Autophagy under glucose starvation enhances protein translation initiation in response to re-addition of glucose in C2C12 myotubes.

Proteolysis is known to play a crucial role in maintaining skeletal muscle mass and function. Autophagy is a conserved intracellular process for the bulk degradation of proteins in lysosomes. Although nutrient starvation is known to induce autophagy, the effect of nutrient repletion following starvation on the mTOR pathway-mediated protein translation remains unclear. In the present study, we examined the effect of glucose starvation on the initiation of protein translation in response to glucose re-addition in C2C12 myotubes. Glucose starvation decreased the phosphorylation of p70 S6 kinase (p70S6K), a bonafide marker for protein translation initiation. Following re-addition of glucose, phosphorylation of p70S6K markedly increased only in glucose-starved cells. Inhibiting autophagy using pharmacological inhibitors diminished the effect of glucose re-addition on the phosphorylation of p70S6K, whereas inhibition of the ubiquitin-proteasome system did not exert any effect. In conclusion, autophagy under glucose starvation partially accounts for the activation of translation initiation by re-addition of glucose.

Distinct Roles of mTOR Targets S6K1 and S6K2 in Breast Cancer.

The mechanistic target of rapamycin (mTOR) is a master regulator of protein translation, metabolism, cell growth and proliferation. It forms two complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2). mTORC1 is frequently deregulated in many cancers, including breast cancer, and is an important target for cancer therapy. The immunosuppressant drug rapamycin and its analogs that inhibit mTOR are currently being evaluated for their potential as anti-cancer agents, albeit with limited efficacy. mTORC1 mediates its function via its downstream targets 40S ribosomal S6 kinases (S6K) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). There are two homologs of S6K: S6K1 and S6K2. Most of the earlier studies focused on S6K1 rather than S6K2. Because of their high degree of structural homology, it was generally believed that they behave similarly. Recent studies suggest that while they may share some functions, they may also exhibit distinct or even opposite functions. Both homologs have been implicated in breast cancer, although how they contribute to breast cancer may differ. The purpose of this review article is to compare and contrast the expression, structure, regulation and function of these two S6K homologs in breast cancer.

No effect of anti-inflammatory medication on postprandial and postexercise muscle protein synthesis in elderly men with slightly elevated systemic inflammation.

BACKGROUND: Based on circulating C-reactive protein (CRP) levels, some individuals develop slightly increased inflammation as they age. In elderly inflamed rats, the muscle response to protein feeding is impaired, whereas it can be maintained by treatment with non-steroidal anti-inflammatory drugs (NSAIDs). It is unknown whether this applies to elderly humans with increased inflammation. Thus, the muscle response to whey protein bolus ingestion with and without acute resistance exercise was compared between healthy elderly individuals and elderly individuals with slightly increased inflammation±NSAID treatment. METHODS: Twenty-four elderly men (>60years) were recruited. Of those, 14 displayed a slightly increased systemic inflammation (CRP>2mg/l) and were randomly assigned to NSAID (Ibuprofen 1800mg/day) or placebo treatment for 1week. The remaining 10 elderly individuals served as healthy controls (CRP<1mg/l). The muscle protein synthetic response was measured as the fractional synthetic rate (FSR) and p70S6K phosphorylation-to-total protein ratio. RESULTS: The basal myofibrillar FSR and the myofibrillar FSR responses to whey protein bolus ingestion with and without acute resistance exercise were maintained in inflamed elderly compared to healthy controls (p>0.05) and so was p70S6K phosphorylation. Moreover, NSAID treatment did not significantly improve the myofibrillar and connective tissue FSR responses or reduce the plasma CRP level in inflamed, elderly individuals (p>0.05). CONCLUSION: A slight increase in systemic inflammation does not affect the basal myofibrillar FSR or the myofibrillar FSR responses, which suggests that elderly individuals with slightly increased inflammation can benefit from protein ingestion and resistance exercise to stimulate muscle protein anabolism. Moreover, the NSAID treatment did not significantly affect the myofibrillar or connective tissue FSR responses to protein ingestion and acute resistance exercise.

Different interactomes for p70-S6K1 and p54-S6K2 revealed by proteomic analysis.

