Mammalian Target of Rapamycin (mTOR) Regulates Cellular Proliferation and Tumor Growth in Urothelial Carcinoma

Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth.Bladder cancer occurs in multiple forms, the most common of which is urothelial carcinoma (UCC), which represents >90% of all bladder cancers.¹ Approximately 30 to 50% of patients with invasive bladder cancer into the muscular wall of the bladder will develop metastatic disease and die within 2 years of diagnosis.² In addition, virtually all patients diagnosed with distant UCC metastases will succumb to disease.³ Currently, the standard treatment modality for muscle-invasive bladder cancer is radical cystectomy; systemic chemotherapy is generally reserved for patients with metastatic disease, although these treatment regimens provide only a limited long-term benefit with only rare reports of complete remission.^4,5 In light of these clinical outcomes, identification of new therapeutic targets is needed to define potential additional treatment avenues for these patients.Activation of the mammalian target of rapamycin (mTOR) signaling pathway occurs in many cancers and has recently been shown to correlate with more aggressive disease behavior,^6,7,8,9 although it has not been examined in great detail in UCC. Activation of mTOR occurs via a multistep process that includes upstream phosphoinositide-3 kinase (PI3K) and AKT activation, leading to phosphorylation and inactivation of the tuberous sclerosis complex 1 and 2 (TSC1/TSC2) heterodimer.^10,11 Inactivation of this heterodimer results in release of Rheb inhibition and subsequent mTOR activation by means of Rheb GTPase activity. Once activated, mTOR can induce increased mRNA translation or regulate the actin cytoskeleton via differential association Rictor and Raptor proteins.^10,11 Ultimately, mTOR activity regulates the effects of a number of downstream molecules important in cellular growth, including p70 S6 kinase-1 (S6K) and elongation-initiation factor 4E binding protein-1 (4E-BP1). Selective inhibition of the mTOR pathway can be achieved using rapamycin or rapamycin analogs temsirolimus (CCI-779, Wyeth Pharmaceuticals) and everolimus (RAD001, Novartis), which are currently in use in numerous clinical trials for solid tumors, with promising results in patients with advanced renal cell carcinoma.^12,13To further investigate the potential role of mTOR signaling and inhibition in UCC of the bladder, we used human cancer specimens, xenograft models, and in vitro analysis to determine the effects of mTOR on cellular proliferation, apoptosis, tumor growth, and clinical outcomes in this cancer population.     

Evaluation of anti-inflammatory and anti-arthritic property of ethanolic extract of Clitoria ternatea

Objective: Clitoria ternatea is a well-known bioactive plant used to treat several inflammatory ailments in Ayurvedic system of medicine in India. The present investigation aimed to determine the anti-inflammatory and anti-arthritic activity of ethanolic extract of Clitoria ternatea roots (EECT) in animal models. Methods: The anti-inflammatory activity of the EECT was evaluated by carrageenan and histamine-induced paw edema. Results: EECT showed a significant reduction in mean paw edema volume in both carrageenan and histamine-induced inflammation. The efficacy of EECT in rheumatoid arthritis was tested against Freund’s complete adjuvant (CFA) induced arthritic models in Wistar rats. The anti-arthritic effect of EECT was determined by systematic scoring of arthritis symptoms and measuring paw edema. A considerable decrease in paw diameter was observed in the EECT (200 and 400 mg/kg) and diclofenac (10 mg/kg) treated groups after day 7. Diclofenac (10 mg/kg) and EECT (400 mg/kg) showed a significant reduction in paw diameter from day 14 compared with CFA control (P < 0.001). The anti-arthritic activity was also confirmed from the altered biochemical, haematological (Hb, RBC and WBC) and anti-oxidant parameters (SOD, MDA, CAT, and GSH). EECT (400 and 200 mg/kg) also showed a marked inhibition of joint destruction. Conclusion: This study provides a pharmacological rationale for the traditional use of C. ternatea against inflammation and rheumatoid arthritis in India.     

The role of semantic complexity in treatment of naming deficits: training semantic categories in fluent aphasia by controlling exemplar typicality.

The effect of typicality of category exemplars on naming was investigated using a single subject experimental design across participants and behaviors in 4 patients with fluent aphasia. Participants received a semantic feature treatment to improve naming of either typical or atypical items within semantic categories, while generalization was tested to untrained items of the category. The order of typicality and category trained was counterbalanced across participants. Results indicated that patients trained on naming of atypical exemplars demonstrated generalization to naming of intermediate and typical items. However, patients trained on typical items demonstrated no generalized naming effect to intermediate or atypical examples. Furthermore, analysis of errors indicated an evolution of errors throughout training, from those with no apparent relationship to the target to primarily semantic and phonemic paraphasias. Performance on standardized language tests also showed changes as a function of treatment. Theoretical and clinical implications regarding the impact of considering semantic complexity on rehabilitation of naming deficits in aphasia are discussed.     

