The hallmarks of cancer.

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

The emergence of order in natural systems is a constant source of inspiration for both physical and biological sciences. While the spatial order characterizing for example the crystals has been the basis of many advances in contemporary physics, most complex systems in nature do not offer such high degree of order. Many of these systems form complex networks whose nodes are the elements of the system and edges represent the interactions between them. 
Traditionally complex networks have been described by the random graph theory founded in 1959 by Paul Erdohs and Alfred Renyi. One of the defining features of random graphs is that they are statistically homogeneous, and their degree distribution (characterizing the spread in the number of edges starting from a node) is a Poisson distribution. In contrast, recent empirical studies, including the work of our group, indicate that the topology of real networks is much richer than that of random graphs. In particular, the degree distribution of real networks is a power-law, indicating a heterogeneous topology in which the majority of the nodes have a small degree, but there is a significant fraction of highly connected nodes that play an important role in the connectivity of the network. 
The scale-free topology of real networks has very important consequences on their functioning. For example, we have discovered that scale-free networks are extremely resilient to the random disruption of their nodes. On the other hand, the selective removal of the nodes with highest degree induces a rapid breakdown of the network to isolated subparts that cannot communicate with each other. 
The non-trivial scaling of the degree distribution of real networks is also an indication of their assembly and evolution. Indeed, our modeling studies have shown us that there are general principles governing the evolution of networks. Most networks start from a small seed and grow by the addition of new nodes which attach to the nodes already in the system. This process obeys preferential attachment: the new nodes are more likely to connect to nodes with already high degree. We have proposed a simple model based on these two principles wich was able to reproduce the power-law degree distribution of real networks. Perhaps even more importantly, this model paved the way to a new paradigm of network modeling, trying to capture the evolution of networks, not just their static topology.

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Statistical mechanics of complex networks

A genetic model for colorectal tumorigenesis

http://www.maths.qmul.ac.uk/%7Elatora/report_06.pdf

Complex networks: Structure and dynamics

DNA replication in SV40-infected cells: VIII. The distribution of replicating molecules at different stages of replication in SV40-infected cells

TSC1 acts coordinately with TSC2 in a complex to inhibit mTOR, an emerging therapeutic target and known promoter of cell growth and cell cycle progression. Perturbation of the mTOR pathway, through abnormal expression or function of pathway genes, could lead to tumorigenesis. TSC1 and TSC2 expression is reduced in invasive breast cancer as compared with normal mammary epithelium. Because single nucleotide polymorphisms (SNPs) in regulatory genes have been implicated in risk and age at diagnosis of breast cancers, systematic SNP association studies were performed on TSC1 and TSC2 SNPs for their associations with clinical features of breast cancer. TSC1 and TSC2 haplotypes were constructed from genotyping of multiple loci in both genes in healthy volunteers. SNPs were selected for further study using a bioinformatics approach based on SNP associations with drug response in NCI-60 cell lines and evidence of selection bias based on haplotype frequencies. Genotyping for five TSC1 and one TSC2 loci were performed on genomic DNA from 1,137 women with breast cancer. This study found that for TSC1 rs7874234, TT variant carriers had a 9-year later age at diagnosis of estrogen receptor positive (ER+), but not ER−, ductal carcinomas (P = 0.0049). No other SNP locus showed an association with age at diagnosis, nor any other breast cancer phenotype. TSC1 rs7874234 is hypothesized to be functional in ER+ breast cancer because the T allele, but not the C allele, may create an estrogen receptor element (ERE) site, resulting in increased TSC1 transcription and subsequent inhibition of mTOR.

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Polymorphic variants in TSC1 and TSC2 and their association with breast cancer phenotypes.

