ABSTRACT
Chromophobe renal cell carcinoma (chRCC) is a histologically and molecularly distinct class of rare renal tumor. TCGA studies revealed low mutational burden, with only TP53 and PTEN recurrently mutated, and discovered alterations in TERT promoter and in the electron transport chain Complex I genes. However, knowledge on drug targetable genes is limited and treatments at metastatic stage do not follow a molecular rationale. In a large series of 92 chRCC enriched with metastatic cases, we performed an in-depth characterization of mTOR pathway alterations through targeted NGS and immunohistochemistry (IHC) of phospho-S6, tuberin, and PTEN. Mutations in mitochondria, telomere maintenance and other renal cancer related genes and p53 IHC, were also assessed. The impact on metastasis development and disease specific survival was determined, using TCGA-KICH series (n = 65) for validation. mTOR pathway mutations (MTOR, TSC1, TSC2) were present in 17% of primary tumors, most of them being classified as pathogenic. Mutations were associated with positive IHC staining of phospho-S6 and PTEN (P = 0.009 and P = 0.001, respectively) and with chRCC eosinophilic variant (P = 0.039), supporting a biological relevance of the pathway. mTOR pathway mutations were associated with worse clinical outcomes. Survival analysis gave a hazard ratio of 5.5 (P = 0.027), and this association was confirmed in TCGA-KICH (HR = 10.3, P = 0.006). TP53 mutations were enriched in metastatic cases (P = 0.018), and mutations in telomere maintenance genes showed a trend in the same direction. p53 IHC staining pattern was associated with the underlying TP53 defect, and negative PTEN IHC staining (82% of cases) suggested PTEN loss as a chRCC hallmark. In conclusion, our study provides with novel genomic knowledge in chRCC and identifies novel markers of poor survival. Furthermore, this is the first study showing that mTOR pathway mutations correlate with poor prognosis, and may help to identify patients with increased sensitivity to mTOR inhibitors.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Renal Cell/genetics , Kidney Neoplasms/genetics , Mutation , TOR Serine-Threonine Kinases/genetics , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/analysis , Carcinoma, Renal Cell/chemistry , Carcinoma, Renal Cell/pathology , Carcinoma, Renal Cell/therapy , DNA Mutational Analysis , Female , Genetic Predisposition to Disease , High-Throughput Nucleotide Sequencing , Humans , Immunohistochemistry , Kidney Neoplasms/chemistry , Kidney Neoplasms/pathology , Kidney Neoplasms/therapy , Male , Middle Aged , PTEN Phosphohydrolase/analysis , Phenotype , Phosphorylation , Ribosomal Protein S6 Kinases/analysis , Tuberous Sclerosis Complex 1 Protein/genetics , Tuberous Sclerosis Complex 2 Protein/analysis , Tuberous Sclerosis Complex 2 Protein/genetics , Tumor Suppressor Protein p53/geneticsABSTRACT
BACKGROUND: Gastric Cancer (GC) is a frequently common malignancy. Recent studies have reported Rab1A as an activator of mTORC1, and the mTOR1 pathway is involved in regulating Gli1 expression in several cancers. Only a few studies have been performed to explore the relationship between Rab1A and p-S6K/Gli1in GC. METHODS: Immunohistochemistry (IHC) was performed to explore the association of Rab1A/p-S6K/Gli1 expression and prognosis in 117 GC tissue samples and adjacent normal tissues. RESULTS: Our results indicated that Rab1A/p-S6K/Gli1 was significantly overexpressed in GC tissues. High expression of Rab1A was closely related to the tumor size and the depth of tumor invasion. In addition, Rab1A expression was closely related with p-S6K/Gli1 expression in GC, and high level of Rab1A/p-S6K/Gli1 caused worse prognosis of GC patients. The univariate and multivariate analysis indicated that the expression of Rab1A was an independent prognostic factor. Moreover, both high Rab1A and p-S6K expression led to a worse prognosis when compared to a single positive expression as well as both high Rab1A/Gli1 expression also led to a worse prognosis than the single positive expression of Rab1A/Gli1. Strikingly, the overexpression of p-S6K also led to a worse prognosis in Rab1A positive patients, as did Gli1. CONCLUSION: Our results indicate that Rab1A/mTOR/S6K/Gli1 axis played a crucial role in GC, which may provide a novel field on targeted therapy of GC, especially for mTORC1-targeted therapy-resistant cancers.
