Metastasis is thought to be governed partially by induction of epithelial–mesenchymal transition. Combination of proteasome and histone deacetylase inhibitors has shown significant promise, but no studies have investigated this in esophageal cancer. This study investigated effects of vorinostat (histone deacetylase inhibitor) and bortezomib (proteasome inhibitor) on esophageal cancer epithelial–mesenchymal transition.
Three-dimensional tumor spheroids mimicking tumor architecture were created with esophageal squamous and adenocarcinoma cancer cells. Cells were treated with tumor necrosis factor α (to simulate proinflammatory tumor milieu) and transforming growth factor β (cytokine critical for induction of epithelial–mesenchymal transition). Tumor models were then treated with vorinostat, bortezomib, or both. Cytotoxic assays assessed cell death. Messenger RNA and protein expressions of metastasis suppressor genes were assessed. After treatment, Boyden chamber invasion assays were performed.
Combined therapy resulted in 3.7-fold decrease in adenocarcinoma cell invasion (P = .002) and 2.8-fold decrease in squamous cell invasion (P = .003). Three-dimensional invasion assays demonstrated significant decrease in epithelial–mesenchymal transition after combined therapy. Quantitative reverse transcriptase polymerase chain reaction and Western blot analyses revealed robust rescue of E-cadherin transcription and protein expression after combined therapy. Importantly, inhibition of the E-cadherin gene resulted in abolition of the salutary benefits of combined therapy, highlighting the importance of this metastasis suppressor gene in the epithelial–mesenchymal transition process.
Combined vorinostat and bortezomib therapy significantly decreased esophageal cancer epithelial–mesenchymal transition. This combined therapeutic effect on esophageal cancer epithelial–mesenchymal transition was associated with upregulation of E-cadherin protein expression.
Chronic renal failure after lung transplantation is associated with significant morbidity. However, the significance of acute kidney injury (AKI) after lung transplantation remains unclear and poorly studied. We hypothesized that hemodialysis (HD)-dependent AKI after lung transplantation is associated with significant mortality.
Materials and methods
We performed a retrospective review of all patients undergoing lung transplantation from July 1991 to July 2009 at our institution. Recipients with AKI (creatinine > 3 mg/dL) were identified. We compared recipients without AKI versus recipients with and without HD-dependent AKI. Kaplan-Meier survival curves were compared by log rank test.
Of 352 lung transplant recipients reviewed at our institution, 17 developed non–HD-dependent AKI (5%) and 16 developed HD-dependent AKI (4.6%). Cardiopulmonary bypass was significantly higher in patients with HD-dependent AKI. None of the recipients who required HD had recovery of renal function. The 30-day mortality was significantly greater in recipients requiring HD (63% versus 0%; P < 0.0001). One-year mortality after transplantation was significantly increased in recipients with HD-dependent AKI compared with those with non–HD-dependent AKI (87.5% versus 17.6%; P < 0.001).
Hemodialysis is associated with mortality after lung transplantation. Fortunately, AKI that does not progress to HD commonly resolves and has a better overall survival. Avoidance, if possible, of cardiopulmonary bypass may attenuate the incidence of AKI. Aggressive measures to identify and treat early postoperative renal dysfunction and prevent progression to HD may improve outcomes after lung transplantation.
Transplantation; Lung; Dialysis; Acute kidney injury; Outcomes; Mortality
The conclusions from the new IASLC/ATS/ERS lung adenocarcinoma classification portend important clinical consequences. The interpretation of the histological, biomolecular and radiological correlates of this classification not only allows for the definitive abandonment of the bronchoalveolar carcinoma definition but provides surgeons with significant clues to better understand the adenocarcinoma subsets and their surgical management. Indeed, the information will benefit surgeons who are fully involved in the lung cancer CT screening programs as well as in the diagnostic and therapeutic pathways of both early and locally advanced lung cancer. Moreover, intriguing perspectives are disclosing on the inclusion of the surgical modality among the ones used in the oligometastatic disease status. On the other hand, the new adenocarcinoma classification also emphasizes the need for surgeons working in a multidisciplinary environment to be thoroughly cognizant of the ever evolving lung cancer biomolecular knowledge and, in particular, of the potentially druggable somatic mutations in line with the modern professional profile of the so-called “surgeon scientist”.
