Determine the Functional Role Dub3 in Breast Cancer Progression and Metastasis

 描述（由申请人提供）：基底样乳腺癌（BLBC）具有侵袭性临床病史、复发发展、远处转移、生存期较短，通常发生于年轻女性。BLBC具有激活的上皮-间充质转化（EMT）程序，其为细胞提供在胚胎发育、组织重塑、伤口愈合和转移期间所需的增加的表型和细胞可塑性。我们的长期目标是发现控制BLBC细胞在转移中的功能可塑性的分子电路，并确定可能作为治疗这种致命疾病的药物靶点的分子。在过去的九年中，我们系统地研究了Snail的功能及其抑制BLBC中E-cadherin表达的分子机制。我们的研究清楚地表明，Snail水平主要通过蛋白质泛素化来调节。高水平的蜗牛，由于蛋白质的稳定，有助于显着 增加的侵袭、治疗抗性、CSC样性质和肿瘤复发。然而，Snail逃避泛素化和降解的机制仍然未知;揭示这种机制对于开发治疗BLBC的新方法至关重要。最近，我们发现Dub 3是Snail的一种真正的去泛素化酶，并有助于Snail在乳腺癌中的稳定。从果蝇到人类，Dub 3在进化上是保守的。在果蝇中，Dub 3基因敲除下调Snail并破坏原肠胚形成（需要EMT的事件）。在BLBC样品和细胞系中，Dub 3的表达升高与Snail表达明确相关。在乳腺肿瘤MCF 7和T47 D细胞系中，Dub 3的过表达诱导向基底样表型的转化，而Dub 3敲低下调Snail并抑制细胞迁移。有趣的是，Dub 3是暴露于炎性细胞因子后出现的早期反应基因。我们假设Dub 3是长期寻找的“缺失分子”，它通过控制EMT和转移过程中Snail的水平来调节细胞可塑性。本提案的目的是表征Dub 3-Snail轴的功能和调节，并探索Dub 3作为治疗转移性乳腺癌的预后生物标志物和可药用靶点的临床价值。在强有力的初步数据的指导下，我们将通过追求三个具体目标来验证这一假设：（1）描绘Dub 3控制EMT的分子机制;（2）确定Dub 3在乳腺癌中的功能和调节;（3）确定Dub 3在体内的功能和治疗效果。我们的建议是创新和重要的，因为它不仅揭示了一个新的范式，显着增加了我们对肿瘤进展和转移过程中细胞可塑性调节的理解，而且还导致了特异性Dub 3抑制剂治疗转移性乳腺癌的发展。

 DESCRIPTION (provided by applicant): Basal-like breast cancer (BLBC) presents with an aggressive clinical history, development of recurrence, distant metastasis, shorter survival, and usually occurs in young women. BLBC has an activated epithelial-mesenchymal transition (EMT) program, which provides cells with the increased phenotypic and cellular plasticity required during embryonic development, tissue remodeling, wound healing and metastasis. Our long-term goal is to discover the molecular circuitry that governs the functional plasticity of BLBC cells in metastasis and to identify molecules that may serve as druggable targets for treating this deadly disease. In the last nine years, we have systematically studied the function of Snail and the molecular mechanism by which Snail represses E-cadherin expression in BLBC. Our studies clearly indicate that Snail levels are regulated predominantly through protein ubiquitination. High levels of Snail, due to protein stabilization, contribute significantly to the increased invasion, therapeutic resistance, CSC-like properties, and tumor recurrence. However, the mechanism by which Snail escapes ubiquitination and degradation remains unknown; uncovering this mechanism is crucially important for the development of novel approaches to treat BLBC. Recently, we discovered that Dub3 is a bona fide deubiquitinase of Snail and contributes to Snail stabilization in breast cancer. Dub3 is evolutionarily conserved from Drosophila to humans. Dub3-knockout downregulated Snail and disrupted gastrulation (an event that requires EMT) in Drosophila. Elevated expression of Dub3 correlates unequivocally with Snail expression in BLBC samples and cell lines. Overexpression of Dub3 in luminal breast tumor MCF7 and T47D cell lines, which contain no detectable endogenous Dub3 and Snail, induces transformation to a basal-like phenotype; whereas, Dub3-knockdown downregulates Snail and suppresses cell migration. Intriguingly, Dub3 was an early response gene that appeared after exposure to inflammatory cytokines. We hypothesize that Dub3 is the long-sought "missing molecule" that regulates cellular plasticity by controlling the level of Snail durig EMT and metastasis. The objective of this proposal is to characterize the function and regulation of the Dub3-Snail axis and explore the clinical value of Dub3 as a prognostic biomarker and a druggable target for treating metastatic breast cancer. Guided by strong preliminary data, we will test this hypothesis by pursuing three specific aims: (1) to delineate th molecular mechanism by which Dub3 controls EMT; (2) to determine the function and regulation of Dub3 in breast cancer; and (3) to define the function and therapeutic efficacy of Dub3 in vivo. Our proposal is innovative and significant, because it will not only uncover a new paradigm that significantly increases our understanding of the regulation of cellular plasticity during tumor progression and metastasis but also leads to the development of specific Dub3 inhibitors for treating metastatic breast cancer.