Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis

Cellular proto-oncogenes can be activated by both point mutations and chromosomal translocations, suggesting that there may be a direct link between exposure to agents which damage DNA and genetic change leading to malignancy. Several groups have therefore analysed mutations found in cellular oncogenes of tumours induced by particular physical or chemical carcinogens. Here, we have analysed the molecular changes at different stages of carcinogenesis in mouse skin tumours induced by initiating and promoting agents. Over 90% of tumours, including premalignant papillomas, initiated with dimethylbenzanthracene (DMBA) have a specific A----T transversion at the second nucleotide of codon 61 of the Harvey-ras (Ha-ras) gene. The frequency of this mutation was dependent on the initiating agent used, but not on the promoter, suggesting that the mutation occurs at the time of initiation. The mutation was heterozygous in most papillomas tested, but was homozygous or amplified in some carcinomas. The development of further chromosomal changes at the c-Ha-ras gene locus is therefore a common feature of tumour progression.     

Somatic deletion and duplication of genes on chromosome 11 in Wilms' tumours

One of the most provocative findings in tumour biology is the relationship between chromosomal changes and embryonal cancers in children. For example, children with the rare paediatric syndrome AGR triad (aniridia, genito-urinary abnormalities and mental retardation) often develop Wilms' tumours at a very early age and carry a germ-line deletion on the short arm of chromosome 11 (11p13). It has been suggested that the germ-line deletion 11p is the first of two or more steps to cancer in AGR children. If this were true, one might expect a similar deletion to arise somatically in the far more common isolated Wilms' tumours of children without AGR, as suggested by Knudson from epidemiological data. However, a chromosomal deletion on 11p was observed in only two of five such cases, while it was absent or seen inconsistently in others. We have now used a molecular genetic approach to determine whether Wilms' tumour cells possess somatic alterations at 11p loci. We have found somatic deletions of specific genes in four of six Wilms' tumours. Surprisingly, in all four cases, the deletions were associated with duplications leading to homozygosity of the non-deleted alleles in the tumour cells. As analogous observations were recently reported in retinoblastoma, the genetic events reported here may underlie the development of many such embryonal tumours in children.     

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.     

Patched acts catalytically to suppress the activity of Smoothened

Mutations affecting the transmembrane proteins Patched (Ptc) or Smoothened (Smo) that trigger ligand-independent activity of the Hedgehog (Hh) signalling pathway are associated with human tumours such as basal cell carcinoma (BCC) and medulloblastoma. Despite extensive genetic studies demonstrating the importance of these receptor components in embryonic patterning and cancer, the mechanism by which Ptc regulates Smo is not understood. Here we report that Ptc and Smo are not significantly associated within Hh-responsive cells. Furthermore, we show that free Ptc (unbound by Hh) acts sub-stoichiometrically to suppress Smo activity and thus is critical in specifying the level of pathway activity. Patched is a twelve-transmembrane protein with homology to bacterial proton-driven transmembrane molecular transporters; we demonstrate that the function of Ptc is impaired by alterations of residues that are conserved in and required for function of these bacterial transporters. These results suggest that the Ptc tumour suppressor functions normally as a transmembrane molecular transporter, which acts indirectly to inhibit Smo activity, possibly through changes in distribution or concentration of a small molecule.     

An artefact explains the apparent association of the transferrin receptor with a ras gene product

There is substantial evidence implicating ras genes in a number of human neoplasms. The ras genes of several human tumours display mutational changes which are likely to be responsible for their transforming activity. Normal cells also express ras genes, over-expression of which can induce cellular transformation. ras genes encode proteins of approximately 21,000 molecular weight (MW) (p21) that are localized to the inner surface of the plasma membrane. Much effort is being focused on the elucidation of the physiological function of ras-encoded proteins in normal and transformed cells, concentrating on interactions between p21 and other cellular elements. Recently, Finkel and Cooper reported that p21 in extracts of human bladder carcinoma cells is involved in a molecular complex with the transferrin receptor of these cells. This report aroused considerable interest, particularly as expression of the transferrin receptor has been linked to cell proliferation. I present here evidence that the apparent association of p21 and the transferrin receptor is an artefact of the immunoprecipitation technique.     

