RESUMEN
Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Fragmentos de Péptidos/farmacología , Animales , Astrocitos , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/patología , Caspasas/metabolismo , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/patología , Humanos , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/uso terapéutico , Dominios Proteicos/genética , Transducción de Señal/efectos de los fármacos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alaninerich Ckinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rhoassociated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracyclineinducible system in PTENnull U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the nonphosphorylatable ED mutant exerted growthsuppressive and radiationsensitizing effects, while the pseudophosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS proteinprotein interactions using coimmunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKSassociated proteins, including importinß and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.