Merkel cell polyomavirus T antigens in tumorigenesis

描述（由申请方提供）：默克尔细胞多瘤病毒T抗原和体内肿瘤转化项目总结默克尔细胞癌（MCC）是一种罕见的神经内分泌皮肤肿瘤，由于无法获得有效治疗，晚期疾病预后不良。大多数MCC携带来自新型多瘤病毒默克尔细胞多瘤病毒（MCPyV）的序列，并表达两种推定的癌蛋白：MCPyV小T抗原（sTAg）和肿瘤特异性截短的大T抗原（tLTAg）。与其他病毒癌蛋白一样，MCPyV转化抗原预计与多种细胞蛋白（包括肿瘤抑制因子）相互作用：tLTAg靶向RB1，而sTAg靶向PP2A复合物。在培养细胞和异种移植物中的至少一些敲低研究支持MCC肿瘤细胞维持中对tLTAg和sTAg抗原的需要。此外，培养的成纤维细胞中的过表达研究指出stAg是主要的转化癌基因，令人惊讶的是，其以PP2A非依赖性方式运作。相比之下，sTAG驱动的成纤维细胞转化严格依赖于最近描述的结构域，该结构域结合SCF E3泛素连接酶复合物的Fbxw7组分，并导致LTAg和作为Fbxw7底物的几种细胞癌蛋白的积累。这些报告提供了有价值的洞察sTAg在培养的成纤维细胞的转化潜力，但研究评估是否MCPyV标签可以作为致癌驱动程序在体内尚未报告。在初步研究中，我们表明，MCPyV的sTAg，但既不是tLTAg也不是全长LTAg，是一个强大的转化癌基因在皮肤和口腔上皮的转基因小鼠，导致显着增生，受损的终末分化，DNA损伤反应，和细胞凋亡。我们提出了一系列的实验，以进一步研究在体内转化潜力的个人 MCPyV标签，并测试它们在诱导型小鼠模型中共表达时的功能相互作用。为了研究MCC肿瘤细胞的起源，我们将比较当靶向1）表皮的增殖与分化细胞区室，和2）默克尔祖细胞与分化的默克尔细胞时的MCPyV TAg转化潜力。我们还将从条件性MCPyV TAg转基因小鼠中分离确定的细胞群，并研究它们对细胞培养物中TAg表达的反应，从而实现旨在确定转化机制的功能研究，然后可以在体内验证。拟议的研究非常重要，因为它们将通过定义完整动物中MCPyV标签的生物活性来帮助填补我们知识中的关键空白，为研究促进MCPyV相关癌症发展和维持的因素提供一套急需的工具。此外，这些研究将有助于确定MCPyV TAg细胞靶点，其通过非病毒机制的失调可能驱动病毒阴性MCC的发展，并且可能更普遍地促进其他类型癌症的发展。最后，拟议的工作可能与MCC患者有直接的翻译相关性，因为它可能导致识别新的治疗靶点，并为功能研究和临床前试验提供急需的小鼠模型。

DESCRIPTION (provided by applicant): Merkel cell polyomavirus T antigens and neoplastic transformation in vivo Project Summary Merkel cell carcinoma (MCC) is a rare neuroendocrine skin tumor with a poor prognosis at advanced disease stages due to the unavailability of effective treatments. Most MCCs carry sequences from a novel polyomavirus, Merkel cell polyomavirus (MCPyV), and express two putative oncoproteins: MCPyV small T antigen (sTAg) and tumor-specific truncated large T antigen (tLTAg). Like other viral oncoproteins, MCPyV transforming antigens are predicted to interact with multiple cellular proteins including tumor suppressors: tLTAg targets RB1 while sTAg targets the PP2A complex. At least some knock-down studies in cultured cells and xenografts support a requirement for tLTAg and sTAg antigens in MCC tumor cell maintenance. In addition, overexpression studies in cultured fibroblasts point to stAg as the predominant transforming oncogene which, surprisingly, operates in a PP2A-independent manner. In contrast, sTAg-driven fibroblast transformation is strictly dependent on a recently-described domain that binds the Fbxw7 component of the SCF E3 ubiquitin ligase complex and leads to accumulation of LTAg and several cellular oncoproteins that are Fbxw7 substrates. These reports have provided valuable insight into the transforming potential of sTAg in cultured fibroblasts, but studies assessing whether MCPyV TAgs can function as oncogenic drivers in vivo have not yet been reported. In preliminary studies we show that MCPyV sTAg, but neither tLTAg nor full-length LTAg, is a potent transforming oncogene in skin and oral epithelia of transgenic mice, leading to striking hyperplasia, impaired terminal differentiation, a DNA damage response, and apoptosis. We propose a series of experiments to further study the in vivo transformation potential of individual MCPyV TAgs, and to test their functional interaction when co-expressed in inducible mouse models. To investigate the MCC tumor cell of origin, we will compare MCPyV TAg transforming potential when targeted to 1) proliferating versus differentiating cellular compartments of epidermis, and 2) Merkel cell progenitors versus differentiated Merkel cells. We will also isolate defined cell populations from conditional MCPyV TAg transgenic mice and study their response to TAg expression in cell culture, enabling functional studies aimed at defining mechanisms of transformation which can then be verified in vivo. The proposed studies are highly significant since they will help fill a critical gap in our knowledge by defining the biological activity of MCPyV TAgs in intact animals, providing a much-needed set of tools for studying factors contributing to the development and maintenance of MCPyV-associated cancer. In addition, these studies will help identify MCPyV TAg cellular targets whose deregulation via non-viral mechanisms may drive the development of virus-negative MCC, and may contribute more generally to the development of other types of cancer. Finally, the proposed work is likely to have direct translational relevance to MCC patients, as it may lead to the identification of new therapeutic targets and yield much-needed mouse models for functional studies and preclinical trials.