Abstract B54: Difluoromethylornithine treatment affects the LIN28/Let-7 axis resulting in reduced glycolytic metabolism in neuroblastoma
Authors: Kendzicky Ann , Rich Maria , Peck Anderson , Ping Zhao , VanSickle Elizabeth , McClung Heather , Chang Anthony , Sholler Giselle 
Published: May 2015

Background: Overexpression of LIN28 correlates with poor outcome in neuroblastoma (NB). The LIN28/Let-7 axis affects many cellular processes including cell differentiation and glycolytic metabolism. MYCN overexpression correlates with decreased Let-7 miRNA which results in an increase in LIN28 protein. Recent studies have shown that ODC inhibition decreases LIN28 levels. We propose that therapy targeting ODC will affect the LIN28/Let-7 axis, thus suppressing the glycolytic metabolic activity of NB tumor cells. We also propose that cells overexpressing LIN28 will have greater sensitivity to Difluoromethylornithine (DFMO) treatment which inhibits ODC and decreases cellular polyamines.

Methods: Two MYCN high-expression cell lines, BE(2)-C and SMS-KCNR, and one MYCN low-expression cell line, CHLA90, were grown in RPMI-1640 medium with 10% fetal bovine serum for 24 hours prior to treatment with DFMO. Cells were treated with 5 mM or 10 mM DFMO from 48-96 hours followed by cell viability assay, ATP per cell, and western blot analysis and 6 hours for qPCR analysis. Cell viability was measured using Calcein AM fluorescence assay. IC50 values were calculated using GraphPad software. ATP per cell was measured by combining Cell Titer GLO luminescence assay with CyQuant fluorescence assay. Western blot analysis was used to measure LIN28B and MYCN protein levels. TaqMan PCR reagents were used to measure Let-7 miRNA levels using qPCR analysis. SMS-KCNR cells were injected subcutaneously into nude mice for in vivo xenograft studies. Mice were drugged with 2% DFMO in drinking water when tumors reached 200mm3. Tumor volumes were measured using both caliper and micro-CT, and tumor glycolytic metabolism was determined by Maximum Standard Uptake Value (SUVMax) in the tumors through longitudinal 18F-FDG micro PET/CT scans on days 19 and 32.

Results: treatment with high and low dose DFMO resulted in decreased LIN28B protein levels in all three cell lines at 48, 72, and 96 hours timepoints. MYCN protein levels decreased in MYCN high-expression cell lines, BE(2)-C and SMS-KCNR, with high and low dose DFMO treatments, but did not change in MYCN low-expression cell line CHLA90. Let-7 miRNA levels were increased in both MYCN high-expression cell lines after 6 hours of high dose DFMO treatment and no change was seen in CHLA90 cells. Sensitivity to DFMO correlated with LIN28B expression levels in all three cell lines (BE(2)-C&gt;SMS-KCNR&gt;CHLA90). BE(2)-C cells were most sensitive to DFMO treatment with an IC50 of 3.01 mM followed by SMS-KCNR cells (10.61 mM), and CHLA90 cells, which showed resistance (25.76 mM). In addition, ATP per cell levels were most significantly reduced in BE(2)-C cells after treatment with DFMO followed by SMS-KCNR and CHLA90 cells. In vivo 18F-FDG PET/CT studies showed decreased SUVMax in the DFMO treatment group indicating reduced glycolytic metabolism.

Conclusions: Treatment with DFMO reverses the LIN28/Let-7 axis with a decrease in LIN28B protein expression and an increase in Let-7 miRNA expression. This axis has been shown to play a role in metabolic activity of cells. Decreased metabolic activity and decreased tumor growth is seen in NB cells both in vitro and in vivo following DFMO treatment. NB cell lines with higher levels of LIN28B and MYCN expression are more sensitive to DFMO treatment. These studies suggest that targeting of the LIN28/Let-7 pathway may offer a new method of treating neuroblastoma.

Citation Format: Ann Kendzicky, Maria Rich, Anderson Peck, Zhao Ping, Elizabeth VanSickle, Heather McClung, Anthony Chang, Giselle Sholler. Difluoromethylornithine treatment affects the LIN28/Let-7 axis resulting in reduced glycolytic metabolism in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B54.