Metformin and simvastatin exert additive antitumour effects in glioblastoma via senescence-state: clinical and translational evidence.

BACKGROUND: Glioblastoma is one of the most devastating and incurable cancers due to its aggressive behaviour and lack of available therapies, being its overall-survival from diagnosis ∼14-months. Thus, identification of new therapeutic tools is urgently needed. Interestingly, metabolism-related drugs (e.g., metformin/statins) are emerging as efficient antitumour agents for several cancers. Herein, we evaluated the in vitro/in vivo effects of metformin and/or statins on key clinical/functional/molecular/signalling parameters in glioblastoma patients/cells. METHODS: An exploratory-observational-randomized retrospective glioblastoma patient cohort (n = 85), human glioblastoma/non-tumour brain human cells (cell lines/patient-derived cell cultures), mouse astrocytes progenitor cell cultures, and a preclinical xenograft glioblastoma mouse model were used to measure key functional parameters, signalling-pathways and/or antitumour progression in response to metformin and/or simvastatin. FINDINGS: Metformin and simvastatin exerted strong antitumour actions in glioblastoma cell cultures (i.e., proliferation/migration/tumoursphere/colony-formation/VEGF-secretion inhibition and apoptosis/senescence induction). Notably, their combination additively altered these functional parameters vs. individual treatments. These actions were mediated by the modulation of key oncogenic signalling-pathways (i.e., AKT/JAK-STAT/NF-κB/TGFß-pathways). Interestingly, an enrichment analysis uncovered a TGFß-pathway activation, together with AKT inactivation, in response to metformin + simvastatin combination, which might be linked to an induction of the senescence-state, the associated secretory-phenotype, and to the dysregulation of spliceosome components. Remarkably, the antitumour actions of metformin + simvastatin combination were also observed in vivo [i.e., association with longer overall-survival in human, and reduction in tumour-progression in a mouse model (reduced tumour-size/weight/mitosis-number, and increased apoptosis)]. INTERPRETATION: Altogether, metformin and simvastatin reduce aggressiveness features in glioblastomas, being this effect significantly more effective (in vitro/in vivo) when both drugs are combined, offering a clinically relevant opportunity that should be tested for their use in humans. FUNDING: Spanish Ministry of Science, Innovation and Universities; Junta de Andalucía; CIBERobn (CIBER is an initiative of Instituto de Salud Carlos III, Spanish Ministry of Health, Social Services and Equality).

Somatostatin Receptor Splicing Variant sst5TMD4 Overexpression in Glioblastoma Is Associated with Poor Survival, Increased Aggressiveness Features, and Somatostatin Analogs Resistance.

Glioblastoma (GBM) is the most malignant and lethal brain tumor. Current standard treatment consists of surgery followed by radiotherapy/chemotherapy; however, this is only a palliative approach with a mean post-operative survival of scarcely ~12-15 months. Thus, the identification of novel therapeutic targets to treat this devastating pathology is urgently needed. In this context, the truncated splicing variant of the somatostatin receptor subtype 5 (sst5TMD4), which is produced by aberrant alternative splicing, has been demonstrated to be overexpressed and associated with increased aggressiveness features in several tumors. However, the presence, functional role, and associated molecular mechanisms of sst5TMD4 in GBM have not been yet explored. Therefore, we performed a comprehensive analysis to characterize the expression and pathophysiological role of sst5TMD4 in human GBM. sst5TMD4 was significantly overexpressed (at mRNA and protein levels) in human GBM tissue compared to non-tumor (control) brain tissue. Remarkably, sst5TMD4 expression was significantly associated with poor overall survival and recurrent tumors in GBM patients. Moreover, in vitro sst5TMD4 overexpression (by specific plasmid) increased, whereas sst5TMD4 silencing (by specific siRNA) decreased, key malignant features (i.e., proliferation and migration capacity) of GBM cells (U-87 MG/U-118 MG models). Furthermore, sst5TMD4 overexpression in GBM cells altered the activity of multiple key signaling pathways associated with tumor aggressiveness/progression (AKT/JAK-STAT/NF-κB/TGF-ß), and its silencing sensitized GBM cells to the antitumor effect of pasireotide (a somatostatin analog). Altogether, these results demonstrate that sst5TMD4 is overexpressed and associated with enhanced malignancy features in human GBMs and reveal its potential utility as a novel diagnostic/prognostic biomarker and putative therapeutic target in GBMs.

SF3B1 inhibition disrupts malignancy and prolongs survival in glioblastoma patients through BCL2L1 splicing and mTOR/ß-catenin pathways imbalances.

BACKGROUND: Glioblastoma is one of the most devastating cancer worldwide based on its locally aggressive behavior and because it cannot be cured by current therapies. Defects in alternative splicing process are frequent in cancer. Recently, we demonstrated that dysregulation of the spliceosome is directly associated with glioma development, progression, and aggressiveness. METHODS: Different human cohorts and a dataset from different glioma mouse models were analyzed to determine the mutation frequency as well as the gene and protein expression levels between tumor and control samples of the splicing-factor-3B-subunit-1 (SF3B1), an essential and druggable spliceosome component. SF3B1 expression was also explored at the single-cell level across all cell subpopulations and transcriptomic programs. The association of SF3B1 expression with relevant clinical data (e.g., overall survival) in different human cohorts was also analyzed. Different functional (proliferation/migration/tumorspheres and colonies formation/VEGF secretion/apoptosis) and mechanistic (gene expression/signaling pathways) assays were performed in three different glioblastomas cell models (human primary cultures and cell lines) in response to SF3B1 blockade (using pladienolide B treatment). Moreover, tumor progression and formation were monitored in response to SF3B1 blockade in two preclinical xenograft glioblastoma mouse models. RESULTS: Our data provide novel evidence demonstrating that the splicing-factor-3B-subunit-1 (SF3B1, an essential and druggable spliceosome component) is low-frequency mutated in human gliomas (~ 1 %) but widely overexpressed in glioblastoma compared with control samples from the different human cohorts and mouse models included in the present study, wherein SF3B1 levels are associated with key molecular and clinical features (e.g., overall survival, poor prognosis and/or drug resistance). Remarkably, in vitro and in vivo blockade of SF3B1 activity with pladienolide B drastically altered multiple glioblastoma pathophysiological processes (i.e., reduction in proliferation, migration, tumorspheres formation, VEGF secretion, tumor initiation and increased apoptosis) likely by suppressing AKT/mTOR/ß-catenin pathways, and an imbalance of BCL2L1 splicing. CONCLUSIONS: Together, we highlight SF3B1 as a potential diagnostic and prognostic biomarker and an efficient pharmacological target in glioblastoma, offering a clinically relevant opportunity worth to be explored in humans.