Objective Platelet-derived growth factor (PDGF) and its receptor (PDGFR) promote fibrosis in scleroderma (SSc) dermal fibroblasts, which produce excessive reactive oxygen species (ROS). PDGFR is phosphorylated upon PDGF stimulation, and dephosphorylated by protein tyrosine phosphatases (PTPs), including PTP1B. In this study we determine whether the thiol-sensitive PTP1B is affected by ROS, thus enhancing PDGFR phosphorylation (p-PDGFR) and collagen I (Col I) synthesis. The effect of a thiol antioxidant, n-acetylcysteine (NAC), was also investigated. Methods Fibroblasts were isolated from skin. A phosphate release assay was used for PTP1B activity. Results ROS and Col I were significantly higher in SSc fibroblasts, accompanied by significantly lower amounts of free thiols compared to normal fibroblasts. After PDGF stimulation, not only were the PDGFR and ERK1/2 phosphorylated to a greater extent, but the ability to produce PTP1B was also hampered in SSc fibroblasts. PTP1B activity was significantly inactivated in SSc fibroblasts, which resulted from cysteine oxidation by higher levels of ROS, since oxidation of multiple PTPs, including PTP1B, was observed. Decreased PTP1B expression in normal fibroblasts led to increased Col I. NAC restored the low PTP1B activity, improved the profile of p-PDGFR, decreased the numbers of tyrosine-phosphorylated proteins and Col I, and scavenged ROS in SSc fibroblasts. Conclusion We introduce a new mechanism by which ROS promote a profibrotic phenotype in SSc fibroblasts through oxidative inactivation of PTP1B leading to pronounced PDGFR activation. Our study also provides a novel molecular mechanism by which NAC therapy may act on ROS and PTP1B to benefit SSc patients.

Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in many forms of cancer including NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested in NB. We propose that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501 will result in enhanced NB growth inhibition. Single and combination drug treatments were conducted on three NB cell lines. DFMO IC 50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC 50 values ranged from 14.13 to 17.72 mM in NB. The combination treatment resulted in hypophosphorylation of retinoblastoma protein (Rb), suggesting growth inhibition via G 1 cell cycle arrest. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hr suggested apoptosis. The combination treatment depleted intracellular polyamine pools and decreased intracellular ATP, further verifying growth inhibition. Given the current lack of effective therapies for patients with relapsed/refractory NB and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB.Neuroblastoma (NB) is the most common extracranial solid pediatric tumor, accounting 8-10% of all pediatric cancers and for 15% of cancer-related deaths in children. 1 Approximately 650 new cases of NB arise in the United States each year. Of the children diagnosed, roughly 70% have disease that has already metastasized to other parts of the body. Children with high-risk NB have a long-term survival of less than 50%, despite the use of intensive multimodality therapy. 2 Of the children with relapsed or refractory disease, there are no known curative measures and the 5-year survival is less than 10%. Biomarkers are powerful tools for determining diagnosis and prognosis for different cancers, including NB. The oncogenic transcription factor MYCN is amplified in roughly 30% of all NBs and is generally associated with high-risk disease and poor survival. 3,4 As a transcription factor, MYCN induces and represses a large number of genes involved in multiple biological processes including cell growth and differentiation. However, the genes necessary or sufficient to initiate neuroblastoma tumorigenesis downstream of MYCN remain to be established. 5 Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis in mammalian cells, is directly activated by c-MYC and MYCN 6-8 and is overexpressed in NB. 9-11 ODC decarboxylates t...

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>SMS-KCNR>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.

Background: Neuroblastoma (NB) is associated with MYCN oncogene amplification occurring in approximately 30% of NBs and is associated with poor prognosis. MYCN is linked to a number of genes including ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. ODC expression is elevated in NB. Alpha-difluoromethylornithine (DFMO), an ODC inhibitor, is currently being used in a Phase I clinical trial for treatment of NB. However, cancer cells treated with DFMO may overcome their polyamine depletion by the uptake of polyamines from extracellular sources. A novel polyamine transport inhibitor, AMXT-1501, has not yet been tested on NB. We proposed that inhibiting ODC with DFMO, coupled with polyamine transport inhibition by AMXT-1501, would result in increased NB growth inhibition. Methods: Single and combination drug treatments were conducted on three NB cell lines in vitro and in a xenograft mouse model in vivo. Cell viability was measured using Calcein AM fluorescent assay. Real-time cell proliferation was measured using the xCELLigence system. Western blot analysis was used to measure cleaved and full PARP, cleaved and full caspase 3, phosphorylated Rb and MYCN levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Reverse-phase HPLC was used to measure polyamine levels in cells. Results: DFMO IC50 values ranged from 20.76 to 33.3 mM, and AMXT-1501 IC50 values ranged from 14.13 to 17.72 μM in NB cell lines. Low dose combination treatment (2.5 μM AMXT-1501, 2.5 mM DFMO) synergistically inhibited cell viability and proliferation in vitro. Combination treatment decreased MYCN expression and resulted in hypophosphorylation of pRb, suggesting cell growth inhibition. Increased expression of cleaved PARP and cleaved caspase 3 in combination-treated cells starting at 48 hours suggested apoptosis. The combination treatment resulted in intracellular polyamine pool depletion and decreased intracellular ATP. The combination treatment also significantly reduced tumor size in NB xenograft mice in vivo. Conclusion: Given the current lack of effective therapies for relapsed/refractory NB patients and the preclinical effectiveness of DFMO with AMXT-1501, this combination treatment provides promising preclinical results. DFMO and AMXT-1501 may be a potential new therapy for children with NB. Citation Format: Ann M. Kendzicky, Ping Zhao, Katherine Samal, Lisette P. Yco, Heather McClung, Eugene W. Gerner, André S. Bachmann, Giselle L. Sholler. Targeting both ornithine decarboxylase and polyamine transport inhibits tumor growth in neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2765. doi:10.1158/1538-7445.AM2013-2765