Glioblastoma is an aggressive form of brain cancer with well-established patterns of intra-tumoral heterogeneity implicated in treatment resistance and progression. While regional and single cell transcriptomic variations of glioblastoma have been recently resolved, downstream phenotype-level proteomic programs have yet to be assigned across glioblastoma’s hallmark histomorphologic niches. Here, we leverage mass spectrometry to spatially align abundance levels of 4,794 proteins to distinct histologic patterns across 20 patients and propose diverse molecular programs operational within these regional tumor compartments. Using machine learning, we overlay concordant transcriptional information, and define two distinct proteogenomic programs, MYC- and KRAS-axis hereon, that cooperate with hypoxia to produce a tri-dimensional model of intra-tumoral heterogeneity. Moreover, we highlight differential drug sensitivities and relative chemoresistance in glioblastoma cell lines with enhanced KRAS programs. Importantly, these pharmacological differences are less pronounced in transcriptional glioblastoma subgroups suggesting that this model may provide insights for targeting heterogeneity and overcoming therapy resistance.
Low levels of oxygen (hypoxia) occurs in many (patho)physiological situations. Adaptation to hypoxia is in part mediated by proteins expressed in the extracellular space that mature in the endoplasmic reticulum (ER) prior to traversing the secretory pathway. The majority of such ER cargo proteins require disulfide bonds for structural stability. Disulfide bonds are formed co- and posttranslationally in a redox relay that requires a terminal electron acceptor such as oxygen. We have previously demonstrated that some ER cargo proteins such as low-density lipoprotein receptor (LDLR) and influenza hemagglutinin (Flu-HA) are unable to complete disulfide bond formation in the absence of oxygen, limiting their ability to pass ER quality control and their ultimate expression. Here, using radioactive pulse-chase immunoprecipitation analysis, we demonstrate that hypoxia-induced ER cargo proteins such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor A (VEGF-A) complete disulfide bond formation and mature with similar kinetics under hypoxia and normoxia. A global in silico analysis of ER cargo revealed that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other ER cargo proteins. These data demonstrate the existence of alternative electron acceptors to oxygen for disulfide bond formation in cellulo . However, the ability of different proteins to utilize an oxygen-independent pathway for disulfide bond formation varies widely, contributing to differential gene expression in hypoxia. The superior ability of hypoxia-induced proteins such as VEGF-A and CA9 to mature in hypoxia may be conferred by a simpler disulfide architecture.
Tumor hypoxia results in poor patient outcome due to treatment resistance as well as biological changes that stimulate angiogenesis, vasculogenesis, migration, invasion and immune suppression. These hypoxia-induced adverse biological changes are often mediated by membrane bound or secreted proteins through transcriptional and translational upregulation. Thus, understanding the regulation of how secreted proteins in hypoxia can therefore reveal novel therapeutic targets. Proteins that traverse through the secretory pathway form disulfide bonds in the endoplasmic reticulum (ER). Recent data from our lab have demonstrated that disulfide bond formation remains incomplete in ER cargo proteins like LDLR and Flu-HA in the absence of oxygen. To address whether hypoxia-induced proteins were likewise impaired, radioactive pulse chase assays were performed to measure disulfide bond formation and secretion capacity under both normoxic and hypoxic conditions. Here, we demonstrate that both hypoxia induced proteins carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) complete disulfide bond formation and are secreted with equal kinetics under hypoxia and normoxia. These proteins hence have a superior ability to be expressed in the absence of oxygen. Additionally, in a global in silico analysis of all proteins that traverse through the ER, we discovered that hypoxia-induced proteins on average contain fewer free cysteines and shorter-range disulfide bonds in comparison to other proteins. These traits may contribute to their superior ability to form correct disulfide bonds in hypoxia. These data show that the ability of proteins to form native disulfide bonds in hypoxia varies widely which can ultimately contribute to their expression in the extracellular space. Citation Format: Sandy Che-Eun Serena Lee, Fiana Levitin, Stephanie Hulme, Ryan Rumantir, Jenna Sykes, Marianne Koritzinsky. Protein secretion rates of VEGF and CA9 in normoxia and hypoxia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3559.
2-aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a role in both metabolism and protein stability. ADO directly metabolizes cysteamine to produce hypotaurine and taurine in mammals. ADO has also been recently identified to promote oxygen dependent stability of a subset of substrates involved in the N-degron pathway in mammals (IL32, RGS4 and RGS5). The ability of ADO to target protein stability of signaling molecules suggests that it may have the potential to transduce rapid responses to hypoxia and affect tumour initiation and progression phenotypes. Here, we have successfully knocked down and knocked out ADO using two independent siRNAs and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system, respectively. We have assessed proliferation and migration through the Incucyte® ZOOM system by imaging cell confluency over time. Survival was assessed through a clonogenic assay. siRNA mediated knockdown of ADO in cervical (HeLa and SiHa), pancreatic (Panc1 and Capan2) and liver (SNU499 and Huh6) cancer cell lines drastically reduced proliferation, survival, and migration in normoxia. These results were also replicated in hypoxia (0.2% O2) across all 6 cell lines. Out of the 6 cell lines, the liver cancer cell lines were most drastically affected by the knockdown of ADO. This phenotype was replicated in the ADO KO cell lines. Taken together, these data suggest that expression of ADO may contribute to phenotypes that induce aggressive tumour phenotypes by targeting the stability of specific proteins and altering cellular metabolism in mammals. Citation Format: Sandy Che-Eun Serena Lee, Andrea Hye An Pyo, Marianne Koritzinsky. ADO contributes to tumour initiating phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1431.
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