The mammalian target of rapamycin pathway is widely activated without PTEN deletion in renal cell carcinoma metastases

BACKGROUND:

Inhibitors of the mammalian target of rapamycin (mTOR) are emerging as promising therapies for metastatic renal cell carcinoma (RCC). Because rational treatment strategies require understanding the activation status of the underlying signaling pathway being targeted at the desired stage of disease, the authors examined the activation status of different components of the mTOR pathway in RCC metastases and matched primary tumors.

METHODS:

The authors immunostained metastatic RCC samples from 132 patients and a subset of 25 matched primary RCCs with antibodies against phosphatidylinositol 3′‐kinase, PTEN, phospho‐Akt, phospho‐mTOR, and p70S6. PTEN genomic status was assessed by fluorescent in situ hybridization. Marker expression was correlated to clinicopathologic variables and to survival.

RESULTS:

The mTOR pathway showed widespread activation in RCC metastases of various sites with strong correlation between different components of this signaling cascade (P<.0001), but without significant PTEN genomic deletion. Only cytoplasmic phospho‐mTOR showed independent prognostic significance (P = .029) and fidelity between primary RCCs and their matched metastases (P = .004).

CONCLUSIONS:

Activation of various components of the mTOR signaling pathway in metastatic RCC lesions across various tumor histologies, nuclear grades, and metastatic sites suggests the potential for vertical blockade of multiple steps of this pathway. Patient selection may be improved by mTOR immunostaining of primary RCC. Cancer 2011. © 2010 American Cancer Society.     

Targeted inhibition of mammalian target of rapamycin for the treatment of advanced renal cell carcinoma

Clinical trials have validated the importance of mammalian target of rapamycin (mTOR) as a targeted mechanism in the treatment of renal cell carcinoma (RCC). Temsirolimus, an mTOR inhibitor that is approved for treatment of advanced RCC, has demonstrated both overall survival benefits and progression‐free survival benefits versus interferon?α as first‐line treatment for patients with poor prognostic features. Exploratory subset analyses indicated that temsirolimus benefits patients with RCC regardless of tumor histology or nephrectomy status. Everolimus, the second mTOR inhibitor to demonstrate activity in RCC, improved progression‐free survival versus placebo in patients whose disease progressed after treatment with vascular endothelial growth factor receptor tyrosine kinase inhibitors (sunitinib, sorafenib, or both); benefit was observed for all risk groups. Deforolimus also exhibited antitumor activity against RCC in early clinical studies. There is now compelling clinical evidence for the effectiveness of targeting mTOR in the treatment of RCC. Cancer 2009. © 2009 American Cancer Society.     

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.     

Oral adverse events associated with tyrosine kinase and mammalian target of rapamycin inhibitors in renal cell carcinoma: a structured literature review

Background.

Oral adverse events (OAEs) associated with multitargeted tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin inhibitors (mTORIs) are underestimated but frequent and novel presentations of mucosal manifestations. Because optimal antitumor activity requires maintaining the optimal dose, it is essential to avoid unintended treatment delays or interruptions.

Methods.

We review the reported prevalence and appearance of OAEs with TKIs and mTORIs and the current oral assessment tools commonly used in clinical trials. We discuss the correlations between OAEs and hand–foot skin reaction (HFSR) and rash.

Results.

The reported prevalence of oral mucositis/stomatitis of any grade is 4% for pazopanib, 28% for sorafenib, 38% for sunitinib, 41% for temsirolimus, and 44% for everolimus. Oral lesions associated with these agents have been reported to more closely resemble aphthous stomatitis than OM caused by conventional agents. In addition, these agents may result in symptoms such as oral mucosal pain, dysgeusia, and dysphagia, in the absence of clinical lesions. Because of these factors, OAEs secondary to targeted agents may be underreported. In addition, a correlation between OAEs and HFSR was identified.

Conclusions.

OAEs caused by TKIs and mTORIs may represent dose-limiting toxicities, especially considering the fact that even low grades of OAEs may be troubling to the patient. We discuss how these novel AEs can be assessed because current mucositis assessment tools have limitations. Prospective studies investigating the pathogenesis, risk factors, and management of OAEs are needed in order to minimize the impact on patient''s health-related quality of life.     