Adenocarcinoma; lung cancer; thoracic surgery
A new histologic classification of lung adenocarcinoma was proposed by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society (IASLC/ATS/ERS) in 2011 to provide uniform terminology and diagnostic criteria for multidisciplinary strategic management. This classification proposed a comprehensive histologic subtyping (lepidic, acinar, papillary, micropapillary, and solid pattern) and a semi-quantitative assessment of histologic patterns (in 5% increments) in an effort to choose a single, predominant pattern in invasive adenocarcinomas. The prognostic value of this classification has been validated in large, independent cohorts from multiple countries. In patients who underwent curative-intent surgery, those with either an adenocarcinoma in situ (AIS) or a minimal invasive adenocarcinoma have nearly 100% disease-free survival and are designated “low grade tumors”. For invasive adenocarcinomas, the acinar and papillary predominant histologic subtypes were usually designated as “intermediate grade” while the solid and micropapillary predominant histologic subtypes were designated “high grade” tumors; this was based on the statistic difference of overall survival. This classification, coupled with additional prognostic factors [nuclear grade, cribriform pattern, high Ki-67 labeling index, thyroid transcription factor-1 (TTF-1) immunohistochemistry, immune markers, and 18F-fluorodeoxyglucose uptake on positron emission tomography (PET)] that we have published on, could further stratify patients into prognostic subgroups and may prove helpful for individual patient care. With regard to Chinese oncologists, the implementation of this new classification only requires hematoxylin and eosin (H&E) stained slides and basic pathologic training, both of which require no additional costs. More importantly, this new classification system could provide informative data for better selection and stratification of clinical trials and molecular studies.
Lung; adenocarcinoma; histologic classification; prognosis
The mechanisms through which the metastasis suppressor gene BRMS1 functions are poorly understood. Herein, we report the identification of a previously undescribed E3 ligase function of BRMS1 on the histone acetyltransferase p300. BRMS1 induces polyubiquitination of p300 resulting in its proteasome-mediated degradation. We identify BRMS1 as the first eukaryote structural mimic of the bacterial IpaH E3 ligase family, and establish that the evolutionarily conserved CXD motif located in in BRMS1 is responsible for its E3 ligase function. Mutation of this E3 ligase motif not only abolishes BRMS1-induced p300 polyubiquitination and degradation, but importantly, dramatically reduces the metastasis suppressor function of BRMS1 in both in vitro and in vivo models of lung cancer metastasis.
BRMS1; metastasis suppressor; p300; E3 ligase; polyubiquitination
In vitro studies have shown that hydrogen peroxide (H2O2) produced by high-concentration ascorbate and cell culture medium iron efficiently kills cancer cells. This provided the rationale for clinical trials of high-dose intravenous ascorbate-based treatment for cancer. A drawback in all the in vitro studies was their failure to take into account the in vivo concentration of iron to supplement cell culture media which are characterized by low iron content. Here we showed, using two prostate cancer cell lines (LNCaP and PC-3) and primary astrocytes, that the anticancer/cytotoxic effects of ascorbate are completely abolished by iron at physiological concentrations in cell culture medium and human plasma. A detailed examination of mechanisms showed that iron at physiological concentrations promotes both production and decomposition of H2O2. The latter is mediated by Fenton reaction and prevents H2O2 accumulation. The hydroxyl radical, which is produced in the Fenton reaction, is buffered by extracellular proteins, and could not affect intracellular targets like H2O2. These findings show that anticancer effects of ascorbate have been significantly overestimated in previous in vitro studies, and that common cell culture media might be unsuitable for redox research.
