Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

为了进一步研究调节肺癌干细胞的表观遗传机制，并确定这些肿瘤的新治疗靶点，我们最近通过慢病毒转导OSKM （Yamanaka）因子，从正常的人小气道上皮细胞（SAEC）中产生了诱导多能干细胞（iPSC）。SAEC来自一名57岁的西班牙裔非吸烟者女性。生成了许多肺iPSC （Lu-iPSC）克隆，其中随机选择了几个进行进一步分析。这些Lu-iPSC表现出多能性特征，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，Nanog、Oct4和SALL4等干细胞基因上调，细胞角蛋白表达降低，以及体外增殖和畸胎瘤形成。光谱核型分析和qRT-PCR分析表明，Lu-iPSCs中没有染色体畸变，重编程基因完全沉默。染色质免疫沉淀(ChIP)-seq、RNA-seq、qRT-PCR和免疫印迹实验表明，DNA甲基化发生了复杂的变化，并向高甲基化状态转变；在对A549和Calu-6肺癌细胞以及其他iPSC和胚胎干细胞（ESC）的公开数据集进行分析后，也观察到这一意想不到的发现。RNAseq分析显示，与SAEC相比，Lu-iPSC中有15000个基因普遍改变。prc -2相关基因明显上调。值得注意的是，ASXL3，一种以前未在重编程细胞或胸部恶性肿瘤中描述的表观遗传修饰因子，在Lu-iPSC中上调了400倍。与SAEC相比，Lu-iPSC中ASXL1或ASXL2的表达未见显著上调。随后的qRT-PCR和免疫印迹实验显示ASXL3在SCLC系中相对于NSCLC系或正常或永活的呼吸上皮细胞过表达。ASXL3在SCLC细胞系中表达特别高，并且与基因拷贝数增加相关。免疫组化实验证实ASXL3在原发性SCLC中显著上调。ASXL3基因的敲除抑制了Lu-iPSC的增殖和畸胎瘤的形成，并在体外和体内显著抑制SCLC细胞的生长。总的来说，这些实验首次描述了人类气道上皮细胞的重编程，并首次证明了ASXL3的上调有助于人类癌症。与这些发现有关的一份全面的手稿已经暂时被接受发表在《癌症研究》杂志上，等待进行小的修改。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的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，米霉素降低了musashi-2 （Msi-2）的表达，musashi-2是一种新的RNA结合蛋白，在正常干细胞和几种人类癌症的侵袭性表型中介导自我更新。qRT-PCR和免疫印迹实验证实，米霉素对肺癌细胞中Msi-2的消耗呈时间和剂量依赖性。Msi-2在非小细胞和小细胞肺癌细胞系中的表达水平相对于正常/永生化人呼吸道上皮细胞显著升高（p < 0.001）。与这些发现一致的是，原发性肺癌中Msi-2 mRNA水平显著高于相邻配对正常肺实质（p 0.0003）。Msi-2在培养的肺癌细胞侧群中表达丰富，在Lu-iPSC中表达显著增加。si- rna介导的Msi-2敲低可降低Oct4、Nanog和Myc的表达，并可短暂抑制肺癌细胞的增殖。试图通过shRNA技术永久敲除Msi-2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。实验正在进行中，利用靶向MSI-2的慢病毒载体，在体外和体内明确表征肺癌细胞中MSI-2耗竭的机制和影响。总的来说，这些数据表明，MSI-2的药物耗竭可能是肺癌治疗的一种新策略。这些研究结果在2017年1月的Keystone表观遗传学会议上发表，一旦基因敲除和基因表达谱实验完成，将提交有关这些研究的手稿发表。

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.