Phase 1 and pharmacokinetic study of everolimus,a mammalian target of rapamycin inhibitor,in combination with docetaxel for recurrent/refractory nonsmall cell lung cancer

BACKGROUND:

Everolimus is a novel inhibitor of the mammalian target of rapamycin pathway, which is aberrantly activated in nonsmall cell lung cancer (NSCLC). The authors conducted a phase 1 and pharmacokinetic study of everolimus and docetaxel for recurrent NSCLC.

METHODS:

Patients with advanced stage NSCLC and progression after prior platinum‐based chemotherapy were eligible. Sequential cohorts were treated with escalating doses of docetaxel (Day 1) and everolimus (orally daily, Days 1‐19) every 3 weeks. Pharmacokinetic sampling of everolimus and docetaxel was done in Cycle 1. The primary endpoint was determination of the recommended phase 2 doses of the combination.

RESULTS:

Twenty‐four patients were enrolled (median age, 62 years; women, 11; number of prior regimens, 1 [n = 13], 2 [n = 6], ≥3 [n = 5]; Eastern Cooperative Oncology Group performance status, 0 [n = 6], 1 [n = 17]). The dose‐limiting toxicities (DLTs) were fever with grade 3/4 neutropenia, grade 3 fatigue, and grade 3 mucositis. None of the 7 patients treated at the recommended phase 2 dose (docetaxel 60 mg/m² and everolimus 5 mg daily) experienced DLT. Everolimus area under the concentration time curve (AUC) was not different with 60 or 75 mg/m² docetaxel. Mean ± standard deviation AUC‐based accumulation factors for everolimus on Days 8 and 15 were 1.16 ± 0.37 and 1.42 ± 0.42, respectively. Docetaxel Day 1 half‐life was 9.4 ± 3.4 hours. Among 21 patients evaluable, 1 had a partial response, and 10 had disease stabilization.

CONCLUSIONS:

The recommended phase 2 doses of docetaxel and everolimus for combination therapy are 60 mg/m² and 5 mg orally daily, respectively. Promising anticancer activity has been noted. Cancer 2010. © 2010 American Cancer Society.     

New developments in mammalian target of rapamycin inhibitors for the treatment of sarcoma

Although sarcomas account for a small portion of solid malignancies, currently, there are few treatment options for sarcomas, particularly for advanced disease. The mammalian target of rapamycin (mTOR), a serine-threonine protein kinase in the phosphatidylinositol 3-kinase/serine/threonine protein kinase Akt signaling pathway, has an important role in the regulation of protein synthesis, cell proliferation, angiogenesis, and metabolism. Alterations of the mTOR signaling pathway are common in malignancies, including several types of sarcoma. Therefore, mTOR is a potentially important therapeutic target in these diseases. Rapamycin and its analogs (rapalogs) are effective anticancer agents in a broad range of preclinical models. Clinical trials with these agents alone and in combination with other anticancer agents, including chemotherapy and targeted therapies, have demonstrated potential clinical benefit in several types of sarcoma. The evidence from both preclinical and clinical studies supports further study of mTOR-targeting rapalogs in the treatment of various subtypes of sarcoma.     

Expression of multiple epigenetically regulated cancer/germline genes in nonsmall cell lung cancer

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized on the X-chromosome may provide effective treatment for an extended number of patients.     

Signaling pathway for aloe-emodin-induced apoptosis in human H460 lung nonsmall carcinoma cell

Aloe-emodin (1,8-dihydroxy-3-(hydroxymethyl)-anthraquinone) is an active component from the root and rhizome of Rheum palmatum that has been reported to exhibit antitumor effects through an unknown mechanism. Our study investigated the mechanisms of aloe-emodin-induced cell death in the human lung nonsmall cell carcinoma cell line H460. Aloe-emodin (40 microM)-induced apoptosis of H460 cells involves modulation of cAMP-dependent protein kinase, protein kinase C, Bcl-2, caspase-3 and p38 protein expression. The relationship of various signals involved in cell death, such as cAMP-dependent protein kinase, protein kinase C, Bcl-2, caspase-3 and p38, has been investigated in the regulation of apoptotic cell death of aloe-emodin. We demonstrated that the expression of p38 is an important determinant of apoptotic death induced by aloe-emodin.     