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis

Angiogenesis and the development of a vascular network are required for tumour progression, and they involve the release of angiogenic factors, including vascular endothelial growth factor (VEGF-A), from both malignant and stromal cell types. Infiltration by cells of the myeloid lineage is a hallmark of many tumours, and in many cases the macrophages in these infiltrates express VEGF-A. Here we show that the deletion of inflammatory-cell-derived VEGF-A attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumours in mice. Vasculature in tumours lacking myeloid-cell-derived VEGF-A was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF-A decreases the phosphorylation of VEGF receptor 2 (VEGFR2) in tumours, even though overall VEGF-A levels in the tumours are unaffected. However, deletion of myeloid-cell VEGF-A resulted in an accelerated tumour progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumour cell death and decreased tumour hypoxia. Furthermore, loss of myeloid-cell VEGF-A increased the susceptibility of tumours to chemotherapeutic cytotoxicity. This shows that myeloid-derived VEGF-A is essential for the tumorigenic alteration of vasculature and signalling to VEGFR2, and that these changes act to retard, not promote, tumour progression.     

IL-23 promotes tumour incidence and growth

Chronic inflammation has long been associated with increased incidence of malignancy and similarities in the regulatory mechanisms have been suggested for more than a century. Infiltration of innate immune cells, elevated activities of matrix metalloproteases and increased angiogenesis and vasculature density are a few examples of the similarities between chronic and tumour-associated inflammation. Conversely, the elimination of early malignant lesions by immune surveillance, which relies on the cytotoxic activity of tumour-infiltrating T cells or intra-epithelial lymphocytes, is thought to be rate-limiting for the risk to develop cancer. Here we show a molecular connection between the rise in tumour-associated inflammation and a lack of tumour immune surveillance. Expression of the heterodimeric cytokine interleukin (IL)-23, but not of its close relative IL-12, is increased in human tumours. Expression of these cytokines antagonistically regulates local inflammatory responses in the tumour microenvironment and infiltration of intra-epithelial lymphocytes. Whereas IL-12 promotes infiltration of cytotoxic T cells, IL-23 promotes inflammatory responses such as upregulation of the matrix metalloprotease MMP9, and increases angiogenesis but reduces CD8 T-cell infiltration. Genetic deletion or antibody-mediated elimination of IL-23 leads to increased infiltration of cytotoxic T cells into the transformed tissue, rendering a protective effect against chemically induced carcinogenesis. Finally, transplanted tumours are growth-restricted in hosts depleted for IL-23 or in IL-23-receptor-deficient mice. Although many strategies for immune therapy of cancer attempt to stimulate an immune response against solid tumours, infiltration of effector cells into the tumour tissue often appears to be a critical hurdle. We show that IL-23 is an important molecular link between tumour-promoting pro-inflammatory processes and the failure of the adaptive immune surveillance to infiltrate tumours.     

Lack of linkage of familial Wilms' tumour to chromosomal band 11p13

Wilms' tumour (WT), a paediatric renal neoplasm, affect approximately 1 in 10,000 children. One or both kidneys can be affected and 5-10% of tumours are bilateral. Most tumours occur sporadically; however, around 1% of the cases are familial, with siblings or cousins most often being affected. Familial cases are more frequently bilateral, and familial and bilateral tumours are diagnosed at an earlier age. On the basis of these observations, it was proposed that the development of WT requires two mutations. In most sporadic unilateral WT, both are somatic; in familial and bilateral tumours the first is thought to be germinal. Cytogenetic and molecular studies have demonstrated germinal mutations in WT/aniridia patients and somatic mutations in sporadic WT at chromosomal band 11p13. To investigate whether familial predisposition to WT is due to a germinal 11p13 mutation, we studied a WT family with seen DNA markers that span the 11p13 region. We found that familial WT predisposition was not genetically linked to any of the 11p13 markers. This suggests that the gene involved in familial WT predisposition is outside 11p13 and is distinct from the gene involved in tumorigensis and in WT predisposition in WT/aniridia 11p13-deletion patients.     

