Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

已经进行了一系列实验，专门研究吸烟暴露是否会促进遗传/表观遗传改变，从而增强胸部恶性肿瘤的多能性。简单地说，Affymetrix微阵列被用于鉴定在临床相关暴露条件下由香烟烟雾冷凝物（CSC）介导的培养的肺癌和食管癌细胞的基因表达谱。ABCG2编码一种在癌症干细胞中高度表达的外源泵蛋白，在CSC暴露后这些细胞中持续上调。qRT-PCR实验显示，CSC暴露后，ABCG2在肺癌和食管癌细胞中呈时间和剂量依赖性诱导，但在正常呼吸道上皮细胞中无诱导作用。免疫印迹和流式细胞术实验证实，CSC增加了肺和食管癌细胞中ABCG2的表达。另外的流式细胞术实验表明，肺癌细胞中ABCG2的上调与多能侧群（SP）的增加相一致。随后的实验表明，转录因子特异性蛋白1 （SP1）在肺癌和食管癌细胞中对csc介导的ABCG2活化起着重要作用。qRT-PCR、免疫印迹和染色质免疫沉淀（ChIP）实验表明，米霉素（Sp1与富含gc的DNA结合的药理学抑制剂）降低了ABCG2的基础水平，并以剂量依赖的方式显著减弱了csc介导的肺和食管癌细胞中ABCG2的诱导。另外，流式细胞术和MTT实验表明，米霉素在体外和体内均能降低SP，并显著抑制肺癌和食管癌细胞的生长。微阵列实验表明，米霉素显著抑制肺癌和食管癌细胞的干细胞信号传导。这些研究结果发表在《癌症研究》杂志上。在最近的研究中，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，Msi-2是一种新的RNA结合蛋白，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，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SP部位表达丰富，在SAEC重编程为多能性后表达显著增加。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。迄今为止，通过shRNA技术永久敲除Msi-2的尝试尚未成功，这表明在这些细胞中存在维持Msi-2表达的强大选择压力。综上所述，这些数据表明，米霉素可以减少肺癌细胞中的Msi-2，并提示从药理学上减少这种多能因子可能是肺癌治疗的一种新策略。这些研究的结果已被选择在2015年9月的世界肺癌大会上发表，有关这些研究的手稿将在不久的将来提交发表。另外的实验已经进行，以检查米霉素对恶性胸膜间皮瘤（MPM）的影响。qRT-PCR实验显示，与培养的正常间皮细胞或正常胸膜相比，Sp1在培养的MPM细胞和原代MPM标本中显著过表达。米特霉素显著抑制MPM细胞的增殖和克隆原性。此外，腹腔注射米霉素介导剂量依赖性生长抑制和已建立的MPM异种移植物的消退。生长抑制与早期G0/G1阻滞和衰老相一致，随后发生细胞凋亡。微阵列实验揭示了培养的MPM细胞和相应的异种移植物中基因表达的剂量依赖性改变。米霉素在MPM细胞/异种移植物中调节的主要途径包括干细胞信号、多能性和p53信号。Sp1敲低/p53过表达的联合表达在MPM细胞中表型化了米霉素的作用。一篇总结MPM实验的手稿已经暂时被接受发表在《临床癌症研究》杂志上。上述数据为正在进行的“米霉素（一种癌症干细胞信号传导抑制剂）在肺、食道、胸膜或纵隔恶性肿瘤患者中的II期评估”以及一项评估不能手术的胸部恶性肿瘤患者24小时米霉素输注的I期方案提供了基本原理，该方案已进行了CCR审查。由于临床中心PDS问题，这两项试验目前都处于暂停状态，它们将利用新的精准医学技术来选择最可能耐受米霉素的患者，并通过药代动力学模拟来确定米霉素输注的剂量和持续时间，以总结临床前实验中暴露条件介导的令人印象深刻的抗肿瘤活性。为了进一步研究促进癌症干细胞启动过程的表观遗传机制，并可能确定肺癌治疗的新靶点，研究人员进行了额外的实验。简单地说，几种不同的正常人小气道上皮细胞（SAECs）被含有OKSM（山中因子）的Stemcca病毒转导，并成功地重编程为多能状态。这些诱导多能干细胞（iPSCs）表现出多能性的特征，包括形态、增殖、表面抗原表达、干性基因表达和体内畸胎瘤形成。谱核型分析显示，iPSCs无染色体畸变。使用5-MC抗体的流式细胞术和免疫组织化学显示，与亲代SAEC相比，iPSC的整体DNA低甲基化。然而，有趣的是，肺癌细胞中经常由DNA去甲基化诱导的癌睾丸基因，如NY-ESO-1、MAGE-A1和MAGE-A3，在iPSC中仍然受到转录抑制。另一方面，NANOG和POU5F1基因在iPSCs中相对于SAEC的低甲基化，与其在iPSCs中的过度表达相关。RNA-Seq分析显示，与亲本SAEC相比，iPSC中编码polycomb - suppression Complex 2 （PRC2）组分的基因上调，DKK1、p16和p21等肿瘤抑制基因下调。几个新的多能性相关基因也被发现在肺iPSC中上调。总的来说，这是首次成功地将正常人类呼吸上皮细胞重编程为多能性。该模型可能有助于阐明肺癌发生的基本表观基因组机制和确定肺癌治疗的新靶点。这些实验的结果已被选择在2015年9月的世界肺癌大会上进行口头报告，并将在不久的将来提交有关这些研究的手稿发表。

A series of experiments have been performed to specifically examine if cigarette smoke exposure promotes genetic/epigenetic alterations that enhance pluripotency in thoracic malignancies. Briefly, Affymetrix microarrays were used to identify gene expression profiles in cultured lung and esophageal cancer cells mediated by cigarette smoke condensate (CSC) under clinically-relevant exposure conditions. ABCG2, which encodes a xenobiotic pump protein highly expressed in cancer stem cells, was consistently up-regulated in these cells following CSC exposure. qRT-PCR experiments demonstrated time and dose-dependent induction of ABCG2 in lung and esophageal cancer cells but not normal respiratory epithelia following CSC exposure. Immunoblot and flow cytometry experiments confirmed that CSC increased ABCG2 expression in lung and esophageal cancer cells. Additional flow cytometry experiments demonstrated that up-regulation of ABCG2 in lung cancer cells coincided with an increase in the pluripotent side population (SP). Subsequent experiments demonstrated that the transcription factor Specificity Protein 1 (SP1) contributed significantly to CSC-mediated activation of ABCG2 in lung and esophageal cancer cells. qRT-PCR, immunoblot and chromatin immunoprecipitation (ChIP) experiments demonstrated that mithramycin, a pharmacologic inhibitor of Sp1 binding to GC-rich DNA, decreased basal levels of ABCG2, and markedly attenuated CSC-mediated induction of ABCG2 in lung and esophageal cancer cells in a dose dependent manner. Additional flow cytometry and MTT experiments demonstrated that mithramycin decreased SP, and dramatically inhibited growth of lung and esophageal cancer cells in-vitro and in-vivo. Micro-array experiments demonstrated that mithramycin significantly inhibited stem cell signaling in lung and esophageal cancer cells. Results of these studies were published in Cancer Research. In more recent 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 SP fractions of cultured lung cancer cells, and was significantly increased in SAEC following reprogramming to pluripotency. 