methylation patterns between separated CD133+ and CD133- cell populations. Furthermore, the CD133 DNA methylation seems to constitute an abnormal promoter signature because it is not found in normal brain and colon but only in cultured and primary tumors. Thus, the DNA methylation is imposed on the transition between the active versus repressed transcription state for CD133 only in tumors. Our findings provide additional insight for the dynamics of aberrant DNA methylation associated with aberrant gene silencing in human tumors.

Disabled-1 is a Large Common Fragile Site Gene, Inactivated in Multiple Cancers

Genes, Chromosomes & Cancer. Feb, 2008  |  Pubmed ID: 18008369

Common fragile sites (CFS) are large, genomically unstable regions, which are hot-spots for deletions and other alterations, especially in cancer cells. Several have been shown to contain genes that span large genomic regions, such as FHIT (1.5 Mb), WWOX (1.0 Mb), GRID2 (1.36 Mb), PARK2 (1.3 Mb), and RORA (730 kb). These genes are frequently inactivated in multiple different cancers, and FHIT and WWOX are shown to function as tumor suppressors. The disabled-1 gene (DAB1) is one of the human homologs of the Drosophila disabled locus, which in mammals is involved in neuronal migration and lamination in the developing cerebral cortex. Mice DAB1 inactivation results in the neurological mutant Scrambler, having similarities to mice with the inactivation of PARK2 (Quaker), GRID2 (Lurcher), and RORA (Staggerer). We were interested in whether DAB1 was another large CFS gene that could have cancer development importance. We demonstrated here that the human DAB1 gene (spanning 1.25 Mb) mapped within FRA1B CFS region on chromosomal band 1p32.2. Real-time RT-PCR analysis revealed that the expression level of DAB1 was decreased in many human cancer samples, including primary tumor tissues and cancer-derived cell lines, from several different cancers, especially in brain and endometrial cancer. Additionally, the introduction of an over-expression DAB1 plasmid into two different cell lines, having insignificant endogenous DAB1 expression, resulted in decreased cell growth. In summary, DAB1 is another gene that resides within an unstable CFS region and might play a role in human tumorigenesis. These data may provide further linkage between neurological development and cancer.

P53 Small-molecule Inhibitor Enhances Temozolomide Cytotoxic Activity Against Intracranial Glioblastoma Xenografts

Cancer Research. Dec, 2008  |  Pubmed ID: 19074867

In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53(wt)) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53(wt) GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53(null) GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.

Phase II Study of Erlotinib Plus Temozolomide During and After Radiation Therapy in Patients with Newly Diagnosed Glioblastoma Multiforme or Gliosarcoma

Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. Feb, 2009  |  Pubmed ID: 19075262

This open-label, prospective, single-arm, phase II study combined erlotinib with radiation therapy (XRT) and temozolomide to treat glioblastoma multiforme (GBM) and gliosarcoma. The objectives were to determine efficacy of this treatment as measured by survival and to explore the relationship between molecular markers and treatment response.

Comparative Analyses of Gene Copy Number and MRNA Expression in Glioblastoma Multiforme Tumors and Xenografts

Neuro-oncology. Oct, 2009  |  Pubmed ID: 19139420

Development of model systems that recapitulate the molecular heterogeneity observed among glioblastoma multiforme (GBM) tumors will expedite the testing of targeted molecular therapeutic strategies for GBM treatment. In this study, we profiled DNA copy number and mRNA expression in 21 independent GBM tumor lines maintained as subcutaneous xenografts (GBMX), and compared GBMX molecular signatures to those observed in GBM clinical specimens derived from the Cancer Genome Atlas (TCGA). The predominant copy number signature in both tumor groups was defined by chromosome-7 gain/chromosome-10 loss, a poor-prognosis genetic signature. We also observed, at frequencies similar to that detected in TCGA GBM tumors, genomic amplification and overexpression of known GBM oncogenes, such as EGFR, MDM2, CDK6, and MYCN, and novel genes, including NUP107, SLC35E3, MMP1, MMP13, and DDX1. The transcriptional signature of GBMX tumors, which was stable over multiple subcutaneous passages, was defined by overexpression of genes involved in M phase, DNA replication, and chromosome organization (MRC) and was highly similar to the poor-prognosis mitosis and cell-cycle module (MCM) in GBM. Assessment of gene expression in TCGA-derived GBMs revealed overexpression of MRC cancer genes AURKB, BIRC5, CCNB1, CCNB2, CDC2, CDK2, and FOXM1, which form a transcriptional network important for G2/M progression and/or checkpoint activation. Our study supports propagation of GBM tumors as subcutaneous xenografts as a useful approach for sustaining key molecular characteristics of patient tumors, and highlights therapeutic opportunities conferred by this GBMX tumor panel for testing targeted therapeutic strategies for GBM treatment.