Subject(s)
Ribosomal Protein S6 Kinases/biosynthesis , Stomach Neoplasms/diagnosis , Zinc Finger Protein GLI1/biosynthesis , rab1 GTP-Binding Proteins/biosynthesis , Adult , Aged , Female , Humans , Male , Middle Aged , Ribosomal Protein S6 Kinases/analysis , Ribosomal Protein S6 Kinases/metabolism , Stomach Neoplasms/metabolism , Zinc Finger Protein GLI1/analysis , Zinc Finger Protein GLI1/metabolism , rab1 GTP-Binding Proteins/analysis , rab1 GTP-Binding Proteins/metabolismABSTRACT
BACKGROUND: The role of the mammalian target of rapamycin (mTOR) in hair follicle tumorigenesis is unclear. mTOR controls cell growth and can be activated through ribosomal S6 kinase. Herein, we sought to evaluate the expression of phospho-S6 in six different benign and malignant follicular tumor types. METHODS: 76 cases were selected (17 fibrofolliculomas, 20 trichoepitheliomas, 10 tricholemmomas, 19 pilomatricomas, 1 malignant proliferating tricholemmal tumor, 8 tricholemmal carcinomas, and 1 trichoblastic carcinoma) and collected over 16 years. Immunohistochemistry with monoclonal antibody for phospho-S6 was performed and analyzed semi-quantitatively; statistical analysis using the χ2 test was performed, with P < 0.05 considered significant. RESULTS: All malignant neoplasms in our series (8/8 [100%] cases of tricholemmal carcinoma, 1/1 [100%] trichoblastic carcinoma, and 1/1 [100%] malignant proliferating tricholemmal tumor) showed a strong and diffuse pattern of staining for phospho-S6 involving 70% to 90% of tumor cells. By contrast, a minority of benign tumors were positive for phospho-S6 and most stained in a patchy pattern including 12/17 (71%) fibrofolliculomas, 9/20 (45%) trichoepitheliomas and 1/10 (10%) tricholemmomas, involving 30% to 50%, 5% to 20%, and 40% to 50% of tumor cells, respectively. Most pilomatricomas (17/19 [89%]) exhibited a stronger, but distinctive staining pattern, staining mostly the basaloid cells with a multifocal distribution, involving 70% to 90% of tumor cells. CONCLUSIONS: Phospho-S6 is differentially expressed among benign and malignant hair follicle tumors (P = 0.0044). While malignant tumors show diffuse expression, only a small subset of benign neoplasms were positive, primarily in a patchy distribution.
Subject(s)
Biomarkers, Tumor/analysis , Hair Diseases/diagnosis , Hair Follicle/pathology , Ribosomal Protein S6 Kinases/analysis , Skin Neoplasms/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Hair Diseases/metabolism , Hair Diseases/pathology , Hair Follicle/metabolism , Humans , Infant , Male , Middle Aged , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , TOR Serine-Threonine Kinases/metabolism , Young AdultABSTRACT
Colorectal cancer (CRC) is one of the most common cancers worldwide. Recent studies have reported that PI3K/AKT/mTOR pathway regulated the GLI1 expression level via SMO-independent pathway in a variety of tumor types. We detected the expression level of GLI1/p-S6K in CRC tissues. We found the expression of GLI1/p-S6K was apparently close with lymph node metastasis and TNM stage and patients with positive GLI1/p-S6K expression had shorter survival time and patients with both GLI1 and p-S6K positive expression had an even worse overall survival than those with single positive expression. Moreover, GLI1 and p-S6K expression was considered to be independent prognostic factors in CRC patient and the positive co-expression of GLI1/p-S6K had greater influence than single expression positive on the prognosis of postoperative patients with tumor size≥5â¯cm, well differentiation, positive lymph node metastasis, venous invasion, neural invasion and TNM III-IV. Meanwhile, the GLI1/p-S6K expression had impact on more clinicopathologic features in colon-side carcinoma than in rectum-side carcinoma and the mTOR/S6K/GLI1 axis played an important role in CRC especially in advanced stage. Hence, further studies are underway to explore the molecular mechanism between GLI1 and p-S6K in CRC, and in addition, it offers novel facilities for molecular targeting therapy for CRC.
Subject(s)
Colorectal Neoplasms/pathology , Ribosomal Protein S6 Kinases/analysis , Zinc Finger Protein GLI1/analysis , Biomarkers, Tumor/analysis , Colon/pathology , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/epidemiology , Female , Humans , Immunohistochemistry , Lymphatic Metastasis/diagnosis , Lymphatic Metastasis/pathology , Male , Middle Aged , Neoplasm Staging , Prognosis , Rectum/pathology , Survival AnalysisABSTRACT
Automatic cell segmentation is a powerful method for quantifying signaling dynamics at single-cell resolution in live cell fluorescence imaging. Segmentation methods for mononuclear and round shape cells have been developed extensively. However, a segmentation method for elongated polynuclear cells, such as differentiated C2C12 myotubes, has yet to be developed. In addition, myotubes are surrounded by undifferentiated reserve cells, making it difficult to identify background regions and subsequent quantification. Here we developed an automatic quantitative segmentation method for myotubes using watershed segmentation of summed binary images and a two-component Gaussian mixture model. We used time-lapse fluorescence images of differentiated C2C12 cells stably expressing Eevee-S6K, a fluorescence resonance energy transfer (FRET) biosensor of S6 kinase (S6K). Summation of binary images enhanced the contrast between myotubes and reserve cells, permitting detection of a myotube and a myotube center. Using a myotube center instead of a nucleus, individual myotubes could be detected automatically by watershed segmentation. In addition, a background correction using the two-component Gaussian mixture model permitted automatic signal intensity quantification in individual myotubes. Thus, we provide an automatic quantitative segmentation method by combining automatic myotube detection and background correction. Furthermore, this method allowed us to quantify S6K activity in individual myotubes, demonstrating that some of the temporal properties of S6K activity such as peak time and half-life of adaptation show different dose-dependent changes of insulin between cell population and individuals.Key words: time lapse images, cell segmentation, fluorescence resonance energy transfer, C2C12, myotube.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Muscle Fibers, Skeletal/enzymology , Ribosomal Protein S6 Kinases/analysis , Single-Cell Analysis/methods , Animals , Enzyme Activation , Image Processing, Computer-Assisted/methods , Mice , Muscle Fibers, Skeletal/ultrastructure , Optical Imaging/methods , Ribosomal Protein S6 Kinases/metabolismABSTRACT
