Identification of ten serum microRNAs from a genome-wide serum microRNA expression profile as novel noninvasive biomarkers for nonsmall cell lung cancer diagnosis

The detection of nonsmall cell lung cancer (NSCLC) at an early stage presents a daunting challenge due to the lack of a specific noninvasive marker. The discovery of microRNAs (miRNAs), particularly those found in serum, has opened a new avenue for tumor diagnosis. To determine whether the expression profile of serum miRNAs can serve as a NSCLC fingerprint, we performed Taqman probe-based quantitative RT-PCR assay to selected differentially expressed serum miRNAs from a sample set including 400 NSCLC cases and 220 controls, and risk score analysis to evaluate the diagnostic value of the serum miRNA profiling system. After a two-phase selection and validation process, 10 miRNAs were found to have significantly different expression levels in NSCLC serum samples compared with the control serum samples. Risk score analysis showed that this panel of miRNAs was able to distinguish NSCLC cases from controls with high sensitivity and specificity. Under ROC curves, the AUC for tumor identification in training set and validation set were 0.966 and 0.972, respectively. Furthermore, the expression profile of the 10-serum miRNAs was correlated with the stage of NSCLC patients, especially in younger patients and patients with current smoking habits. More importantly, the serum miRNA-based biomarker for early NSCLC detection was supported by a retrospective analysis in which the 10-serum miRNA profile could accurately classify serum samples collected up to 33 months ahead of the clinical NSCLC diagnosis. Taken together, we demonstrate that the profiling of 10-serum miRNAs provides a novel noninvasive biomarker for NSCLC diagnosis.     

Hsp90表达上调与肺癌细胞化疗耐药的相关性研究

背景与目的热休克蛋白(heatshockprotein,Hsp)90是Hsps家族中的重要成员,其高表达与肿瘤的发生、发展及化疗耐药密切相关。本研究旨在探讨替普瑞酮(geranylgeranylacetone,GGA)作用下人肺癌细胞SPCA-1及H446中Hsp90的表达水平变化及与肺癌细胞对顺铂化疗耐药的相关性。方法将细胞分成实验组与对照组,分别用含有不同浓度(0μM,10μM,50μM,100μM,500μM,1,000μM)的诱导剂GGA处理6h。采用免疫荧光细胞化学及West-ernblot方法检测各组细胞中Hsp90在蛋白水平的表达;应用MTT法测定细胞在化疗药物顺铂作用下生存率,并分析Hsp90的表达在两种肺癌细胞对顺铂耐药中的作用。结果 SPCA-1及H446实验组细胞中Hsp90的表达水平均明显高于相应的对照组细胞,且与GGA具有一定的浓度依赖性。MTT显示两种实验组细胞对顺铂的生存率均明显高于相应的对照组细胞,亦呈一定的GGA浓度依赖性。结论 GGA可以诱导人肺癌细胞SPCA-1及H446中的Hsp90的表达上调,且其表达水平都与GGA具有一定的浓度依赖性。Hsp90高表达的细胞对顺铂的生存率明显高于低表达的细胞,表明Hsp90的表达上调与肺癌细胞对顺铂的耐药有一定的相关性。     

Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells

Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the “non-oncogene” addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded “client” proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G₂-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.     

Oxidative stress measured by thioredoxin reductase level as potential biomarker for prostate cancer

