DEVELOPMENT OF A STOCHASTIC MOUSE TO STUDY BREAST CANCER

尽管70%的乳腺癌患者死于溶骨性骨转移，但由于缺乏合适的动物模型来模拟乳腺癌原发肿瘤转移到骨的临床经验，对骨转移的生物学特性仍难以彻底了解。小鼠已经被用来作为转移级联中的一些单独步骤的模型，但还没有开发出整个过程的小鼠模型，即乳腺原发肿瘤的发展到其转移到骨骼等远处器官的小鼠模型。虽然，一些乳腺癌转基因小鼠模型已被证明广泛转移，但没有一个转移到骨。原癌基因neu(c-erbB2，ERBB2)的过度表达和扩增与人类侵袭性乳腺癌的发生有关。靶向表达neu原癌基因到小鼠乳腺(在MMTV启动子的控制下)在平均潜伏期205天后诱导转移疾病。另一方面，人们普遍认为，在肿瘤细胞从骨髓扩展到骨之前，破骨细胞性骨吸收是必要的。对于乳腺癌来说，这最后一步是由破骨细胞介导的，破骨细胞受到肿瘤多肽甲状旁腺激素相关肽(PTH-RP)的局部产生的刺激。支持这一假设的一些证据是：PTH-RP阳性乳腺癌患者更有可能发生骨转移。此外，在转移到骨的乳腺癌中，超过90%的病例表达PTH-RP，而在转移到非骨部位的乳腺癌中，这一比例仅为17%。也有报道称，PTHrP在MDA231人乳腺癌细胞中的表达增加了溶骨性骨转移，而中和PTHrP抗体则显著抑制了它们。因此，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，如果将Neu表达的乳腺癌细胞改造为过表达PTHrP，它们可能能够附着在骨骼上并转移。有了这一假设，这项建议的总体目标是建立乳腺癌向骨转移的随机模型，并确定在乳腺癌转移过程中介导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neu/PTHrP的双基因小鼠来验证这一假说。我们的具体目标是：特定目标1：利用体外小鼠细胞培养模型建立乳腺癌从乳腺到骨的转移模型，该模型被设计为同时过表达neu和PTHrP。特定目的2：确定靶向表达PTHrP和neu的小鼠乳腺上皮细胞是否促进这些动物的骨转移形成。开发这样一种过度表达原癌基因neu并过度产生PTHrP的模型将最接近于模拟人类乳腺癌的发展，并为治疗药物的临床前研究提供一个改进的模型。它还将提供一个独特的平台，以调查涉及乳腺癌部位特异性转移到骨的细胞事件。

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.