EGFR Signals to MTOR Through PKC and Independently of Akt in Glioma

Science Signaling. 2009  |  Pubmed ID: 19176518

Amplification of the gene encoding the epidermal growth factor (EGF) receptor (EGFR) occurs commonly in glioblastoma, leading to activation of downstream kinases including phosphatidylinositol 3'-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Here, we show that phosphorylation of mTOR and its downstream substrate rpS6 (ribosomal protein S6) are robust biomarkers for the antiproliferative effect of EGFR inhibitors. Inhibition of EGFR signaling correlated with decreased abundance of phosphorylated mTOR (p-mTOR) and rpS6 (p-rpS6) in cells wild type for the gene encoding PTEN (phosphatase and tensin homolog on chromosome 10), a negative regulator of PI3K. In contrast, inhibition of EGFR signaling failed to affect p-mTOR or p-rpS6 in cells mutant for PTEN, which are resistant to EGFR inhibitors. Although the abundance of phosphorylated Akt (p-Akt) decreased in response to inhibition of EGFR signaling, Akt was dispensable for signaling between EGFR and mTOR. We identified an Akt-independent pathway linking EGFR to mTOR that was critically dependent on protein kinase C (PKC). Consistent with these observations, the abundance of EGFR generally correlated with phosphorylation of rpS6 and PKC in primary human glioblastoma tumors, and correlated poorly with phosphorylation of Akt. Inhibition of PKC led to decreased viability of glioma cells regardless of PTEN or EGFR status, suggesting that PKC inhibitors should be tested in glioma. These findings underline the importance of signaling between EGFR and mTOR in glioma, identify PKCalpha as essential to this network, and question the necessity of Akt as a critical intermediate coupling EGFR and mTOR in glioma.

Stem Cells for Treating Glioblastoma: How Close to Reality?

Neuro-oncology. Apr, 2009  |  Pubmed ID: 19237733

Pathologic Correlates of Primary Central Nervous System Lymphoma Defined in an Orthotopic Xenograft Model

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Mar, 2009  |  Pubmed ID: 19276270

The prospect for advances in the treatment of patients with primary central nervous system lymphoma (PCNSL) is likely dependent on the systematic evaluation of its pathobiology. Animal models of PCNSL are needed to facilitate the analysis of its molecular pathogenesis and for the efficient evaluation of novel therapeutics.

Fyn and SRC Are Effectors of Oncogenic Epidermal Growth Factor Receptor Signaling in Glioblastoma Patients

Cancer Research. Sep, 2009  |  Pubmed ID: 19690143

Activating epidermal growth factor receptor (EGFR) mutations are common in many cancers including glioblastoma. However, clinical responses to EGFR inhibitors are infrequent and short-lived. We show that the Src family kinases (SFK) Fyn and Src are effectors of oncogenic EGFR signaling, enhancing invasion and tumor cell survival in vivo. Expression of a constitutively active EGFR mutant, EGFRvIII, resulted in activating phosphorylation and physical association with Src and Fyn, promoting tumor growth and motility. Gene silencing of Fyn and Src limited EGFR- and EGFRvIII-dependent tumor cell motility. The SFK inhibitor dasatinib inhibited invasion, promoted tumor regression, and induced apoptosis in vivo, significantly prolonging survival of an orthotopic glioblastoma model expressing endogenous EGFRvIII. Dasatinib enhanced the efficacy of an anti-EGFR monoclonal antibody (mAb 806) in vivo, further limiting tumor growth and extending survival. Examination of a large cohort of clinical samples showed frequent coactivation of EGFR and SFKs in glioblastoma patients. These results establish a mechanism linking EGFR signaling with Fyn and Src activation to promote tumor progression and invasion in vivo and provide rationale for combined anti-EGFR and anti-SFK targeted therapies.

Radiosensitizing Effects of Temozolomide Observed in Vivo Only in a Subset of O6-methylguanine-DNA Methyltransferase Methylated Glioblastoma Multiforme Xenografts

International Journal of Radiation Oncology, Biology, Physics. Sep, 2009  |  Pubmed ID: 19695438

Concurrent temozolomide (TMZ) and radiation therapy (RT) followed by adjuvant TMZ is standard treatment for patients with glioblastoma multiforme (GBM), although the relative contribution of concurrent versus adjuvant TMZ is unknown. In this study, the efficacy of TMZ/RT was tested with a panel of 20 primary GBM xenografts.

