PIM1 Inhibition Affects Glioblastoma Stem Cell Behavior and Kills Glioblastoma Stem-like Cells.

Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.

Preoperative Thrombocytosis is Not Associated with Overall Survival in 309 Glioblastoma Patients.

BACKGROUND: In recent years, a correlation of thrombocytosis and a worse prognosis was shown for many solid cancers, including glioblastoma multiforme (GBM). METHODS: A retrospective review was performed for all patients with a histologically proven and first-diagnosed GBM between 2005 and 2015 in our department. Clinical and paraclinical parameters were acquired from patient documentation and structured for subsequent data analysis. The association of potential risk factors with overall survival was assessed using the Kaplan-Meier survival analysis and Cox regression. RESULTS: The present study includes 309 patients first diagnosed with primary GBM. Our analyses validate well-known risk factors of a decreased overall survival such as higher patient age, a larger preoperative tumor volume, Karnofsky performance status, extent of resection, tumor localization, and adjuvant treatment. However, no correlation was observed between a preoperative thrombocytosis, the mean platelet volume, leucocyte count, activated partial thromboplastin time (apTT), fibrinogen level, and acetylsalicylic acid 100 co-medication. Patients with preoperative hemoglobin below 7.5 mmol/L had decreased overall survival. CONCLUSION: The present study, enrolling the largest numbers of patients assessing this topic to date, did not find any association between a preoperative thrombocytosis and overall survival in 309 patients with GBM.

L-Carnitine-Mediated Tumor Cell Protection and Poor Patient Survival Associated with OCTN2 Overexpression in Glioblastoma Multiforme.

PURPOSE: Apoptotic dysregulation, redox adaptive mechanisms, and resilience to hypoxia are major causes of glioblastoma (GBM) resistance to therapy. Commonly known as crucial factors in energy metabolism, OCTN2 (SLC22A5) and its substrate L-carnitine (LC) are increasingly recognized as actors in cytoprotection. This study provides a comprehensive expression and survival analysis of the OCTN2/LC system in GBM and clarifies the system's impact on GBM progression. EXPERIMENTAL DESIGN: OCTN2 expression and LC content were measured in 121 resected human GBM specimens and 10 healthy brain samples and analyzed for prognostic significance. Depending on LC administration, the effects of hypoxic, metabolic, and cytotoxic stress on survival and migration of LN18 GBM cells were further studied in vitro. Finally, an orthotopic mouse model was employed to investigate inhibition of the OCTN2/LC system on in vivo GBM growth. RESULTS: Compared with healthy brain, OCTN2 expression was increased in primary and even more so in recurrent GBM on mRNA and protein level. High OCTN2 expression was associated with a poor overall patient survival; the unadjusted HR for death was 2.7 (95% CI, 1.47-4.91; P < 0.001). LC administration to GBM cells increased their tolerance toward cytotoxicity, whereas siRNA-mediated OCTN2 silencing led to a loss of tumor cell viability. In line herewith, OCTN2/LC inhibition by meldonium resulted in reduced tumor growth in an orthotopic GBM mouse model. CONCLUSIONS: Our data indicate a potential role of the OCTN2/LC system in GBM progression and resistance to therapy, and suggests OCTN2 as a prognostic marker in patients with primary GBM.

Platelet activation parameters and platelet-leucocyte-conjugate formation in glioblastoma multiforme patients.

Patients with glioblastoma multiforme (GBM) suffer from an increased incidence of vascular thrombotic events. However, key influencing factors of the primary hemostasis have not been characterized in GBM patients to date. Thus, the present study determines the activation level of circulating platelets in GBM patients, in-vitro reactivity to agonist-induced platelet stimulation and the formation of circulating platelet-leucocyte conjugates as well as the plasma levels of the proinflammatory lipid mediator sphingosine-1-phosphate (S1P). The endogenous thrombin potential (ETP) was determined as global marker for hemostasis. The 21 GBM patients and 21 gender and age matched healthy individuals enrolled in this study did not differ in mean total platelet count. Basal surface expression of platelet CD63 determined by flow cytometry was significantly increased in GBM patients compared to controls as was observed for the concentration of soluble P-selectin in the plasma of GBM patients. While the ETP was not affected, the immunomodulatory lipid S1P was significantly decreased in peripheral blood in GBM. Interestingly, monocyte expression of PSGL-1 (CD162) was decreased in GBM patient blood, possibly explaining the rather decreased formation of platelet-monocyte conjugates. Our study reveals an increased CD63 expression and P-selectin expression/ secretion of circulating platelets in GBM patients. In parallel a down-modulated PSGL-1 expression in circulating monocytes and a trend towards a decreased formation of heterotypic platelet-monocyte conjugates in GBM patients was seen. Whether this and the observed decreased plasma level of the immunomodulatory S1P reflects a systemic anti-inflammatory status needs to be addressed in future studies.

Expression of S1P metabolizing enzymes and receptors correlate with survival time and regulate cell migration in glioblastoma multiforme.

A signaling molecule which is involved in proliferation and migration of malignant cells is the lipid mediator sphingosine-1-phosphate (S1P). There are hints for a potential role of S1P signaling in malignant brain tumors such as glioblastoma multiforme (GBM) which is characterized by a poor prognosis. Therefore, a comprehensive expression analysis of S1P receptors (S1P1-S1P5) and S1P metabolizing enzymes in human GBM (n = 117) compared to healthy brain (n = 10) was performed to evaluate their role for patient´s survival. Furthermore, influence of S1P receptor inhibition on proliferation and migration were studied in LN18 GBM cells. Compared to control brain, mRNA levels of S1P1, S1P2, S1P3 and S1P generating sphingosine kinase-1 were elevated in GBM. Kaplan-Meier analyses demonstrated an association between S1P1 and S1P2 with patient´s survival times. In vitro, an inhibitory effect of the SphK inhibitor SKI-II on viability of LN18 cells was shown. S1P itself had no effect on viability but stimulated LN18 migration which was blocked by inhibition of S1P1 and S1P2. The participation of S1P1 and S1P2 in LN18 migration was further supported by siRNA-mediated silencing of these receptors. Immunoblots and inhibition experiments suggest an involvement of the PI3-kinase/AKT1 pathway in the chemotactic effect of S1P in LN18 cells.In summary, our data argue for a role of S1P signaling in proliferation and migration of GBM cells. Individual components of the S1P pathway represent prognostic factors for patients with GBM. Perspectively, a selective modulation of S1P receptor subtypes could represent a therapeutic approach for GBM patients and requires further evaluation.