Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition

The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. Rapamycin and its derivatives that specifically inhibit mTOR are now being actively evaluated in clinical trials. Recently, the inhibition of mTOR has been shown to reverse Akt-dependent prostate intraepithelial neoplasia. However, many cancer cells are resistant to rapamycin and its derivatives. The mechanism of this resistance remains a subject of major therapeutic significance. Here we report that the inhibition of mTOR by rapamycin triggers the activation of two survival signaling pathways that may contribute to drug resistance. Treatment of human lung cancer cells with rapamycin suppressed the phosphorylation of p70S6 kinase and 4E-BP1, indicating an inhibition of mTOR signaling. Paradoxically, rapamycin also concurrently increased the phosphorylation of both Akt and eIF4E. The rapamycin-induced phosphorylation of Akt and eIF4E was suppressed by the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002, suggesting the requirement of PI3K in this process. The activated Akt and eIF4E seem to attenuate rapamycin's growth-inhibitory effects, serving as a negative feedback mechanism. In support of this model, rapamycin combined with LY294002 exhibited enhanced inhibitory effects on the growth and colony formation of cancer cells. Thus, our study provides a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor.     

Analysis of surgical margins with the molecular marker eIF4E: a prognostic factor in patients with head and neck cancer.

PURPOSE: Complete excision of cancer is guided by histologic assessment of surgical margins. Molecular markers may be more sensitive in identifying malignant cells. eIF4E, a eukaryotic protein synthesis initiation factor, is found elevated in all head and neck squamous cell cancers (HNSCC). In a preliminary study using Western blots and a retrospective study using immunohistochemistry, eIF4E elevation in histologically tumor-free surgical margins correlated with a higher local-regional recurrence. We wanted to confirm this hypothesis in a prospective study. PATIENTS AND METHODS: Immunohistochemical analysis of surgical margins and tumors with an antibody to eIF4E was performed on all newly diagnosed HNSCC patients who underwent surgical resection for their disease between January 1996 and December 1997. RESULTS: All 65 patients had elevated levels of eIF4E in the tumors. Thirty-six patients (55%) had elevated eIF4E in histologically tumor-free margins, and 20 of these patients (56%) have had local-regional recu rrences. Twenty-nine patients (45%) had no elevation of eIF4E in the margins, and only two of these patients (6.9%) have had recurrences. Cox regression analysis showed that elevated eIF4E in the margins was an independent prognostic factor (P =.009) for recurrence. The Kaplan-Meier curves for the probability of nonrecurrence were significantly different for positive and negative eIF4E margins (P =. 0001, log-rank test). CONCLUSION: In histologically tumor-free surgical margins, elevated levels of eIF4E predict a significantly increased risk of recurrence. Elevated levels of eIF4E in tumor margins may identify patients who could benefit from additional therapy.     

Phosphoprotein pathway mapping: Akt/mammalian target of rapamycin activation is negatively associated with childhood rhabdomyosarcoma survival

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains approximately 60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser(473) (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr(37/46) (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser(1108) (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr(389) (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy.     

Dissecting the Akt/mammalian target of rapamycin signaling network: emerging results from the head and neck cancer tissue array initiative.

PURPOSE: As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from head and neck squamous cell carcinoma (HNSCC) patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high-density HNSCC tissue microarray, with a high representation of oral squamous cell carcinoma. EXPERIMENTAL DESIGN: These tissue arrays were constructed by acquiring cylindrical biopsies from multiple individual tumor tissues and transferring them into tissue microarray blocks. From a total of 1,300 cases, 547 cores, including controls, were selected and used to build the array. RESULTS: Emerging information by the use of phosphospecific antibodies detecting the activated state of signaling molecules indicates that the Akt-mammalian target of rapamycin (mTOR) pathway is frequently activated in HNSCC, but independently from the activation of epidermal growth factor receptor or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of epidermal growth factor receptor activation. Furthermore, we have also identified a small subgroup of patients in which the mTOR pathway is activated but not Akt, suggesting the existence of an Akt-independent signaling route stimulating mTOR. CONCLUSIONS: These findings provide important information about the nature of the dysregulated signaling networks in HNSCC and may also provide the rationale for the future development of novel mechanism-based therapies for HNSCC patients.     

