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 the third to seventh most commonly up-regulated gene 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 in addition to aryl hydrocarbon receptor (AhR) signaling, 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. In addition, mithramycin decreased basal levels, and attenuated CSC-mediated increases in Sp1 and AhR expression in these cells. Additional flow cytometry and MTT experiments demonstrated that mithramycin decreased SP, and dramatically inhibited in-vitro proliferation of lung and esophageal cancer cells. Furthermore, intraperitoneal administration of mithramycin decreased ABCG2 expression, and significantly inhibited growth of established subcutaneous lung cancer xenografts in athymic nude mice. Affymetrix micro-array experiments demonstrated that mithramycin mediated dose-dependent alterations in gene expression in lung cancer cells and corresponding xenografts. Sixteen canonical cancer pathways were down-regulated by mithramycin in vitro and in-vivo;eight of these pathways were related to stem cell signaling. Top molecular and cellular functions of these differentially-expressed genes include stem cell pluripotency, cell cycle progression, gene expression, cellular morphology, and death signaling. Additional experiments have been undertaken to examine the effects of mithramycin on stem cell signaling 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 was primarily due to induction of senescence and autophagy, rather than 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 recapitulated the effects of mithramycin in MPM cells. Results of the MPM experiments have been submitted for peer review. Collectively, these data 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"""""""" in TGIB, as well as additional translational efforts in our lab to target stem cell signaling for treatment of thoracic malignancies.