The aims of this study were to determine if Thioredoxin reductase (TR) is detected in the serum, and to establish the sensitivity and specificity of serum TR for diagnosing prostate cancer (PC). We assessed serum TR in 380 participants in the training cohort: 160 patients with PC, 120 with benign prostatic hyperplasia and 100 healthy individuals. The validation cohort comprised 320 participants: 120 with PC, 100 with BPH and 100 healthy individuals. TR was measured in serum by ELISA by independent researchers. The patients with PC were graded using the Gleason system. Receiver operating characteristic (ROC) curves were utilized to evaluate the accuracy of biomarkers to diagnose PC. The influence of serum levels of TR on tumor grade and metastasis was performed by binary logistic regression analysis. The median levels of serum TR in PC were significantly higher than that of healthy subjects and patients with BPH (P < 0.0001). Based on the ROC curve, the optimal cutoff value of serum TR levels as an indicator for auxiliary diagnosis of PC from BPH was projected to be 8.2 U/ml, which yielded a sensitivity of 81.8% and a specificity of 68.9%, with the area under the curve at 0.862 (95% CI, 0.821-0.903). Combined model (TR and PSA) showed a significantly greater discriminatory ability as compared with those markers alone. In regression analysis, after adjusting for other significant predictors, TR remained an independent metastasis predictor with an adjusted OR of 4.99 (95% CI, 2.64-8.09). Similarly, TR also was an independent High-grade tumors (HGT) predictor with an adjusted OR of 5.15 (95% CI, 2.52-9.14). Our study has demonstrated the additional benefit of TR measurement in the diagnosis of PC in the Chinese population. Further studies of the application of TR in this region may be beneficial.     

Hsp90潜在抑制剂姜黄素诱导人宫颈癌Hela细胞凋亡的机制

目的:研究姜黄素对人宫颈癌细胞Hela的抑制作用及对Hsp90信号通路的影响.方法:MTT法检测姜黄素对Hela细胞的增殖抑制作用.划痕实验检测姜黄素对细胞浸润侵袭的影响.流式细胞术检测姜黄素促进细胞凋亡的效果.Western blotting检测姜黄素对Hela细胞内Hsp90、Hsp70、AKT及Her-2蛋白表达的影响.结果:作用48h后,姜黄素对Hela细胞的增殖抑制呈现出显著的剂量依赖性(P＜0.01),当药物浓度为100 μmol/L时抑制率达到(87.5 ±3.5)％,IC50值为(41.8 ±2.3)μmol/L.划痕实验表明,姜黄素能抑制细胞的浸润和迁移.流式细胞结果显示,姜黄素能有效的促进Hela细胞凋亡,40 μmol/L组细胞的凋亡率即从原来的(10.3±0.6)％提高至(19.1±1.5)％(P＜0.05),当药物浓度达到100 μmol/L时凋亡率达到(55.8±2.6)％.Westem blotting结果显示,姜黄素能引起Hsp70蛋白表达上调,并促进Hsp90下游客户蛋白AKT及Her-2降解.结论:姜黄素表现出较强的抗Hela细胞活性,其机制可能是通过抑制Hsp90活性,影响相关信号通路引起的.     

Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease

Background:

The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic.

Methods:

We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours.

Results:

Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol.

Conclusions:

The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer.     

Assessment of a fragment of e-cadherin as a serum biomarker with predictive value for prostate cancer

In prostate cancer, biomarkers may provide additional value above standard clinical and pathology parameters to predict outcome after specific therapy. The purpose of this study is to evaluate an 80 kDa fragment of the cell adhesion molecule e-cadherin as a serum biomarker. A broad spectrum of prostate cancer serum samples, representing different stages of prostate cancer disease, including benign prostatic hyperplasia (BPH), localised (Loc PCA) and metastatic prostate cancer (Met PCA), was examined for the cleaved product. There is a significant difference in the expression level of the 80 kDa fragment in the serum of healthy individuals vs patients with BPH and between BPH vs Loc PCA and Met PCA (P<0.001). Highest expression levels are observed in advanced metastatic disease. In the cohort of Loc PCA cases, there was no association between the 80 kDa serum concentration and clinical parameters. Interestingly, patients with an 80 kDa level of >7.9 microg l(-1) at the time of diagnosis have a 55-fold higher risk of biochemical failure after surgery compared to those with lower levels. This is the first report of the application of an 80 kDa fragment of e-cadherin as a serum biomarker in a broad spectrum of prostate cancer cases. At an optimised cutoff, high expression at the time of diagnosis is associated with a significantly increased risk of biochemical failure, potentially supporting its use for a tailored follow-up protocol for those patients.     

