Investigating the role of p73 and its isoforms in tumorigenesis and metastasis

描述（由申请人提供）：尽管p53长期以来被认为是一种肿瘤抑制基因，但其最近发现的家族成员p73是否也是一种肿瘤抑制基因仍存在争议。来自一些人类肿瘤的数据，包括肺腺癌、膀胱移行细胞癌、骨肉瘤、乳腺腺癌和骨髓性白血病，表明p73的缺失与肿瘤的形成有关，而其他数据表明p73过表达，因此作为癌基因。关于p73作用的许多争议是由于存在许多具有潜在相反功能的p73异构体，并且缺乏区分它们的抗体。已经产生了p73缺失小鼠，但这些小鼠大多数在大约3周龄时死亡，这妨碍了肿瘤分析。我们之前已经证明，活到成年的p73-/-小鼠患肺腺癌的频率很高（60%）。有趣的是,p53 + / -;P73 +/-小鼠发展出复杂的肿瘤谱，包括肺腺癌，其中许多是转移性的。这表明p73在肿瘤抑制和转移中的作用。我们希望确定p73及其异构体如何调节导致肿瘤发生和转移的细胞过程。然而，由于该基因存在多种亚型，因此对p73的研究具有挑战性。这些同工异构体主要分为两大类：具有反活化结构域的反活化（TA）同工异构体和？N个同工异构体，缺少反激活结构域。这些异构体在癌症中的作用尚不清楚，来自人类肿瘤的数据是相关的。来自其他实验室的数据表明，TA同工型的活性更像p53，而？N亚型对p53、TAp63和TAp73起显性阴性作用。有趣的是，最近的一些报告表明？Np73本身可以反激活靶基因，并且在生理水平上表达该亚型不会导致细胞生长增加。这表明？N异构体除了促进活性外，还可能具有抑制肿瘤的作用。我们假设TAp73抑制肿瘤发生和转移，而？Np73可能根据细胞背景抑制或促进肿瘤发生。本应用的特定目的旨在阐明p73及其同工型在肿瘤抑制和转移中的作用，使用基因工程小鼠携带TA或？p73的N个同工异构体。现有的p73-/-小鼠模型不适合我们的研究，因为这些小鼠缺乏p73的所有异构体，使我们无法确定每种异构体对肿瘤发生和转移的遗传贡献，并且因为大多数p73-/-小鼠在生命早期（~ 3周龄）死亡，因此很难理解成年小鼠p73缺失的后果。我们计划生成新的鼠标模型来克服这些限制。本建议的具体目标是：1。确定p73的哪个异构体(TAp73和/或？Np73)在肿瘤抑制和转移中发挥作用，通过表征小鼠原代细胞分别表达这些亚型。2. 确定p73的哪个异构体(TAp73和/或？Np73)在肿瘤发生的小鼠体内模型和人类患者肿瘤样本中发挥肿瘤抑制和转移作用。3. 利用小鼠肺腺癌模型和人类患者肿瘤样本，寻找p73及其同工异构体在肿瘤发生、肿瘤进展和转移过程中的直接转录靶点基因。这些同种异构体特异性p73敲除小鼠的产生以及小鼠和人肺肿瘤的分析将清楚地定义TA和？np73在肿瘤发生和转移中的作用。该提案的数据将有助于p53/p73通路突变的癌症患者的新疗法。公共卫生相关性：p53在人类肿瘤中经常发生突变。p53家族成员p73在肿瘤发生中的作用尚不清楚。p73在肿瘤发生中的功能一直难以确定，因为存在多个功能明显相反的亚型。这项拨款提案的目标是使用我的实验室产生的基因工程小鼠，分别表达这些同种异构体，以确定它们在肿瘤发生和转移中的功能。一种全基因组的方法将用于确定p73及其在肿瘤发生和转移中的同种异构体的直接靶点。此外，p73亚型和靶点的状态将在人类肺肿瘤中被确定，并将与p53状态和对化疗的反应相关。最后，p73靶点在转移中的功能将通过体外和体内技术来确定。这些研究将揭示p73亚型在肿瘤发生和转移中的功能，并对p53/p73通路突变患者具有重要的临床意义。

DESCRIPTION (provided by applicant): Although p53 has long been known as a tumor suppressor, whether its recently discovered family member, p73, is also a tumor suppressor gene remains controversial. Data from some human tumors, including lung adenocarcinoma, transitional cell carcinoma of the bladder, osteosarcoma, mammary adenocarcinoma and myelogenous leukemia, indicate that loss of p73 correlates with tumor formation while other data has shown that it is overexpressed and thus behaves as an oncogene. Much of the controversy regarding the role of p73 is due to the existence of numerous p73 isoforms with potentially opposing functions and the lack of antibodies to distinguish between them. p73-null mice have been generated, but most of these mice die at approximately 3 weeks of age, which precludes tumor analysis. We have shown previously that p73-/- mice that live to adulthood develop lung adenocarcinomas at high frequency (60%). Interestingly, p53+/-;p73+/- mice develop a complex tumor spectrum including lung adenocarcinoma, many of which are metastatic. This points to a role for p73 in tumor suppression and metastasis. We wish to determine how p73 and its isoforms may regulate cellular processes that lead to tumorigenesis and metastasis. Study of p73 is challenging, though, because of the existence of multiple isoforms of this gene. These isoforms fall into two main categories-the transactivation (TA) isoforms, which have a transactivation domain, and the ?N isoforms, which lack the transactivation domain. The role of these isoforms in cancer is unclear, and data from human tumors are correlative. Data from other labs have shown that the TA isoforms have activities more like those of p53 whereas