MicroRNAs in Gliomas: Small Regulators of a Big Problem

Neuromolecular Medicine. 2009  |  Pubmed ID: 19731102

Gliomas are the most common form of primary brain tumors and are associated with a poor clinical outcome. The molecular mechanisms that contribute to gliomagenesis have become increasingly clear in recent years, yet much remains to be learned. This is particularly true for the role of microRNAs in gliomagenesis, as an appreciation for the significance of aberrant miRNA expression in human cancer has only emerged in the last 5 years. It is now evident that microRNAs regulate a wide variety of tumorigenic processes including cellular proliferation, differentiation, angiogenesis, invasion, and apoptosis. Here we review the current state of knowledge related to the role of microRNAs in glial tumor development. This is a rapidly evolving field and it is likely that we have only begun to appreciate the involvement of microRNAs in relation to glioma formation, and the therapeutic potential of microRNAs to improve outcome for glioma patients.

Immunological Considerations of Modern Animal Models of Malignant Primary Brain Tumors

Journal of Translational Medicine. 2009  |  Pubmed ID: 19814820

Recent advances in animal models of glioma have facilitated a better understanding of biological mechanisms underlying gliomagenesis and glioma progression. The limitations of existing therapy, including surgery, chemotherapy, and radiotherapy, have prompted numerous investigators to search for new therapeutic approaches to improve quantity and quality of survival from these aggressive lesions. One of these approaches involves triggering a tumor specific immune response. However, a difficulty in this approach is the the scarcity of animal models of primary CNS neoplasms which faithfully recapitulate these tumors and their interaction with the host's immune system. In this article, we review the existing methods utilized to date for modeling gliomas in rodents, with a focus on the known as well as potential immunological aspects of these models. As this review demonstrates, many of these models have inherent immune system limitations, and the impact of these limitations on studies on the influence of pre-clinical therapeutics testing warrants further attention.

Induction of MGMT Expression is Associated with Temozolomide Resistance in Glioblastoma Xenografts

Neuro-oncology. Jun, 2009  |  Pubmed ID: 18952979

Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM tumors, and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all, GBM tumors. In this study, the relationship between MGMT protein expression and tumor response to TMZ was evaluated in four GBM xenograft lines that had been established from patient specimens and maintained by serial subcutaneous passaging in nude mice. Three MGMT unmethylated tumors displayed elevated basal MGMT protein expression, but only two of these were resistant to TMZ therapy (tumors GBM43 and GBM44), while the other (GBM14) displayed a level of TMZ sensitivity that was similar in extent to that seen in a single MGMT hypermethylated line (GBM12). In tissue culture and animal studies, TMZ treatment resulted in robust and prolonged induction of MGMT expression in the resistant GBM43 and GBM44 xenograft lines, while MGMT induction was blunted and abbreviated in GBM14. Consistent with a functional significance of MGMT induction, treatment of GBM43 with a protracted low-dose TMZ regimen was significantly less effective than a shorter high-dose regimen, while survival for GBM14 was improved with the protracted dosing regimen. In conclusion, MGMT expression is dynamically regulated in some MGMT nonmethylated tumors, and in these tumors, protracted dosing regimens may not be effective.

Evaluation of MGMT Promoter Methylation Status and Correlation with Temozolomide Response in Orthotopic Glioblastoma Xenograft Model

Journal of Neuro-oncology. Mar, 2009  |  Pubmed ID: 19011762

CpG methylation within the O6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in approximately 50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kgx5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r=-0.75; P=0.003) and directly correlated with qMS-PCR (r=0.72; P=0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r=0.78, P=0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.

Reversing HOXA9 Oncogene Activation by PI3K Inhibition: Epigenetic Mechanism and Prognostic Significance in Human Glioblastoma

Cancer Research. Jan, 2010  |  Pubmed ID: 20068170

HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using reverse transcription-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrently activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a phosphoinostide 3-kinase (PI3K) inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with tumor necrosis factor-related apoptosis-including ligand resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent, and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.

Oncogenic BRAF Mutation with CDKN2A Inactivation is Characteristic of a Subset of Pediatric Malignant Astrocytomas

