Results of our aforementioned experiments clearly demonstrate that a major theme of tobacco-induced pulmonary carcinogenesis is upregulation of stem cell related signaling pathways via epigenetic mechanisms. These alterations enhance the malignant phenotype of lung cancer cells. Recently, a series of experiments have been performed to extend these observations and specifically examine if CSC exposure promotes genetic/epigenetic alterations that might inhibit senescence of normal SAEC in culture. Interestingly, following exposure to CSC for approximately three weeks as the cells stopped proliferating, spheroids formed above background SAEC, which remained firmly attached to the culture plates. This phenomenon was not observed in untreated control SAEC cultured for three weeks. Subsequent qRT-PCR, immunohistochemistry and micro-array experiments demonstrated increased expression of a variety of stem cell-related genes, such as BMP-3, Aldh, and CD133, Oct4, SOX-2, NANOG, Myc and TERT, and activation of a variety of stem cell related signaling pathways in spheroids from CSC-treated SAEC. These findings suggest that CSC induces reprogramming of differentiated respiratory epithelial cells toward pluripotency. Alternatively, oxidative and genotoxic stress induced by CSC selects for emergence of pluripotent cells present within the SAEC population. Collectively, these results support continued evaluation of our model system to delineate mechanisms coinciding with epigenomic reprogramming in respiratory epithelia mediated by cigarette smoke. Affymetrix microarrays were used to identify gene expression profiles in cultured lung (Calu-6, A549) and esophageal cancer (EsC1, EsC2) cells mediated by 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 cancer cells. A549 and EsC2 cells had relatively high basal expression of ABCG2, which was increased 2-4 fold and 8 fold, respectively, by CSC treatment. In contrast, Calu-6 and EsC1 exhibited relatively low level basal expression of ABCG2, which was augmented approximately 25-30 fold and 6 fold, respectively, by CSC. Normal SAEC and immortalized esophageal squamous cells (HET1A) exhibited very low basal levels of ABCG2, and minimal induction of ABCG2 by CSC. Immunoblot and flow cytometry experiments confirmed that CSC increased ABCG2 expression in lung and esophageal cancer cells. Additional flow cytometry experiments utilizing Hoechst staining with and without verapamil demonstrated that up-regulation of ABCG2 in A549 and Calu-6 cells coincided with an increase in the side population (SP), which is believed to be enriched with cancer stem cells (183, 184). Subsequent experiments were undertaken to examine mechanisms mediating up-regulation of ABCG2 by cigarette smoke. Software guided analysis revealed that in addition to xenobiotic response elements (XRE) that are binding sites for aryl hydrocarbon receptor (AhR) signaling, the ABCG2 promoter contains a number of Sp1 sites that could mediate activation of this gene in response to cigarette smoke. Transient transfection experiments using ABCG2 promoter-reporter constructs revealed that in addition to AhR, Sp1 contributes significantly to CSC-mediated activation of ABCG2 in lung and esophageal cancer cells. Consistent with these latter observations, qRT-PCR and immunoblot 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. Furthermore, mithramycin decreased basal levels and inhibited CSC-mediated upregulation of Nuclear Factor Erythroid Related Factor 2 (Nrf2), which has been shown recently to modulate ABCG2 expression. Quantitative ChIP experiments demonstrated that CSC-mediated activation of ABCG2 coincided with recruitment of Sp1, AhR, and Nrf2 to the ABCG2 promoter;these results were most dramatic for Sp1. Mithramycin diminished CSC-mediated occupancy of these transcription factors within the ABCG2 promoter;these effects coincided with appropriate alterations in RNA polymerase II, H3K9Ac, and H3K9Me3 (activation and repression marks, respectively). 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. Because Sp1 had diverse targets, Affymetrix micro-array experiments were performed to comprehensively examine gene expression profiles in Calu-6 and A549 cells cultured in NM with or without mithramycin for 24 hours, as well as A549 xenografts from control and mithramycin-treated mice. Under exposure conditions potentially achievable in clinical settings, mithramycin mediated dramatic dose-dependent alterations in gene expression in lung cancer cells and 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. Approximately 340 genes were simultaneously modulated in cultured A549 and Calu-6 cells and A549 xenografts under comparable exposure conditions. 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 stem cell signaling in malignant pleural mesotheliomas. Affymetrix micro-array and qRT-PCR experiments demonstrated up-regulation of ABCG2 in normal mesothelial cells and MPM lines following exposure to CSC. Further analysis demonstrated over-expression of Specificity protein (Sp1)- a transcription factor, which regulates a variety of genes mediating stemness, proliferation, invasion and metastasis in cultured pleural mesothelioma cells, as well as primary mesothelioma specimens compared to cultured normal mesothelia. Knock-down of Sp1 as well as mithramycin treatment under clinically relevant exposure conditions significantly inhibited proliferation and clonogenicity of MPM cells. Furthermore, intraperitoneal mithramycin mediated dose dependent growth inhibition and regression of established MPM xenografts. Micro-array experiments revealed dose-dependent alterations in mRNA as well as micro-RNA expression profiles in MPM cells following in-vitro and in-vivo mithramycin exposures. Gene expression and microRNA signatures coinciding with response to mithramycin were identified. Results of these studies will be submitted for peer review in the near future.

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