Targeting Notch, PI3K-AKT and other novel pathways in breast cancer stem cells

描述（由申请人提供）：尽管对全身治疗有初始反应，但许多患者随时间复发。一种解释是，具有致瘤潜力的罕见癌症干细胞亚群本质上对治疗具有抗性。与此一致，我们首次在临床上证明了人类乳腺癌患者化疗后的残留肿瘤1）富含致瘤性CD 44 +/CD 24-/低群体，2）显示出增强的乳腺球形成效率（MSFE），3）在免疫功能低下的SCID/Beige小鼠中显示出异种移植物移植物的生长增加，从而表明其致瘤性增加。我们最近从配对的人乳腺癌样本中获得的数据表明，标准治疗消除了分裂的子细胞，因此治疗后获得的样本富含CD 44 +/CD 24-/低推定的“乳腺癌干细胞”，这些干细胞具有在乳腺球培养物中自我更新的能力，并在异种移植物移植后产生肿瘤。我们已经确定了一个癌症干细胞的签名CD 44 +/CD 24-/低乳腺球形成细胞来源于人类乳腺癌活检。确定的顶级经典途径包括Notch和PI 3-AKT以及其他信号传导途径。我们现在提出了一系列临床前和临床研究，以直接测试乳腺癌干细胞可以被Notch，PI 3-AKT和其他途径的抑制剂特异性靶向的假设。我们研究的一个独特组成部分是在术前（新辅助）治疗后残留疾病的乳腺癌患者靶向治疗前后获得的人类活检样本的可用性。具体目标和研究设计1.在临床前模型中，确定抑制已识别的自我更新和治疗耐药途径是否可以改善现有的癌症治疗。从Notch和PI 3-AKT抑制剂开始，我们将使用MSFE和人乳腺癌异种移植模型确定这些抑制剂是否会改善常规治疗的疗效。接下来，我们将通过有序的基于慢病毒的shRNA文库来靶向我们的干细胞特征中差异表达的前300个基因，该文库旨在允许人类基因组中每个基因的遗传“敲低”，以及开发干细胞自我更新的高通量功能基因组测定。2.进行新的临床试验，以确定抑制干细胞自我更新和治疗耐药途径是否可以改善乳腺癌患者的现有癌症治疗。已计划进行新型干细胞自我更新抑制剂（Notch和PI 3）的临床试验。我们建议纳入常规治疗难治性晚期乳腺癌患者，因为这些女性的预期临床结局较差，并且最有可能从靶向自我更新途径的治疗中获益。3.使用来自这些临床试验的乳腺癌活检标本进行相关性研究。将使用来自这些试验的人类癌症活检进行相关研究，例如干细胞标志物和致瘤潜力的降低，以及相关通路抑制的下游效应。

DESCRIPTION (provided by applicant): Many patients relapse over time despite initial response to systemic therapy. One explanation is that a rare sub-population of cancer stem cells with tumorigenic potential is intrinsically resistant to therapy. Consistent with this, we have shown for the first time clinically in human breast cancer patients that residual tumors after chemotherapy are 1) enriched for the tumorigenic CD44+/CD24-/low population, 2) show enhanced mammosphere-forming efficiency (MSFE), and 3) display an increase in outgrowths in xenograft transplants in immunocompromised SCID/Beige mice, thus suggesting their increased tumorigenicity. Our recent data from paired human breast cancer samples indicates that standard therapy eliminates dividing daughter cells, so that samples obtained after therapy are enriched for CD44+/CD24-/low putative "breast cancer stem cells" that have the ability to self-renew in mammosphere cultures, and to give rise to tumors upon xenograft transplantation. We have identified a cancer stem cell signature of CD44+/CD24-/low mammosphere-forming cells derived from human breast cancer biopsies. The top canonical pathways identified include Notch and PI3-AKT, and other signaling pathways. We now propose a series of preclinical and clinical studies to directly test the hypothesis that breast cancer stem cells can be specifically targeted by inhibitors of the Notch, PI3-AKT and other pathways. A unique component of our studies is the availability of human biopsy samples obtained before and after targeted therapy in breast cancer patients with residual disease after preoperative (neoadjuvant) therapy. Specific Aims and Study Design 1. To determine whether suppression of identified self-renewal and treatment resistance pathways can improve existing cancer therapies in preclinical models. Beginning with Notch and PI3-AKT inhibitors, we will determine if these will improve efficacy of conventional therapy, using MSFE and human breast cancer xenograft models. Next, we will target the top ~300 genes differentially expressed in our stem cell signature by ordered lentivirus-based shRNA libraries designed to allow genetic "knockdown" of every gene in the human genome, as well as the development of high-throughput functional genomic assays of stem cell self-renewal. 2. To conduct novel clinical trials to determine whether suppressing stem cell self-renewal and treatment resistance pathways can improve existing cancer therapies in breast cancer patients. Clinical trials with novel inhibitors of stem cell self-renewal (Notch and PI3) have been planned. We propose to include patients with advanced breast cancers refractory to conventional therapy, as these women have a poor expected clinical outcome, and who are most likely to benefit from therapies targeting self-renewal pathways. 3. To perform correlative studies using breast cancer biopsy specimens from these clinical trials. Correlative studies using human cancer biopsies from these trials will be conducted, e.g. a decrease in stem cell markers and tumorigenic potential, as well as downstream effects of inhibition of the relevant pathways.