Charles A. Whittaker

Functional annotation of a full-length mouse cDNA collection

The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.The RIKEN Mouse Gene Encyclopaedia Project, a systematic approach to determining the full coding potential of the mouse genome, involves collection and sequencing of full-length complementary DNAs and physical mapping of the corresponding genes to the mouse genome. We organized an international functional annotation meeting (FANTOM) to annotate the first 21,076 cDNAs to be analysed in this project. Here we describe the first RIKEN clone collection, which is one of the largest described for any organism. Analysis of these cDNAs extends known gene families and identifies new ones.

Aneuploidy affects proliferation and spontaneous immortalization in mammalian cells.

Aneuploidy, an incorrect number of chromosomes, is the leading cause of miscarriages and mental retardation in humans and is a hallmark of cancer. We examined the effects of aneuploidy on primary mouse cells by generating a series of cell lines that carry an extra copy of one of four mouse chromosomes. In all four trisomic lines, proliferation was impaired and metabolic properties were altered. Immortalization, the acquisition of the ability to proliferate indefinitely, was also affected by the presence of an additional copy of certain chromosomes. Our data indicate that aneuploidy decreases not only organismal but also cellular fitness and elicits traits that are shared between different aneuploid cells.

Identification of Aneuploidy-Tolerating Mutations

Aneuploidy causes a proliferative disadvantage in all normal cells analyzed to date, yet this condition is associated with a disease characterized by unabated proliferative potential, cancer. The mechanisms that allow cancer cells to tolerate the adverse effects of aneuploidy are not known. To probe this question, we identified aneuploid yeast strains with improved proliferative abilities. Their molecular characterization revealed strain-specific genetic alterations as well as mutations shared between different aneuploid strains. Among the latter, a loss-of-function mutation in the gene encoding the deubiquitinating enzyme Ubp6 improves growth rates in four different aneuploid yeast strains by attenuating the changes in intracellular protein composition caused by aneuploidy. Our results demonstrate the existence of aneuploidy-tolerating mutations that improve the fitness of multiple different aneuploidies and highlight the importance of ubiquitin-proteasomal degradation in suppressing the adverse effects of aneuploidy.

Suppression of lung adenocarcinoma progression by Nkx2-1

Despite the high prevalence and poor outcome of patients with metastatic lung cancer the mechanisms of tumour progression and metastasis remain largely uncharacterized. Here we modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS and inactivation of the p53 pathway, using conditional alleles in mice. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of KrasLSL-G12D/+;p53flox/flox mice initiates lung adenocarcinoma development. Although tumours are initiated synchronously by defined genetic alterations, only a subset becomes malignant, indicating that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK2-related homeobox transcription factor Nkx2-1 (also called Ttf-1 or Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1 negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1-regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically restricted chromatin regulator Hmga2. Whereas focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor.

Stage-specific sensitivity to p53 restoration during lung cancer progression

Tumorigenesis is a multistep process that results from the sequential accumulation of mutations in key oncogene and tumour suppressor pathways. Personalized cancer therapy that is based on targeting these underlying genetic abnormalities presupposes that sustained inactivation of tumour suppressors and activation of oncogenes is essential in advanced cancers. Mutations in the p53 tumour-suppressor pathway are common in human cancer and significant efforts towards pharmaceutical reactivation of defective p53 pathways are underway. Here we show that restoration of p53 in established murine lung tumours leads to significant but incomplete tumour cell loss specifically in malignant adenocarcinomas, but not in adenomas. We define amplification of MAPK signalling as a critical determinant of malignant progression and also a stimulator of Arf tumour-suppressor expression. The response to p53 restoration in this context is critically dependent on the expression of Arf. We propose that p53 not only limits malignant progression by suppressing the acquisition of alterations that lead to tumour progression, but also, in the context of p53 restoration, responds to increased oncogenic signalling to mediate tumour regression. Our observations also underscore that the p53 pathway is not engaged by low levels of oncogene activity that are sufficient for early stages of lung tumour development. These data suggest that restoration of pathways important in tumour progression, as opposed to initiation, may lead to incomplete tumour regression due to the stage-heterogeneity of tumour cell populations.

Gene Expression Analysis of Macrophages That Facilitate Tumor Invasion Supports a Role for Wnt-Signaling in Mediating Their Activity in Primary Mammary Tumors

The tumor microenvironment modifies the malignancy of tumors. In solid tumors, this environment is populated by many macrophages that, in genetic studies that depleted these cells from mouse models of breast cancer, were shown to promote tumor progression to malignancy and increase metastatic potential. Mechanistic studies showed that these tumor-promoting effects of macrophages are through the stimulation of tumor cell migration, invasion, intravasation, and enhancement of angiogenesis. Using an in vivo invasion assay, it was demonstrated that invasive carcinoma cells are a unique subpopulation of tumor cells whose invasion and chemotaxis is dependent on the comigration of tumor-associated macrophages (TAMs) with obligate reciprocal signaling through an epidermal growth factor–CSF-1 paracrine loop. In this study, these invasion-promoting macrophages were isolated and subjected to analysis of their transcriptome in comparison with TAMs isolated indiscriminately to function using established macrophage markers. Unsupervised analysis of transcript patterns showed that the invasion-associated TAMs represent a unique subpopulation of TAMs that, by gene ontology criteria, have gene expression patterns related to tissue and organ development. Gene set enrichment analysis showed that these macrophages are also specifically enriched for molecules involved in Wnt-signaling. Previously, it was shown that macrophage-derived Wnt molecules promote vascular remodeling and that tumor cells are highly motile and intravasate around perivascular TAM clusters. Taken together, we conjecture that invasive TAMs link angiogenesis and tumor invasion and that Wnt-signaling plays a role in mediating their activity.

