Targeting pERK1/2 in Human Mammary Cancer Stem Cells

描述（由申请人提供）：乳腺癌是一种与特定形态和临床特征相关的复杂疾病。据报道，非裔美国人 (AA) 患者的健康状况存在显着差异，且死亡率较高，他们患有独特且高度侵袭性的乳腺肿瘤。与白人女性相比，AA 三阴性 (TN) 乳腺癌患者的化疗结果较差。侵袭性 TN 乳腺肿瘤含有分化不良的细胞并表达胚胎干细胞特异性基因集。这些分化差的乳腺癌干细胞（MCSC）是最具致瘤性的细胞类型，可驱动乳腺癌的发生和进展。研究发现 RAS/Raf/ERK1/2 信号级联可促进乳腺肿瘤进展的各个方面，包括高血管生成和运动等侵袭行为。我们假设“乳腺癌干细胞通过持续的 ERK1/2 信号传导在 AA 女性中驱动侵袭性 TN 乳腺肿瘤。”我们将通过以下具体目标来检验这一假设：1) 确定来自 AA TN 乳腺肿瘤的乳腺癌干细胞是否在裸鼠中形成侵袭性异种移植物。 2) 检查持续的 ERK1/2 是否会增加来自 AA TN 乳腺肿瘤的富含乳腺癌干细胞的乳腺球的侵袭行为。 3) 检查抑制 ERK1/2 信号传导是否能有效减弱来自 AA TN 乳腺肿瘤的乳腺癌干细胞的侵袭特性。将使用荧光激活细胞分选（FACS）从TN AA和高加索乳腺肿瘤中富集MCSC（Lin-/CD44/CD24-/ALDH1），将其注射到裸鼠中形成异种移植。这些异种移植物将用于比较两组之间的肿瘤体积、血管生成刺激因子（VEGF、CD31、MMP9）的表达和细胞运动性（Boyden Chamber Assay）。 MCSC 也将被富集为乳腺球，并且将分析 pERK1/2 介导的细胞运动和血管生成刺激因子表达的影响。 Nup153 shRNA 将减弱 MCSC 中的 ERK1/2 信号传导，并将在体外和体内分析其对肿瘤促进行为的影响。加深对 AA 患者 MCSC 中 ERK1/2 信号转导关键作用的了解，并可能为治疗药物设计提供新靶点。 公共健康相关性：该项目将测试磷酸化 ERK1/2 靶向对从非裔美国人和白种人患者中分离出的人类乳腺癌干细胞的有效性。将从废弃的患者新鲜组织样本中分离出乳腺癌干细胞，并在抑制 ERK1/2 信号传导之前和之后测试它们在裸鼠中形成肿瘤的能力。如果我们的初步数据得到验证，我们的研究可能会为临床实践提供独特的方法，通过设计专门针对乳腺癌干细胞中的 pERK1/2 的治疗药物，pERK1/2 高度稳定，并提供关键的致癌触发因素。

DESCRIPTION (provided by applicant): Breast cancer is a complex disease associated with specific morphological and clinical features. Significant health disparity and high mortality rate is reported in African American (AA) patients, who suffer from unique and highly aggressive breast tumors. AA triple negative (TN) breast cancer patients suffer worse outcomes to chemotherapy compared with Caucasian women. Aggressive TN breast tumors contain poorly differentiated cells and express embryonic stem cell specific gene sets. These poorly differentiated mammary cancer stem cells (MCSCs) are the most tumorigenic cell types that drive initiation and progression of breast cancers. RAS/Raf/ERK1/2 signaling cascade has been found to promote every aspect of breast tumor progression including aggressive behavior like high angiogenesis and motility. We hypothesize that "Mammary cancer stem cells drive aggressive TN breast tumors in AA women through sustained ERK1/2 signaling." We will test this hypothesis with the following Specific Aims: 1) Establish whether mammary cancer stem cells from AA TN breast tumors form aggressive xenografts in nude mice. 2) Examine whether sustained ERK1/2 increases aggressive behavior of mammary cancer stem cells enriched mammospheres from AA TN breast tumors. 3) Examine whether inhibition of ERK1/2 signaling is effective in attenuating aggressive properties of mammary cancer stem cells from AA TN breast tumors. Fluorescence-activated cell sorting (FACS) will be used to enrich MCSCs (Lin-/CD44+/CD24- /ALDH1+) from TN AA and Caucasian breast tumors, which will injected into the nude mice to form xenotransplants. These xenografts will be used to compare tumor volume, expression of angiogenesis stimulating factors (VEGF, CD31, MMP9), and motility of cells (Boyden Chamber Assay) between the two groups. MCSCs will also be enriched as mammospheres and pERK1/2-mediated effect on cell motility and expression of angiogenesis stimulating factors will be analyzed. ERK1/2 signaling will be attenuated in MCSCs by Nup153 shRNA and its effect on tumor promoting behavior will be analyzed both in vitro and in vivo. Increased understanding of the critical role of ERK1/2 signaling in MCSCs from AA patients and may provide novel targets for therapeutic drug design. PUBLIC HEALTH RELEVANCE: This project will test the effectiveness of phospho-ERK1/2 targeting in human mammary cancer stem cells isolated from African American and Caucasian patients. Mammary cancer stem cells will be isolated from the discarded fresh tissue samples from patient and their ability to form tumors in Nude mice will be tested before and after inhibition of ERK1/2 signaling. Our studies if validated by our preliminary data may provide unique approach to clinical practice by designing therapeutic drugs to specifically target pERK1/2 in mammary cancer stem cells, which are highly stable and provide key oncogenic trigger.