Loss of genes on the short arm of chromosome 11 in bladder cancer

Recent studies have shown that normal cellular sequences on chromosome 13 are lost during the development of retinoblastomas and that sequences on chromosome 11 are similarly lost during the development of Wilms' kidney tumours and embryonal tumours. Cells from these tumors have been found to contain either the paternal or maternal copies of loci on the affected chromosome, but not both. Thus, the somatic loss of heterozygosity for sequences on chromosome 13 or 11 is hypothesized to result in homozygosity for a recessive mutant allele on these chromosomes, and in this way the chromosomal loss may contribute to the development of these tumours. We sought to investigate whether similar losses of heterozygosity for chromosome 11 sequences occurred in a common adult tumour. We chose to analyse bladder cancers, since such cancers are common in the adult population and are derived from urogenital tissue, as are Wilms' tumours. We examined constitutional and tumour genotypes at loci on the short arm of chromosome 11 (11p) in 12 patients with transitional cell carcinomas. In five tumours, we observed the somatic loss of genes on 11p resulting in homozygosity or hemizygosity of the non-deleted alleles in the tumour cells. Our results show that the frequency of loss of 11p sequences in bladder cancer approaches that seen in Wilms' tumour (42% compared with 55%), and suggest that recessive genetic changes involving sequences on 11p may contribute to the development of bladder neoplasms.     

Imaging in the era of molecular oncology

New technologies for imaging molecules, particularly optical technologies, are increasingly being used to understand the complexity, diversity and in vivo behaviour of cancers. 'Omic' approaches are providing comprehensive 'snapshots' of biological indicators, or biomarkers, of cancer, but imaging can take this information a step further, showing the activity of these markers in vivo and how their location changes over time. Advances in experimental and clinical imaging are likely to improve how cancer is understood at a systems level and, ultimately, should enable doctors not only to locate tumours but also to assess the activity of the biological processes within these tumours and to provide 'on the spot' treatment.     

Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes

Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.     

Cancer-related inflammation

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumours. In some types of cancer, inflammatory conditions are present before a malignant change occurs. Conversely, in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumours. Regardless of its origin, 'smouldering' inflammation in the tumour microenvironment has many tumour-promoting effects. It aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to hormones and chemotherapeutic agents. The molecular pathways of this cancer-related inflammation are now being unravelled, resulting in the identification of new target molecules that could lead to improved diagnosis and treatment.     

The landscape of cancer genes and mutational processes in breast cancer

All cancers carry somatic mutations in their genomes. A subset, known as driver mutations, confer clonal selective advantage on cancer cells and are causally implicated in oncogenesis, and the remainder are passenger mutations. The driver mutations and mutational processes operative in breast cancer have not yet been comprehensively explored. Here we examine the genomes of 100 tumours for somatic copy number changes and mutations in the coding exons of protein-coding genes. The number of somatic mutations varied markedly between individual tumours. We found strong correlations between mutation number, age at which cancer was diagnosed and cancer histological grade, and observed multiple mutational signatures, including one present in about ten per cent of tumours characterized by numerous mutations of cytosine at TpC dinucleotides. Driver mutations were identified in several new cancer genes including AKT2, ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1 and TBX3. Among the 100 tumours, we found driver mutations in at least 40 cancer genes and 73 different combinations of mutated cancer genes. The results highlight the substantial genetic diversity underlying this common disease.     

Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy

The molecular complexity of tissues and the inaccessibility of most cells within a tissue limit the discovery of key targets for tissue-specific delivery of therapeutic and imaging agents in vivo. Here, we describe a hypothesis-driven, systems biology approach to identifying a small subset of proteins induced at the tissue-blood interface that are inherently accessible to antibodies injected intravenously. We use subcellular fractionation, subtractive proteomics and bioinformatics to identify endothelial cell surface proteins exhibiting restricted tissue distribution and apparent tissue modulation. Expression profiling and gamma-scintigraphic imaging with antibodies establishes two of these proteins, aminopeptidase-P and annexin A1, as selective in vivo targets for antibodies in lungs and solid tumours, respectively. Radio-immunotherapy to annexin A1 destroys tumours and increases animal survival. This analytical strategy can map tissue- and disease-specific expression of endothelial cell surface proteins to uncover novel accessible targets useful for imaging and therapy.     

Loss of heterozygosity in three embryonal tumours suggests a common pathogenetic mechanism

Children with the Beckwith-Wiedemann syndrome have a greatly increased potential for the specific development of the embryonal tumours hepatoblastoma, rhabdomyosarcoma and Wilms' tumour. Data obtained with molecular probes suggest that the association between these disparate, rare tumour types reflects a common pathogenetic mechanism that entails the somatic development of homozygosity for a mutant allele at a locus on human chromosome 11.