The role of semantic complexity in treatment of naming deficits: training semantic categories in fluent aphasia by controlling exemplar typicality.

The effect of typicality of category exemplars on naming was investigated using a single subject experimental design across participants and behaviors in 4 patients with fluent aphasia. Participants received a semantic feature treatment to improve naming of either typical or atypical items within semantic categories, while generalization was tested to untrained items of the category. The order of typicality and category trained was counterbalanced across participants. Results indicated that patients trained on naming of atypical exemplars demonstrated generalization to naming of intermediate and typical items. However, patients trained on typical items demonstrated no generalized naming effect to intermediate or atypical examples. Furthermore, analysis of errors indicated an evolution of errors throughout training, from those with no apparent relationship to the target to primarily semantic and phonemic paraphasias. Performance on standardized language tests also showed changes as a function of treatment. Theoretical and clinical implications regarding the impact of considering semantic complexity on rehabilitation of naming deficits in aphasia are discussed.     

Reevaluation of the role of LIP-1 as an ERK/MPK-1 dual specificity phosphatase in the C. elegans germline

The fidelity of a signaling pathway depends on its tight regulation in space and time. Extracellular signal-regulated kinase (ERK) controls wide-ranging cellular processes to promote organismal development and tissue homeostasis. ERK activation depends on a reversible dual phosphorylation on the TEY motif in its active site by ERK kinase (MEK) and dephosphorylation by DUSPs (dual specificity phosphatases). LIP-1, a DUSP6/7 homolog, was proposed to function as an ERK (MPK-1) DUSP in the Caenorhabditis elegans germline primarily because of its phenotype, which morphologically mimics that of a RAS/let-60 gain-of-function mutant (i.e., small oocyte phenotype). Our investigations, however, reveal that loss of lip-1 does not lead to an increase in MPK-1 activity in vivo. Instead, we show that loss of lip-1 leads to 1) a decrease in MPK-1 phosphorylation, 2) lower MPK-1 substrate phosphorylation, 3) phenocopy of mpk-1 reduction-of-function (rather than gain-of-function) allele, and 4) a failure to rescue mpk-1–dependent germline or fertility defects. Moreover, using diverse genetic mutants, we show that the small oocyte phenotype does not correlate with increased ectopic MPK-1 activity and that ectopic increase in MPK-1 phosphorylation does not necessarily result in a small oocyte phenotype. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the C. elegans germline. Our results caution against overinterpretation of the mechanistic underpinnings of orthologous phenotypes, since they may be a result of independent mechanisms, and provide a framework for characterizing the distinct molecular targets through which LIP-1 may mediate its several germline functions.

