J. Graeme Hodgson

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

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.

A Human-Like Senescence-Associated Secretory Phenotype Is Conserved in Mouse Cells Dependent on Physiological Oxygen

Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP), which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that “senescent” mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen), do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3%) oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-α. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

Oncogenic BRAF mutation with CDKN2A inactivation is characteristic of a subset of pediatric malignant astrocytomas.

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.

Syngeneic mouse mammary carcinoma cell lines: Two closely related cell lines with divergent metastatic behavior

Two cell lines, Met-1fvb2 and DB-7fvb2, with different metastatic potential, were derived from mammary carcinomas in FVB/N-Tg(MMTV-PyVmT) and FVB/N-Tg(MMTV-PyVmTY315F/Y322F) mice, transplanted into syngeneic FVB/N hosts and characterized. The lines maintain a stable morphological and biological phenotype after multiple rounds of in vitro culture and in vivo transplantation. The Met-1fvb2 line derived from a FVB/N-Tg(MMTV-PyVmT) tumor exhibits invasive growth and 100% metastases when transplanted into the females FVB/N mammary fat pad. The DB-7fvb2 line derived from the FVB/N-Tg(MMTV-PyVmTY315F/Y322F) with a “double base” modification at Y315F/Y322F exhibits more rapid growth when transplanted into the mammary fat pad, but a lower rate of metastasis (17%). The Met1fvb2 cells show high activation of AKT, while DB-7fvb2 cells show very low levels of AKT activation. The DNA content and gene expression levels of both cell lines are stable over multiple generations. Therefore, these two cell lines provide a stable, reproducible resource for the study of metastasis modulators, AKT molecular pathway interactions, and gene target and marker discovery.

Glioblastoma multiforme regional genetic and cellular expression patterns: Influence on anatomic and physiologic MR imaging

PURPOSE To determine whether magnetic resonance (MR) imaging is influenced by genetic and cellular features of glioblastoma multiforme (GBM) aggressiveness. MATERIALS AND METHODS In this HIPAA-compliant institutional review board-approved study, multiple enhancing and peritumoral nonenhancing stereotactic neurosurgical biopsy samples from treatment-naïve GBMs were collected prospectively, with guidance from cerebral blood volume (CBV) MR imaging measurements. By using monoclonal antibodies, tissue specimens were examined for microvascular expression, hypoxia, tumor and overall cellular density, and histopathologic features of GBM aggressiveness. Genetic expression patterns were investigated with RNA microarrays. Imaging and histopathologic variables were compared with the Welch t test and Pearson correlations. Microarray analysis was performed by using false discovery rate (FDR) statistics. RESULTS Tumor biopsy of 13 adult patients yielded 16 enhancing and 14 peritumoral nonenhancing specimens. Enhancing regions had elevated relative CBV and reduced relative apparent diffusion coefficient (ADC) measurements compared with peritumoral nonenhancing biopsy regions (P < .01). A positive correlation was found between relative CBV and all histopathologic features of aggressiveness (P < .04). An inverse correlation was found between relative ADC and all histopathologic features of aggressiveness (P < .05). RNA expression patterns between tumor regions were found to be significantly different (FDR < 0.05), with hierarchical clustering by biopsy region only. CONCLUSION These findings suggest MR imaging is significantly influenced by GBM genetic and cellular biologic features of aggressiveness and imply physiologic MR imaging may be useful in pinpointing regions of highest malignancy within heterogeneous tissues, thus facilitating histologic grading of primary glial brain tumors.

Comparative analyses of gene copy number and mRNA expression in glioblastoma multiforme tumors and xenografts

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.

Genome amplification of chromosome 20 in breast cancer

Recurrent gain and amplification of the long arm of chromosome 20 (20q) has been observed in a wide variety of cancers. This suggests that a gene or genes encoded on 20q play important roles in contributing to the cancer phenotype when overexpressed. In the quest to discover cancer genes, this region of the genome has been exhaustively studied, and the results demonstrate remarkable complexity. Multiple regions of low and high-level 20q copy number gain correlate with poor clinical prognosis and appear to contribute to the cancer phenotype, especially aspects of immortalization, genome instability, apoptosis, and increased proliferation. Gene discovery efforts have revealed a number of interesting candidate genes on chromosome 20 that may contribute to oncogenic progression. The study of 20q serves as a model for positional cloning enthusiasts, demonstrating the path typically taken when moving from initial discovery of an important genomic abnormality to identification of genes likely to be significant players in disease progression. This review will summarize approximately a decade of study on 20q and is structured as moving from an introduction to the techniques used in 20q analyses, to the details of 20q genomic complexity and its involvement with cancer, and finally to a detailed gene-specific look at this region.

