Cell cycle analyses A. Dansylcadaverine-mediated nIGF1R depletion arrested CCH1 cells at the G1-phase and reduced the percentage of cells in the S-phase. B. Western blots show that Cyclin A2 and Cyclin D1 levels were high in CCH5 cells with nIGF1R relative to NCH6 with mIGF1R. C. Cell cycle analyses shows higher percentages of CCH1 and CCH5 cells in S-phase than CCH2 and NCH6 cells. D. CCH5 cells with nIGF1R divided faster, had higher percentages of S-phase cells with shorter S-phase duration than NCH6 with mIGF1R. E. EdU/BrdU double labelling-based S-phase duration analysis was carried out. Top panel shows that cells showed EdU-AF55+ and BrdU-FITC+ signals in comparison to unstained cells. F. Percentages of EdU+BrdU+ cells (CCH1, CCH5, CCH2 and NCH6 cells), at indicated time points, are shown. G.Timings of S-phase exit (red) for all cells were calculated using linear regression.
ARTICLE ABSTRACT
Targeted cancer therapeutics have not significantly benefited patients with Ewing sarcoma with metastatic or relapsed disease. Understanding the molecular underpinnings of drug resistance can lead to biomarker-driven treatment selection.
Receptor tyrosine kinase (RTK) pathway activation was analyzed in tumor cells derived from a panel of Ewing sarcoma tumors, including primary and metastatic tumors from the same patient. Phospho-RTK arrays, Western blots, and IHC were used. Protein localization and the levels of key markers were determined using immunofluorescence. DNA damage tolerance was measured through PCNA ubiquitination levels and the DNA fiber assay. Effects of pharmacologic inhibition were assessed in vitro and key results validated in vivo using patient-derived xenografts.
Ewing sarcoma tumors fell into two groups. In one, IGF1R was predominantly nuclear (nIGF1R), DNA damage tolerance pathway was upregulated, and cells had low replication stress and RRM2B levels and high levels of WEE1 and RAD21. These tumors were relatively insensitive to IGF1R inhibition. The second group had high replication stress and RRM2B, low levels of WEE1 and RAD21, membrane-associated IGF1R (mIGF1R) signaling, and sensitivity to IGF1R or WEE1-targeted inhibitors. Moreover, the matched primary and metastatic tumors differed in IGF1R localization, levels of replication stress, and inhibitor sensitivity. In all instances, combined IGF1R and WEE1 inhibition led to tumor regression.
IGF1R signaling mechanisms and replication stress levels can vary among Ewing sarcoma tumors (including in the same patient), influencing the effects of IGF1R and WEE1 treatment. These findings make the case for using biopsy-derived predictive biomarkers at multiple stages of Ewing sarcoma disease management.
