Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

为了进一步研究调节肺癌中干细胞的表观遗传机制并确定这些肿瘤中的新治疗靶点，我们最近通过OSKM（Yamanaka）因子的慢病毒转导从正常人小气道上皮细胞（SAEC）产生诱导多能干细胞（iPSC）。SAEC来源于一名57岁的西班牙裔女性非吸烟者。产生了许多肺iPSC（Lu-iPSC）克隆，随机选择其中几个进行进一步分析。这些Lu-iPSC表现出多能性的标志，包括形态学、细胞质碱性磷酸酶表达、SSEA-3、SSEA-4、TRA-1 - 60和TRA-1 - 81的细胞表面表达、干细胞基因（包括Nanog、Oct4和SALL4）的上调、细胞角蛋白的表达降低以及体外增殖和畸胎瘤形成。光谱核型分析和qRT-PCR分析证明Lu-iPSC中没有染色体畸变和重编程转基因的完全沉默。染色质免疫沉淀（ChIP）-seq、RNA-seq、qRT-PCR和免疫印迹实验证明了DNA甲基化的复杂变化，并向更高的甲基化状态转变;在分析A549和Calu-6肺癌细胞以及其他iPSC和胚胎干细胞（ESC）的实验可用数据集后，也观察到了这一意外发现。RNAseq分析表明，相对于SAEC，Lu-iPSC通常改变了15，000个基因。观察到PRC-2相关基因的显著上调。值得注意的是，ASXL3，一种先前在重编程细胞或胸部恶性肿瘤中未描述的表观遗传修饰剂，在Lu-iPSC中上调400倍。相对于SAEC，在Lu-iPSC中未观察到ASXL1或ASXL2的显著上调。随后的qRT-PCR和免疫印迹实验证明，相对于NSCLC系或正常或永生化呼吸道上皮细胞，ASXL3在SCLC系中过表达。ASXL3表达在SCLC细胞系的子集中特别高，并且与增加的基因拷贝数相关。免疫组化实验证实ASXL3在原发性SCLC中显著上调。ASXL3的敲低抑制Lu-iPSC的增殖和畸胎瘤形成，并显著减少体外和体内SCLC细胞的生长。总的来说，这些实验是人类气道上皮细胞重编程的第一次描述，也是ASXL3上调导致人类癌症的第一次证明。一份关于这些发现的综合性手稿已被《癌症研究》杂志暂时接受，有待微小的修订。我们的研究结果强调了这种Lu-iPSC模型在阐明肺癌发生的表观基因组机制和鉴定肺癌治疗的其他新表观遗传靶点方面的潜在效用。在其他研究中，培养的肺癌细胞和异种移植物的微阵列分析表明，光辉霉素降低了musashi-2（Msi-2）的表达，这是一种新型RNA结合蛋白，可介导正常干细胞的自我更新和几种人类癌症的侵袭性表型。qRT-PCR和免疫印迹实验证实，光辉霉素以时间和剂量依赖性方式消耗肺癌细胞中的Msi-2。相对于正常/永生化人呼吸道上皮细胞，非小细胞和小细胞肺癌细胞系中Msi-2的表达水平显著升高（p <0.001）。与这些结果一致，原发性肺癌中的Msi-2 mRNA水平显著高于相邻配对正常肺实质中检测到的水平（p 0.0003）。Msi-2表达在培养的肺癌细胞的侧群中富集，并且在Lu-iPSC中显著增加。si-RNA介导的Msi-2敲低降低了Oct4、Nanog和Myc的表达，并短暂抑制了肺癌细胞的增殖。通过shRNA技术永久敲除Msi-2的尝试是不成功的，这表明在这些细胞中维持Msi-2表达的强大选择压力。正在进行使用靶向MSI-2的tet诱导型慢病毒载体的实验，以明确表征体外和体内肺癌细胞中MSI-2耗竭的机制和作用。总的来说，这些数据表明，MSI-2的药理学耗竭可能是肺癌治疗的新策略。这些研究的结果在2017年1月的Keystone表观遗传学会议上发表，一旦敲除和基因表达谱实验完成，与这些研究有关的手稿将提交出版。

To further examine epigenetic mechanisms regulating stemness in lung cancers and identify novel therapeutic targets in these neoplasms, we recently generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) by lentiviral transduction of OSKM (Yamanaka) factors. SAEC were derived from a 57 year old Hispanic female non-smoker. Numerous lung iPSC (Lu-iPSC) clones were generated, several of which were randomly selected for further analysis. These Lu-iPSC demonstrated hallmarks of pluripotency including morphology, cytoplasmic alkaline phosphatase expression, cell surface expression of SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81, up-regulation of stem cell genes including Nanog, Oct4, and SALL4, with decreased expression of cytokeratins, as well as in-vitro proliferation and teratoma formation. Spectral karyotyping and qRT-PCR analyses demonstrated no chromosomal aberrations and complete silencing of reprogramming transgenes in Lu-iPSCs. Chromatin immunoprecipitation (ChIP)-seq, RNA-seq, qRT-PCR, and immunoblot experiments demonstrated complex alterations in DNA methylation with a shift toward a more hypermethylated state; this unexpected finding was also observed following analysis of A549 and Calu-6 lung cancer cells as well as publically available data sets for other iPSC as well as embryonic stem cells (ESC). RNAseq analysis demonstrated that 15,000 genes were commonly altered Lu-iPSC relative to SAEC. A marked up-regulation of PRC-2-related genes was observed. Notably, ASXL3, an epigenetic