Successful control of heavily pretreated metastatic gastric cancer with the mTOR inhibitor everolimus (RAD001) in a patient with PIK3CA mutation and pS6 overexpression.

BACKGROUND: Everolimus (RAD001) is an orally administered mTOR inhibitor that is well known for its antitumor efficacy and that has been approved for the treatment of several solid tumors, including renal cell carcinoma. In gastric cancer (GC), despite previous preclinical and phase I/II studies suggesting the promising efficacy of everolimus in previously treated AGC, more recent trials revealed that only certain subsets of patients might benefit from treatment with everolimus. CASE PRESENTATION: A 26-year-old man with metastatic gastric cancer with multiple liver lesions was treated with everolimus after failure of 1st-line and 2nd-line chemotherapy. A durable partial response was achieved for over 2 years. After progression from initial everolimus treatment, sequential cytotoxic chemotherapies were tried but failed rapidly. Everolimus was re-tried as salvage chemotherapy (re-treatment), and the patient achieved stable disease for 1 year until his death. Subsequent mutational analysis and immunohistochemical (IHC) staining with the tumor tissues just before re-treatment with everolimus revealed a PIK3CA hotspot mutation and pS6 overexpression in the primary tumor. After two cycles of everolimus re-treatment, the overexpression of pS6 became nearly absent in follow-up IHC staining. CONCLUSIONS: Everolimus monotherapy was satisfactory in a patient with refractory metastatic GC harboring PIK3CA and pS6 aberrations. These molecular alterations might be potential biomarkers that can predict the treatment response of everolimus, particularly in the terms of durable disease control. This case suggests and emphasizes that close evaluation of biomarkers in tumor tissue may be essential for identifying highly favorable groups among various subpopulations with AGC.

Central administration of metformin into the third ventricle of C57BL/6 mice decreases meal size and number and activates hypothalamic S6 kinase.

Administration of metformin is known to reduce both body weight and food intake. Although the hypothalamus is recognized as a critical regulator of energy balance and body weight, there is currently no evidence for an effect of metformin in the hypothalamus. Therefore, we sought to determine the central action of metformin on energy balance and body weight, as well as its potential involvement with key hypothalamic energy sensors, including adenosine monophosphate-activated protein kinase (AMPK) and S6 kinase (S6K). We used meal pattern analysis and a conditioned taste aversion (CTA) test and measured energy expenditure in C56BL/6 mice administered metformin (0, 7.5, 15, or 30 µg) into the third ventricle (I3V). Furthermore, we I3V-administered either control or metformin (30 µg) and compared the phosphorylation of AMPK and S6K in the mouse mediobasal hypothalamus. Compared with the control, I3V administration of metformin decreased body weight and food intake in a dose-dependent manner and did not result in CTA. Furthermore, the reduction in food intake induced by I3V administration of metformin was accomplished by decreases in both nocturnal meal size and number. Compared with the control, I3V administration of metformin significantly increased phosphorylation of S6K at Thr(389) and AMPK at Ser(485/491) in the mediobasal hypothalamus, while AMPK phosphorylation at Thr(172) was not significantly altered. Moreover, I3V rapamycin pretreatment restored the metformin-induced anorexia and weight loss. These results suggest that the reduction in food intake induced by the central administration of metformin in the mice may be mediated by activation of S6K pathway.

Epigallocatechin-3-gallate potentiates curcumin's ability to suppress uterine leiomyosarcoma cell growth and induce apoptosis.