Mammaglobin peptide as a novel biomarker for breast cancer detection

Among the different types of tests used for cancer diagnosis, molecular tests have been increrasingly incorporated because of their ability to detect either expression or functional changes in the molecules associated with the disease. Mammaglobin is a protein found in mammary tissue and can be detected in serum. This protein has been proposed as a biomarker to diagnose breast cancer, given that patients exhibit an increased amount of the protein in serum and tumor tissue, in comparison to healthy individuals. The ELISA test was used in the present study to detect mammaglobin in blood samples from 51 breast cancer patients and 51 control individuals. Antibodies against mamaglobin were generated in rabbits by using the following synthetic peptides: A (amino acids 13 to 21), B (amino acids 31 to 39), C (amino acids 56 to 64) and a D peptide, corresponding to the protein isoform without three amino acids (59, 60 and 61 amino acids) from peptide C. All peptides were immunogenic and allowed generation of antibodies that were able to discriminate patients from controls. The best results were obtained for antiserum B, achieving the best sensitivity (86.3%) and specificity (96%).     

Telomere length variation: A potential new telomere biomarker for lung cancer risk

ObjectivesIn this report the associations between telomere length variation (TLV), mean telomere length in blood lymphocytes and lung cancer risk were examined.Materials and methodsThe study design is case–control. Cases (N = 191) were patients newly diagnosed with histologically confirmed non-small cell lung cancer. Controls (N = 207) were healthy individuals recruited from the same counties as cases and matched to cases on age and gender. Telomere fluorescent in situ hybridization was used to measure telomere features using short-term cultured blood lymphocytes. Logistic regression was used to estimate the strength of association between telomere features and lung cancer risk.ResultsTelomere length variation across all chromosomal ends was significantly associated with lung cancer risk; adjusted odds ratios 4.67 [95% confidence interval (CI): 1.46–14.9] and 0.46 (95% CI: 0.25–0.84) for younger (age ≤ 60) and older (age > 60) individuals, respectively. TLV and mean telomere length jointly affected lung cancer risk: when comparing individuals with short telomere length and high TLV to those with long telomere length and low TLV, adjusted odd ratios were 8.21 (95% CI: 1.71–39.5) and 0.33 (95% CI: 0.15–0.72) for younger and older individuals, respectively.ConclusionsTLV in blood lymphocytes is significantly associated with lung cancer risk and the associations were modulated by age. TLV in combination with mean telomere length might be useful in identifying high risk population for lung cancer computerized tomography screening.     

Identification of plasma microRNA-21 as a biomarker for early detection and chemosensitivity of non-small cell lung cancer

Studies have shown cell-free microRNA(miRNA) circulating in the serum and plasma with specific expression in cancer,indicating the potential of using miRNAs as biomarkers for cancer diagnosis and therapy.This study was to investigate whether plasma miRNA-21(miR-21) can be used as a biomarker for the early detection of non-small cell lung cancer(NSCLC) and to explore its association with clinicopathologic features and sensitivity to platinum-based chemotherapy.We used real-time RT-PCR to investigate the expr...     

