Green tea is widely consumed throughout the world and is known to possess various beneficial properties that may affect carcinogen metabolism, free radical scavenging, or formation of DNA adducts. Therefore, it is plausible that green tea extract may modify BPDE-induced DNA damage. In this report, we utilized the comet assay to (1) evaluate BPDE-induced DNA damage as a potential marker of cancer susceptibility and (2) assess the ability of green tea to modify BPDE-induced DNA damage. DNA damage in individual comet cells was quantified by (1) visually measuring the proportion of cells exhibiting migration versus those without and (2) the length of damaged DNA migration (comet tail). We detected a dose-response between BDPE concentration and mean comet tail length in EBV-immortalized lymphoblastiod (lymphoid) cell lines. As the concentration of BPDE increased from 0.5 to 3 microM, the length of the mean comet tail length increased proportionally in the 3590P (derived from a healthy subject) and 3640P (derived from a patient with head and neck cancer) cell lines. In separate experiments using lymphoid cells from 21 lung cancer cases and 12 healthy subjects, the mean comet tail length was significantly higher in the lung cancer cases (80.19 +/- 15.55) versus the healthy subjects (59.94 +/- 14.23) (P < 0.01). Similar findings were observed when analyzing the mean percentage of comet induced cells (84.57 +/- 8.85 and 69.04 +/- 12.50, respectively) (P < 0.01). When green tea extract was added in conjunction with BPDE, there was a notable reduction of the mean comet tail length (13.29 +/- 0.97) as compared to BPDE treatment alone (80.19 +/- 15.55) (P < 0.01) in lung cancer cases. There were no statistical differences between the baseline (no treatments) (12.74 +/- 0.63) and the green tea extract treatment (13.06 +/- 0.97) (P = 0.21). These data suggest the modification of lung cancer susceptibility by the green tea extract. Similar results were observed for the percentage of induced comet cells and the statistical trends were similar for the 12 healthy subjects. This preliminary study demonstrated that the detection of BPDE-induced DNA damage via the comet assay may be a useful biologic marker of lung cancer susceptibility. The differential effects in BPDE-induced DNA damage between lung cancer cases and healthy subjects suggests predisposed cancer susceptibility to lung cancer risk. This reports also demonstrated the chemopreventive effects of green tea extract on BPDE-induced DNA damage. These observations provide further support for the application of the comet assay in molecular epidemiologic studies.

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells. Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Lung cancers express lower levels of prostacyclin than normal lung tissues. Prostacyclin prevents lung cancer in a variety of mouse models. A randomized phase II trial comparing oral iloprost (a prostacyclin analog) with placebo in high-risk subjects showed improvement in bronchial histology in former, but not current, smokers. This placebo-controlled study offered the opportunity for investigation of other potential intermediate endpoint and predictive biomarkers to incorporate into chemoprevention trials. Matched bronchial biopsies were obtained at baseline and at 6-month follow-up from 125 high-risk individuals who completed the trial: 31/29 and 37/28 current/former smokers in the iloprost and placebo arm, respectively. We analyzed the expression of 14 selected miRNAs by Real Time PCR in 496 biopsies. The expression of seven miRNAs was significantly correlated with histology at baseline. The expression of miR-34c was inversely correlated with histology at baseline (P < 0.0001) and with change in histology at follow-up (P = 0.0003), independent of treatment or smoking status. Several miRNAs were also found to be differentially expressed in current smokers as compared with former smokers. In current smokers, miR-375 was upregulated at baseline (P < 0.0001) and downregulated after treatment with iloprost (P = 0.0023). No miRNA at baseline reliably predicted a response to iloprost. No biomarker predictive of response to iloprost was found. MiR-34c was inversely correlated with baseline histology and with histology changes. Mir-34c changes at follow-up could be used as a quantitative biomarker that parallels histologic response in formalin-fixed bronchial biopsies in future lung cancer chemoprevention studies.

Epigenetic targets are exciting new avenues for cancer drug discovery. Zhang and colleagues have designed the open-source EZH2 inhibitor JQEZ5 and shown antitumor efficacy in vitro and in vivo in preclinical studies in murine and human lung adenocarcinoma models expressing high levels of EZH2. Cancer Discov; 6(9); 949-52. ©2016 AACRSee related article by Zhang and colleagues, p. 1006.

