Disrupting long noncoding RNA methylation to elicit antimorph behavior in breast cancer

项目总结 在乳腺癌中过度表达的特定的长非编码RNA(LncRNAs)有助于诊断和治疗 促进推动乳腺癌进展的分子机制。我们最近的研究发现了一种乳房- 通过单个突变将癌基因转化为肿瘤抑制基因的癌症相关lncRNA 核苷酸。这种“反变形”效应是由防止RNA甲基化在这一个部位引起的，使得 过度表达转换后的lncRNA以减少癌症表型的作用比耗尽野生- 键入Version。这一令人兴奋的新发现促使我们更深入地研究这是如何发生的，最终的 目的是作为一种新的治疗方法特异性地干扰这一M6A通路。 建议的乳腺癌相关lncRNAs治疗干预措施主要针对 耗尽促进癌症的RNA，假设这将是最有效的方法 减少癌症表型。我们的结果提示了一种新的治疗方法来治疗致癌的lncRNA，其中 仅干扰RNA甲基化就比简单地消除RNA具有更大的影响。海流 该提案提供了一个路线图，以了解如何在致癌的lncRNA中诱导抗形态效应。我们的目标是 将这一原则证明项目作为未来工作的跳板，这将导致这种治疗方法 去诊所。 我们假设特定乳腺癌相关lncRNAs上的m6A修饰位点可能是 定向和中断，以将致癌行为逆转到基线以下，为潜在的新的 治疗方法。在我们的第一个目标中，我们将研究一种具有抗变形作用的突变体HOTAIR lncRNA 使用蛋白质组学方法在三阴性的MDA-MB-231乳腺癌细胞中进行物理相互作用。 物理和功能上的相互作用将在过度表达该基因的乳腺癌细胞系中耗尽。 以确定它们在抗变形机制中的作用。新近衍生的原代细胞系 乳腺癌患者来源的异种移植将被用来确定这一机制的普适性。在 第二个目的，反义寡核苷酸(ASO)将被用来破坏HOTAIR上的m6A位点以诱导 反变形行为。在一种并行的方法中，我们将开发一种优化的策略来交付反变形 HOTAIR RNA直接抑制肿瘤表型。在以下情况下，这两种方法是互补的 HOTAIR过度表达的乳腺癌和不过度表达的乳腺癌。 我们的工作将是对一种新的乳腺癌治疗方法的基础测试，通过诱导 M6A修饰的lncRNA的抗晶型行为。总的来说，基于ASO的策略是一种新的方法 乳腺癌治疗学。RNA疗法现在已经成为现实，并得到了广泛的接受。我们的项目 利用这种令人兴奋的氛围，用一种雄心勃勃的、新颖的方法来进行非编码RNA干预 乳腺癌。通过利用M6A提供的分子开关，我们可以逆转 致癌的lncRNA逆转乳腺癌的特性。

PROJECT SUMMARY Specific long noncoding RNAs (LncRNAs) that are overexpressed in breast cancer aid in diagnosis and promote molecular mechanisms that drive breast cancer progression. Our recent work has identified a breast- cancer associated LncRNA that converts from an oncogene to a tumor suppressor by mutation of one single nucleotide. This “antimorphic” effect is caused by the prevention of RNA methylation at this one site, allowing overexpression of the converted LncRNA to decrease cancer phenotypes more so than depletion of the wild- type version. This exciting new finding prompts us to delve deeper into how this is occurring, with the ultimate goald of specifically disrupting this m6A pathway as a novel therapeutic approach. Proposed therapeutic intervention for breast cancer-associated LncRNAs has mainly been aimed at depleting the cancer-promoting RNA, with the assumption that this would be the most effective approach to reduce cancer phenotypes. Our results suggest a novel therapeutic approach to oncogenic LncRNAs, where disruption of RNA methylation alone has a greater impact than simple elimination of the RNA. The current proposal provides a roadmap to understand how to elicit antimorphic effects in an oncogenic LncRNA. We aim to use this proof-of-principle project as a springboard for future work that would lead this therapeutic approach to the clinic. We hypothesize that m6A modification sites on a specific breast cancer-associated LncRNAs can be targeted and disrupted to reverse oncogenic behavior below baseline, providing a roadmap for a potential new therapeutic approach. In our first aim, a mutant HOTAIR LncRNA that behaves as an antimorph will be studied for physical interactions in triple negative MDA-MB-231 breast cancer cells using proteomic approaches. Physical and functional interactors will then be depleted in a breast cancer cell line that overexpresses the antimorph HOTAIR to determine their role in the antimorph mechanism. Recently-derived primary cell lines from breast cancer patient-derived xenografts will be used to determine the generalizability of this mechanism. In the second aim, anti-sense oligonucleotides (ASOs) will be used to disrupt the m6A site on HOTAIR to elicit antimorph behavior. In a parallel approach, we will developed an optimized strategy to deliver the antimorph HOTAIR RNA directly to suppress cancer phenotypes. These two approaches are complementary in cases of breast cancer where HOTAIR is overexpressed and those where it is not. Our work would be a foundational test of a novel therapeutic approach in breast cancer by inducing antimorphic behavior of an m6A-modified LncRNA. In general, the ASO-based strategy is a novel approach to breast cancer therapeutics. RNA therapeutics are now a reality and are gaining wide acceptance. Our project capitalizes on this exciting atmosphere with an ambitious, novel approach to noncoding RNA interventions in breast cancer. By taking advantage of a molecular switch provided by m6A, we can reverse the activity of an oncogenic LncRNA to reverse breast cancer properties.