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.