Inactivation of LLC1 gene in nonsmall cell lung cancer

Serial analysis of gene expression studies led us to identify a previously unknown gene, c20orf85, that is present in the normal lung epithelium but absent or downregulated in most primary nonsmall cell lung cancers and lung cancer cell lines. We named this gene LLC1 for Low in Lung Cancer 1. LLC1 is located on chromosome 20q13.3 and has a 70% GC content in the promoter region. It has 4 exons and encodes a protein containing 137 amino acids. By in situ hybridization, we observed that LLC1 message is localized in normal lung bronchial epithelial cells but absent in 13 of 14 lung adenocarcinoma and 9 out of 10 lung squamous carcinoma samples. Methylation at CpG sites of the LLC1 promoter was frequently observed in lung cancer cell lines and in a fraction of primary lung cancer tissues. Treatment with 5-aza deoxycytidine resulted in a reduced methylation of the LLC1 promoter concomitant with the increase of LLC1 expression. These results suggest that inactivation of LLC1 by means of promoter methylation is a frequent event in nonsmall cell lung cancer and may play a role in lung tumorigenesis.     

Serum fibrinogen is an independent prognostic factor in operable nonsmall cell lung cancer

Serum fibrinogen converted to insoluble fibrin by activated thrombin, plays an important role in the coagulation system. Increased fibrinogen considerably influences cancer cell growth, progression and metastasis. In nonsmall cell lung cancer (NSCLC), however, the association between serum fibrinogen concentration and prognosis has not been fully examined. We enlisted 567 operable NSCLC patients in our study. Preoperative serum fibrinogen was measured by the Clauss method. The association of serum fibrinogen concentration with clinical pathological factors and patient outcome was evaluated. Survival analysis indicated that serum fibrinogen was an independent prognostic factor in operable NSCLC. Patients with hyperfibrinogenemia had an elevated risk of disease progression and death compared to patients with normal fibrinogen levels. The hazard ratio was 1.49 (95% confidence interval [CI] 1.07–2.05) for disease progression and 1.64 (95% CI 1.06–2.53) for death. The trend linking increasing fibrinogen levels with risk was also statistically significant for both outcomes (p < 0.05). These analyses were adjusted for patient age, sex, smoking behavior, disease stage, tumor grade and histology. Kaplan–Meier survival curves showed similar results. Preoperative serum fibrinogen is a novel independent prognostic biomarker in operable NSCLC.     

Preliminary characterization of oral lesions associated with inhibitors of mammalian target of rapamycin in cancer patients

BACKGROUND:

Mammalian target of rapamycin (mTOR) inhibitors may have efficacy as an intervention for advanced malignancies. Oral ulceration (OU), reported as mucositis, has been a dose‐limiting toxicity for this new class of agents. An analysis of the appearance, course, and toxicity associations of mTOR inhibitor‐associated stomatitis (mIAS) demonstrated that the condition is distinct from conventional mucositis (CM) and more closely resembles aphthous stomatitis.

METHODS:

Safety data from 78 solid tumor patients enrolled in 2 Phase 1, multicenter trials of the mTOR inhibitor deforolimus (AP23573, MK‐8669) were evaluated. Adverse events (AEs) based on National Cancer Institute Common Toxicity Criteria for National Cancer Institute Common Terminology Criteria for Adverse Events (version 3.0) criteria were coded, consolidated, and stratified according to the presence or absence and duration of concordant OU. The relation between OU and other AEs was analyzed.

RESULTS:

Treatment‐emergent AEs were reported in 91% of 78 study participants. OUs were reported in 66%, appeared within 5 days of deforolimus administration, and were discrete, ovoid, superficial, well demarcated, and surrounded by an erythematous halo. Their clinical appearance and distribution were similar to that of aphthous stomatitis but inconsistent with CM. Patients with OU were more likely to have nonspecific rashes and acneiform dermatitis but not gastrointestinal AEs.