Metastatic breast cancer (MBC) is essentially incurable despite recent improvements in systemic therapies. We often encounter difficulties in choosing the most appropriate treatments, with optimal timing, for individual patients. Everolimus, one of the mTOR inhibitors, is usually used with endocrine therapy for MBC. Identification of predictive markers for everolimus-based treatment remains a major issue, but to date, no predictive markers have been established. We retrospectively investigated predictive markers for treatments with everolimus plus exemestane in patients with ER-positive and HER2-negative breast cancer. Clinicopathological features of 18 patients, with locally advanced disease or MBC given everolimus plus exemestane treatments, were examined in relation to treatment effects. Also, primary breast cancer specimens, all ER positive and HER2 negative, were immunohistochemically investigated for phospho-S6 (pS6) and PTEN, to evaluate the mTOR and PIK3CA/Akt pathways. Those showing a good clinical response had a significantly lower Ki67 labeling index than the poor responders. A similar trend was observed in pS6 level but without statistical significance. Interestingly, there was no correlation between the Ki67 labeling index and pS6, and when both indexes were low, the good clinical response rate was high. The median progression-free survival was longer in the group showing a low Ki67 labeling index (109 weeks) than in that with high Ki67 (19 weeks). There was no trend between PTEN expression and treatment effects. Our results suggest that the primary tumor in luminal HER2-negative breast cancer patients with a low Ki67 labeling index and pS6 level has the potential to respond well to everolimus plus exemestane.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/analysis , Breast Neoplasms/drug therapy , Adult , Aged , Androstadienes/administration & dosage , Breast Neoplasms/pathology , Disease-Free Survival , Everolimus/administration & dosage , Female , Humans , Ki-67 Antigen/analysis , Middle Aged , PTEN Phosphohydrolase/analysis , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/genetics , Receptors, Estrogen/biosynthesis , Receptors, Estrogen/genetics , Ribosomal Protein S6 Kinases/analysisABSTRACT
PI3K/Akt/mTOR pathway activation is a hallmark of high-grade gliomas, which prompted clinical trials for the use of PI3K and mTOR inhibitors. However, the poor results in the original trials suggested that better patient profiling was needed for such drugs. Thus, accurate and reproducible monitoring of mTOR complexes can lead to improved therapeutic strategies. In this work, we evaluated the expression and phosphorylation of mTOR, RAPTOR, and rpS6 in 195 human astrocytomas and 30 normal brain tissue samples. The expression of mTOR increased in glioblastomas, whereas mTOR phosphorylation, expression of RAPTOR, and expression and phosphorylation of rpS6 were similar between grades. Interestingly, the overexpression of total and phosphorylated mTOR as well as phosphorylated rpS6 (residues 240-244) were associated with wild-type IDH1 only glioblastomas. The expression and phosphorylation of mTOR and phosphorylation of rpS6 at residues 240-244 were associated with a worse prognosis in glioblastomas. Our results suggest that mTOR and rpS6 could be used as markers of overactivation of the PI3K-mTOR pathway and are predictive factors for overall survival in glioblastomas. Our study thus suggests that patients who harbor IDH1 wild-type glioblastomas might have increased benefit from targeted therapy against mTOR.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Isocitrate Dehydrogenase/analysis , Ribosomal Protein S6 Kinases/analysis , TOR Serine-Threonine Kinases/analysis , Up-Regulation , Adult , Brain Neoplasms/diagnosis , Brain Neoplasms/epidemiology , Child , Female , Glioblastoma/diagnosis , Glioblastoma/epidemiology , Humans , Immunohistochemistry/methods , Male , Middle Aged , Phosphorylation , Prognosis , Survival RateABSTRACT
Dermatofibrosarcoma protuberans (DFSP) is a cutaneous mesenchymal tumor of intermediate malignancy and fibroblastic/myofibroblastic differentiation. Fibrosarcomatous (FS) component is a high-grade component of DFSP. The detailed oncogenic difference between DFSP and FS components is not clear. We thus investigated the Akt-mTOR pathway in both components. We used 65 tumor samples obtained from 65 patients. The phosphorylation of Akt-mTOR pathway proteins (Akt, mTOR, 4EBP1, and S6RP) and PDGFRα/ß was assessed by immunohistochemical staining, the results of which were confirmed by Western blotting. The immunohistochemical results were as follows: in ordinary DFSP components, p-PDGFRα-positive tumors were 41.9% (18/43 cases), p-PDGFRß 55.8% (24/43 cases), p-Akt 51.2% (22/43 cases), p-mTOR 39.5% (17/43 cases), p-4EBP1 46.5% (20/43 cases), and p-S6RP 41.8% (18/43 cases); in DFSP components of FS-DFSP, 52.6% (10/19 cases), 47.4% (9/19 cases), 52.6% (10/19 cases), 36.8% (7/19 cases), 52.6% (10/19 cases), and 52.6% (10/19 cases); and in FS components, 45.5% (10/22 cases), 36.4% (8/22 cases), 72.7% (16/22 cases), 54.5% (12/22 cases), 72.7% (16/22 cases), and 68.2% (15/22 cases), respectively. There were significant positive correlations of the phosphorylation of most of the Akt-mTOR pathway proteins (p-Akt, p-mTOR, p-4EBP1, and p-S6RP) with each other (P < .05). Phospho-PDGFRß was well correlated with the phosphorylation of Akt-mTOR pathway proteins in DFSP components of ordinary and FS-DFSPs, but these correlations were weaker in FS components. This study suggested the association of activation of Akt-mTOR pathway proteins and PDGFR with the progression of DFSP to FS. The Akt-mTOR pathway is thus a potential therapeutic target in imatinib-resistant DFSP/FS.