BACKGROUND: Uterine leiomyosarcoma (LMS) has an unfavorable response to standard chemotherapeutic regimens. Two natural occurring compounds, curcumin and epigallocatechin gallate (EGCG), are reported to have anti-cancer activity. We previously reported that curcumin reduced uterine LMS cell proliferation by targeting the AKT-mTOR pathway. However, challenges remain in overcoming curcumin's low bioavailability. METHODS: The human LMS cell line SKN was used. The effect of EGCG, curcumin or their combination on cell growth was detected by MTS assay. Their effect on AKT, mTOR, and S6 was detected by Western blotting. The induction of apoptosis was determined by Western blotting using cleaved-PARP specific antibody, caspase-3 activity and TUNEL assay. Intracellular curcumin level was determined by a spectrophotometric method. Antibody against EGCG cell surface receptor, 67-kDa laminin receptor (67LR), was used to investigate the role of the receptor in curcumin's increased potency by EGCG. RESULTS: In this study, we showed that the combination of EGCG and curcumin significantly reduced SKN cell proliferation more than either drug alone. The combination inhibited AKT, mTOR, and S6 phosphorylation, and induced apoptosis at a much lower curcumin concentration than previously reported. EGCG enhanced the incorporation of curcumin. 67LR antibody partially rescued cell proliferation suppression by the combination treatment, but was not involved in the EGCG-enhanced intracellular incorporation of curcumin. CONCLUSIONS: EGCG significantly lowered the concentration of curcumin required to inhibit the AKT-mTOR pathway, reduce cell proliferation and induce apoptosis in uterine LMS cells by enhancing intracellular incorporation of curcumin, but the process was independent of 67LR.

Mammalian target of rapamycin (mTOR) and S6 kinase down-regulate phospholipase D2 basal expression and function.

The mammalian target of rapamycin (mTOR) and S6 kinase (S6K) pathway is essential for cell differentiation, growth, and survival. Phospholipase D2 (PLD2) plays a key role in mTOR/S6K mitogenic signaling. However, the impact of PLD on mTOR/S6K gene expression is not known. Here we show that interleukin-8 (IL-8) increases mRNA expression levels for PLD2, mTOR, and S6K, with PLD2 preceding mTOR/S6K in time. Silencing of PLD2 gene expression abrogated IL-8-induced mTOR/S6K mRNA expression, whereas silencing of mTOR or S6K gene expression resulted in large (>3-fold and >5-fold, respectively) increased levels of PLD2 RNA, which was paralleled by increases in protein expression and lipase activity. Treatment of cells with 0.5 nm rapamycin induced a similar trend. These results suggest that, under basal conditions, PLD2 expression and concomitant activity is negatively regulated by the mTOR/S6K signaling pathway. Down-regulation of PLD2 was confirmed in differentiated HL-60 leukocytes overexpressing an mTOR-wild type, but not an mTOR kinase-dead construct. At the cellular level, overexpression of mTOR-wild type resulted in lower basal cell migration, which was reversed by treatment with IL-8. We propose that IL-8 reverses an mTOR/S6K-led down-regulation of PLD2 expression and enables PLD2 to fully function as a facilitator for cell migration.

Prognosis, Significance and Positive Correlation of Rab1A and p-S6K/Gli1 Expression in Gastric Cancer.

BACKGROUND: Gastric Cancer (GC) is a frequently common malignancy. Recent studies have reported Rab1A as an activator of mTORC1, and the mTOR1 pathway is involved in regulating Gli1 expression in several cancers. Only a few studies have been performed to explore the relationship between Rab1A and p-S6K/Gli1in GC. METHODS: Immunohistochemistry (IHC) was performed to explore the association of Rab1A/p-S6K/Gli1 expression and prognosis in 117 GC tissue samples and adjacent normal tissues. RESULTS: Our results indicated that Rab1A/p-S6K/Gli1 was significantly overexpressed in GC tissues. High expression of Rab1A was closely related to the tumor size and the depth of tumor invasion. In addition, Rab1A expression was closely related with p-S6K/Gli1 expression in GC, and high level of Rab1A/p-S6K/Gli1 caused worse prognosis of GC patients. The univariate and multivariate analysis indicated that the expression of Rab1A was an independent prognostic factor. Moreover, both high Rab1A and p-S6K expression led to a worse prognosis when compared to a single positive expression as well as both high Rab1A/Gli1 expression also led to a worse prognosis than the single positive expression of Rab1A/Gli1. Strikingly, the overexpression of p-S6K also led to a worse prognosis in Rab1A positive patients, as did Gli1. CONCLUSION: Our results indicate that Rab1A/mTOR/S6K/Gli1 axis played a crucial role in GC, which may provide a novel field on targeted therapy of GC, especially for mTORC1-targeted therapy-resistant cancers.

