cis-splicing of adjacent genes in prostate cancer

描述(申请人提供)：基因融合被认为是肿瘤形成的标志。基因融合的研究为许多癌症的诊断和治疗奠定了理论基础。最近，我们和其他人报道了前列腺癌中涉及两个相邻基因SLC45A3和ELK4的嵌合融合RNA。嵌合RNA表达水平与前列腺癌的进展相关。此外，让嵌合体沉默会导致速度变慢 雄激素依赖和去势抵抗的前列腺癌细胞的增殖和CDKN1A的高水平表达。有趣的是，我们发现SLC45A3-ELK4是由相邻基因的顺式剪接(cis-sage)/通读机制产生的，而不是染色体重排。尽管SLC45A3-ELK4具有生物学和临床意义，但顺式SAGE的机制尚未阐明，也没有鉴定出其他顺式SAGE的例子。在我们的初步研究中，我们假设并积累了多条证据，即锌指蛋白CTCF与相邻两个基因之间绝缘子位置的结合在SLC45A3和ELK4之间顺式SAGE的产生中起着重要作用。我们还假设，通过控制CTCF水平可以识别更多顺式事件，这对前列腺癌生物学和一般肿瘤生物学都有意义。我们建议从三个方面为这些假说提供更多的证据。在Aim1中，我们将以SLC45A3-ELK4为模型来研究顺式SAGE的机制。我们推断，顺式剪接本质上是连续转录产物通过基因边界的选择性剪接。我们假设SLC45A3和ELK4之间发生顺式SAGE事件必须满足三个因素：5‘SLC45A3基因的激活，减少CTCF与两个基因之间绝缘子区域的结合，以及增强选择性剪接。在AIM1中，我们将首先在LNCaP前列腺癌细胞系中研究所有三个调控因素，然后在其他细胞系和临床样本中；在AIM2中，我们将操纵CTCF水平并使用成对末端转录组测序来确定其他顺式融合。我们使用LNCaP细胞进行的初步研究已经产生了许多用于研究的融合候选细胞。我们将进一步验证这些候选为真正的顺式事件，并扩展到其他细胞系。在Aim3中，我们将研究新发现的顺式鼠尾融合的生物学和临床意义。这项拟议的研究将阐明顺式SAGE的机制，并发现与癌症相关的新型嵌合转录本。新型融合基因产物可能成为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.