MECHANISMS OF METASTASIS IN EXPERIMENTAL PROSTATE CANCER

转移是一个复杂的生物学过程， 在分子和细胞水平上的理解。虽然差 据了解，转移通常被描述为肿瘤转移中最重要的步骤。 恶性进展，因为癌症死亡率通常直接由 扩散的转移细胞的生长。这种深刻的影响， 转移性疾病显然是前列腺癌的情况， 1994年，200，000名美国男性被确诊，38，000名美国男性死亡， 男人 最近，我们改进了小鼠前列腺重建（MPR）模型， 使用来自p53“敲除”小鼠的尿生殖窦组织作为 作为ras和myc癌基因导入的靶点。使用这个在 在体内模型系统中，我们研究了ras+myc的致瘤作用 在野生型p53、杂合p53突变体或 纯合子p53突变体。我们证明ras+ myc启动的野生型 129/Sv小鼠主要产生低频率的增生 （约10%）为非转移性癌。相比之下， 杂合p53以及纯合p53 MPR产生转移性 几乎100%的病例都是癌症。ras+myc +杂合子的研究进展 p53“敲除”MPR与完全丢失、部分丢失或 野生型等位基因的表达丧失。总的来说，我们的数据清楚地表明， 表明p53功能完全丧失导致这种肿瘤转移， 实验小鼠前列腺癌模型。有趣的是， 转移与人类前列腺中所见的非常相似，包括广泛的 转移到肺、肠系膜淋巴结、骨，偶尔 肝脏我们已经扩展了这个模型，以促进机械研究， 在分子和细胞水平上，通过产生一系列细胞系， 来源于原发部位癌以及转移性疾病 来自不同的器官部位到目前为止，我们已经证明TGF β 1 抑制所有（n=6）原发部位肿瘤衍生细胞的体外生长 细胞系（8天后平均抑制率为45+/- 9%）以及对照 尿生殖窦上皮（CUGE）细胞，但仅抑制3/6 来源于肺转移瘤的细胞系（敏感性细胞系中的平均抑制率） 线20 +/- 1%）。对TGF-β 1也有类似的差异反应， 观察集落形成和运动性测定。的结果 对TGF-β 1的交联研究表明， 在所有原发部位肿瘤中结合1、2和3型TGF-β受体- 和转移来源的细胞系表明，这些TGF-β 1 在转移来源的细胞系中诱导的反应是 受体结合我们建议使用这种新的前列腺癌转移 模型系统，以调查遗传机制的基础损失 TGF β 1反应及其与转移进展的关系。在 此外，与以下相关的纯合缺失的特定区域 转移性进展将被鉴定和克隆， 代表性差异分析和杂合性缺失区 在体内转移过程中发生的变化将使用 微卫星探针 从过去使用MPR模型系统的研究中， 可以预测，这些研究的结果也将与 人类疾病

Metastasis is a complex biological process for which there is minimal understanding at the molecular and cellular level. Although poorly understood, metastasis is often described as the most significant step in malignant progression as cancer mortality in general results directly from the growth of disseminated metastatic cells. This profound impact of metastatic disease is clearly the case in prostate cancer which will be diagnosed in 200,000 US males in 1994 and cause mortality in 38,000 US men. Recently, we have modified the mouse prostate reconstitution (MPR) model system to use urogenital sinus tissue derived from p53 "knock-out" mice as target for the introduction of the ras and myc oncogenes. Using this in vivo model system, we have studied the tumorigenic effects of ras+myc initiation in a background of wild-type p53, heterozygous p53 mutants or homozygous p53 mutants. We demonstrated that ras+myc-initiated wild-type 129/Sv mice produce predominantly hyperplasia with low frequency (approximately 10%) of focal nonmetastatic cancer. In contrast, both heterozygous p53 as well as homozygous p53 MPRs produce metastatic carcinoma in nearly 100% of the cases. Progress of ras+myc + heterozygote p53 "knock-out" MPRs correlated with either complete loss, partial loss or loss of expression of the wild-type allele. Overall, our data clearly indicate that complete loss of p53 function leads to metastasis in this experimental mouse prostate cancer model. Interestingly, the pattern of metastasis closely mimics that seen in human prostate including widespread metastasis to the lung, mesenteric lymph nodes, bone and occasionally liver. We have extended this model to facilitate mechanistic studies at the molecular and cellular level, by generating a series of cell lines derived from both primary site carcinomas as well as metastatic disease from different organ sites. We have thus far demonstrated that TGFbeta1 inhibited growth in vitro in all (n=6) primary site tumor-derived cell lines (mean inhibition of 45+/- 9% after 8 days) as well as control urogenital sinus epithelial (CUGE) cells but only inhibited 3 out of 6 cell lines derived from lung metastases (mean inhibition in sensitive lines 20 +/- 1%). Similar differential responses to TGF-beta1 were also observed for colony formation and motility assays. The results of crosslinking studies for TGF-beta1 which demonstrated specific TGF-beta1 binding for type l, 2 and 3 TGF-beta receptors in all primary site tumor- and metastasis-derived cell lines suggest that loss of these TGF-beta1 induced responses in metastasis-derived cell lines is downstream of receptor binding. We propose to use this novel prostate cancer metastasis model system to investigate the genetic mechanisms which underlie the loss of TGFbeta1 response and its relationship to metastatic progression. In addition, specific regions of homozygous deletion associated with metastatic progression will be identified and cloned using representational difference analysis and regions of loss of heterozygosity that occur during metastasis in vivo will be determined using microsatellite probes. From past studies using the MPR model system we can predict that the results of these studies will also be relevant to the human disease.