The pharmacological inhibition of general transcriptional regulators has the potential to block growth through targeting multiple tumorigenic signalling pathways simultaneously. Here, using an innovative cell-based screen, we identify a structurally unique small molecule (named JIB-04) that specifically inhibits the activity of the Jumonji family of histone demethylases in vitro, in cancer cells, and in tumours in vivo. Unlike known inhibitors, JIB-04 is not a competitive inhibitor of α-ketoglutarate. In cancer, but not in patient-matched normal cells, JIB-04 alters a subset of transcriptional pathways and blocks viability. In mice, JIB-04 reduces tumour burden and prolongs survival. Importantly, we find that patients with breast tumours that overexpress Jumonji demethylases have significantly lower survival. Thus, JIB-04, a novel inhibitor of Jumonji demethylases in vitro and in vivo, constitutes a unique potential therapeutic and research tool against cancer, and validates the use of unbiased cellular screens to discover chemical modulators with disease relevance.
Prospectively identifying who will benefit from adjuvant chemotherapy (ACT) would improve clinical decisions for non-small cell lung cancer (NSCLC) patients. In this study, we aim to develop and validate a functional gene set that predicts the clinical benefits of ACT in NSCLC.
An 18-hub-gene prognosis signature was developed through a systems biology approach, and its prognostic value was evaluated in six independent cohorts. The 18-hub-gene set was then integrated with genome-wide functional (RNAi) data and genetic aberration data to derive a 12-gene predictive signature for ACT benefits in NSCLC.
Using a cohort of 442 stage I to III NSCLC patients who underwent surgical resection, we identified an 18-hub-gene set that robustly predicted the prognosis of patients with adenocarcinoma in all validation datasets across four microarray platforms. The hub genes, identified through a purely data-driven approach, have significant biological implications in tumor pathogenesis, including NKX2-1, Aurora Kinase A, PRC1, CDKN3, MBIP, and RRM2. The 12-gene predictive signature was successfully validated in two independent datasets (n = 90 and 176). The predicted benefit group showed significant improvement in survival after ACT (UT Lung SPORE data: HR = 0.34, P = 0.017; JBR.10 clinical trial data: HR = 0.36, P = 0.038), whereas the predicted nonbenefit group showed no survival benefit for 2 datasets (HR = 0.80, P = 0.70; HR = 0.91, P = 0.82).
This is the first study to integrate genetic aberration, genome-wide RNAi data, and mRNA expression data to identify a functional gene set that predicts which resectable patients with non-small cell lung cancer will have a survival benefit with ACT.
We have developed an algorithm that predicted 11,265 potentially polymorphic tandem repeats within transcribed sequences. We estimate that 22% (2,207/9,717) of the annotated clusters within UniGene contain at least one potentially polymorphic locus. Our predictions were tested by allelotyping a panel of approximately 30 individuals for 5% of these regions, confirming polymorphism for more than half the loci tested. Our study indicates that tandem-repeat polymorphisms in genes are more common than is generally believed. Approximately 8% of these loci are within coding sequences and, if polymorphic, would result in frameshifts. Our catalogue of putative polymorphic repeats within transcribed sequences comprises a large set of potentially phenotypic or disease-causing loci. In addition, from the anomalous character of the repetitive sequences within unannotated clusters, we also conclude that the UniGene cluster count substantially overestimates the number of genes in the human genome. We hypothesize that polymorphisms in repeated sequences occur with some baseline distribution, on the basis of repeat homogeneity, size, and sequence composition, and that deviations from that distribution are indicative of the nature of selection pressure at that locus. We find evidence of selective maintenance of the ability of some genes to respond very rapidly, perhaps even on intragenerational timescales, to fluctuating selective pressures.
EML4-ALK fusions define a subset of lung cancers that can be effectively treated with anaplastic lymphoma kinase (ALK) inhibitors. Unfortunately, the duration of response is heterogeneous and acquired resistance limits their ultimate efficacy. Thus, a better understanding of resistance mechanisms will help to enhance tumor control in EML4-ALK-positive tumors.
By applying orthogonal functional mutagenesis screening approaches, we screened for mutations inducing resistance to the aminopyridine PF02341066 (crizotinib) and/or the diaminopyrimidine TAE684.
Here, we show that the resistance mutation, L1196M, as well as other crizotinib resistance mutations (F1174L and G1269S), are highly sensitive to the structurally unrelated ALK inhibitor TAE684. In addition, we identified two novel EML4-ALK resistance mutations (L1198P and D1203N), which unlike previously reported mutations, induced resistance to both ALK inhibitors. An independent resistance screen in ALK-mutant neuroblastoma cells yielded the same L1198P resistance mutation but defined two additional mutations conferring resistance to TAE684 but not to PF02341066.
