Endocrine response in invasive lobular carcinoma is characterized by unique estrogen-mediated gene expression and de novo tamoxifen resistance (SUM44)

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer that is frequently associated with favorable outcomes, as ~90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that ILC patients receiving adjuvant endocrine therapy may not benefit from improved outcomes versus other breast cancer patients. Based on these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome in vitro via gene expression microarray analyses and ER ChIP-Seq. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. Response to endocrine therapy was also examined in ILC cell lines. We identified 915 genes that were uniquely E2-regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also regulated in vivo in HCI-013. We observed that MM134 were de novo tamoxifen resistant, and were induced to grow by 4-hydroxytamoxifen, as well as other anti-estrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required FGFR1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance. Cells were hormone deprived by replacing growth medium with IMEM+2% charcoal stripped serum for 3 days. Following deprivation, cells were treated for 3 or 24 hours with 1nM E2 or vehicle (0.01% EtOH) in biological quadruplicate. Following treatment, cells were lysed and RNA was harvested using the Illustra RNAspin Mini kit (GE Health). cRNA synthesis and labeling was performed using the Ambion MessageAmp Premier Kit (Life Technologies), and cRNA was hybridized to U133A 2.0 arrays (Affymetrix, Santa Clara, CA). cRNA synthesis and labeling, hybridization, and scanning were performed by the University of Pittsburgh Cancer Biomarkers Facility.

浸润性小叶癌（Invasive lobular carcinoma, ILC）是乳腺癌的一种组织学亚型，通常预后较好，约90%的ILC表达雌激素受体（estrogen receptor, ER）。然而近期的回顾性分析显示，与其他乳腺癌患者相比，接受辅助内分泌治疗的ILC患者并未从预后改善中获益。基于上述观察，我们对ILC模型中的ER功能及内分泌应答进行了表征。本研究采用ER阳性的ILC细胞系MDA MB 134VI（简称MM134）与SUM44PE，通过基因表达芯片分析及ER染色质免疫沉淀测序（ChIP-Seq）在体外检测ER调控的转录组。同时，在患者来源的ILC异种移植瘤HCI-013体内评估雌激素应答。我们还在ILC细胞系中检测了对内分泌治疗的应答情况。研究共鉴定出915个在ILC细胞系中受雌二醇（E2）特异性调控的基因，且其中部分基因在HCI-013体内也存在调控现象。我们发现MM134细胞天生对他莫昔芬耐药，且4-羟基他莫昔芬及其他抗雌激素药物可作为部分激动剂促进其生长。该生长过程伴随他莫昔芬对ER介导的基因表达的激动活性。尽管他莫昔芬可诱导细胞增殖，但MM134细胞的存活依赖FGFR1信号通路，且对内分泌治疗联合FGFR1抑制的方案敏感。我们的研究结果表明，ER在ILC细胞中调控独特的基因表达程序，这与他莫昔芬诱导此类细胞增殖的能力相关。使用FGFR1抑制剂靶向生长因子或可阻断ILC存活所需的信号通路，进而逆转他莫昔芬耐药。实验中，我们将细胞更换为IMEM培养基+2%活性炭吸附血清培养3天，以剥夺激素环境。激素剥夺后，以1nM E2或溶剂（0.01%乙醇）处理细胞3小时或24小时，每组设置4个生物学重复。处理结束后裂解细胞，采用Illustra RNAspin Mini试剂盒（GE Health）提取RNA。采用Ambion MessageAmp Premier试剂盒（Life Technologies）进行cRNA合成与标记，随后将cRNA与U133A 2.0芯片（Affymetrix, 加州圣克拉拉）进行杂交。cRNA合成、标记、杂交及扫描流程均由匹兹堡大学癌症生物标志物中心完成。