Generation of pulmonary neuro-endocrine cells and tumors resembling small cell lung cancers from human embryonic stem cells

Human cancers arising from different cell lineages display genotypes with characteristic features, but the lineage-specific factors that influence the differences in genetic profiles have not been identified. Such differences could be explained by models in which a cellular phenotype limits a cells vulnerability to certain mutations or by models in which observed mutations determine the ultimate cell phenotype. To evaluate the relative merits of these proposals, we are studying the initiation of cancers by genetically modifying cells at discrete stages of differentiation after derivation from human embryonic stem cells (hESCs). We have focused initially on small cell lung cancer (SCLC), the most aggressive type of human lung cancer, characterized by a poor prognosis, the rapid development of resistance to treatment, and nearly universal loss of function of multiple tumor suppressor genes, especially TP53 and RB. Earlier studies with mouse models indicate that the normal cell precursors to SCLCs are likely to be pulmonary neuro-endocrine cells (PNECs). Building on existing methods for sequentially differentiating hESCs in culture into several types of lung cells, we have now produced PNECs in significant numbers for the first time by blocking signaling through NOTCH receptors (using inhibitors of gamma-secretase) and by interfering with expression of the RB gene (using inhibitory RNAs) in lung progenitor cells. In contrast, expression of mutated EGFR or KRAS genes, common in human lung adenocarcinomas that arise from the alveolar cell lineage, has no evident effects on the formation of PNECs. Although PNECs induced by blocking NOTCH and RB signaling do not form xenografts in immune-deficient mice, the cells form subcutaneous tumors resembling early stage human SCLC and bearing neuroendocrine markers if expression of the TP53 gene is also impaired. These findings imply that PNECs or their committed precursors are vulnerable to oncogenic mutations found specifically in human SCLC, resulting in a significant increase in number of PNECs and conversion to neoplasia. This experimental system provides opportunities to study tumor progression and cancer drug susceptibility and resistance, using human lung cells grown in culture.源自不同细胞谱系的人类癌症，均携带有特征性的基因型谱，但调控其遗传特征差异的谱系特异性因子至今尚未被鉴定。此类遗传谱差异可通过两类假说模型解释：其一为细胞表型限制细胞对特定突变的易感性；其二为已观测到的突变决定细胞最终表型。为评估这两种假说的相对优劣，本研究针对从人类胚胎干细胞（human embryonic stem cells, hESCs）中分离、并处于不同分化阶段的细胞开展基因修饰操作，以此探究癌症的起始机制。 我们最初聚焦于小细胞肺癌（small cell lung cancer, SCLC）——这是恶性程度最高的人类肺癌亚型，其临床特征为预后极差、治疗耐药性快速出现，且几乎普遍存在多个肿瘤抑制基因的功能失活，尤以TP53与RB基因的失活最为典型。此前基于小鼠模型的研究表明，小细胞肺癌的正常细胞前体大概率为肺神经内分泌细胞（pulmonary neuro-endocrine cells, PNECs）。基于现有可将体外培养的hESCs依次定向分化为多种肺细胞的技术方法，我们首次通过阻断NOTCH受体信号通路（使用γ-分泌酶抑制剂），并在肺祖细胞中干扰RB基因的表达（使用抑制性RNA），成功获得了大量的PNECs。 与之相反，在源自肺泡细胞谱系的人类肺腺癌中常见的突变型EGFR或KRAS基因的过表达，对PNECs的形成无明显影响。尽管仅通过阻断NOTCH与RB信号通路诱导得到的PNECs，无法在免疫缺陷小鼠体内形成异种移植瘤，但当同时使TP53基因表达受抑时，这些细胞可形成皮下肿瘤，其形态与人类早期小细胞肺癌相似，且表达神经内分泌细胞标志物。 上述研究结果表明，PNECs或其定向分化的前体细胞，对人类小细胞肺癌中特异性存在的致癌突变具有易感性，可导致PNECs数量显著增加并向肿瘤组织转化。该实验体系为利用体外培养的人类肺细胞研究肿瘤进展、癌症药物敏感性与耐药性提供了研究契机。