Cerebrospinal fluid proteomic analysis reveals dysregulation of methionine aminopeptidase-2 expression in human and mouse neurofibromatosis 1-associated glioma

Individuals affected with the neurofibromatosis 1 (NF1) tumor predisposition syndrome are prone to the development of multiple nervous system tumors, including optic pathway gliomas (OPG). The NF1 tumor suppressor gene product, neurofibromin, functions as a Ras GTPase-activating protein, and has been proposed to regulate cell growth by inhibiting Ras activity. Recent studies from our laboratory have shown that neurofibromin also regulates the mammalian target of rapamycin activity in a Ras-dependent fashion, and that the rapamycin-mediated mammalian target of rapamycin inhibition ameliorates the Nf1-/- astrocyte growth advantage. Moreover, Nf1-deficient astrocytes exhibit increased protein translation. As part of a larger effort to identify protein markers for NF1-associated astrocytomas that could be exploited for therapeutic drug design, we did an objective proteomic analysis of the cerebrospinal fluid from genetically engineered Nf1 mice with optic glioma. One of the proteins found to be increased in the cerebrospinal fluid of OPG-bearing mice was the eukaryotic initiation factor-2alpha binding protein, methionine aminopeptidase 2 (MetAP2). In this study, we show that Nf1 mouse OPGs and NF1-associated human astrocytic tumors, but not sporadic pilocytic or other low-grade astrocytomas, specifically expressed high levels of MetAP2. In addition, we show that Nf1-deficient astrocytes overexpress MetAP2 in vitro and in vivo, and that treatment with the MetAP2 inhibitor fumagillin significantly reduces Nf1-/- astrocyte proliferation in vitro. These observations suggest that MetAP2 is regulated by neurofibromin, and that MetAP2 inhibitors could be potentially employed to treat NF1-associated tumor proliferation.     

Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors

Disruption of the TSC1 or TSC2 gene leads to the development of tumors in multiple organs, most commonly affecting the kidney, brain, lung, and heart. Recent genetic and biochemical studies have identified a role for the tuberous sclerosis gene products in phosphoinositide 3-kinase signaling. On growth factor stimulation, tuberin, the TSC2 protein, is phosphorylated by Akt, thereby releasing its inhibitory effects on p70S6K. Here we demonstrate that primary tumors from tuberous sclerosis complex (TSC) patients and the Eker rat model of TSC expressed elevated levels of phosphorylated mammalian target of rapamycin (mTOR) and its effectors: p70S6K, S6 ribosomal protein, 4E-BP1, and eIF4G. In the Eker rat, short-term inhibition of mTOR by rapamycin was associated with a significant tumor response, including induction of apoptosis and reduction in cell proliferation. Surprisingly, these changes were not accompanied by significant alteration in cyclin D1 and p27 levels. Our data provide in vivo evidence that the mTOR pathway is aberrantly activated in TSC renal pathology and that treatment with rapamycin appears effective in the preclinical setting.     

Clinical activity of mammalian target of rapamycin inhibitors in solid tumors

The phosphatidylinositol 3-kinase (PI3K)–Akt–mammalian target of rapamycin (mTOR) pathway is vital for cell metabolism, growth, and proliferation. mTOR is frequently upregulated in many tumor types and hence has become an important target in cancer treatment. Sirolimus and its derivatives (rapalogs) interact with the intracellular receptor FK506 binding protein 12 (FKBP12), forming a complex with high affinity for mTOR and thus disrupting its activity. Rapalogs are being evaluated extensively in cancer patients with different formulations and schedules. Significant clinical activity has led to their approval for the treatment of kidney cancer, mantle cell lymphoma, and subependymal giant cell astrocytoma; however, despite increasing knowledge about cancer cell biology, their activity in other malignancies is unclear. Further research is needed to identify optimal dosage, administration and targeted combination as well as the subset of patients likely to respond to mTOR/PI3K inhibition. This review focuses on a discussion of the pathway, its implications in cancer biology and results of clinical trials of rapalogs alone or in combination, organizing them by common malignancy type.     

