Novel Proteins Regulated by mTOR in Subependymal Giant Cell Astrocytomas of Patients with Tuberous Sclerosis Complex and New Therapeutic Implications

Abstract

Subependymal giant cell astrocytomas (SEGAs) are rare brain tumors associated with tuberous sclerosis complex (TSC), a disease caused by mutations in TSC1 or TSC2, resulting in enhancement of mammalian target of rapamycin (mTOR) activity, dysregulation of cell growth, and tumorigenesis. Signaling via mTOR plays a role in multifaceted genomic responses, but its effectors in the brain are largely unknown. Therefore, gene expression profiling on four SEGAs was performed with Affymetrix Human Genome arrays. Of the genes differentially expressed in TSC, 11 were validated by real-time PCR on independent tumor samples and 3 SEGA-derived cultures. Expression of several proteins was confirmed by immunohistochemistry. The differentially-regulated proteins were mainly involved in tumorigenesis and nervous system development. ANXA1, GPNMB, LTF, RND3, S100A11, SFRP4, and NPTX1 genes were likely to be mTOR effector genes in SEGA, as their expression was modulated by an mTOR inhibitor, rapamycin, in SEGA-derived cells. Inhibition of mTOR signaling affected size of cultured SEGA cells but had no influence on their proliferation, morphology, or migration, whereas inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways led to significant alterations of these processes. For the first time, we identified genes related to the occurrence of SEGA and regulated by mTOR and demonstrated an effective modulation of SEGA growth by pharmacological inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways, which could represent a novel therapeutic approach.

Combination of Imatinib Mesylate and AKT Inhibitor Provides Synergistic Effects in Preclinical Study of Gastrointestinal Stromal Tumor

“…The WTS results for both BEX1 and NPTX1 were confirmed by qRT-PCR in the xenografts from the study, and the induction of BEX1 by the drug combination was also confirmed in IM-sensitive GIST-T1 and IM-resistant GIST430 cell lines, providing cellular models for exploring this finding. Numerous reports in the literature implicate BEX1 and NPTX1 as tumor-suppressor genes or biomarkers in various cancers (33-35,37,38). Both genes have been implicated in connection with pro-apoptotic pathways in different cellular contexts.…”

Cyr61, a Matricellular Protein, Is Needed for Dendritic Arborization of Hippocampal Neurons

“…Because effects of Cyr61 on dendrites were not previously reported, to additionally confirm that in case of Cyr61 knockdown our morphological criteria for dendrite selection are correct, we independently repeated experiment using either pSuper or Cyr61 shRNA pool transfection and quantified the number of MAP2-positive/Tau (PHF-1)-negative neurites. With such criteria we also showed that Cyr61 knockdown results in a significant decrease in the number of dendrites (pSuper/GFP (n ϭ 50), 22.58 Ϯ 0.72; Cyr61sh-mix (n ϭ 46), 16.09 Ϯ 0.61; p Ͻ 0.001; Mann-Whitney test). Because embryonic hippocampal cultures are mixtures of a large population of excitatory and a small population of inhibitory (glutamic acid decarboxylase (GAD67)-positive) neurons, we tested if knockdown of Cyr61 affects selectively one of them.…”

“…Recent phosphoproteomic studies identified several regulators of transcription and mRNA processing among mTOR substrates (43,44). Therefore, we decided to test whether the genes that encode mRNAs whose levels were previously shown to depend on mTOR activity (8,16,17) are involved in dendritic arbor development. To achieve this, we designed a minilibrary of shRNAs that target 46 such mRNAs (supplemental Table 1) using algorithms that proved to be accurate in over 66% of the cases in our previous studies (i.e.…”

“…YY-1, sterol regulatory element-binding protein, and HIF1␣ were also shown to play important roles in physiological or pathological neuronal transmission (13)(14)(15). Moreover, the transcriptional profiling of cells with increased mTOR signaling, like neuroepithelial cells that lack TSC2 or subependymal giant-cell astrocytomas derived from tuberous sclerosis patients revealed differences in the expression of several genes that are important for neuronal development (16,17).…”

Tuberous sclerosis and epilepsy: Role of astrocytes

“…The molecular changes that occur in SEGAs may be distinct from other TSC lesions and could account for their often relentless growth. In a recent study, gene array analysis in SEGAs identified numerous differentially expressed genes, in particular secreted frizzled-related protein 4, lactotransferrin, glycoprotein (transmembrane) nmb, annexin A1, Rho family GTPase 3, and S100 calcium binding protein a11 (Tyburczy et al, 2010). Another study using cDNA array and immunohistochemical analysis showed that epidermal growth factor (EGF), EGF receptor (EGFR), hepatocyte growth factor, c-Met, and vascular endothelial growth factor (VEGF), but not Flt-1, mRNA, and protein expression was upregulated in Tsc1 conditional knockout mouse brain, and these alterations closely predicted enhanced expression of these proteins in SEGAs (Parker et al, 2011;Fig.…”