Determine the Functional Role Dub3 in Breast Cancer Progression and Metastasis

 描述(申请人提供)：基底细胞样乳腺癌(BLBC)表现为侵袭性的临床病史，复发，远处转移，生存期较短，通常发生在年轻女性。BLBC有一个激活的上皮-间充质转化(EMT)计划，该计划为细胞提供胚胎发育、组织重塑、伤口愈合和转移所需的表型和细胞可塑性增加。我们的长期目标是发现在转移过程中控制BLBC细胞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。在过去的九年里，我们系统地研究了Snail的功能以及Snail抑制BLBC中E-钙粘蛋白表达的分子机制。我们的研究清楚地表明，蜗牛的水平主要是通过蛋白质泛素化来调节的。由于蛋白质的稳定，蜗牛的高水平对 侵袭性、治疗耐受性、肿瘤干细胞样特性和肿瘤复发增加。然而，Snail逃脱泛素化和降解的机制仍不清楚；揭示这一机制对于开发治疗BLBC的新方法至关重要。最近，我们发现Dub3是一种真正的Snail脱泛素酶，在乳腺癌中起到稳定Snail的作用。Dub3在进化上从果蝇到人类都是保守的。在果蝇中，Dub3基因敲除下调了蜗牛的表达，扰乱了原肠形成(这是一个需要EMT的事件)。在BLBC样本和细胞系中，Dub3的高表达与Snail的表达明确相关。在未检测到内源性Dub3和Snail的乳腺肿瘤MCF7和T47D细胞系中过表达Dub3可诱导向基底样表型转化；而Dub3基因敲除下调Snail的表达并抑制细胞迁移。有趣的是，Dub3是一种在接触炎性细胞因子后出现的早期反应基因。我们假设Dub3是长期寻找的“缺失分子”，它通过控制蜗牛在EMT和转移期间的水平来调节细胞的可塑性。本研究的目的是研究Dub3-Snail轴的功能和调控，并探讨Dub3作为预测预后的生物标志物和治疗转移性乳腺癌的药物靶点的临床价值。在强大的初步数据的指导下，我们将通过追求三个具体目标来检验这一假说：(1)描述Dub3控制EMT的分子机制；(2)确定Dub3在乳腺癌中的功能和调节；(3)确定Dub3在体内的功能和治疗效果。我们的建议具有创新性和重要意义，因为它不仅将揭示一种新的范式，显著提高我们对肿瘤进展和转移过程中细胞可塑性调节的理解，而且还将导致用于治疗转移性乳腺癌的特定Dub3抑制剂的开发。

 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.