S6Ks are major effectors of the mTOR (mammalian target of rapamycin) pathway, signaling for increased protein synthesis and cell growth in response to insulin, AMP/ATP levels, and amino acids. Deregulation of this pathway has been related to disorders and diseases associated with metabolism, such as obesity, diabetes, and cancer. S6K family is composed of two main members, S6K1 and S6K2, which comprise different isoforms resulted from alternative splicing or alternative start codon use. Although important molecular functions have been associated with p70-S6K1, the most extensively studied isoform, the S6K2 counterpart lacks information. In the present study, we performed immunoprecipitation assays followed by mass spectrometry (MS) analysis of FLAG-tagged p70-S6K1 and p54-S6K2 interactomes, after expression in HEK293 cells. Protein lists were submitted to CRAPome (Contaminant Repository for Affinity Purification) and SAINT (Significance Analysis of INTeractome) analysis, which allowed the identification of high-scoring interactions. By a comparative approach, p70-S6K1 interacting proteins were predominantly related to "cytoskeleton" and "stress response," whereas p54-S6K2 interactome was more associated to "transcription," "splicing," and "ribosome biogenesis." Moreover, we have found evidences for new targets or regulators of the S6K protein family, such as proteins NCL, NPM1, eIF2α, XRCC6, PARP1, and ILF2/ILF3 complex. This study provides new information about the interacting networks of S6Ks, which may contribute for future approaches to a better understanding of the mTOR/S6K pathway.

Bronchial involvement in advanced stage lymphangioleiomyomatosis: histopathologic and molecular analyses.

Lymphangioleiomyomatosis (LAM), a rare progressive disease that almost exclusively affects women, is characterized by pulmonary cysts and neoplastic proliferation of smooth muscle-like cells (LAM cells). Airflow obstruction is a physiologic consequence that is commonly observed in LAM and has been attributed to narrowing of peripheral airways. However, histopathologic examinations of the entire airway have been precluded by the limited availability of such specimens. Here, we used explanted lung tissues from 30 LAM patients for a thorough histologic analysis with a special emphasis on the bronchi. We found bronchial involvement by LAM cells and lymphatics in all patients examined. Furthermore, a moderate to severe degree of chronic inflammation (73%), goblet cell hyperplasia (97%), squamous cell metaplasia (83%) of the epithelium, and thickening of basal lamina (93%) were identified in the bronchi. Because LAM cells are transformed by the functional loss of the TSC genes leading to a hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling pathway, we confirmed the expression of phospho-p70S6K, phospho-S6, phospho-4E-BP1, and vascular endothelial growth factor (VEGF)-D in LAM cells from all of the patients examined. In contrast, no protein expression of hypoxia-inducible factor 1α, a downstream molecule indicative of mTORC1 activation and leading to VEGF production, was detected in any patient. Our study indicates that late-stage LAM patients commonly have bronchi involved by the proliferation of both LAM cells and lymphatics and that chronic inflammation complicated their disease. Furthermore, the up-regulation of hypoxia-inducible factor 1α, a common event in mTORC1-driven tumor cells, does not occur in LAM cells and plays no role in VEGF-D expression in LAM cells.

Branched-chain amino acid and lysine deficiencies exert different effects on mammary translational regulation.

Deficiencies and imbalances of specific group II essential amino acids (EAA) were created in lactating cows by an infusion subtraction protocol to explore effects on milk production and abundance and phosphorylation state of regulators of mRNA translation in the mammary glands. Five lactating cows on a diet of 11.2% crude protein were infused abomasally for 5d with saline, 563 g/d of a complete EAA mix, or EAA mixes without the branched-chain amino acids (BCAA), Leu, or Lys in a 5 × 5 Latin square design. Milk protein yield was stimulated by EAA infusion and returned to saline levels upon subtraction of BCAA, Leu, or Lys. Mammary abundance of phosphorylated S6K1 was measured as an indicator of mammalian target of rapamycin complex 1 (mTORC1) activity and was found not to be affected by the complete EAA mix but was increased by the mixture lacking Lys. Total S6K1 abundances in mammary tissue were elevated by complete and BCAA-lacking infusions. All of the EAA treatments except the one lacking BCAA upregulated mammary eIF2Bε and eIF2α abundances, which is stimulatory to global mRNA translation. Phosphorylation state of eIF2Bε tended to decrease when complete or Lys-lacking EAA mixtures were infused. Phosphorylation state of eIF2α was not affected by treatment. We detected a correlation of 0.62 between phosphorylation state of S6K1 and total eIF2Bε abundance, and a correlation of 0.58 between phosphorylation state of S6K1 and total eIF2α abundance, suggesting that mTORC1 activation may have upregulated eIF2Bε and eIF2α expression. Despite maintenance of mammary eIF2Bε and eIF2α abundances during Leu and Lys deficiencies, milk protein yield declined, suggesting that other factors are responsible for mediating effects of Lys and Leu. A deficiency of all 3 BCAA may impair milk protein yield through deactivation of mTORC1-mediated upregulation of eIF2Bε and eIF2α abundances.

