Farnesylthiosalicylic acid blocks mammalian target of rapamycin signaling in breast cancer cells

Estradiol (E2) stimulates proliferation of hormone-dependent breast cancer and exerts downstream effects on growth factors and their receptors. Key among the pathways' mediating growth factor action is the MAP kinase signaling cascade and the PI-3 kinase pathway with its downstream effector mTOR. We postulated that farnesylthiosalicylic acid (FTS), a novel anti-Ras drug, could effectively inhibit hormone-dependent breast cancer because Ras activates both the MAP kinase and the PI3 kinase pathways. Wild-type MCF-7 cells and a long-term estrogen-deprived subline (LTED) were used to examine the effect of FTS on cell growth and on several biochemical parameters. FTS inhibited growth of both cell lines by reducing proliferation and inducing apoptosis. These effects correlated best with blockade of phosphorylation of PHAS-I and p70 S6 kinase, 2 downstream effectors of mTOR. We observed only minimal inhibition of Akt, an effector upstream of mTOR. Taken together, these findings demonstrate a novel effect of FTS to inhibit mTOR signaling and also suggest that mTOR has a key role in breast cancer cell proliferation. Unexpectedly, only minimal inhibition of MAP kinase occurred in response to FTS at concentrations that markedly reduced cell growth. These later data provide support for the concept that FTS exerts its effects predominantly by blocking mTOR and to a lesser effect by inhibition of MAP kinase in breast cancer cells.     

Mechanistic / mammalian target protein of rapamycin signaling in hematopoietic stem cells and leukemia

Mechanistic/mammalian target protein of rapamycin (mTOR) is an evolutionarily conserved kinase that plays a critical role in sensing and responding to environmental determinants such as nutrient availability, energy sufficiency, stress, and growth factor concentration. mTOR participates in two complexes, designated mTOR complex 1 (mTORC1) and 2 (mTORC2), both of which phosphorylate multiple substrates. Recent studies have revealed that the fine‐tuning activity of mTOR complexes contributes to both maintenance of hematopoietic stem cells (HSCs) and suppression of leukemogenesis. Dysregulation of mTORC1 activity results in impaired HSC homeostasis. Abnormalities of mTOR signaling are observed in many patients with leukemia and genetic studies clearly show that the leukemogenesis associated with Pten deficiency involves both mTORC1 and mTORC2. Although the several mTOR inhibitors have been developed for cancer therapy, effectiveness of the inhibitors for eradication of leukemia stem cells (LSCs) is unknown. Advances in understanding of how mTOR signaling is involved in mechanisms of normal HSC and LSC homeostasis may lead to novel therapeutic approaches that can successfully eradicate leukemia.     

Suppression of Peutz-Jeghers polyposis by targeting mammalian target of rapamycin signaling.

PURPOSE: Peutz-Jeghers syndrome (PJS) is a unique disorder characterized by the development of hamartomas in the gastrointestinal tract as well as increased risks for variety of malignancies. Germ-line mutations of LKB1 cause PJS. We have generated Lkb1+/- mice, which model human PJS. Rapamycin and its analogues are promising preventive and therapeutic agents that specifically inhibit signaling from mammalian target of rapamycin (mTOR). Hyperactivation of mTOR signaling has been associated with PJS. The objective of the study is to investigate the efficacy of mTOR inhibition in suppressing Peutz-Jeghers polyposis in Lkb1+/- mice. EXPERIMENTAL DESIGN: We initiated a trial of rapamycin in Lkb1+/- mice at 9 months of age (after the onset of polyposis) at the dose of 2 mg/kg/d for a 2-month period. We assessed the efficacy of rapamycin by measuring polyp sizes and tumor burden. To examine the effect of rapamycin on mTOR signaling, phosphorylation levels of S6 were evaluated by immunostaining. RESULTS: We observed a significant decrease in mean tumor burden (Student's t test, P = 0.023) as well as total tumor burden in rapamycin-treated group compared with control group. Comparison of the polyp size observed in both rapamycin-treated and control groups showed that rapamycin efficiently decreased the tumor burden of large polyps (> 8 mm). This inhibition of rapamycin was associated with a decrease in phosphorylated S6 levels in the polyps. CONCLUSIONS: Rapamycin effectively suppresses Peutz-Jeghers polyposis in a mouse model, suggesting that rapamycin or its analogues may represent a new targeted therapy for the treatment of PJS.     

Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells

The mammalian target of rapamycin (mTOR) plays a central role in regulating the proliferation of malignant glioma cells, and mTOR-specific inhibitors such as rapamycin analogs are considered as promising therapy for malignant gliomas. However, the efficacy of mTOR inhibitors alone in the treatment of patients with malignant gliomas is only modest, potentially because these agents rather than acting as mTOR kinase inhibitors instead interfere with the function of only mTOR/raptor (regulatory-associated protein of mTOR) complex and thus do not perturb all mTOR functions. The purpose of this study was to determine whether global inhibition of the mTOR molecule enhances the antitumor effect of rapamycin on malignant glioma cells. We showed that rapamycin induced autophagy and that inhibition of autophagy by small interfering RNA (siRNA) directed against autophagy-related gene Beclin 1 attenuated the cytotoxicity of rapamycin in rapamycin-sensitive tumor cells, indicating that the autophagy was a primary mediator of rapamycin's antitumor effect rather than a protective response. Exogenous expression of an mTOR mutant interfering with its kinase activity markedly enhanced the incidence of rapamycin-induced autophagy. Moreover, silencing of mTOR with siRNA augmented the inhibitory effect of rapamycin on tumor cell viability by stimulating autophagy. Importantly, not only rapamycin-sensitive malignant glioma cells with PTEN mutations but also rapamycin-resistant malignant glioma cells with wild-type PTEN were sensitized to rapamycin by mTOR siRNA. These results indicate that rapamycin-induced autophagy is one of the agent's antitumor effects and that silencing or inhibiting mTOR kinase activity could enhance the effectiveness of rapamycin.     

Metastatic role of mammalian target of rapamycin signaling activation by chemoradiotherapy in advanced rectal cancer

Postoperative distant metastasis dramatically affects rectal cancer patients who have undergone neoadjuvant chemoradiotherapy (NACRT). Here, we clarified the association between NACRT‐mediated mammalian target of rapamycin (mTOR) signaling pathway activation and rectal cancer metastatic potential. We performed immunohistochemistry for phosphorylated mTOR (p‐mTOR) and phosphorylated S6 (p‐S6) on surgical specimen blocks from 98 rectal cancer patients after NACRT (cohort 1) and 80 colorectal cancer patients without NACRT (cohort 2). In addition, we investigated the association between mTOR pathway activity, affected by irradiation, and the migration ability of colorectal cancer cells in vitro. Based on the results of the clinical study, p‐mTOR was significantly overexpressed in cohort 1 (with NACRT) as compared to levels in cohort 2 (without NACRT) (P < .001). High p‐mTOR and p‐S6 levels correlated with the development of distant metastasis only in cohort 1. Specifically, high p‐S6 expression (HR 4.51, P = .002) and high pathological T‐stage (HR 3.73, P = .020) after NACRT were independent predictors of the development of distant metastasis. In vitro, p‐S6 levels and migration ability increased after irradiation in SW480 cells (TP53 mutation‐type) but decreased in LoVo cells (TP53 wild‐type), suggesting that irradiation modulates mTOR signaling and migration through cell type‐dependent mechanisms. We next assessed the expression level of p53 by immunostaining in cohort 1 and demonstrated that p‐S6 was overexpressed in samples with high p53 expression as compared to levels in samples with low p53 expression (P = .008). In conclusion, p‐S6 levels after NACRT correlate with postoperative distant metastasis in rectal cancer patients, suggesting that chemoradiotherapy might modulate the mTOR signaling pathway, promoting metastasis.     

