Identification of long stress-induced non-coding transcripts that have altered expression in cancer.

It has recently become clear that the transcriptional output of the human genome is far more abundant than previously anticipated, with the vast majority of transcripts not coding for protein. Utilizing whole-genome tiling arrays, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) and NHBE cells exposed to the tobacco carcinogen NNK. Our efforts focused on the characterization of non-coding transcripts that were greater than 300 nucleotides in length and whose expression was increased in response to NNK. We identified 12 Long Stress-Induced Non-coding Transcripts that we term LSINCTs. Northern blot analysis revealed that these transcripts were larger than predicted from the tiling array data. Quantitative real-time RT-PCR performed across a panel of normal cell lines indicates that these transcripts are more abundantly expressed in rapidly growing tissues or in tissues that are more prone to cellular stress. These transcripts that have increased expression after exposure to NNK also had increased expression in a number of lung cancer cell lines and also in many breast cancer cell lines. Collectively, our results identified a new class of long stress responsive non-coding transcripts, LSINCTs, which have increased expression in response to DNA damage induced by NNK. LSINCTs interestingly also have increased expression in a number of cancer-derived cell lines, indicating that the expression is increased in both, correlating cellular stress and cancer.

Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

Long, abundantly expressed non-coding transcripts are altered in cancer.

Recent studies with tiling arrays have revealed more genomic transcription than previously anticipated. Whole new groups of non-coding transcripts (NCTs) have been detected. Some of these NCTs, including miRNAs, can regulate gene expression. To date, most known NCTs studied have been relatively short, but several important regulatory NCTs, including XIST, MALAT-1, BC1 and BC200, are considerably larger in length and represent a novel class of long, non-coding RNA species. Whole-genome tiling arrays were utilized to identify novel long NCTs across the entire human genome. Our results have identified a new group of long (>400 nt), abundantly expressed NCTs and have found that a subset of these are also highly evolutionarily conserved. In this report, we have begun to characterize 15 long, conserved NCTs. Quantitative real-time RT-PCR was used to analyze their expression in different normal human tissue and also in breast and ovarian cancers. We found altered expression of many of these NCTs in both cancer types. In addition, several of these NCTs have consistent mutations when sequences of normal samples were compared with a panel of cancer-derived cell lines. One NCT was found to be consistently mutated in a panel of endometrial cancers compared with matched normal blood. These NCTs were among the most abundantly expressed transcripts detected. There are probably many long, conserved NCTs, albeit with lower levels of expression. Although the function of these NCTs is currently unknown, our study indicates that they may play an important function in both normal cells and in cancer development.

Disabled-1 is a large common fragile site gene, inactivated in multiple cancers.

Common fragile sites (CFS) are large, genomically unstable regions, which are hot-spots for deletions and other alterations, especially in cancer cells. Several have been shown to contain genes that span large genomic regions, such as FHIT (1.5 Mb), WWOX (1.0 Mb), GRID2 (1.36 Mb), PARK2 (1.3 Mb), and RORA (730 kb). These genes are frequently inactivated in multiple different cancers, and FHIT and WWOX are shown to function as tumor suppressors. The disabled-1 gene (DAB1) is one of the human homologs of the Drosophila disabled locus, which in mammals is involved in neuronal migration and lamination in the developing cerebral cortex. Mice DAB1 inactivation results in the neurological mutant Scrambler, having similarities to mice with the inactivation of PARK2 (Quaker), GRID2 (Lurcher), and RORA (Staggerer). We were interested in whether DAB1 was another large CFS gene that could have cancer development importance. We demonstrated here that the human DAB1 gene (spanning 1.25 Mb) mapped within FRA1B CFS region on chromosomal band 1p32.2. Real-time RT-PCR analysis revealed that the expression level of DAB1 was decreased in many human cancer samples, including primary tumor tissues and cancer-derived cell lines, from several different cancers, especially in brain and endometrial cancer. Additionally, the introduction of an over-expression DAB1 plasmid into two different cell lines, having insignificant endogenous DAB1 expression, resulted in decreased cell growth. In summary, DAB1 is another gene that resides within an unstable CFS region and might play a role in human tumorigenesis. These data may provide further linkage between neurological development and cancer.

Gene expression changes associated with altered growth and differentiation in benzo[a]pyrene or arsenic exposed normal human epidermal keratinocytes.

Both arsenic and benzo[a]pyrene (BaP) inhibit terminal differentiation and alter growth potential in normal human epidermal keratinocytes (NHEK) in vitro. To identify molecular alterations that may be involved in these cellular processes, microarray analysis was carried out on NHEK treated with BaP or arsenic. The gene expression microarray results measuring mRNA levels were as follows: (1) in total, the expression of 85 genes was induced and 17 genes was suppressed by 2.0 microm BaP. (2) Arsenic at an equitoxic dose (5.0 microm) induced the expression of 106 and suppressed 15 genes. Quantitative real-time RT-PCR was used subsequently to confirm microarray findings on selected genes involved in keratinocyte growth and differentiation pathways. These studies confirmed increased mRNA levels in NHEK by BaP of alpha-integrin binding protein 63 (AIBP63) (2.48-fold), retinoic acid- and interferon-inducible protein (IFIT5) (2.74-fold), interleukin-1 alpha (IL1A) (2.64-fold), interleukin-1 beta (IL1B) (2.84-fold) and Ras guanyl releasing protein 1 (RASGRP1) (3.14-fold). Real-time RT-PCR confirmed that arsenic increased mRNA levels of the following genes: retinoblastoma 1 (RB1) (5.4-fold), retinoblastoma-binding protein 1 (ARID4A) (6.8-fold), transforming growth factor beta-stimulated protein (TSC22D1) (6.84-fold), MAX binding protein (MNT) (2.44-fold), and RAD50 (4.24-fold). Collectively, these results indicate that these chemicals target different genes and molecular pathways involved in the regulatory processes controlling NHEK proliferation and differentiation. Mechanistic studies with a subset of genes may allow the correlation of alterations in these molecular markers with chemical-specific blocks to differentiation in NHEK.

Large common fragile site genes and cancer.

The common fragile sites are large regions of genomic instability that are found in all individuals and are hot spots for chromosomal rearrangements and deletions. A number of the common fragile sites have been found to span genes that are encoded by very large genomic regions. Two of these genes, FHIT and WWOX, have already been demonstrated to function as tumor suppressors. In this review we will discuss the large common fragile site genes that have been identified to date, and the role that these genes appear to play both in cellular responses to stress and in the development of cancer.