TY  - JOUR
AU  - Stillger, Maren Nicole
AU  - Chen, Chia-Yi
AU  - Lai, Zon Weng
AU  - Li, Mujia
AU  - Schäfer, Agnes
AU  - Pagenstecher, Axel
AU  - Nimsky, Christopher
AU  - Bartsch, Jörg Walter
AU  - Schilling, Oliver
TI  - Changes in calpain-2 expression during glioblastoma progression predisposes tumor cells to temozolomide resistance by minimizing DNA damage and p53-dependent apoptosis.
JO  - Cancer cell international
VL  - 23
IS  - 1
SN  - 1475-2867
CY  - London
PB  - BioMed Central
M1  - DKFZ-2023-00559
SP  - 49
PY  - 2023
AB  - Glioblastoma 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.Mass 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 ɣH2AX 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.Calcium-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-κB signaling. TP53 showed increased protein abundance, but no transcriptional regulation.TMZ-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.
KW  - Calpain-1 (Other)
KW  - Calpain-2 (Other)
KW  - DNA damage (Other)
KW  - Glioblastoma (Other)
KW  - TP53 (Other)
KW  - Temozolomide resistance (Other)
KW  - U251N (Other)
LB  - PUB:(DE-HGF)16
C6  - pmid:36932402
C2  - pmc:PMC10022304
DO  - DOI:DOI: 10.1186/s12935-023-02889-8
UR  - https://inrepo02.dkfz.de/record/274357
ER  -