Alternative splicing and epithelial-mesenchymal plasticity in prostate tumors

描述(申请人提供)：在上皮细胞中，成纤维细胞生长因子受体-2(FGFR2)转录本的选择性剪接导致FGFR2(IIIb)亚型的表达，而在间充质细胞中，同样的过程导致FGFR2(IIIc)的合成，其中包括外显子IIIc。这种受调控的剪接在前列腺癌进展过程中被破坏，导致FGFR2亚型的不适当表达。为了可视化FGFR2外显子IIIc在同基因大鼠的AT3肿瘤中的作用，我们构建了报告选择性剪接的微型基因结构。成像这些选择性剪接决定揭示了这些原发肿瘤中意想不到的间充质-上皮转变(MET)。理解这种由剪接差异所揭示的上皮-间充质可塑性，是这项应用的首要目标。为此，我们建议完成以下特定目标：特定目标1.研究FGFR2剪接与前列腺肿瘤上皮-间充质可塑性之间的关系。我们开发了一种新的双色报告，可以用来检测MET和上皮-间充质转化(EMT)。我们还将我们的观察扩展到培养中具有良好特性的人前列腺肿瘤细胞。在这一目标上完成的工作将检验上皮可塑性是人类侵袭性前列腺癌的一般特性的假设。具体目的2.研究剪接改变所揭示的原发前列腺癌的上皮-间充质可塑性。在这个目标中，我们将描述原发肿瘤中MET簇的分子特征和行为。具体目的3.研究上皮-间充质可塑性与肿瘤转移行为之间的关系。我们将研究上皮-间充质可塑性与转移行为之间的联系。最后，我们将从AT3大鼠肿瘤和患者中分离出循环中的前列腺癌细胞，并确定它们的FGFR2剪接状态和它们的上皮状态。在目标二和这个目标中完成的工作将检验上皮-间充质可塑性导致肿瘤适合性增加的假设。这项工作的意义是双重的：首先，我们将揭示前列腺癌进展过程中受选择性剪接控制的基因表达程序的基本信息。其次，前列腺癌作为一个健康问题的严重性，突显了它对人类健康的潜在意义。识别将进展为转移性疾病的肿瘤/细胞是一项挑战和优先事项。这里提出的工作应该确定有用的标记物，甚至是治疗靶点。与公共健康相关：前列腺癌作为一个健康问题的严重性，突显了我们应用于人类健康的潜在意义。美国癌症协会(ACS)预测，仅在美国，前列腺癌就将困扰约20万男性，并导致2.5万人死亡。此外，前列腺癌在选择治疗方案和预测最终结果方面给临床医生和患者带来了独特的问题(参见2月26日《纽约时报》的文章《前列腺癌回顾让男性陷入混乱》)。这里提出的工作应该确定有用的肿瘤行为标记物，甚至是新疗法的靶标。

DESCRIPTION (provided by applicant): In epithelial cells alternative splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells the same process results in the synthesis of FGFR2(IIIc), which include exon IIIc. This regulated splicing is disrupted during progression of prostate carcinomas leading to the inappropriate expression of FGFR2 isoforms. To visualize the use of FGFR2 exon IIIc in Dunning AT3 tumors in syngeneic rats we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions (MET) in these primary tumors. The understanding of this epithelial-mesenchymal plasticity, revealed by differences in splicing, is the overarching goal of this application. To this end we propose to accomplish the following specific aims: Specific Aim 1. To characterize the connections between FGFR2 splicing and epithelial-mesenchymal plasticity in prostate tumors. We have developed a new bichromatic reporter that can be used to detect both MET and epithelial-mesenchymal transitions (EMT). We have also extended our observations to well characterized human prostate tumor cells in culture. Work accomplished in this aim will test the hypothesis that epithelial plasticity is a general property of aggressive prostate carcinomas in humans. Specific Aim 2. To characterize the epithelial-mesenchymal plasticity in primary prostate tumors revealed by alterations in splicing. In this aim we will characterize the molecular signatures and behavior of the MET clusters in primary tumors. Specific aim 3. To characterize the connections between epithelial-mesenchymal plasticity and metastatic behavior. We will investigate the connections between epithelial-mesenchymal plasticity and metastatic behavior. Finally, we will isolate circulating prostate tumor cells from both the AT3 rat tumors and patients, and we will determine their FGFR2 splicing status and their epithelial state. Work accomplished in aim two and in this aim will test the hypothesis that epithelial-mesenchymal plasticity leads to increased tumor fitness. The significance of this work is twofold: First we will unravel basic information about gene expression programs controlled by alternative splicing during prostate tumor progression. Second, the potential significance to human health is underscored by the magnitude of prostate cancer as a health problem. Identifying the tumors/cells that will progress to metastatic disease is a challenge and a priority. The work proposed here should identify useful markers and perhaps even therapeutic targets. PUBLIC HEALTH RELEVANCE: The potential significance of our application to human health is underscored by the magnitude of prostate cancer as a health problem, which The American Cancer Society (ACS) predicts will afflict ~200,000 men and will kill >25,000 in the US alone. In addition, prostate cancer presents the clinician and the patient with unique problems in terms of choosing among therapeutic options and predicting eventual outcomes (see New York Times 26 February article "A Review of Prostate Cancer Leaves Men in a Muddle"). The work proposed here should identify useful markers of tumor behavior and perhaps even targets for novel therapies.