A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.

Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.

PERK-mediated expression of peptidylglycine α-amidating monooxygenase supports angiogenesis in glioblastoma.

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells.

Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.

Liposomal sphingomyelin influences the cellular lipid profile of human lymphoblastic leukemia cells without effect on P-glycoprotein activity.

Sphingomyelin (SM)/cholesterol liposomes are currently investigated as drug carriers in cancer therapy. However, no data is available on the influence of SM itself on P-glycoprotein (P-gp) mediated multidrug resistance. P-gp is at least partly located in sphingolipid-enriched lipid raft domains of the plasma membrane, and its activity depends on the lipid profile of the membrane, which could be altered by therapeutical SM liposomes. Therefore, the aim of this study was to analyze the effect of liposomal SM on P-gp activity, P-gp distribution in microdomains, SM content of the membrane domains, and sensitivity of human lymphoblastic CEM cells toward cytotoxic drugs in vitro. Assays were conducted in CEM and multidrug resistant CEM/ADR5000 cells. SM-only liposomes were prepared by a newly developed ethanol injection protocol and thoroughly characterized. Inclusion of SM into the membrane was analyzed by fluorescence microscopy and flow cytometry. Influence of SM liposomes on P-gp activity was assessed by rhodamine efflux and calcein assay, and sensitivity toward cytotoxic drugs was analyzed by flow cytometric 7-AAD staining. Influence on P-gp distribution was analyzed by Western blot after density gradient centrifugation. SM 16:0, 18:0, and 24:1 were quantified by liquid chromatography coupled to tandem mass spectrometry. P-gp was mainly located in nonraft fractions, which did not change upon liposome treatment. Liposomes increased SM 16:0 and SM 24:1 content in nonraft domains, but not in raft domains of multidrug resistant cells. SM-only liposomes did not influence P-gp activity and chemosensitivity. In conclusion, SM-only liposomes in therapeutic amounts did not influence P-gp mediated multidrug resistance in CEM cells.