Cancer Research. Jan, 2010  |  Pubmed ID: 20068183

Malignant astrocytomas are a deadly solid tumor in children. Limited understanding of their underlying genetic basis has contributed to modest progress in developing more effective therapies. In an effort to identify such alterations, we performed a genome-wide search for DNA copy number aberrations (CNA) in a panel of 33 tumors encompassing grade 1 through grade 4 tumors. Genomic amplifications of 10-fold or greater were restricted to grade 3 and 4 astrocytomas and included the MDM4 (1q32), PDGFRA (4q12), MET (7q21), CMYC (8q24), PVT1 (8q24), WNT5B (12p13), and IGF1R (15q26) genes. Homozygous deletions of CDKN2A (9p21), PTEN (10q26), and TP53 (17p3.1) were evident among grade 2 to 4 tumors. BRAF gene rearrangements that were indicated in three tumors prompted the discovery of KIAA1549-BRAF fusion transcripts expressed in 10 of 10 grade 1 astrocytomas and in none of the grade 2 to 4 tumors. In contrast, an oncogenic missense BRAF mutation (BRAF(V600E)) was detected in 7 of 31 grade 2 to 4 tumors but in none of the grade 1 tumors. BRAF(V600E) mutation seems to define a subset of malignant astrocytomas in children, in which there is frequent concomitant homozygous deletion of CDKN2A (five of seven cases). Taken together, these findings highlight BRAF as a frequent mutation target in pediatric astrocytomas, with distinct types of BRAF alteration occurring in grade 1 versus grade 2 to 4 tumors.

Integrated Genomic Analysis Identifies Clinically Relevant Subtypes of Glioblastoma Characterized by Abnormalities in PDGFRA, IDH1, EGFR, and NF1

Cancer Cell. Jan, 2010  |  Pubmed ID: 20129251

The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM). We describe a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural subtypes, respectively. Gene signatures of normal brain cell types show a strong relationship between subtypes and different neural lineages. Additionally, response to aggressive therapy differs by subtype, with the greatest benefit in the Classical subtype and no benefit in the Proneural subtype. We provide a framework that unifies transcriptomic and genomic dimensions for GBM molecular stratification with important implications for future studies.

Noninvasive Detection of Target Modulation Following Phosphatidylinositol 3-kinase Inhibition Using Hyperpolarized 13C Magnetic Resonance Spectroscopy

Cancer Research. Feb, 2010  |  Pubmed ID: 20145128

Numerous mechanism-based anticancer drugs that target the phosphatidylinositol 3-kinase (PI3K) pathway are in clinical trials. However, it remains challenging to assess responses by traditional imaging methods. Here, we show for the first time the efficacy of hyperpolarized (13)C magnetic resonance spectroscopy (MRS) in detecting the effect of PI3K inhibition by monitoring hyperpolarized [1-(13)C]lactate levels produced from hyperpolarized [1-(13)C]pyruvate through lactate dehydrogenase (LDH) activity. In GS-2 glioblastoma cells, PI3K inhibition by LY294002 or everolimus caused hyperpolarized lactate to drop to 42 +/- 12% and to 76 +/- 5%, respectively. In MDA-MB-231 breast cancer cells, hyperpolarized lactate dropped to 71 +/- 15% after treatment with LY294002. These reductions were correlated with reductions in LDH activity to 48 +/- 4%, 63 +/- 4%, and 69 +/- 12%, respectively, and were associated with a drop in levels of LDHA mRNA and LDHA and hypoxia-inducible factor-1alpha proteins. Supporting these findings, tumor growth inhibition achieved by everolimus in murine GS-2 xenografts was associated with a drop in the hyperpolarized lactate-to-pyruvate ratio detected by in vivo MRS imaging, whereas an increase in this ratio occurred with tumor growth in control animals. Taken together, our findings illustrate the application of hyperpolarized (13)C MRS of pyruvate to monitor alterations in LDHA activity and expression caused by PI3K pathway inhibition, showing the potential of this method for noninvasive imaging of drug target modulation.

Hyperpolarized 13C Magnetic Resonance Metabolic Imaging: Application to Brain Tumors

Neuro-oncology. Feb, 2010  |  Pubmed ID: 20150380

In order to compare in vivo metabolism between malignant gliomas and normal brain, (13)C magnetic resonance (MR) spectroscopic imaging data were acquired from rats with human glioblastoma xenografts (U-251 MG and U-87 MG) and normal rats, following injection of hyperpolarized [1-(13)C]-pyruvate. The median signal-to-noise ratio (SNR) of lactate, pyruvate, and total observed carbon-13 resonances, as well as their relative ratios, were calculated from voxels containing Gadolinium-enhanced tissue in T(1) postcontrast images for rats with tumors and from normal brain tissue for control rats. [1-(13)C]-labeled pyruvate and its metabolic product, [1-(13)C]-lactate, demonstrated significantly higher SNR in the tumor compared with normal brain tissue. Statistical tests showed significant differences in all parameters (P < .0004) between the malignant glioma tissue and normal brain. The SNR of lactate, pyruvate, and total carbon was observed to be different between the U-251 MG and U-87 MG models, which is consistent with inherent differences in the molecular characteristics of these tumors. These results suggest that hyperpolarized MR metabolic imaging may be valuable for assessing prognosis and monitoring response to therapy for patients with brain tumors.

