Characterization of prostatic stem cells and prostate cancer-initiating cells

描述(申请人提供)：前列腺癌是美国男性癌症相关死亡的第二大原因。我们已经使用SCA-1表面抗原增强了前列腺干细胞的活性，并证明了具有丰富干细胞活性的细胞是肿瘤启动的靶点。我们的长期目标是充分鉴定前列腺癌中干细胞和致癌细胞的特性。这项研究将为干细胞生物学和癌症生物学的研究提供一般性的见解。 我们的具体目标是：(1)全面鉴定前列腺干细胞。(A)我们将利用分离的前列腺细胞再生系统研究干细胞是否存在于特定的前列腺上皮细胞谱系中。我们将(I)分离单个谱系并测试其再生能力，以及(Ii)永久标记单个谱系并确定其后代的谱系状态。这可以通过建立前列腺基底细胞特异性绿色荧光蛋白标记的转基因小鼠模型或通过受前列腺谱系特异性启动子调控的Cre-loxP标记系统来实现。(B)我们将继续使用表面抗原筛选分离的前列腺细胞中表面抗原的表达，并利用再生系统鉴定具有丰富干细胞活性的部分(S)。(2)前列腺癌始发细胞的鉴定。(A)我们将评估基底细胞和腔细胞对致癌转化的敏感性。我们将通过用慢病毒感染PTENIoxp/loxP转基因小鼠模型中的前列腺上皮细胞来诱导单个谱系的Pten缺失，慢病毒表达由谱系特异性启动子调控的Cre重组酶。被感染的细胞将在再生系统中进行测试，以确定哪些细胞谱系(S)发生了转化。(B)我们将确定在Aim1B中分离的每个细胞群对单一致癌刺激或联合致癌刺激诱导的恶性转化的敏感性。来自野生型和p53-/-小鼠的代表干细胞、短期祖细胞和终末分化细胞的细胞部分将被慢病毒感染，这些慢病毒介导不同的致癌信号，如myc、pRb家族蛋白的失活、PTEN-AKT信号通路的扰动等。被感染的细胞将被显微注射到免疫缺陷的宿主小鼠前列腺中，或在再生系统中进行测试，以确定它们引发癌症的能力。

DESCRIPTION (provided by applicant): Prostate cancer is the second-leading cause of the cancer-related death in men in the United States. We have enriched prostate stem cell activity using the Sca-1 surface antigen and demonstrated that cells with enriched stem cell activity serve as a target for tumor initiation. Our long-term goals are to fully characterize the identity of stem cells and cancer-initiating cells in prostate. This study will provide general insights for the studies on stem cell biology and cancer biology. Our specific aims are: (1) Fully characterize the prostatic stem cells. (A) We will investigate whether stem cells reside in a specific prostatic epithelial cell lineage using the dissociated prostate cell regeneration system. We will (i) isolate individual lineages and test their regenerative capacity, and (ii) permanently mark individual lineages and determine the lineage status of their progeny. These can be achieved by creating a prostate basal cell-specific green fluorescence protein-marked transgenic mouse model or through the Cre-loxp marking system regulated by prostate lineage-specific promoters. (B) We will continue to screen the expression of surface antigens in prostate, fractionate prostate cells using surface antigens and identify the fraction(s) with enriched stem cell activity using the regeneration system. (2) Identify the prostate cancer-initiating cells. (A) We will evaluate the susceptibility of basal cells and luminal cells to oncogenic transformation. We will induce Pten deletion in individual lineages by infecting prostate epithelial cells from the PTENIoxp/loxp transgenic mouse model with lentivirus that express the Cre recombinase regulated by lineage-specific promoters. Infected cells will be tested in the regeneration system to determine which cell lineage(s) have been transformed. (B) We will determine the susceptibility of each cell population fractionated in Aim1 B to malignant transformation induced by single or a combination of oncogenic stimuli. Cell fractions from the wild type and P53-/- mice that represent stem cells, short-term progenitor cells and terminally differentiated cells will be infected with lentivirus that mediate distinct oncogenic signals, such as myc, inactivation of the pRB family proteins, perturbations in the PTEN-AKT signaling pathway and others. The infected cells will be microinjected into immunodeficient host mouse prostate or tested in the regeneration system to determine their capacity to initiate cancer.