Genetics of neurofibromatosis 1-associated peripheral nerve sheath tumors

Neurofibromatosis 1, an inherited disorder that affects 1/3500 individuals worldwide, predisposes to the development of benign and malignant peripheral nerve sheath tumors. The disorder results from inactivation of one of the NFI genes. The second NFI gene is typically inactivated in Schwann cells during tumor formation. This article reviews the different types of genetic alterations in NFI in both constitutional and tumor tissues and genetic alterations of other genes that may affect tumorigenesis. These studies have provided insight into the genetic basis of both the variable expression of the disorder and of benign and malignant peripheral nerve sheath tumorigenesis.     

The mammalian target of rapamycin pathway as a potential target for cancer chemoprevention.

The mammalian target of rapamycin (mTOR) is a key signaling node coordinating cell cycle progression and cell growth in response to genetic, epigenetic, and environmental conditions. Pathways involved in mTOR signaling are dysregulated in precancerous human tissues. These findings, together with the intriguing possibility that mTOR suppression may be associated with antitumor actions of caloric restriction, suggest that mTOR signaling may be an important target for chemopreventive drugs.     

哺乳动物雷帕霉素靶蛋白的反馈激活机制在肿瘤中的研究进展

哺乳动物雷帕霉素靶蛋白（mTOR）作为生长因子和氨基酸特别是亮氨酸所激发的信号通路的汇合点,在肿瘤细胞中起中心调控作用.近些年,人们逐渐认识到mTOR的反馈激活机制是影响雷帕霉素及其类似物抗肿瘤疗效的重要因素之一.因此,针对mTOR的反馈激活机制及联合应用相关抑制剂协同阻断这一机制的研究成为肿瘤防治的新热点.     

Differential effects of rapamycin on mammalian target of rapamycin signaling functions in mammalian cells

Rapamycin and its analogues have shown promising anticancer activities in preclinical and clinical studies. However, the mechanism whereby rapamycin inhibits signaling through the mammalian target of rapamycin (mTOR) remains poorly understood. Here, we show that the FKBP12/rapamycin complex is an essentially irreversible inhibitor of mTOR kinase activity in vitro. However, we observe no suppression of mTOR catalytic activity after immunoprecipitation from rapamycin-treated cells. These results suggest either that rapamycin acts as a reversible kinase inhibitor in intact cells or that the cellular effects of rapamycin are not mediated through global suppression in mTOR kinase activity. To better understand the cellular pharmacology of rapamycin, we compared the individual and combined effects of rapamycin and kinase-inactive mTOR expression on a panel of mTOR-dependent cellular responses. These studies identified glycolytic activity, amino acid transporter trafficking, and Akt kinase activity as novel, mTOR-modulated functions in mammalian cells. Whereas kinase-inactive mTOR did not enhance the decreases in cell size and glycolysis induced by rapamycin, expression of this mTOR mutant significantly enhanced the inhibitory effects of rapamycin on cell proliferation, 4EBP1 phosphorylation, and Akt activity. Unexpectedly, amino acid transporter trafficking was perturbed by kinase-inactive mTOR but not by rapamycin, indicating that this process is rapamycin insensitive. These results indicate that rapamycin exerts variable inhibitory actions on mTOR signaling functions and suggest that direct inhibitors of the mTOR kinase domain will display substantially broader anticancer activities than rapamycin.     

Inhibition of the phosphatidylinositol 3-kinase/mammalian target of rapamycin pathway in hematologic malignancies

Opinion statement The phosphatidylinositol 3-kinase (PI3-K)/mammalian target of rapamycin (mTOR) signal transduction pathway integrates signals from multiple receptor tyrosine kinases to control cell proliferation and survival. Key components of the pathway are the lipid kinase PI3-K, the small guanosine triphosphate-binding protein Rheb, and the protein kinases Akt and mTOR. Important natural inhibitors of the pathway include the lipid phosphatase PTEN and the tuberous sclerosis complex. Several components of this pathway are targeted by investigational antineoplastic agents. Rapamycin (sirolimus), the prototypic mTOR inhibitor, exhibits activity in acute myeloid leukemia. Three rapamycin analogs, temsirolimus, everolimus, and AP23573, are in clinical trials for various hematologic malignancies. Temsirolimus has produced a 38% overall response rate in relapsed mantle cell lymphoma, and AP23573 has demonstrated activity in acute leukemia. Everolimus is undergoing clinical testing in lymphoma (Hodgkin and non-Hodgkin) and multiple myeloma. In addition, perifosine, an inhibitor of Akt activation that exhibits substantial antimyeloma activity in preclinical models, is being examined in relapsed multiple myeloma. Based on results obtained to date, it appears that inhibitors of the PI3-K/mTOR pathway hold promise as single agents and in combination for hematologic malignancies.     

AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway

Cisplatin [cis-diaminodichloroplatinum (II) (CDDP)] is one of the most widely used and effective therapeutic agents for many kinds of cancers. However, its efficiency is limited due to development of drug resistance. In this study, we showed that CDDP resistance was associated with AKT1 overexpression and gene amplification in human lung cancer cells that acquired the drug resistance. We showed that AKT1 forced expression in the cells was sufficient to render the cells CDDP resistant, and that AKT1 inhibition by its dominant negative mutant reversed the CDDP-resistant cells to be CDDP sensitive. These results show that AKT1 activity is essential for regulating CDDP resistance in cultured lung cancer cells. To study whether these results were correlated with human lung cancer tumors, we randomly selected tumor samples from human lung cancer patients to study the correlation of AKT activation and CDDP resistance in clinical tumor samples. We showed that AKT activation was highly related to CDDP chemosensitivity in human tumor tissues. Our results further showed that AKT1 induced lung cancer cells to become resistant to CDDP through the mammalian target of the rapamycin (mTOR) signaling pathway. These studies conclude that AKT amplification and the mTOR pathway play an important role in human lung cancer cells acquiring CDDP resistance, which represents a new mechanism for acquiring CDDP resistance and a potential novel therapeutic target for overcoming CDDP resistance in human cancer in the future.     

Bile acid exposure up-regulates tuberous sclerosis complex 1/mammalian target of rapamycin pathway in Barrett's-associated esophageal adenocarcinoma

Barrett's esophagus, a columnar metaplasia of the lower esophagus epithelium related to gastroesophageal reflux disease, is the strongest known risk factor for the development of esophageal adenocarcinoma (EAC). Understanding the signal transduction events involved in esophageal epithelium carcinogenesis may provide insights into the origins of EAC and may suggest new therapies. To elucidate the molecular pathways of bile acid-induced tumorigenesis, the newly identified inflammation-associated signaling pathway involving I kappaB kinases beta (IKK beta), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1) was confirmed to be activated in immortalized Barrett's CPC-A and CPC-C cells and esophageal cancer SEG-1 and BE3 cells. Phosphorylation of TSC1 and S6K1 was induced in response to bile acid stimulation. Treatment of these cells with the mTOR inhibitor rapamycin or the IKK beta inhibitor Bay 11-7082 suppressed bile acid-induced cell proliferation and anchorage-independent growth. We next used an orthotopic rat model to evaluate the role of bile acid in the progression of Barrett's esophagus to EAC. Of interest, we found high expression of phosphorylated IKK beta (pIKK beta) and phosphorylated S6K1 (pS6K1) in tumor tissues and the Barrett's epithelium compared with normal epithelium. Furthermore, immunostaining of clinical EAC tissue specimens revealed that pIKK beta expression was strongly correlated with pS6K1 level. Together, these results show that bile acid can deregulate TSC1/mTOR through IKK beta signaling, which may play a critical role in EAC progression. In addition, Bay 11-7082 and rapamycin may potentially be chemopreventive drugs against Barrett's esophagus-associated EAC.     

Regulation of microtubule-dependent protein transport by the TSC2/mammalian target of rapamycin pathway

Protein transport plays a critical role in the interaction of the cell with its environment. Recent studies have identified TSC1 and TSC2, two tumor suppressor genes involved in tuberous sclerosis complex, as regulators of the mammalian target of rapamycin (mTOR) pathway. Cells deficient in TSC1 or TSC2 possess high levels of Rheb-GTP resulting in constitutive mTOR activation. We have shown previously that the TSC1/TSC2 complex is involved in post-Golgi transport of VSVG and caveolin-1 in mammalian cells. Here, we show that modulation of mTOR activity affects caveolin-1 localization and that this effect is independent of p70S6K. Tsc1- and Tsc2-null cells exhibit abnormal caveolin-1 localization that is accompanied by disorganized microtubules in the subcortical region. Analyses of green fluorescent protein-EB1 and tubulin in live mutant cells suggest a failure of the plus-ends to sense cortical signals and to halt microtubule growth. Down-regulation of CLIP-170, a putative mTOR substrate with microtubule-binding properties, rescued the abnormal microtubule arrangement and caveolin-1 localization in Tsc2-/- cells. Together, these findings highlight a novel role of the TSC2/mTOR pathway in regulating microtubule-dependent protein transport.     

