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

描述（由申请人提供）：具有CD44细胞表面标记表达谱的乳腺癌细胞？或表达较高水平的醛脱氢酶1被称为癌症干细胞（CSCs）。最近的研究发现了可以进一步区分多个CD44亚群的额外标记。抗放射或化疗的细胞。比如CD44？细胞分裂后保留较高水平PKH26染料或具有较低蛋白体活性的细胞对辐射具有抗性。CD44吗?表达乳腺球体富集基因的细胞对抗雌激素和化疗具有抗性。尽管取得了巨大的进步，但很少有CSC标记物是直接的治疗靶点。因此，进一步提纯CD44？基于附加标记的细胞对于表征和靶向本质上耐药或易转移的癌细胞至关重要。我们的研究已经确定了CD44？表达炭疽毒素受体1 （ANTXR1）的细胞富集。与管腔祖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也升高。在原发性乳腺癌中，只有一小部分乳腺癌细胞表达ANTXR1。通过其天然配体C5A激活ANTXR1，可以通过GSK3的磷酸化测量Wnt活性，并增加乳腺球形成。基于这些观察，我们假设ANTXR1是CSCs/耐药癌细胞的功能标记物，通过Wnt途径控制CSCs的自我更新。提出了两个目的：1)破坏正常和癌性乳腺上皮细胞中的ANTXR1信号，研究干细胞和对化疗的敏感性。2)探讨乳腺癌中ANTXR1阳性细胞是否富集Wnt活性，癌细胞中ANTXR1和Wnt通路分子的共同表达是否与疾病参数相关。第一个目的，我们将使用ANTXR1拮抗剂，如对ANTXR1具有高亲和力的突变型炭疽保护抗原或ANTXR1的可溶性细胞外结构域，在体外和体内研究对乳房球形成能力、自我更新和对化疗药物多西他赛敏感性的影响。在第二个目标中，我们将通过免疫组化检测原发性乳腺癌中ANTXR1、phospho-LRP6 （Wnt受体）和核2-catenin的表达，并将表达与乳腺癌亚型、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常规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。先前关于ANTXR1在癌症中的研究主要集中在肿瘤相关的内皮细胞上。本研究将在CSC假说和Wnt通路在癌细胞中的调节的背景下描述其在癌细胞中的功能。在此基础上，我们将进一步提出R01类型的建议，重点关注与ANTXR1:LRP6结合并抑制癌细胞中Wnt信号传导的小分子。

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.