Subject(s)
Cell Transformation, Neoplastic/chemistry , Dermatofibrosarcoma/enzymology , Proto-Oncogene Proteins c-akt/analysis , Receptor, Platelet-Derived Growth Factor beta/analysis , Skin Neoplasms/enzymology , TOR Serine-Threonine Kinases/analysis , Adaptor Proteins, Signal Transducing/analysis , Adolescent , Adult , Aged , Biopsy , Blotting, Western , Cell Cycle Proteins , Cell Transformation, Neoplastic/metabolism , Child , Child, Preschool , Dermatofibrosarcoma/pathology , Disease Progression , Female , Humans , Immunohistochemistry , Infant , Male , Middle Aged , Phosphoproteins/analysis , Phosphorylation , Receptor, Platelet-Derived Growth Factor alpha/analysis , Ribosomal Protein S6 Kinases/analysis , Signal Transduction , Skin Neoplasms/pathology , Young AdultABSTRACT
Brain metastases (BMs) are common complications of adenocarcinomas (ADCs) of the lung and are associated with a poor prognosis. Although an increasing amount of data indicates that dysregulated activity of mammalian target of rapamycin (mTOR) can influence the metastatic potential of various tumors, the role of mTOR complexes in the development of BMs from ADCs of the lung is largely unknown. To estimate mTOR activity, we studied the expression of mTOR-related proteins (mTORC1: p-mTOR, p-S6; mTORC2: p-mTOR, Rictor) in primary (n=67) and brain metastatic (n=67) lung ADCs, including 15 paired tissue samples, using immunohistochemistry and tissue microarrays. Correlation with clinicopathological parameters was also analyzed. Increased p-mTOR, p-S6, and Rictor expressions were observed in 34%, 33%, and 37% of primary ADCs and in 79%, 70%, and 66% of BMs, respectively. Expression of these markers was significantly higher in BMs as compared with primary carcinomas (P<.0001, P<.0001, P<.001). Rictor expression was significantly higher in primary ADCs of the paired cases with BMs as compared with primary ADCs without BMs (67% versus 28%; P<.01). No other statistically significant correlations were found between mTOR activity and clinicopathological parameters. The increased mTORC1/C2 activity in a subset of pulmonary ADCs and the higher incidence of increased mTORC1/C2 activity in BMs suggest that the immunohistochemistry panel for characterizing mTOR activity and its potential predictive and prognostic role warrants further investigations.
Subject(s)
Adenocarcinoma/enzymology , Biomarkers, Tumor/analysis , Brain Neoplasms/enzymology , Lung Neoplasms/enzymology , Multiprotein Complexes/analysis , TOR Serine-Threonine Kinases/analysis , Adenocarcinoma/secondary , Adenocarcinoma/surgery , Adenocarcinoma of Lung , Aged , Brain Neoplasms/secondary , Brain Neoplasms/surgery , Carrier Proteins/analysis , Female , Humans , Hungary , Immunohistochemistry , Lung Neoplasms/pathology , Lung Neoplasms/surgery , Male , Mechanistic Target of Rapamycin Complex 1 , Mechanistic Target of Rapamycin Complex 2 , Middle Aged , Phosphorylation , Prognosis , Rapamycin-Insensitive Companion of mTOR Protein , Ribosomal Protein S6 Kinases/analysis , Tissue Array Analysis , Up-RegulationABSTRACT
OBJECTIVE: Aggressive cutaneous squamous cell carcinoma (cSCC) patients are at increased risk of metastasis. Currently, there are no accepted criteria or biomarkers for reliably predicting individuals at risk for recurrence and metastasis. Our objective is to determine if pS6 and pERK can predict cSCC aggressiveness and to identify primary tumor characteristics that may predict parotid metastasis. STUDY DESIGN: Retrospective case series. SETTINGS: Tertiary care center. SUBJECTS AND METHODS: An Institutional Review Board-approved retrospective review was performed for patients with facial cSCC, with and without metastasis to the parotids. Subjects for the study were identified through the Louisiana Tumor Registry, Veterans Medical Records, and LSU Health-Shreveport pathology database. Tumor specimens from patients with cSCC and cSCC with parotid metastasis were analyzed for pERK and pS6 expression through immunohistochemistry. To identify risk factors for tumor aggressiveness, multiple logistic regression analysis was used to evaluate patients with cSCC that was metastatic to the parotid and managed surgically. RESULTS: cSCC with parotid metastasis specimens exhibited significantly higher average pS6 but not pERK positivity than those from cSCC without metastasis (P < .05). Primary lesion-positive margins (P < .01), size of the skin tumor (P < .01) and degree of tumor differentiation (P < .01) were significantly associated with parotid metastasis. CONCLUSION: Surgical history of cSCC, primary lesion-positive margins, degree of differentiation, and lesion size together with pS6 positivity appear to be predictors of cSCC aggressiveness and should prompt increased monitoring or elective parotidectomy.
