ALDH1A3阳性胰腺导管腺癌亚型再分类及致癌分子网络探索

Pancreatic ductal adenocarcinoma (PDAC) is a histopathologically defined tumor entity with devastating prognosis. A number of clinically relevant molecular subtypes have already been identified. One squamous subtype also known as quasi-mesenchymal subtype (QM-PDA) contributes particularly to the dismal mortality rates of PDAC. However, the clinical outcome of squamous (QM-PDA) subtype is still heterogeneous and subtyping of squamous (QM-PDA) subtype is necessary to offer more concise patient stratification. Using a reverse-translational approach, we previously defined a highly aggressive PDAC subtype labeled by ALDH1A3 (aldehyde dehydrogenase 1 family member A3) expression. Furthermore, ALDH1A3-positive PDAC was found to be partially overlapping with a previously defined squamous (QM-PDA) subtype. Application of 2-mean clustering for ALDH1A3 expression across microarray datasets for cell lines, patient-derived xenograft (PDX), and micro-dissected tumor tissues identified eight subtype-specific markers. Based on dynamic transcriptional data generated from a small-scale lentiviral shRNA library, we constructed an oncogenic network from these eight markers. Our analysis revealed five mutually promotive interactions between ALDH1A3 and network components which can potentially be used to sub-classify ALDH1A3-positive PDAC. In vivo functional relevance of mutual promotions and network dynamics under different targeted pharmaceutical inhibition will be explored to develop potential combination therapies specific for this subtype. Combination therapies effective in inactivating those mutual promotions will be further tested in a number of preclinical platforms including in PDX and subcutaneous tumor model of nude mice. Finally the clinical relevance of ALDH1A3-positive PDAC will be analyzed.

胰腺导管腺癌（PDAC）是一类具有明显异质性且预后极差的实体肿瘤。不同分子亚型的PDAC预后差异明显，其中准间质型（QM-PDA）预后最差。本课题组前期研究中通过逆向转化医学策略鉴定出一类具有高转移潜能且预后差的PDAC分子亚型，该亚型以高表达ALDH1A3分子为特征，故命名为ALDH1A3阳性PDAC。本课题组前期研究发现该亚型与QM-PDA亚型有部分重叠，其存在包括ALDH1A3在内的8个分子标记物，运用慢病毒shRNA文库初步构建了其相互作用的动态致癌网络，该网络中5个组分有望成为ALDH1A3阳性PDAC亚型再分类标记。后续研究将运用不同靶向抑制剂，以期发现潜在对各子亚型有效的靶向联合治疗方案，并在病人来源的小鼠异种移植瘤（PDX）模型、小鼠皮下成瘤模型上进行多层次验证。最终明确ALDH1A3阳性PDAC亚型的再分类及致癌分子网络，并对各子亚型的靶向联合治疗方案进行探索和验证。

胰腺导管腺癌(PDAC)是一类具有明显异质性且预后极差的实体肿瘤，不同分子亚型的PD AC预后差异明显。本课题组前期研究中通过逆向转化医学策略鉴定出一类具有高转移潜能且预后差的PDAC分子亚型，该亚型以高表达ALDH1A3分子为特征，故命名为ALDH1A3阳性PDAC。我们进一步对已经发表的显微切割的病人PDAC组织、人PDAC细胞系及PDX模型的基因芯片数据进行分析，通过双均数聚类分析方法将样本分为ALDH1A3阳性与阴性。在这三个基因芯片数据中均存在包含ALDH1A3在内的8个分子的稳定协同高表达。进一步通过构建shRNA文库发现ALDH1A3阳性PDAC中的8个分子协同表达、相互调控构成紧密的分子网络，对ALDH1A3阳性PDAC的侵袭转移至关重要。基于分子网络中8个基因的表达，我们构建ALDH1A3评分系统并且发现DH1A3阳性PDAC亚型属于现有胰腺癌经典分子分型中Basal-like PDAC亚型。接着，我们通过转录因子预测工具发现AP-1可能是调控核心分子网络的转录因子。在TCGA的胰腺癌数据库、病人的石蜡切片及人胰腺癌细胞系中，AP-1家族尤其是FOS家族亚基与ALDH1A3表达密切相关。在Aldh1a3过表达和敲除的转基因小鼠模型中也验证了Ap-1与Aldh1a3表达的显著正相关性。进一步我们通过对体内外测序数据分析发现，ALDH1A3可导致染色质可及性显著改变。Motif富集分析结果示ALDH1A3引起的差异染色质可及性区域存在大量AP-1结合位点，且结合区域同时也存在大量增强子标记物H3K4me1、H3K27ac结合位点。提示ALDH1A3可通过AP-1介导增强子区域的染色质重构。最后，我们发现ALDH1A3可显著促进胰腺癌细胞及组织的SAM生成。并且利用SAM抑制剂EPZ5676可有效抑制ALDH1A3阳性PDAC亚型的分子网络表达，并且可能对预后较差的Basal-like PDAC分子亚型具有较好的治疗效果。