Abstract A34: Hypoxia signaling pathway is frequently involved in pediatric osteosarcoma microenvironment, as diagnostic and prognostic biomarkers, but also as new therapeutic targets
Authors: Pierrevelcin Marina , Grain Audrey , Tripp Aurelien , Obrecht Adeline , Lhermitte Benoit , Weingertner Noelle , Gaspar Nathalie , Villa Pascal , Lelong-Rebel Isabelle , Redini Françoise , Dontenwill Monique , Entz-Werlé Natacha 
Published: August 2020

Abstract
Osteosarcoma is the first bone cancer diagnosed in adolescent and young adults. Multiple studies involved a deregulation of osteoblast, osteoclast, or microenvironment genes in their development and progression. Nevertheless, no focus was already done in the oxygen-modulated environment of those cancers. Our objectives in this study were to determine, first, the presence of hypoxia deregulation in a cohort of pediatric osteosarcomas (pOS at diagnosis) and in 4 PDCLs (patient-derived cell lines from diagnostic samples). For this purpose, we performed CGHarray for 67 samples and a validation by semiquantitative PCR in another cohort of 20 samples. We also explored by immunohistochemistry in 30 tumor specimens protein expressions of key biomarkers from the hypoxic signaling pathway. Those biomarkers were validated and assessed functionally by immunofluorescence and Western blotting in PDCLs and paired xenografts. All those results in the pOS collections were correlated to survivals and response to first-line therapies. Secondly, the role of hypoxia modulation in PDCLs was determined using variations of oxygen levels from 21% to 5% on cell proliferation characteristics and protein parameters. Finally, to understand how the process of hypoxia is a potential target for pOS treatment, we inhibited different targets of the mTor/HIF1alpha pathway in those PDCLs. As expected, the hypoxia signaling pathway was highly expressed in pOS (tumor samples and PDCLs). Several biomarkers (for example, ULK1, USP33, VEGFR2, CCL7, etc.) were overexpressed in specific groups, linking them significantly to prognosis and a response to chemotherapy. Most of them were involved in the metabolism, autophagy, or angiogenic processes. The PDCLs also expressed in vitro and in xenografts several proteins of this hypoxic pathway as in tumors themselves (pS6, phosphor-mTor, pAKT, HIF1alpha, HIF2alpha). The induction of HIF1alpha during oxygen decrease is early and constant in PDCLs. mTor and HIF2alpha were not induced by the variation of oxygen and were constantly expressed in the cells. Surprisingly, most of the PDCLs increase their proliferation rate in hypoxic conditions, but do not change their microscopic aspects. Finally, the inhibition of mTor and HIF1alpha with rapamycine and irinotecan, respectively, decreases the cell proliferation with a complete inhibition of the targets. In conclusion, hypoxia seems to be frequently involved in pOS through different downstream signaling processes and might be a new potential way of treatment.
Citation Format: Marina Pierrevelcin, Audrey Grain, Aurelien Tripp, Adeline Obrecht, Benoit Lhermitte, Noelle Weingertner, Nathalie Gaspar, Pascal Villa, Isabelle Lelong-Rebel, Françoise Redini, Monique Dontenwill, Natacha Entz-Werlé. Hypoxia signaling pathway is frequently involved in pediatric osteosarcoma microenvironment, as diagnostic and prognostic biomarkers, but also as new therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A34.