Network meta-analysis (NMA) enables simultaneous comparison of multiple treatments while preserving randomisation. When summarising evidence to inform an economic evaluation, it is important that the analysis accurately reflects the dependency structure within the data, as correlations between outcomes may have implication for estimating the net benefit associated with treatment. A multivariate NMA offers a framework for evaluating multiple treatments across multiple outcome measures while accounting for the correlation structure between outcomes.
The standard NMA model is extended to multiple outcome settings in two stages. In the first stage, information is borrowed across outcomes as well across studies through modelling the within-study and between-study correlation structure. In the second stage, we make use of the additional assumption that intervention effects are exchangeable between outcomes to predict effect estimates for all outcomes, including effect estimates on outcomes where evidence is either sparse or the treatment had not been considered by any one of the studies included in the analysis. We apply the methods to binary outcome data from a systematic review evaluating the effectiveness of nine home safety interventions on uptake of three poisoning prevention practices (safe storage of medicines, safe storage of other household products, and possession of poison centre control telephone number) in households with children. Analyses are conducted in WinBUGS using Markov Chain Monte Carlo (MCMC) simulations.
Univariate and the first stage multivariate models produced broadly similar point estimates of intervention effects but the uncertainty around the multivariate estimates varied depending on the prior distribution specified for the between-study covariance structure. The second stage multivariate analyses produced more precise effect estimates while enabling intervention effects to be predicted for all outcomes, including intervention effects on outcomes not directly considered by the studies included in the analysis.
Accounting for the dependency between outcomes in a multivariate meta-analysis may or may not improve the precision of effect estimates from a network meta-analysis compared to analysing each outcome separately.
Network meta-analysis; Mixed treatment comparisons; Multiple outcomes; Multivariate; WinBUGS
To determine to what extent underlying data published as part of Quality and Outcomes Framework (QOF) can be used to estimate smoking prevalence within practice populations and local areas and to explore the usefulness of these estimates.
Cross-sectional, observational study of QOF smoking data. Smoking prevalence in general practice populations and among patients with chronic conditions was estimated by simple manipulation of QOF indicator data. Agreement between estimates from the integrated household survey (IHS) and aggregated QOF-based estimates was calculated. The impact of including smoking estimates in negative binomial regression models of counts of premature coronary heart disease (CHD) deaths was assessed.
Primary care in the East Midlands.
All general practices in the area of study were eligible for inclusion (230). 14 practices were excluded due to incomplete QOF data for the period of study (2006/2007–2012/2013). One practice was excluded as it served a restricted practice list.
Estimates of smoking prevalence in general practice populations and among patients with chronic conditions.
Median smoking prevalence in the practice populations for 2012/2013 was 19.2% (range 5.8–43.0%). There was good agreement (mean difference: 0.39%; 95% limits of agreement (−3.77, 4.55)) between IHS estimates for local authority districts and aggregated QOF register estimates. Smoking prevalence estimates in those with chronic conditions were lower than for the general population (mean difference −3.05%), but strongly correlated (Rp=0.74, p<0.0001). An important positive association between premature CHD mortality and smoking prevalence was shown when smoking prevalence was added to other population and service characteristics.
Published QOF data allow useful estimation of smoking prevalence within practice populations and in those with chronic conditions; the latter estimates may sometimes be useful in place of the former. It may also provide useful estimates of smoking prevalence in local areas by aggregating practice based data.
Epidemiology; Statistics & Research Methods; Public Health; Primary Care
Despite complete surgical resection survival in early stage non-small cell lung cancer (NSCLC) remains poor. Based on prior pre-clinical evaluations, we hypothesized that combined induction proteasome and histone deacetylase inhibitor therapy, followed by tumor resection, is feasible.