Cyclin-dependent Kinase 5 (Cdk5)-dependent Phosphorylation of p70 Ribosomal S6 Kinase 1 (S6K) Is Required for Dendritic Spine Morphogenesis.

The maturation and maintenance of dendritic spines depends on neuronal activity and protein synthesis. One potential mechanism involves mammalian target of rapamycin, which promotes protein synthesis through phosphorylation of eIF4E-binding protein and p70 ribosomal S6 kinase 1 (S6K). Upon extracellular stimulation, mammalian target of rapamycin phosphorylates S6K at Thr-389. S6K also undergoes phosphorylation at other sites, including four serine residues in the autoinhibitory domain. Despite extensive biochemical studies, the importance of phosphorylation in the autoinhibitory domain in S6K function remains unresolved, and its role has not been explored in the cellular context. Here we demonstrated that S6K in neuron was phosphorylated at Ser-411 within the autoinhibitory domain by cyclin-dependent kinase 5. Ser-411 phosphorylation was regulated by neuronal activity and brain-derived neurotrophic factor (BDNF). Knockdown of S6K in hippocampal neurons by RNAi led to loss of dendritic spines, an effect that mimics neuronal activity blockade by tetrodotoxin. Notably, coexpression of wild type S6K, but not the phospho-deficient S411A mutant, could rescue the spine defects. These findings reveal the importance of cyclin-dependent kinase 5-mediated phosphorylation of S6K at Ser-411 in spine morphogenesis driven by BDNF and neuronal activity.

Maternal low-protein diet affects myostatin signaling and protein synthesis in skeletal muscle of offspring piglets at weaning stage.

PURPOSE: We tested the hypothesis that maternal low-protein (LP) diet during gestation and lactation can program myostatin (MSTN) signaling and protein synthesis in skeletal muscle of offspring at weaning stage (35 days). METHODS: Fourteen Meishan sows were fed either LP or standard-protein diets throughout gestation and lactation, male offspring piglets were killed at weaning stage and longissimus dorsi (LD) muscles were taken. The cross-sectional areas (CSA) of LD muscles were measured by hematoxylin and eosin staining. The levels of free amino acids in plasma were measured by amino acid auto-analyzer. Proteins and mRNA were determined by Western blot and RT-qPCR, respectively. RESULTS: Body weight, LD muscle weight and the myofiber CSA were significantly decreased (P < 0.05) in LP piglets; meanwhile, the concentration of branched-chain amino acids was also significantly decreased (P < 0.001). MSTN protein content tended to be higher (P = 0.098) in LP piglets, while the expression of MSTN receptors, activin type II receptor-beta and transforming growth factor type-beta type I receptor kinase, was significantly up-regulated (P < 0.05). Furthermore, p38 mitogen-activated protein kinase, the downstream signaling factor of MSTN, was also enhanced significantly (P < 0.05). In addition, key factors of translation initiation, phosphorylated eukaryotic initiation factor 4E and the 70 kDa ribosomal protein S6 kinase, were significantly decreased (P < 0.05) in LP piglets. CONCLUSIONS: Our results suggest that maternal LP diet during gestation and lactation affects MSTN signaling and protein synthesis in skeletal muscle of offspring at weaning stage.

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy.

BACKGROUND: Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential. RESULTS: The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude. CONCLUSIONS: Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.

Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

BACKGROUND: Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential. RESULTS: The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude. CONCLUSIONS: Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.

Overexpression of RPS6KB1 predicts worse prognosis in primary HCC patients.

