DEVELOPMENT OF A STOCHASTIC MOUSE TO STUDY BREAST CANCER

尽管70%的死于乳腺癌的患者有溶骨性骨转移，但由于缺乏适当的动物模型来模拟原发性乳腺肿瘤向骨转移的临床经验，对骨骼转移的生物学仍然难以彻底理解。小鼠已被用作转移级联过程中若干个别步骤的模型，但用于整个过程的小鼠模型，即乳房原发肿瘤发展到转移到远处器官（如骨）的小鼠模型尚未开发。虽然，一些转基因乳腺癌小鼠模型已被证明可以广泛转移，但它们都没有转移到骨骼。新的（c-erbB2, ERBB2）原癌基因的过度表达和扩增与侵袭性人类乳腺癌的发展有关。新的原癌基因特异性表达到小鼠乳腺（在MMTV启动子的控制下）在平均潜伏期205天后诱导转移性疾病。另一方面，人们普遍认为，在肿瘤细胞从骨髓向骨扩张之前，破骨性骨吸收是必要的。在乳腺癌的情况下，这最后一步是由破骨细胞介导的，破骨细胞被局部产生的肿瘤肽甲状旁腺激素相关肽（PTH-rp）刺激。支持这一假设的一些证据是：PTH-rp阳性乳腺癌患者更容易发生骨转移。此外，超过90%的乳腺癌骨转移病例表达PTH-rp，相比之下，只有17%的乳腺癌转移到非骨部位。也有报道称，PTHrp在MDA 231人乳腺癌细胞中的表达增加了骨溶解性骨转移，而PTHrp的中和抗体则显著抑制了这种转移。由此可见，PTHrp在骨转移过程中起重要作用。综上所述，临床和实验数据都表明，如果新表达的乳腺癌细胞被改造成过度表达PTHrp，它们可能会回到骨中并转移。根据这一假设，本研究的总体目标是建立乳腺癌骨转移的随机模型，并确定在乳腺癌转移进展过程中介导ERBB2和PTHrp活性的下游靶点。我们建议在体外使用细胞培养模型和在体内通过产生表达new /PTHrp的双基因小鼠来验证这一假设。我们的具体目标是：具体目标1：利用体外小鼠细胞培养模型建立乳腺癌从乳腺转移到骨的小鼠模型，该模型被设计为过表达new和PTHrp。特异性目的2：确定PTHrp和neu在小鼠乳腺上皮细胞中的靶向表达是否促进了这些动物骨转移的形成。开发这种过度表达原癌基因neu并过量产生PTHrp的模型将最接近地模拟人类乳腺癌的进展，并为治疗药物的临床前研究提供改进的模型。它也将提供一个独特的平台来研究参与乳腺癌骨转移的部位特异性细胞事件。

Even though 70 percent patients dying with breast cancer have osteolytic bone metastasis, a thorough understanding of the biology of skeletal metastasis remains elusive due to the lack of appropriate animal models that mimic the clinical experience of metastasis of primary breast tumors to the bone. The mouse has been used as a model for a number of individual steps in the metastatic cascade, but a mouse model for the entire process i.e. development of the primary tumor in the breast to its metastases to distant organs like bone, has not been developed. Although, some transgenic mouse models of breast cancer have been shown to metastasize extensively, none of them metastasize to the bone. Overexpression and amplification of the neu (c-erbB2, ERBB2) proto-oncogene have been implicated in the development of aggressive human breast cancer. Targeted expression of the neu proto-oncogene specifically to the mouse mammary gland (under the control of a MMTV promotor) induces metastatic disease after a mean latency of 205 days. On the other hand, it is generally recognized that ostoclastic bone resorption is necessary before expansion of tumor cells from bone marrow to bone. In the case of breast cancer, this final step is mediated by osteoclasts that are stimulated by local production of the tumor peptide parathyroid hormone - related peptide (PTH-rp). Some of the evidence which supports this hypothesis are: patients with PTH-rp positive breast carcinomas are more likely to develop bone metastasis. In addition, breast carcinoma metastatic to bone express PTH-rp in greater than 90 percent of cases, compared with only 17 percent of metastases to nonbone sites. It has also been reported that expression of PTHrp in MDA 231 human breast cancer cells increased osteolytic bone metastasis while neutralising antibodies to PTHrp markedly suppressed them. Thus, PTHrp plays an important role in the process of bone metastasis. Taken together, both the clinical and experimental data suggest that if neu-expressing breast carcinoma cells are engineered to overexpress PTHrp, they may be able to home to the bone and metastasize. With this hypothesis, the overall objective of this proposal is to develop a stochastic model of breast cancer metastasis to the bone and to identify downstream targets that mediate the activity of ERBB2 and PTHrp during breast cancer metatatic progression. We propose to test this hypothesis both in vitro, using a cell culture model and in vivo by generating neu/PTHrp expressing bigenic mice. Our specific aims are: Specific Aim 1: To develop a mouse model of breast cancer metastasis from the mammary gland to the bone using an in vitro mouse cell culture model that is engineered to overexpress both neu and PTHrp. Specific Aim 2: To determine, if, targeted expression of PTHrp and neu specifically to the mouse mammary epithelial cells facilitates bone metastases formation in these animals. Development of such a model that overexpresses proto-oncogene neu and is engineered to overproduce PTHrp would most closely mimic human breast cancer progresion and provide an improved model for preclinical studies of therapeutic agents. It will also provide a unique platform to investigate cellular events involved in site-specific metastasis of breast cancer to the bone.