Expression of an activated mammalian target of rapamycin (mTOR) in gastroenteropancreatic neuroendocrine tumors

Aims

Gastroenteropancreatic neuroendocrine tumors are rare, and the current WHO classification divides this tumor entity into well-differentiated (neuro)endocrine tumors, well-differentiated (neuro)endocrine carcinomas, and poorly differentiated (neuro)endocrine carcinomas. Poorly differentiated (neuro)endocrine carcinoma is extremely aggressive, and no appropriate therapeutic approach has been established. The mammalian target of rapamycin (mTOR), an important regulator of cell proliferation and protein translation, is activated in various malignancies. Recent phase II trial has revealed the efficacy of mTOR inhibitor (RAD001; everolimus) against low-to-intermediate grade neuroendocrine tumors. However, the beneficial role of mTOR inhibitor against poorly neuroendocrine carcinoma remains uncertain. The purpose of the present study was to determine the activation of mTOR in gastropancreatic neuroendocrine tumors, especially in poorly differentiated neuroendocrine carcinomas.

Methods

Expression of p-mTOR(Ser2448) was assessed by immunohistochemistry in 20 gastropancreatic neuroendocrine tumors (seven well-differentiated neuroendocrine tumors, four well-differentiated neuroendocrine carcinomas, and nine poorly differentiated neuroendocrine carcinomas). Double immunohistochemistry was performed with p-Akt for patients with high p-mTOR expression.

Results

Expression of mTOR was seen in 9 (45%) of 20 gastroenteropancreatic neuroendocrine tumors. High expression of p-mTOR was seen in 6 (67%) of 9 poorly differentiated neuroendocrine carcinomas which was higher than the expression rate of well-differentiated neuroendocrine tumors and carcinomas, 3 (27%) of 11. All large cell neuroendocrine carcinomas showed high p-mTOR expression. Some tumor cells showed positive staining for p-mTOR co-expressed p-Akt.

Conclusions

High expression rate of p-mTOR in poorly differentiated neuroendocrine carcinomas (large-cell type) may suggest the potential role of mTOR inhibitors as effective therapeutic agents for this highly malignant disease.     

Activation of mammalian target of rapamycin pathway confers adverse outcome in nonsmall cell lung carcinoma

BACKGROUND:

Dysregulation of the mammalian target of rapamycin (mTOR) pathway has been shown to contribute to tumorigenesis. This study explored protein expression profiles of mTOR pathway and the relationship with prognosis in patients with nonsmall cell lung carcinoma (NSCLC).

METHODS:

The protein expression profiles of mTOR/phosphorylated (p‐)mTOR, phosphoinositide‐dependent kinase 1 (PDK1)/p‐PDK1, p‐Akt1, and P70 ribosomal protein S6 kinase (P70S6K)/p‐P70S6K were determined via immunohistochemical staining assay. The clinical prognostic values of both single and combined protein expression were investigated with univariate and multivariate survival analysis.

RESULTS:

Compared with normal lung tissues, the protein levels of mTOR/p‐mTOR, p‐Akt1 Ser473/Thr308, and P70S6K/p‐P70S6K were higher (all P < .05), whereas p‐PDK1 was lower (P < .05) in tumor tissues. p‐mTOR expression was associated with histological differentiation, histological type, lymph node invasion, and stage (all P < .05). Overall survival in NSCLC patients was significantly shorter in cases with positive phenotype for p‐mTOR, p‐PDK1, and p‐P70S6K (all P < .05). Subjects with coexpression of any 2 of p‐mTOR, p‐PDK1, p‐Akt1 Ser473, and p‐P70S6K demonstrated worse prognosis than those expressing no biomarker or any 1 biomarker alone (all P < .05). Multivariate analysis showed that the combination of p‐mTOR/p‐P70S6K is an independent prognostic factor in addition to tumor stage.

CONCLUSIONS:

This study provides clinical evidence that activated components of mTOR pathway, not total protein, are predictors of poor prognosis in NSCLC. Moreover, evaluating protein‐expression profiles of these molecules might be a new strategy for individual therapy in subjects with NSCLC. Cancer 2011;. © 2011 American Cancer Society.     

