Abstract 3178: Up-regulation of GLI1 expression in drug resistant rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines
Authors: Yoon Joon Won , Smith Iris , Manzoor Sumeed , Lamm Marilyn , Iannaccone Philip , Walterhouse David 
Published: August 2018

Abstract
Outcome for pediatric patients with recurrent sarcomas remains poor. Therefore, understanding mechanisms that lead to drug resistance and ways to overcome drug resistance therapeutically are essential. Recent reports have suggested a role for the Hedgehog signaling pathway and its downstream mediator GLI1 in acquisition of a multidrug resistance phenotype in esophageal adenocarcinoma, glioblastoma, and myeloid leukemia. Since GLI1 is expressed in the majority of RMS and EWS cases, we hypothesized that GLI1 up-regulation occurs as cells become drug resistant in vitro and in vivo. We established vincristine (VCR) resistant RMS cell lines (Embryonal RMS: RD, Ruch-2; Alveolar RMS: Rh30, Rh41) by serial exposure to increasing concentrations of VCR. Drug-resistance was defined as an IC50 of ≥30 fold of the baseline that was established using parental cells. In addition, we obtained EWS cell lines, representing untreated cases (CHLA-9 and TC-32) and cases following recurrence (CHLA-258 and TC-71). We used an 86-gene cancer drug resistance PCR array (Qiagen), to characterize gene expression differences between the parental vs. VCR-resistant RMS cells lines and between the untreated and recurrent EWS cell lines. GLI1 expression was significantly increased (p≤0.05) in VCR-resistant alveolar RMS cell lines (Rh30 [2.3 fold], and Rh41 [10.3 fold]) compared with parental cells. Expression of 2 additional genes (MDR1 [13,307 fold in Rh30 and 1,755 fold in Rh41 cells] and MVP [2.4 fold in Rh30 and 8.8 fold in Rh41]) was also significantly up-regulated in these cells. Gel mobility shifts showed interaction of GLI1 with the MVP promoter, and cotransfection assays showed that GLI1 up-regulates reporter gene expression through the MVP promoter. GLI1 expression was significantly up-regulated in Ruch-2 embryonal RMS cells (2 fold). We also showed higher GLI1 expression in recurrent EWS cell lines (TC-71 [3.4 fold] and CHLA-258 [4.8 fold]) compared with untreated CHLA-9 cells. Expression of 1 additional gene (androgen receptor [11.1 fold in CHLA-258 cells and 274.6 fold in TC-71 cells]) was also significantly up-regulated in CHLA-258 and TC-71 cells. Treatment of VCR-resistant UKF Rhb-1 RMS cells with VCR together with either GANT61 or Cpd#33 (GLI1 inhibitors) significantly decreased cell viability by MTT assay at doses that did not reduce cell viability individually. Our results suggest that GLI1 up-regulation contributes to development of drug-resistance in RMS and EWS and that GLI1-inhibitors may reduce multidrug resistance.
Citation Format: Joon Won Yoon, Iris Smith, Sumeed Manzoor, Marilyn Lamm, Philip Iannaccone, David Walterhouse. Up-regulation of GLI1 expression in drug resistant rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3178.