RPS6KB1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) plays a key role in regulating protein translation. The role of RPS6KB1 in HCC (hepatocellular carcinoma) has not been fully investigated. This study was undertaken to determine the clinicopathological features and prognostic value of RPS6KB1 in HCC. We examined RPS6KB1 expression in 30 paired liver cancer tissues and adjacent noncancerous tissues by reverse transcription real-time PCR and Western blotting. In addition, we analyzed RPS6KB1 expression in 87 HCC samples by immunohistochemistry. The expression of RPS6KB1 was significantly increased in cancer tissues. Clinicopathological analysis showed that the expression of RPS6KB1 was significantly correlated with tumor size, histopathologic classifications, and serum alpha-fetoprotein (AFP). Kaplan-Meier survival curves revealed that increased expression of RPS6KB1 correlated with poor prognosis of HCC patients. RPS6KB1 expression was an independent prognostic marker for overall survival of HCC patients by multivariate analysis. Our data suggest that RPS6KB1 may play an important role in the progression of HCC and could serve as a potential molecular target for HCC therapy.

Spatio-temporal expression study of phosphorylated 70-kDa ribosomal S6 kinase (p70S6k) in mesial temporal lobe epilepsy.

OBJECTIVE: To determine the spatio-temporal expression of p70S6k activation in hippocampus in mesial temporal lobe epilepsy. METHODS: Temporal lobe epilepsy model was established by stereotaxically unilateral and intrahippocampal injection of kainite acid (KA) in adult male C57BL/6 mice. Latent and chronic epileptogenesis were represented by mice 5 days after KA injection (n = 5) and mice 5 weeks after KA injection (n = 8), respectively. Control mice (n = 5) were injected with saline. Immunohistochemical assays were performed on brain sections of the mice. RESULTS: Hippocampus both ipsilateral and contralateral to the KA injection displayed significantly up-regulated pS6 immunoreactivity in dispersed granule cells in 5-day and 5-week model mice. CONCLUSION: The activation of p70S6k is mainly located in the dentate gyrus in KA-induced mouse model of temporal lobe epilepsy, indicating that the activation may be related with the disperse degree and hypertrophy of granule cells.

The expression of phospho-AKT1 and phospho-MTOR is associated with a favorable prognosis independent of PTEN expression in intrahepatic cholangiocarcinomas.

AKT1 signaling pathway is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. In this study, we explored the prognostic significance of AKT1 pathway in intrahepatic cholangiocarcinomas. We investigated the status of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphorylated (p) AKT1 (p-AKT1), p-mammalian target of rapamycin (p-MTOR), p-p70 ribosomal protein S6 kinase (p-RPS6KB2) and p-eukaryotic initiation factor 4E-binding protein-1 (p-EIF4EBP1) in 101 intrahepatic cholangiocarcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-AKT1 and p-MTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. The overexpression of p-AKT1, p-MTOR, and PTEN was associated with a better survival in patients with intrahepatic cholangiocarcinoma (P=0.0137, 0.0194, and 0.0337, respectively). In a multivariate analysis, PTEN was an independent prognostic factor, and p-AKT1 showed tendency (P=0.032 and 0.051, respectively). The overexpression of p-MTOR was correlated with well-to-moderately differentiated tumors (P<0.001) and tumors without metastasis (P=0.046). Expression levels of the AKT1 signaling pathway proteins in this study showed positive correlations with each other, except for PTEN. Aberrant expressions of p-AKT1 and p-MTOR in intrahepatic cholangiocarcinoma were associated with a favorable prognosis, possibly in a PTEN-independent manner. Our results indicate that dysregulation of the AKT1 pathway may have an important role in the development of intrahepatic cholangiocarcinoma, but not necessarily in the progression of the disease.

Morphoproteomics demonstrates activation of mammalian target of rapamycin pathway in papillary thyroid carcinomas with nuclear translocation of MTOR in aggressive histological variants.