Critical and diverse involvement of Akt/mammalian target of rapamycin signaling in human lung carcinomas

BACKGROUND: Aberrant signaling cascades emanating from epidermal growth factor receptor (EGFR) are involved in the complex network of oncogenic signaling in lung carcinomas. One representative cascade is the phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. METHODS: The authors investigated the involvement of mTOR in the pathobiologic profiles of 150 specimens of lung carcinoma by immunohistochemistry and immunoblotting in correlation with the upstream and downstream proteins Akt and p70S6‐kinase (S6K), respectively. RESULTS: Immunohistochemistry revealed Akt activation in 44% of tumors and mTOR expression in 68.7% of tumors, and the preponderance of activation was observed in adenocarcinoma (AC) (100%). Phosphorylated mTOR (p‐mTOR) was observed in 53.3% of tumors and had the highest frequency in AC (89.7%). In AC, the frequency of p‐mTOR staining was higher in the well differentiated subtype, in particular, in the acinar structure. However, little correlation was observed between the activation of mTOR and Akt, except in the 5 AC specimens that harbored an EGFR gene mutation, which exhibited constitutive activation of both Akt and mTOR. Conversely, in squamous cell carcinomas, mTOR activation was associated with a significantly higher frequency of lymph node metastasis. CONCLUSIONS: The results of this study suggested the dual functions of mTOR. First, mTOR may function not only in the proliferation of tumor cells as an effector molecule downstream of EGFR but also possibly in the morphogenesis of AC. Second, the activation of mTOR may play a key role in metastasis in squamous cell carcinoma. Overall, the current results demonstrated the potential for the application of rapamycin, an mTOR inhibitor, as an additional novel component of chemotherapy for a defined subset of patients with lung carcinoma. Cancer 2009. © 2008 American Cancer Society.     

Common toxicities of mammalian target of rapamycin inhibitors

The toxicities of newer targeted therapies are different from those seen with the traditional chemotherapy. Mammalian target of rapamycin (mTOR) inhibitors are evolving into an important class of drugs in oncology, and this class of drugs presents with a variety of different toxicities. Although similar to the toxicities seen in transplantation, these rapamycin analogs have unique side effects when compared to traditional chemotherapy agents. While most of the toxicities are mild, few can be severe and require routine monitoring. Mucositis and rash are the most common side effects. The metabolic toxicities, hyperglycemia, hyperlipidemia, and hypophosphatemia are different from the side effects traditionally seen with chemotherapy. This review will focus on the common toxicities seen with the mTOR inhibitors.     

Clinical development of mammalian target of rapamycin inhibitors

Rapamycin and CCI-779 have significant in vitro and in vivo anti-proliferative activity against a broad range of human tumor cell lines, justifying the clinical evaluation of this class of agent in cancer patients. Preliminary results from phase I studies of CCI-779 suggest that the agent is well tolerated and has anti-tumor activity. The challenge to investigators is to efficiently determine what role this class of agent will play in the treatment of cancer patients.     

A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells

The microbially derived antiproliferative agent rapamycin inhibits cell growth by interfering with the signaling functions of the mammalian target of rapamycin (mTOR). In this study, we demonstrate that interleukin-3 stimulation induces a wortmannin-sensitive increase in mTOR kinase activity in a myeloid progenitor cell line. The involvement of phosphoinositide 3'-kinase (PI3K) in the regulation of mTOR activity was further suggested by findings that mTOR was phosphorylated in vitro and in vivo by the PI3K-regulated protein kinase, AKT/PKB. Although AKT phosphorylated mTOR at two COOH-terminal sites (Thr2446 and Ser2448) in vitro, Ser2448 was the major phosphorylation site in insulin-stimulated or -activated AKT-expressing human embryonic kidney cells. Transient transfection assays with mTOR mutants bearing Ala substitutions at Ser2448 and/or Thr2446 indicated that AKT-dependent mTOR phosphorylation was not essential for either PHAS-I phosphorylation or p70S6K activation in HEK cells. However, a deletion of amino acids 2430-2450 in mTOR, which includes the potential AKT phosphorylation sites, significantly increased both the basal protein kinase activity and in vivo signaling functions of mTOR. These results demonstrate that mTOR is a direct target of the PI3K-AKT signaling pathway in mitogen-stimulated cells, and that the identified AKT phosphorylation sites are nested within a "repressor domain" that negatively regulates the catalytic activity of mTOR. Furthermore, the activation status of the PI3K-AKT pathway in cancer cells may be an important determinant of cellular sensitivity to the cytostatic effect of rapamycin.     

Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells.

