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

 说明(申请人提供)尽管在治疗方面取得了重大进展，但对常规化疗的耐药性的产生仍然是一个尚未解决的问题。因此，深入了解化疗耐药发生的机制对于改进治疗方案至关重要。最近的证据表明，上皮向间充质转化(EMT)是参与肿瘤侵袭和转移的转分化过程，也可能与化疗耐药有关。然而，由于无法跟踪肿瘤进展过程中的EMT事件，EMT在体内的重要性一直备受争议。为了解决这一关键问题，我们结合多种转基因模型(MMTV-PYMT/Fsp1-CRE/rosa26-GRFP)建立了一种新的乳腺癌EMT谱系追踪系统。在这个模型中，间质特异性Cre介导的重组启动了接受EMT的肿瘤细胞中荧光标记的永久切换，这使我们能够跟踪EMT肿瘤细胞在乳腺肿瘤进展过程中的整个过程。对该模型的初步研究发现，在以上皮性为主的原发肿瘤中，一小部分(约.2%)的肿瘤细胞接受了EMT。值得注意的是，这些EMT细胞没有引起转移，因为转移的主要是非EMT肿瘤细胞(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肿瘤细胞在化疗中存活的原因是增殖减少、对凋亡的耐受性降低以及化疗耐药相关基因的表达增加。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在化疗耐药中的直接证据，并为研究EMT靶向策略作为克服乳腺癌化疗耐药的一种手段打开了窗口。在这个方案中，我们将利用EMT谱系追踪模型，进一步研究一系列合理指导的抗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介导的化疗耐药中的重要性。此外，基于我们的初步转录组分析，我们将创建一个针对EMT肿瘤细胞差异表达基因的CRISPR gRNA文库。EMT谱系追踪模型将为CRISPR筛查提供一个独特的平台，以确定EMT介导的化疗耐药的新治疗靶点(AIM 3)。最后，我们将在人类乳腺癌中验证我们的发现，并在一系列转移性乳腺癌患者来源的异种移植(PDX)模型中评估联合治疗的有效性(AIM 4)。我们预见，这种基于机制的靶向EMT介导的化疗耐药将成为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.