Targeting the epithelial to mesenchymal transition-mediated chemoresistance in breast cancer

 描述（由申请人提供）尽管在治疗方面取得了显著进展，但对常规化疗的耐药性的发展仍然是一个未解决的问题。因此，深入了解耐药性产生的机制对于改善治疗方案至关重要。最近的证据表明，上皮间质转化（EMT），涉及肿瘤侵袭和转移的转分化程序，也可能有助于化疗耐药性。然而，由于无法跟踪肿瘤进展期间的EMT事件，EMT在体内的重要性受到激烈争论。为了解决这个关键问题，我们建立了一个新的EMT谱系跟踪系统，结合多种转基因模型（MMTV-PyMT/Fsp 1-Cre/Rosa 26-GRFP）在乳腺癌。在该模型中，间充质特异性Cre介导的重组启动了经历EMT的肿瘤细胞中荧光标记物的永久转换，这使得我们能够在整个乳腺肿瘤进展中追踪EMT肿瘤细胞。使用该模型的初步研究发现，在主要为上皮的原发性肿瘤中，一小部分（约10%）是由上皮细胞引起的。2%的肿瘤细胞发生EMT。引人注目的是，这些EMT细胞没有引起转移，因为转移主要由非EMT肿瘤细胞组成（没有荧光开关）。重要的是，我们发现EMT肿瘤细胞在化疗后显著促进肺转移。EMT肿瘤细胞由于增殖减少、凋亡耐受和化疗耐药相关基因表达升高而在化疗中存活。通过过表达miR-200家族抑制EMT可消除这种耐药性。这些结果提供了EMT在化疗耐药性中的直接证据，并打开了研究EMT靶向策略作为克服乳腺癌化疗耐药性的手段的窗口。在本研究中，我们将利用EMT谱系追踪模型，进一步研究一系列合理引导的抗EMT方法，如靶向EMT诱导的自分泌/旁分泌信号（Aim 1）和EMT促进转录因子（Aim 2）。我们将使用遗传学和药理学方法来评估它们在EMT介导的化疗耐药性中的重要性。此外，基于我们初步的转录组分析，我们将产生一个CRISPR gRNA文库，靶向EMT肿瘤细胞中差异表达的基因。EMT谱系追踪模型将为CRISPR筛选提供一个独特的平台，以确定EMT介导的化学抗性的新治疗靶点（目的3）。最后，我们将验证我们在人类乳腺癌中的发现，并在一系列转移性乳腺癌患者来源的异种移植物（PDX）模型中评估联合治疗的疗效（目标4）。我们设想这种基于EMT介导的化学抗性的机制的靶向将构成乳腺癌治疗的可行策略。

 DESCRIPTION (provided by applicant) Despite significant advances in the treatment, development of resistance to conventional chemotherapy remains an unresolved problem. Consequently an in-depth understanding of the mechanisms underlying the development of chemoresistance is of utmost importance for improving the therapeutic regimens. Recent evidence indicates that epithelial to mesenchymal transition (EMT), the transdifferentiation program involved in tumor invasion and metastasis, may also contribute to chemoresistance. However, the importance of EMT in vivo is fiercely debated due to the inability of tracking EMT events during tumor progression. To solve this critical issue, we have established a novel EMT lineage tracing system in breast cancer by combining multiple transgenic models (MMTV-PyMT/Fsp1-Cre/Rosa26-GRFP). In this model, mesenchymal- specific Cre-mediated recombination initiates a permanent switch of fluorescent markers in tumor cells undergoing EMT, which enable us to track the EMT tumor cells throughout the breast tumor progression. The initial studies with this model found that within a predominantly epithelial primary tumor, a small portion (approx. 2%) of tumor cells underwent EMT. Strikingly, these EMT cells did not give rise to metastasis as the metastases were mainly comprised of non-EMT tumor cells (no fluorescence switch). Importantly, we discovered that EMT tumor cells significantly contributed to lung metastasis after chemotherapy. The EMT tumor cells survived chemotherapy due to reduced proliferation, apoptotic tolerance, and elevated expression of chemoresistance- related genes. Inhibiting EMT by overexpressing miR-200 family abrogated this resistance. These results provide direct evidence of EMT in chemoresistance and open the window to investigate EMT-targeting strategies as a means to overcome chemoresistance in breast cancer. In this proposal, we will take advantage of the EMT lineage tracing model, to further investigate a series of rationally guided anti-EMT approaches such as targeting EMT-induced autocrine/paracrine signals (Aim 1) and EMT-promoting transcription factors (Aim 2). We will use both genetic and pharmacological approaches to evaluate their importance in EMT-mediated chemoresistance. Moreover, based on our preliminary transcriptome analysis we will generate a CRISPR gRNA library targeting the differentially expressed genes in EMT tumor cells. The EMT lineage tracing model will provides a unique platform for the CRISPR screen to identify novel therapeutic targets for the EMT-mediated chemoresistance (Aim 3). At the end, we will validate our findings in human breast cancer and evaluate efficacy of combination therapies in a series of metastatic breast cancer patient-derived xenograft (PDX) models (Aim 4). We envision that this mechanism-based targeting of EMT- mediated chemoresistance will constitute feasible strategies for breast cancer therapy.