Subject(s)
Biomarkers, Tumor/analysis , Carcinoma, Squamous Cell/secondary , Parotid Neoplasms/secondary , Skin Neoplasms/pathology , Adult , Aged , Carcinoma, Squamous Cell/surgery , Female , Humans , Immunohistochemistry , Louisiana/epidemiology , Male , Middle Aged , Neoplasm Metastasis/pathology , Neoplasm Recurrence, Local/pathology , Parotid Neoplasms/surgery , Registries , Retrospective Studies , Ribosomal Protein S6 Kinases/analysis , Risk Assessment , Risk Factors , eIF-2 Kinase/analysisABSTRACT
AT-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancers, and loss of ARID1A expression is considered a poor prognostic factor in various cancers. However, in practice, ARID1A shows various expression patterns, and our understanding of its significance is limited. We performed immunohistochemistry for ARID1A, MLH1, and pS6 using whole tissue blocks of 350 gastric cancers and classified the ARID1A expression as follows: retained (63.7%), reduced (17.7%), complete loss (14.9%), and partial loss (3.7%). Complete/partial loss was more common in poorly differentiated histology (P < .001), and reduced or complete loss of ARID1A was frequent in cases with MLH1 loss (P < .001). The ARID1A-reduced group showed only slightly inferior disease-free survival (DFS; P = .254) and overall survival (OS; P = .377) compared to those of the ARID1A-retained group, whereas the group with complete loss showed significantly worse DFS (hazard ratio [HR], 1.732; P = .015) and OS (HR, 1.751; P = .013). Worse DFS (HR, 2.672; P = .005) and OS (HR, 2.531; P = .002) were also noted in the group with partial loss. High expression of pS6 was observed more frequently in groups showing altered ARID1A expression patterns (P < .001). In conclusion, reduced ARID1A expression is not a major prognostic determinant, although it may lead to AKT pathway activation. Tumor cells lacking ARID1A expression may influence the prognosis even if they constitute only a small proportion of the tumor sample. Our data provide an enhanced roadmap for understanding ARID1A with implications for future research and therapeutics.
Subject(s)
Adenocarcinoma/chemistry , Biomarkers, Tumor/analysis , Nuclear Proteins/analysis , Stomach Neoplasms/chemistry , Transcription Factors/analysis , Adaptor Proteins, Signal Transducing/analysis , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adenocarcinoma/surgery , Adult , Aged , Aged, 80 and over , Biopsy , Cell Differentiation , DNA-Binding Proteins , Disease-Free Survival , Down-Regulation , Female , Gastrectomy , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Male , Middle Aged , MutL Protein Homolog 1 , Predictive Value of Tests , Proportional Hazards Models , Ribosomal Protein S6 Kinases/analysis , Risk Factors , Stomach Neoplasms/mortality , Stomach Neoplasms/pathology , Stomach Neoplasms/surgery , Time Factors , Treatment Outcome , Young AdultABSTRACT
BACKGROUND: Dysregulation of mammalian target of rapamycin (mTOR) pathway has been linked with malignant tumorigenesis. This study explored the expression profiles of proteins involved in the mTOR pathway and their relationships with clinicopathologic characteristics in stage-I non-small-cell lung carcinoma (NSCLC). METHODS: The protein expression profiles of PTEN, p-Akt, p-mTOR, p-S6, and eIF4E were examined using immunohistochemical staining and tissue microarray method in 408 patients with stage-I NSCLC (250 adenocarcinomas [ADC] and 158 squamous cell carcinomas). RESULTS: Retained PTEN expression (P<0.001), p-mTOR expression (P<0.001), and p-S6 expression (P=0.007) were associated with ADC histology. Expression of PTEN (P=0.001), p-Akt (P=0.005), p-mTOR (P=0.007), p-S6 (P<0.001) were correlated with lower pathologic T stage. PTEN loss was correlated with male gender and smoking history and p-mTOR expression was inversely correlated with these factors (P<0.001). Subgroup analysis of ADCs indicated that male gender, high pT stage, lymphovascular invasion, and PTEN loss were poor prognostic factors. Multivariate analysis revealed that the PTEN(-)/p-Akt(+)/p-mTOR(+) combination more effectively determined the prognosis of ADC (hazard ratio=2.2, P=0.004) than PTEN alone. CONCLUSIONS: Activation of the mTOR pathway in early-stage ADCs suggests a significant role for the mTOR axis in early carcinogenesis. The combination of PTEN(-)/p-Akt(+)/p-mTOR(+) expression was correlated with poor overall survival in patients with stage-I lung ADC.
