Targeting pERK1/2 in Human Mammary Cancer Stem Cells

描述（由申请人提供）：乳腺癌是一种复杂的疾病，具有特定的形态学和临床特征。据报道，非洲裔美国人（AA）患者的健康差异很大，死亡率很高，他们患有独特的、高度侵袭性的乳房肿瘤。AA三阴性（TN）乳腺癌患者与白种人妇女相比，化疗结果更差。侵袭性TN乳腺肿瘤含有低分化细胞和表达胚胎干细胞特异性基因组。这些低分化的乳腺癌干细胞（MCSCs）是驱动乳腺癌发生和发展的最具致瘤性的细胞类型。RAS/Raf/ERK1/2信号级联被发现促进乳腺肿瘤进展的各个方面，包括侵略性行为，如高血管生成和运动。我们假设“乳腺癌干细胞通过持续的ERK1/2信号驱动AA女性的侵袭性TN乳腺肿瘤。”我们将通过以下具体目的来验证这一假设：1)确定来自AA TN乳腺肿瘤的乳腺癌干细胞是否在裸鼠体内形成侵袭性异种移植物。2)研究持续的ERK1/2是否会增加AA TN乳腺肿瘤中富含乳腺癌干细胞的乳腺球的侵袭行为。3)研究抑制ERK1/2信号是否能有效减弱AA TN乳腺肿瘤中乳腺癌干细胞的侵袭性。利用荧光活化细胞分选（FACS）从TN - AA和高加索乳腺肿瘤中富集MCSCs (Lin-/CD44+/CD24- /ALDH1+)，将其注射到裸鼠体内形成异种移植。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）。MCSCs也将作为乳腺微球富集，并分析perk1 /2介导的对细胞运动和血管生成刺激因子表达的影响。Nup153 shRNA将减弱MCSCs中的ERK1/2信号，并将在体内和体外分析其对促瘤行为的影响。进一步了解ERK1/2信号在AA患者MCSCs中的关键作用，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. 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.