A phase I clinical trial using a two-staged multiple agent design of bortezomib and vorinostat as induction therapy followed by consolidative surgery in patients with NSCLC was performed. Standard toxicity and MTD were examined. Pre- and post-treatment tumor gene expression arrays were performed and analyzed. Pre- and post-treatment FDG-PET imaging was used to assess tumor metabolism. Finally, serum 20S proteasome levels were analyzed with ELISA, and selected intratumoral proteins were assessed via immunohistochemistry.
Thirty-four patients were consented with 21 patients enrolling in the trial. One patient withdrew early secondary to disease progression. The MTD was bortezomib 1.3 mg/m2 and vorinostat 300 mg BID given. There were (2) grade III dose-limiting toxicities of fatigue and hypophosphatemia that were self-limited. There was no mortality. Thirty percent (6/20) of patients had greater than 60% histologic necrosis of their tumor following treatment, with two having ≥90% tumor necrosis. Tumor metabolism, 20S proteasome activity, and specific protein expression did not demonstrate consistent results. Gene expression arrays comparing pre- and post-therapy NSCLC specimens revealed robust intratumoral changes in specific genes.
Induction bortezomib and vorinostat therapy followed by surgery in patients with operable NSCLC is feasible. Correlative gene expression studies suggest new targets and cell signaling pathways that may be important in modulating this combined therapy.
Histone deacetylase; proteasome inhibitor; lung cancer
Expression of the breast cancer metastasis suppressor 1 (BRMS1) protein is dramatically reduced in non-small cell lung cancer (NSCLC) cells and in primary human tumors. Although BRMS1 is a known suppressor of metastasis, the mechanisms through which BRMS1 functions to regulate cell migration and invasion in response to specific NSCLC driver mutations are poorly understood. To experimentally address this, we utilized immortalized human bronchial epithelial cells in which p53 was knocked down in the presence of oncogenic K-RasV12 (HBEC3-p53KD-K-RasV12). These genetic alterations are commonly found in NSCLC and are associated with a poor prognosis. To determine the importance of BRMS1 for cytoskeletal function, cell migration and invasion in our model system we stably knocked down BRMS1. Here, we report that loss of BRMS1 in HBEC3-p53KD-K-RasV12 cells results in a dramatic increase in cell migration and invasion compared to controls that expressed BRMS1. Moreover, the loss of BRMS1 resulted in additional morphological changes including F-actin re-distribution, paxillin accumulation at the leading edge of the lamellapodium, and cellular shape changes resembling mesenchymal phenotypes. Importantly, re-expression of BRMS1 restores, in part, cell migration and invasion; however it does not fully reestablish the epithelial phenotype. These finding suggests that loss of BRMS1 results in a permanent, largely irreversible, mesenchymal phenotype associated with increased cell migration and invasion. Collectively, in NSCLC cells without p53 and expression of oncogenic K-Ras our study identifies BRMS1 as a key regulator required to maintain a cellular morphology and cytoskeletal architecture consistent with an epithelial phenotype.
Video-assisted thoracic surgery (VATS) lobectomy has become the standard of care for early stage lung cancer throughout the world. Teaching this complex procedure requires adequate case volume, adequate instrumentation, a committed operating room team and baseline experience with open lobectomy. We outline what key maneuvers and steps are required to teach and learn VATS lobectomy. This is most easily performed as part of a thoracic surgery training program, but with adequate commitment and proctoring, there is no reason experienced open surgeons cannot become proficient VATS surgeons. We provide videos showing the key portions of a subcarinal lymph node dissection, posterior hilar dissection of the right upper lobe, fissureless right middle lobectomy, and fissureless left lower lobectomy. These videos highlight what we feel are important principals in VATS lobectomy, i.e., N2 and N1 lymph node dissection, fissureless techniques, and progressive responsibility of the learner. Current literature in simulation of VATS lobectomy is also outlined as this will be the future of teaching in VATS lobectomy.