Pharmacogenomics in cancer drug discovery and development: inhibitors of the Hsp90 molecular chaperone

Drug discovery is being revolutionised by a number of technological developments. These include high throughput screening, combinatorial chemistry and genomics. The impact of the new technologies is to accelerate the pace of anticancer discovery. The completion of the Human Genome Project and the ongoing high throughput sequencing of cancer genomes will facilitate the identification of a range of new molecular targets for drug discovery. Over the next few years we will have a complete molecular understanding of the various combinations of genes and cognate pathways that drive the malignant phenotype and tumour progression. The vision for postgenomic cancer drug discovery must be to identify therapeutic agents that correct or exploit each of these molecular abnormalities. In this way, it will be possible to develop personalised drug combinations that are targeted to the molecular make up of individual tumours. It is anticipated that these therapies will be more effective and less toxic than current approaches, although combinations of novel agents with existing cytotoxic therapies are likely to continue for some time. Examples of postgenomic, mechanism-based drugs include Glivec, Herceptin and Iressa, with many more agents undergoing preclinical and clinical development. An interesting new approach involves the development of inhibitors of heat shock protein (Hsp90) molecular chaperone. Because Hsp90 is required for the correct folding, stability and function of a range of oncoproteins that are mutated or over expressed in cancer, Hsp90 inhibitors have the potential to provide a simultaneous, combinatorial attack on multiple oncogenic pathways. By depleting the levels of multiple oncoproteins in cancer cells and blocking a wide range of oncogenic pathways, Hsp90 inhibitors have the potential to inhibit all of the hallmark characteristics of cancer cells. Progress in the preclinical and clinical development of Hsp90 inhibitors will be described, including an update on clinical studies with the first-in-class agent 17AAG. The use of the postgenomic technology of gene expression microarrays in cancer pharmacology and drug development will be exemplified.     

AKAP4 is a circulating biomarker for non-small cell lung cancer

Cancer testis antigens (CTAs) are widely expressed in tumor tissues, circulating tumor cells (CTCs) and in cancer derived exosomes that are frequently engulfed by lymphoid cells. To determine whether tumor derived CTA mRNAs could be detected in RNA from purified peripheral blood mononuclear cells (PBMC) of non-small cell lung cancer (NSCLC) patients, we assayed for the expression of 116 CTAs in PBMC RNA in a discovery set and identified AKAP4 as a potential NSCLC biomarker. We validated AKAP4 as a highly accurate biomarker in a cohort of 264 NSCLCs and 135 controls from 2 different sites including a subset of controls with high risk lung nodules. When all (264) lung cancers were compared with all (135) controls the area under the ROC curve (AUC) was 0.9714. When 136 stage I NSCLC lung cancers are compared with all controls the AUC is 0.9795 and when all lung cancer patients were compared to 27 controls with histologically confirmed benign lung nodules, a comparison of significant clinical importance, the AUC was 0.9825. AKAP4 expression increases significantly with tumor stage, but independent of age, gender, smoking history or cancer subtype. Follow-up studies in a small number of resected NSCLC patients revealed a decrease of AKAP4 expression post-surgical resection that remained low in patients in remission and increased with tumor recurrence. AKAP4 is a highly accurate biomarker for the detection of early stage lung cancer.     

Clinical significance of serum thymus and activation‐regulated chemokine in gastric cancer: Potential as a serum biomarker

Thymus and activation‐regulated chemokine (TARC) can stimulate cancer cell proliferation and migration. The present study evaluated the clinical significance of serum TARC in gastric cancer (GC). We measured serum TARC, macrophage‐derived chemokine, monocyte chemotactic protein‐1 and stem cell factor (SCF) levels using a chemiluminescent immunoassay along the GC carcinogenesis (normal, high‐risk, early GC [EGC] and advanced GC [AGC]) in both training (N = 25 per group) and independent validation datasets (90 normal, 30 high‐risk, 50 EGC and 50 AGC). Serum levels were compared among groups using one‐way analysis of variance. To evaluate the diagnostic potential of serum TARC for GC, receiver operating characteristic curve and logistic regression analyses were performed. Correlations between serum TARC and GC clinicopathological features were analyzed using Spearman's correlation. In the training dataset, serum TARC correlated with serum MDC, MCP‐1 and SCF. However, only serum TARC and SCF were significantly higher in cancer groups than non‐cancer groups (P < 0.001). In the validation dataset, serum TARC also increased along the GC carcinogenesis; the AGC group (167.2 ± 111.1 ng/mL) had significantly higher levels than the EGC (109.1 ± 67.7 ng/mL), the high‐risk (66.2 ± 47.7 ng/mL) and the normal (67.5 ± 36.2 ng/mL) groups (Bonferroni, all P < 0.001). Receiver operating characteristic curves and logistic regression demonstrated the remarkable diagnostic potential of serum TARC as a single marker (72.0% sensitivity and 71.1% specificity; cutoff point, 0.37; logistic regression) and in a multiple‐marker panel (72.6% sensitivity and 88.2% specificity; cutoff point, 0.54). Spearman's correlation showed that serum TARC was closely correlated with tumor size (γ_s = 0.227, P = 0.028), T‐stage (γ_s = 0.340, P = 0.001), N‐stage (γ_s = 0.318, P = 0.002) and M‐stage (γ_s = 0.346, P = 0.001). Serum TARC is a promising serum biomarker for GC.     