The Role of p53 in Suppression of KSHV Cyclin-induced Lymphomagenesis

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a cyclin D homolog, K cyclin, that is thought to promote viral oncogenesis. However, expression of K cyclin in cultured cells not only triggers cell cycle progression but also engages the p53 tumor suppressor pathway, which probably restricts the oncogenic potential of K cyclin. Therefore, to assess the tumorigenic properties of K cyclin in vivo, we transgenically targeted expression of K cyclin to the B and T lymphocyte compartments via the Eμ promoter/enhancer. Around 17% of Eμ-K cyclin animals develop lymphoma by 9 months of age, and all such lymphomas exhibit loss of p53. A critical role of p53 in suppressing K cyclin-induced lymphomagenesis was confirmed by the greatly accelerated onset of B and T lymphomagenesis in all Eμ-K cyclin/p53−/− mice. However, absence of p53 did not appear to accelerate K cyclin-induced lymphomagenesis by averting apoptosis: Eμ-K cyclin/p53−/− end-stage lymphomas contained abundant apoptotic cells, and transgenic Eμ-K cyclin/p53−/− lymphocytes in vitro were not measurably protected from DNA damage-induced apoptosis compared with Eμ-K cyclin/p53wt cells. Notably, whereas aneuploidy was frequently evident in pre-lymphomatous tissues, end-stage Eμ-K cyclin/p53−/− tumors showed a near-diploid DNA content with no aberrant centrosome numbers. Nonetheless, such tumor cells did harbor more restricted genomic alterations, such as single-copy chromosome losses or gains or high-level amplifications. Together, our data support a model in which K cyclin-induced genome instability arises early in the pre-tumorigenic lymphocyte population and that loss of p53 licenses subsequent expansion of tumorigenic clones.

Shared Epigenetic Mechanisms in Human and Mouse Gliomas Inactivate Expression of the Growth Suppressor SLC5A8

Tumors arise in part from the deleterious effects of genetic and epigenetic mechanisms on gene expression. In several mouse models of human tumors, the tumorigenic phenotype is reversible, suggesting that epigenetic mechanisms also contribute significantly to tumorigenesis in mice. It is not known whether these are the same epigenetic mechanisms in human and mouse tumors or whether they affect homologous genes. Using an integrated approach for genome-wide methylation and copy number analyses, we identified SLC5A8 on chromosome 12q23.1 that was affected frequently by aberrant methylation in human astrocytomas and oligodendrogliomas. SLC5A8 encodes a sodium monocarboxylate cotransporter that was highly expressed in normal brain but was significant down-regulated in primary gliomas. Bisulfite sequencing analysis showed that the CpG island was unmethylated in normal brain but frequently localized methylated in brain tumors, consistent with the tumor-specific loss of gene expression. In glioma cell lines, SLC5A8 expression was also suppressed but could be reactivated with a methylation inhibitor. Expression of exogenous SLC5A8 in LN229 and LN443 glioma cells inhibited colony formation, suggesting that it may function as a growth suppressor in normal brain cells. Remarkably, 9 of 10 murine oligodendroglial tumors (from p53+/- or ink4a/arf+/- animals transgenic for S100beta-v-erbB) showed a similar tumor-specific down-regulation of mSLC5A8, the highly conserved mouse homologue. Taken together, these data suggest that SLC5A8 functions as a growth suppressor gene in vitro and that it is silenced frequently by epigenetic mechanisms in primary gliomas. The shared epigenetic inactivation of mSLC5A8 in mouse gliomas indicates an additional degree of commonality in the origin and/or pathway to tumorigenesis between primary human tumors and these mouse models of gliomas.

TIMP-1 Alters Susceptibility to Carcinogenesis

Tissue inhibitors of metalloproteinases (TIMPs) are a family of multifunctional proteins known to possess a broad range of biological activities, including inhibition of metalloproteinase activity, regulation of proliferation and apoptosis of a variety of cell types, and, depending on the context, differential regulation of angiogenic and inflammatory responses. Elevated mRNA expression of TIMP family members correlates with malignancy and clinical outcome in many human cancer types; however, a protective role for TIMPs also has been observed in various mouse models of human cancer. In the current study, we found distinct spatial-temporal expression patterns for the mRNA of TIMP family members in a mouse model of epithelial carcinogenesis [i.e., keratin 14-human papillomavirus 16 (K14-HPV16) transgenic mice]. To test the hypothesis that elevated expression of TIMP-1 functionally regulates epithelial carcinogenesis, we introduced a human TIMP-1 transgene into K14-HPV16 transgenic mice and assessed neoplastic progression. Results from these studies suggest that TIMP-1 enhances tumorgenicity by potentiating keratinocyte hyperproliferation and appearance of chromosomal aberrations in premalignant cells, thereby increasing their risk to undergo malignant conversion. In addition, TIMP-1 inhibits tissue gelatinolytic activity in tumor stroma, affects stabilization of collagen fibrils, but does not inhibit malignant conversion of dysplasias into carcinomas or development of metastases. The combined implications of these studies suggest that TIMP-1 is an important contributor to epithelial neoplastic progression and supports the concept that TIMP-1 exerts differential regulation on tissues in a stage-dependent manner.