No abstract

Genes or their encoded products are not expected to mingle with each other unless in some disease situations. In cancer, a frequent mechanism that can produce gene fusions is chromosomal rearrangement. However, recent discoveries of RNA trans-splicing and cis-splicing between adjacent genes (cis-SAGe) support for other mechanisms in generating fusion RNAs. In our transcriptome analyses of 28 prostate normal and cancer samples, 30% fusion RNAs on average are the transcripts that contain exons belonging to same-strand neighboring genes. These fusion RNAs may be the products of cis-SAGe, which was previously thought to be rare. To validate this finding and to better understand the phenomenon, we used LNCaP, a prostate cell line as a model, and identified 16 additional cis-SAGe events by silencing transcription factor CTCF and paired-end RNA sequencing. About half of the fusions are expressed at a significant level compared to their parental genes. Silencing one of the in-frame fusions resulted in reduced cell motility. Most out-of-frame fusions are likely to function as non-coding RNAs. The majority of the 16 fusions are also detected in other prostate cell lines, as well as in the 14 clinical prostate normal and cancer pairs. By studying the features associated with these fusions, we developed a set of rules: 1) the parental genes are same-strand-neighboring genes; 2) the distance between the genes is within 30kb; 3) the 5′ genes are actively transcribing; and 4) the chimeras tend to have the second-to-last exon in the 5′ genes joined to the second exon in the 3′ genes. We then randomly selected 20 neighboring genes in the genome, and detected four fusion events using these rules in prostate cancer and non-cancerous cells. These results suggest that splicing between neighboring gene transcripts is a rather frequent phenomenon, and it is not a feature unique to cancer cells.

Neighboring genes transcribing in the same direction can form chimeric RNAs via cis-splicing (cis-SAGe). Previously, we reported 16 novel cis-SAGe chimeras in prostate cancer cell lines, and performed in silico validation on 14 pairs of normal and tumor samples from Chinese patients. However, whether these fusions exist in different populations, as well as their clinical implications, remains unclear. To investigate, we developed a bioinformatics pipeline using modified Spliced Transcripts Alignment to a Reference (STAR) to quantify these fusion RNAs simultaneously in silico. From RNA-Seq data of 100 paired normal and prostate cancer samples from TCGA, we find that most fusions are not specific to cancer. However, D2HGDH-GAL3ST2 is more frequently seen in cancer samples, and seems to be enriched in the African American group. Further validation with our own collection as well as from commercial sources did not detect this fusion RNA in 29 normal prostate samples, but in 19 of 93 prostate cancer samples. It is more frequently detected in late stage cancer, suggesting a role in cancer progression. Consistently, silencing this fusion resulted in dramatic reduction of cell proliferation rate and cell motility.

The chimeric RNA, SLC45A3-ELK4, was found to be a product of cis-splicing between the two adjacent genes (cis-SAGe). Despite the biological and clinical significance of SLC45A3-ELK4, its generating mechanism has not been elucidated. It was shown in one cell line that the binding of transcription factor CTCF to the insulators located at or near the gene boundaries, inversely correlates with the level of the chimera. To investigate the mechanism of such cis-SAGe events, we sequenced potential regions that may play a role in such transcriptional read-through. We could not detect mutations at the transcription termination site, insulator sites, splicing sites, or within CTCF itself in LNCaP cells, thus suggesting a “soft-wired” mechanism in regulating the cis-SAGe event. To investigate the role CTCF plays in regulating the chimeric RNA expression, we compared the levels of CTCF binding to the insulators in different cell lines, as well as clinical samples. Surprisingly, we did not find an inverse correlation between CTCF level, or its bindings to the insulators and SLC45A3-ELK4 expression among different samples. However, in three prostate cancer cell lines, different environmental factors can cause the expression levels of the chimeric RNA to change, and these changes do inversely correlate with CTCF level, and/or its bindings to the insulators. We thus conclude that CTCF and its bindings to the insulators are not the primary reasons for differential SLC45A3-ELK4 expression in different cell lines, or clinical cases. However, they are the likely mechanism for the same cells to respond to different environmental cues, in order to regulate the expression of SLC45A3-ELK4 chimeric RNA. This response to different environmental cues is not general to other cis-SAGe events, as we only found one out of 16 newly identified chimeric RNAs showing a pattern similar to SLC45A3-ELK4.

Background: Chimeric RNAs, comprising two or more different transcripts from distinct chromosomal regions, are common features in many cancers and play important roles in carcinogenesis. Prostate cancer is the most important non-skin cancer and a leading cause of cancer-related deaths in American men. During the progression of prostate cancer, various fusion transcripts generated as a result of chromosomal rearrangement have been identified. However, we recently reported that a chimeric RNA SLC45A3-ELK4 was generated in the absence of DNA rearrangement by cis-splicing of adjacent genes(Cis-SAGe)/read-through. CCCTC-binding factor (CTCF) acts as chromatin barrier, with CTCF binding at boundaries between the two parental genes, SLA45A3 and ELK4 to prevent the Cis-SAGe events in normal prostate cells. On the other hand, silencing CTCF resulted in an induction of the fusion RNA expression. These findings promoted us to manipulate CTCF level in combination with deep RNA sequencing (RNA-seq) to discover more Cis-SAGe fusions. Methods: To identify more Cis-SAGe chimeric RNAs in prostate cancer, we sequenced LNCaP cells transfected with negative control siRNA and siRNA against CTCF. The samples were processed in two different companies using Illumina Hi-seq platform to generate two output sequences: paired-end 50-nucleotide and 101-nucleotide reads. SOAPfuse (BGI, China) software was used to identify chimeric RNAs. To filter out non Cis-SAGe events, we required all the candidates to have the features: induced by siCTCF; neighboring parental genes; same transcribing direction; CTCF binding sites at gene boundaries. Majority of the fusions were validated with real-time PCR and Sanger sequencing. Results: Using SOAPfuse, we identified 95 chimeric RNAs from the combined 50- and 100-bp paired RNA sequencing data. For all 95 candidates, 56 are intra-chromosomal same strand neighboring gene fusions, 13 inter-chromosomal, and 26 other intra-chromosomal fusions. We considered the 56 (from 48 pairs of unique parental genes) to be candidates for Cis-SAGe. qPCR results showed 25 candidates were up-regulated more than 2 folds by knocking down CTCF and 4 candidates were down-regulated. Sanger sequencing confirmed 22 out of the 25 candidates. Intergenic transcripts for all these 22 candidates were detected, supporting that these chimeric transcripts could be products of Cis-SAGe. To evaluate their differential expression in cancers vs. benign tissues, we compared the RNA expression of these candidates in LNCaP, PC-3, another prostate cancer cell line and RWPE-1, a benign prostate cell line. Most of the verified chimeric RNAs appeared to be highly expressed in all three cell lines. Four chimeric RNAs are expressed only in the cancer cells, one fusion transcript detected only in RWPE-1 and four other fusions only present in LNCaP cells. Together, our results suggest that many more Cis-SAGe chimeric RNAs are expressed in prostate cancer and they may represent a unique class of bio-markers. Citation Format: Fujun Qin, Yansu Song, Henry F. Frierson, Hui Li. Discovering Cis-SAGe chimeric RNAs in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2014-2374