Deciphering Potential Role of Unfolded Protein Response in Glioblastoma

The unfolded protein response (UPR) plays a significant role in reducing the burden of protein from the endoplasmic reticulum (ER) of cells by enhancing the expression of factors which facilitate protein folding, such as chaperones and isomerases, while simultaneously reducing translation and initiating ER-associated degradation pathways. When confronted with adverse conditions in their microenvironment, tumor cells need to adapt their proteomes by producing and secreting factors which support their survival and growth. This results in the generation of ER stress conditions that leads to activation of the UPR pathway. UPR has been linked to almost every type of cancer including grade IV glioma, glioblastoma, a devastating disease with patients surviving on average only 15 months after diagnosis. One of the reasons for this poor prognosis is the ability of glioblastoma to induce early angiogenesis followed by invasion of the normal brain parenchyma. This PhD thesis focuses on understanding how UPR is regulated in glioblastoma and how this contributes to tumor angiogenesis. To this end, the UPR pathway in glioblastoma cell lines was first characterized using artificial ER stress inducers. As hypoxia is the leading physiological inducer of UPR while also regulating the early events of glioblastoma progression, the UPR branches that are activated under hypoxia were investigated. PKR-like kinase (PERK), a UPR sensor protein which controls the translation machinery of tumor cells under ER stress was examined in detail, and it was determined whether this protein is involved in the regulation of expression and secretion of any angiogenic proteins in glioblastoma. For this purpose, proteomic analysis of the conditioned media of LN308 glioblastoma cells with and without PERK inhibition was performed and PERK-regulated secretory factors were identified. Among the hits was Peptidyl-glycine alpha-amidating monooxygenase (PAM), a transmembrane protein which plays a role in the rate-limiting final step of the activation of various neuropeptides, including the angiogenic peptide adrenomedullin (ADM). PAM was validated as a pro-angiogenic factor for glioblastoma, its regulation by hypoxia (HIF1α) was characterized, and the importance of PERK kinase activity for the generation of a small soluble cytosolic cleaved product of PAM (PAM-sfCD), which has the ability to induce potential gene expression changes favoring glioblastoma progression, was shown. PAM was also found to have mesenchymal-subtype specific expression in glioblastoma, where AP-1 was found to be the leading transcription factor regulating PAM transcripts. The in vivo study in this thesis illustrates the importance of PAM for glioblastoma growth kinetics in an orthotopic xenotransplanted mouse model, revealing an increased overall survival of animals upon PAM knockdown. Supporting this, clinical data suggest better survival of glioblastoma patients with lower expression of PAM. Thus, this work reveals the importance of the PERK-mediated expression and post-translational modification of PAM for angiogenesis and tumor progression in glioblastoma, introducing the protein as a novel anti-angiogenic therapeutic target in this deadly disease