Oncogene-specific changes in cellular signaling have been widely observed in lung cancer. Here, we investigated how these alterations could affect signaling heterogeneity and suggest novel therapeutic strategies. We compared signaling changes across six human bronchial epithelial cell (HBEC) strains that were systematically transformed with various combinations of TP53, KRAS, and MYC-oncogenic alterations commonly found in non-small cell lung cancer (NSCLC). We interrogated at single-cell resolution how these alterations could affect classic readouts (β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1, and phospho-ERK1/2) of key pathways commonly affected in NSCLC. All three oncogenic alterations were required concurrently to observe significant signaling changes, and significant heterogeneity arose in this condition. Unexpectedly, we found two mutually exclusive altered subpopulations: one with STAT3 upregulation and another with SMAD2/3 downregulation. Treatment with a STAT3 inhibitor eliminated the upregulated STAT3 subpopulation, but left a large surviving subpopulation with downregulated SMAD2/3. A bioinformatics search identified BCL6, a gene downstream of SMAD2/3, as a novel pharmacologically accessible target of our transformed HBECs. Combination treatment with STAT3 and BCL6 inhibitors across a panel of NSCLC cell lines and in xenografted tumors significantly reduced tumor cell growth. We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (STAT3 and BCL6) downstream of common oncogenes, and tumor suppressors may provide a potent way to defeat intratumor heterogeneity. Cancer Res; 77(11); 3070-81. ©2017 AACR.

Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003). The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al., 2003; de Lange, 2002; Karlseder et al., 1999). The method we describe here can be used in normal human and cancer cells. Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.

The acyltransferase Porcupine (Porcn) is essential for the secretion of Wnt proteins which contribute to embryonic development, tissue regeneration, and tumorigenesis. We have previously discovered four molecular scaffolds harboring Porcn-inhibitory activity. Comparison of their structures led to the identification of a general scaffold that can be readily assembled by modular synthesis. We report herein the development of a triazole version of this new class of Porcn inhibitors. This study yielded IWP-O1, a Porcn inhibitor with an EC50 value of 80pM in a cultured cell reporter assay of Wnt signaling. Additionally, IWP-O1 has significantly improved metabolic stability over our previously reported Porcn inhibitors.

Use of molecular profiles and clinical information can help predict which treatment would give the best outcome and survival for each individual patient, and thus guide optimal therapy, which offers great promise for the future of clinical trials and practice. High prediction accuracy is essential for selecting the best treatment plan. The gold standard for evaluating the prediction models is prospective clinical studies, in which patients are enrolled sequentially. However, there is no statistical method using this sequential feature to adapt the prediction model to the current patient cohort. In this article, we propose a reweighted random forest (RWRF) model, which updates the weight of each decision tree whenever additional patient information is available, to account for the potential heterogeneity between training and testing data. A simulation study and a lung cancer example are used to show that the proposed method can adapt the prediction model to current patients' characteristics, and, therefore, can improve prediction accuracy significantly. We also show that the proposed method can identify important and consistent predictive variables. Compared with rebuilding the prediction model, the RWRF updates a well-tested model gradually, and all of the adaptive procedure/parameters used in the RWRF model are prespecified before patient recruitment, which are important practical advantages for prospective clinical studies.

We consider analysis of clustered binary data from multiple observations for each subject in which any two observations from a subject are assumed to have a common correlation coefficient. In the weighted sign test on proportion in clustered binary data, three weighting schemes are considered: equal weights to observations, equal weights to clusters and the optimal weights that minimize the variance of the estimator. Since the distribution of cluster sizes may not be exactly specified before the trial starts, the sample size is usually determined using an average cluster size without taking into account any potential imbalance in cluster size even though cluster size usually varies among clusters. In this paper we investigate the relative efficiency (RE) of unequal versus equal cluster sizes for clustered binary data using the weighted sign test estimators. The REs are computed as a function of correlation among observations within each subject and the various cluster size distributions. The required sample size for unequal cluster sizes will not exceed the sample size for an equal cluster size multiplied by the maximum RE. It is concluded that the maximum RE for various cluster size distributions considered here does not exceed 1.50, 1.61 and 1.12 for equal weights to observations, equal weights to clusters and optimal weights, respectively. It suggests sampling 50%, 61% and 12% more clusters depending on the weighting schemes than the number of clusters computed using an average cluster size.

The effects of radiation in two-dimensional (2D) cell culture conditions may not recapitulate tissue responses as modeled in three-dimensional (3D) organotypic culture. In this study, we determined if the frequency of radiation-induced transformation and cancer progression differed in 3D compared to 2D culture. Telomerase immortalized human bronchial epithelial cells (HBECs) with shTP53 and mutant KRas expression were exposed to various types of radiation (gamma, (+)H, (56)Fe) in either 2D or 3D culture. After irradiation, 3D structures were dissociated and passaged as a monolayer followed by measurement of transformation, cell growth and expression analysis. Cells irradiated in 3D produced significantly fewer and smaller colonies in soft agar than their 2D-irradiated counterparts (gamma P = 0.0004; (+)H P = 0.049; (56)Fe P < 0.0001). The cell culture conditions did not affect cell killing, the ability of cells to survive in a colony formation assay, and proliferation rates after radiation-implying there was no selection against cells in or dissociated from 3D conditions. However, DNA damage repair and apoptosis markers were increased in 2D cells compared to 3D cells after radiation. Ideally, expanding the utility of 3D culture will allow for a better understanding of the biological consequences of radiation exposure.