MELK as Functional Marker for Mammary Tumors

描述(申请人提供)：这项建议的目标是确定敲除母体胚胎亮氨酸拉链激酶(Melk)的表达是否会抑制体内乳腺肿瘤的形成。与正常组织相比，Melk在人类乳腺癌组织中过度表达。用siRNA下调Melk转录本的水平可以抑制几种类型的肿瘤的体外生长，包括乳腺癌。然而，Melk在体内乳腺肿瘤发生中的作用还没有被证明。我们将使用竞争性移植试验来验证我们的假设，即Melk功能是乳腺肿瘤在体内发生所必需的。目的：1.确定Melk功能是否在体内多瘤中T细胞(PYMT)诱导的乳腺肿瘤的发生中是必需的。我们将从MMTV-PYMT小鼠中分离乳腺肿瘤细胞，并使用表达Melk shRNA和红色荧光蛋白mCherry的慢病毒敲除Melk转录本。将共表达的shRNA与EGFP共表达的shRNA感染对照细胞，将感染的肿瘤细胞与对照细胞混合，移植到同基因清除的乳腺脂肪垫中，形成肿瘤。将对由此产生的肿瘤进行分析，以确定是否存在感染Melk shRNA的供体细胞。如果Melk shRNA抑制体内肿瘤的发展，我们希望找不到或很少发现这样的细胞。目的2：确定在体内Wnt诱导的乳腺肿瘤的发生是否需要Melk功能。在MMTV-WNT模型中诱导的乳腺肿瘤明显比PYMT肿瘤更具异质性，并且在大多数人类乳腺癌中更典型。我们将使用MMTV-WNT1转基因小鼠在这个模型中测试Melk假说，就像在PYMT模型中描述的那样。在这两种情况下，我们将：1)建立原发肿瘤的有限稀释移植效率；2)分离肿瘤细胞，用Melk shRNA或混杂的对照shRNA慢病毒感染它们，并将它们移植到透明的乳腺脂肪垫中；3)结合荧光显微镜、FACS和Q-PCR分析移植动物中形成的肿瘤，以确定是否存在Melk shRNA(mCherry/red)感染细胞和内源性Melk转录本。目前迫切需要有效的乳腺癌抑制药物。如果Melk功能是乳腺肿瘤体内生长的先决条件，则可以使用小鼠肿瘤细胞和动物模型来筛选和验证在乳腺肿瘤模型中特异性抑制Melk功能的化合物(例如，PI的实验室已经开发出的特定激酶抑制剂)。鉴于人和小鼠的Melk激酶结构域具有非常高的同源性，在小鼠模型中验证的化合物将成为开发针对人类乳腺癌的临床相关抑制剂的先导。项目简介：美国女性患乳腺癌的终身风险为八分之一，每年约有20万新确诊病例。目前迫切需要有效的乳腺癌抑制药物。一个候选基因，母体胚胎亮氨酸拉链酶(Melk)，在大多数乳腺癌中过度表达，特别是在侵袭性、低分化肿瘤中。尽管有迹象表明，在培养中敲除Melk会抑制肿瘤细胞的生长，但缺乏Melk在肿瘤发生中的体内作用的证据。我们的假设是，Melk功能是体内乳腺肿瘤发生所必需的。在目标1中，我们将使用已建立的最好的乳腺肿瘤模型之一，多瘤中T(PYMT)诱导的肿瘤。在目标2中，我们将利用最近建立的WNT诱导的肿瘤模型。我们已经证明，在体内，Melk的表达标志着PyMT肿瘤，并且在体外，下调Melk的表达可以抑制乳腺肿瘤细胞系的生长。PYMT诱导的肿瘤是同种的，并且可以非常有效地在体内移植，这使得PYMT模型成为检验我们假设的最有利的模型。PYMT诱导的肿瘤类似于人类乳腺肿瘤的一个子集。然而，其他人类肿瘤更加异质性，更好地被WNT诱导的肿瘤模型捕获。WNT诱导的肿瘤是异质性的，可能起源于前体细胞，因此保留了乳腺前体细胞的一些特征。我们的初步数据表明，含有这些祖细胞的乳腺末端芽表达高水平的Melk。因此，在WNT1诱导的乳腺肿瘤中，Melk的表达可能被保留在转化的细胞中，并在肿瘤的发生中发挥作用。在这两个小鼠肿瘤模型中，我们将进行一组类似的实验。首先，我们将确定原发肿瘤的移植效率；其次，我们将用抑制性小发夹RNA(Melk ShRNAs)或杂乱的对照shRNA感染肿瘤，并将这些肿瘤移植到乳房脂肪垫中。第三，我们将结合荧光显微镜和流式细胞术分析移植动物体内形成的肿瘤，以确定感染Melk shRNA的供体细胞是否形成肿瘤。如果Melk shRNA在体内抑制肿瘤的形成，我们预计不会发现这些细胞。如果Melk功能是乳腺肿瘤体内生长的先决条件，则可以使用小鼠肿瘤细胞和动物模型来筛选和验证乳腺肿瘤模型中特定抑制Melk功能的化合物(例如，PI的实验室已经开发出的特定激酶抑制剂)。鉴于人和小鼠的Melk激酶结构域具有非常高的同源性，在小鼠模型中验证的化合物将成为开发针对人类乳腺癌的临床相关抑制剂的先导。

DESCRIPTION (provided by applicant): The goal of this proposal is to determine if knocking down Maternal Embryonic Leucine zipper Kinase (MELK) expression will inhibit mammary tumor formation in vivo. MELK is overexpressed in human breast cancers compared to normal tissue. Knocking down levels of MELK transcript with siRNA inhibits the in vitro growth of several types of tumors, including mammary tumors. However, the role of MELK in mammary tumorigenesis in vivo has not been demonstrated. We will use a competitive transplantation assay to test our hypothesis that MELK function is required for mammary tumorigenesis in vivo. Aim1. Determine if MELK function is required for development of Polyoma Middle T (PyMT) induced mammary tumors in vivo. We will isolate mammary tumor cells from MMTV-PyMT mice and knock down MELK transcripts using lentivirus expressing MELK shRNA and the red fluorescent protein mCherry. Control cells will be infected with scrambled shRNA co-expressed with eGFP MELK shRNA-infected tumor cells will be admixed with control cells and transplanted into syngenic cleared mammary fat pads to generate tumors. The resulting tumors will be analyzed for the presence of donor cells infected with MELK shRNA. We expect to find no/few such cells if MELK shRNA inhibits tumor development in vivo. Aim 2: Determine if MELK function is required for development of Wnt-induced mammary tumors in vivo. Mammary tumors induced in the MMTV-Wnt model are significantly more heterogeneous than are PyMT tumors and are more typical of the majority of human breast cancers. We will use MMTV-Wnt1 transgenic mice to test the MELK hypothesis in this model exactly as described for the PyMT model. In both cases we will: 1) establish limited dilution transplantation efficiency of primary tumors; 2) isolate