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学和临床特征相关的复杂疾病。据报道，非裔美国人（AA）患者患有独特的高度侵袭性乳腺肿瘤，存在显著的健康差异和高死亡率。与白人女性相比，AA三阴性（TN）乳腺癌患者的化疗结局更差。侵袭性TN乳腺肿瘤含有低分化细胞并表达胚胎干细胞特异性基因集。这些低分化的乳腺癌干细胞（MCSC）是最具致瘤性的细胞类型，可驱动乳腺癌的发生和进展。RAS/Raf/ERK 1/2信号级联已被发现促进乳腺肿瘤进展的各个方面，包括侵袭性行为，如高血管生成和运动性。我们假设“乳腺癌干细胞通过持续的ERK 1/2信号传导驱动AA女性的侵袭性TN乳腺肿瘤。“我们将用以下具体目的来检验这一假设：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ERK 1/2是否增加来自AA TN乳腺肿瘤的乳腺癌干细胞富集的乳腺球的侵袭行为。3)检查ERK 1/2信号传导的抑制是否有效地减弱来自AA TN乳腺肿瘤的乳腺癌干细胞的侵袭性。将使用活化细胞分选（FACS）来富集来自TNAA和高加索人乳腺肿瘤的MCSC（Lin-/CD 44 +/CD 24- /ALDH 1+），其将被注射到裸鼠中以形成异种移植物。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、CD 31、MMP 9）的表达和细胞运动性（Boyden小室试验）。MCSC也将富集为乳腺球，并将分析pERK 1/2介导的对细胞运动性和血管生成刺激因子表达的影响。通过Nup 153 shRNA将减弱MCSC中的ERK 1/2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ERK 1/2信号传导在AA患者MCSC中的关键作用的了解，并可能为治疗药物设计提供新的靶点。

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.