Role of Splicing Factors in Breast Cancer

描述(申请人提供)：癌细胞经常表现出选择性剪接的异常特征，导致产生能增加细胞增殖、迁移和抗凋亡能力的蛋白质异构体。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错误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开发。K99/R00职业生涯奖将帮助我实现这一目标，它将推动我在以下方面的培训：在我的主要导师禤浩焯·克莱纳博士的指导下进行反义寡核苷酸技术的培训；在Michael Schatz博士的共同指导下进行下一代测序；在Mikala Egeblad博士的共同指导下进行转移性乳腺癌模型的培训。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。冷泉港实验室非常令人兴奋的科学环境不仅将为我提供完成该项目指导阶段所需的专业知识和设施，还将为我顺利过渡到独立的教员职位做好准备。在K99指导阶段，我将确定剪接因子和剪接错误在乳腺癌中的作用。在随后的R00独立阶段，我将确定可能构成治疗靶点的致癌剪接事件 我未来的独立研究。我们先前已经证明，剪接因子SRSF1的过表达可以在体外和体内转化人乳腺上皮细胞。我们还表明，SRFS1水平直接受MYC癌蛋白的调节。然而，额外的剪接因子也在人类乳腺肿瘤中过度表达，这表明它们可能在乳腺癌中也起到了作用。在目标1中，在K99阶段，我将确定 利用相关的细胞和动物模型模拟肿瘤发生的生物学背景，研究乳腺癌中的特定剪接因子。这包括：(1)确定备选方案中的变化 在人类乳腺上皮细胞的三维培养中，通过下一代RNA测序，剪接因子介导的转化的剪接事件；以及(Ii)使用活体小鼠模型确定这些致癌剪接因子的转移潜力。在目标2中，在K99阶段，我将确定MYC在乳腺癌选择性剪接调控中的作用，通过在MYC诱导的细胞培养系统中通过下一代RNA测序来确定剪接因子表达和选择性剪接特征的变化。在目标3中，在R00阶段，我将确定剪接因子在获得对 酪氨酸激酶抑制剂在乳腺癌中，通过识别剪接因子水平和I替代剪接事件的变化。最后，从K99期开始，进入R00期，我将确定使用反义寡核苷酸来调节AIMS 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。 这一计划将为我的独立研究计划奠定基础，在该计划中，我计划通过调节剪接因子或其靶点的表达和活性来为开发新的癌症疗法做出贡献。

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.