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.

The common fragile sites are regions of profound genomic instability found in all individuals. The full size of each region of instability ranges from under one megabase (Mb) to greater than 10 Mbs. At least half of the CFS regions have been found to span extremely large genes that spanned from 600 kb to greater than 2.0 Mbs. The large CFS genes are also very interesting from a cancer perspective as several of them, including FHIT and WWOX, have already demonstrated the capacity to function as tumor suppressor genes, both in vitro and in vivo. We estimate that there may be 40–50 large genes localized in CFS regions. The expression of a number of the large CFS genes has been previously shown to be lost in many different cancers and this is frequently associated with a worse clinical outcome for patients. To determine if there was selection for the inactivation of different large CFS genes in different cancers, we examined the expression of 13 of the 20 known large CFS genes: FHIT, WWOX, PARK2, GRID2, NBEA, DLG2, RORA isoforms 1 and 4, DAB1, CNTNAP2, DMD, IL1RAPL1, IMMP2L and LARGE in breast, ovarian, endometrial and brain cancers using real-time RT-PCR analysis. Each cancer had a distinct profile of different large CFS genes that were inactivated. Interestingly, in breast, ovarian and endometrial cancers there were some cancers that had inactivation of expression of none or only one of the tested genes, while in other specimens there was inactivation of multiple tested genes. Brain cancers had inactivation of many of the tested genes, a number of which function in normal neurological development. We find that there is no relationship between the frequency that any specific CFS is expressed and the frequency that the gene from that region is inactivated in different cancers. Instead, it appears that different cancers select for the inactivation of different large CFS genes.

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.

The number of non-coding transcripts (NCTs) exceeds the number of transcripts that code for protein. Their diverse and important functions are now being identified and utilized in the lab as well as in the clinic. Utilizing the Affymetrix Gene Chip Human Tiling 1.0R Array Set, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) alone and cells exposed to the tobacco carcinogen (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNK. We focused our efforts on the identification and function of novel long non-coding transcripts that had increased expression after exposure to NNK. We report on the identification of 12 long NNK-induced non-coding transcripts (NiTs) that are 300 nucleotides and larger in length. We first validated that these transcripts were indeed stress responsive using quantitative real-time RT-PCR (qPCR) and by Northern blot analysis revealed that these transcripts were between two and four kilobases in length. We also show using qPCR that these transcripts are more abundantly expressed in normal rapidly growing tissues or tissues that are under cellular stress. We show that there is altered expression of NiTs in a panel of lung cancer cell lines and many of these transcripts had increased expression in many breast cancer cell lines. We further analyzed one of these transcripts, NiT5 and identified it to be a 2.5 kb antisense, polyadenylated transcript by RLM-RACE and by Northern Blot analysis. Isolating nuclear and cytoplasmic fractions in BT474 cells, we identified Nit5 to be localized in the nucleus and found it to be transcribed by RNA polymerase III. NiT5 also has increased expression in breast, ovarian and cervical cancer-derived cell lines relative to normals from these three sites, but not increased expression in lung cancer cell lines. In summary, our results suggest that these novel long non-coding transcripts play an important role in stress also may play a role in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4083.