Role of Splicing Factors in Breast Cancer

描述（由申请人提供）：癌细胞通常显示异常的选择性剪接谱，导致产生可增加细胞增殖、迁移和凋亡抗性的蛋白质同种型。我的长期目标是建立一个独立的研究实验室，在那里我将阐明选择性剪接失调通过改变各种癌基因和肿瘤抑制基因的表达在癌症中发挥作用的分子机制。重要的是，这些发现将转化为新的治疗策略的发展。K99/R 00职业奖将有助于通过推进我在以下方面的培训来实现这一目标：在我的主要导师Adrian Krainer博士的指导下的反义寡核苷酸技术;在Michael Schatz博士的共同指导下的下一代测序;以及在Mikala Egeblad博士的共同指导下的转移性乳腺癌模型。这次培训将补充我以前在乳腺癌研究和RNA剪接机制方面的专业知识。在冷泉港实验室非常刺激的科学环境将不仅为我提供必要的专业知识和设施，完成本项目的指导阶段，但也将准备我顺利过渡到一个独立的教师职位。在K99指导阶段，我将定义剪接因子和剪接失调在乳腺癌中的作用。在随后的R 00独立阶段，我将确定致癌剪接事件，这些事件可能构成治疗靶点， 我未来的独立研究我们先前已经证明，剪接因子SRSF 1的过表达可以在体外和体内转化人乳腺上皮细胞。我们还发现SRFS 1水平直接受MYC癌蛋白调节。然而，其他剪接因子也在人类乳腺肿瘤中过表达，这表明它们也可能在乳腺癌中发挥作用。在目标1中，在K99阶段，我将确定 使用相关的细胞和动物模型，模拟肿瘤发生的生物学背景，研究乳腺癌中的特异性剪接因子。这包括：（一）确定替代方案的变化 在人乳腺上皮细胞的3-D培养物中通过下一代RNA测序的潜在剪接因子介导的转化的剪接事件;和（ii）使用体内小鼠模型定义这些致癌剪接因子的转移潜力。在目标2中，在K99阶段，我将通过在MYC诱导的细胞培养系统中通过下一代RNA测序鉴定剪接因子表达和选择性剪接谱的变化来确定MYC在乳腺癌中调节选择性剪接中的作用。在目标3中，在R 00阶段，我将确定剪接因子在获得抗 酪氨酸激酶抑制剂在乳腺癌中的作用，通过确定剪接因子水平和选择性剪接事件的变化。最后，从K99期开始并进入R 00期，我将确定使用反义寡核苷酸调节目标1-3中确定的特定致癌选择性剪接事件的治疗潜力。这项拟议中的研究不仅将确定与乳腺癌有关的剪接因子，还将确定它们的调节因子和特定靶点。 该计划将为我的独立研究项目奠定基础，在该项目中，我计划为基于调节剪接因子或其靶点的表达和活性的新癌症疗法的开发做出贡献。

DESCRIPTION (provided by applicant): Cancer cells often display aberrant profiles of alternative splicing, leading to the production of protein isoforms that can increase cell proliferation, migration, and apoptotic resistance. My long-term goal is to establish an independent research lab, where I will elucidate molecular mechanisms by which alternative splicing misregulation plays a role in cancer by altering the expression of various oncogenes and tumor-suppressor genes. Importantly, these findings will translate into the development of novel therapeutic strategies. The K99/R00 career award will help in achieving this goal by advancing my training in: antisense oligonucleotide technologies under the guidance of my primary mentor Dr. Adrian Krainer; next-generation sequencing under the co-mentorship of Dr. Michael Schatz; and metastatic breast cancer models under the co-mentorship of Dr. Mikala Egeblad. This training will complement my previous expertise in breast cancer research and RNA splicing mechanisms. The very stimulating scientific environment at Cold Spring Harbor Laboratory will not only provide me with the expertise and facilities necessary for the completion of the mentored phase of this project, but will also prepare me to transition smoothly into an independent faculty position. During the K99 mentored phase, I will define the role of splicing factors and splicing misregulation in breast cancer. In the subsequent R00 independent phase, I will indentify oncogenic splicing events that could constitute therapeutic targets to pursue during my future independent research. We have previously demonstrated that overexpression of the splicing factor SRSF1 can transform human mammary epithelial cells in vitro and in vivo. We have also shown that SRFS1 levels are directly regulated by the MYC oncoprotein. However, additional splicing factors are also overexpressed in human breast tumors, suggesting that they may also play a role in breast cancer. In Aim 1, during the K99 phase, I will determine the role of specific splicing factors in breast cancer using relevant cell and animal models that mimic the biological context in which the tumors arise. This includes: (i) identifying changes in alternative splicing events underlying splicing-factor- mediated transformation by next-generation RNA-sequencing in 3-D cultures of human mammary epithelial cells; and (ii) defining the metastatic potential of these oncogenic splicing factors using in vivo mouse models. In Aim 2, during the K99 phase, I will determine the role of MYC in the regulation of alternative splicing in breast cancer, by identifying changes in both splicing-factor expression and in alternative splicing profiles by next-generation RNA-sequencing in a MYC-inducible cell culture system. In Aim 3, during the R00 phase, I will determine the role of splicing factors in acquisition of resistance to tyrosine kinase inhibitors in breast cancer, by identifying changes in splicing-factor levels and i alternative splicing events. Finally, starting in the K99 phase and leading into the R00 phase, I will determine the therapeutic potential of using antisense oligonucleotides to modulate specific oncogenic alternative splicing events identified in Aims 1-3. The proposed research will identify not only splicing factors involved in breast cancer but also their regulators and specific targets. This plan will establish the basis for my independent research program, in which I plan to contribute to the development of new cancer therapies based on modulating the expression and activity of splicing factors or their targets.