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Epithelioid glioblastoma is a recognized glioblastoma variant, recently added to the World Health Organization brain tumor classification, with similar prognosis as the classic variant and B-Raf V600E mutations in 50% of the cases. We identified a new subset of epithelioid glioblastoma with periventricular location and subependymal giant cell astrocytoma (SEGA)-like morphology. Genomic profiling of these tumors revealed driver mutations in NF1, subclonal mutations in TSC1, and a novel driver mutation in MTOR, suggesting upregulation of the MAPK/TSC1/mTOR pathway. Strong mTOR activation was confirmed by immunohistochemistry for the mTOR kinase target 4E-BP1. TSC1 and MTOR mutations have been previously described in low-grade glioma, such as SEGA, and focal cortical dysplasia, respectively, that display large cells with abundant cytoplasm, most likely resulting from the biogenetic signaling of mTOR. Unlike these, the mutations in SEGA-like glioblastoma occurred in the context of other genetic aberrations present in high-grade neoplasms, including in the CDKN2A/B, PIK3R1, PIK3CA and EGFR genes. For one patient with two temporally distinct specimens, the subclonal TSC1 pathogenic mutation was detected only in the specimen showing SEGA-like morphology, indicating requirement for mTOR activation as trigger for specific epithelioid/SEGA-like morphology. As FDA-approved kinase inhibitors are available and target many steps of the MAPK/mTOR pathway, recognition of this new subset of periventricular high-grade gliomas with clear phenotypic-genotypic correlates is essential for prompt biomarker testing and appropriate targeted therapeutic management of these patients.

The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.

The cap-dependent translation is frequently deregulated in a variety of cancers associated with tumor progression. However, the molecular basis of the translation activation for metastatic progression of cancer remains largely elusive. Here, we demonstrate that activation of cap-dependent translation by silencing the translational repressor 4E-BP1 causes cancer epithelial cells to undergo epithelial-mesenchymal transition (EMT), which is associated with selective upregulation of the EMT inducer Snail followed by repression of E-cadherin expression and promotion of cell migratory and invasive capabilities as well as metastasis. Conversely, inhibition of cap-dependent translation by a dominant active mutant 4E-BP1 effectively downregulates Snail expression and suppresses cell migration and invasion. Furthermore, dephosphorylation of 4E-BP1 by mTORC1 inhibition or directly targeting the translation initiation also profoundly attenuates Snail expression and cell motility, whereas knockdown of 4E-BP1 or overexpression of Snail significantly rescues the inhibitory effects. Importantly, 4E-BP1-regulated Snail expression is not associated with its changes in the level of transcription or protein stability. Together, these findings indicate a novel role of 4E-BP1 in the regulation of EMT and cell motility through translational control of Snail expression and activity, and suggest that targeting cap-dependent translation may provide a promising approach for blocking Snail-mediated metastatic potential of cancer.

Suzan K. Chao 1 , Susan Band Horwitz 1 , and Hayley M. McDaid 1,2 1 Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 2 Department of Medicine (Oncology) Albert Einstein College of Medicine, Bronx, NY 10461 Keywords: Senescence, 4E-BP1, mTORC1, p53, discodermolide Received: February 22, 2011; Accepted: February 22, 2011; Published: February 22, 2011; Correspondence: Susan Band Horwitz, e-mail: // Hayley McDaid, e-mail: // // Abstract Senescence is a valid tumor suppressive mechanism in cancer. Accelerated cell senescence describes the growth arrested state of cells that have been treated with anti-tumor drugs, such as doxorubicin that induce a DNA damage response. Discodermolide, a microtubule-stabilizing agent, is a potent inducer of accelerated cell senescence. Resistance to discodermolide is mediated via resistance to accelerated cell senescence, and is associated with reduced expression of the mTORC1 substrate, 4E-BP1 and increased expression of p53 [1]. Although the association of p53 with senescence induction is well-characterized, senescence reversion in the presence of high expression of p53 has not been well-documented. Furthermore, studies addressing the role of mTOR signaling in regulating senescence have been limited and recent data implicate a novel, senescence-associated role for 4E-BP1 in crosstalk with the transcription factor p53. This research perspective will address these somewhat contradictory findings and summarize recent research regarding senescence and mTORC1 signaling.

Chronic lymphocytic leukemia (CLL), the most common adult leukemia, remains incurable despite advancements in treatment regimens over the past decade. Several expression profile studies have been pursued to better understand CLL pathogenesis. However, these large-scale studies only provide information at the transcriptional level. To better comprehend the differential protein changes that take place in CLL, we performed a reverse-phase protein array (RPPA) analysis using 167 different antibodies on B-cell lysates from 18 CLL patients and 6 normal donors. From our analysis, we discovered an enrichment of protein alterations involved with mRNA translation, specifically upregulation of the translation initiator eIF4G and phosphorylation of the cap-dependent translation inhibitor 4E-BP1 at serine 65. Interestingly, 4E-BP1 phosphorylation occurred independently of AKT phosphorylation, suggesting a disconnect between PI3K/AKT pathway activation and 4E-BP1 phosphorylation. Based on these results, we treated primary CLL samples with NVP-BEZ235, a PI3K/mTOR dual inhibitor, and compared its apoptotic-inducing potential against the BTK inhibitor Ibrutinib and the PI3Kδ inhibitor Idelalisib. We demonstrated that treatment with NVP-BEZ235 caused greater apoptosis, greater apoptotic cleavage of eIF4G, and greater dephosphorylation of 4E-BP1 in primary CLL cells. Taken together, these results highlight the potential dependence of eIF4G overexpression and 4E-BP1 phosphorylation in CLL survival.

An important strategy for improving advanced PCa treatment is targeted therapies combined with chemotherapy. PC-1, a prostate Leucine Zipper gene (PrLZ), is specifically expressed in prostate tissue as an androgen-induced gene and is up-regulated in advanced PCa. Recent work confirmed that PC-1 expression promotes PCa growth and androgen-independent progression. However, how this occurs and whether this can be used as a biomarker is uncertain. Here, we report that PC-1 overexpression confers PCa cells resistance to rapamycin treatment by antagonizing rapamycin-induced cytostasis and autophagy (rapamycin-sensitivity was observed in PC-1-deficient (shPC-1) C4-2 cells). Analysis of the mTOR pathway in PCa cells with PC-1 overexpressed and depressed revealed that eukaryotic initiation factor 4E-binding protein 1(4E-BP1) was highly regulated by PC-1. Immunohistochemistry assays indicated that 4E-BP1 up-regulation correlates with increased PC-1 expression in human prostate tumors and in PCa cells. Furthermore, PC-1 interacts directly with 4E-BP1 and stabilizes 4E-BP1 protein via inhibition of its ubiquitination and proteasomal degradation. Thus, PC-1 is a novel regulator of 4E-BP1 and our work suggests a potential mechanism through which PC-1 enhances PCa cell survival and malignant progression and increases chemoresistance. Thus, the PC-1-4E-BP1 interaction may represent a therapeutic target for treating advanced PCa.