video-assisted thoracic surgery (VATS) lobectomy; teaching; simulation
Delayed engraftment is a significant limitation of umbilical cord blood (UCB) transplantation due to low stem cell numbers. Inhibition of dipeptidyl peptidase (DPP)-4 enhanced engraftment in murine transplants. We evaluated the feasibility of systemic DPP-4 inhibition using sitagliptin to enhance engraftment of single-unit UCB grafts in adults with hematological malignancies. Twenty-four patients (21–58 years) received myeloablative conditioning, followed by sitagliptin 600 mg orally days −1 to +2, and single UCB grafts day 0. Seventeen receiving red cell-depleted (RCD) grafts, matched at 4 (n=10) or 5 (n=7) of 6 human leucocyte antigen (HLA) loci with median nucleated cell dose 3.6 (2.5–5.2)×107/kg, engrafted at median of 21 (range, 13–50) days with cumulative incidence of 94% (95% confidence interval, 84%–100%) at 50 days. Plasma DDP-4 activity was reduced to 23%±7% within 2 h. Area under DPP-4 activity-time curve (AUCA) correlated with engraftment; 9 of 11 with AUCA <6,000 activity·h engrafted within ≤21 days, while all 6 with higher AUCA engrafted later (P=0.002). Seven patients receiving red cell replete grafts had 10-fold lower colony forming units after thawing compared with RCD grafts, with poor engraftment. Systemic DPP-4 inhibition was well tolerated and may enhance engraftment. Optimizing sitagliptin dosing to achieve more sustained DPP-4 inhibition may further improve outcome.
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5 hours and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10 hours. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation.
The best current noninvasive surrogate for tumor biology is fluorodeoxyglucose positron emission tomography (FDG–PET). Both FDG–PET maximal standardized uptake values and selected tumor markers have been shown to correlate with stage, nodal disease, and survival in non–small cell lung cancer (NSCLC). However, there are limited data correlating FDG–PET with tumor markers. The purpose of this study was to determine the correlation of tumor marker expression with FDG–PET maximal standardized uptake values in NSCLC.
FDG–PET maximal standardized uptake values were calculated in patients with NSCLC (n = 149). No patient had induction chemoradiotherapy. Intraoperative NSCLC tissue was obtained and tissue microarrays were created. Immunohistochemical analysis was performed for 5 known NSCLC tumor markers (glucose transporter 1, p53, cyclin D1, epidermal growth factor receptor, and vascular endothelial growth factor). Each tumor marker was assessed independently by two pathologists using common grading criteria. Subgroup analysis based on histologic characteristics and regional nodal status was performed.
FDG–PET correlated with T classification (P<.0001), N stage (P = .002), and greatest tumor dimension (P<.0001). In addition, increasing maximal standardized uptake values correlated with increased expression of glucose transporter 1 (P<.0001) and p53 (P =.04) in adenocarcinoma. Epidermal growth factor receptor expression correlated with maximal standardized uptake values without predilection for histologic subtype (P = .004).
FDG–PET maximal standardized uptake values correlate with an increased expression of glucose transporter 1 and p53 in lung adenocarcinoma, but not squamous cell cancer. Future studies attempting to correlate FDG–PET with tumor biology will need to consider the effect of different tumor histologic types.
There is a paucity of studies evaluating the change in liver metabolism in subjects receiving hemodialysis. The purpose of this study was to compare the effect of uremic toxins on hepatic cytochrome P450 (CYP)3A4 and CYP2D6 metabolism before and after a 4-hour hemodialysis session. Midazolam and dextromethorphan were incubated with uremic serum collected from subjects before and after the 4-hour hemodialysis session. Analysis and quantification of the 1′-OH-midazolam and 4-OH-midazolam and dextrorphan metabolites were performed by high-pressure liquid chromatography/mass spectrometry. Statistical analysis using the Student’s t-test (paired) was used to compare the amount of metabolite formed. The mean amount of 1′-OH-midazolam, 4-OH-midazolam, and dextrorphan metabolites formed before and after hemodialysis did not significantly differ. There was no significant difference in CYP3A4 and CYP2D6 metabolic activity in uremic serum before and after hemodialysis.
hemodialysis; uremia; CYP3A4; CYP2D6; metabolism
The survival of patients with non–small-cell lung cancer (NSCLC), even when resectable, remains poor. Several small studies suggest that occult metastases (OMs) in pleura, bone marrow (BM), or lymph nodes (LNs) are present in early-stage NSCLC and are associated with a poor outcome. We investigated the prevalence of OMs in resectable NSCLC and their relationship with survival.
