(PQB5) Does the timing of Pten and Rb1 mutation affect prostate cancer phenotypes

描述（由申请人提供）：癌症的进展是由基因突变、表型变异和选择的进化过程驱动的。因此，癌细胞的遗传背景是不断变化的。遗传背景将决定未来突变的性质，这些突变可以被容忍和选择。特定新突变的影响将取决于它们发生的遗传背景。因此，突变的时间顺序预计会影响癌症表型。然而，突变的时间如何影响癌症表型仍然是一个悬而未决的问题。回答这个具有争议性的问题（PQB5）具有重要意义，因为癌症预后和有效的个性化癌症治疗不仅取决于特定癌症中存在的突变的性质，还取决于它们获得的顺序。基因工程小鼠癌症模型在阐明癌症的遗传病因方面至关重要，但很少用于解决PQB5问题。这是由于通常使用的基因工程方法固有的局限性；当产生多个突变时，突变的顺序要么是未知的，要么是不可控制的，要么是不局限于早期癌细胞。目前进展的一个障碍是小鼠癌症模型的可用性，该模型允许精确控制多个基因突变的时间和顺序。我们建议通过开发一种基于lox-neo/stop-lox FlpO-ERT2转基因的新型小鼠癌症模型来克服这一障碍。该基因编码FlpO重组酶，ERT2雌激素受体融合蛋白，其活性可由他莫昔芬诱导。该转基因与组织特异性Cre转基因、癌症起始突变的弯曲等位基因和继发突变的弯曲等位基因结合。Cre的表达产生癌症启动突变，并从lox-neo/stop-lox FlpO-ERT2中移除neo/stop盒，限制其在启动癌细胞中的表达。然后，在实验控制的时间内，通过给药他莫昔芬来诱导FlpO-ERT2活性，通过删除前置基因等位基因来产生继发性突变。该小鼠模型可用于确定在确定和可控的时间顺序中发生的2个突变如何影响体内的癌症表型。我们将使用该小鼠模型来测试Pten和Rb1突变的时间是否会改变前列腺癌的表型。PTEN和RB1突变在人类前列腺癌中很常见，PTEN丢失发生早，RB1丢失发生晚。晚期RB1的丢失和早期一样令人费解

DESCRIPTION (provided by applicant): Cancer progression is driven by an evolutionary process of genetic mutation, phenotypic variation, and selection. The genetic background of cancer cells is thus continuously changing. Genetic background will dictate the nature of future mutations that can be tolerated and selected for. The effects of specific new mutations will depend on the genetic background in which they occur. Thus the temporal order of mutations is expected to influence cancer phenotype. How the timing of mutation affects cancer phenotypes remains a largely unanswered question, however. Answering this provocative question (PQB5) is significant because cancer prognosis and effective personalized cancer treatment will depend not only on the nature of mutations present within a particular cancer, but also on the order in which they are acquired. Genetically engineered mouse cancer models have been vital in elucidating the genetic etiology of cancer, but have rarely been used to address PQB5. This is due to limitations inherent in commonly used genetic engineering methodology; when multiple mutations are created, the order of mutations is either unknown, is not controllable, or is not restricted to incipient cancer cells. A current barrier to progress is the availability of mouse cancer models that allow precise control over the timing and order of multiple genetic mutations. We propose to overcome this barrier by developing a novel mouse cancer model based on a lox-neo/stop-lox FlpO-ERT2 transgene. This transgene encodes an FlpO recombinase, ERT2 estrogen receptor fusion protein whose activity is tamoxifen inducible. The transgene is combined with tissue specific Cre transgenes, floxed alleles of the cancer initiating mutation, and frted alleles of the secondary mutation. Cre expression creates the cancer initiating mutation and removes the neo/stop cassette from lox-neo/stop-lox FlpO-ERT2, restricting its expression to initiated cancer cells. FlpO-ERT2 activity can then be induced at experimentally controlled times by tamoxifen administration, creating a secondary mutation by deleting frted gene alleles. This mouse model can be used to determine how 2 mutations occurring in a defined and controllable temporal order affect cancer phenotypes in vivo. We will use this mouse model to test whether the timing of Pten and Rb1 mutation alters prostate cancer phenotype. PTEN and RB1 mutation are common in human prostate cancer, with PTEN loss occurring early and RB1 loss occurring late. The late loss of RB1 is puzzling as its early loss is known to initiate other human cancers. It is unknown whether PTEN and RB1 loss cooperate to drive prostate cancer progression or whether the temporal pattern of mutation influences prostate cancer phenotype. We postulate that the timing of Pten and Rb1 mutation will alter prostate cancer phenotypes in vivo because the effects of these mutations are dependent on genetic background. Two specific aims are proposed to create the lox-neo/stop-lox FlpO-ERT2 allele, to use it to alter the timing of Pten and Rb1 mutation in the mouse prostate cancer model, and to characterize the effects of these mutations on prostate cancer phenotypes in vivo.