The Akt/mammalian target of rapamycin pathway is activated and associated with adverse prognosis in soft tissue leiomyosarcomas

BACKGROUND:

The Akt/mammalian target of rapamycin (mTOR) pathway mediates cell survival and proliferation and contributes to tumor progression. Soft tissue leiomyosarcoma continues to show poor prognosis, and little is known about its mechanisms of tumor progression. Here the authors investigated the significance of activation of the Akt/mTOR pathway in soft tissue leiomyosarcomas.

METHODS:

The phosphorylation status of Akt, mTOR, S6, and the eukaryotic translation initiation factor 4E‐binding protein (4E‐BP1) and the protein expression of phosphatase and tensin homologue (PTEN) were assessed by immunohistochemistry in 145 formalin‐fixed paraffin‐embedded samples of soft tissue leiomyosarcoma including 129 primary tumors. The expression of phosphorylated Akt and mTOR in comparison with their total forms was assessed by Western blot analysis in 13 frozen samples, which were paired with normal tissue samples. Moreover, 39 frozen tumor samples were analyzed for PIK3CA and AKT1 gene mutation.

RESULTS:

Immunohistochemically, phosphorylated forms of Akt, mTOR, S6, and 4E‐BP1 were positive in 78.3%, 72.6%, 74.5%, and 70.5% of the samples, respectively. These results were correlated with each other, and associated with higher mitotic activity and adverse prognosis. Decreased expression of PTEN was recognized in only 19.7% and had no statistically significant correlation with Akt or other molecules. Immunoblotting showed a high degree of Akt and mTOR phosphorylation in tumor samples compared with that in non‐neoplastic tissue. Mutational analysis failed to reveal any PIK3CA or AKT1 mutations around the hot spots.

CONCLUSIONS:

The Akt/mTOR pathway was activated in most cases of soft tissue leiomyosarcoma and associated with worse clinical behavior and aggressive pathological findings. Cancer 2011;. © 2011 American Cancer Society.     

Nitric oxide in physiologic concentrations targets the translational machinery to increase the proliferation of human breast cancer cells: involvement of mammalian target of rapamycin/eIF4E pathway

Nitric oxide (NO) in nanomolar (nmol/L) concentrations is consistently detected in tumor microenvironment and has been found to promote tumorigenesis. The mechanism by which NO enhances tumor progression is largely unknown. In this study, we investigated the possible mechanisms and identified cellular targets by which NO increases proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7. DETA-NONOate, a long acting NO donor, with a half-life of 20 h, was used. We found that NO (nmol/L) dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 and also increased cell proliferation. NO specifically increased the translation of cyclin D1 and ornithine decarboxylase (ODC) without altering their mRNA levels or half-lives. Critical components in the translational machinery, such as phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were up-regulated following NO treatment, and inhibition of mTOR with rapamycin attenuated NO induced increase of cyclin D1 and ODC. Activation of translational machinery was mediated by NO-induced up-regulation of the Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK (Raf/MEK/ERK) and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt signaling pathways. Up-regulation of the Raf/MEK/ERK and PI-3 kinase/Akt pathways by NO was found to be mediated by activation of Ras, which was cyclic guanosine 3',5'-monophosphate independent. Furthermore, inactivation of Ras by farnesyl transferase inhibitor or K-Ras small interfering RNA attenuated NO-induced increase in proliferation signaling and cyclin D1 and ODC translation, further confirming the involvement of Ras activation during NO-induced cell proliferation.     

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.     

Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers.