AMPK represses TOP mRNA translation but not global protein synthesis in liver.

The AMP-activated protein kinase (AMPK) represses signaling through the mammalian target of rapamycin complex 1 (mTORC1). In muscle, repression of mTORC1 leads to a reduction in global protein synthesis. In contrast, repression of mTORC1 in the liver has no immediate effect on global protein synthesis. In the present study, signaling through mTORC1 and translation of specific mRNAs such as those bearing a 5'-terminal oligopyrimidine (TOP) tract and were examined in rat liver following activation of AMPK after treadmill running. Activation of AMPK repressed translation of the TOP mRNAs encoding rpS6, rpS8, and eEF1alpha. In contrast, neither global protein synthesis nor translation of mRNAs encoding GAPDH or beta-actin was changed. Basal phosphorylation of the mTORC1 target 4E-BP1, but not S6K1 or rpS6, was reduced following activation of AMPK. Thus, in liver, AMPK activation repressed translation of TOP mRNAs through a mechanism distinct from downregulated phosphorylation of S6K1 or rpS6.

Insulin-like growth factor-1 (IGF-1) and leucine activate pig myogenic satellite cells through mammalian target of rapamycin (mTOR) pathway.

Myogenic satellite cells are adult stem cells and have important roles in skeletal muscle growth, repair, and regeneration. Both insulin-like growth factor-1 (IGF-1) and leucine stimulate skeletal muscle growth, which link to the activation and proliferation of myogenic satellite cells in skeletal muscle. Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis and cell proliferation. Thus, IGF-1 and leucine may stimulate activation of myogenic satellite cells through mTOR signaling. In this study, myogenic satellite cells were isolated from 6-month-old pigs and subjected to IGF-1 and leucine treatments. Both IGF-1 and leucine upregulated mTOR signaling in myogenic satellite cells. The phosphorylation of mTOR at Ser(2448) increased 83.8 +/- 7.7% by IGF-1 (P < 0.05) and 83.4 +/- 5.7% by leucine (P < 0.05). The downstream targets of mTOR, S6 kinase, and 4E-binding protein 1 (4EBP1) were also phosphorylated due to IGF-1 and leucine treatments. Treatment with IGF-1 and leucine induced the phosphorylation of tuburin (TSC2), a key mediator upstream of mTOR signaling, by 272.8 +/- 26.4% and 94.2 +/- 28.7%, respectively. Treatment of cells with both IGF-1 and leucine did not show synergistic effect on mTOR signaling. Inhibition of mTOR by rapamycin abolished the protein synthesis and cell proliferation stimulated by both IGF-1 and leucine. In summary, our data showed that in preliminary cultured myogenic satellite cells mTOR signaling was activated due to IGF-1 and leucine treatments, and this mTOR activation is necessary for the activation of myogenic satellite cells.

Adding protein to a carbohydrate supplement provided after endurance exercise enhances 4E-BP1 and RPS6 signaling in skeletal muscle.

To examine the role of both endurance exercise and nutrient supplementation on the activation of mRNA translation signaling pathways postexercise, rats were subjected to a 3-h swimming protocol. Immediately following exercise, the rats were provided with a solution containing either 23.7% wt/vol carbohydrates (CHO), 7.9% wt/vol protein (Pro), 31.6% wt/vol (23.7% wt/vol CHO + 7.9% wt/vol Pro) carbohydrates and Pro (CP), or a placebo (EX). The rats were then killed at 0, 30, and 90 min postexercise, and phosphorylation states of mammalian target of rapamycin (mTOR), ribosomal S6 kinase (p70(S6K)), ribosomal protein S6 (rpS6), and 4E-binding protein 1 (4E-BP1), were analyzed by immunoblot analysis in the red and white quadriceps muscle. Results demonstrated that rat groups provided with any of the three nutritional supplements (CHO, Pro, CP) transiently increased the phosphorylation states of mTOR, 4E-BP1, rpS6, and p70(S6K) compared with EX rats. Although CHO, Pro, and CP supplements phosphorylated mTOR and p70(S6K) after exercise, only CP elevated the phosphorylation of rpS6 above all other supplements 30 min postexercise and 4E-BP1 30 and 90 min postexercise. Furthermore, the phosphorylation states of 4E-BP1 (r(2) = 0.7942) and rpS6 (r(2) = 0.760) were highly correlated to insulin concentrations in each group. These results suggest that CP supplementation may be most effective in activating the mTOR-dependent signaling pathway in the postprandial state postexercise, and that there is a strong relationship between the insulin concentration and the activation of enzymes critical for mRNA translation.

