Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

Glioblastoma multiforme (GBM), which is classified as grade IV astrocytoma by the WHO, is the most common and aggressive primary brain tumor in adults. Despite advances in treatment options combining surgical resection, radiotherapy, and concomitant alkylating chemotherapy, the prognosis for GBM patients still remains dismal with a median survival of 14.2 months.1 The disproportionate malignancy of GBM is due to its invasive growth pattern and high inter-and intratumoral genetic heterogeneity.2,3 Frequent genetic alterations in GBM affect 3 major cancer pathways: (i) the receptor tyrosine kinase (RTK) pathway and its downstream oncogenic signaling by PI3K/AKT and Ras/MAPK, (ii) the p53, and (iii) the retinoblastoma protein (pRb) signaling network.4 Cellular senescence refers to the state of irreversible cell cycle arrest that occurs at the end of cells’ replicative life span or as a physiological response to different types of cellular stress including aberrant oncogenic activation, DNA damage, and oxidative stress. Depending on the type of senescence-inducing stimulus and their genetic background, different cell types display differential senescence responses. In addition to long-term exit from cell cycle, common characteristic features of the senescent phenotype are the induction of senescence-associated β-galactosidase (SA-β-Gal) activity, chromatin remodeling accompanied by the formation of senescence-associated heterochromatin foci, and morphological transformation, including vacuolization and change in morphology to a flat and enlarged cell shape. At molecular level, the senescence response can be triggered through several genetic effectors converging on the activation of tumor suppressor networks p53 and pRb.5,6 During early stages of tumorigenesis, senescence acts as an important anticancer mechanism. Aberrant activation of oncogenes, such as Ras, or the loss of tumor suppressors, as described for PTEN, can trigger senescence in vitro and in vivo. For further progression, tumor cells have to bypass the senescent arrest by additional mutations of tumor suppressor genes, including p53 and p16, or reactivation of telomerase. Thus, re-induction of the senescence program in tumor cells could represent an additional therapeutic approach. Potential intervention targets for prosenescence therapies are telomerase inhibition, modulation of cyclin-dependent kinase (CDK) activities, the reactivation of tumor suppressor genes (eg, p53) and the inactivation of oncogenes, as demonstrated for c-Myc addicted tumors.6–8 The protein kinase D (PRKD) family belongs to a subgroup of serine/threonine kinases within the calcium/calmodulin-dependent protein kinase superfamily and comprises 3 abundantly expressed mammalian isoforms. The 3 isoforms, PRKD1, PRKD2, and PRKD3, contain highly conserved structural motifs within their regulatory domains and are directly activated by phorbolesters, diacylglycerol (DAG), or DAG-activated protein kinase C (PKC) isoforms. By integrating downstream signaling of tyrosine kinase and G protein-coupled receptors, PRKDs have emerged as central players in tumor-promoting processes including proliferation, migration, invasion, and angiogenesis.9 PRKDs have been linked mechanistically to targets that are implicated in the regulation of cell proliferation, survival, and apoptosis including the Ras/MAPK pathway, HDAC, CREB, JNK, and AKT.10,11 Within a large-scale RNAi screen of human kinases, PRKD2, but not PRKD1 and PRKD3, was identified as an essential regulator of cell survival.12 In human cancers, PRKD2 is positively involved in dedifferentiation,13 survival,14 angiogenesis,15 and invasion.16 In astrocytoma, a positive correlation between PRKD2 expression and tumor grading was shown.17 This study further demonstrated that inhibition of PRKD2 results in an apoptosis-independent reduction of glioma cell proliferation in vitro and prevents tumor formation in a chicken chorioallantoic membrane assay. Just recently, we have identified PRKD2 as a potential kinase target to reduce glioblastoma cell motility and invasion.18 The present study aimed to investigate molecular pathways underlying PRKD2-silencing induced cell-cycle inhibition in glioma. We elucidated the effects of PRKD2 depletion on senescence-associated and RTK-mediated signaling in vitro and confirmed the role of PRKD2 in glioma growth in a xenograft model. Here we show for the first time that PRKD2 silencing induces a senescence-like program in p53wt and p53mut glioma cells and may represent a promising approach for prosenescence therapy in glioma.