Cancer genomics

Kim H, Huang W, Jiang X, Pennicooke B, Park PJ, Johnson MD. Integrative genome analysis reveals an oncomir/oncogene cluster regulating glioblastoma survivorship. Proc Natl Acad Sci U S A 2010;107(5):2183-8.Abstract

Using a multidimensional genomic data set on glioblastoma from The Cancer Genome Atlas, we identified hsa-miR-26a as a cooperating component of a frequently occurring amplicon that also contains CDK4 and CENTG1, two oncogenes that regulate the RB1 and PI3 kinase/AKT pathways, respectively. By integrating DNA copy number, mRNA, microRNA, and DNA methylation data, we identified functionally relevant targets of miR-26a in glioblastoma, including PTEN, RB1, and MAP3K2/MEKK2. We demonstrate that miR-26a alone can transform cells and it promotes glioblastoma cell growth in vitro and in the mouse brain by decreasing PTEN, RB1, and MAP3K2/MEKK2 protein expression, thereby increasing AKT activation, promoting proliferation, and decreasing c-JUN N-terminal kinase-dependent apoptosis. Overexpression of miR-26a in PTEN-competent and PTEN-deficient glioblastoma cells promoted tumor growth in vivo, and it further increased growth in cells overexpressing CDK4 or CENTG1. Importantly, glioblastoma patients harboring this amplification displayed markedly decreased survival. Thus, hsa-miR-26a, CDK4, and CENTG1 comprise a functionally integrated oncomir/oncogene DNA cluster that promotes aggressiveness in human cancers by cooperatively targeting the RB1, PI3K/AKT, and JNK pathways.

Lee E, Iskow R, Yang L, Gokcumen O, Haseley P, Luquette LJ, Lohr JG, Harris CC, Ding L, Wilson RK, Wheeler DA, Gibbs RA, Kucherlapati R, Lee C, Kharchenko PV**, Park PJ**, Cancer Genome Atlas Research Network TCGA. Landscape of somatic retrotransposition in human cancers. Science 2012;337(6097):967-71.Abstract

Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.

Liu F*, Park PJ*, Lai W, Maher E, Chakravarti A, Durso L, Jiang X, Yu Y, Brosius A, Thomas M, Chin L, Brennan C, DePinho RA, Kohane I, Carroll RS, Black PM, Johnson MD. A genome-wide screen reveals functional gene clusters in the cancer genome and identifies EphA2 as a mitogen in glioblastoma. Cancer Res 2006;66(22):10815-23.Abstract

A novel genome-wide screen that combines patient outcome analysis with array comparative genomic hybridization and mRNA expression profiling was developed to identify genes with copy number alterations, aberrant mRNA expression, and relevance to survival in glioblastoma. The method led to the discovery of physical gene clusters within the cancer genome with boundaries defined by physical proximity, correlated mRNA expression patterns, and survival relatedness. These boundaries delineate a novel genomic interval called the functional common region (FCR). Many FCRs contained genes of high biological relevance to cancer and were used to pinpoint functionally significant DNA alterations that were too small or infrequent to be reliably identified using standard algorithms. One such FCR contained the EphA2 receptor tyrosine kinase. Validation experiments showed that EphA2 mRNA overexpression correlated inversely with patient survival in a panel of 21 glioblastomas, and ligand-mediated EphA2 receptor activation increased glioblastoma proliferation and tumor growth via a mitogen-activated protein kinase-dependent pathway. This novel genome-wide approach greatly expanded the list of target genes in glioblastoma and represents a powerful new strategy to identify the upstream determinants of tumor phenotype in a range of human cancers.

Yang HW, Kim T-M, Song SS, Shrinath N, Park R, Kalamarides M, Park PJ, Black PM, Carroll RS, Johnson MD. Alternative splicing of CHEK2 and codeletion with NF2 promote chromosomal instability in meningioma. Neoplasia 2012;14(1):20-8.Abstract

Mutations of the NF2 gene on chromosome 22q are thought to initiate tumorigenesis in nearly 50% of meningiomas, and 22q deletion is the earliest and most frequent large-scale chromosomal abnormality observed in these tumors. In aggressive meningiomas, 22q deletions are generally accompanied by the presence of large-scale segmental abnormalities involving other chromosomes, but the reasons for this association are unknown. We find that large-scale chromosomal alterations accumulate during meningioma progression primarily in tumors harboring 22q deletions, suggesting 22q-associated chromosomal instability. Here we show frequent codeletion of the DNA repair and tumor suppressor gene, CHEK2, in combination with NF2 on chromosome 22q in a majority of aggressive meningiomas. In addition, tumor-specific splicing of CHEK2 in meningioma leads to decreased functional Chk2 protein expression. We show that enforced Chk2 knockdown in meningioma cells decreases DNA repair. Furthermore, Chk2 depletion increases centrosome amplification, thereby promoting chromosomal instability. Taken together, these data indicate that alternative splicing and frequent codeletion of CHEK2 and NF2 contribute to the genomic instability and associated development of aggressive biologic behavior in meningiomas.

