Julie Hong

Abstract 2507: Mithramycin depletes stem-like cells in lung adenocarcinoma via repression of mastermind-like 2 and mastermind-like 3

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 ALDEFLUOR TM 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.

Abstract 4250: Cigarette smoke induces expression of the putative stem cell marker ABCG2 in cultured esophageal adenocarcinoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Limited information is available regarding epigenetic mechanisms by which cigarette smoke mediates initiation and progression of esophageal cancer. In order to examine this issue, esophageal adenocarcinoma cell (EsC) lines (NCI-EsC1 and NCI-EsC2) were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) under clinically relevant exposure conditions. Microarray analysis revealed that CSC exposure significantly upregulated expression of ABCG2, which encodes a xenobiotic pump protein believed to be highly expressed in cancer stem cells, and recently shown to correlate with aggressive phenotype of primary esophageal squamous cell carcinomas. Analysis revealed an average of 11 fold upregulation of ABCG2 as well as a 4 fold upregulation of another ABC transporter, ABCC3. Quantitative reverse transcription-PCR (RT-PCR), western blot, and immunohistochemistry experiments confirmed upregulation of ABCG2 in esophageal cancer cells exposed to CSC. Interestingly, basal as well as CSC-induced levels of ABCG2 were considerably higher in NCI-EsC2 derived from a smoker, relative to NCI-EsC1, which was established from a never-smoker. Time course experiments revealed that induction of ABCG2 commenced approximately 8 hours and peaked 48 hours following initiation of CSC exposure; thereafter, ABCG2 expression was sustained at these levels with continuous CSC exposure. HDAC inhibitors such as TSA and depsipeptide repressed basal levels of ABCG2 expression, and reduced CSC-mediated induction of ABCG2 in EsC cells. HDAC inhibitors in combination with verapamil (a ABCC3 inhibitor) and Depsipeptide virtually abolished basal, as well as CSC-induced ABCG2 expression in these cells. Preliminary pyrosequencing and chromatin immunoprecipitation (ChIP) experiments revealed that CSC-mediated ABCG2 induction was independent of promoter DNA demethylation, and coincided with hyperacetylation of H3K9 within the ABCG2 promoter. Flow cytometry analysis utilizing hoescht dye with and without fumitremorgin C, an ABCG2 inhibitor, revealed an increased side population in CSC-treated ESC cells relative to untreated controls. Experiments are in progress to further delineate the mechanisms by which CSC induces ABCG2 expression, and to ascertain if ABCG2 induction enhances the malignant phenotype of esophageal adenocarcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4250.