Endocrine response in invasive lobular carcinoma is characterized by unique estrogen-mediated gene expression and de novo tamoxifen resistance (MM134)

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+10% 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，其通常预后较好，约90%的ILC表达雌激素受体（estrogen receptor, ER）。不过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患者并未获得更优的预后获益。基于上述观察结果，本研究对ILC模型中的ER功能与内分泌应答进行了系统表征。

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。与此同时，在患者来源的ILC异种移植模型HCI-013中体内评估雌激素应答，并在ILC细胞系中检测其对内分泌治疗的应答。

本研究鉴定出915个仅在ILC细胞系中受雌激素（E2）调控的基因，其中部分基因在HCI-013模型体内也存在表达调控。研究发现MM134细胞存在原发性他莫昔芬耐药，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增殖，该增殖过程伴随他莫昔芬对ER介导的基因表达的激动活性。尽管他莫昔芬可促进细胞增殖，但MM134细胞的存活依赖成纤维细胞生长因子受体1（FGFR1）信号通路，且对内分泌治疗联合FGFR1抑制敏感。本研究观察到ER在ILC细胞中调控独特的基因表达程序，这与他莫昔芬促进此类细胞增殖的能力相一致。采用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。

实验流程如下：将细胞更换为IMEM培养基（添加10%活性炭吸附血清）培养3天，以完成激素剥夺处理。激素剥夺后，设置四组生物学重复，分别用1nM E2或溶剂对照（0.01%乙醇）处理细胞3小时或24小时。处理结束后裂解细胞，采用Illustra RNAspin Mini试剂盒（GE Health）提取总RNA。采用Ambion MessageAmp Premier试剂盒（Life Technologies）完成cRNA的合成与标记，随后将cRNA与U133A 2.0基因芯片（Affymetrix，美国加利福尼亚州圣克拉拉）进行杂交。所有cRNA合成、标记、杂交及扫描操作均由匹兹堡大学癌症生物标志物实验室完成。