Identification of serum angiopoietin-2 as a biomarker for clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy

Background:

The combination of chemotherapy with the vascular endothelial growth factor (VEGF) antibody bevacizumab is a standard of care in advanced colorectal cancer (CRC). However, biomarkers predicting outcome of bevacizumab-containing treatment are lacking. As angiopoietin-2 (Ang-2) is a key regulator of vascular remodelling in concert with VEGF, we investigated its role as a biomarker in metastatic CRC.

Methods:

Serum Ang-2 levels were measured in 33 healthy volunteers and 90 patients with CRC. Of these, 34 had metastatic disease and received bevacizumab-containing therapy. To determine the tissue of origin of Ang-2, quantitative real-time PCR was performed on microdissected cryosections of human CRC and in a murine xenograft model of CRC using species-specific amplification.

Results:

Ang-2 originated from the stromal compartment of CRC tissues. Serum Ang-2 levels were significantly elevated in patients with metastatic CRC compared with healthy controls. Amongst patients receiving bevacizumab-containing treatment, low pre-therapeutic serum Ang-2 levels were associated with a significant better response rate (82 vs 31% P<0.01), a prolonged median progression-free survival (14.1 vs 8.5 months; P<0.01) and a reduction of 91% in the hazard of death (P<0.05).

Conclusion:

Serum Ang-2 is a candidate biomarker for outcome of patients with metastatic CRC treated with bevacizumab-containing therapy, and it should be further validated to customise combined chemotherapeutic and anti-angiogenic treatment.     

Sensitization of multidrug-resistant human cancer cells to Hsp90 inhibitors by down-regulation of SIRT1

The effectiveness of Hsp90 inhibitors as anticancer agents was limited in multidrug-resistant (MDR) human cancer cells due to induction of heat shock proteins (Hsps) such as Hsp70/Hsp27 and P-glycoprotein (P-gp)-mediated efflux. In the present study, we showed that resistance to Hsp90 inhibitors of MDR human cancer cells could be overcome with SIRT1 inhibition. SIRT1 knock-down or SIRT1 inhibitors (amurensin G and EX527) effectively suppressed the resistance to Hsp90 inhibitors (17-AAG and AUY922) in several MDR variants of human lymphoblastic leukemia and human breast cancer cell lines. SIRT1 inhibition down-regulated the expression of heat shock factor 1 (HSF1) and subsequently Hsps and facilitated Hsp90 multichaperone complex disruption via hyperacetylation of Hsp90/Hsp70. These findings were followed by acceleration of ubiquitin ligase CHIP-mediated mutant p53 (mut p53) degradation and subsequent down-regulation of P-gp in 17-AAG-treated MDR cancer cells expressing P-gp and mut p53 after inhibition of SIRT1. Therefore, combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be a more effective therapeutic approach for Hsp90 inhibitor-resistant MDR cells via down-regulation of HSF1/Hsps, mut p53 and P-gp.