Park PJ, Tian L, Kohane IS. Linking gene expression data with patient survival times using partial least squares. Bioinformatics 2002;18 Suppl 1:S120-7.Abstract

There is an increasing need to link the large amount of genotypic data, gathered using microarrays for example, with various phenotypic data from patients. The classification problem in which gene expression data serve as predictors and a class label phenotype as the binary outcome variable has been examined extensively, but there has been less emphasis in dealing with other types of phenotypic data. In particular, patient survival times with censoring are often not used directly as a response variable due to the complications that arise from censoring. We show that the issues involving censored data can be circumvented by reformulating the problem as a standard Poisson regression problem. The procedure for solving the transformed problem is a combination of two approaches: partial least squares, a regression technique that is especially effective when there is severe collinearity due to a large number of predictors, and generalized linear regression, which extends standard linear regression to deal with various types of response variables. The linear combinations of the original variables identified by the method are highly correlated with the patient survival times and at the same time account for the variability in the covariates. The algorithm is fast, as it does not involve any matrix decompositions in the iterations. We apply our method to data sets from lung carcinoma and diffuse large B-cell lymphoma studies to verify its effectiveness.

Gurumurthy S, Xie SZ, Alagesan B, Kim J, Yusuf RZ, Saez B, Tzatsos A, Ozsolak F, Milos P, Ferrari F, Park PJ, Shirihai OS, Scadden DT, Bardeesy N. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature 2010;468(7324):659-63.Abstract

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.

Yang L, Luquette LJ, Gehlenborg N, Xi R, Haseley PS, Hsieh C-H, Zhang C, Ren X, Protopopov A, Chin L, Kucherlapati R, Lee C, Park PJ. Diverse mechanisms of somatic structural variations in human cancer genomes. Cell 2013;153(4):919-29.Abstract

Identification of somatic rearrangements in cancer genomes has accelerated through analysis of high-throughput sequencing data. However, characterization of complex structural alterations and their underlying mechanisms remains inadequate. Here, applying an algorithm to predict structural variations from short reads, we report a comprehensive catalog of somatic structural variations and the mechanisms generating them, using high-coverage whole-genome sequencing data from 140 patients across ten tumor types. We characterize the relative contributions of different types of rearrangements and their mutational mechanisms, find that ~20% of the somatic deletions are complex deletions formed by replication errors, and describe the differences between the mutational mechanisms in somatic and germline alterations. Importantly, we provide detailed reconstructions of the events responsible for loss of CDKN2A/B and gain of EGFR in glioblastoma, revealing that these alterations can result from multiple mechanisms even in a single genome and that both DNA double-strand breaks and replication errors drive somatic rearrangements.

Kim T-M, Luquette LJ, Xi R, Park PJ. rSW-seq: algorithm for detection of copy number alterations in deep sequencing data. BMC Bioinformatics 2010;11:432.Abstract

BACKGROUND: Recent advances in sequencing technologies have enabled generation of large-scale genome sequencing data. These data can be used to characterize a variety of genomic features, including the DNA copy number profile of a cancer genome. A robust and reliable method for screening chromosomal alterations would allow a detailed characterization of the cancer genome with unprecedented accuracy. RESULTS: We develop a method for identification of copy number alterations in a tumor genome compared to its matched control, based on application of Smith-Waterman algorithm to single-end sequencing data. In a performance test with simulated data, our algorithm shows >90% sensitivity and >90% precision in detecting a single copy number change that contains approximately 500 reads for the normal sample. With 100-bp reads, this corresponds to a ~50 kb region for 1X genome coverage of the human genome. We further refine the algorithm to develop rSW-seq, (recursive Smith-Waterman-seq) to identify alterations in a complex configuration, which are commonly observed in the human cancer genome. To validate our approach, we compare our algorithm with an existing algorithm using simulated and publicly available datasets. We also compare the sequencing-based profiles to microarray-based results. CONCLUSION: We propose rSW-seq as an efficient method for detecting copy number changes in the tumor genome.

