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

描述（由申请人提供）：癌症进展是由遗传突变、表型变异和选择的进化过程驱动的。 因此，癌细胞的遗传背景不断变化。 遗传背景将决定未来可以耐受和选择的突变的性质。 特定新突变的影响将取决于它们发生的遗传背景。 因此，预期突变的时间顺序会影响癌症表型。 然而，突变的时间如何影响癌症表型仍然是一个很大程度上没有答案的问题。 提出这个挑衅性的问题（PQB 5）是重要的，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，而且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学方面至关重要，但很少用于解决PQB 5。 这是由于常用的基因工程方法固有的限制;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，要么不限于初期的癌细胞。 目前进展的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。 我们建议通过开发基于lox-neo/stop-lox FlpO-ERT 2转基因的新型小鼠癌症模型来克服这一障碍。 该转基因编码FlpO重组酶ERT 2雌激素受体融合蛋白，其活性是他莫昔芬诱导的。 将转基因与组织特异性Cre转基因、癌症起始突变的floxed等位基因和继发突变的frted等位基因组合。 Cre表达产生癌症起始突变并从lox-neo/stop-lox FlpO-ERT 2中去除neo/stop盒，将其表达限制于起始的癌细胞。 FlpO-ERT 2活性然后可以通过他莫昔芬给药在实验控制的时间诱导，通过删除frted基因等位基因产生二次突变。 该小鼠模型可用于确定以确定和可控的时间顺序发生的2个突变如何影响体内癌症表型。 我们将使用这种小鼠模型来测试Pten和Rb 1突变的时间是否会改变前列腺癌的表型。 PTEN和RB 1突变在人类前列腺癌中很常见，其中PTEN丢失发生在早期，RB 1丢失发生在晚期。 RB 1的后期损失令人困惑，因为它的早期损失是 已知会引发其他人类癌症 目前尚不清楚PTEN和RB 1缺失是否协同驱动前列腺癌进展，或者突变的时间模式是否影响前列腺癌表型。 我们假设Pten和Rb 1突变的时间将改变体内前列腺癌的表型，因为这些突变的影响取决于遗传背景。 提出了两个具体的目标，以创建lox-neo/stop-lox FlpO-ERT 2等位基因，使用它来改变小鼠前列腺癌模型中Pten和Rb 1突变的时间，并表征这些突变对体内前列腺癌表型的影响。

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.