Transcription profiling of human breast cancer brain metastases in a mouse xenograft model with vehicle of vorinostat treatment

Gene Expression Profiling of a Mouse Xenograft Model of “Triple-Negative” Breast Cancer Brain Metastases With and Without Vorinostat Treatment. Purpose: As chemotherapy and molecular therapy improve the systemic survival of breast cancer patients, the incidence of brain metastases increases. Few therapeutic strategies exist for the treatment of brain metastases because the blood-brain barrier severely limits drug access. We report the pharmacokinetic, efficacy, and mechanism of action studies for the histone deactylase inhibitor vorinostat (suberoylanilide hydroxamic acid) in a preclinical model of brain metastasis of triple-negative breast cancer. Experimental Design: The 231-BR brain trophic subline of the MDA-MB-231 human breast cancer cell line was injected into immunocompromised mice for pharmacokinetic and metastasis studies. Pharmacodynamic studies compared histone acetylation, apoptosis, proliferation, and DNA damage in vitro and in vivo. Results: Following systemic administration, uptake of [14C]vorinostat was significant into normal rodent brain and accumulation was up to 3-fold higher in a proportion of metastases formed by 231-BR cells. Vorinostat prevented the development of 231-BR micrometastases by 28% (P = 0.017) and large metastases by 62% (P < 0.0001) compared with vehicle-treated mice when treatment was initiated on day 3 post-injection. The inhibitory activity of vorinostat as a single agent was linked to a novel function in vivo: induction of DNA double-strand breaks associated with the down-regulation of the DNA repair gene Rad52. Conclusions: We report the first preclinical data for the prevention of brain metastasis of triple-negative breast cancer. Vorinostat is brain permeable and can prevent the formation of brain metastases by 62%. Its mechanism of action involves the induction of DNA double-strand breaks, suggesting rational combinations with DNA active drugs or radiation. Experiment Overall Design: We performed gene expression profiling on metastases from vehicle- or vorinostat-treated mice to determine if alterations in gene expression were observable that were consistent with the phenotypes observed. Brain metastases from five vehicle-treated mice and six 150 mg/kg vorinostat-treated mice were procured by laser capture microdissection. RNA was extracted from the captured tumor cells from each brain and two rounds of linear amplification was done. The amplified RNA from each mouse was processed separately through microarray hybridization and analysis.

有/无伏立诺他（Vorinostat）处理的三阴性乳腺癌（Triple-Negative Breast Cancer）脑转移小鼠异种移植模型（Xenograft Model）的基因表达谱分析。
研究背景：随着化疗与分子疗法不断改善乳腺癌患者的系统生存期，脑转移的发生率亦随之升高。由于血脑屏障（Blood-Brain Barrier）会严重限制药物的递送通路，目前针对脑转移的临床治疗策略仍十分有限。本研究针对三阴性乳腺癌脑转移的临床前模型，报道了组蛋白去乙酰化酶抑制剂（Histone Deacetylase Inhibitor）伏立诺他（Vorinostat，辛二酰苯胺异羟肟酸，suberoylanilide hydroxamic acid）的药代动力学（Pharmacokinetic）、疗效及作用机制相关研究结果。
实验设计：将MDA-MB-231人乳腺癌细胞系的231-BR脑亲和亚株注射至免疫缺陷小鼠体内，以开展药代动力学与肿瘤转移研究。药效动力学（Pharmacodynamic）研究分别在体外与体内环境中，对比了组蛋白乙酰化、细胞凋亡、细胞增殖及DNA损伤（DNA damage）的水平变化。
实验结果：系统给药后，[¹⁴C]标记伏立诺他可有效渗透至正常啮齿类动物脑组织，且在231-BR细胞形成的部分脑转移灶中，药物蓄积量可达正常脑组织的3倍。与赋形剂处理组小鼠相比，若于肿瘤细胞注射后第3天启动伏立诺他治疗，可分别抑制28%的231-BR微转移灶形成（P=0.017），并减少62%的大体积转移灶（P<0.0001）。伏立诺他单药的抗肿瘤转移活性与一项全新的体内功能相关：其可诱导DNA双链断裂，并伴随DNA修复基因Rad52的表达下调。
研究结论：本研究首次报道了用于预防三阴性乳腺癌脑转移的临床前数据。伏立诺他具备脑穿透性，可降低62%的脑转移灶形成率。其作用机制涉及DNA双链断裂的诱导，提示该药物可与DNA活性药物或放射疗法联合使用，具备合理的联合用药开发潜力。
整体实验设计：本研究对赋形剂或伏立诺他处理小鼠的脑转移灶开展基因表达谱分析，以验证是否可观测到与前期表型结果一致的基因表达改变。通过激光捕获显微切割（Laser Capture Microdissection）技术，获取5只赋形剂处理组小鼠及6只150mg/kg伏立诺他处理组小鼠的脑转移灶组织。从每例捕获的肿瘤细胞中提取总RNA，并进行两轮线性扩增。每只小鼠的扩增RNA均通过独立的微阵列杂交（Microarray Hybridization）与数据分析流程进行处理。