Kim T-M, Xi R, Luquette LJ, Park RW, Johnson MD, Park PJ. Functional genomic analysis of chromosomal aberrations in a compendium of 8000 cancer genomes. Genome Res 2013;23(2):217-27.Abstract

A large database of copy number profiles from cancer genomes can facilitate the identification of recurrent chromosomal alterations that often contain key cancer-related genes. It can also be used to explore low-prevalence genomic events such as chromothripsis. In this study, we report an analysis of 8227 human cancer copy number profiles obtained from 107 array comparative genomic hybridization (CGH) studies. Our analysis reveals similarity of chromosomal arm-level alterations among developmentally related tumor types as well as a number of co-occurring pairs of arm-level alterations. Recurrent ("pan-lineage") focal alterations identified across diverse tumor types show an enrichment of known cancer-related genes and genes with relevant functions in cancer-associated phenotypes (e.g., kinase and cell cycle). Tumor type-specific ("lineage-restricted") alterations and their enriched functional categories were also identified. Furthermore, we developed an algorithm for detecting regions in which the copy number oscillates rapidly between fixed levels, indicative of chromothripsis. We observed these massive genomic rearrangements in 1%-2% of the samples with variable tumor type-specific incidence rates. Taken together, our comprehensive view of copy number alterations provides a framework for understanding the functional significance of various genomic alterations in cancer genomes.

Yoon SS, Stangenberg L, Lee Y-J, Rothrock C, Dreyfuss JM, Baek K-H, Waterman PR, Nielsen PG, Weissleder R, Mahmood U, Park PJ, Jacks T, Dodd RD, Fisher CJ, Ryeom S, Kirsch DG. Efficacy of sunitinib and radiotherapy in genetically engineered mouse model of soft-tissue sarcoma. Int J Radiat Oncol Biol Phys 2009;74(4):1207-16.Abstract

PURPOSE: Sunitinib (SU) is a multitargeted receptor tyrosine kinase inhibitor of the vascular endothelial growth factor and platelet-derived growth factor receptors. The present study examined SU and radiotherapy (RT) in a genetically engineered mouse model of soft tissue sarcoma (STS). METHODS AND MATERIALS: Primary extremity STSs were generated in genetically engineered mice. The mice were randomized to treatment with SU, RT (10 Gy x 2), or both (SU+RT). Changes in the tumor vasculature before and after treatment were assessed in vivo using fluorescence-mediated tomography. The control and treated tumors were harvested and extensively analyzed. RESULTS: The mean fluorescence in the tumors was not decreased by RT but decreased 38-44% in tumors treated with SU or SU+RT. The control tumors grew to a mean of 1378 mm(3) after 12 days. SU alone or RT alone delayed tumor growth by 56% and 41%, respectively, but maximal growth inhibition (71%) was observed with the combination therapy. SU target effects were confirmed by loss of target receptor phosphorylation and alterations in SU-related gene expression. Cancer cell proliferation was decreased and apoptosis increased in the SU and RT groups, with a synergistic effect on apoptosis observed in the SU+RT group. RT had a minimal effect on the tumor microvessel density and endothelial cell-specific apoptosis, but SU alone or SU+RT decreased the microvessel density by >66% and induced significant endothelial cell apoptosis. CONCLUSION: SU inhibited STS growth by effects on both cancer cells and tumor vasculature. SU also augmented the efficacy of RT, suggesting that this combination strategy could improve local control of STS.

Jiang X, Xing H, Kim T-M, Jung Y, Huang W, Yang HW, Song S, Park PJ, Carroll RS, Johnson MD. Numb regulates glioma stem cell fate and growth by altering epidermal growth factor receptor and Skp1-Cullin-F-box ubiquitin ligase activity. Stem Cells 2012;30(7):1313-26.Abstract

Glioblastoma contains a hierarchy of stem-like cancer cells, but how this hierarchy is established is unclear. Here, we show that asymmetric Numb localization specifies glioblastoma stem-like cell (GSC) fate in a manner that does not require Notch inhibition. Numb is asymmetrically localized to CD133-hi GSCs. The predominant Numb isoform, Numb4, decreases Notch and promotes a CD133-hi, radial glial-like phenotype. However, upregulation of a novel Numb isoform, Numb4 delta 7 (Numb4d7), increases Notch and AKT activation while nevertheless maintaining CD133-hi fate specification. Numb knockdown increases Notch and promotes growth while favoring a CD133-lo, glial progenitor-like phenotype. We report the novel finding that Numb4 (but not Numb4d7) promotes SCF(Fbw7) ubiquitin ligase assembly and activation to increase Notch degradation. However, both Numb isoforms decrease epidermal growth factor receptor (EGFR) expression, thereby regulating GSC fate. Small molecule inhibition of EGFR activity phenocopies the effect of Numb on CD133 and Pax6. Clinically, homozygous NUMB deletions and low Numb mRNA expression occur primarily in a subgroup of proneural glioblastomas. Higher Numb expression is found in classical and mesenchymal glioblastomas and correlates with decreased survival. Thus, decreased Numb promotes glioblastoma growth, but the remaining Numb establishes a phenotypically diverse stem-like cell hierarchy that increases tumor aggressiveness and therapeutic resistance.