CONCLUSIONS:

OU associated with mTOR inhibitor therapy differed from CM. Lesions more closely resembled those of aphthous stomatitis. The lack of other gastrointestinal involvement but the presence of a higher incidence of concomitant cutaneous AEs provided additional evidence to suggest a distinction between mIAS and CM. Treatment strategies for aphthous stomatitis may be a rational approach for the prevention and control of mIAS. Cancer 2010. © 2010 American Cancer Society.     

NV‐128, a novel isoflavone derivative,induces caspase‐independent cell death through the Akt/mammalian target of rapamycin pathway

BACKGROUND:

Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase‐independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV‐128 as an inducer of cell death through a caspase‐independent pathway.

METHODS:

Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV‐128, and the concentration that caused 50% growth inhibition (GI₅₀) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein‐protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model.

RESULTS:

NV‐128 was able to induce significant cell death in both paclitaxel‐resistant and carboplatin‐resistant EOC cells with a GI₅₀ between 1 μg/mL and 5 μg/mL. Cell death was characterized by chromatin condensation but was caspase‐independent. The activated pathway involved the down‐regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin‐1 followed by nuclear translocation of endonuclease G.

CONCLUSIONS:

The authors characterized a novel compound, NV‐128, which inhibits mTOR and promotes caspase‐independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase‐dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV‐128, which may have beneficial effects in patients with chemoresistant ovarian cancer. Cancer 2009. © 2009 American Cancer Society.     

Modulation of the activities of AMP-activated protein kinase, protein kinase B, and mammalian target of rapamycin by limiting energy availability with 2-deoxyglucose

2-Deoxyglucose (2-DG), which has been shown to inhibit mammary carcinogenesis, was used as a metabolic probe to investigate effects of limiting energy availability (reduced cellular ATP) on patterns of proteins' phosphorylation that play a role in the development of cancer. Experiments were conducted using a human breast cancer cell line, MDA-MB-468, and 1-methyl-1-nitrosourea-induced rat model for mammary carcinogenesis. Under in vitro conditions in which cellular ATP concentration decreased rapidly with increasing 2-DG in a dose and time dependent manner, levels of phosphorylated mammalian target of rapamycin (P-mTOR) decreased in parallel to decreases in ATP concentration. Concomitantly, phosphorylation of two upstream regulators of mTOR, AMP-activated protein kinase (AMPK) and Akt/protein kinase B were increased and decreased, respectively, with increased levels of phosphorylated acetyl-CoA carboxylase as an indicator of AMPK activation. Levels of insulin like growth factor 1-receptor and phosphoinositide-3 kinase p110 alpha were also reduced. Similar effects were observed in mammary carcinomas in vivo at concentration of 0.03% (w/w) dietary 2-DG that inhibited carcinogenesis. In vitro, downregulation of mTOR was accompanied by decreases in phosphorylation of two of mTOR's targets, 70-kDa ribosomal protein S6 kinase and eukaryote initiation factor 4E binding protein 1. Glucose treatment reversed 2-DG effects. When cells were transfected with dominant-negative AMPK alpha 2, effects of 2-DG on mTOR and its downstream effectors were diminished, providing evidence of a link between AMPK and mTOR when energy availability was limited. This work indicates that AMPK, Akt, and mTOR are candidate targets for efforts to inhibit the carcinogenic process by limiting energy availability.     

Clinical and histopathologic characteristics of rash in cancer patients treated with mammalian target of rapamycin inhibitors

BACKGROUND:

Dermatologic adverse events stemming from anticancer therapies have become an increasingly frequent clinical problem. Inhibitors of mammalian target of rapamycin (mTOR), such as temsirolimus and everolimus, have been associated with a high rate of skin eruptions, but their clinical and histopathologic characteristics have not been explored.

METHODS:

A retrospective analysis of patients who were referred to the Dermatology Service for diagnosis and management of rash in the setting of therapy with the mTOR inhibitors everolimus and temsirolimus was performed. The parameters that were studied included the time to onset, clinical presentation at the time of dermatologic evaluation, associated symptoms, evolution, results of microbiologic studies, concomitant medications, the need for dose reduction and/or treatment interruption because of rash, and routine histopathology.

