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

Abstract

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.

Focus on Hypoxia-Related Pathways in Pediatric Osteosarcomas and Their Druggability

“…Recent studies have linked hypoxia and immunomodulation scores in OTS [ 77 , 85 ]. This score also supports the idea that hypoxia-driven immunity is associated with glycolytic metabolic switch, collagen biosynthesis and redox regulation.…”

“…When focusing on HIFs, several levels of inhibition can be listed and are summarized in Figure 3 . In fact, a growing number of molecules have been demonstrated to inhibit HIFs by reducing mRNA or protein levels, DNA-binding activity or the trans-activation of some HIFs related genes [ 85 ]. They can also block HIFs/HIF-1β dimerization.…”

“…To date, no specific inhibitors of HIF-1α have been brought to the market, but the use of indirect or partial inhibitors are currently increasing in trials and recently in OTS protocols. Oppositely, recent direct inhibitors of HIF-2α have been developed and commercialized, such as PT2385 or PT2977, but are not used directly in OTS and only preclinical data show that HIF-2α targeting can attenuate the proliferation, migration and invasion of OTS cells [ 61 , 85 , 86 ]. Those specific inhibitors of HIF-2α destabilize heterodimerization of HIF-2α with ARNT, leading to inhibition of target genes and tumor regression.…”

“…Those specific inhibitors of HIF-2α destabilize heterodimerization of HIF-2α with ARNT, leading to inhibition of target genes and tumor regression. These treatments were used recently in clinical trials for patients with clear cell renal cell carcinoma, where the majority of patients had a partial response or a stable disease associated with a good tolerance [ 61 , 85 , 86 ]. The HIF-1α inhibitors are mostly decreasing HIF-1α expression with a proven mRNA downregulation in the OTS preclinical models or in cancer trials [ 85 , 86 , 87 , 88 , 89 , 90 ].…”

Targeting Mechanotransduction in Osteosarcoma: A Comparative Oncology Perspective

“…Proteomic analysis comparing primary and metastatic canine OSA cells under normoxic or hypoxic conditions found that P4HA1, PLOD1 and 2 and LOX were all increased by hypoxia, with a more dramatic effect seen in the metastatic cell line [ 103 ]. Contrasting these observations, LOX was reported to have tumour suppressive effects in human OSA cell lines, although this capacity was not tested in vivo [ 104 ].…”