Molecular Function and Mechanism of ARID4B in ERalpha Signaling and Breast Cancer

项目总结/摘要 超过70%的乳腺癌是ERα+，内分泌治疗是这些患者的标准治疗。 不幸的是，随着时间的推移，对内分泌治疗的耐药性仍然是一个主要问题。的原因 耐药性尚不完全清楚，但异常ERα激活是一个潜在因素。因此，确定新的 ERα信号的组成部分，并了解其在乳腺癌中的作用， ERα+癌症，特别有利于晚期和转移性肿瘤患者， 目前的内分泌治疗。我们的初步结果表明，染色质重塑蛋白AT丰富 相互作用结构域4 B（ARID 4 B）是ERα的一个必需的转录辅激活因子（而不是辅抑制因子）， 乳腺特异性消融Arid 4 b抑制肿瘤发生。大规模基因组数据集分析 (TCGA和其他乳腺癌队列），全基因组转录组和IHC分析显示，ARID 4 B 在ERα+乳腺癌中扩增并表达升高。有趣的是，ERα+中ARID 4 B高表达 (but非ERα-）乳腺癌与癌症复发风险增加和生存率降低相关， 这表明ARID 4 B参与癌症发展和向治疗抗性的进展。获得 为了深入了解其功能，初步结果显示，ARID 4 B被募集到 ERα靶向基因远离已鉴定的ERα结合增强子。此外，还鉴定了DHX 9 作为一种新的ARID 4 B相互作用蛋白，参与ERα的激活。DHX 9是一种NTP依赖性解旋酶 能够解析抑制生产性转录的转录偶联的“R环”。基于这些 研究结果表明，我们的中心假设是ARID 4 B激活ERα信号以促进肿瘤发生， 内分泌治疗抗性通过：（1）通过与ERα相互作用介导启动子-增强子环和（2）通过 募集DHX 9以解析启动子和增强子上的转录偶联R环，以促进生产性 转录。在目的1中，ARID 4 B在肿瘤起始、癌症生长和内分泌治疗中的体内功能 将使用肿瘤异种移植模型中的几种乳腺癌细胞、患者来源的 PDX中的耐药肿瘤，以及新产生的ARID 4 B基因敲入小鼠模型。在目标2中，我们将研究 ARID 4 B介导的增强子-启动子环如何调节ERα激活， ARID 4 B上的结构域，以深入了解ARID 4 B-ER α和ARID 4 B-DHX 9相互作用。 此外，我们将研究DHX 9对启动子和增强子上的“R环”的解析是否是 激活ERα所必需的。最后，我们将证明，通过以下方式瞄准ARID 4 B-DHX 9轴： 抑制DHX 9表达或活性抑制肿瘤发生和对抗雌激素的抗性。我们的研究将 填补了理解新出现的致癌活性的功能和机制的关键空白， ARID 4 B和建立ARID 4 B-DHX 9轴作为乳腺癌的治疗靶点。

Project Summary/Abstract Over 70% of breast cancer are ERα+ and endocrine therapy is the standard treatment for these patients. Unfortunately, resistance to endocrine therapy develops over time and remains a major problem. The cause for resistance is not fully understood, but aberrant ERα activation is an underlying factor. Therefore, identifying new component of the ERα signaling and understanding its role in breast cancer holds a great promise for treatment of ERα+ cancer and will particularly benefit patients with advanced and metastatic tumors that are refractory to current endocrine therapies. Our preliminary results showed that chromatin remodeling protein AT-rich interaction domain 4B (ARID4B) is an essential transcription co-activator (not a co-repressor) for ERα and mammary gland-specific ablation of Arid4b inhibits tumorigenesis. Analyses of large-scale genomic datasets (TCGA and other breast cancer cohorts), genome-wide transcriptome, and IHC analyses showed that ARID4B is amplified and its expression elevated in ERα+ breast cancer. Interestingly, high ARID4B expression in ERα+ (but not ERα-) breast cancers is associated with increased risk of cancer recurrence and decreased survival, suggesting that ARID4B is involved in the cancer development and progression to therapy resistance. To gain mechanistic insight into its function, preliminary results revealed that ARID4B is recruited to the promoters of ERα target genes that are far away from the identified ERα-bound enhancers. In addition, DHX9 was identified as a novel ARID4B-interacting protein and is involved in ERα activation. DHX9 is an NTP-dependent helicase capable of resolving transcription-coupled ‘R-loops’ that is inhibitory to productive transcription. Based on these findings, our central hypothesis is that ARID4B activates ERα signaling to promote tumorigenesis and endocrine therapy resistance by: (1) mediating promoter-enhancer looping via interaction with ERα and (2) by recruiting DHX9 to resolve the transcription-coupled R-loops on promoters and enhancers to promote productive transcription. In Aim 1, the in vivo function of ARID4B in tumor initiation, cancer growth, and endocrine therapy resistance will be investigated using several breast cancer cells in tumor xenograft models, patient-derived resistant tumors in PDX, and a newly generated ARID4B knock-in mouse model. In Aim 2, we will investigate how ARID4B-mediated enhancer-promoter looping regulates ERα activation and identify the important functional domain(s) on ARID4B to provide in-depth understanding of ARID4B-ERα and ARID4B-DHX9 interactions. Furthermore, we will investigate whether resolution of “R-loops” on the promoter and enhancer by DHX9 is required for the activation of ERα. Finally, we will demonstrate that targeting the ARID4B-DHX9 axis by suppressing DHX9 expression or activity inhibits tumorigenesis and resistance to antiestrogens. Our study will fill the crucial gap in understanding the function and mechanism underlying the emerging oncogenic activity of ARID4B and establish ARID4B-DHX9 axis as a therapeutic target for breast cancer.