Retinoblastoma is a rare pediatric tumor of the retina, caused by the homozygous loss of the Retinoblastoma 1 (RB1) tumor suppressor gene. Previous microarray studies have identified changes in the expression profiles of coding genes; however, our understanding of how non-coding genes change in this tumor is absent. This is an important area of research, as in many adult malignancies, non-coding genes including LNC-RNAs are used as biomarkers to predict outcome and/or relapse. To establish a complete and in-depth RNA profile, of both coding and non-coding genes, in Retinoblastoma tumors, we conducted RNA-seq from a cohort of tumors and normal retina controls. This analysis identified widespread transcriptional changes in the levels of both coding and non-coding genes. Unexpectedly, we also found rare RNA fusion products resulting from genomic alterations, specific to Retinoblastoma tumor samples. We then determined whether these gene expression changes, of both coding and non-coding genes, were also found in a completely independent Retinoblastoma cohort. Using our dataset, we then profiled the potential effects of deregulated LNC-RNAs on the expression of neighboring genes, the entire genome, and on mRNAs that contain a putative area of homology. This analysis showed that most deregulated LNC-RNAs do not act locally to change the transcriptional environment, but potentially function to modulate genes at distant sites. From this analysis, we selected a strongly down-regulated LNC-RNA in Retinoblastoma, DRAIC, and found that restoring DRAIC RNA levels significantly slowed the growth of the Y79 Retinoblastoma cell line. Collectively, our work has generated the first non-coding RNA profile of Retinoblastoma tumors and has found that these tumors show widespread transcriptional deregulation.

The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.

BackgroundFusion proteins have unique oncogenic properties and their identification can be useful either as diagnostic or therapeutic targets. Next generation sequencing data have previously shown a fusion gene formed between Rad51C and ATXN7 genes in the MCF7 breast cancer cell line. However, the existence of this fusion gene in colorectal patient tumor tissues is largely still unknown.MethodsWe evaluated for the presence of Rad51C-ATXN7 fusion gene in colorectal tumors and cells by RT-PCR, PCR, Topo TA cloning, Real time PCR, immunoprecipitation and immunoblotting techniques.ResultsWe identified two forms of fusion mRNAs between Rad51C and ATXN7 in the colorectal tumors, including a Variant 1 (fusion transcript between Rad51C exons 1–7 and ATXN7 exons 6–13), and a Variant 2 (between Rad51C exons 1–6 and ATXN7 exons 6–13). In silico analysis showed that the Variant 1 produces a truncated protein, whereas the Variant 2 was predicted to produce a fusion protein with molecular weight of 110 KDa. Immunoprecipitation and Western blot analysis further showed a 110 KDa protein in colorectal tumors. 5-Azacytidine treatment of LS-174 T cells caused a 3.51-fold increase in expression of the fusion gene (Variant 2) as compared to no treatment controls evaluated by real time PCR.ConclusionIn conclusion we found a fusion gene between DNA repair gene Rad51C and neuro-cerebral ataxia Ataxin-7 gene in colorectal tumors. The in-frame fusion transcript of Variant 2 results in a fusion protein with molecular weight of 110 KDa. In addition, we found that expression of fusion gene is associated with functional impairment of Fanconi Anemia (FA) DNA repair pathway in colorectal tumors. The expression of Rad51C-ATXN7 in tumors warrants further investigation, as it suggests the potential of the fusion gene in treatment and predictive value in colorectal cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0527-1) contains supplementary material, which is available to authorized users.

Introduction: A key response mechanism to DNA damage is the Fanconi Anemia repair pathway (FA), which involves homologous recombination DNA repair and is activated through mono- ubiquitination of FANCD2. FA deficiency is considered to increase the sensitivity of tumors to particular DNA-targeted agents, and may prove to be a target of cancer treatment. We hypothesize that FA deficient tumors have a low growth rate and reduced ability for DNA repair compared to FA functioning tumors. Given that genetic modifications can interfere with FA functionality, we aim to explore the association between the FA pathways and downstream genes that influence tumor growth. To date, few studies have examined gene expression associated with FA deficiency in lung cancer cells. Identification of the FA downstream genes may provide insight on DNA repair networks that impact cancer treatment. Methods: To generate FANCD2 knockdown cells, human lung cancer cell lines A549 and H1299 were transduced with FANCD2-specific short hairpin RNA expressing and puromycin-resistant lentiviral particles or control shRNA lentiviral particles. The cells were cultured in growth medium, and successful FANCD2 knockdown was confirmed by western immunoblot analysis. RNA deep sequencing was completed with Illumina RNA-Seq. We compared gene expression between knockdown FANCD2 and control samples across three cell lines and ranked significant gene expression changes, defined as a five-fold change in upregulation or downregulation. The fold change was calculated by dividing FANCD2 deficient expression by FANCD2 efficient expression. Results and discussion: 13436 genes were evaluated across three cell lines and 17 genes demonstrated gene expression change by at least 5-fold with FANCD2 knockdown in three cell lines. FANCD2 knockdown resulted in 14 downregulated genes and 3 upregulated genes. The downregulated genes RP11-618G20.1, RP5-1021I20.4, RP11-219A15.1, XXbac-BPG32J3.20, and BMS1P17 demonstrated significant expression change across three cell lines. Of the 14 downregulated genes, 13 genes had literature supporting oncogenic function. FA downstream genes confers oncogenic function. As FANCD2 is considered to promote cell proliferation, downregulation of oncogenic genes expression was expected with FANCD2 knockdown. However, the literature suggested that the 3 upregulated genes with FANCD2 knockdown also have oncogenic function. These genes may have functioning beyond the scope of carcinogenesis which may explain gene upregulation with FANCD2 knockdown. Pinpointing genes related to FA pathway deficiency may provide insight into genetic phenomena that drive cancer. Our results provide a starting point for developing targets to specific downstream genes associated with FA deficient tumors, which may prove to limit cancer progression. Further investigation is needed to determine how FANCD2 interacts with these genes to promote cell proliferation. Citation Format: Bianca Nguyen, Li Gao, Abeer Almiman, Shirley Tang, Kathleen Dotts, Miguel A. Villalona-Calero, Wenrui Duan. Investigation of Fanconi Anemia pathway downstream genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2568.

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.