Delineation of hypoxia-induced proteome shifts in osteosarcoma cells with different metastatic propensities.

Osteosarcoma (OS) is the most common bone cancer in children and young adults. Solid tumors are characterized by intratumoral hypoxia, and hypoxic cells are associated with the transformation to aggressive phenotype and metastasis. The proteome needed to support an aggressive osteosarcoma cell phenotype remains largely undefined. To link metastatic propensity to a hypoxia-induced proteotype, we compared the protein profiles of two isogenic canine OS cell lines, POS (low metastatic) and HMPOS (highly metastatic), under normoxia and hypoxia. Label-free shotgun proteomics was applied to comprehensively characterize the hypoxia-responsive proteome profiles in the OS cell phenotypes. Hypothesis-driven parallel reaction monitoring was used to validate the differential proteins observed in the shotgun data and to monitor proteins of which we expected to exhibit hypoxia responsiveness, but which were absent in the label-free shotgun data. We established a "distance" score (|z _HMPOS  - z _POS |), and "sensitivity" score (|z _Hypoxia  - z _Normoxia ) to quantitatively evaluate the proteome shifts exhibited by OS cells in response to hypoxia. Evaluation of the sensitivity scores for the proteome shifts observed and principal component analysis of the hypoxia-responsive proteins indicated that both cell types acquire a proteome that supports a Warburg phenotype with enhanced cell migration and proliferation characteristics. Cell migration and glucose uptake assays combined with protein function inhibitor studies provided further support that hypoxia-driven adaption of pathways associated with glycolytic metabolism, collagen biosynthesis and remodeling, redox regulation and immunomodulatory proteins typify a proteotype associated with an aggressive cancer cell phenotype. Our findings further suggest that proteins involved in collagen remodeling and immune editing may warrant further evaluation as potential targets for anti-metastatic treatment strategies in osteosarcoma.