RESULTS:

In total, 13 patients were analyzed. Most rashes were mild (grade 1; 31%) and moderate (grade 2; 54%) in severity, and grade 3 rashes were observed only in 2 patients (15%). The trunk was the most frequently affected region (77%), with the scalp (23%), face (38%), neck (54%), and extremities (69%) also commonly involved. Erythematous papules and pustules constituted the predominant primary lesion morphology (62%). No unique or uniform histopathologic reaction pattern was observed. The most common reaction pattern was that of a mixed, spongiotic interface and perivascular dermatitis, which was observed in 7 of 11 patients (63%).

CONCLUSIONS:

Although mTOR inhibitors may commonly induce erythematous papules and pustules, they are associated with a spectrum of lesion morphologies and a variety of histopathologic findings. Further clinicohistologic correlation studies are needed. Cancer 2012. © 2012 American Cancer Society.     

Prognostic implications of cell cycle-related proteins in primary resectable pathologic N2 nonsmall cell lung cancer

BACKGROUND: Patients who have pathologic N2 (pN2) nonsmall cell lung cancer (pN2 NSCLC) represent a heterogeneous group with regard to prognosis and treatment. Molecular features of NSCLC seem to be of interest. For the current study, to select an appropriate therapeutic strategy for each patient, patients with N2 NSCLC were stratified into homogenous subgroups according to the expression profiles of cell cycle-related markers. METHODS: The expression levels of retinoblastoma protein (pRb), cyclin D1, p16, p53, and p21 proteins and values of the Ki-67 labeling index were evaluated in 61 primary surgically resected tumor specimens from patients with pN2 NSCLC using immunohistochemistry. The prognostic impact of these markers on overall survival was analyzed in both univariate and multivariate analyses. RESULTS: In univariate analysis, p21, p16, and Ki-67 were correlated significantly with survival. In multivariate analysis, only p21 and p16 influenced survival. Indeed, the group of patients with pN2 NSCLC who were positive for p21 and p16 had the most favorable overall survival (P = .001) and were correlated significantly with the clinical lymph node (cN) status (cN2 disease; P = .008). Moreover, no significant difference in survival was observed between patients with cN0/cN1 disease and patients with cN2 disease within the group (P = .4333). CONCLUSIONS: Loss of control of cell-cycle checkpoints is a common occurrence in pN2 NSCLC. Functional cooperation between different cell-cycle regulators constitutes another level of regulation in cell growth control and tumor suppression. Preoperative patients with pN2 NSCLC, even those with cN2 disease, who have positive p21 and p16 protein expression in their primary tumors are expected to have a favorable postoperative prognosis and may be candidates for primary resection.     

Role of mammalian target of rapamycin complex 2 in primary and secondary liver cancer

The mammalian target of rapamycin (mTOR) acts in two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Upon deregulation, activated mTOR signaling is associated with multiple processes involved in tumor growth and metastasis. Compared with mTORC1, much less is known about mTORC2 in cancer, mainly because of the unavailability of a selective inhibitor. However, existing data suggest that mTORC2 with its two distinct subunits Rictor and mSin1 might play a more important role than assumed so far. It is one of the key effectors of the PI3K/AKT/mTOR pathway and stimulates cell growth, cell survival, metabolism, and cytoskeletal organization. It is not only implicated in tumor progression, metastasis, and the tumor microenvironment but also in resistance to therapy. Rictor, the central subunit of mTORC2, was found to be upregulated in different kinds of cancers and is associated with advanced tumor stages and a bad prognosis. Moreover, AKT, the main downstream regulator of mTORC2/Rictor, is one of the most highly activated proteins in cancer. Primary and secondary liver cancer are major problems for current cancer therapy due to the lack of specific medical treatment, emphasizing the need for further therapeutic options. This review, therefore, summarizes the role of mTORC2/Rictor in cancer, with special focus on primary liver cancer but also on liver metastases.