The gas5gene was identified in a screen for mRNAs induced in NIH3T3 fibroblasts following growth arrest (Schneider et al. 1988). gas5 message levels decline upon serum stimulation of quiescent mouse fibroblasts by what appears to be a posttranscriptional mechanism that may be dependent on protein kinase activity (Ciccarelli et al. 1990). gas5message levels may also be subject to transcriptional regulation that occurs in response to cellular differentiation (Coccia et al. 1992). Abundant levels ofgas5message have been observed in most adult mouse tissues examined, the exceptions being liver and spleen (Coccia et al.1992). Neural tube-specific expression of gas5 message during neural tube closure has been reported to occur in embryos of the SWV/Fnn mouse strain, but not in embryos of the LM/Bc/Fnn mouse strain. SWV/Fnn embryos are also more susceptible to hyperthermia-induced exencephaly than are LM/Bc/ Fnn embryos, which suggests a possible link between gas5 expression and susceptibility to neural tube defects (Vacha et al. 1997). Two predominantgas5messages are produced by alternative splicing, which eliminates exon 7 from one of the forms. There are only short open reading frames (ORFs) within the gas5message, the longest being either 123 or 117 base pairs depending on the splice variant (Coccia et al. 1992). Peptides encoded by these two ORFs, both with apparent molecular weights of about 8 kDa, have been produced in an in vitro translation system (Coccia et al. 1992); however, no evidence corroborating expression of these proteins in cells has been reported. Amplification of gas5cDNA by RT-PCR from the C57Bl/6J and 129Sv+++/J mouse strains resulted in a consistently smaller product from the 129 strain (data not shown). Sequence analysis revealed five fewer base pairs within exon 5 of the 129 derivedgas5cDNA owing to the absence of half of the 5 base pair tandem repeat TAGAATAGAA (Fig. 1A). Primers flanking this polymorphic region were designed to enable the analysis of genomic DNA. With these primers, tail DNA was assayed from an additional 11 inbred mouse strains and from outbred Swiss Webster andMus spretusmice (Fig. 1B). Of the 11 additional inbred strains tested, 5 produced PCR products of a size consistent with the 129 sequence. These strains were A/J, C3H/HeJ, LP/J, LT/SvA, and NZW/LacJ. The other 6 inbred strains produced PCR products of a size consistent with the C57Bl/6 sequence. These strains were AKR/J, BALB/cJ, DBA/2J, FVB/NJ, NOD/LtJ, and NON/ LtJ. The size of the PCR products from the outbred Swiss and spretusmice were both consistent with the C57Bl/6 sequence. Since the intact tandem repeat was found in the species Mus spretus, this seems more likely to represent the wild-type sequence. However, the high proportion of inbred mouse strains harboring the 5-bp deletion suggests either that this sequence has been remarkably unstable within the inbred strains or that this polymorphism was present within and inherited from the founder population. Three markers, D12Mit46, D12Mit64,and D12Mit54, which should flank thegas5gene based upon its previously reported map position on mouse Chromosome (Chr) 12 (Dietrich et al. 1996), failed to show evidence of linkage on a panel of 42 C57B16×129 Correspondence to: A.J. Levine Fig. 1. (A) Nucleotide sequence of the 5-bp polymorphism identified within exon 5 of thegas5 gene. Capital letters indicate the nucleotide sequence of exon 5 of the gas5 gene, while lower-case letters indicate flanking intron/exon boundary sequences (Coccia et al. 1992). Asterisks (*) denote the 5-bp that are absent in the 129 strain. The top and bottom arrows del ineate the sequences of the forward primer 5 8TAACTATTTGTTTGTTGTAGGTGC-38 and of the reverse primer 5 8TGACTTTACCATTTCATTTTCTGG-38 used for PCR amplification from genomic DNA.(B) Genomic PCR analysis of exon 5 of the gas5gene from 13 different inbred mouse strains 129Sv+ ++/J, C57Bl/6J, A/J, AKR/J, BALB/cJ, C3H/HeJ, DBA/2J, FVB/NJ, LP/J, LT/Sv-A, NOD/ LtJ, NON/LtJ, NZW/LacJ and from outbred Swiss Webster and Mus spretus mice. The PCR products of either 55 or 60 bp were resolved on 15% acrylamide gels and detected by ethidium bromide staining. Mammalian Genome 9, 773–774 (1998).

The gas5 gene is disrupted by a frameshift mutation within its longest open reading frame in several inbred mouse strains and maps to murine chromosome 1.