The Akt/mammalian target of rapamycin (mTOR)/4E-BP1 pathway is considered to be a central regulator of protein synthesis, involving the regulation of cell proliferation, differentiation, and survival. The inhibitors of mTOR as anticancer reagents are undergoing active evaluation in various malignancies including breast cancer. However, the activation status of the Akt/mTOR/4E-BP1 pathway and its potential roles in breast cancers remain unknown. Thus, we examined 165 invasive breast cancers with specific antibodies for the phosphorylation of Akt, mTOR, and 4E-BP1 by immunohistochemistry and compared them with normal breast epithelium, fibroadenoma, intraductal hyperplasia, and ductal carcinoma in situ. We discovered that the phosphorylation of Akt, mTOR, and 4E-BP1 increased progressively from normal breast epithelium to hyperplasia and abnormal hyperplasia to tumor invasion. Phosphorylated Akt, mTOR, and 4E-BP1 were positively associated with ErbB2 overexpression. Survival analysis showed that phosphorylation of each of these three markers was associated with poor disease-free survival independently. In vitro, we further confirmed the causal relationship between ErbB2 overexpression and mTOR activation, which was associated with enhanced invasive ability and sensitivity to a mTOR inhibitor, rapamycin. Our results, for the first time, demonstrate the following: (a) high levels of phosphorylation of Akt, mTOR, and 4E-BP1 in breast cancers, indicating activation of the Akt/mTOR/4E-BP1 pathway in breast cancer development and progression; (b) a link between ErbB2 and the Akt/mTOR/4E-BP1 pathway in breast cancers in vitro and in vivo, indicating the possible role of Akt/mTOR activation in ErbB2-mediated breast cancer progression; and (c) a potential role for this pathway in predicting the prognosis of patients with breast cancer, especially those treated with mTOR inhibitors.     

The Akt/mammalian target of rapamycin signal transduction pathway is activated in high-risk myelodysplastic syndromes and influences cell survival and proliferation

The Akt/mammalian target of rapamycin (mTOR) signaling pathway is important for both cell growth and survival. In particular, an impaired regulation of the Akt/mTOR axis has been strongly implicated in mechanisms related to neoplastic transformation, through enhancement of cell proliferation and survival. Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic stem cell disorders characterized by ineffective hematopoiesis and by a high risk of evolution into acute myelogenous leukemia (AML). The pathogenesis of the MDS evolution into AML is still unclear, although some recent studies indicate that aberrant activation of survival signaling pathways could be involved. In this investigation, done by means of immunofluorescent staining, we report an activation of the Akt/mTOR pathway in high-risk MDS patients. Interestingly, not only mTOR was activated but also its downstream targets, 4E-binding protein 1 and p70 ribosomal S6 kinase. Treatment with the selective mTOR inhibitor, rapamycin, significantly increased apoptotic cell death of CD33(+) (but not CD33(-)) cells from high-risk MDS patients. Rapamycin was ineffective in cells from healthy donors or low-risk MDS. Moreover, incubation of high-risk MDS patient CD34(+) cells with rapamycin decreased the in vitro clonogenic capability of these cells. In contrast, the phosphoinositide 3-kinase inhibitor, LY294002, did not significantly affect the clonogenic activity of high-risk MDS cells. Taken together, our results indicate that the Akt/mTOR pathway is critical for cell survival and proliferation in high-risk MDS patients. Therefore, this signaling network could become an interesting therapeutic target for treating more advanced MDS cases.     