Extracellular signal-regulated kinases (ERKs) are a group of serine/threonine protein kinases and classical members of mitogen activated protein kinases (MAPKs). The ERK MAPKs are terminal enzymes of a highly conserved three-tiered kinase signaling cascade, the RAS–ERK pathway (1, 2). Extracellular stimuli, including growth factors and insulin signaling induce the sequential activation of RAS–ERK pathway that orchestrates a wide range of cellular processes such as gene expression, proliferation, differentiation, and apoptosis to regulate tissue and organismal homeostasis (Fig. 1A) (1–3). Because the ERK MAPK signaling pathway regulates a myriad of developmental processes for controlled and ordered execution of the pathway, ERK activity is tightly monitored in space and time (4). MEK (also known as MAPK/ERK kinase) phosphorylates ERK at threonine and tyrosine residues (TEY motif), thus activating its function (1). Active ERK is then inactivated by dual specificity MAPK phosphatases (MKPs or DUSPs) that remove the phosphate residues. Together, MEK and DUSPs shape the magnitude, duration, and spatiotemporal profile of ERK activity (1, 4–6).Open in a separate windowFig. 1.lip-1 mutants are defective in pachytene exit and oocyte formation. (A) Schematic view of the conserved LET-60 (RAS)–MPK-1 (ERK) pathway showing that the regulation of ERK/MPK-1 activation depends on upstream kinase cascade and dephosphorylation depends on DUSPs. (B) Schematic view of a hermaphroditic C. elegans germline displaying the spatiotemporal nature of MPK-1 activation. The germline is oriented in a distal (*) to proximal direction from left to right. Proliferative PZ cells are in the distal region, capped by the distal tip cell (DTC). Germ cells enter meiotic prophase at the transition zone (TZ), followed by progression through different stages of meiotic prophase. The “loop region” is the anatomic bend in the U-shaped gonad. The −1 marks the oldest oocyte at the proximal end. Active MPK-1 is visualized by a specific dpMPK-1 antibody in two distinct regions of the germline: midpachytene, termed as zone 1, and proximal few oocytes, termed as zone 2. The intensity of the color (red) correlates with strength of MPK-1 di-phosphorylation. (C) Predicted activation of MPK-1 in the absence of DUSP: either distal to zone 1, called “precocious” activation, or in the late-pachytene/early-diplotene region (anatomically in the loop region), called “ectopic” activation. (D–I) Differential interference contrast microscopy images of germlines from indicated genotypes, age, and temperature to visualize germline morphology. The loop region is on the right in the photographs and oocytes on the ventral side. Oocytes are numbered from proximal to distal polarity (toward loop). The most proximal oocyte is labeled as −1. Arrowheads indicate oocytes, and arrows indicate pachytene-stage germ cells. (J) Quantification of germlines of the indicated genotypes, with pachytene-progression–defective phenotypes expressed as a percentage. (K–P) Dissected DAPI-stained germlines of the indicated genotypes (mid-L4 + 24 h at 25 °C) displaying germline morphology. Insets are magnified views of germ cell(s) at the proximal gonad (after loop region). The dissected germlines are oriented with the distal on the left (*) to proximal on the right of the photograph, according to the meiotic progression. Arrowheads indicate oocytes, and arrows indicate pachytene germ cells. The total number of germlines (n) analyzed per genotype is indicated in each panel (scale bars, 25 μm).The Caenorhabditis elegans oogenic germline, like most complex biological systems, displays a controlled spatiotemporal pattern of ERK (MPK-1 in C. elegans) activity (7–11). Active MPK-1, as assayed using an antibody that detects dual phosphorylated MPK-1 at threonine and tyrosine of the TEY motif (7, 12), is visualized in midpachytene (termed as zone 1 of activation). However, MPK-1 is dephosphorylated, and thus, its activity is very low in the late-pachytene and early-diplotene region of the germline, which corresponds to the anatomic “loop” of the C. elegans U-shaped gonad (Fig. 1B). MPK-1 phosphorylation is again visible in the proximal diakinesis oocytes (termed as zone 2) in a hermaphroditic germline (7–9). Zone 2 activation is mediated by a secreted sperm signal (major sperm protein, or MSP), which antagonizes the VAB-1 Ephrin receptor (13). Thus, zone 2 activation is absent in C. elegans females, which do not produce sperm (7). In a wild-type oogenic hermaphroditic germline, active MPK-1 has not been visualized in the distal germline, from the progenitor zone (PZ) to midpachytene, and is very low in the loop region of the germline. Because total MPK-1 protein is expressed throughout the germline (8), the striking spatiotemporal activation pattern of MPK-1 observed using the dual-phosphospecific antibody suggests localized activation and inactivation of MPK-1 through MEK and DUSPs.In the oogenic hermaphroditic germline, the phenotypic consequences of MPK-1 activation are complex. In genetic mutants of the mpk-1 pathway, changes to the MPK-1 activation pattern along the spatiotemporal axis, as well as alterations to signal strength, produce distinct phenotypes. For example, a complete loss of MPK-1 activity in a null allele causes the oogenic germ cells to arrest in early- to midpachytene (8, 14). In the absence of MPK-1 activity, the germ cells fail to launch the apoptotic program because they do not progress into midpachytene, the stage in which meiotic checkpoint activation culls errors (9, 15). Reduction of MPK-1 signal strength using temperature-sensitive (ts) alleles, however, produced different phenotypes depending on the