Bartsch, J. W. | Search bioRxiv

bioRxiv2024

Proteomic profiling of IDH-wildtype Glioblastoma Tissue and Serum uncovers prognostic Subtypes and Marker Candidates

BackgroundIDH-wildtype glioblastoma (GBM) is the most prevalent primary brain cancer with a 5-year survival rate below 10%. Despite combined treatment through extensive resection and radiochemotherapy, nine out of ten patients develop recurrences. The lack of targeted treatment options and reliable diagnostic markers for recurrent tumors remain major challenges. Methods & AimsIn this study, we present the proteomic characterization of tissue and serum from 55 initial GBM tumors and five matching recurrences, which we investigated for proteomic tumor subtypes and proteomic signatures associated with recurrence. ResultsPrimary tumors revealed four distinct subgroups through hierarchical clustering: a neuronal cluster with elevated mature neuron markers, an innate immunity cluster with increased protease expression, a mixed cluster, and a stem-cell cluster. Neurodevelopmental and inflammatory processes were identified as key factors influencing clustering, with proteolytic activity increasing relative to the degree of inflammation. An analysis comprising proteins with lower coverage confirmed and expanded this pattern. Patients in the neuronal cluster exhibited significantly longer survival compared to those in the stem-cell cluster. In a patient-matched differential expression analysis, five recurrent tumors displayed significantly altered protein expression compared to their primary counterparts, emphasizing the proteomic plasticity of recurrent tumors. Investigation of serum proteomes before and after surgery, using a depletion-based protocol, revealed highly patient-specific and stable proteome compositions, despite a notable increase in inflammation markers post-surgery. However, the levels of circulating proteolytic products matched to the proteolytic activity within the tissue and one fragment of proteolysis activated receptor 2 (PAR2) consistently dropped in abundance after removal of inflamed tumors. ConclusionOverall, we describe a large proteomic GBM cohort. We identified distinct tumor subgroups, molecular patterns of recurrence, and matching proteomic patterns in the bloodstream, which may improve risk prediction for recurrent GBM.

cancer biology↗

bioRxiv2022

Changes in calpain-2 expression during glioblastoma progression predisposes tumor cells to temozolomide resistance by minimizing DNA damage and p53-dependent apoptosis

BackgroundGlioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance. MethodsMass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ +/- calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and {gamma}H2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation. ResultsCalcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-{kappa}B signaling. TP53 showed increased protein abundance, but no transcriptional regulation. ConclusionTMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via "priming" GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.

cancer biology↗