The expression of mammalian target of rapamycin in Ishikawa and HEC-1A cells.

The activation of mammalian target of rapamycin (mTOR) signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-1A cells respectively (P<0.01). There was mTOR expression in both Ishikawa and HEC-1A cells and the phosphorylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells (P<0.05). mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.

Alterations in mammalian target of rapamycin signaling pathways after traumatic brain injury.

In response to traumatic brain injury (TBI), neurons initiate neuroplastic processes through the activation of intracellular signaling pathways. However, the molecular mechanisms underlying neuroplasticity after TBI are poorly understood. To study this, we utilized the fluid-percussion brain injury (FPI) model to investigate alterations in the mammalian target of rapamycin (mTOR) signaling pathways in response to TBI. Mammalian target of rapamycin stimulates mRNA translation through phosphorylation of eukaryotic initiation factor 4E binding protein-1 (4E-BP1), p70 ribosomal S6 kinase (p70S6K), and ribosomal protein S6 (rpS6). These pathways coordinate cell growth and neuroplasticity via dendritic protein synthesis. Rats received sham surgery or moderate parasagittal FPI on the right side of the parietal cortex, followed by 15 mins, 30 mins, 4 h, 24 h, or 72 h of recovery. Using Western blot analysis, we found that mTOR, p70S6K, rpS6, and 4E-BP1 phosphorylation levels were significantly increased in the ipsilateral parietal cortex and hippocampus from 30 mins to 24 h after TBI, whereas total protein levels were unchanged. Using confocal microscopy to localize these changes, we found that rpS6 phosphorylation was increased in the parietal cortex and all subregions of the hippocampus. In accordance with these results, eIF4E, a key, rate-limiting mRNA translation factor, was also phosphorylated by mitogen-activated protein kinase-interacting kinase 1 (Mnk1) 15 mins after TBI. Together, these results suggest that changes in mRNA translation may be one mechanism that neurons use to respond to trauma and may contribute to the neuroplastic changes observed after TBI.

Inhibition of mammalian target of rapamycin reverses alveolar epithelial neoplasia induced by oncogenic K-ras.

The serine/threonine kinase AKT and its downstream mediator mammalian target of rapamycin (mTOR) are activated in lung adenocarcinoma, and clinical trials are under way to test whether inhibition of mTOR is useful in treating lung cancer. Here, we report that mTOR inhibition blocked malignant progression in K-ras(LA1) mice, which undergo somatic activation of the K-ras oncogene and display morphologic changes in alveolar epithelial cells that recapitulate those of precursors of human lung adenocarcinoma. Levels of phospho-S6(Ser236/235), a downstream mediator of mTOR, increased with malignant progression (normal alveolar epithelial cells to adenocarcinoma) in K-ras(LA1) mice and in patients with lung adenocarcinoma. Atypical alveolar hyperplasia, an early neoplastic change, was prominently associated with macrophages and expressed high levels of phospho-S6(Ser236/235). mTOR inhibition in K-ras(LA1) mice by treatment with the rapamycin analogue CCI-779 reduced the size and number of early epithelial neoplastic lesions (atypical alveolar hyperplasia and adenomas) and induced apoptosis of intraepithelial macrophages. LKR-13, a lung adenocarcinoma cell line derived from K-ras(LA1) mice, was resistant to treatment with CCI-779 in vitro. However, LKR-13 cells grown as syngeneic tumors recruited macrophages, and those tumors regressed in response to treatment with CCI-779. Lastly, conditioned medium from primary cultures of alveolar macrophages stimulated the proliferation of LKR-13 cells. These findings provide evidence that the expansion of lung adenocarcinoma precursors induced by oncogenic K-ras requires mTOR-dependent signaling and that host factors derived from macrophages play a critical role in adenocarcinoma progression.

