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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. Gioblastoma tumours consist of different niches defined by histology. Here, the authors use proteomics and machine learning to assign protein expression programs to these niches, and reveal that KRAS and hypoxia are associated with drug resistance.

The aryl hydrocarbon receptor (AHR) mediates many toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone does not explain the widely different outcomes among organisms. To identify the other factors involved, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS) providing widely different resistance to TCDD toxicity, as well as C57BL/6 and DBA/2 mice which exhibit a ~ tenfold divergence in TCDD sensitivity (exposures of 5-1000 μg/kg TCDD). We supplement these with whole-genome sequencing, together with transcriptomic and proteomic analyses of the corresponding rat models, Long–Evans (L–E) and Han/Wistar (H/W) rats (having a ~ 1000-fold difference in their TCDD sensitivities; 100 μg/kg TCDD), to identify genes associated with TCDD-response phenotypes. Overall, we identified up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (33.8%, 11.7%, 5.2% and 0.3% of 3076 genes altered unique to rWT, DEL, C57BL/6 and INS respectively following 1000 μg/kg TCDD). Hepatic Pxdc1 was significantly repressed in all three TCDD-sensitive animal models (C57BL/6 and rWT mice, and L–E rat) after TCDD exposure. Three genes, including Cxxc5, Sugp1 and Hgfac, demonstrated different AHRE-1 (full) motif occurrences within their promoter regions between rat strains, as well as different patterns of mRNA abundance. Several hepatic proteins showed parallel up- or downward alterations with their RNAs, with three genes (SNRK, IGTP and IMPA2) showing consistent, strain-dependent changes. These data show the value of integrating genomic, transcriptomic and proteomic evidence across multi-species models in toxicologic studies.

The aryl hydrocarbon receptor (AHR) mediates many of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone is insufficient to explain the widely different outcomes among organisms. Attempts to identify unknown factor(s) have been confounded by genetic variability of model organisms. Here, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS), as well as C57BL/6 and DBA/2 mice. We supplement these with whole-genome sequencing and transcriptomic analyses of the corresponding rat models: Long-Evans (L-E) and Han/Wistar (H/W) rats. These integrated multi-species genomic and transcriptomic data were used to identify genes associated with TCDD-response phenotypes. We identified several genes that show consistent transcriptional changes in both transgenic mice and rats. Hepatic Pxdc1 was significantly repressed by TCDD in C57BL/6, rWT mice, and in L-E rat. Three genes demonstrated different AHRE-1 (full) motif occurrences within their promoter regions: Cxxc5 had fewer occurrences in H/W, as compared with L-E; Sugp1 and Hgfac (in either L-E or H/W respectively). These genes also showed different patterns of mRNA abundance across strains. The AHR isoform explains much of the transcriptional variability: up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (30% and 10% unique to DEL and rWT respectively following 500 μg/kg TCDD). Genomic and transcriptomic evidence allowed identification of genes potentially involved in phenotypic outcomes: Pxdc1 had differential mRNA abundance by phenotype; Cxxc5 had altered AHR binding sites and differential mRNA abundance.

Low levels of oxygen (hypoxia) in solid tumors contributes to therapy resistance and poor patient prognosis. How the cells in the tumor microenvironment (TME) adapt to hypoxia at a transcriptional, epigenetic, and translational level has been extensively explored. Here, we provide a comprehensive review on how molecular signaling in hypoxia represents an intricate, coordinated response triggered by oxygen dependent enzymes. Our work also highlights the lack of understanding of the requirement for oxygen in protein folding and the biological roles of an oxygen dependent enzyme, 2-aminoethanethiol dioxygenase (ADO).Secreted proteins contribute to aggressive cancer phenotypes in the TME. These secreted proteins are thought to utilize an oxygen dependent mechanism for disulfide bond formation required for proper folding, which represents a paradox when they are expressed under hypoxic conditions. Here we confirm the existence of oxygen independent pathways for disulfide bond formation. For the first time we demonstrate that key hypoxia-induced proteins such as vascular endothelial growth factor (VEGF-A) and carbonic anhydrase 9 (CA9) remain fully competent at disulfide bond formation in the absence of oxygen, supporting their efficient expression in hypoxia. This work highlights that the ability of individual proteins to undergo protein folding in the absence of oxygen ultimately determines their expression in the extracellular space in hypoxia.In parallel, we explored the cellular and physiological roles of a relatively uncharacterized oxygen sensing enzyme, ADO. ADO synthesizes hypotaurine and directly regulates protein stability through catalyzing a post translational modification in an oxygen dependent manner. Here we show that ADO is essential for the growth and survival of cancer cells and promotes tumor xenograft growth. ADO mitigates reactive oxygen species (ROS) through regulating proline metabolism. These data provide novel insights into how depletion of an oxygen dependent enzyme may elicit a metabolic imbalance leading to loss of redox homeostasis.Taken together, this thesis explores the role and the importance of oxygen and oxygen dependent enzymes that drive acute responses such as affecting the disulfide bond formation of protein folding and deregulation of the cancer metabolome.

As cancer cells evade therapeutic pressure and adopt alternate lineage identities not commonly observed in the tissue of origin, they likely adopt alternate metabolic programs to support their evolving demands. Targeting these alternative metabolic programs in distinct molecular subtypes of aggressive prostate cancer may lead to new therapeutic approaches to combat treatment-resistance. We identify the poorly studied metabolic enzyme Oxoglutarate Dehydrogenase-Like (OGDHL), named for its structural similarity to the tricarboxylic acid (TCA) cycle enzyme Oxoglutarate Dehydrogenase (OGDH), as an unexpected regulator of tumor growth, treatment-induced lineage plasticity, and DNA Damage in prostate cancer. While OGDHL has been described as a tumor-suppressor in various cancers, we find that its loss impairs prostate cancer cell proliferation and tumor formation. Loss of OGDHL profoundly alters Androgen Receptor inhibition-induced plasticity, including suppressing the neuroendocrine markers DLL3 and HES6, induces accumulation of the DNA damage response marker ƔH2AX, and reduces nucleotide synthesis. Our data suggest that OGDHL has minimal impact on TCA cycle activity, and that mitochondrial localization is not required for its regulation of prostate cancer plasticity and nucleotide metabolism. Finally, we demonstrate that OGDHL expression is tightly correlated with neuroendocrine differentiation in clinical prostate cancer. These findings underscore the importance of investigating poorly characterized metabolic genes as potential regulators of distinct molecular subtypes of aggressive cancer.