Enhancing mammalian target of rapamycin (mTOR)-targeted cancer therapy by preventing mTOR/raptor inhibition-initiated, mTOR/rictor-independent Akt activation

It has been shown that mammalian target of rapamycin (mTOR) inhibitors activate Akt while inhibiting mTOR signaling. However, the underlying mechanisms and the effect of the Akt activation on mTOR-targeted cancer therapy are unclear. The present work focused on addressing the role of mTOR/rictor in mTOR inhibitor-induced Akt activation and the effect of sustained Akt activation on mTOR-targeted cancer therapy. Thus, we have shown that mTOR inhibitors increase Akt phosphorylation through a mechanism independent of mTOR/rictor because the assembly of mTOR/rictor was inhibited by mTOR inhibitors and the silencing of rictor did not abrogate mTOR inhibitor-induced Akt activation. Moreover, Akt activation during mTOR inhibition is tightly associated with development of cell resistance to mTOR inhibitors. Accordingly, cotargeting mTOR and phosphatidylinositol 3-kinase/Akt signaling prevents mTOR inhibition-initiated Akt activation and enhances antitumor effects both in cell cultures and in animal xenograft models, suggesting an effective cancer therapeutic strategy. Collectively, we conclude that inhibition of the mTOR/raptor complex initiates Akt activation independent of mTOR/rictor. Consequently, the sustained Akt activation during mTOR inhibition will counteract the anticancer efficacy of the mTOR inhibitors.     

Clinical significance of molecular alterations in histologically negative surgical margins of head and neck cancer patients

The development of locoregional recurrence is the main reason for treatment failure in head and neck squamous cell carcinomas (HNSCC) and the remaining of tumor cells in surgical margins is associated with recurrence. Surgical margins are considered negative based on histologic assessment of the pathological specimen. However, this method lacks sensitivity in identifying cells that already started malignant transformation but have not yet developed a pathologic phenotype. We investigated the usefulness of assessing the expression of PTHLH, EPCAM, MMP9, LGALS1 and MET for the detection of molecular alterations in histologically negative surgical margins and determine the correlation of these tumor-related alterations with clinical and prognostic parameters. Differential gene expression was determined by quantitative RT-PCR analyses in normal mucosa, HNSCC and negative margin samples. Thirty-eight percent of the histologically negative surgical margins examined were margin-positive for overexpression of at least one of the genes evaluated. Moreover, MMP9 and PTHLH overexpression in the surgical margins was associated with the development of second primary tumors (p=0.002) and lower rates of local control (log rank test p=0.022; HR=4.186; p=0.035), respectively. These findings demonstrate that the overexpression of tumor-related genes in histologically negative surgical margins is a frequent event. The use of qRT-PCR may be an useful tool in detecting actually negative HNSCC surgical margins and the overexpression of specific genes in these margins could be helpful in the identification of patients with a higher risk of developing second primary tumors and local recurrences, thus aiding the surgeon in the delineation of the HNSCC resection extent and helping in the planning of adjuvant therapy.     

Estrogen-induced activation of mammalian target of rapamycin is mediated via tuberin and the small GTPase Ras homologue enriched in brain

Inhibitors of the mammalian target of rapamycin (mTOR) are currently in clinical trials for the treatment of breast cancer. The mechanisms through which mTOR are activated in breast cancer and the relationship of mTOR activation to steroid hormones, such as estrogen, that are known to influence breast cancer pathogenesis, are not yet understood. Using MCF-7 cells as a model, we found that 17-beta estradiol (E(2)) rapidly increased the phosphorylation of downstream targets of mTOR: p70 ribosomal protein S6 kinase, ribosomal protein S6, and eukaryotic initiation factor 4E-binding protein 1. The phosphoinositide-3-kinase inhibitor, wortmannin, and the mTOR inhibitor, rapamycin, blocked E(2)-induced activation of p70 ribosomal protein S6 kinase. We hypothesized that tuberin and the small GTPase Ras homologue enriched in brain (Rheb), regulators of the mTOR pathway, mediate E(2)-induced activation of mTOR. Consistent with this hypothesis, E(2) rapidly (within 5 minutes) stimulated tuberin phosphorylation at T1462, a site at which Akt phosphorylates and inactivates tuberin. E(2) also rapidly decreased the inactive, GDP-bound form of Rheb. Finally, we found that small interfering RNA down-regulation of endogenous Rheb blocked the E(2)-stimulated proliferation of MCF-7 cells, demonstrating that Rheb is a key determinant of E(2)-dependent cell growth. Taken together, these data reveal that the TSC/Rheb/mTOR pathway plays a critical role in the regulation of E(2)-induced proliferation, and highlight Rheb as a novel molecular target for breast cancer therapy.     

