Abstract
BackgroundOsteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents. The current treatment approach includes neoadjuvant and adjuvant chemotherapy; however, drug resistance often hinders therapy in OS patients. Also, the post-relapse survival of OS patients is as low as 20%. We therefore planned to understand the molecular cause for its poor prognosis and design an appropriate therapeutic strategy to combat the disease.MethodsWe analyzed OS patient dataset from Gene Expression Omnibus (GEO) and identified the differentially expressed genes and the top deregulated pathways in OS. Subsequently, drugs targeting the major de-regulated pathways were selected and the following assays were conducted- MTT assay to assess cytotoxicity of drugs in OS cells; immunoblotting and immunostaining to analyze key protein expression and localization after drug treatment; LysoTracker staining to monitor lysosomes; Acridine Orange to label acidic vesicles; and DCFDA to measure Reactive Oxygen Species (ROS).ResultsThe differential gene expression analysis from OS patient dataset implicated the striking involvement of cellular processes linked to autophagy and protein processing in the development of OS. We therefore selected the FDA approved drugs, chloroquine (CQ) and verteporfin (VP) known for autophagy inhibitory and proteotoxic functions to explore against OS. Importantly, VP, but not CQ, showed an extensive dose-dependent cytotoxicity. It resulted in autophagy disruption at multiple steps extending from perturbation of early autophagic processes, inhibition of autophagic flux to induction of lysosomal instability. Interestingly, VP treated protein lysates showed a ROS-dependent high molecular weight (HMW) band when probed for P62 and P53 protein. Further, VP triggered accumulation of ubiquitinated proteins as well. Since VP had a pronounced disruptive effect on cellular protein homeostasis, we explored the possibility of simultaneous inhibition of the ubiquitin-proteasomal system (UPS) by MG-132 (MG). Addition of a proteasomal inhibitor significantly aggravated VP induced cytotoxicity. MG co-treatment also led to selective targeting of P53 to the lysosomes.ConclusionHerein, we propose VP and MG induce regulation of autophagy and protein homeostasis which can be exploited as an effective therapeutic strategy against osteosarcoma.
Highlights
Osteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents
Autophagy associated pathways are frequently dysregulated in OS patients To understand the cause of the poor prognosis of OS, we analyzed the gene expression data of OS patients, extracted from Gene Expression Omnibus (GEO)
Autophagy, endocytosis and phagosome were found to harbor a high percentage of differentially expressed genes implicating their probable involvement in the disease (Fig. 1b)
Summary
Osteosarcoma (OS) is a malignant tumor of the bone mostly observed in children and adolescents. Saini et al Cancer Cell Int (2021) 21:52 often includes a multi-modal therapeutic procedure with a pre-operative poly-chemotherapy followed by surgery, and a post-operative chemotherapy again [2] Even with such an aggressive treatment regime, chemotherapy often fails in patients with OS. This despondency demands further careful analysis of the molecular signature of OS and design of an appropriate therapeutic strategy to tame the disease In this context, analysis of the existing literature indicates a plausible role of the cellular homeostatic process- autophagy in OS pathogenesis [4,5,6,7]. It is reported to mediate contrasting cellular effects, like, cell survival or cell death in a context dependent manner This implicates the need for further exploration of the role of autophagy in cancer [8]. Further research in this direction is required to enable introduction of a prospective treatment strategy, targeting autophagy, against cancers like OS
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