Inhibiting p21-Activated Kinase Induces Cell Death in Vestibular Schwannoma and Meningioma via Mitotic Catastrophe

Abstract

Hypothesis p21-Activated Kinase (PAK) regulates signaling pathways that promote cell survival and proliferation; therefore, pharmacological inhibition of PAK will induce cell death in vestibular schwannomas (VS) and meningiomas. Background All VS and many meningiomas result from loss of the neurofibromatosis type 2 (NF2) gene product merlin, with ensuing PAK hyperactivation and increased cell proliferation/survival. Methods The novel small molecule PAK inhibitors PI-8 and PI-15 – tested in schwannoma and meningioma cells – perturb molecular signaling and induce cell death. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), flow cytometry and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay analyzed PAK inhibitors’ effect on cell viability, cell cycle and cell death, respectively. Western blots evaluated activation and expression of cell proliferation, apoptotic, and mitotic catastrophe markers. Light microscopy evaluated cell morphology, and immunocytochemistry analyzed cellular localization of phospho-Merlin and ATG5 (autophagy-related protein). Results Treatment with PI-8 and PI-15 decreased cell viability at 0.65–3.7 μM IC50 in schwannoma and meningioma cells. TUNEL and immunocytochemistry studies show that PI-8 and PI-15 induce mitotic catastrophe but not apoptosis in HEI193 cells while in BenMen1 cells, PI-8 induces autophagy and mitotic catastrophe. PI-15 induces apoptosis in BenMen1 cells. PAK inhibitor treated cells show phospho-Merlin localized to over-duplicated centrosomes of dividing cells, multiple enlarged nuclei, and misaligned/missegregated chromosomes – markers for mitotic catastrophe. Increased ATG5 levels in the nucleus confirmed this cell death type. PI-8 and PI-15 inhibits PAK in both cell lines. However, only PI-15 inhibits AKT (v-Akt Murine Thymoma Viral Oncogene Homolog) in BenMen1 cells. Conclusion PAK inhibitors induce cell death in schwannoma and meningioma cells, at least in part, by mitotic catastrophe.

Potential Molecular Biomarkers of Vestibular Schwannoma Growth: Progress and Prospects

“…Activated Rac expression induces merlin phosphorylation and decreases the association between merlin and cytoskeleton ( 60 ). Based on the study of the role of Ras pathway signal transduction in the growth of VS, preclinical assessment of PAK inhibitors ( 88 ), MEK1/2 inhibitors ( 81 ) and Ailanthone, the down regulator of Ras and Raf, all exhibited certain antitumor properties ( 89 ).…”

“…An increasing body of literature suggests that merlin counteract Ras-induced transformation at multiple levels. Merlin interferes with the expression of endogenous growth factor receptor binding 2 (GRB2) protein ( 21), directly reduces the GTP-loading of Ras and Rac to restrain their activation (86), and directly binds to the p21 binding domain (PBD) of PAK1 to interrupt PAK1 activation and its recruitment to focal adhesions (22,87). Merlin competitively binds to angiomotin and releases the Rac1 negative regulator Rich1, which ultimately attenuates Rac1 signaling (23).…”

PAK4-relevant proliferation reduced by cell autophagy via p53/mTOR/p-AKT signaling

“…It is well-known that impaired cell proliferation results from not only cell cycle arrest but enhanced death. Since the death was not from apoptosis in PAK4-knockdown hepatocarcinoma cells in our previous study (33), and p53 (25,26,34) and PAKs (16,19,21) were relevant with autophagy in cancer cells, the present study focused on autophagy. To date, main functional forms in autophagy are proposed to be cytotoxic, cytostatic, and cytoprotective in the context of antitumor therapy: cytotoxic autophagy leads to cancer cell death, cytostatic autophagy inhibits cancer cell propagation, and cytoprotective autophagy enhances cancer cells' drug sensitivity (35).…”

“…Indeed, PAK-relative factors induced cancer cell cycle arrest but also the autophagy following being coupled to acetyltransferases (15). PAK inhibitors in meningioma cells induce cell death with increased autophagy-related protein levels and chromosomes-markers for mitotic catastrophe (16). The inhibition of potential PAK1 interactors revealed the importance of genes related to the autophagy in PAK1-mediated glioma cell proliferation (17).…”

Simultaneous inhibition of PI3K and PAK in preclinical models of neurofibromatosis type 2-related schwannomatosis

“…Moreover, in merlin-deficient tumors the Rac/PAK pathway was described as contributing to the loss of contact inhibition and activation of WNT/β-catenin signaling pathway [ 26 ]. Later PAK inhibitors have been shown to induce schwannoma cell death via mitotic catastrophe [ 27 ]. In our in vivo study the most significant decrease in tumor growth was observed with pictilisib and its use in combination treatment.…”