Anthrax Toxin Receptor as a marker and target of breast cancer stem cells

描述(申请人提供)：细胞表面标志物表达谱CD44的乳腺癌细胞？或表达较高水平的乙醛脱氢酶1被描述为癌症干细胞(CSCs)。最近的研究已经发现了更多的标记，可以进一步区分CD44？耐受辐射或化疗的细胞。例如，CD44？细胞分裂后保持较高水平的PKH26染料或较低的蛋白质小体活性的细胞对辐射具有抵抗力。CD44？表达乳房浓缩基因的细胞对抗雌激素和化疗具有抵抗力。尽管取得了如此巨大的进展，但很少有CSC标记物是直接治疗的靶点。因此，CD44的进一步提纯？基于其他标记物的细胞对于表征和靶向内在耐药或易转移的癌细胞是必不可少的。我们的研究已经确定了CD44的一个亚群？富含炭疽毒素受体1(ANTXR1)的细胞。与腔前体细胞或成熟细胞相比，具有干细胞表型的原代乳腺上皮细胞中ANTXR1的表达也升高。在原发乳腺癌中，只有一小部分乳腺癌细胞表达ANTXR1。ANTXR1通过其天然配体C5a的激活增加了Wnt的活性，通过GSK3的磷酸化来衡量，并增加了乳房的形成。基于这些观察结果，我们推测ANTXR1是CSCs/耐药癌细胞的一个功能标志，它通过Wnt途径控制CSCs的自我更新。我们提出了两个目标：1)阻断正常和癌变乳腺上皮细胞的ANTXR1信号，并研究干细胞和化疗的敏感性。2)研究乳腺癌组织中ANTXR1阳性细胞是否富含Wnt活性，以及ANTXR1和Wnt通路分子在癌细胞中的共表达是否与疾病参数有关。对于第一个目标，我们将使用ANTXR1拮抗剂，如与ANTXR1高亲和力的突变型炭疽保护抗原或ANTXR1的可溶性胞外区，在体内外观察其对乳房形成能力、自我更新和对化疗药物多西紫杉醇敏感性的影响。在第二个目标中，我们将通过免疫组织化学方法检测ANTXR1、磷酸化LRP6(Wnt受体)和核2-连环素在乳腺癌中的表达，并将其与乳腺癌亚型、淋巴结状态、分级和Oncotype DX复发评分相关联。这项研究的结果将对预测对传统治疗的反应以及开发针对耐药乳腺癌的治疗方法产生重大影响。以往关于ANTXR1在癌症中的研究主要集中在肿瘤相关的内皮细胞上。这项研究将在CSC假说的背景下描述其在癌细胞中的功能以及在癌细胞中Wnt途径的调控。基于这些结果，未来的R01型方案将被开发出来，它将专注于与ANTXR1：LRP6结合并抑制癌细胞中Wnt信号的小分子。 公共卫生相关性：该提案将确定炭疽毒素受体1(ANTXR1)在乳腺癌干细胞中的作用。与成熟细胞相比，ANTXR1在乳腺癌干细胞中的表达水平较高，可能控制了肿瘤干细胞中Wnt通路的功能。ANTXR1的抑制剂，如炭疽的突变型保护性抗原或可溶性的ANTXR1，可能会抑制ANTXR1的活性，并使肿瘤干细胞对化疗敏感。

DESCRIPTION (provided by applicant): Breast cancer cells with the cell surface marker expression profile of CD44? or expressing higher levels of aldehyde dehydrogenase 1 are described as cancer stem cells (CSCs). Recent studies have identified additional markers that can further distinguish multiple subpopulations of CD44? cells that are resistant to radiation or chemotherapy. For example, CD44? cells retaining higher levels of the dye PKH26 after cell division or with lower proteosome activity are resistant to radiation. CD44? cells expressing mammosphere-enriched genes are resistant to anti-estrogen and chemotherapies. Despite this enormous progress, very few CSC markers are direct therapeutic targets. Therefore, further refining of CD44? cells based on additional markers is essential to characterize and target intrinsically drug-resistant or metastasis-prone cancer cells. Our studies have identified a subpopulation of CD44? cells enriched for the expression of anthrax toxin receptor 1 (ANTXR1). ANTXR1 was also elevated in primary breast epithelial cells with stem cell phenotype compared to luminal progenitor or mature cells. Only a subpopulation of breast cancer cells in primary breast cancer expresses ANTXR1. Activation of ANTXR1 through its natural ligand C5A increased Wnt activity as measured by phosphorylation of GSK3 and increased mammosphere formation. Based on these observations, we hypothesize that ANTXR1 is a functional marker of CSCs/drug-resistant cancer cells that controls self-renewal of CSCs through Wnt pathway. Two aims are proposed: 1) Disrupt ANTXR1 signaling in normal and cancerous breast epithelial cells and investigate stemness and sensitivity to chemotherapy. 2) Investigate whether ANTXR1-positive cells are enriched for Wnt activity in breast cancers and whether co- expression of ANTXR1 and Wnt pathway molecules in cancer cells is associated with disease parameters. For the first aim, we will use ANTXR1 antagonists such as mutant anthrax protective antigen with high affinity for ANTXR1 or soluble extracellular domain of ANTXR1 to investigate the effects on mammosphere forming ability, self-renewal and sensitivity to the chemotherapeutic drug docetaxel both in vitro and in vivo. In the second aim, we will determine ANTXR1, phospho-LRP6 (Wnt receptor) and nuclear 2-catenin expression by immunohistochemistry in primary breast cancers and correlate the expression with breast cancer subtypes, nodal status, grade, and the Oncotype DX recurrence score. Results of this study will have a significant impact in predicting response to conventional therapy as well as developing therapies against drug-resistant breast cancer. Prior studies on ANTXR1 in cancer were focused mainly on tumor-associated endothelial cells. This study will delineate its function in cancer cells in the context of CSC hypothesis as well as Wnt pathway modulation in cancer cells. Based on the results, future R01 type proposals will be developed, which will focus on small molecules that bind to ANTXR1:LRP6 and inhibit Wnt signaling in cancer cells. PUBLIC HEALTH RELEVANCE: This proposal will determine the role of anthrax toxin receptor 1 (ANTXR1) in breast cancer stem cells. ANTXR1 is expressed at a higher level in breast cancer stem cells compared to mature cells and may control the function of Wnt pathway in cancer stem cells. Inhibitors of ANTXR1, such as mutant protective antigen of anthrax or soluble ANTXR1 may inhibit ANTXR1 activity and sensitize cancer stem cells to chemotherapy.