Activating KRAS mutations and p16 Ink4a inactivation are near universal events in human pancreatic ductal adenocarcinoma (PDAC). In mouse models, Kras G12D initiates formation of premalignant pancreatic ductal lesions, and loss of either Ink4a͞Arf (p16 Ink4a ͞p19 Arf ) or p53 enables their malignant progression. As recent mouse modeling studies have suggested a less prominent role for p16 Ink4a in constraining malignant progression, we sought to assess the pathological and genomic impact of inactivation of p16 Ink4a , p19 Arf , and͞or p53 in the Kras G12D model. Rapidly progressive PDAC was observed in the setting of homozygous deletion of either p53 or p16 Ink4a , the latter with intact germ-line p53 and p19 Arf sequences. Additionally, Kras G12D in the context of heterozygosity either for p53 plus p16 Ink4a or for p16 Ink4a ͞p19 Arf produced PDAC with longer latency and greater propensity for distant metastases relative to mice with homozygous deletion of p53 or p16 Ink4a ͞p19 Arf . Tumors from the double-heterozygous cohorts showed frequent p16 Ink4a inactivation and loss of either p53 or p19 Arf . Different genotypes were associated with specific histopathologic characteristics, most notably a trend toward less differentiated features in the homozygous p16 Ink4a ͞p19 Arf mutant model. High-resolution genomic analysis revealed that the tumor suppressor genotype influenced the specific genomic patterns of these tumors and showed overlap in regional chromosomal alterations between murine and human PDAC. Collectively, our results establish that disruptions of p16 Ink4a and the p19 ARF -p53 circuit play critical and cooperative roles in PDAC progression, with specific tumor suppressor genotypes provocatively influencing the tumor biological phenotypes and genomic profiles of the resultant tumors.array comparative genomic hybridization ͉ mouse models ͉ pancreatic cancer ͉ KRAS ͉ tumor suppressor P ancreatic ductal adenocarcinoma (PDAC) ranks as the fourth leading cause of cancer mortality in the United States and causes Ͼ200,000 deaths worldwide annually (1, 2). Histopathological analyses have identified precursor lesions, pancreatic intraepithelial neoplasias (PanIN), which appear to progress through increasingly severe stages of cellular atypia leading to invasive PDAC (3). These lesions show multistep molecular progression that includes early activating KRAS mutations and telomere attrition, and subsequent inactivation of p16 Ink4a , p14 ARF , p53, and͞or SMAD4 tumor suppressors in a high percentage of cases (4-6).The Ink4a͞Arf locus (hereafter denoted p16 Ink4a ͞p19 Arf ) encodes tumor suppressors p16 INK4A and p14 ARF (p19 Arf in the mouse). p16 INK4A is a G 1 cyclin-dependent kinase (CDK) inhibitor that binds to CDK4 and CDK6 and prevents their association with D-type cyclins (7), thereby facilitating CDK4͞6-cyclin D-mediated phosphorylation and inactivation of retinoblastoma protein (RB) and S-phase entry. p16 INK4A -mediated tumor suppression may relate to its induction by activated oncogenes and consequen...

Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells. We identified genes previously implicated in proliferative control and epithelial cell function including two established tumor suppressors, TGFBR2 and PTEN. In addition, we uncovered a previously unrecognized tumor suppressor role for REST/NRSF, a transcriptional repressor of neuronal gene expression. Array-CGH analysis identified REST as a frequent target of deletion in colorectal cancer. Furthermore, we detect a frameshift mutation of the REST gene in colorectal cancer cells that encodes a dominantly acting truncation capable of transforming epithelial cells. Cells lacking REST exhibit increased PI(3)K signaling and are dependent upon this pathway for their transformed phenotype. These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer.

To identify genetic events underlying the genesis and progression of multiple myeloma (MM), we conducted a high-resolution analysis of recurrent copy number alterations (CNAs) and expression profiles in a collection of MM cell lines and outcome-annotated clinical specimens. Attesting to the molecular heterogeneity of MM, unsupervised classification using nonnegative matrix factorization (NMF) designed for array comparative genomic hybridization (aCGH) analysis uncovered distinct genomic subtypes. Additionally, we defined 87 discrete minimal common regions (MCRs) within recurrent and highly focal CNAs. Further integration with expression data generated a refined list of MM gene candidates residing within these MCRs, thereby providing a genomic framework for dissection of disease pathogenesis, improved clinical management, and initiation of targeted drug discovery for specific MM patients.