Morphologic and Molecular Characterization of ATRT Xenografts Adapted for Orthotopic Therapeutic Testing

Neuro-oncology. Apr, 2010  |  Pubmed ID: 20308314

Atypical teratoid rhabdoid tumor (ATRT) is a malignant tumor of the central nervous system that most commonly arises in young children. The aggressive growth and propensity for early dissemination throughout the neuraxis confers a dismal prognosis. Large clinical trials that could test new therapeutic agents are difficult to conduct due to the low incidence of this cancer. For this reason, high throughput preclinical testing with suitable animal models for ATRT would serve a critical need for identifying the most efficacious treatments. In response to this need, we have adapted ATRT cell lines for bioluminescence imaging (BLI) of intracranial (orthotopic) xenografts established in athymic mice. Our results indicate that following supratentorial or infratentorial injection in athymic mice, ATRT cells produce rapidly growing tumors, often with intraventricular spread or neuraxis dissemination. When established as orthotopic xenografts, the tumors predominantly display cells with a rhabdoid-like cellular morphology that show a spectrum of immunophenotypes similar to primary ATRT tumors. To demonstrate the feasibility of this orthotopic ATRT xenograft model for therapeutic testing with correlation to biomarker analysis, we examined the responses of luciferase-modified ATRT cells to temozolomide (TMZ). These xenografts, which highly express MGMT, are resistant to TMZ treatment when compared with an orthotopic glioblastoma xenograft that is MGMT deficient and responsive to TMZ. These data suggest that an orthotopic ATRT xenograft model, in which BLI is used for monitoring tumor growth and response to therapy, should contribute to the identification of effective therapeutics and regimens for treating this highly aggressive pediatric brain tumor.

Pharmacologic Inhibition of Cyclin-dependent Kinases 4 and 6 Arrests the Growth of Glioblastoma Multiforme Intracranial Xenografts

Cancer Research. Apr, 2010  |  Pubmed ID: 20354191

Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study, we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6-specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G(1) cell cycle arrest and induction of senescence in each of 16 retinoblastoma protein (Rb)-proficient cell lines regardless of other genetic lesions, whereas 5 cell lines with homozygous inactivation of Rb were completely resistant to treatment. Short hairpin RNA depletion of Rb expression conferred resistance of GBM cells to PD-0332991, further demonstrating a requirement of Rb for sensitivity to cdk4/6 inhibition. PD-0332991 was found to efficiently cross the blood-brain barrier and proved highly effective in suppressing the growth of intracranial GBM xenograft tumors, including those that had recurred after initial therapy with temozolomide. Remarkably, no mice receiving PD-0332991 died as a result of disease progression while on therapy. Additionally, the combination of PD-0332991 and radiation therapy resulted in significantly increased survival benefit compared with either therapy alone. In total, our results support clinical trial evaluation of PD-0332991 against newly diagnosed as well as recurrent GBM, and indicate that Rb status is the primary determinant of potential benefit from this therapy.

A Hierarchy of Self-renewing Tumor-initiating Cell Types in Glioblastoma

Cancer Cell. Apr, 2010  |  Pubmed ID: 20385361

The neural stem cell marker CD133 is reported to identify cells within glioblastoma (GBM) that can initiate neurosphere growth and tumor formation; however, instances of CD133(-) cells exhibiting similar properties have also been reported. Here, we show that some PTEN-deficient GBM tumors produce a series of CD133(+) and CD133(-) self-renewing tumor-initiating cell types and provide evidence that these cell types constitute a lineage hierarchy. Our results show that the capacities for self-renewal and tumor initiation in GBM need not be restricted to a uniform population of stemlike cells, but can be shared by a lineage of self-renewing cell types expressing a range of markers of forebrain lineage.

Pleiotropic Role for MYCN in Medulloblastoma

Genes & Development. May, 2010  |  Pubmed ID: 20478998

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in approximately 5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

Tumor-initiating Cells: an Influential Paradigm for Xenograft Research

Neuro-oncology. Jun, 2010  |  Pubmed ID: 20530515

Identification of Internalizing Human Single-chain Antibodies Targeting Brain Tumor Sphere Cells