Molecular diagnosis of surgical margins and local recurrence in head and neck cancer patients: a prospective study.

PURPOSE: Approximately 10-30% of surgically treated head and neck cancer patients develop local recurrences while the resection margins are histologically tumor free. These recurrences may arise from cancer cells left behind but not detected by the pathologist, or they may develop from precursor lesions adjacent to the tumor that were not completely resected. We have investigated whether TP53-mutated DNA in the surgical margins is suitable to identify patients with head and neck squamous cell carcinoma at risk for local and locoregional recurrence. EXPERIMENTAL DESIGN: In a prospective cohort study of 76 patients with histologically tumor-free margins, the presence of TP53-mutated DNA was determined in the surgical margins using the phage plaque assay and correlated to clinical outcome. Immunostaining of the molecular-positive margins for mutated p53 protein was used to identify whether unresected precursor lesions or residual tumor cells were left behind. RESULTS: The absence of TP53-mutated DNA in surgical margins was significantly associated with remaining free of local and locoregional recurrence (P = 0.027 and P = 0.028, respectively). Moreover, the presence of TP53-mutated DNA in the surgical margins was an independent prognosticator for locoregional recurrence (relative risk = 7.1; P = 0.021; 95% confidence interval, 0.9-56). In 20% of the cases, the presence of TP53-mutated DNA in the surgical margins was found to be related to the presence of tumor-related precursor lesions. CONCLUSIONS: This study shows the value of TP53-mutated DNA as a molecular marker to predict locally recurrent head and neck squamous cell carcinoma. The observation that all patients who were negative for TP53-mutated DNA in the surgical margins remained free of local recurrence raises hope that molecular analysis of histologically tumor-free surgical margins can be exploited to decide on postoperative radiotherapy. Furthermore, our data provide evidence that local recurrences originate mainly from tumor cells left behind but also originate, in part, from unresected precursor lesions.     

Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma.

PURPOSE: Statins are pharmacologic inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase with potent regulatory effects on cholesterol biosynthesis in vitro and in vivo. There is accumulating evidence that, beyond their cholesterol-lowering properties, statins inhibit cell proliferation and promote apoptosis of malignant cells in vitro, but the mechanisms by which they generate such responses remain to be defined. EXPERIMENTAL DESIGN: Combinations of experimental approaches were used, including immunoblotting and cell proliferation and apoptosis assays. RESULTS: We provide evidence that fluvastatin is a potent inducer of apoptosis and suppresses proliferation of renal cell carcinoma (RCC) cells in vitro. Such effects are mediated by direct targeting of the Akt/mammalian target of rapamycin (mTOR) pathway, as evidenced by the suppression of phosphorylation/activation of Akt, resulting in inhibition of its downstream effectors, mTOR and p70 S6 kinase. In addition, fluvastatin blocks the mTOR-dependent phosphorylation/deactivation of the translational repressor eukaryotic initiation factor 4E (eIF4E)-binding protein, leading to the formation of eIF4E-binding protein-eIF4E complexes that suppress initiation of cap-dependent mRNA translation. Importantly, inhibition of p70 S6 kinase activity by fluvastatin results in the up-regulation of expression of programmed cell death 4 (PDCD4), a tumor suppressor protein with inhibitory effects on the translation initiation factor eIF4A, suggesting a mechanism for the generation of antitumor responses. CONCLUSIONS: Altogether, our findings establish that fluvastatin exhibits potent anti-RCC activities via inhibitory effects on the Akt/mTOR pathway and raise the possibility that combinations of statins and Akt inhibitors may be of future therapeutic value in the treatment of RCC.     

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.     

Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis

Mammalian target of rapamycin complex (mTORC) regulates a variety of cellular responses including proliferation, growth, differentiation and cell migration. In this study, we show that mammalian target of rapamycin complex 2 (mTORC2) regulates invasive cancer cell migration through selective activation of Akt1. Insulin-like growth factor-1 (IGF-1)-induced SKOV-3 cell migration was completely abolished by phosphatidylinositol 3-kinase (PI3K) (LY294002, 10?μM) or Akt inhibitors (SH-5, 50?μM), whereas inhibition of extracellular-regulated kinase by an ERK inhibitor (PD98059, 10?μM) or inhibition of mammalian target of rapamycin complex 1 (mTORC1) by an mTORC1 inhibitor (Rapamycin, 100?nM) did not affect IGF-1-induced SKOV-3 cell migration. Inactivation of mTORC2 by silencing Rapamycin-insensitive companion of mTOR (Rictor), abolished IGF-1-induced SKOV-3 cell migration as well as activation of Akt. However, inactivation of mTORC1 by silencing of Raptor had no effect. Silencing of Akt1 but not Akt2 attenuated IGF-1-induced SKOV-3 cell migration. Rictor was preferentially associated with Akt1 rather than Akt2, and over-expression of Rictor facilitated IGF-1-induced Akt1 activation. Expression of PIP3-dependent Rac exchanger1 (P-Rex1), a Rac guanosine exchange factor and a component of the mTOR complex, strongly stimulated activation of Akt1. Furthermore, knockdown of P-Rex1 attenuated Akt activation as well as IGF-1-induced SKOV-3 cell migration. Silencing of Akt1 or P-Rex1 abolished IGF-1-induced SKOV-3 cell invasion. Finally, silencing of Akt1 blocked in vivo metastasis, whereas silencing of Akt2 did not. Given these results, we suggest that selective activation of Akt1 through mTORC2 and P-Rex1 regulates cancer cell migration, invasion and metastasis.     

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.     

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.     

High expression levels of centromere protein A plus upregulation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway affect chemotherapy response and prognosis in patients with breast cancer

Centromere proteins (CENPs) are involved in mitosis, and CENP gene expression levels are associated with chemotherapy responses in patients with breast cancer. The present study aimed to examine the roles and underlying mechanisms of the effects of CENP genes on chemotherapy responses and breast cancer prognosis. Using data obtained from the Gene Expression Omnibus (GEO) database, correlation and Cox multivariate regression analyses were used to determine the CENP genes associated with chemotherapy responses and survival in patients with breast cancer. Weighted gene co-expression network and correlation analyses were used to determine the gene modules co-expressed with the identified genes and the differential expression of gene modules associated with the pathological complete response (PCR) and residual disease (RD) subgroups. CENPA, CENPE, CENPF, CENPI, CENPJ and CENPN were associated with a high nuclear grade and low estrogen and progesterone receptor expression levels. In addition, CENPA, CENPB, CENPC and CENPO were independent factors affecting the distant relapse-free survival (DRFS) rates in patients with breast cancer. Patients with high expression levels of CENPA or CENPO exhibited poor prognoses, whereas those with high expression levels of CENPB or CENPC presented with favorable prognoses. For validation between databases, the Cancer Genome Atlas (TCGA) database analysis also revealed that CENPA, CENPB and CENPO exerted similar effects on overall survival. However, according to the multivariate analyses, only CENPA was an independent risk factor associated with DRFS in GEO database. In addition, in the RD subgroup, patients with higher CENPA expression levels had a worse prognosis compared with those with lower CENPA expression levels. Among patients with high expression levels of CENPA, the PI3K/Akt/mTOR pathway was more likely to be activated in the RD compared with the PCR subgroup. The same trend was observed in TCGA data. These results suggested that high CENPA expression levels plus upregulation of the PI3K/Akt/mTOR signaling pathway may affect DRFS in patients with breast cancer.