17q23 amplifications in breast cancer involve the PAT1, RAD51C, PS6K, and SIGma1B genes.

Amplification of the 17q23 region occurs frequently in breast tumors. To characterize the structure of 17q23 amplicons and to identify oncogene targets associated with this alteration, we performed a copy number analysis of 87 17q23 localized expressed sequence tags in seven breast cancer cell lines. Three major regions of amplification were detected in the MCF7 and BT474 cell lines. Amplification of at least one of four known genes (PAT1, PS6K, RAD51C, and SIGMA1B) was detected in the cell lines and in 28% of 94 breast tumors. In most cases, these four genes were overexpressed when amplified, but there was a particularly good association between amplification of the SIGMA1B gene and elevated expression in tumors, which suggested a possible role for this gene in tumor progression. Our data show that this region contains at least four independent targets of amplification, which suggests that there is considerable variability in the structure of the 17q23 amplicon.

Multiple genes at 17q23 undergo amplification and overexpression in breast cancer.

Studies by comparative genomic hybridization imply that amplification of the chromosomal region 17q22-q24 is common in breast cancer. Here, amplification and expression levels of six known genes located at 17q23 were examined in breast cancer cell lines. Four of them (RAD51C, S6K, PAT1, and TBX2) were found to be highly amplified and overexpressed. To investigate the involvement of these genes in vivo, fluorescence in situ hybridization analysis of a tissue microarray containing 372 primary breast cancers was used. S6K, PAT1, and TBX2 were coamplified in about 10% of tumors, whereas RADS1C amplification was seen in only 3% of tumors. Expression analysis in 12 primary tumors showed that RAD51C and S6K were consistently expressed in all cases in which they were amplified and also in some tumors without amplification. These data suggest that 17q23 amplification results in simultaneous up-regulation of several genes, whose increased biological activity may jointly contribute to the more aggressive clinical course observed in patients with 17q23-amplified tumors.

RhoA-dependent murine prostate cancer cell proliferation and apoptosis: role of protein kinase Czeta.

We previously showed that RhoA played an important role in the proliferation of murine We prostate cancer (TRAMP) cells (P. M. Ghosh et al., Oncogene, 18: 4120-4130, 1999). Untransfected TRAMP cells as well as those expressing constitutively active RhoA (Q63L) mutant protein (Q63L cells) were highly proliferative. In contrast, TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein (T19N cells) were slow growing. In this study, we showed, in addition, that T19N cells displayed reduced rates of apoptotic cell death in response to serum deprivation, compared with TRAMP and Q63L cells, and we studied the mechanisms of the effects of RhoA on TRAMP cell proliferation and apoptosis. Both proliferation and apoptosis of TRAMP and Q63L cells were dependent on the activation of phosphatidylinositol 3-kinase (PI3K). The ubiquitous mitogen-activated Ser/Thr kinase, p70S6 kinase, a downstream effector of PI3K, was overexpressed in TRAMP and Q63L cells. Another PI3K effector, the cell survival protein Akt, displayed increased activity in T19N cells, which did not express active RhoA, compared with TRAMP and Q63L cells. The atypical protein kinase C (PKC) isoform PKCzeta, which is downstream of PI3K, was activated in cells expressing active RhoA. In addition, expression of constitutively activated PKCzeta in TRAMP cells enhanced proliferation and p70S6 kinase phosphorylation, whereas the inhibition of PKCzeta activation resulted in activation of Akt and enhanced cell survival. Thus, the effects of RhoA on TRAMP cell proliferation and apoptosis may be mediated by PKCzeta.

Activation of mTOR/S6K But Not MAPK Pathways Might Be Associated With High Ki-67, ER(+), and HER2(-) Breast Cancer.