PURPOSE: The serine-threonine kinase mammalian target of rapamycin has emerged as a potential target for cancer therapy. Rapamycin and rapamycin analogs are undergoing clinical trials and have induced clinical responses in a subgroup of patients. Rapamycin has also been reported to enhance the efficacy of several cytotoxic agents. The aim of this study was to determine the nature of the interactions between rapamycin and chemotherapeutic agents used as first- and second-line agents against breast cancer. EXPERIMENTAL DESIGN: We performed a multiple drug effect/combination index isobologram analysis in cells sensitive and resistant to rapamycin alone in vitro, and we evaluated the in vivo efficacy of combination therapy in a rapamycin-sensitive model. RESULTS: In vitro, synergistic interactions were observed in combinations with paclitaxel, carboplatin, and vinorelbine. Additive effects were observed in combinations with doxorubicin and gemcitabine. Rapamycin dramatically enhanced paclitaxel- and carboplatin-induced apoptosis. This effect was sequence dependent and mediated at least partly through caspase activation. Furthermore, rapamycin enhanced chemosensitivity to paclitaxel and carboplatin in HER2/neu-overexpressing cells, suggesting a potential approach to these poorly behaving tumors. Cell lines that are resistant to the growth-inhibitory effect of rapamycin were also resistant to rapamycin-mediated chemosensitization. In vivo, rapamycin combined with paclitaxel resulted in a significant reduction in tumor volume compared with either agent alone in rapamycin-sensitive tumors. CONCLUSIONS: Rapamycin potentiates the cytotoxicity of selected chemotherapeutic agents in cell lines sensitive to the effects of rapamycin due to aberrations in the phosphatidylinositol 3'-kinase/Akt pathway, suggesting that combination therapy may be effective in patients selected for aberrations in this pathway.     

Inhibition of the mammalian target of rapamycin (mTOR) by rapamycin increases chemosensitivity of CaSki cells to paclitaxel

Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.     

AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4(+) T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL.     

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.     

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.     

Curcumin inhibits the mammalian target of rapamycin-mediated signaling pathways in cancer cells

Curcumin (diferuloylmethane), a polyphenol natural product of the plant Curcuma longa, is undergoing early clinical trials as a novel anticancer agent. However, the anticancer mechanism of curcumin remains to be elucidated. Here we show that curcumin inhibited growth of rhabdomyosarcoma cells (Rh1 and Rh30) (IC50 = 2-5 microM) and arrested cells in G1 phase of the cell cycle. Curcumin also induced apoptosis and inhibited the basal or type I insulin-like growth factor-induced motility of the cells. At physiological concentrations (2.5 microM), curcumin rapidly inhibited phosphorylation of the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1), in a panel of cell lines (Rh1, Rh30, DU145, MCF-7 and Hela). Curcumin also inhibited phosphorylation of Akt in the cells, but only at high concentrations (>40 microM). The data suggest that curcumin may execute its anticancer activity primarily by blocking mTOR-mediated signaling pathways in the tumor cells.     

Current status of mammalian target of rapamycin inhibitors in lung cancer

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a critical role in the control of cell growth and proliferation. The mTOR integrates mitogenic signals and intracellular nutrient levels to activate eukaryotic initiation factor 4E-binding protein-1 and the 40S ribosomal protein S6 kinase, which controls protein translation and cell cycle progression. Abnormal activation of signaling pathways proximal and distal to mTOR appears to occur frequently in human cancer, making mTOR an attractive target for anticancer drug development. Inhibitors of mTOR, including the naturally occurring inhibitor rapamycin as well as newer agents against this target, are currently in clinical development for cancer treatment. In preclinical studies, these agents have shown significant effects against a variety of preclinical models of cancer. In early clinical studies, mTOR inhibitors have been well tolerated, resulted in plasma levels able to inhibit mTOR in normal and tumor tissues of patients treated with the drug, and resulted in antitumor responses in patients with different tumor types including lung cancer. These agents are now in late phases of clinical development. As with other targeted agents, the key issues in the future will be to elucidate the molecular factors predicting a favorable response to the drugs as well as the rational integration with other targeted agents with activity in lung cancer, such as inhibitors of the epidermal growth factor receptor tyrosine kinase.     

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.