Subject(s)
Adenocarcinoma/chemistry , Carcinoma, Non-Small-Cell Lung/chemistry , Lung Neoplasms/chemistry , TOR Serine-Threonine Kinases/analysis , Adenocarcinoma/pathology , Aged , Carcinoma, Non-Small-Cell Lung/pathology , Eukaryotic Initiation Factor-4E/analysis , Female , Humans , Immunohistochemistry , Lung Neoplasms/pathology , Male , Middle Aged , Neoplasm Invasiveness , Neoplasm Staging , PTEN Phosphohydrolase/analysis , Phosphorylation , Prognosis , Proto-Oncogene Proteins c-akt/analysis , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases/analysis , Ribosomal Protein S6 Kinases/metabolism , Sex Factors , Signal Transduction , Smoking , Survival Rate , TOR Serine-Threonine Kinases/metabolism , Tissue Array AnalysisABSTRACT
PURPOSE OF THE STUDY: Aberrant activation of the Akt/mTOR/pS6 signaling pathway has been identified in various types of cancer and is under investigation in cervical cancer. The purpose of this study was to assess the expression of the phosphorylated/activated forms of Akt (upstream molecule), 4E-BP1 and pS6 (downstream molecules) in biopsy samples of cervical low grade squamous intraepithelial lesions (LSIL), high grade squamous intraepithelial lesions (HSIL), and squamous cell carcinoma (Ca) compared to normal cervical epithelium. MATERIAL AND METHODS: The study included 38 cases diagnosed as LSIL, 31 cases as HSIL, 29 cases as Ca, and eight control cases from normal cervix. Immunohistochemistry was used to assess the expression of pAkt, p4E-BP1 and pS6. RESULTS: Statistical analysis revealed significant differences between HSIL and Ca groups compared to controls regarding intensity, positivity, and total scores for all three molecules (p < 0.001). A trend for higher expression with increasing grade of dysplasia was demonstrated. CONCLUSION: These results strongly support the view that the mTOR signaling pathway is involved in cervical carcinogenesis.
Subject(s)
Carcinoma, Squamous Cell/chemistry , Precancerous Conditions/chemistry , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Uterine Cervical Dysplasia/chemistry , Uterine Cervical Neoplasms/chemistry , Adaptor Proteins, Signal Transducing/analysis , Carcinoma, Squamous Cell/metabolism , Cell Cycle Proteins , Female , Humans , Phosphoproteins/analysis , Phosphorylation , Precancerous Conditions/metabolism , Proto-Oncogene Proteins c-akt/analysis , Ribosomal Protein S6 Kinases/analysis , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Dysplasia/metabolismABSTRACT
BACKGROUND & AIMS: Obesity is associated with neoplasia, possibly via insulin-mediated cell pathways that affect cell proliferation. Metformin has been proposed to protect against obesity-associated cancers by decreasing serum insulin. We conducted a randomized, double-blind, placebo-controlled, phase 2 study of patients with Barrett's esophagus (BE) to assess the effect of metformin on phosphorylated S6 kinase (pS6K1), a biomarker of insulin pathway activation. METHODS: Seventy-four subjects with BE (mean age, 58.7 years; 58 men [78%; 52 with BE >2 cm [70%]) were recruited through 8 participating organizations of the Cancer Prevention Network. Participants were randomly assigned to groups given metformin daily (increasing to 2000 mg/day by week 4, n = 38) or placebo (n = 36) for 12 weeks. Biopsy specimens were collected at baseline and at week 12 via esophagogastroduodenoscopy. We calculated and compared percent changes in median levels of pS6K1 between subjects given metformin vs placebo as the primary end point. RESULTS: The percent change in median level of pS6K1 did not differ significantly between groups (1.4% among subjects given metformin vs -14.7% among subjects given placebo; 1-sided P = .80). Metformin was associated with an almost significant reduction in serum levels of insulin (median -4.7% among subjects given metformin vs 23.6% increase among those given placebo, P = .08) as well as in homeostatic model assessments of insulin resistance (median -7.2% among subjects given metformin vs 38% increase among those given placebo, P = .06). Metformin had no effects on cell proliferation (on the basis of assays for KI67) or apoptosis (on the basis of levels of caspase 3). CONCLUSIONS: In a chemoprevention trial of patients with BE, daily administration of metformin for 12 weeks, compared with placebo, did not cause major reductions in esophageal levels of pS6K1. Although metformin reduced serum levels of insulin and insulin resistance, it did not discernibly alter epithelial proliferation or apoptosis in esophageal tissues. These findings do not support metformin as a chemopreventive agent for BE-associated carcinogenesis. ClinicalTrials.gov number, NCT01447927.
Subject(s)
Barrett Esophagus/complications , Barrett Esophagus/drug therapy , Cell Proliferation/drug effects , Esophageal Neoplasms/prevention & control , Metformin/administration & dosage , Adult , Aged , Aged, 80 and over , Barrett Esophagus/pathology , Double-Blind Method , Endoscopy, Digestive System , Female , Humans , Insulin/blood , Male , Middle Aged , Placebos/administration & dosage , Prospective Studies , Ribosomal Protein S6 Kinases/analysis , Young AdultABSTRACT
OBJECTIVES: The epidermal growth factor receptor (EGFR) is a validated target in head and neck squamous cell carcinoma (HNSCC). In recurrent and/or metastatic (R/M) HNSCC, resistance to anti-EGFR therapy inevitably occurs. Downstream activation of the PI3K/Akt/mTOR pathway is an established resistance mechanism. Concurrent mTOR blockade may improve efficacy of anti-EGFR therapy. MATERIALS AND METHODS: Erlotinib 150 mg daily and temsirolimus 15 mg weekly were administered to patients with platinum-refractory R/M HNSCC and ECOG performance status 0-2. The primary endpoint was progression-free survival (PFS). Correlative studies determined PIK3CA and HRAS mutation status; p16, EGFR, pS6K, pAkt and PTEN expression; and pre- and post-treatment plasma levels of 20 immunomodulatory cytokines. RESULTS: Twelve patients enrolled; six withdrew within 6 weeks due to toxicity or death, prompting early closure of the trial. Grade ≥ 3 toxicities included fatigue, diarrhea, gastrostomy tube infection, peritonitis, pneumonia, dyspnea, and HN edema. Median PFS was 1.9 months. Median overall survival was 4.0 months. Six/12 tumors were p16(+), 9/11 lacked measurable PTEN expression, and 1/12 harbored a PIK3CA mutation. On exploratory analysis, high baseline plasma VEGF and interferon-gamma levels marginally associated with tumor progression. CONCLUSIONS: The combination of erlotinib and temsirolimus was poorly tolerated. Low prevalence of PTEN expression and 8% incidence of PIK3CA mutations indicate biological relevance of this pathway in R/M disease. Investigation of more tolerable combinations of EGFR and PI3K/Akt/mTOR pathway inhibitors in selected HNSCC patients is warranted.