modifier not previously described in reprogrammed cells or thoracic malignancies, was up-regulated 400-fold in Lu-iPSC. No significant up-regulation of ASXL1 or ASXL2 was observed in Lu-iPSC relative to SAEC. Subsequent qRT-PCR and immunoblot experiments demonstrated over-expression of ASXL3 in SCLC lines relative to NSCLC lines or normal or immortalized respiratory epithelial cells. ASXL3 expression was particularly high in a subset of SCLC lines and correlated with increased gene copy number. Immunohistochemistry experiments confirmed significant up-regulation of ASXL3 in primary SCLC. Knock-down of ASXL3 inhibited proliferation and teratoma formation by Lu-iPSC, and significantly diminished growth of SCLC cells in-vitro and in-vivo. Collectively these experiments are the first description of reprogramming of human airway epithelial cells, and the first demonstration that up-regulation of ASXL3 contributes to human cancer. A comprehensive manuscript pertaining to these findings has been tentatively accepted for publication in Cancer Research pending minor revisions. Our findings highlight the potential utility of this Lu-iPSC model for elucidation of epigenomic mechanisms of pulmonary carcinogenesis and identification of other novel epigenetic targets for lung cancer therapy. In additional studies, microarray analysis of cultured lung cancer cells and xenografts demonstrated that mithramycin decreased expression of musashi-2 (Msi-2), a novel RNA binding protein which mediates self-renewal in normal stem cells and aggressive phenotype of several human cancers. qRT-PCR and immunoblot experiments confirmed that mithramycin depletes Msi-2 in lung cancer cells in a time and dose-dependent manner. Expression levels of Msi-2 were significantly elevated in non-small cell as well as small-cell lung cancer lines relative to normal/immortalized human respiratory epithelial cells (p 0.001). Consistent with these findings, Msi-2 mRNA levels in primary lung cancers were significantly higher than those detected in adjacent paired normal lung parenchyma (p 0.0003). Msi-2 expression was enriched in side populations of cultured lung cancer cells, and was significantly increased in Lu-iPSC. si-RNA-mediated knock-down of Msi-2 decreased expression of Oct4, Nanog and Myc, and transiently inhibited proliferation of lung cancer cells. Attempts to permanently knockdown Msi-2 by shRNA techniques were unsuccessful, suggesting strong selection pressure to maintain Msi-2 expression in these cells. Experiments are underway using tet-inducible lentiviral vectors targeting MSI-2 to definitively characterize mechanisms and effects of MSI-2 depletion in lung cancer cells in-vitro and in-vivo. Collectively, these data suggest that pharmacologic depletion of MSI-2 may be a novel strategy for lung cancer therapy. Results of these studies were presented at a Keystone Epigenetics conference in January 2017, and a manuscript pertaining to these studies will be submitted for publication once the knock-down and gene expression profiling experiments have been completed.