Patients and Methods
Eligible patients had previously untreated, potentially resectable NSCLC. Saline lavage of the pleural space, performed before and after pulmonary resection, was examined cytologically. Rib BM and all histologically negative LNs (N0) were examined for OM, diagnosed by cytokeratin immunohistochemistry (IHC). Survival probabilities were estimated using the Kaplan-Meier method. The log-rank test and Cox proportional hazards regression model were used to compare survival of groups of patients. P < .05 was considered significant.
From July 1999 to March 2004, 1,047 eligible patients (538 men and 509 women; median age, 67.2 years) were entered onto the study, of whom 50% had adenocarcinoma and 66% had stage I NSCLC. Pleural lavage was cytologically positive in only 29 patients. OMs were identified in 66 (8.0%) of 821 BM specimens and 130 (22.4%) of 580 LN specimens. In univariate and multivariable analyses OMs in LN but not BM were associated with significantly worse disease-free survival (hazard ratio [HR], 1.50; P = .031) and overall survival (HR, 1.58; P = .009).
In early-stage NSCLC, LN OMs detected by IHC identify patients with a worse prognosis. Future clinical trials should test the role of IHC in identifying patients for adjuvant therapy.
The majority of patients with lung cancer present with metastatic disease. Chronic inflammation and subsequent activation of NF-κB have been associated the development of cancers. The RelA/p65 subunit of NF-κB is typically associated with transcriptional activation. In this report we show that RelA/p65 can function as an active transcriptional repressor through enhanced methylation of the BRMS1 metastasis suppressor gene promoter via direct recruitment of DNMT-1 to chromatin in response to TNF. TNF-mediated phosphorylation of S276 on RelA/p65 is required for RelA/p65-DNMT-1 interactions, chromatin loading of DNMT-1, and subsequent BRMS1 promoter methylation and transcriptional repression. The ability of RelA/65 to function as an active transcriptional repressor is promoter specific as the NF-κB-regulated gene cIAP2 is transcriptionally activated while BRMS1 is repressed under identical conditions. Small molecule inhibition of either of the minimal interacting domains between RelA/p65-DNMT-1 and RelA/p65-BRMS1 promoter abrogates BRMS1 methylation and its transcriptional repression. The ability of RelA/p65 to directly recruit DNMT-1 to chromatin resulting in promoter-specific methylation and transcriptional repression of tumor metastasis suppressor gene BRMS1 highlights a new mechanism through which NF-κB can regulate metastatic disease, and offers a potential target for newer generation epigenetic oncopharmaceuticals.
DNMT-1; Phosphorylation; RelA-p65; TNF; Transcription
Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. A detailed understanding of PTP functions in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific, cell-permeable small molecule agents. We present a stepwise focused library approach that transforms a weak and general nonhydrolyzable pTyr mimetic (F2Pmp, phosphonodifluoromethyl phenylalanine) into a highly potent and selective inhibitor of PTP-MEG2, an antagonist of hepatic insulin signaling. The crystal structures of the PTP-MEG2-inhibitor complexes provide direct evidence that potent and selective PTP inhibitors can be obtained by introducing molecular diversity into the F2Pmp scaffold to engage both the active site and unique nearby peripheral binding pockets. Importantly, the PTP-MEG2 inhibitor possesses highly efficacious cellular activity and is capable of augmenting insulin signaling and improving insulin sensitivity and glucose homeostasis in diet-induced obese mice. The results indicate that F2Pmp can be converted into highly potent and selective PTP inhibitory agents with excellent in vivo efficacy. Given the general nature of the approach, this strategy should be applicable to other members of the PTP superfamily.