Subject(s)
Antineoplastic Agents/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Squamous Cell/drug therapy , ErbB Receptors/antagonists & inhibitors , Head and Neck Neoplasms/drug therapy , Neoplasm Recurrence, Local/drug therapy , Protein Kinase Inhibitors/administration & dosage , Quinazolines/administration & dosage , Sirolimus/analogs & derivatives , Aged , Aged, 80 and over , Antineoplastic Agents/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carcinoma, Squamous Cell/secondary , Class I Phosphatidylinositol 3-Kinases , Cyclin-Dependent Kinase Inhibitor p16/analysis , Cytokines/analysis , Drug Resistance, Neoplasm , Erlotinib Hydrochloride , Female , Humans , Male , Middle Aged , Mutation/genetics , Oncogene Protein v-akt/analysis , PTEN Phosphohydrolase/analysis , Phosphatidylinositol 3-Kinases/analysis , Phosphatidylinositol 3-Kinases/genetics , Platinum , Proto-Oncogene Proteins p21(ras)/analysis , Proto-Oncogene Proteins p21(ras)/genetics , Quinazolines/adverse effects , Ribosomal Protein S6 Kinases/analysis , Sirolimus/administration & dosage , Sirolimus/adverse effects , Survival Rate , TOR Serine-Threonine Kinases/antagonists & inhibitors , Tumor Suppressor Proteins/analysisABSTRACT
The dimensions of neuronal dendrites, axons, and synaptic terminals are reproducibly specified for each neuron type, yet it remains unknown how these structures acquire their precise dimensions of length and diameter. Similarly, it remains unknown how active zone number and synaptic strength are specified relative the precise dimensions of presynaptic boutons. In this paper, we demonstrate that S6 kinase (S6K) localizes to the presynaptic active zone. Specifically, S6K colocalizes with the presynaptic protein Bruchpilot (Brp) and requires Brp for active zone localization. We then provide evidence that S6K functions downstream of presynaptic PDK1 to control synaptic bouton size, active zone number, and synaptic function without influencing presynaptic bouton number. We further demonstrate that PDK1 is also a presynaptic protein, though it is distributed more broadly. We present a model in which synaptic S6K responds to local extracellular nutrient and growth factor signaling at the synapse to modulate developmental size specification, including cell size, bouton size, active zone number, and neurotransmitter release.
Subject(s)
Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6 Kinases/analysis , Synapses/enzymology , 3-Phosphoinositide-Dependent Protein Kinases , Animals , Animals, Genetically Modified , Drosophila/embryology , Drosophila/metabolism , Immunohistochemistry , Microscopy, Electron , Presynaptic Terminals , Protein Serine-Threonine Kinases/genetics , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction , Synaptic Transmission/physiologyABSTRACT
Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.
Subject(s)
Depressive Disorder, Major/metabolism , Prefrontal Cortex/physiopathology , TOR Serine-Threonine Kinases/metabolism , Adult , Aged , Aged, 80 and over , Autopsy , Depressive Disorder, Major/genetics , Depressive Disorder, Major/immunology , Eukaryotic Initiation Factors/analysis , Eukaryotic Initiation Factors/biosynthesis , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/immunology , Family , Female , Humans , Male , Middle Aged , Prefrontal Cortex/immunology , Prefrontal Cortex/pathology , Ribosomal Protein S6 Kinases/analysis , Ribosomal Protein S6 Kinases/biosynthesis , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/immunology , Ribosomal Protein S6 Kinases, 70-kDa/analysis , Ribosomal Protein S6 Kinases, 70-kDa/biosynthesis , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Ribosomal Protein S6 Kinases, 70-kDa/immunology , Signal Transduction/genetics , Signal Transduction/immunology , TOR Serine-Threonine Kinases/analysis , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/immunologyABSTRACT
Cell-based arrays offer powerful tools for genomics/proteomics and drug discovery, and are widely applicable for most cell lines. However, it is challenging to apply cell-based arrays for in vitro diagnosis due to limited amount of patient samples. Here, we utilized and demonstrated microfluidic image cytometry (MIC), capable of quantitative, single-cell profiling of multiple signaling molecules using only 300-3,000 cells from clinical brain tumor specimens for in vitro molecular diagnosis. First, we characterized the PI3K/AKT/mTOR pathway, which is often over-activated in the brain tumors, in U87 brain tumor cell lines by measuring EGFR, PTEN, pAKT, and pS6 with a MIC platform, and applied this measurement to clinical brain tumor specimens. In conjunction with statistical analysis, we were able to characterize extensive inter- and intra-tumoral molecular heterogeneity.
