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

描述(申请人提供)：许多患者随着时间的推移复发，尽管对系统治疗有初步反应。一种解释是，具有致瘤潜力的罕见癌症干细胞亚群天生对治疗具有抵抗力。与此一致，我们首次在临床上表明，在人类乳腺癌患者中，化疗后的残余肿瘤1)富含致癌CD44/CD24-/低群体，2)显示增强的乳房形成效率(MSFE)，以及3)在免疫受损的SCID/Beige小鼠的异种移植中显示外围生长增加，从而表明它们的致瘤性增加。我们最近从配对的人类乳腺癌样本中获得的数据表明，标准疗法消除了子代细胞的分裂，因此治疗后获得的样本可以丰富CD44/CD24-/低假设的“乳腺癌干细胞”，这些干细胞具有在乳房培养中自我更新的能力，并在异种移植后产生肿瘤。我们已经从人类乳腺癌活检组织中鉴定出CD44/CD24-/低乳房形成细胞的癌症干细胞特征。已发现的最典型的信号通路包括Notch和PI3-AKT，以及其他信号通路。我们现在提出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、PI3-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部分是靶向治疗前后乳腺癌患者在术前(新辅助)治疗后有残留病变时获得的人体活检样本的可用性。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抵抗途径是否可以改善现有的癌症治疗。从Notch和PI3-AKT抑制剂开始，我们将使用MSFE和人乳腺癌异种移植模型来确定这些药物是否会改善传统治疗的疗效。接下来，我们将通过有序的基于慢病毒的shRNA文库来定位在我们的干细胞签名中差异表达的前300个基因，该文库旨在允许对人类基因组中的每个基因进行基因敲除，以及高通量干细胞自我更新的功能性基因组分析的发展。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通路是否可以改善乳腺癌患者的现有癌症治疗。干细胞自我更新的新型抑制剂(Notch和PI3)的临床试验已经计划进行。我们建议纳入常规治疗难治的晚期乳腺癌患者，因为这些女性的预期临床结果很差，他们最有可能从针对自我更新途径的治疗中受益。3.使用这些临床试验中的乳腺癌活检标本进行相关研究。将使用这些试验中的人类癌症活检组织进行相关研究，例如干细胞标记物和致瘤潜力的减少，以及相关通路抑制的下游影响。

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.