The epithelial-mesenchymal transition (EMT) is a de-differentiation process required for wound healing and development. In tumors of epithelial origin aberrant induction of EMT contributes to cancer progression and metastasis. Studies have begun to implicate epigenetic reprogramming in EMT; however, the relationship between reprogramming and the coordination of cellular processes is largely unexplored. We have previously developed a system to study EMT in a canonical non-small cell lung cancer (NSCLC) model. In this system we have shown that the induction of EMT results in constitutive NF-κB activity. We hypothesized a role for chromatin remodeling in the sustained deregulation of cellular signaling pathways.
We mapped sixteen histone modifications and two variants for epithelial and mesenchymal states. Combinatorial patterns of epigenetic changes were quantified at gene and enhancer loci. We found a distinct chromatin signature among genes in well-established EMT pathways. Strikingly, these genes are only a small minority of those that are differentially expressed. At putative enhancers of genes with the ‘EMT-signature’ we observed highly coordinated epigenetic activation or repression. Furthermore, enhancers that are activated are bound by a set of transcription factors that is distinct from those that bind repressed enhancers. Upregulated genes with the ‘EMT-signature’ are upstream regulators of NF-κB, but are also bound by NF-κB at their promoters and enhancers. These results suggest a chromatin-mediated positive feedback as a likely mechanism for sustained NF-κB activation.
There is highly specific epigenetic regulation at genes and enhancers across several pathways critical to EMT. The sites of these changes in chromatin state implicate several inducible transcription factors with critical roles in EMT (NF-κB, AP-1 and MYC) as targets of this reprogramming. Furthermore, we find evidence that suggests that these transcription factors are in chromatin-mediated transcriptional feedback loops that regulate critical EMT genes. In sum, we establish an important link between chromatin remodeling and shifts in cellular reprogramming.
EMT; Epigenetics; Chromatin; Reprogramming; Feedback
atients undergoing resections for suspicious pulmonary lesions have a 9-55% benign rate. Validated prediction models exist to estimate the probability of malignancy in a general population and current practice guidelines recommend their use. We evaluated these models in a surgical population to determine the accuracy of existing models to predict benign or malignant disease.
We conducted a retrospective review of our thoracic surgery quality improvement database (2005-2008) to identify patients who underwent resection of a pulmonary lesion. Patients were stratified into subgroups based on age, smoking status and fluorodeoxyglucose positron emission tomography (PET) results. The probability of malignancy was calculated for each patient using the Mayo and SPN prediction models. Receiver operating characteristic (ROC) and calibration curves were used to measure model performance.
89 patients met selection criteria; 73% were malignant. Patients with preoperative PET scans were divided into 4 subgroups based on age, smoking history and nodule PET avidity. Older smokers with PET-avid lesions had a 90% malignancy rate. Patients with PET- non-avid lesions, or PET-avid lesions with age<50 years or never smokers of any age had a 62% malignancy rate. The area under the ROC curve for the Mayo and SPN models was 0.79 and 0.80, respectively; however, the models were poorly calibrated (p<0.001).
Despite improvements in diagnostic and imaging techniques, current general population models do not accurately predict lung cancer among patients ref erred for surgical evaluation. Prediction models with greater accuracy are needed to identify patients with benign disease to reduce non-therapeutic resections.
