Mutations truncating the EP300 acetylase in human cancers.

The EP300 protein is a histone acetyltransferase that regulates transcription via chromatin remodelling and is important in the processes of cell proliferation and differentiation. EP300 acetylation of TP53 in response to DNA damage regulates its DNA-binding and transcription functions. A role for EP300 in cancer has been implied by the fact that it is targeted by viral oncoproteins, it is fused to MLL in Leukaemia and two missense sequence alterations in EP300 were identified in epithelial malignancies. Nevertheless, direct demonstration of the role of EP300 in tumorigenesis by inactivating mutations in human cancers has been lacking. Here we describe EP300 mutations, which predict a truncated protein, in 6(3%) of 193 epithelial cancers analysed. Of these six mutations, two were in primary tumours (a colorectal cancer and a breast cancer) and four were in cancer cell lines (colorectal, breast and pancreatic). In addition, we identified a somatic in-frame insertion in a primary breast cancer and missense alterations in a primary colorectal cancer and two cell lines (breast and pancreatic). Inactivation of the second allele was demonstrated in five of six cases with truncating mutations and in two other cases. Our data show that EP300 is mutated in epithelial cancers and provide the first evidence that it behaves as a classical tumour-suppressor gene.

Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours.

Intragenic deletions of TSG101, the human homolog of a mouse gene (tsg101) that acts to suppress malignant cell growth, were reported in human breast tumours. We screened TSG101 for somatic mutations in DNA and RNA samples isolated from a variety of common human malignancies, EBV-immortalised B-cells, and normal lung parenchyma. Intragenic TSG101 deletions in RNA transcripts were frequently found in all types of samples. Analysis of DNA failed to show genomic rearrangements corresponding to transcripts containing deletions in the same samples. The breakpoints of most transcript deletions coincide with genuine or cryptic splice site sequences, suggesting that they result from alternative or aberrant splicing. A similar spectrum of transcript deletions has previously been described in the putative tumour suppressor gene FHIT. We analysed FHIT in the same series of RNA samples and detected truncated FHIT transcripts frequently in both tumour and normal tissues. In addition, transcripts from TSG101, FHIT and seven other genes were analysed in RNA isolated from normal peripheral blood lymphocytes. Large TSG101 and FHIT intragenic transcript deletions were detected and these appeared to be the predominant transcript in 'aged' lymphocytes. Similar alterations were not detected in transcripts of the other genes which were analysed. Our findings demonstrate that truncated TSG101 and FHIT transcripts are commonly detected in both normal and malignant tissues and that a significant fraction of these are likely to be the result of aberrant splicing. While we cannot exclude that alterations in TSG101 and FHIT occur during cancer development, our data indicate that in this context the commonly observed transcript abnormalities are misleading.

MLL2, the second human homolog of the Drosophila trithorax gene, maps to 19q13.1 and is amplified in solid tumor cell lines.

The Mixed Lineage Leukemia (MLL) gene is commonly involved in translocations in infantile leukemia and is amplified in some cases of adult myeloid leukemia. A homolog of MLL denoted MLL2, which represents the second human homolog of the Drosophila trithorax gene, was characterized by assembling ESTs, the KIAA0304 cDNA clone, RT - PCR fragments and a new clone isolated from a cDNA phage library and compared to the available genomic sequence. The MLL2 gene maps to 19q13.1, a region of frequent rearrangement or amplification in solid tumors. MLL2 consists of an 8.5 - 9 kb transcript and spans 20 kb of genomic DNA. The predicted MLL2 protein possesses all of the major domains defined in MLL and the two genes have a similar genomic structure. We find that MLL2 is amplified in two of 14 pancreatic carcinoma cell lines and one of five glioblastoma cell lines and is a likely critical gene in 19q13.1 amplifications. It is also a candidate for chromosomal rearrangements involving this chromosome locus. MLL2 is one additional mammalian trithorax-group gene with involvement in human cancer.

Expression of HGF/SF, HGF1/MSP, and c-met suggests new functions during early chick development.

We report the cloning of fulllength cDNAs for a plasminogen-related growth factor, hepatocyte growth factor/scatter factor (HGF/SF), its tyrosine kinase receptor, c-met, and a close member of the same family, hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), from the chick. We have used these cDNAs to provide the first report of the expression of this family of growth factors and the c-met receptor at early stages of vertebrate development. RNAase protection and wholemount in situ hybridization were used on chick embryos between formation of the primitive streak and early organogenesis. We find patterns of expression for HGF/SF and its receptor c-met consistent with their known roles in epithelial-mesenchymal transformation and angiogenesis. In addition, these genes and HGF1/MSP are expressed in discrete locations within developing somites, suggesting a role in paraxial mesodermal development. Very strong and early expression of HGF/SF in the elevating limb buds suggests its involvement in limb outgrowth. HGF1/MSP is expressed in the notochord and then in the prospective floor plate region and could play a role in development of the neural tube. Interestingly, c-met is often more closely associated with HGF1/MSP than with its known ligand, HGF/SF, raising the possibility that c-met expression may be induced by HGF1/MSP.

Mutation analysis of CBP and PCAF reveals rare inactivating mutations in cancer cell lines but not in primary tumours.

In this study we screened the histone acetyltransferases CBP and PCAF for mutations in human epithelial cancer cell lines and primary tumours. We identified two CBP truncations (both in cell lines), seven PCAF missense variants and four CBP intronic microdeletions. These data suggest that neither gene is commonly inactivated in human epithelial cancers.