Molecular Cancer Therapeutics. Jul, 2010  |  Pubmed ID: 20587664

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain tumor for which there is no curative treatment to date. Resistance to conventional therapies and tumor recurrence pose major challenges to treatment and management of this disease, and therefore new therapeutic strategies need to be developed. Previous studies by other investigators have shown that a subpopulation of GBM cells can grow as neurosphere-like cells when cultured in restrictive medium and exhibits enhanced tumor-initiating ability and resistance to therapy. We report here the identification of internalizing human single-chain antibodies (scFv) targeting GBM tumor sphere cells. We selected a large naive phage antibody display library on the glycosylation-dependent CD133 epitope-positive subpopulation of GBM cells grown as tumor spheres and identified internalizing scFvs that target tumor sphere cells broadly, as well as scFvs that target the CD133-positive subpopulation. These scFvs were found to be efficiently internalized by GBM tumor sphere cells. One scFv GC4 inhibited self-renewal of GBM tumor sphere cells in vitro. We have further developed a full-length human IgG1 based on this scFv, and found that it potently inhibits proliferation of GBM tumor sphere cells and GBM cells grown in regular nonselective medium. Taken together, these results show that internalizing human scFvs targeting brain tumor sphere cells can be readily identified from a phage antibody display library, which could be useful for further development of novel therapies that target subpopulations of GBM cells to combat recurrence and resistance to treatment.

Akt and Autophagy Cooperate to Promote Survival of Drug-resistant Glioma

Science Signaling. 2010  |  Pubmed ID: 21062993

Although the phosphatidylinositol 3-kinase to Akt to mammalian target of rapamycin (PI3K-Akt-mTOR) pathway promotes survival signaling, inhibitors of PI3K and mTOR induce minimal cell death in PTEN (phosphatase and tensin homolog deleted from chromosome 10) mutant glioma. Here, we show that the dual PI3K-mTOR inhibitor PI-103 induces autophagy in a form of glioma that is resistant to therapy. Inhibitors of autophagosome maturation cooperated with PI-103 to induce apoptosis through the mitochondrial pathway, indicating that the cellular self-digestion process of autophagy acted as a survival signal in this setting. Not all inhibitors of mTOR synergized with inhibitors of autophagy. Rapamycin delivered alone induced autophagy, yet cells survived inhibition of autophagosome maturation because of rapamycin-mediated activation of Akt. In contrast, adenosine 5'-triphosphate-competitive inhibitors of mTOR stimulated autophagy more potently than did rapamycin, with inhibition of mTOR complexes 1 and 2 contributing independently to induction of autophagy. We show that combined inhibition of PI3K and mTOR, which activates autophagy without activating Akt, cooperated with inhibition of autophagy to cause glioma cells to undergo apoptosis. Moreover, the PI3K-mTOR inhibitor NVP-BEZ235, which is in clinical use, synergized with the lysosomotropic inhibitor of autophagy, chloroquine, another agent in clinical use, to induce apoptosis in glioma xenografts in vivo, providing a therapeutic approach potentially translatable to humans.

Inhibition of PI3K/mTOR Pathways in Glioblastoma and Implications for Combination Therapy with Temozolomide

Neuro-oncology. Apr, 2011  |  Pubmed ID: 21317208

Due to its molecular heterogeneity and infiltrative nature, glioblastoma multiforme (GBM) is notoriously resistant to traditional and experimental therapeutics. To overcome these hurdles, targeted agents have been combined with conventional therapy. We evaluated the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor (XL765) in in vitro and in vivo studies. In vivo serially passaged human GBM xenografts that are more genetically stable than GBM cell lines in culture were used for all experiments. Biochemical downstream changes were evaluated by immunoblot and cytotoxicity by colorimetric ATP-based assay. For in vivo experiments, human xenograft GBM 39 grown intracranially in nude mice was altered to express luciferase to monitor tumor burden by optical imaging. XL765 resulted in concentration-dependent decreases in cell viability in vitro. Cytotoxic doses resulted in specific inhibition of PI3K signaling. Combining XL765 with temozolomide (TMZ) resulted in additive toxicity in 4 of 5 xenografts. In vivo, XL765 administered by oral gavage resulted in greater than 12-fold reduction in median tumor bioluminescence compared with control (Mann-Whitney test p = 0.001) and improvement in median survival (logrank p = 0.05). TMZ alone showed a 30-fold decrease in median bioluminescence, but the combination XL765 + TMZ yielded a 140-fold reduction in median bioluminescence (Mann-Whitney test p = 0.05) with a trend toward improvement in median survival (logrank p = 0.09) compared with TMZ alone. XL765 shows activity as monotherapy and in combination with conventional therapeutics in a range of genetically diverse GBM xenografts.

Detection of Early Response to Temozolomide Treatment in Brain Tumors Using Hyperpolarized 13C MR Metabolic Imaging

Journal of Magnetic Resonance Imaging : JMRI. Jun, 2011  |  Pubmed ID: 21590996

To demonstrate the feasibility of using DNP hyperpolarized [1-(13)C]-pyruvate to measure early response to temozolomide (TMZ) therapy using an orthotopic human glioblastoma xenograft model.