Lung Cancer; Lung Cancer; diagnosis; cancer staging; Positron Emission Tomography (PET)
The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has been implicated in metastasis and the generation of cancer initiating cells (CICs) in solid tumors. To examine EMT in non-small cell lung cancer (NSCLC), we utilized a three dimensional (3D) cell culture system in which cells were co-stimulated with tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGFβ). NSCLC spheroid cultures display elevated expression of EMT master-switch transcription factors, TWIST1, SNAI1/Snail1, SNAI2/Slug and ZEB2/Sip1, and are highly invasive. Mesenchymal NSCLC cultures show CIC characteristics, displaying elevated expression of transcription factors KLF4, SOX2, POU5F1/Oct4, MYCN, and KIT. As a result, these putative CIC display a cancer “stem-like” phenotype by forming lung metastases under limiting cell dilution. The pleiotropic transcription factor, NF-κB, has been implicated in EMT and metastasis. Thus, we set out to develop a NSCLC model to further characterize the role of NF-κB activation in the development of CICs. Here, we demonstrate that induction of EMT in 3D cultures results in constitutive NF-κB activity. Furthermore, inhibition of NF-κB resulted in the loss of TWIST1, SNAI2, and ZEB2 induction, and a failure of cells to invade and metastasize. Our work indicates that NF-κB is required for NSCLC metastasis, in part, by transcriptionally upregulating master-switch transcription factors required for EMT.
The ability to predict the efficacy of molecularly-targeted therapies for non-small cell lung cancer (NSCLC) for an individual patient remains problematic. The purpose of this study was to identify tumor biomarkers, using a refined “coexpression extrapolation (COXEN)” algorithm with a continuous spectrum of drug activity, that predict drug sensitivity and therapeutic efficacy in NSCLC to Vorinostat, a histone deacetylase inhibitor, and Velcade, a proteasome inhibitor. Using our refined COXEN algorithm, biomarker prediction models were discovered and trained for Vorinostat and Velcade based on in vitro drug activity profiles of 9 NSCLC cell lines (NCI-9). Independently, a panel of 40 NSCLC cell lines (UVA-40) was treated with Vorinostat or Velcade to obtain 50% growth inhibition values. Genome-wide expression profiles for both the NCI-9 and UVA-40 cell lines were determined using HG-U133A Affymetrix platform. Modeling generated multi-gene expression signatures for Vorinostat (45-gene, p=0.002) and Velcade (15-gene, p=0.0002), with one overlapping gene (CFLAR). Examination of Vorinostat gene ontogeny revealed a predilection for cellular replication and death, whereas those of Velcade suggested involvement in cellular development and carcinogenesis. Multivariate regression modeling of the refined COXEN scores significantly predicted the activity of combination therapy in NSCLC cells (p=0.007). Through the refinement of the COXEN algorithm, we provide an in silico method to generate biomarkers that predict tumor sensitivity to molecularly-targeted therapies. Use of this refined COXEN methodology has significant implications for the a priori examination of targeted therapies to more effectively streamline subsequent clinical trial design and cost.
Lung cancer; histone deacetylase inhibitor; proteasome inhibitor; tumor biomarker; molecularly-targeted agents; chemotherapy
The University HealthSystem Consortium (UHC) mortality risk adjustment models are increasingly being used as benchmarks for quality assessment. But these administrative database models may include postoperative complications in their adjustments for preoperative risk. The purpose of this study was to compare the performance of the UHC with the Society of Thoracic Surgeons (STS) risk-adjusted mortality models for adult cardiac surgery and evaluate the contribution of postoperative complications on model performance.
We identified adult cardiac surgery patients with mortality risk estimates in both the UHC and Society of Thoracic Surgeons databases. We compared the predictive performance and calibration of estimates from both models. We then reestimated both models using only patients without any postoperative complications to determine the relative contribution of adjustments for postoperative events on model performance.
In the study population of 2,171 patients, the UHC model explained more variability (27% versus 13%, p < 0.001) and achieved better discrimination (C statistic = 0.88 versus 0.81, p < 0.001). But when applied in the population of patients without complications, the UHC model performance declined severely. The C statistic decreased from 0.88 to 0.49, a level of discrimination equivalent to random chance. The discrimination of the Society of Thoracic Surgeons model was unchanged (C statistic of 0.79 versus 0.81).
Although the UHC model demonstrated better performance in the total study population, this difference in performance reflects adjustments for conditions that are postoperative complications. The current UHC models should not be used for quality benchmarks.