Abstract 2507: Mithramycin depletes stem-like cells in lung adenocarcinoma via repression of mastermind-like 2 and mastermind-like 3
Authors: Haobin Chen, Mary Zhang, Sichuan Xi, Yin Xiong, Julie Hong, David Schrump
Journal: Cancer Research (2016)

Abstract Background: Targeting cancer stem-like cells is a potential strategy to prevent development of drug resistance and tumor recurrence. Our group has previously demonstrated that mithramycin 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 lung adenocarcinoma cell lines (H2228, H358 and HCC827) were detected based on stem cell markers, CD133 or ALDH, using flow cytometry or ALDEFLUORTM assay. Sphere-formation assays were performed using low attachment plates and stem-cell media (serum-free DMEM/F12 media supplemented with epidermal growth factor and fibroblast growth factor). qRT-PCR and western blot techniques were used to evaluate gene and protein expression levels, respectively. SiRNA transfection was performed to knockdown target gene(s) of interest. Results: A small CD133+ or ALDH+ cell fraction was detected in untreated H2228, H358 and HCC827 cells, respectively. The chemotherapeutic agent cisplatin enriched CD133+ fraction in H2228 cells and ALDH+ fraction in H358 cells, while the EGFR inhibitor erlotinib remarkably increased ALDH+ fraction in HCC827 cells. Consistent with the notion that stem-like cells are present in these cancer lines, H2228 and H358 cells formed pulmospheroids when cultured in stem cell media in low attachment plates, and these pulmospheroids showed increased expression of stem-cell marker genes (Aldh1a1 and Prominin) as well as stemness-related genes (Oct4, Sox2 and Nanog). Relative to untreated controls, mithramycin decreased the cancer stem-like cell fractions in all three cell lines, and abolished their enrichment after cisplatin or erlotinib treatment. Analysis of stem-cell signaling pathways revealed that maintenance of cancer stem-cell like fractions in H358 and H2228 cells requires Notch signaling. Mithramycin decreased gene and protein expression of Mastermind-like 2 and 3 (MAML2 and MAML3), both of which are important transcription co-activators of canonical Notch signaling. Knockdown of MAML2 and MAML3 remarkably decreased the stem-like cell fraction in H358 cells, which recapitulates the phenotype of mithramycin treatment. Conclusion: Mithramycin depletes cancer stem-like cells via repression of MAML2 and MAML3 and inhibition of Notch signaling. These findings support evaluation of mithramycin as a strategy to eliminate stem-like cells emerging in lung cancers after cisplatin or EGFR targeted therapy. Citation Format: Haobin Chen, Mary Zhang, Sichuan Xi, Yin Xiong, Julie Hong, David Schrump. Mithramycin depletes stem-like cells in lung adenocarcinoma via repression of mastermind-like 2 and mastermind-like 3. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2507.