UNLABELLED: We determined the activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways in 108 cases of estrogen receptor-positive and human epidermal growth factor receptor 2-negative breast cancer with high and low Ki-67 expression. The expression levels of Ki-67, p53, phosphorylated MAPK (pMAPK), and protein S6 (pS6; downstream molecule of PI3K/Akt/mammalian target of rapamycin/S6 kinase pathway) were determined immunohistochemically. pS6 positivity, but not pMAPK positivity, was significantly associated with the high Ki-67 expression subset. BACKGROUND: Evaluation of luminal A and luminal B characteristics of estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer is considered important. Although the phosphoinositide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways are thought to be involved in the luminal B subtype, the details of their contribution to breast cancer remain unclear. MATERIALS AND METHODS: We determined the activation of these pathways (phosphorylated MAPK [pMAPK] and protein S6 [pS6; a downstream molecule of PI3K/Akt/mammalian target of rapamycin (mTOR)/S6 kinase (S6K)]) in 108 ER(+), HER2(-) breast cancer cases with high and low Ki-67 expression. The ER, progesterone receptor (PgR), Ki-67, p53 expression levels were also determined immunohistochemically. The cutoff value for Ki-67 was set at 15%. RESULTS: A significantly greater percentage of cancer cases with high Ki-67 expression showed pS6 positivity than did those with low Ki-67 expression (53.2% vs. 19.7%; P = .0003). No significant differences were found between the cases with high and low expression levels were detected for p53 (23.4% vs. 11.5%; P = .12) or pMAPK (36.2% vs. 34.4%; P = .85) positivity. Multivariate analysis showed that pS6 positivity (odds ratio 5.16, 95% confidence interval 1.95-13.63; P = .0009), nuclear grade 2 and 3, and low PgR expression (≤ 20%) were independently associated with the high Ki-67 subset. CONCLUSION: From our findings, we have concluded that the pS6 expression level is associated with the characteristics of breast cancer with high Ki-67 expression. Because these associations were observed, irrespective of menopausal status, the biologic difference seems to be less affected by estrogen signaling than by activation of S6 protein, especially in terms of proliferation. Our findings have also indicated that targeting the mTOR/S6K pathway might be a useful strategy for the treatment of ER(+)/HER2(-) breast cancer with high Ki-67 expression.

Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages.

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (> or = 30 nM) and rapamycin (> or = 3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70s6k in CSF-1-mediated induction of c-fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

Glucocorticoid-induced insulin resistance of protein synthesis is independent of the rapamycin-sensitive pathways in rat skeletal muscle.

This study was designed to evaluate the role of p70 S6 kinase (p70(S6K) ), p90 S6 kinase (p90(RSK)) and mitogen-activated protein (MAP) kinase pathways in the insulin resistance of muscle protein synthesis observed during glucocorticoid treatment. Dexamethasone treatment decreased the effect of insulin on protein synthesis (-35. 2%) in epitrochlearis muscle incubated in vitro. This resistance is associated with a total blockage of the stimulation of p70(S6K) by insulin without any significant decrease in the amount of the kinase. However, the effect of rapamycin (inhibitor of several intracellular pathways including p70(S6K) pathways) on muscle protein synthesis was not modified by dexamethasone in rat muscles. This suggested that 'rapamycin-sensitive pathways' associated with the insulin stimulation of protein synthesis were not altered by glucocorticoids and thus are not responsible for the insulin resistance observed. As incubation of muscles with a MAP kinase inhibitor (PD98059) did not modify the stimulation of protein synthesis by insulin and as glucocorticoids did not alter the effect of insulin on p90(RSK )activity, our results provide evidence that glucocorticoid-induced alterations in muscle protein synthesis regulation by insulin do not involve factors or kinases that are dependent on MAP kinase and/or p90(RSK).

Toxin-induced tail phosphorylation of hepatocellular S6 kinase: evidence for a dual involvement of the AMP-activated protein kinase in S6 kinase regulation.