Evolution of Mouse Models for Studying CNS Cancer: a Decade of Progress

Neuro-oncology. Jul, 2011  |  Pubmed ID: 21727207

Mutational Inactivation of STAG2 Causes Aneuploidy in Human Cancer

Science (New York, N.Y.). Aug, 2011  |  Pubmed ID: 21852505

Most cancer cells are characterized by aneuploidy, an abnormal number of chromosomes. We have identified a clue to the mechanistic origins of aneuploidy through integrative genomic analyses of human tumors. A diverse range of tumor types were found to harbor deletions or inactivating mutations of STAG2, a gene encoding a subunit of the cohesin complex, which regulates the separation of sister chromatids during cell division. Because STAG2 is on the X chromosome, its inactivation requires only a single mutational event. Studying a near-diploid human cell line with a stable karyotype, we found that targeted inactivation of STAG2 led to chromatid cohesion defects and aneuploidy, whereas in two aneuploid human glioblastoma cell lines, targeted correction of the endogenous mutant alleles of STAG2 led to enhanced chromosomal stability. Thus, genetic disruption of cohesin is a cause of aneuploidy in human cancer.

Phase I Study of Temsirolimus in Combination with EKB-569 in Patients with Advanced Solid Tumors

Investigational New Drugs. Sep, 2011  |  Pubmed ID: 21881915

Purpose Activation of EGFR can stimulate proliferative and survival signaling through mTOR. Preclinical data demonstrates synergistic activity of combined EGFR and mTOR inhibition. We undertook a phase I trial of temsirolimus (T, an mTOR inhibitor) and EKB-569 (E, an EGFR inhibitor) to determine the safety and tolerability. Methods The primary aim was to determine the maximally tolerated dose (MTD) of this combination in adults with solid tumors. Following the dose-escalation phase, (Cohort A), two subsequent cohorts were used to assess any pharmacokinetic (PK) interaction between the agents. Results Forty eight patients were enrolled. The MTD of this combination was E, 35 mg daily and T, 30 mg on days 1-3 and 15-17 using a 28-day cycle. The most common toxicities were nausea, diarrhea, fatigue, anorexia, stomatitis, rash, anemia, neutropenia, thrombocytopenia, and hypertriglyceridemia. Sixteen patients (36%) had at least one grade 3 toxicity. The most frequent grade 3/4 toxicities were diarrhea, dehydration, and nausea and vomiting (19% each). No grade 5 events were seen. Four patients had a partial response and 15 had stable disease. Clinical benefit was seen across a range of tumor types and in all cohorts. PK analysis revealed no significant interaction between E and T. Conclusions This combination of agents is associated with tolerable toxicities at doses that induced responses. PK studies revealed no interaction between the drugs. Further investigations of this targeting strategy may be attractive in renal cell carcinoma, non-small cell lung cancer, alveolar sarcoma, and carcinoid tumor.

Investigation of Intravenous Delivery of Nanoliposomal Topotecan for Activity Against Orthotopic Glioblastoma Xenografts

Neuro-oncology. Dec, 2011  |  Pubmed ID: 21954443

Achieving effective treatment outcomes for patients with glioblastoma (GBM) has been impeded by many obstacles, including the pharmacokinetic limitations of antitumor agents, such as topotecan (TPT). Here, we demonstrate that intravenous administration of a novel nanoliposomal formulation of TPT (nLS-TPT) extends the survival of mice with intracranial GBM xenografts, relative to administration of free TPT, because of improved biodistribution and pharmacokinetics of the liposome-formulated drug. In 3 distinct orthotopic GBM models, 3 weeks of biweekly intravenous therapy with nLS-TPT was sufficient to delay tumor growth and significantly extend animal survival, compared with treatment with free TPT (P ≤ .03 for each tumor tested). Analysis of intracranial tumors showed increased activation of cleaved caspase-3 and increased DNA fragmentation, both indicators of apoptotic response to treatment with nLS-TPT. These results demonstrate that intravenous delivery of nLS-TPT is a promising strategy in the treatment of GBM and support clinical investigation of this therapeutic approach.

Evidence for Sequenced Molecular Evolution of IDH1 Mutant Glioblastoma from a Distinct Cell of Origin

Journal of Clinical Oncology : Official Journal of the American Society of Clinical Oncology. Dec, 2011  |  Pubmed ID: 22025148

Mutation in isocitrate dehydrogenase 1 (IDH1) at R132 (IDH1(R132MUT)) is frequent in low-grade diffuse gliomas and, within glioblastoma (GBM), has been proposed as a marker for GBMs that arise by transformation from lower-grade gliomas, regardless of clinical history. To determine how GBMs arising with IDH1(R132MUT) differ from other GBMs, we undertook a comprehensive comparison of patients presenting clinically with primary GBM as a function of IDH1(R132) mutation status.

