Regulation of epithelial-mesenchymal transition and stem cell activity by PTEN in breast cancer

 描述（由申请人提供）：上皮-间质转化（EMT）与促进乳腺癌侵袭和转移有关。最近的研究表明，这一重要过程也是乳腺癌干细胞（CSC）产生的原因，这些干细胞对常规治疗有抵抗力，并有助于肿瘤转移和复发。与这些发现无关，BRCA 1/2、TP 53和PTEN突变已成为高风险易感基因，并与乳腺癌风险和预后的确定具有临床相关性。在这些基因中，PTEN失活促进EMT和CSC富集。然而，这些活动背后的潜在分子机制在很大程度上是未知的。在我们对PTEN在上皮形态发生中的功能的遗传分析中，我们已经确定了ABI 1，WAVE调节复合物（WRC）的关键组分，作为PTEN的新底物。蛋白质相互作用研究进一步证明了SWI/SNF染色质重塑复合物的Abi 1和Smarcc 1之间的新型相互作用，其将PTEN与SWI/SNF介导的癌症表观遗传调控联系起来。PTEN使Abi 1去磷酸化，从而下调Abi 1和Smarcc 1。Abi 1或Smarcc 1在人乳腺上皮细胞中的过表达促进EMT并增强干细胞活性。基于这些发现，我们假设在乳腺癌中，PTEN缺失通过Abi 1和Smarcc 1诱导EMT和CSC富集。为了验证这一假设，我们提出了两个具体目标。在目标1中，Abi 1在PTEN缺失诱导的EMT、CSC富集、致瘤性和转移中的作用将通过乳腺癌细胞和小鼠异种移植乳腺脂肪垫模型中的功能获得和丧失方法来分析。此外，我们将在PTEN缺失小鼠乳腺中删除一个或两个Abi 1等位基因，以确定Abi 1升高是否介导PTEN缺失诱导的乳腺肿瘤形成。为了阐明PTEN-Abi 1下游的信号事件，我们将重点关注Abi 1与WRC组分和Abl激酶的相互作用及其在EMT和CSC富集中的作用。在目标2中，我们将通过定点诱变来定位介导Abi 1-Smarcc 1相互作用的结合位点。然后，我们将分析在SWI/SNF复合物组装和染色质重塑活性的EMT诱导转录因子的启动子的Abi 1-Smarcc 1相互作用的功能后果。将在乳腺癌细胞和小鼠异种移植物中分析对EMT和CSC活性的影响。此外，我们将测试这一假设，即Smarcc 1升高与Smarcc 2在SWI/SNF复合物中竞争，从而通过过表达Smarcc 2并在PTEN缺陷的乳腺癌细胞中敲低Smarcc 1来诱导EMT和CSC活性。成功完成拟议的研究将为开发针对PTEN缺陷乳腺癌中EMT和CSC的治疗策略奠定基础。它也将阐明新的PTEN-Abi 1-Smarcc 1通路。

 DESCRIPTION (provided by applicant): Epithelial-mesenchymal transition (EMT) has been implicated in promoting breast cancer invasion and metastasis. Recent studies have shown that this important process is also responsible for the generation of breast cancer stem cells (CSCs), which are resistant to conventional therapy and contribute to tumor metastasis and relapse. Independent of these findings, mutations in BRCA1/2, TP53 and PTEN have emerged as high- penetrance susceptibility genes and are clinically relevant for the determination of breast cancer risk and prognosis. Among these genes, PTEN inactivation promotes both EMT and CSC enrichment. However, the underlying molecular mechanisms behind these activities are largely unknown. In our genetic analysis of PTEN functions in epithelial morphogenesis, we have identified Abi1, a key component of the WAVE regulatory complex (WRC), as a new substrate for PTEN. Protein interaction studies have further demonstrated a novel interaction between Abi1 and Smarcc1 of the SWI/SNF chromatin remodeling complex, which links PTEN to SWI/SNF-mediated epigenetic regulation of cancer. PTEN dephosphorylates Abi1 and consequently downregulates Abi1 and Smarcc1. Overexpression of Abi1 or Smarcc1 in human mammary epithelial cells promotes EMT and enhances stem cell activity. Based on these findings, we hypothesize that PTEN loss induces EMT and CSC enrichment through Abi1 and Smarcc1 in breast cancer. To test this hypothesis, we propose two specific aims. In Aim 1, the role of Abi1 in PTEN loss-induced EMT, CSC enrichment, tumorigenicity and metastasis will be analyzed by gain- and loss-of-function approaches in breast cancer cells and a mouse xenograft mammary fat pad model. In addition, we will delete one or both Abi1 alleles in PTEN- null mouse mammary glands to determine if Abi1 elevation mediates PTEN loss-induced breast tumor formation. To elucidate the signaling events downstream of PTEN-Abi1, we will focus on the interaction of Abi1 with WRC components and Abl kinase and their role in the EMT and CSC enrichment. In Aim 2, we will map the binding sites that mediate the Abi1-Smarcc1 interaction by site-directed mutagenesis. We will then analyze the functional consequences of the Abi1-Smarcc1 interaction in SWI/SNF complex assembly and chromatin remodeling activity at the promoters of the EMT-inducing transcription factors. The impact on EMT and CSC activity will be analyzed both in breast cancer cells and mouse xenografts. Furthermore, we will test the hypothesis that elevated Smarcc1 competes with Smarcc2 in the SWI/SNF complex and thus induces EMT and CSC activity by overexpressing Smarcc2 and knocking down Smarcc1 in PTEN-deficient breast cancer cells. Successful completion of the proposed studies will lay the foundation for developing therapeutic strategies targeting EMT and CSCs in PTEN-deficient breast cancer. It will also shed light on the new PTEN- Abi1-Smarcc1 pathway.