time at which MPK-1 activation was reduced during oogenic development. These mpk-1(ts) germlines exhibit increased apoptosis (due to higher levels of meiotic asynapsis defects; Ref. 11), a pachytene-progression defect in which pachytene-stage cells linger and are observed in the loop region, and fewer oocytes with an increased size relative to wild-type animals (8). Conversely, in RAS/let-60 gain-of-function mutants, the spatiotemporal pattern of MPK-1 activation is different from the wild-type in two regions: 1) in midpachytene, the germline displays “precocious” activation of MPK-1, and 2) the loop region exhibits “ectopic” MPK-1 activation (Fig. 1C). These animals, unlike the wild-type, display multiple small oocytes (8). Because of the striking increase in oocyte number in the RAS/let-60 gain-of-function mutants, an increase in oocyte number has been considered as a readout for MPK-1 activation. Mutants displaying multiple small oocytes are thus interpreted to be a consequence of increased MPK-1 activity.The C. elegans genome has 29 predicted DUSPs, of which LIP-1 (lateral signal induced phosphatase-1) bears homology with mammalian DUSP6/7 (16, 17). Genetic evidence suggested that loss of lip-1 negatively regulates MPK-1 during somatic vulval development (17). In vitro, in mammalian Cos-1 cultured cells, Myc-tagged LIP-1 protein was shown to dephosphorylate mammalian ERK1/2 (16). Coupled with the homology to mammalian DUSPs, the authors concluded that LIP-1 functions as an MPK-1 DUSP in vivo. In the C. elegans germline, immunofluorescence staining using an anti-LIP-1 antibody showed that the total protein is expressed from the proximal one-third of the PZ region and throughout the pachytene as membrane-associated bright puncta (18). LIP-1 was proposed to function as an MPK-1 DUSP, in the germline, from two lines of evidence (18), which we reevaluated based on the reasoning outlined below. First, in the prior report, the authors showed that in a feminized germline, which does not produce any sperm signal, loss of lip-1 led to an increase in phosphorylated MPK-1 in zone 2 (Fig. 1B). However, in the absence of the sperm signal, MPK-1 cannot be phosphorylated in zone 2 to begin with (7, 13) (Fig. 1A). In this context, inactivation or absence of a DUSP (LIP-1, in this case) should not lead to an increase in the level of phosphorylated MPK-1 since it was never phosphorylated. Second, the authors observed that loss of lip-1 led to ectopic (loop region) MPK-1 activation in hermaphrodites coupled with an increase in oocyte numbers. The authors interpreted this phenotype to be similar to that of RAS/let-60 gain-of-function mutant germlines (18). However, recent work has revealed that the increased oocyte production in RAS/let-60 gain-of-function animals is due to the “precocious” activation of MPK-1 in the early-pachytene, rather than the “ectopic” MPK-1 activation in the loop region (Fig. 1C) (11). Together, these two lines of reasoning led us to reinvestigate the role of LIP-1 as an MPK-1 DUSP in the C. elegans germline and to determine where in the germline spatiotemporal axis LIP-1 might function to regulate oocyte formation, using cytology, genetics, and phenotypic analyses.Contrary to what was previously published (18), our results show that 1) precocious or ectopic MPK-1 activity is not detected in the absence of lip-1—in fact, we found that loss of lip-1 led to lower MPK-1 activation; 2) loss of lip-1 fails to rescue the pachytene-progression and fertility defects observed upon reducing mpk-1 function; and 3) germlines with loss of lip-1 displayed an mpk-1 loss-of-function–like oocyte phenotype, rather than a gain-of-function–like oocyte phenotype, and 4) led to lower MPK-1 substrate phosphorylation. Moreover, we show that mutants in other genes, such as ooc-5 (human ortholog of torsinA AAA+ ATPase), also exhibit multiple small oocytes (19, 20) but do not present with ectopic MPK-1 activity, suggesting that increased oocyte number is not invariably equivalent to, or due to, increased MPK-1 phosphorylation. In support of this, we observed that loss of rskn-1 (human ortholog of RPS6KA, ribosomal protein S6 kinase A), which results in increased ectopic activation of MPK-1 in the loop region of the germline, does not exhibit increased oocyte numbers. This finding demonstrates that ectopic MPK-1 activation does not necessarily cause oocyte numbers to increase. Finally, in wild-type C. elegans diplotene oocytes, the synaptonemal complex (SC) central proteins are removed from the long arm of the chromosome axis to allow for accurate chromosome segregation (21). However, RAS/let-60(ga89ts) gain-of-function mutants have been shown to retain the SC central proteins on the long arm (10). Nadarajan et al. (10) reported that loss of lip-1 also leads to retention of the SC central protein to the long chromosomal arm and proposed that this was because of an increase in MPK-1 activation. We found that while the SC central element proteins are retained on the long arm of the chromosome in diplotene oocytes in both RAS/let-60(ga89ts) gain-of-function and lip-1 mutant oocytes, they are not retained in the rskn-1 mutant germlines, which display increased MPK-1 activation in oocytes. Thus, the retention of the SC central proteins in lip-1 mutant germlines likely occurs through MPK-1–independent mechanisms, suggesting that multiple regulatory processes, both independent of and dependent on ectopic MPK-1 phosphorylation, control SC disassembly. Together, these data demonstrate that LIP-1 does not function as an MPK-1 DUSP in the context of the C. elegans germline and may have multiple other targets through which it mediates its several germline functions.