Targeted Therapy for BRAFV600E Malignant Astrocytoma

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Dec, 2011  |  Pubmed ID: 22038996

Malignant astrocytomas (MA) are aggressive central nervous system tumors with poor prognosis. Activating mutation of BRAF (BRAF(V600E)) has been reported in a subset of these tumors, especially in children. We have investigated the incidence of BRAF(V600E) in additional pediatric patient cohorts and examined the effects of BRAF blockade in preclinical models of BRAF(V600E) and wild-type BRAF MA.

An LXR Agonist Promotes GBM Cell Death Through Inhibition of an EGFR/AKT/SREBP-1/LDLR-dependent Pathway

Cancer Discovery. Sep, 2011  |  Pubmed ID: 22059152

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. EGFR mutations (EGFRvIII) and PI3K hyperactivation are common in GBM, promoting tumor growth and survival, including through SREBP-1-dependent-lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. Here, studies in GBM cell lines, xenograft models and GBM clinical samples, including from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the LDL receptor. Targeting LDLR with the Liver X Receptor (LXR) agonist GW3965 caused IDOL (Inducible Degrader Of LDLR)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results demonstrate that EGFRvIII can promote tumor survival through PI3K-SREBP-1 dependent up-regulation of LDLR, and suggest a role for LXR agonists in the treatment of GBM patients.

Stimulating Anti-tumor Immune Response: the Problem of Regulatory T-cells

Neuro-oncology. Dec, 2011  |  Pubmed ID: 22113488

Reduced Phosphocholine and Hyperpolarized Lactate Provide Magnetic Resonance Biomarkers of PI3K/Akt/mTOR Inhibition in Glioblastoma

Neuro-oncology. Dec, 2011  |  Pubmed ID: 22156546

The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

Clinical Stratification of Glioblastoma Based on Alterations in Retinoblastoma Tumor Suppressor Protein (RB1) and Association with the Proneural Subtype

Journal of Neuropathology and Experimental Neurology. Jan, 2012  |  Pubmed ID: 22157621

A recent study of CDK4/6 inhibitors in glioblastoma (GBM) xenografts identified retinoblastoma tumor suppressor protein RB1 status as a determinant of tumor therapeutic efficacy. Because of the need for clinically applicable RB1 testing, we assessed the utility of 2 complementary methods for determining RB1 status in GBM. Using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), we analyzed 34 GBMs that had also undergone molecular characterization as part of The Cancer Genome Atlas (TCGA). By IHC, 4 tumors (11.8%) had complete loss of RB protein expression, including 2 with homozygous deletion of RB1 by FISH and 1 with hemizygous deletion of RB1 by FISH combined with a novel nonsense mutation in RB1. Consistent with these results, in an independent set of 51 GBMs tested by IHC, we demonstrated loss of RB1 protein in 5 (9.8%). In GBM molecular subtype analysis of TCGA data, complete loss of RB1 transcript expression was seen in 18 (10.6%) of 170 tumors, and these were highly enriched for, but not exclusive to, the proneural subtype (p < 0.01). These data support the use of IHC for determining RB1 status in clinical GBM specimens and suggest that RB1 alterations may be more common in certain GBM subgroups.

An Experimental Xenograft Mouse Model of Diffuse Pontine Glioma Designed for Therapeutic Testing

Journal of Neuro-oncology. Jan, 2012  |  Pubmed ID: 22231932

The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P < 0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG.

Challenges in Drug Delivery to Tumors of the Central Nervous System: An Overview of Pharmacological and Surgical Considerations

Advanced Drug Delivery Reviews. Jan, 2012  |  Pubmed ID: 22306489

The majority of newly diagnosed brain tumors are treated with surgery, radiation, and the chemotherapeutic temozolomide. Development of additional therapeutics to improve treatment outcomes is complicated by the blood-brain barrier (BBB), which acts to protect healthy tissue from chemical insults. The high pressure found within brain tumors adds a challenge to local delivery of therapy by limiting the distribution of bolus injections. Here we discuss various drug delivery strategies, including convection-enhanced delivery, intranasal delivery, and intrathecal delivery, as well as pharmacological strategies for improving therapeutic efficacy, such as blood-brain barrier disruption.

Hyperpolarized 13C MR Spectroscopic Imaging Can Be Used to Monitor Everolimus Treatment in Vivo in an Orthotopic Rodent Model of Glioblastoma

NeuroImage. Jan, 2012  |  Pubmed ID: 21807103

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the potential of HP lactate-to-pyruvate ratio as an early biomarker of response, thereby supporting further investigation of this non-invasive imaging approach for eventual clinical application.