Subject(s)
Image Cytometry/methods , Microfluidic Analytical Techniques/methods , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , ErbB Receptors/analysis , ErbB Receptors/metabolism , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/metabolism , Microfluidic Analytical Techniques/instrumentation , Microscopy, Fluorescence , PTEN Phosphohydrolase/analysis , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/analysis , Proto-Oncogene Proteins c-akt/metabolism , Reproducibility of Results , Ribosomal Protein S6 Kinases/analysis , Ribosomal Protein S6 Kinases/metabolism , Tissue Array Analysis/methodsABSTRACT
BACKGROUND: The clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors. METHODS: We investigated the levels of telomerase activity, and inhibitor of apoptosis proteins (IAPs) family (c-IAP1, c-IAP2, XIAP) and c-Myc mRNA expression levels in 20 specimens of Bowenoid papulosis and 36 specimens of condyloma acuminatum in anogenital areas. Overall, phosphorylated (p-) AKT, p-ribosomal protein S6 (S6) and p-4E-binding protein 1 (4EBP1) expression levels were examined by immunohistochemistry in anogenital tumors both with and without positive telomerase activity. RESULTS: Positive telomerase activity was detected in 41.7% of Bowenoid papulosis and 27.3% of condyloma acuminatum compared to normal skin (p < 0.001). In contrast, the expression levels of Bowenoid papulosis indicated that c-IAP1, c-IAP2 and XIAP mRNA were significantly upregulated compared to those in both condyloma acuminatum samples (p < 0.001, p < 0.001, p = 0.022, respectively) and normal skin (p < 0.001, p = 0.002, p = 0.034, respectively). Overall, 30% of Bowenoid papulosis with high risk HPV strongly promoted IAPs family and c-Myc but condyloma acuminatum did not significantly activate those genes. Immunohistochemically, p-Akt and p-S6 expressions were associated with positive telomerase activity but not with p-4EBP1 expression. CONCLUSION: Combined analysis of the IAPs family, c-Myc mRNA expression, telomerase activity levels and p-Akt/p-S6 expressions may provide clinically relevant molecular markers in HPV associated anogenital tumors.
Subject(s)
Anus Neoplasms/chemistry , Bowen's Disease/chemistry , Cell Cycle Proteins/analysis , Condylomata Acuminata/metabolism , Genital Neoplasms, Female/chemistry , Genital Neoplasms, Male/chemistry , Inhibitor of Apoptosis Proteins/analysis , Papillomavirus Infections/complications , Skin Neoplasms/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Anus Neoplasms/genetics , Anus Neoplasms/pathology , Anus Neoplasms/virology , Apoptosis , Baculoviral IAP Repeat-Containing 3 Protein , Blotting, Western , Bowen's Disease/genetics , Bowen's Disease/pathology , Bowen's Disease/virology , Cell Cycle Proteins/genetics , Cell Proliferation , Condylomata Acuminata/genetics , Condylomata Acuminata/pathology , Condylomata Acuminata/virology , Female , Genital Neoplasms, Female/genetics , Genital Neoplasms, Female/pathology , Genital Neoplasms, Female/virology , Genital Neoplasms, Male/genetics , Genital Neoplasms, Male/pathology , Genital Neoplasms, Male/virology , Humans , Immunohistochemistry , Inhibitor of Apoptosis Proteins/genetics , Japan , Male , Papillomavirus Infections/genetics , Papillomavirus Infections/metabolism , Papillomavirus Infections/pathology , Phosphoproteins/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/analysis , Proto-Oncogene Proteins c-myc/analysis , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein S6 Kinases/analysis , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Skin Neoplasms/virology , Telomerase/analysis , Ubiquitin-Protein Ligases , X-Linked Inhibitor of Apoptosis Protein/analysisABSTRACT
The cytotoxic effects and mechanism of action of cisplatin and the mTOR inhibitor rapamycin on Hep-2 laryngeal cancer cells were investigated. Hep-2 cells were cultured in the presence of different concentrations of rapamycin, cisplatin, or the two combined. Inhibition of cell growth, apoptosis, and AKT, mTOR, S6K, and ERCC1 protein levels were assessed. All combinations of rapamycin and cisplatin resulted in synergistic inhibition of cell growth (as indicated by q values determined using Jin's formula > 1.15). Rapamycin inhibited Hep-2 cell growth, induced G1 arrest, and when combined with cisplatin, enhanced apoptosis. p-mTOR and S6K expressions were significantly downregulated by rapamycin. ERCC1 expression was significantly upregulated with cisplatin treatment. Combined cisplatin and rapamycin treatment resulted in significant downregulated p-mTOR and S6K expression, but no change in ERCC1 expression. Rapamycin and cisplatin act in a synergistic manner, increasing the cytotoxic effect on Hep-2 cells. Rapamycin may facilitate increased Hep-2 cell apoptosis with cisplatin via inhibiting downstream expression of proteins in the AKT-mTOR signaling pathway.