APC mutations occur early during colorectal tumorigenesis.

Human tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumour suppressor genes. But in no common adult cancer have the mutations that are critical in the early stages of the tumorigenic process been defined. We have attempted to determine if mutations of the APC gene play such a role in human colorectal tumours, which evolve from small benign tumours (adenomas) to larger malignant tumours (carcinomas) over the course of several decades. Here we report that sequence analysis of 41 colorectal tumours revealed that the majority of colorectal carcinomas (60%) and adenomas (63%) contained a mutated APC gene. Furthermore, the APC gene met two criteria of importance for tumour initiation. First, mutations of this gene were found in the earliest tumours that could be analysed, including adenomas as small as 0.5 cm in diameter. Second, the frequency of such mutations remained constant as tumours progressed from benign to malignant stages. These data provide strong evidence that mutations of the APC gene play a major role in the early development of colorectal neoplasms.     

Melanoma genome sequencing reveals frequent PREX2 mutations

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5-55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)--a PTEN-interacting protein and negative regulator of PTEN in breast cancer--as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.     

Somatic mutations affect key pathways in lung adenocarcinoma

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers--including NF1, APC, RB1 and ATM--and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.     

Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene

The FBXW7/hCDC4 gene encodes a ubiquitin ligase implicated in the control of chromosome stability. Here we identify the mouse Fbxw7 gene as a p53-dependent tumour suppressor gene by using a mammalian genetic screen for p53-dependent genes involved in tumorigenesis. Radiation-induced lymphomas from p53+/- mice, but not those from p53-/- mice, show frequent loss of heterozygosity and a 10% mutation rate of the Fbxw7 gene. Fbxw7+/- mice have greater susceptibility to radiation-induced tumorigenesis, but most tumours retain and express the wild-type allele, indicating that Fbxw7 is a haploinsufficient tumour suppressor gene. Loss of Fbxw7 alters the spectrum of tumours that develop in p53 deficient mice to include a range of tumours in epithelial tissues such as the lung, liver and ovary. Mouse embryo fibroblasts from Fbxw7-deficient mice, or wild-type mouse cells expressing Fbxw7 small interfering RNA, have higher levels of Aurora-A kinase, c-Jun and Notch4, but not of cyclin E. We propose that p53-dependent loss of Fbxw7 leads to genetic instability by mechanisms that might involve the activation of Aurora-A, providing a rationale for the early occurrence of these mutations in human cancers.     

Lung cancer: intragenic ERBB2 kinase mutations in tumours

The protein-kinase family is the most frequently mutated gene family found in human cancer and faulty kinase enzymes are being investigated as promising targets for the design of antitumour therapies. We have sequenced the gene encoding the transmembrane protein tyrosine kinase ERBB2 (also known as HER2 or Neu) from 120 primary lung tumours and identified 4% that have mutations within the kinase domain; in the adenocarcinoma subtype of lung cancer, 10% of cases had mutations. ERBB2 inhibitors, which have so far proved to be ineffective in treating lung cancer, should now be clinically re-evaluated in the specific subset of patients with lung cancer whose tumours carry ERBB2 mutations.     

Expression of N-myc in teratocarcinoma stem cells and mouse embryos

The N-myc gene, which is distantly related to the proto-oncogene c-myc, was first detected as an amplified sequence in human neuroblastoma cell lines and tumours. It has since been revealed that there is up to a 300-fold amplification of N-myc DNA in almost 50% of advanced metastatic human neuroblastomas, whereas amplification is not detected in less advanced tumours that have a better prognosis (ref.3 and M.S., unpublished data). Although expression of N-myc is detectable in all neuroblastoma cell lines and tumours examined, its level is greatly enhanced when the N-myc gene is amplified. Recently, it has been shown that on co-transfection with the c-Ha-ras (EJ) gene, N-myc can induce the malignant transformation of rat embryo fibroblasts. Taken together, these data imply a function for N-myc in the development and/or progression of human neuroblastomas. Surveys indicate that N-myc also may be amplified and/or expressed in two other types of human tumours and cell lines derived from them: retinoblastomas and small cell lung cancers. Here, we report that N-myc is expressed at high levels in mouse and human teratocarcinoma stem cells, thus identifying another tumour cell type that expresses the N-myc gene. In addition, we found that N-myc is abundantly expressed in mouse embryos at mid-gestation and that its expression appears to decrease as the embryo approaches term. In the adult mouse, N-myc is expressed at an approximately fivefold lower level in the brain than in teratocarcinoma stem cells and embryos, and at even lower levels in the adult testis and kidney. Our data represent the first demonstration of expression of the N-myc gene in normal cells, and suggest that N-myc may be involved in mammalian embryogenesis.     

The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.     

Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice.

CDKN2A (INK4a/ARF) is frequently disrupted in various types of human cancer, and germline mutations of this locus can confer susceptibility to melanoma and other tumours. However, because CDKN2A encodes two distinct cell cycle inhibitory proteins, p16INK4a and p14ARF (p19Arf in mice), the mechanism of tumour suppression by CDKN2A has remained controversial. Genetic disruption of Cdkn2a(p19Arf) (hereafter Arf) alone predisposes mice to tumorigenesis, demonstrating that Arf is a tumour-suppressor gene in mice. We mutated mice specifically in Cdkn2a(p16Ink4a) (hereafter Ink4a). Here we demonstrate that these mice, designated Ink4a*/*, do not show a significant predisposition to spontaneous tumour formation within 17 months. Embryo fibroblasts derived from them proliferate normally, are mortal, and are not transformed by oncogenic HRAS. The very mild phenotype of the Ink4a*/* mice implies that the very strong phenotypes of the original Ink4a/ArfDelta2,3 mice were primarily or solely due to loss of Arf. However, Ink4a*/Delta2,3 mice that are deficient for Ink4a and heterozygous for Arf spontaneously develop a wide spectrum of tumours, including melanoma. Treatment of these mice with the carcinogen 7,12-dimethylbenzanthracene (DMBA) results in an increased incidence of melanoma, with frequent metastases. Our results show that, in the mouse, Ink4a is a tumour-suppressor gene that, when lost, can recapitulate the tumour predisposition seen in humans.     

Novel mutations target distinct subgroups of medulloblastoma

Medulloblastoma is a malignant childhood brain tumour comprising four discrete subgroups. Here, to identify mutations that drive medulloblastoma, we sequenced the entire genomes of 37 tumours and matched normal blood. One-hundred and thirty-six genes harbouring somatic mutations in this discovery set were sequenced in an additional 56 medulloblastomas. Recurrent mutations were detected in 41 genes not yet implicated in medulloblastoma; several target distinct components of the epigenetic machinery in different disease subgroups, such as regulators of H3K27 and H3K4 trimethylation in subgroups 3 and 4 (for example, KDM6A and ZMYM3), and CTNNB1-associated chromatin re-modellers in WNT-subgroup tumours (for example, SMARCA4 and CREBBP). Modelling of mutations in mouse lower rhombic lip progenitors that generate WNT-subgroup tumours identified genes that maintain this cell lineage (DDX3X), as well as mutated genes that initiate (CDH1) or cooperate (PIK3CA) in tumorigenesis. These data provide important new insights into the pathogenesis of medulloblastoma subgroups and highlight targets for therapeutic development.     

LKB1 modulates lung cancer differentiation and metastasis

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz-Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.     

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.     

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.     

The clonal and mutational evolution spectrum of primary triple-negative breast cancers

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.     

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.