We used morphoproteomics to investigate mammalian target of rapamycin (MTOR) signaling pathway in papillary thyroid carcinomas and correlated the results with clinicopathological parameters. Archival paraffin-embedded tissue of papillary thyroid carcinomas was obtained from 30 patients, including 15 classical type and 8 follicular, 4 tall-cell, 1 columnar-cell, 1 diffuse sclerosing and 1 cribriform variants. Immunohistochemical stains were performed for three phosphorylated (p) protein analytes: p-MTOR (Ser2448), p-Akt (Ser473) and p-p70S6K (Thr389). Chromogenic signals and subcellular compartmentalization (nuclear, cytoplasmic and plasmalemmal) were evaluated. Clinicopathological parameters were reviewed. Immunoreactivities for p-MTOR, p-Akt and p-p70S6K were observed in all papillary thyroid carcinomas. In addition to an expression of p-MTOR in cytoplasmic location, nuclear translocation of p-MTOR with variable loss of plasmalemmal expression, and with concomitant nuclear expression of p-Akt, was also identified in all tall-cell, columnar-cell and diffuse sclerosing variants of papillary thyroid carcinoma. There were no significant differences in the clinicopathological parameters, including tumor size, extrathyroidal extension, angioinvasion and nodal metastases between the groups with and without nuclear expression of p-MTOR (P>0.05). The expressions of p-MTOR in cytoplasmic and/or plasmalemmal locations with the concomitant immunoreactivity for p-p70S6K in all papillary thyroid carcinomas indicate the activation of MTOR complex 1 pathway. The nuclear translocation of p-MTOR evidences the activation of MTOR complex 2 and is identified only in the known aggressive histological variants of papillary thyroid carcinoma, including tall-cell, columnar-cell and diffuse sclerosing variants. Thus, these results suggest the constitutive activation of MTOR signaling pathway in papillary thyroid carcinomas and provide a new insight of biogenetic basis for the aggressive histological variants of papillary thyroid carcinoma. The pattern of expression of p-MTOR in papillary thyroid carcinomas may serve as a diagnostic/prognostic marker and a potential therapeutic target.

The mTOR signaling pathway in the prefrontal cortex is compromised in major depressive disorder.

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.

Proteomic profiling identifies pathways dysregulated in non-small cell lung cancer and an inverse association of AMPK and adhesion pathways with recurrence.

INTRODUCTION: The identification of key pathways dysregulated in non-small cell lung cancer (NSCLC) is an important step toward understanding lung pathogenesis and developing new therapeutic approaches. METHODS: Toward this goal, reverse-phase protein lysate arrays (RPPA) were used to compare signaling pathways between NSCLC tumors and paired normal lung tissue from 46 patients and assess their association with clinical outcome. RESULTS: After RPPA quantification of 63 proteins and phosphoproteins, tissue pairs were randomized to a training set (n = 25 pairs) and test set (n = 21 pairs). In the training set, 15 protein markers were differentially expressed between tumors and normal lung (p ≤ 0.01), including markers in the PI3K/AKT and p38 MAPK signaling pathways (e.g., p70S6K, S6, p38, and phospho-p38), as well as caveolin-1 and ß-catenin. A four-protein signature (p70S6K, cyclin B1, pSrc(Y527), and caveolin-1) independent of histology classified specimens as tumor versus normal with a predicted accuracy of 83%, sensitivity of 67%, and specificity of 100%. The signature was validated in the test set, correctly classifying all normal tissues and 14 of 21 tumor tissues. RPPA results were confirmed by immunohistochemistry for caveolin-1 and p70S6K. In tumors from patients with resected NSCLC, expression of proteins in the energy-sensing AMPK pathway (pLKB1, AMPK, p-Acetyl-CoA, pTSC2), adhesion, EGFR, and Rb signaling pathways was inversely associated with NSCLC recurrence. CONCLUSIONS: These data provide evidence for dysregulation of several pathways including those involving energy sensing and adhesion that are potentially associated with NSCLC pathogenesis and disease recurrence.

EGFR-dependent and independent activation of Akt/mTOR cascade in bone and soft tissue tumors.

To gain the insight into the involvement of signaling mediated by the mammalian target of rapamycin (mTOR) in the phenotype and biological profiles of tumors and tumor-like lesions of the bone and soft tissue, we analyzed the expression and phosphorylation (activation) of mTOR and its correlation with the status of upstream and downstream modulator proteins Akt, p70S6-kinase (S6K), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), which we refer to collectively as mTOR cassette proteins. Immunohistochemical analysis of 140 cases showed activation of Akt in 55% (61% in malignant and 27% in benign), and mTOR expression in 61% (66% in malignant and 39% in benign). The preponderance of mTOR activation was found in tumors of peripheral nerve sheath (malignant peripheral nerve sheath tumor and schwannoma), skeletal muscle origin (rhabdomyosarcoma), and in those exhibiting epithelial nature (chordoma and synovial sarcoma). Together with the result of immunoblotting analysis, it was shown that many of those particular tumors with mTOR activation exhibited activation of Akt, S6K, and 4E-BP1, suggesting the constitutive activation of the Akt/mTOR pathway. In addition, although activation of the Akt/mTOR pathway was largely independent of activation of epidermal growth factor receptor (EGFR), mutation of EGFR was frequently accompanied by constitutive activation of Akt-mTOR-S6K/4E-BP1. By clinicopathological analysis, activation of Akt correlates with statistically higher probability of metastasis. We conclude that mTOR-mediated signaling proteins function not only in the proliferation of the tumor cells, but also in the differentiation and/or maintenance of morphological phenotypes in tumors of rhabdomyoblastic and nerve sheath cell origin. Furthermore, mTOR signaling may also modulate morphogenesis of tumors exhibiting epithelial nature. Additionally, activated Akt may have a function in metastasis. Overall, these results suggest that inhibitors of mTOR cassette may be useful as novel components of combined chemotherapy for a defined subset of bone and soft tissue sarcomas.