Fifty years ago, when the authors of the reviews published here were in various stages of training for a future in medical research, the causes of cancers in humans were, at best, enigmatic. We knew that some viruses could cause cancers, some chemicals could cause cancers, and genetic predispositions could be observed for some cancers. But just what the relationships were among viruses, chemicals, and genes were matters of heated debate. The decade of the 1970s uncovered cellular oncogenes captured in viruses, the role of chemical mutagens in converting proto-oncogenes to oncogenes, and the discoveries of tumor suppressor genes (retinoblastoma, p53). As it became clear that genetic mutations, and even a few viruses, could cause cancers in animals and humans, defects in DNA repair processes were shown to contribute to a higher mutation rate and inherited and spontaneous cancers in humans. These hard won facts became the foundations of oncology. What has been surprising is howmany different genes in the human genome, altered by mutations, contribute to the formation of cancers over a lifetime. Different cancers have very heterogeneous genetic defects, even when they are of the same tissue and tumor types. But, not all oncogenes, tumor suppressor genes, or DNA repair defects, are created or function equally, nor do they contribute equally to the origins and propagation of cancers. All of the genomic studies searching for common features in thousands of different cancers conclude that about 50% of all cancers have Tp53 mutations and exclude all copies of the wild-type Tp53 gene. In addition, a variable number of cancers inactivate p53 functions by amplifying or overexpressing the negative regulators of p53 (MDM-2, WIP-1). Rather clearly, the most common feature of cancers in humans is the loss of some functions of the wild-type Tp53 gene. That is the justification for, and the importance of, the reviews presented here. But, more important, the nine reviews presented here do not look back over the 38 years of research about the p53 protein and the Tp53 gene. Instead, these reviews address unanswered questions, providing a background, and elucidating the paths to answers that will come over the next few years. This collection of manuscripts reviews the future. Although Tp53 mutations are the most common feature of human cancers, the nature of the Tp53 mutations (mostly missense mutations) and the mutational alleles observed in the p53 gene of many cancer types, are not uniform. Rather, there are hot spot missense mutations, where mutations at 10 codons (out of 190 different mutations observed in the p53 DNA-binding domain) account for 33% of all the p53mutations found in cancers (reviewed in Baugh et al.). One reason for this is the presence of methylated CpG dinucleotides in the Tp53 gene at 7 of these 10 hot spot mutations where methylated CpG dinucleotides have a 10-fold higher mutation frequency than unmethylated CpGs. Indeed, C to T transitions that result from the presence of methylated CpG dinucleotides are the most common point mutations observed in all human cancers at many different genetic loci. In addition, these seven Tp53 codons encode arginines that are conserved over large evolutionary time spans in the Tp53 genes, and so changes at these sites might be expected to contribute to a loss of function. There is some evidence that these alleles are selected for in tumors. One reason why only some mutant alleles are selected for in a tumor is the ‘gain of function(s)’ that some missense mutant alleles confer upon tumors (reviewed in Kim et al.).What are themechanisms that mediate a gain of a function in the p53 mutant protein, and do different mutant alleles produce proteins with different mechanisms for this gain of function? Do diverse tumor tissue types influence the properties of a gain of function? Do the advantages of a gain of function change with the stage of a tumor, the treatment protocols employed, or the other mutations in cancer cells? These questions need to be answered if we are to understand the biology of the p53 gene and its protein. The wild-type p53 protein is a transcription factor that selectively regulates the transcriptional activation of4200 genes in diverse cells (reviewed in Engeland and Sullivan et al.). Activation of p53-mediated transcription by various stress signals results in the expression of a number of pathways that accomplish diverse phenotypes that include cell cycle arrest, senescence, apoptosis and other types of cell death, metabolic alterations, DNA repair processes, and other prosurvival mechanisms and tumor suppression (reviewed in refs. 3–7). There is an active debate about which p53-regulated genes and pathways are responsible for the tumor suppressor phenotype. The answer to this question may reside in a better understanding of how stress activates the p53 protein and how a particular stress initiates a transcriptional pathway resulting in an improved fidelity in the process of cellular replication. P53-regulated gene products interact with many other signal transduction pathways involved in metabolism and growth (PI3K-mTOR pathways), stem cells (the Wnt

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Reviewing the future of the P53 field

A 2-day meeting to explore why we do not yet have a human immunodeficiency virus (HIV) vaccine was held under the joint sponsorship of the Simons Center for Systems Biology at the Institute for Advanced Study and the International AIDS Vaccine Initiative, with additional support from Merck & Co., at

/pdf/why-do-we-not-yet-have-a-human-immunodeficiency-virus-5eqeaj6f1m.pdf

Arnold J. Levine

Papers

DNA replication in SV40-infected cells: VIII. The distribution of replicating molecules at different stages of replication in SV40-infected cells

Polymorphic variants in TSC1 and TSC2 and their association with breast cancer phenotypes.

The gas5 gene is disrupted by a frameshift mutation within its longest open reading frame in several inbred mouse strains and maps to murine chromosome 1.

Reviewing the future of the P53 field

Why do we not yet have a human immunodeficiency virus vaccine