the ?N isoforms act as dominant-negatives against p53, TAp63, and TAp73. Interestingly, some recent reports indicate that ?Np73 itself can transactivate target genes and that expression of this isoform in cells at physiologic levels does not lead to increased cellular growth. This indicates that the ?N isoform may have tumor suppressing in addition to promoting activities. We hypothesize that TAp73 suppresses tumorigenesis and metastasis whereas ?Np73 may suppress or promote tumorigenesis depending on cellular context. The Specific Aims in this application are designed to elucidate the roles of p73 and its isoforms in tumor suppression and metastasis using genetically engineered mice carrying conditional knockout alleles of either the TA or ?N isoforms of p73. The existing p73-/- mouse models are not suitable for our research because these mice lack all isoforms of p73, making it impossible for us to determine the genetic contribution of each isoform to tumorigenesis and metastasis, and because most p73-/- mice die early in life (~ 3 weeks of age), making it difficult to understand the consequences of p73 loss in adult mice. We plan to generate new mouse models to overcome these limitations. The Specific Aims of this proposal are: 1. To determine which isoform(s) of p73 (TAp73 and/or ?Np73) play a role in tumor suppression and metastasis by characterizing primary mouse cells expressing each of these isoforms individually. 2. To determine which isoform(s) of p73 (TAp73 and/or ?Np73) play a role in tumor suppression and metastasis by characterizing in vivo mouse models of tumorigenesis and human patient tumor samples. 3. To identify genes that are direct transcriptional targets of p73 and its isoforms in the process of tumorigenesis, tumor progression and metastasis using a mouse model of lung adenocarcinoma and human patient tumor samples. The generation of these isoform specific p73 knockout mice and the analyses of mouse and human lung tumors will clearly define the mechanisms employed by TA and ?N p73 in tumorigenesis and metastasis. Data from this proposal will aid in novel therapies for cancer patients with mutations in the p53/p73 pathway. PUBLIC HEALTH RELEVANCE: p53 is frequently mutated in human tumors. The role of the p53 family member, p73, in tumorigenesis is poorly understood. The function of p73 in tumorigenesis has been difficult to determine due to the existence of multiple isoforms with apparently opposing functions. The goals of this grant proposal are to use genetically engineered mice generated in my laboratory that express these isoforms individually to determine their function in tumorigenesis and metastasis. A genome wide approach will be used to identify direct targets of p73 and its isoforms in tumorigenesis and metastasis. Additionally, the status of the p73 isoforms and targets will be ascertained in human lung tumors and will be correlated with p53 status and response to chemotherapy. Lastly, the function of p73 targets in metastasis will be determined using in vitro and in vivo techniques. These studies will unveil the functions of the p73 isoforms in tumorigenesis and metastasis and have important clinical implication for patients with mutations in the p53/p73 pathway.