Several protein phosphatase-inhibitory toxins (okadaic acid, microcystin, calyculin A, cantharidin, tautomycin) administered to isolated rat hepatocytes were found to induce phosphorylation in the tail region of S6 kinase (S6K; p70S6K1) as detected with a phosphospecific antibody against doubly phosphorylated Thr-421/Ser424. 5-Aminoimidazole-4-carboxamide riboside (AICAR), an adenosine analogue that elicits activation of the hepatocellular AMP-activated protein kinase (AMPK), similarly stimulated S6K tail phosphorylation. The flavonoid naringin prevented the effects of AICAR, okadaic acid, and microcystin on AMPK activation as well as on S6K tail phosphorylation, suggesting AMPK as a mediator of the latter. The effects of AICAR and the toxins were rapamycin resistant; in contrast, amino acids induced an S6K tail phosphorylation that was rapamycin sensitive, suggesting mediation by the protein kinase mammalian target of rapamycin (mTOR). Amino acids activated S6K by phosphorylation at Thr-389, but the toxins did not, and AICAR in fact suppressed the activating phosphorylation induced by the amino acids. The possibility thus must be considered that the phosphorylated S6K tail may transmit a toxin-induced signal independently of S6K enzymatic activity. Despite their inability to activate S6K, the toxins (but not AICAR) stimulated phosphorylation of the ribosomal protein S6, presumably by activating some other S6-phosphorylating protein kinase.

S19-mRNA expression in squamous cell carcinomas of the upper aerodigestive tract.

BACKGROUND: The differential display method showed altered expression of ribosomal protein S19 gene in human head and neck squamous cell carcinoma (HNSCC) cell lines. MATERIALS AND METHODS: To verify these results, RT-PCR analysis was carried out in 18 HNSCC and 17 benign epithelial cell lines as well as 30 HNSCC and 8 reference tissue samples. In the HNSCC cells S19 mRNA expression was significantly reduced as compared to benign epithelial cells. RESULTS: Change of the S19 gene expression in surgical samples was detectable but not significant although the histopathological grading of the HNSCC biopsies correlated significantly with the S19 mRNA expression levels. The expression of ribosomal protein S6 and S14 genes were additionally analyzed using the same methods. CONCLUSION: High correlation was found between the expression of S6/S14 and S19 suggesting that changes in S19 gene expression might be the result of loss of ribosomes in HNSCC cells.

Expression and activation of P38 MAP kinase in invasive ductal breast cancers: correlation with expression of the estrogen receptor, HER2 and downstream signaling phosphorylated proteins.

MAP kinase signaling proteins have major implications in the molecular oncogenesis of breast cancers and have been extensively investigated as putative targets for therapy. This study reports the investigation of the expression of P38 MAPK and its phosphorylated form (p-P38 MAPK) in clinical specimens of invasive breast carcinomas and their correlation with estrogen receptor (ER) and HER2 expression, as well as MAPK and PI3 kinase-AKT pathway signaling phosphorylated proteins. Expression levels of P38 MAPK and p-P38 MAPK as well as p-AKT, p-GSK3ß, p-S6 kinase, p-MEK1 and p-ERK1/2 were quantitatively assessed using multiplex bead immunoassay in frozen specimens from 45 invasive ductal breast cancers. Twenty-nine specimens were ER+, 15 were HER2+ and 10 were triple­negative breast cancers (TNBCs). P38 MAPK was found to be expressed in all tumor specimens and was significantly (P=0.002) overexpressed in ER+ tumors. P38 MAPK expression was lower in TNBCs than in all of the other tumors. The median expression of p-P38 MAPK was also higher in ER+ tumors while lower in the TNBCs. HER2 status had no effect on P38 MAPK and p-P38 MAPK expression. No variation in the phosphorylation rate of P38 MAPK was observed in relation with ER, HER2 or TNBC status. Significantly higher (P=0.0048) expression of p-AKT was observed in HER2+ tumors. No significant difference in p-MEK1, p-GSK3ß and p-S6K expression was found in any other comparisons based on ER and HER2 expression subtypes. Investigation of the expression of multiple phosphorylated signaling proteins can be used for personalized targeted therapy. In invasive breast cancer, the overexpression of P38 MAPK may serve as a biomarker for the evaluation of P38 MAPK inhibitors.