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 thus far have been unsuccessful, suggesting a strong selective pressure to maintain Msi-2 expression in these cells. Collectively these data demonstrate that mithramycin depletes Msi-2 in lung cancer cells, and suggest that pharmacologic depletion of this pluripotency factor may be a novel strategy for lung cancer therapy. Results of these studies have been selected for presentation at the World Lung Cancer Conference in September 2015, and a manuscript pertaining to these studies will be submitted for publication in the near future. Additional experiments have been undertaken to examine the effects of mithramycin in malignant pleural mesotheliomas (MPM). qRT-PCR experiments demonstrated significant over-expression of Sp1 in cultured MPM cells, as well as primary MPM specimens compared to cultured normal mesothelial cells or normal pleura. Mithramycin dramatically inhibited proliferation and clonogenicity of MPM cells. Furthermore, intraperitoneal mithramycin mediated dose dependent growth inhibition and regression of established MPM xenografts. Growth inhibition coincided with early G0/G1 arrest and senescence with subsequent apoptosis. Micro-array experiments revealed dose-dependent alterations in gene expression in cultured MPM cells and corresponding xenografts. Top canonical pathways modulated by mithramycin in MPM cells/xenografts included stem cell signaling, pluripotency and p53 signaling. Combined Sp1 knockdown/p53 overexpression phenocopied the effects of mithramycin in MPM cells. A manuscript summarizing the MPM experiments has been tentatively accepted for publication in Clinical Cancer Research. The aforementioned data have provided the rationale for an ongoing "Phase II Evaluation of Mithramycin, an Inhibitor of Cancer Stem Cell Signaling, in Patients with Malignancies Involving Lungs, Esophagus, Pleura, or Mediastinum", as well as a phase I protocol evaluating 24 hour mithramycin infusions in patients with inoperable thoracic malignancies which has undergone CCR review. Both of these trials, which are currently on hold due to Clinical Center PDS issues, will utilize novel precision medicine techniques to select for accrual only those patients most likely to tolerate mithramycin, as well as pharmacokinetic simulations to define doses and durations of mithramycin infusions in an effort to recapitulate exposure conditions mediating impressive antitumor activity in preclinical experiments. Additional experiments have been performed to further investigate epigenetic mechanisms contributing to the cancer stem cell initiation process, and possibly identify novel targets for lung cancer therapy. Briefly, several different stocks of normal human small airway epithelial cells (SAECs) were transduced with Stemcca virus containing OKSM (Yamanaka factors) and successfully reprogrammed to a pluripotent state. These induced pluripotent stem cells (iPSCs) demonstrated hallmarks of pluripotency including morphology, proliferation, expression of surface antigens, stemness gene expression, and in vivo teratoma formation. Spectral karyotyping revealed no chromosomal aberrations in iPSCs. Flow cytometry and immunohistochemistry using an antibody to 5-MC revealed global DNA hypomethylation in iPSC compared to parental SAEC. Interestingly however, cancer-testis genes such as NY-ESO-1, MAGE-A1 and MAGE-A3, which are frequently induced by DNA demethylation in lung cancer cells, remained transcriptionally repressed in iPSC. On the other hand, NANOG and POU5F1 genes were hypomethylated in iPSCs relative to SAEC, correlating with their over-expression in iPSCs. RNA-Seq analysis revealed up-regulation of genes encoding components of Polycomb-Repressive Complex 2 (PRC2), and down-regulation of several tumor suppressor genes such as DKK1, p16 and p21 in iPSC relative to parental SAEC. Several novel pluripotency associated genes were also noted to be up-regulated in pulmonary iPSC. Collectively, this is the first demonstration of successful reprogramming of normal human respiratory epithelia to pluripotency. This model may prove useful for elucidating fundamental epigenomic mechanisms of pulmonary carcinogenesis and identification of novel targets for lung cancer therapy. Results of these experiments have been selected for oral presentation at the World Lung Cancer Conference in September 2015, and a manuscript pertaining to these studies will be submitted for publication in the near future.