cis-splicing of adjacent genes in prostate cancer

描述（由申请人提供）：基因融合被认为是瘤形成的标志。基因融合的研究为许多肿瘤的诊断和治疗奠定了理论基础。最近，我们和其他人报道了一种嵌合融合RNA，涉及两个相邻的基因，SLC 45 A3和ELK 4，在前列腺癌。嵌合RNA表达水平与前列腺癌进展相关。此外，沉默嵌合体导致较慢的 在雄激素依赖性和去势抵抗性前列腺癌细胞中的增殖和更高水平的CDKN 1A表达。有趣的是，我们发现SLC 45 A3-ELK 4是通过相邻基因的顺式剪接（cis-SAGe）/通读机制而不是染色体重排产生的。尽管SLC 45 A3-ELK 4具有生物学和临床意义，但顺式SAGe的机制尚未阐明，也没有鉴定出顺式SAGe的其他实例。在我们的初步研究中，我们假设，并积累了多条证据，锌指蛋白CTCF的绝缘子网站之间的两个相邻的基因之间的结合在SLC 45 A3和ELK 4之间的顺式SAGe的产生中起着重要的作用。我们还假设，更多的顺式SAGe事件可以通过操纵CTCF水平，与前列腺癌生物学和肿瘤生物学的影响，一般可以确定。我们建议从三个方面为这些假设提供更多的证据。在本研究中，我们将以SLC 45 A3-ELK 4为模型，研究顺式SAGe的作用机制。我们推断顺式SAGe基本上是通过基因边界的连续转录物的选择性剪接。我们假设SLC 45 A3和ELK 4之间发生顺式SAGe事件必须满足三个因素：5'SLC 45 A3基因的激活，CTCF与两个基因之间绝缘子区域的结合减少，以及增强选择性剪接。在Aim 1中，我们将首先在LNCaP前列腺癌细胞系中研究所有三个调节因子，然后在其他细胞系和临床样品中研究;在Aim 2中，我们将操纵CTCF水平并使用配对末端转录组测序来鉴定额外的cis-SAGe融合。我们使用LNCaP细胞的初步研究已经产生了许多用于研究的融合候选者。我们将进一步验证这些候选人作为真正的顺式SAGe事件，并扩展到其他细胞系。在Aim 3中，我们将研究新鉴定的顺式SAGe融合体的生物学和临床意义。这项研究将有助于阐明顺式SAGe的作用机制，并发现新的肿瘤相关嵌合转录物。新的融合基因产物可能是新的疾病生物标志物和/或治疗靶点的潜在候选者。同样的方法可以很容易地适用于其他类型的癌症。

DESCRIPTION (provided by applicant): Gene fusions have been considered a hallmark of neoplasia. The study of gene fusions has founded the theoretical backgrounds for many cancer diagnosis and therapeutics. Recently, we and others reported a chimeric fusion RNA involving two neighboring genes, SLC45A3 and ELK4, in prostate cancer. The chimeric RNA expression level correlates with prostate cancer progression. In addition, silencing the chimera led to slower proliferation and higher levels of CDKN1A expression in both androgen-dependent and castration- resistant prostate cancer cells. Intriguingly, we found that SLC45A3-ELK4 is generated by a mechanism of cis- splicing of adjacent genes (cis-SAGe)/read-through, instead of chromosomal rearrangement. Despite the biological and clinical significance of SLC45A3-ELK4, neither has the mechanism of cis-SAGe been elucidated, nor have other examples of cis-SAGe been identified. In our preliminary studies we hypothesized, and have accumulated multiple lines of evidence, that the binding of the zinc finger protein CTCF to insulator sites in-between the two neighboring genes plays an important role in the generation of cis-SAGe between SLC45A3 and ELK4. We also hypothesize that more cis-SAGe events can be identified through manipulating CTCF level, with implications for both prostate cancer biology and tumor biology in general. We propose to provide more evidence for these hypotheses in three aims. In Aim1, we will investigate the mechanism of cis- SAGe using SLC45A3-ELK4 as a model. We reasoned that the cis-SAGe is essentially alternative splicing of a continuous transcript passing through gene boundaries. We hypothesized that three factors have to be met for the cis-SAGe event to happen between SLC45A3 and ELK4: activation of the 5' SLC45A3 gene, reduced CTCF binding to the insulator regions in-between two genes, and enhanced alternative splicing. In Aim1, we will investigate all three factors of regulation first in LNCaP prostate cancer cell line, then in other cell lines and in clinical samples; In Aim2, we will manipulate CTCF level and use paired-end transcriptome sequencing to identify additional cis-SAGe fusions. Our preliminary study using LNCaP cells already generated many fusion candidates for study. We will further validate these candidates as true cis-SAGe events and expand to other cell lines. In Aim3, we will investigate the biological and clinical significance o the newly identified cis-SAGe fusions. The proposed study will shed light on the mechanism of cis-SAGe and discover novel cancer- associated chimeric transcripts. The novel fusion gene products may be potential candidates for new disease biomarkers and/or therapeutic targets. The same approach can be easily adapted in other cancer types.