Simmons Matthew J, Serra Ryan, Hermance Nicole, Kelliher Michelle A
Breast Cancer Res. 2012 Sep 19;14(5):R126. doi: 10.1186/bcr3321.
NOTCH activation has been recently implicated in human breast cancers, associated with a poor prognosis, and tumor-initiating cells are hypothesized to mediate resistance to treatment and disease relapse. To address the role of NOTCH1 in mammary gland development, transformation, and mammary tumor-initiating cell activity, we developed a doxycycline-regulated mouse model of NOTCH1-mediated mammary transformation.
Mammary gland development was analyzed by using whole-mount analysis and by flow cytometry in nulliparous transgenic mice maintained in the presence/absence of doxycycline (or intracellular NOTCH1). Mammary tumors were examined histologically and immunophenotyped by staining with antibodies followed by flow cytometry. Tumors were transplanted into mammary fat pads under limiting dilution conditions, and tumor-initiating cell frequency was calculated. Mammary tumor cells were also plated in vitro in a tumorsphere assay in the presence/absence of doxycycline. RNA was isolated from mammary tumor cell lines cultured in the presence/absence of doxycycline and used for gene-expression profiling with Affymetrix mouse arrays. NOTCH1-regulated genes were identified and validated by using quantitative real-time polymerase chain reaction (PCR). Mammary tumor-bearing mice were treated with doxycycline to suppress NOTCH1 expression, and disease recurrence was monitored.
Similar to published studies, we show that constitutive expression of human intracellular NOTCH1 in the developing mouse mammary gland inhibits side branching and promotes luminal cell fate. These mice develop mammary adenocarcinomas that express cytokeratin (CK) 8/18. In vivo limiting-dilution analyses revealed that these mammary tumors exhibit functional heterogeneity and harbor a rare (1/2,978) mammary tumor-initiating cell population. With this dox-regulated NOTCH1 mammary tumor model, we demonstrate that NOTCH1 inhibition results in mammary tumor regression in vivo and prevents disease recurrence in four of six tumors tested. Consistent with the in vivo data, NOTCH1 inhibition reduces mammary tumorsphere activity in vitro. We also identify the embryonic stem cell transcription factor Nanog as a novel NOTCH1-regulated gene in tumorspheres and in mouse and human breast cancer cell lines.
These data indicate that NOTCH1 inhibition results in mammary tumor regression in vivo and interferes with disease recurrence. We demonstrate that NOTCH1-transformed mouse mammary tumors harbor a rare mammary tumor-initiating population and that NOTCH1 contributes to mammary tumor-initiating activity. This work raises the possibility that NOTCH therapeutics may target mammary tumor-initiating cells in certain human breast cancer subtypes.
NOTCH激活最近被认为与人类乳腺癌有关，与预后不良相关，并且肿瘤起始细胞被认为介导了对治疗的抗性和疾病复发。为了研究NOTCH1在乳腺发育、转化和乳腺肿瘤起始细胞活性中的作用，我们构建了一种强力霉素调控的NOTCH1介导的乳腺转化小鼠模型。
通过整体分析和流式细胞术，对在有/无强力霉素（或细胞内NOTCH1）的情况下饲养的未生育转基因小鼠的乳腺发育进行分析。通过组织学检查和用抗体染色后进行流式细胞术对乳腺肿瘤进行免疫表型分析。在有限稀释条件下将肿瘤移植到乳腺脂肪垫中，并计算肿瘤起始细胞频率。在有/无强力霉素的情况下，也在体外肿瘤球测定中接种乳腺肿瘤细胞。从在有/无强力霉素的情况下培养的乳腺肿瘤细胞系中分离RNA，并用于Affymetrix小鼠阵列的基因表达谱分析。通过定量实时聚合酶链反应（PCR）鉴定和验证NOTCH1调控的基因。用强力霉素处理荷乳腺肿瘤小鼠以抑制NOTCH1表达，并监测疾病复发情况。
与已发表的研究相似，我们表明在发育中的小鼠乳腺中组成型表达人细胞内NOTCH1会抑制侧支分支并促进管腔细胞命运。这些小鼠会发生表达细胞角蛋白（CK）8/18的乳腺腺癌。体内有限稀释分析表明，这些乳腺肿瘤表现出功能异质性，并含有罕见的（1/2,978）乳腺肿瘤起始细胞群体。利用这种强力霉素调控的NOTCH1乳腺肿瘤模型，我们证明NOTCH1抑制会导致体内乳腺肿瘤消退，并在六个测试肿瘤中的四个中预防疾病复发。与体内数据一致，NOTCH1抑制会降低体外乳腺肿瘤球活性。我们还鉴定出胚胎干细胞转录因子Nanog是肿瘤球以及小鼠和人类乳腺癌细胞系中一种新的NOTCH1调控基因。
这些数据表明NOTCH1抑制会导致体内乳腺肿瘤消退并干扰疾病复发。我们证明NOTCH1转化的小鼠乳腺肿瘤含有罕见的乳腺肿瘤起始群体，并且NOTCH1有助于乳腺肿瘤起始活性。这项工作提出了NOTCH疗法可能靶向某些人类乳腺癌亚型中的乳腺肿瘤起始细胞的可能性。