The VEGF pathway and the AKT/mTOR/p70S6K1 signalling pathway in human epithelial ovarian cancer.

Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples (N=86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) (R=0.29; P=0.002). Additional gene expression analysis in an independent cDNA microarray dataset (N=24) showed a negative correlation (R=-0.73, P<0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites (chi(2), P=0.002) and reduced overall survival of cisplatin-taxane-based patients with serous histology (N=32, log-rank test, P=0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

Expression and alterations of the PTEN / AKT / mTOR pathway in ameloblastomas.

OBJECTIVES: Recently, an allelic loss of phosphatase and tensin homologue (PTEN) was shown to occur in ameloblastomas. In carcinogenesis, loss of PTEN allows for overactivity of the phosphatidylinositol-3-kinase/protein kinase B (PI3K / AKT) pathway inducing an upregulation of mammalian-target of rapamycin (mTOR) and its downstream effector ribosomal-subunit-6 kinase (S6K); allowing for uncontrolled cell proliferation, apoptosis inhibition and cell cycle deregulation. METHODS: Thirty ameloblastomas and five dental follicles were studied, looking at the immunohistochemical expression of total PTEN and AKT, as well as their phosphorylated (p) active forms, and the downstream effector and indicator of mTOR activity p70 ribosomal-subunit-6 kinase (pS6K). Also assessed was the expression of extracellular-signal-regulated kinase (ERK), which cross talks with AKT. RESULTS: Total PTEN was absent in 33.3% of ameloblastomas, while its stabilized, phosphorylated(ser380 / thr382 / thr383) form was absent in 83.3% of tumors. In contrast, AKT was expressed in 83.3% of ameloblastomas, showing high expression of the p-thr(308)AKT and p-ser(473) AKT forms in 93.3% and 56.6% of cases, respectively. Further, the mTOR activated pS6K(ser240 / 244) was detected in 86.7% of ameloblastomas, while ERK was overexpressed in 70.0% of the cases. CONCLUSION: Immunohistochemical analysis of aberrant signaling in the PI3K/AKT/mTOR pathway in ameloblastomas may represent a valuable tool for elucidating pathogenesis, aggressiveness and selecting optimal therapeutics.

Activation of Akt-mTOR-p70S6K pathway in angiogenesis in hepatocellular carcinoma.

Angiogenesis is an essential process for progression of hepatocellular carcinoma (HCC). The Akt-mTOR-p70S6K signal pathway has been recognized for its roles in regulating neoangiogenesis. The role of activation of the pathway in HCC progression is poorly understood. This study aimed to evaluate the immunohistochemical expression of the phosphorylated forms of the three key constituent proteins (Akt, mTOR and p70S6K) of the Akt-mTOR-p70S6K signal pathway in HCC and non-HCC tissue. Formalin-fixed paraffin-embedded tissue sections of 51 HCC, 9 hepatocellular adenoma (HCA), 48 cirrhotic nodules (CN) and 17 normal liver tissues (NLT) were immunostained for p-Akt, p-mTOR and p-p70S6K. The number of p-Akt and p-p70S6K-positive sinusoidal endothelial cells (SEC) and the intensity of immunostaining were significantly increased in HCC compared with HA, CN and NLT (p<0.01). p-mTOR in SEC tended to have an increased expression in SEC in HCC versus non-HCC tissue (p>0.05). There was a significant correlation between a high p-Akt and p-p70S6K expression, and a venous and capsular invasion of HCC. Our results suggest that activation of the Akt-mTOR-p70S6K pathway plays a significant role in HCC progression by promoting neoangiogenesis. Molecular strategies aimed at inhibiting this signal pathway may be of therapeutic use for the treatment of HCC.