The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

Maximizing the potential of aggressive mouse tumor models in preclinical drug testing.

Atypical teratoid rhabdoid tumor (ATRT) is an aggressive embryonal brain tumor among infants and young children. Two challenges exist for preclinical testing in ATRT. First, genetically quiet, ATRT is a difficult tumor to target molecularly. Tumor cells need to divide to propagate tumor growth-intercepting the common crossroads in cell cycle progression is a feasible strategy. KIF11 is needed for bipolar spindle formation in metaphase. We identified KIF11 as a universal target of all ATRT-molecular-subtypes. Ispinesib, a KIF11-inhibitor, effectively inhibited tumor proliferation in all seven cell lines. A second challenge-a major challenge in preclinical drug testing in-vivo among aggressive tumor models, is the narrow therapeutic window to administer drugs within the limited murine lifespan. Our most aggressive ATRT tumor model was lethal in all mice within ~ 1 month of tumor implantation. Such short-surviving mouse models are difficult to employ for preclinical drug testing due to the narrow time window to administer drugs. To overcome this time restriction, we developed a clinical staging system which allowed physically-fit mice to continue treatment, in contrast to the conventional method of fixed drug-dose-duration regimen in preclinical testing which will not be feasible in such short-surviving mouse models. We validated this approach in a second embryonal brain tumor, medulloblastoma. This is a clinically relevant, cost-efficient approach in preclinical testing for cancer and non-cancer disease phenotypes. Widely used preclinical mouse models are not the most accurate and lack the aggressive tumor spectrum found within a single tumor type. Mice bearing the most aggressive tumor spectrum progress rapidly in the limited murine life-span, resulting in a narrow therapeutic window to administer drugs, and are thus difficult to employ in preclinical testing. Our approach overcomes this challenge. We discovered ispinesib is efficacious against two embryonal brain tumor types.

Relevance of a TCGA-derived Glioblastoma Subtype Gene-Classifier among Patient Populations.

Glioblastoma multiforme (GBM), a deadly cancer, is the most lethal and common malignant brain tumor, and the leading cause of death in adult brain tumors. While genomic data continues to rocket, clinical application and translation to patient care are lagging behind. Big data now deposited in the TCGA network offers a window to generate novel clinical hypotheses. We hypothesized that a TCGA-derived gene-classifier can be applied across different gene profiling platforms and population groups. This gene-classifier validated three robust GBM-subtypes across six different platforms, among Caucasian, Korean and Chinese populations: Three Caucasian-predominant TCGA-cohorts (Affymetrix U133A = 548, Agilent Custom-Array = 588, RNA-seq = 168), and three Asian-cohorts (Affymetrix Human Gene 1.0ST-Array = 61, Illumina = 52, Agilent 4 × 44 K = 60). To understand subtype-relevance in patient therapy, we investigated retrospective TCGA patient clinical sets. Subtype-specific patient survival outcome was similarly poor and reflected the net result of a mixture of treatment regimens with/without surgical resection. As a proof-of-concept, in subtype-specific patient-derived orthotopic xenograft (PDOX) mice, Classical-subtype demonstrated no survival difference comparing radiation-therapy versus temozolomide monotherapies. Though preliminary, a PDOX model of Proneural/Neural-subtype demonstrated significantly improved survival with temozolomide compared to radiation-therapy. A larger scale study using this gene-classifier may be useful in clinical outcome prediction and patient selection for trials based on subtyping.

Genome-wide analysis of DNA copy number alterations and loss of heterozygosity in intracranial germ cell tumors.

BACKGROUNDS: Intracranial germ cell tumors (GCTs) are rare and heterogeneous with very little is known about their pathogenesis and underlying genetic abnormalities. PROCEDURES: In order to identify candidate genes and pathways which are involved in the pathogenesis of these tumors, we have profiled 62 intracranial GCTs for DNA copy number alterations (CNAs) and loss of heterozygosity (LOH) by using single nucleotide polymorphism (SNP) array and quantitative real time PCR (qPCR). RESULTS: Initially 27 cases of tumor tissues with matched blood samples were fully analyzed by SNP microarray and qPCR. Statistical analysis using the genomic identification of significant targets in cancer (GISTIC) tool identified 10 regions of significant copy number gain and 11 regions of significant copy number loss. While overall pattern of genomic aberration was similar between germinoma and nongerminomatous germ cell tumors (NGGCTs), a few subtype-specific peak regions were identified. Analysis by SNP array and qPCR was replicated using an independent cohort of 35 cases. CONCLUSIONS: Frequent aberrations of CCND2 (12p13) and RB1 (13q14) suggest that Cyclin/CDK-RB-E2F pathway might play a critical role in the pathogenesis of intracranial GCTs. Frequent gain of PRDM14 (8q13) implies that transcriptional regulation of primordial germ cell specification might be an important factor in the development of this tumor.

Implications of tumor location on subtypes of medulloblastoma.

BACKGROUND: Medulloblastoma (MB) comprises of four molecular subtypes, Sonic hedgehog (SHH), Wingless (WNT), Groups 3 and 4. WNT-subtype MBs were found to arise from midline of the brainstem occupying the fourth ventricle while SHH-subtype occupied the cerebellar hemisphere in a small subset of patients. PROCEDURE: We tested this hypothesis in a large cohort of pediatric MBs comprising of all four molecular subtypes. RESULTS: We validated in the first comprehensive analysis of tumor location of 60 human MBs representative of the four molecular subtypes, that hemispheric tumors are significantly associated with SHH-subtype MBs while midline tumors with WNT-subtype, Group 3 and 4 MBs (P < 0.001). Nearly half of SHH-subtype MBs were midline. CONCLUSIONS: Tumor location should not be generalized to MB subtypes. SHH-subtype MBs are not exclusively hemispheric and hemispheric MBs are not always SHH-activated. It is imperative to identify subtypes in conjunction with tumor location when exploring currently available targeted therapy.