Pathogenic mechanisms of sinonasal sarcoma, a novel gender dimorphic cancer

项目总结 双表型鼻窦肉瘤(SNS)是一种新发现的肉瘤，其发病率是女性的三倍。 比男人更频繁。这种高度侵袭性病变的唯一治疗方法是毁容面部手术。我们最近 在SNS中发现了一种新的融合，它创建了一种新的嵌合体PAX3-MAML3，它融合了DNA结合 PAX3转录因子的结构域与MAML3转录共激活因子(MAML3)。这个 转录程序，PAX3-MAML3触发，诱导恶性转化和侵袭性生长，以及 SNS性别二态的基础是完全没有特征的。初步数据显示， PAX3-MAML3的表达足以推动组织学上模拟SNS的肿瘤的形成，并 诱导SNS中观察到的基因表达模式。值得注意的是，PAX3-MAML3诱导雌激素的表达 受体β(ERβ)和ERα的磷酸化，并刺激ER活性。此外，肿瘤是由 异种移植的PAX3-MAML3表达细胞在雌性受体小鼠中显著大于雄性受体小鼠。 对原发SNS肿瘤的转录组分析证实了体内雌激素反应信号的激活。 这些结果使我们推测内质网在PAX3-MAML3‘S致病活性中起重要作用 在社交网络中，这是这种癌症的性别二型性的基础。我们进一步假设，反- 雌激素可以作为一种补充甚至替代的方法来进行毁容手术 这种高度侵袭性癌症的治疗。这将通过以下方式进行检验：目标一：确定 内质网在PAX3-MAML3体外和体内致病机制中的作用 CRISPR和抗雌激素药物(如他莫昔芬、弗维斯特和芳香酶抑制剂)，多方面 PAX3-MAML3诱导的转化：(A)在体外，我们将监测对细胞增殖和存活的影响， 锚定-独立的生长，和侵入性。(B)在体内，我们将测试ER和雌激素的贡献 PAX3-MAML3介导的肿瘤发生的信号转导我们将评估ER失活的影响(通过CRISPR- 介导的ERα/ERβ耗尽或通过抗雌激素药物治疗的小鼠)，比较肿瘤生长 在雄鼠和雌鼠之间。目的II：确定PAX3-MAML3和ER如何协调基因表达以 我们将：(A)通过RNA-seq确定PAX3-MAML3诱导的转录程序， 并确定它是如何被ER调节的(通过使用ER激动剂，以及CRISPR介导的 ERα/β)；以及(B)定义PAX3-MAML3和ER染色质占用景观，以获得机械性 了解PAX3-MAML3和ER如何协调这个转录程序。转录组和芯片- SEQ数据集将被交叉，以区分PAX3-MAML3和ER的直接和间接靶基因，以及 来洞察转录上的协同效应。这种综合分析的长期目标是促进 识别PAX3-MAML3/ER靶点及其下游通路 转化的、侵袭性的表型，并最终可能作为额外的治疗靶点。 好了！

PROJECT SUMMARY Bi-phenotypic sinonasal sarcoma (SNS) is a newly identified sarcoma that affects women three times more frequently than men. The only treatment for this highly invasive lesion is disfiguring facial surgery. We recently identified a novel fusion in SNS, which creates a novel chimera PAX3-MAML3 that fuses the DNA-binding domain of the PAX3 transcription factor with the Mastermind-like 3 (MAML3) transcriptional co-activator. The transcriptional program that PAX3-MAML3 triggers to elicit malignant transformation and invasive growth, and the basis of the gender dimorphism of SNS is completely uncharacterized. Preliminary data demonstrate that expression of PAX3-MAML3 is sufficient to drive formation of tumors that histologically mimic SNS, and to induce gene expression patterns observed in SNS. Notably, PAX3-MAML3 induced expression of estrogen receptor β (ERβ) and phosphorylation of ERα, and stimulated ER activity. Furthermore, tumors formed by xenografted PAX3-MAML3-expressing cells were significantly larger in female than male recipient mice. Transcriptome analysis of primary SNS tumors confirmed activation of an estrogen response signature in vivo. These results lead us to hypothesize that the ER plays an essential role in PAX3-MAML3's pathogenic activity in SNS, and that this underlies the gender dimorphic nature of this cancer. We further posit that anti- estrogens might serve as a complementary or even alternative approach to disfiguring surgery for the treatment of this highly invasive cancer. This will be tested through the following: Aim I: Determine role of ER in PAX3-MAML3 pathogenesis in vitro and in vivo We will examine the effects of ER inactivation, using CRISPR and anti-estrogenic agents (i.e. tamoxifen, fulvestrant, and aromatase inhibitor), on multiple aspects of PAX3-MAML3-induced transformation: (A) In vitro, we will monitor effects on cell proliferation and survival, anchorage-independent growth, and invasiveness. (B) In vivo, we will test the contribution of ER and estrogen signaling to PAX3-MAML3-mediated tumorigenesis. We will assess the effects of ER inactivation (by CRISPR- mediated depletion of ERα/ERβ or by treatment of mice with anti-estrogenic agents), comparing tumor growth in male vs. female mice. Aim II: Determine how PAX3-MAML3 and ER coordinate gene expression to drive invasive growth We will: (A) define the transcriptional program induced by PAX3-MAML3 by RNA-seq, and determine how it is modulated by ER (through use of ER agonists, and CRISPR-mediated deletion of ERα/β); and (B) define the PAX3-MAML3 and ER chromatin occupancy landscapes to gain a mechanistic understanding of how PAX3-MAML3 and ER coordinate this transcriptional program. Transcriptome and ChIP- seq data sets will be intersected to distinguish direct from indirect target genes of PAX3-MAML3 and ER, and to gain insight into cooperative effects on transcription. A longterm goal of this integrated analysis is to facilitate identification of PAX3-MAML3/ER targets and their downstream pathways that are essential for the transformed, invasive phenotype, and may ultimately serve as additional therapeutic targets. !