Loss of p53 synthesis in zebrafish tumors with ribosomal protein gene mutations

Zebrafish carrying heterozygous mutations for 17 different ribosomal protein (rp) genes are prone to developing malignant peripheral nerve sheath tumors (MPNSTs), a tumor type that is seldom seen in laboratory strains of zebrafish. Interestingly, the same rare tumor type arises in zebrafish that are homozygous for a loss-of-function point mutation in the tumor suppressor gene p53. For these reasons, and because p53 is widely known to be mutated in the majority of human cancers, we investigated the status of p53 in the rp+/− MPNSTs. Using monoclonal antibodies that we raised to zebrafish p53, we found that cells derived from rp+/− MPNSTs are significantly impaired in their ability to produce p53 protein even in the presence of a proteasome inhibitor and γ-irradiation. Although the coding regions of the p53 gene remain wild type, the gene is transcribed, and overall protein production rates appear normal in rp+/− MPNST cells, p53 protein does not get synthesized. This defect is observed in all MPNSTs we examined that were derived from our 17 zebrafish lines with rp gene mutations. To date, studies of p53 in malignancies have focused predominantly on either p53 gene mutations or the aberrant posttranslational regulation of the p53 protein. Our results show that the appropriate amount of numerous ribosomal proteins is required for p53 protein production in vivo and that disruption of this regulation most likely contributes to tumorigenesis.

DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage

Chromosome 17 is unusual among the human chromosomes in many respects. It is the largest human autosome with orthology to only a single mouse chromosome, mapping entirely to the distal half of mouse chromosome 11. Chromosome 17 is rich in protein-coding genes, having the second highest gene density in the genome. It is also enriched in segmental duplications, ranking third in density among the autosomes. Here we report a finished sequence for human chromosome 17, as well as a structural comparison with the finished sequence for mouse chromosome 11, the first finished mouse chromosome. Comparison of the orthologous regions reveals striking differences. In contrast to the typical pattern seen in mammalian evolution, the human sequence has undergone extensive intrachromosomal rearrangement, whereas the mouse sequence has been remarkably stable. Moreover, although the human sequence has a high density of segmental duplication, the mouse sequence has a very low density. Notably, these segmental duplications correspond closely to the sites of structural rearrangement, demonstrating a link between duplication and rearrangement. Examination of the main classes of duplicated segments provides insight into the dynamics underlying expansion of chromosome-specific, low-copy repeats in the human genome.

Single cell sequencing reveals low levels of aneuploidy across mammalian tissues

Significance Aneuploidy refers to the gain or loss of individual chromosomes within a cell. Typically, aneuploidy is associated with detrimental consequences at both the cellular and organismal levels. However, reports of high levels of aneuploidy in the brain and liver suggested that aneuploidy might play a positive role in these organs. Here we use single cell sequencing to determine the prevalence of aneuploidy in somatic tissues. We find that aneuploidy is a rare occurrence in the liver and brain and is no more prevalent in these tissues than in skin. Our results demonstrate high karyotypic stability in somatic tissues, arguing against a role for aneuploidy in organ function and reinforcing its adverse effects at the cellular and organismal levels. Whole-chromosome copy number alterations, also known as aneuploidy, are associated with adverse consequences in most cells and organisms. However, high frequencies of aneuploidy have been reported to occur naturally in the mammalian liver and brain, fueling speculation that aneuploidy provides a selective advantage in these organs. To explore this paradox, we used single cell sequencing to obtain a genome-wide, high-resolution assessment of chromosome copy number alterations in mouse and human tissues. We find that aneuploidy occurs much less frequently in the liver and brain than previously reported and is no more prevalent in these tissues than in skin. Our results highlight the rarity of chromosome copy number alterations across mammalian tissues and argue against a positive role for aneuploidy in organ function. Cancer is therefore the only known example, in mammals, of altering karyotype for functional adaptation.

Endothelial α5 and αv integrins cooperate in remodeling of the vasculature during development

Integrin cell adhesion receptors and fibronectin, one of their extracellular matrix ligands, have been demonstrated to be important for angiogenesis using functional perturbation studies and complete knockout mouse models. Here, we report on the roles of the α5 and αv integrins, which are the major endothelial fibronectin receptors, in developmental angiogenesis. We generated an integrin α5-floxed mouse line and ablated α5 integrin in endothelial cells. Unexpectedly, endothelial-specific knockout of integrin α5 has no obvious effect on developmental angiogenesis. We provide evidence for genetic interaction between mutations in integrin α5 and αv and for overlapping functions and compensation between these integrins and perhaps others. Nonetheless, in embryos lacking both α5 and αv integrins in their endothelial cells, initial vasculogenesis and angiogenesis proceed normally, at least up to E11.5, including the formation of apparently normal embryonic vasculature and development of the branchial arches. However, in the absence of endothelial α5 and αv integrins, but not of either alone, there are extensive defects in remodeling of the great vessels and heart resulting in death at ~E14.5. We also found that fibronectin assembly is somewhat affected in integrin α5 knockout endothelial cells and markedly reduced in integrin α5/αv double-knockout endothelial cell lines. Therefore, neither α5 nor αv integrins are required in endothelial cells for initial vasculogenesis and angiogenesis, although they are required for remodeling of the heart and great vessels. These integrins on other cells, and/or other integrins on endothelial cells, might contribute to fibronectin assembly and vascular development.