Current status of sporadic and neurofibromatosis type 1-associated malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas that rarely occur in the general population but have a lifetime incidence of 8% to 13% in those with neurofibromatosis type 1 (NF1). Complete surgical resection is the standard treatment for MPNSTs. Unresectable MPNSTs carry a poor prognosis, and survival appears to be worse in NF1-associated tumors than in sporadic tumors. The response rate of MPNSTs to standard chemotherapeutic agents used to treat pediatric and adult soft tissue sarcomas is unknown and is currently undergoing evaluation in a multi-institutional clinical trial. With an increasing understanding of the molecular pathogenesis of MPNSTs, clinical trials with targeted agents have become available and have established that histology-specific trials in this rare malignancy are feasible. This knowledge, coupled with the availability of preclinical MPNST models, likely will accelerate the development of effective treatments for this malignancy.     

The mammalian target of rapamycin signaling pathway: twists and turns in the road to cancer therapy.

The immunosuppressive drug rapamycin played a key role in the functional characterization of mammalian target of rapamycin (mTOR), an unusual protein kinase that coordinates growth factor and nutrient availability with cell growth and proliferation. Several rapamycin-related compounds are now in various stages of clinical development as anticancer agents. This article highlights recent advances in our understanding of the mTOR signaling pathway and the implications of these findings for the clinical application of mTOR inhibitors in cancer patients.     

Targeting the AIB1 oncogene through mammalian target of rapamycin inhibition in the mammary gland

Amplified in breast cancer 1 (AIB1), an estrogen receptor (ER) coactivator, is frequently amplified or overexpressed in human breast cancer. We previously developed a transgenic mouse model in which AIB1 can act as an oncogene, giving rise to a premalignant hyperplastic mammary phenotype as well as to a high incidence of mammary tumors that are primarily ER(+). In this model, the AIB1 transgene is responsible for continued activation of the insulin-like growth factor-I receptor, suggesting a role for the activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway in the premalignant phenotype and tumor development. Here we show that treatment of AIB1 transgenic mice with the mTOR inhibitor RAD001 reverts the premalignant phenotype. Furthermore, treatment of cell lines derived from AIB1-dependent mammary tumors with RAD001 in culture leads to a G(1) cell cycle arrest. Lastly, tumor growth after injection of ER(+) AIB1 tumor cell lines into wild-type animals is inhibited by RAD001 treatment. In this ER(+) model, inhibition of tumor growth by RAD001 was significantly better than inhibition by the antiestrogen 4-hydroxytamoxifen alone, whereas a combination of both RAD001 and 4-hydroxytamoxifen was most effective. Based on these results, we propose that the combination of mTOR inhibition and ER-targeted endocrine therapy may improve the outcome of the subset of ER(+) breast cancers overexpressing AIB1. These studies provide preclinical support for the clinical development of RAD001 and suggest that AIB1 may be a predictive factor of RAD001 response.     

Visual outcomes in children with neurofibromatosis type 1-associated optic pathway glioma following chemotherapy: a multicenter retrospective analysis

Optic pathway gliomas (OPGs) occur in 15%-20% of children with neurofibromatosis type 1 (NF1); up to half become symptomatic. There is little information regarding ophthalmologic outcomes after chemotherapy. A retrospective multicenter study was undertaken to evaluate visual outcomes following chemotherapy for NF1-associated OPG, to identify risks for visual loss, and to ascertain indications for treatment. Subjects included children undergoing initial treatment for OPGs with chemotherapy between January 1997 and December 2007. Of 115 subjects, visual acuity (VA) decline and tumor progression were the primary reasons to initiate treatment, although there were significant differences in the pattern of indications cited among the institutions. Eighty-eight subjects and 168 eyes were evaluable for VA outcome. At completion of chemotherapy, VA improved (32% of subjects), remained stable (40%), or declined (28%). Tumor location was the most consistent prognostic factor for poor VA outcome. There was poor correlation between radiographic and VA outcomes. Although visual outcomes for NF1-associated OPG are not optimal, approximately one-third of children regain some vision with treatment. Since radiographic outcomes do not predict visual outcomes, their use as the primary measure of treatment success is in question. The lack of consensus regarding the indications for treatment underlines the need for better standardization of care. Future clinical trials for OPG require standardized visual assessment methods and clear definitions of visual outcomes.