Osteoprotegerin in breast cancer cells: role in tumor growth and metastasis

描述（由申请方提供）：本研究的目的是研究分泌蛋白骨保护素（OPG）的促肿瘤作用。人乳腺癌细胞系体外表达OPG抑制肿瘤坏死因子相关凋亡诱导配体（TRAIL）介导的细胞死亡。乳腺癌细胞可能产生OPG来阻断TRAIL的作用，抵抗被杀死。我们的初步数据表明OPG也促进转移。本研究将探讨OPG在乳腺癌细胞中的表达调控，OPG对转移的影响以及OPG影响肿瘤细胞行为的机制。所产生的数据将揭示OPG抑制剂是否应该被开发为乳腺癌的治疗方法。 我们的初步数据链接OPG表达与核因子κ B（NF-？B）活性。这种联系将进行调查，以确定OPG介导的NF-？B。人类乳腺癌细胞将与NF-？测定B抑制剂和对OPG mRNA和蛋白表达的影响。为了在体内研究OPG，将用shRNA处理人乳腺癌细胞系以敲低OPG或用非编码shRNA处理人乳腺癌细胞系以产生对照细胞。然后将这些细胞用于研究鸡胚转移模型中OPG敲低对转移的后果。鸡胚模型概括了在人类肿瘤中观察到的转移阶段，并代表了获得有关促进肿瘤发生的分子因子的数据的快速方法。将细胞直接引入鸡绒毛尿囊膜上以允许原发性肿瘤形成和转移，或IV注射以专门研究转移。孵育一周后，可通过人DNA的真实的时间PCR测量人乳腺癌细胞向鸡组织的转移。这些研究将在存在和不存在TRAIL的情况下进行，以揭示OPG的抑制是否使细胞对TRAIL介导的细胞死亡敏感和/或OPG的缺乏是否减少转移，而不依赖于其与TRAIL的相互作用。为了确定OPG影响转移的机制，将分析来自鸡模型的原发性肿瘤的基因表达的变化，其将导致肿瘤促进作用。此外，将进行体外试验，以确定在存在和不存在TRAIL以及核因子-κ B受体激活剂配体（RANKL）（已知结合OPG的另一种分子）的情况下，OPG敲低对细胞凋亡、增殖、迁移和侵袭的影响。这将使我们能够确定OPG在其两种特征性配体存在下对肿瘤行为的影响。 这项研究与乳腺癌健康相关，因为它将提供证据表明，阻断OPG的治疗策略可与TRAIL或其他类型的乳腺癌治疗联合使用，以预防转移，改善预后并提高乳腺癌患者的生存率。 公共卫生相关性：本申请将研究乳腺癌细胞产生蛋白质骨保护素（OPG）促进肿瘤进展和转移的假设。这将证明OPG是否代表一种可以作为乳腺癌靶向治疗的一部分被阻断的分子。由于许多患者对目前的靶向治疗没有反应或产生耐药性，因此确定新的治疗靶点（如OPG）将增加治疗选择并改善乳腺癌患者的预后。

DESCRIPTION (provided by applicant): The goal of this study is to investigate tumor promoting effects of the secreted protein osteoprotegerin (OPG). OPG expression by human breast cancer cell lines in vitro inhibits TNF- related apoptosis inducing ligand (TRAIL)-mediated cell death. Breast cancer cells may produce OPG to block effects of TRAIL and resist being killed. Our preliminary data suggests OPG also promotes metastasis. The current study will investigate regulation of OPG expression in breast cancer cells, the impact of OPG on metastasis and the mechanisms whereby OPG impacts tumor cell behavior. Data generated will reveal whether OPG inhibitors should be developed as a therapy for breast cancer. Our preliminary data links OPG expression with nuclear factor kappa B (NF-?B) activity. This link will be investigated to determine if OPG mediates tumor promoting effects of NF-?B. Human breast cancer cells will be treated with NF-?B inhibitors and effects on OPG mRNA and protein expression measured. To study OPG in vivo, human breast cancer cell lines will be treated with shRNA to knockdown OPG or non-coding shRNA to generate control cells. These cells will then be used to investigate the consequences of OPG knockdown on metastasis in the chick embryo metastasis model. The chick embryo model recapitulates the stages of metastasis observed in human tumors and represents a rapid way to gain data regarding molecular factors that promote tumorigenesis. Cells will either be introduced directly onto the chick chorioallantoic membrane to allow primary tumor formation and metastasis or injected IV to specifically study metastasis. After one week incubation, metastasis of human breast cancer cells to chick tissues can be measured by real time PCR for human DNA. These studies will be performed in the presence and absence of TRAIL to reveal whether inhibition of OPG sensitizes cells to TRAIL-mediated cell death and/or if a lack of OPG reduces metastasis independent of its interaction with TRAIL. To determine the mechanism whereby OPG impacts metastasis, primary tumors from the chick model will be analyzed for changes in the expression of genes that would result in a tumor promoting effect. In addition in vitro assays will be performed to determine the impact of OPG knockdown on apoptosis, proliferation, migration and invasion in the presence and absence of TRAIL and also Receptor Activator of Nuclear Factor-kappaB ligand (RANKL), the other molecule known to bind OPG. This will allow us to determine the effect of OPG on tumor behavior in the presence of both of its characterized ligands. This study is of relevance to publi health as it will provide evidence that treatment strategies to block OPG could be used in combination with TRAIL or other types of breast cancer therapy to prevent metastasis, improve prognosis and increase survival in breast cancer patients. PUBLIC HEALTH RELEVANCE: The current application will investigate the hypothesis that the production of the protein osteoprotegerin (OPG) by breast cancer cells promotes tumor progression and metastasis. This will demonstrate whether OPG represents a molecule that could be blocked as part of targeted treatment for breast cancer. Since many patients do not respond or become resistant to current targeted therapies then identification of new therapeutic targets such as OPG will increase treatment options and improve prognosis for breast cancer patients.