tumor cells, infect them with MELK shRNA or scrambled control shRNA lentivirus and transplant them into cleared mammary fat pads; 3) analyze tumors formed in transplanted animals using a combination of fluorescence microscopy, FACS and Q- PCR for the presence of MELK shRNA (mCherry/red) infected cells and endogenous MELK transcripts. There is a crucial need for potent inhibitors of breast cancers. If MELK function is a prerequisite for mammary tumor growth in vivo, mouse tumor cells and animal models can be used to screen and validate compounds that specifically inhibit MELK function in mammary tumor models (e.g., specific kinase inhibitors that have been developed in the PI's laboratory). Given the very high homology between human and mouse MELK kinase domains, compounds validated in a mouse model will be the leads for developing clinically relevant inhibitors to target human breast cancer. PROJECT NARRATIVE: The lifetime risk of breast cancer for women in the USA is 1 in 8, with about 200,000 new cases diagnosed each year. There is a crucial need for potent inhibitors of breast cancers. A candidate gene, Maternal Embryonic Leucine zipper Kinase (MELK), is overexpressed in most breast cancers, particularly in aggressive, poorly differentiated tumors. Despite indications that knocking down MELK inhibits tumor cell growth in culture, evidence for an in vivo role for MELK in tumorigenesis is missing. Our hypothesis is that MELK function is required for mammary tumorigenesis in vivo. In Aim 1 we will use one of the best established mammary tumor models, Polyoma Middle T (PyMT) induced tumors. In Aim 2 we will take advantage of the more recently established Wnt-induced tumor model. We have shown that MELK expression marks PyMT tumors in vivo and that knocking down MELK expression inhibits growth of a mammary tumor cell line in vitro. PyMT-induced tumors are homogeneous and can be very efficiently transplanted in vivo making the PyMT model the most favorable one in which to test our hypothesis. PyMT-induced tumors resemble a subset of human breast tumors. However, other human tumors are more heterogeneous and better captured by the Wnt-induced tumor model. Wnt-induced tumors are heterogeneous and likely initiated in the progenitor compartment, thus retaining some features of mammary gland progenitor cells. Our preliminary data suggest that mammary gland end buds containing those progenitor cells express high levels of MELK. Thus, MELK expression may be retained in transformed cells and play a functional role in tumor initiation in Wnt1-induced mammary tumors. In both mouse tumor models we will perform a similar set of experiments. First, we will establish the transplantation efficiency of primary tumors; second, we will infect tumors with inhibitory small hairpin RNAs (MELK shRNAs) or a scrambled control shRNA and transplant those tumors into mammary fat pads. Third, we will analyze tumors formed in transplanted animals using a combination of fluorescence microscopy and flow cytometry to determine if donor cells infected with MELK shRNA form tumors. If MELK shRNA inhibits tumor formation in vivo we expect not to find these cells. If MELK function is a prerequisite for mammary tumor growth in vivo, mouse tumor cells and animal models can be used to screen and validate compounds specifically inhibiting MELK function in mammary tumor models (e.g., specific kinase inhibitors that have been developed in the PI's laboratory). Given the very high homology between human and mouse MELK kinase domains, compounds validated in a mouse model will serve as the leads for developing clinically relevant inhibitors to target human breast cancers.