Genomes of human cancer cells are characterized by numerous chromosomal aberrations of uncertain pathogenetic significance. Here, in an inducible mouse model of melanoma, we characterized metastatic variants with an acquired focal chromosomal amplification that corresponds to a much larger amplification in human metastatic melanomas. Further analyses identified Nedd9, an adaptor protein related to p130CAS, as the only gene within the minimal common region that exhibited amplification-associated overexpression. A series of functional, biochemical, and clinical studies established NEDD9 as a bona fide melanoma metastasis gene. NEDD9 enhanced invasion in vitro and metastasis in vivo of both normal and transformed melanocytes, functionally interacted with focal adhesion kinase and modulated focal contact formation, and exhibited frequent robust overexpression in human metastatic melanoma relative to primary melanoma. Thus, comparative oncogenomics has enabled the identification and facilitated the validation of a highly relevant cancer gene governing metastatic potential in human melanoma.

Lung cancer is the leading cause of cancer mortality worldwide, yet there exists a limited view of the genetic lesions driving this disease. In this study, an integrated high-resolution survey of regional amplifications and deletions, coupled with gene-expression profiling of non-small-cell lung cancer subtypes, adenocarcinoma and squamous-cell carcinoma (SCC), identified 93 focal copynumber alterations, of which 21 span <0.5 megabases and contain a median of five genes. Whereas all known lung cancer genes͞loci are contained in the dataset, most of these recurrent copy-number alterations are previously uncharacterized and include high-amplitude amplifications and homozygous deletions. Notably, despite their distinct histopathological phenotypes, adenocarcinoma and SCC genomic profiles showed a nearly complete overlap, with only one clear SCC-specific amplicon. Among the few genes residing within this amplicon and showing consistent overexpression in SCC is p63, a known regulator of squamous-cell differentiation. Furthermore, intersection with the published pancreatic cancer comparative genomic hybridization dataset yielded, among others, two focal amplicons on 8p12 and 20q11 common to both cancer types. Integrated DNA-RNA analyses identified WHSC1L1 and TPX2 as two candidates likely targeted for amplification in both pancreatic ductal adenocarcinoma and non-small-cell lung cancer.array comparative genomic hybridization ͉ expression profiling ͉ lung adenocarcinoma ͉ squamous-cell lung carcinoma ͉ TP73L L ung cancer is the leading cause of cancer-related mortality in the United States, accounting for more than one-fourth of all cancer fatalities in 2004. Lung cancer is classified into two major subtypes, small-cell and non-small-cell lung cancer (NSCLC). NSCLC constitutes 75% of lung cancer cases and is subdivided further into three major histological subtypes: adenocarcinoma (AC), squamous-cell carcinoma (SCC), and large-cell carcinoma. The AC and SCC subtypes represent Ͼ85% of NSCLC cases. Although these NSCLC subtypes exhibit distinct pathological characteristics, the treatment approaches have remained generic and largely ineffective, despite advances in cytotoxic drugs, radiotherapy, and clinical management. For all stages of NSCLC, the 5-year survival rate has remained fixed at 15% for the last 15 years. The recent success of molecularly targeted therapies for a limited subset of cancer genotypes (1) has solidified the view that a more detailed knowledge of the spectrum of genetic lesions in lung cancer will, in turn, lead to meaningful therapeutic progress.To date, the majority of lung cancer genetic studies have cataloged mutations or the promoter methylation status of known cancer genes, performed genome-wide loss-of-heterozygosity surveys, and applied comparative genomic hybridization (CGH) to audit regional copy-number alterations (CNAs) on metaphase chromosomes or small-scale bacterial artificial chromosome (BAC) arrays. These concerted efforts have identified a core set of lesions, including activating...

Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the neoplastic transformation process. Here we engineered lymphoma-prone mice with chromosomal instability to assess the usefulness of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Along with targeted re-sequencing, our comparative oncogenomic studies identified FBXW7 and PTEN to be commonly deleted both in murine lymphomas and in human T-cell acute lymphoblastic leukaemia/lymphoma (T-ALL). The murine cancers acquire widespread recurrent amplifications and deletions targeting loci syntenic to those not only in human T-ALL but also in diverse human haematopoietic, mesenchymal and epithelial tumours. These results indicate that murine and human tumours experience common biological processes driven by orthologous genetic events in their malignant evolution. The highly concordant nature of genomic events encourages the use of genomically unstable murine cancer models in the discovery of biological driver events in the human oncogenome.

