The 8q24 Gene Desert: An Oasis of Non-Coding Transcriptional Activity

Understanding the functional effects of the wide-range of aberrant genetic characteristics associated with the human chromosome 8q24 region in cancer remains daunting due to the complexity of the locus. The most logical target for study remains the MYC proto-oncogene, a prominent resident of 8q24 that was first identified more than a quarter of a century ago. However, many of the amplifications, translocation breakpoints, and viral integration sites associated with 8q24 are often found throughout regions surrounding large expanses of the MYC locus that include other transcripts. In addition, chr.8q24 is host to a number of single nucleotide polymorphisms associated with cancer risk. Yet, the lack of a direct correlation between cancer risk alleles and MYC expression has also raised the possibility that MYC is not always the target of these genetic associations. The 8q24 region has been described as a “gene desert” because of the paucity of functionally annotated genes located within this region. Here we review the evidence for the role of other loci within the 8q24 region, most of which are non-coding transcripts, either in concert with MYC or independent of MYC, as possible candidate gene targets in malignancy

Pvt1-encoded microRNAs in oncogenesis

<p>Abstract</p> <p>Background</p> <p>The functional significance of the <it>Pvt1 </it>locus in the oncogenesis of Burkitt's lymphoma and plasmacytomas has remained a puzzle. In these tumors, <it>Pvt1 </it>is the site of reciprocal translocations to immunoglobulin loci. Although the locus encodes a number of alternative transcripts, no protein or regulatory RNA products were found. The recent identification of non-coding microRNAs encoded within the <it>PVT1 </it>region has suggested a regulatory role for this locus.</p> <p>Results</p> <p>The mouse <it>Pvt1 </it>locus encodes several microRNAs. In mouse T cell lymphomas induced by retroviral insertions into the locus, the <it>Pvt1 </it>transcripts, and at least one of their microRNA products, mmu-miR-1204 are overexpressed. Whereas up to seven co-mutations can be found in a single tumor, in over 2,000 tumors none had insertions into both the <it>Myc </it>and <it>Pvt1 </it>loci.</p> <p>Conclusion</p> <p>Judging from the large number of integrations into the <it>Pvt1 </it>locus – more than in the nearby <it>Myc </it>locus – <it>Pvt1 </it>and the microRNAs encoded by it are as important as <it>Myc </it>in T lymphomagenesis, and, presumably, in T cell activation. An analysis of the co-mutations in the lymphomas likely place <it>Pvt1 </it>and <it>Myc </it>into the same pathway.</p

Systems-wide RNAi analysis of CASP8AP2/FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>Colorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.</p> <p>Results</p> <p>A small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently <it>CASP8AP2</it>/<it>FLASH</it>. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of <it>CASP8AP2</it>/<it>FLASH </it>resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of <it>CASP8AP2</it>/<it>FLASH </it>also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of <it>CASP8AP2</it>/<it>FLASH </it>silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.</p> <p>Conclusions</p> <p>We have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene <it>NEFH</it>.</p

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (∼800 siRNAs, ∼400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive

Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

BACKGROUND: North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA) accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. METHODS: We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop) from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. RESULTS: Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55%) tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1) and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. CONCLUSIONS: Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies