Overview of essential intracellular members of the mTORC1-pathway and respective targeted therapies.

<p>Signaling from growth factor receptors such as EGFR or a nutrient rich environment leads to an activation of the mTOR-pathway, while nutrient depletion inhibits signaling. The mTORC1-complex consists of the mTOR-protein itself and the subunits raptor (regulatory associated protein of mTOR), mLST8 (mammalian lethal with sec thirteen 8) and PRAS40 (proline-rich Akt1 substrate 1). Activation of the pathway leads to phosphorylation of the ribosomal protein S6 at phospho-sites Ser235/236 and Ser240/244 by the S6-kinase (S6K). Additionally, the translational repressor 4EBP1 (eukaryotic initiation factor 4E binding protein-1) is phosporylated at Thr37/Thr46, thereby relieving its translational inhibition and promoting translation especially of mRNAs with a TOP motif [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127123#pone.0127123.ref014" target="_blank">14</a>]. First generation mTORC1-inhibitors are rapamycin and its derivatives temsirolimus and everolimus. Mechanistically these compounds bind the intracellular adaptor protein FKBP12 to form an allosteric mTORC1 inhibitory complex. 4EBP1 phosphorylation though mTORC1-dependent is largely resistant to the mTORC1 inhibitor rapamycin and its derivatives. Recently, ATP-competitive mTORC (2^nd generation) inhibitors like torin1 and torin2 which are efficient in dephosphorylating 4EBP1 have been developed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127123#pone.0127123.ref015" target="_blank">15</a>].</p

Timeline of in vivo and in vitro assessment of phospho-signals before formalin-fixation.

<p>(A) <i>In vitro</i> detection of phospho-RPS6 (Ser235/236), phospho-RPS6 (Ser240/244) and phospho-4EBP1 (Thr37/46) 0–60 minutes after harvesting LNT-229 glioma cells. Cells were kept at room temperature without medium in an open 15 ml plastic tube. Considerable reduction of p4EBP1 (Thr37/46) signal intensity was observed after 60 minutes. (B) <i>In vivo</i> detection of phospho-signals 35–230 minutes after surgical resection of a GBM. As a control to show general immunogenicity of the tissue samples we used an antibody against GFAP. While staining signals of phospho-RPS6 (Ser235/236) and phospho-RPS6 (Ser240/244) did not show considerable decrease over the respective time intervals, we hardly detected any signal for phospho-4EBP1 (Thr37/46) in the tissue specimens.</p

Overview of a selection of clinical trials targeting the mTOR-pathway in brain tumor patients.

<p>Some study protocols point on the issue of using tissue biomarkers for additional evaluation of treatment-response as follows:</p><p>¹Exploration of associations with pS6 positivity and outcome is planned</p><p>²Correlation of phosphorylated PKB/Akt and PTEN expression with response is planned</p><p>³Correlation of tumor objective response rate to established immunohistochemical biomarkers of mTOR pathway activation, including pS6, p4EBP1, pPRAS40, pp70S6K and PTEN is planned</p><p>⁴Use of Biomarkers: Phosphatase and tensin homolog (PTEN) and Epidermal Growth Factor Receptor (EGFR)</p><p>⁵ Comparison of clinical outcome compared to immunohistochemical markers related to the EGFR and PTEN-PI3K-AKT pathways at baseline is planned</p><p>⁶ Evaluation of laboratory variables (phospho-Akt, PTEN status, MGMT expression and promoter methylation status)</p><p>⁷Correlation of the activity of the treatment regimen with expression of selected intra-tumoral biomarkers</p><p>(AZD8055: ATP-competitive mammalian target of rapamycin kinase inhibitor)</p><p>Overview of a selection of clinical trials targeting the mTOR-pathway in brain tumor patients.</p

Detection of phospho-RPS6 (Ser235/236), phospho-RPS6 (Ser240/244) and phospho-4EBP1 (Thr37/46) in malignant brain tumors.

<p>(A) Although pleomorphic tumor cells showed staining for phospho-RPS6 (Ser235/236) and phospho-RPS6 (Ser240/244) we also observed perivascular cells with microglia/macrophage morphology that were strongly positive for both markers (black arrowheads in the first and second row of glioblastoma). We also detected strong expression of both phosphorylated antigens in brain metastases of NSCLC tumor cells (black arrowheads in the upper row, right column indicating multinucleated tumor cells, white arrowheads indicating mitotic figures). In contrast to the more heterogenous staining for phospho-RPS6 (Ser235/236) and phospho-RPS6 (Ser240/244), phospho-4EBP1 (Thr37/46) was strongly detected in the majority of tumor cells. Vessel-associated cells are also a source for mTORC1-signaling (black arrowhead indicating a mitotic figure within the endothelial layer, lower middle image). (B) Immunofluorescent double staining of glioblastomas against phospho-RPS6 (Ser235/236) and (Ser240/244) showing colocalisation with GFAP in some tumor cells (white arrowheads) while numerous GFAP-positive cells did not show phospho-signals for RPS6. (C) Phospho-RPS6 (Ser235/236) and (Ser240/244) were detected in CD68-positive cells in glioblastomas (white arrowheads).</p

Overview of brain tumor patients for statistical analysis.

<p>Overview of brain tumor patients for statistical analysis.</p

Establishment of mTORC1-specific immunohisto- and immunocytochemistry.

<p>(A) LNT-229 glioma cells cultured in DMEM containing 10% FCS and 25 mM glucose displayed strong signals in phospho-specific ICC stainings with all tested antibodies, while serum-free culture condititons resulted in reduced signal intensity and frequency. Treatment with the ATP-competitive mTORC-inhibitor torin2 resulted in a profound reduction of the phospho-spcific signal for all antibodies tested, while treatment with rapamycin only reduced the signal of RPS6 phospho-sites. Total RPS6 and 4EBP1 protein counterparts did not show considerable differences in the tested conditions (B) Immunoblotting using the same antibodies, cell lines and culture conditions confirmed the ICC results presented in (A), 4EBP1 exists in states of different electrophoretic mobility depending on its phosphorylation state [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127123#pone.0127123.ref019" target="_blank">19</a>]. (C) Subependymal giant cell astrocytomas (SEGA) served as a positive control for human routine diagnostic specimens. Although mTORC1-signaling was detectable in SEGA tumor cells, the staining intensity was considerably reduced in freshly cut archived FFPE material especially for the RPS6 phospho-sites.</p

Quantification of mTORC1-signaling in glioma patients during tumor progression.

<p>All graphs show expression results of normal appearing grey matter (NAGM) tissue samples, WHO grade II gliomas, WHO grade III gliomas and glioblastomas (WHO grade IV). (A) Full 4EBP protein expression (NAGM vs. WHO grade III p = 0.001; NAGM vs. WHO grade IV p = 0.0003). (B) Full RPS6 protein expression (NAGM vs. WHO grade II p = 0.034; NAGM vs. WHO grade III p = 0.0072). (C) phospho-4EBP1 (Thr37/46) expression (NAGM vs. WHO grade IV p = 0.002). (D) phospho-RPS6 (Ser235/236) expression (NAGM vs. WHO grade IV p = 0.0018). (E) phospho-RPS6 (Ser240/244) expression. All other comparisons did not reveal statistically significant differences.</p

A Functional Yeast Survival Screen of Tumor-Derived cDNA Libraries Designed to Identify Anti-Apoptotic Mammalian Oncogenes

<div><p>Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for <i>de novo</i> purine biosynthesis, the long non-coding RNA <i>MALAT1</i> and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.</p></div