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H. Chen
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-098 - Mithramycin Is a Potential Therapeutic Agent for Elimination of Stem-Like Cells in Lung Cancer (ID 2165)
09:30 - 17:00 | Author(s): H. Chen
- Abstract
Background:
There have been several studies demonstrating existence of cancer stem-like cells in lung cancers, and resistance of such cells to conventional chemotherapy or targeted agents. As such, targeting cancer stem-like cells is a potential strategy to prevent development of drug resistance and tumor recurrence. Previously our group has demonstrated that mithramycin, a specific inhibitor of transcription factor SP1, attenuates induction of side population (a phenotype of cancer stem-like cells) by cigarette smoke condensate, and modulates expression of multiple genes regulating stem-cell related pathways in lung cancer cells. The present study was performed to further examine the effects of mithramycin on stem cell signaling pathways, and ascertain if mithramycin can eliminate stem-like cells in lung cancer following exposure to conventional chemotherapeutic or targeted agents.
Methods:
Stem-like cell populations in cultured H358 and H2228 lung adenocarcinoma cells were identified based on expression of stem cell markers, ALDH1 and CD133 using ALDEFLUOR[TM] assay and flow cytometry, respectively. Sphere-formation assays were used to examine clonogenic growth of stem-like cells. qRT-PCR techniques were used to evaluate expression levels of stemness-related genes. Western blot techniques were utilized to assess activation of stemness-related (WNT/β-catenin and NOTCH) signaling pathways.
Results:
Small CD133[+] or ALDH1[+] fractions were detected in untreated H2228 and H358 cells, respectively. Consistent with notion that stem-like cells are present in these two lines, H2228 and H358 cells formed pulmospheroids when cultured in stem cell media in low attachment plates; these phenotypic changes were accompanied by increased expression of stemness-related genes including Oct4, Sox2 and Nanog. Cisplatin treatment enriched CD133[+] fraction in H2228 cells and ALDH[+] fraction in H358 cells. Mithramycin abolished this enrichment, and mediated dose-dependent decreases in Oct4, Sox2 and Nanog expression in a dose-dependent manner. Preliminary analysis demonstrated that mithramycin decreased total as well as active forms of β-catenin, but did not affect levels of cleaved NOTCH1, suggesting that mithramycin eliminates lung cancer stem-like cells partially through suppression of WNT/β-catenin signaling. The effects of mithramycin on lung cancer stem-like cells induced by targeted agents are currently under investigation.
Conclusion:
Mithramycin suppresses stemness-related signaling, and is a potential therapeutic agent for elimination of stem-like cells emerging in lung cancers after cisplatin therapy.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-051 - The Pluripotency Factor Musashi-2 Is a Potential Target for Lung Cancer Therapy (ID 2973)
09:30 - 17:00 | Author(s): H. Chen
- Abstract
Background:
Recent studies have demonstrated that mithramycin represses multiple pathways critical for stem cell signaling and pluripotency in lung cancer cells. This phenomenon coincides with decreased side population (SP) fraction, and dramatic dose-dependent growth arrest of lung cancer cells in-vitro and in-vivo. The present study was performed to further examine the effects of mithramycin on stem cell signaling in an attempt to identify novel targets for lung cancer therapy.
Methods:
Microarray, quantitative RT-PCR (qRT-PCR) and immunoblot techniques were used to examine stem cell gene expression and proliferation of human lung cancer cells and normal/immortalized human respiratory epithelial cells (SAEC/NHBE/HBEC) cultured in the presence or absence of mithramycin, or lung cancer cells following stem cell gene knockdown. Micro-array and qRT-PCR techniques were used to assess effects of systemic mithramycin exposure on stem cell gene expression in subcutaneous lung cancer xenografts in athymic nude mice. qRT-PCR and immunoblot techniques were used to examine endogenous levels of selected stem cell genes in induced pluripotent stem cells (iPSC) derived from SAEC, as well as primary lung cancers and paired normal respiratory tissues. siRNA techniques were used to knockdown Msi-2 to confirm potential mechanisms of action of mithramycin-mediated cytotoxicity in lung cancer cells.
Results:
Preliminary microarray analysis of cultured lung cancer cells and xenografts demonstrated that mithramycin decreased expression of musashi-2 (Msi-2), a RNA binding protein which mediates self-renewal in normal stem cells and aggressive phenotype of several human cancers. Subsequent 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.
Conclusion:
Mithramycin depletes Msi-2 in lung cancer cells. Pharmacologic depletion of this pluripotency factor may be a novel strategy for lung cancer therapy.