The pancreatic adenocarcinoma genome harbors multiple amplifications and deletions, pointing to the existence of numerous oncogenes and tumor suppressor genes driving the genesis and progression of this lethal cancer. Here, array comparative genomic hybridization on a cDNA microarray platform and informatics tools have been used to define the copy number alterations in a panel of 24 pancreatic adenocarcinoma cell lines and 13 primary tumor specimens. This high-resolution genomic analysis has identified all known regional gains and losses as well as many previously uncharacterized highly recurrent copy number alterations. A systematic prioritization scheme has selected 64 focal minimal common regions (MCRs) of recurrent copy number change. These MCRs possess a median size of 2.7 megabases (Mb), with 21 (33%) MCRs spanning 1 Mb or less (median of 0.33 Mb) and possessing an average of 15 annotated genes. Furthermore, complementary expression profile analysis of a significant fraction of the genes residing within these 64 prioritized MCRs has enabled the identification of a subset of candidates with statistically significant association between gene dosage and mRNA expression. Thus, the integration of DNA and RNA profiles provides a highly productive entry point for the discovery of genes involved in the pathogenesis of pancreatic adenocarcinoma.array comparative genomic hybridization ͉ expression profile P ancreatic adenocarcinoma is among the most lethal of human cancers, typically presenting as advanced inoperable disease with a rapidly progressive clinical course characterized by intense resistance to all therapeutic modalities. Significant effort has been directed toward charting the molecular genetic events in this cancer with the goals of improving early detection and providing new therapeutic targets. The current compendium of validated genetic mutations has provided a multistep model for the initiation and progression of pancreatic adenocarcinoma that is typified by the near-universal and early occurrence of activating mutations in KRAS and frequent later-stage inactivation of p16 INK4A , p53, and͞or SMAD4 (1).These stereotypic genetic lesions take place against the backdrop of a high level of genomic instability that is evident in the earliest stages of the disease (2-4). Indeed, a hallmark genomic feature of this cancer is the presence of numerous complex chromosome structural aberrations, including nonreciprocal translocations, amplifications, and deletions. To date, karyotype analyses (5-10), chromosomal comparative genomic hybridization (CGH) (11-17), and loss of heterozygosity mapping (18-20) have identified recurrent regions of copy number change or allelic loss. In particular, frequent gains have been mapped to 3q, 5p, 7p, 8q, 11q, 12p, 17q, and 20q and losses to 3p, 4q, 6q, 8p, 9p, 10q, 12q, 13q, 17p, 18q, 21q, and 22q. In some instances, validated oncogenes and tumor suppressor genes residing within these loci have been identified, including MYC (8q24), p16 INK4A (9p21), p53 (17p13), SMAD4 (18q21...

A cardinal feature of malignant melanoma is its metastatic propensity. An incomplete view of the genetic events driving metastatic progression has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for melanoma patients. In this study, we conducted global genomic characterization of primary and metastatic melanomas to examine the genomic landscape associated with metastatic progression. In addition to uncovering three genomic subclasses of metastastic melanomas, we delineated 39 focal and recurrent regions of amplification and deletions, many of which encompassed resident genes that have not been implicated in cancer or metastasis. To identify progression-associated metastasis gene candidates, we applied a statistical approach, Integrative Genome Comparison (IGC), to define 32 genomic regions of interest that were significantly altered in metastatic relative to primary melanomas, encompassing 30 resident genes with statistically significant expression deregulation. Functional assays on a subset of these candidates, including MET, ASPM, AKAP9, IMP3, PRKCA, RPA3, and SCAP2, validated their pro-invasion activities in human melanoma cells. Validity of the IGC approach was further reinforced by tissue microarray analysis of Survivin showing significant increased protein expression in thick versus thin primary cutaneous melanomas, and a progression correlation with lymph node metastases. Together, these functional validation results and correlative analysis of human tissues support the thesis that integrated genomic and pathological analyses of staged melanomas provide a productive entry point for discovery of melanoma metastases genes.