BRAF Activation Induces Transformation and Then Senescence in Human Neural Stem Cells: A Pilocytic Astrocytoma Model

Abstract

Purpose BRAF is frequently activated by gene fusion or point mutation in pilocytic astrocytoma, the most common pediatric brain tumor. We investigated the functional effect of constitutive BRAF activation in normal human neural stem and progenitor cells to determine its role in tumor induction in the brain. Experimental Design The constitutively active BRAFV600E allele was introduced into human neurospheres, and its effects on MAPK (mitogen-activated protein kinase) signaling, proliferation, soft agarose colony formation, stem cell phenotype, and induction of cellular senescence were assayed. Immunohistochemistry was used to examine p16INK4a levels in pilocytic astrocytoma. Results BRAFV600E expression initially strongly promoted colony formation but did not lead to significantly increased proliferation. BRAFV600E-expressing cells subsequently stopped proliferating and induced markers of oncogene-induced senescence including acidic β-galactosidase, PAI-1, and p16INK4a whereas controls did not. Onset of senescence was associated with decreased expression of neural stem cell markers including SOX2. Primary pilocytic astrocytoma cultures also showed induction of acidic β-galactosidase activity. Immunohistochemical examination of 66 pilocytic astrocytomas revealed p16INK4a immunoreactivity in the majority of cases, but patients with tumors negative for p16INK4a had significantly shorter overall survival. Conclusions BRAF activation in human neural stem and progenitor cells initially promotes clonogenic growth in soft agarose, suggesting partial cellular transformation, but oncogene-induced senescence subsequently limits proliferation. Induction of senescence by BRAF may help explain the low-grade pathobiology of pilocytic astrocytoma, whereas worse clinical outcomes associated with tumors lacking p16INK4a expression could reflect failure to induce senescence or an escape from oncogene-induced senescence.

Pediatric glioma-associated KIAA1549:BRAF expression regulates neuroglial cell growth in a cell type-specific and mTOR-dependent manner

“…(*) P < 0.05; (**) P < 0.01; (***) P < 0.001. gliogenesis (Lee et al 2010(Lee et al , 2012. Consistent with prior BRAF results (Raabe et al 2011) and our recent findings regarding NSC brain region heterogeneity (Lee et al 2012), no change in neocortex NSC proliferation was seen following f-BRAF expression (Supplemental Fig. S2F).…”

“…Although f-BRAF can transform NIH3T3 cells (Jones et al 2008), previous studies have demonstrated that BRAF does not increase astrocyte proliferation (Jacob et al 2009), induces senescence of astrocytes (Jacob et al 2011) and neural stem cells (NSCs) in vitro (Raabe et al 2011), and does not result in gliomagenesis when ectopically expressed in mouse brains in vivo (Robinson et al 2010).…”

Pilocytic astrocytomas of the optic nerve and their relation to pilocytic astrocytomas elsewhere in the central nervous system

“…Indeed, a worse clinical outcome has been reported for pilocytic astrocytoma lacking CDKN2A expression. 42 The extent of follow-up in our study permitted only limited evaluation of survival and correlation with the molecular features of pilocytic astrocytoma of the optic nerve, and future studies will be needed to address the relationship between CDKN2A activation and clinical behavior in these tumors. Nevertheless, the presence of an active MAPK pathway and indirect evidence of increased oncogene-induced senescence in pilocytic astrocytoma of the optic nerve can explain their indolent behavior.…”

Ciliated muconodular papillary tumor of the lung harboring BRAF V600E mutation and p16INK4a overexpression without proliferative activity may represent an example of oncogene-induced senescence

“…INK4a show significantly shorter survival or aggressive behavior (15,16). Rare cases of acquired melanocytic nevi progress to malignant melanoma when they acquire additional mutations (8).…”