Our results show that different ALK resistance mutations as well as different ALK inhibitors impact the therapeutic efficacy in the setting of EML4-ALK fusions and ALK mutations.
Non-small cell lung cancer (NSCLC) often expresses mutant KRAS together with tumor-associated mutations of the CDKN2A locus, which are associated with aggressive, therapy-resistant tumors. Here, we unravel specific requirements for the maintenance of NSCLC that carries this genotype. We establish that the extracellular signal-regulated kinase (ERK)/RHOA/focal adhesion kinase (FAK) network is deregulated in high-grade lung tumors. Suppression of RHOA or FAK induces cell death selectively in mutant KRAS;INK4A/ARF-deficient lung cancer cells. Furthermore, pharmacologic inhibition of FAK caused tumor regression specifically in the high-grade lung cancer that developed in mutant Kras;Cdkn2a-null mice. These findings provide a rationale for the rapid implementation of genotype-specific targeted therapies using FAK inhibitors in patients with cancer.
Targeted therapies are effective for only a small fraction of patients with cancer. We report that FAK inhibitors exert potent antitumor effects in NSCLCs that express mutant KRAS in association with INK4A/ARF deficiency. These results reveal a novel genotype-specific vulnerability of cancer cells that can be exploited for therapeutic purposes.
RNA interference (RNAi)--using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein--is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly-L-lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ~47% and ~49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay.
The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RT-PCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for the tumour suppressor gene (TSG) at 3p12, a region defined by hemi and homozygous deletions and functional analysis.
The disabled homolog 2 (DAB2) gene was recently identified as a tumor suppressor gene with its expression downregulated in multiple cancer types. The role of DAB2 in lung tumorigenesis, however, is not fully characterized, and the mechanisms of DAB2 dysregulation in lung cancer are not defined. Here we show that low DAB2 levels in lung tumor specimens are significantly correlated with poor patient survival, and that DAB2 overexpression significantly inhibits cell growth in cultured lung cancer cells, indicating its potent tumor suppressor function. We next identify that microRNA miR-93 functions as a potent repressor of DAB2 expression by directly targeting the 3'UTR of the DAB2 mRNA. Using in vitro and in vivo approaches, we demonstrate that miR-93 overexpression has an important role in promoting lung cancer cell growth, and that its oncogenic function is primarily mediated by downregulating DAB2 expression. Our clinical investigations further indicate that high tumor levels of miR-93 are correlated with poor survival of lung cancer patients. The correlations of both low DAB2 and high miR-93 expression levels with poor patient survival strongly support the critical role of the miR-93/DAB2 pathway in determining lung cancer progression.
It has been hypothesised that clinically evident lung cancers have accumulated many different genetic or epigenetic abnormalities in oncogenes and/or tumour suppressor genes. This notion has important clinical ramifications. Recent developments in our knowledge of the molecular biology of lung cancer are reviewed, with particular reference to genetic abnormalities in tumour suppressor gene inactivation and overactivity of growth promoting oncogenes. These changes lead to the "hallmarks of lung cancer". These hallmarks are the new rational targets for early detection, prevention, and treatment of lung cancer.
SPARC (secreted protein acidic and rich in cysteine) is an extracellular Ca2+-binding matricellular glycoprotein associated with the regulation of cell adhesion and growth. We investigated loss of expression of SPARC gene and promoter methylation in lung cancers and correlated the data with clinicopathological features. We observed loss of SPARC expression in 12 of 20 (60%) lung cancer cell lines. Treatment of expression-negative cell lines with a demethylating agent restored expression in all cases. Methylation frequencies of SPARC gene were 55% in 20 lung cancer cell lines. Primary tumours had methylation at a rate of 69% (119 of 173), while nonmalignant lung tissues (n=60) had very low rates (3%). In lung adenocarcinomas, SPARC methylation correlated with a negative prognosis (P=0.0021; relative risk 4.65, 95% confidence interval 1.75-12.35, multivariate Cox's proportional-hazard model). Immunostaining revealed protein expression in bronchial epithelium (weak intensity) and in juxtatumoral stromal tissues (strong intensity) accompanied by frequent loss in cancer cells that correlated with the presence of methylation (P<0.001). Our findings are of biological interest and potentially of clinical importance in human lung cancers.
Small cell lung cancer (SCLC) is a highly aggressive lung neoplasm with extremely poor clinical outcomes and no approved targeted treatments. To elucidate the mechanisms responsible for driving the SCLC phenotype in hopes of revealing novel therapeutic targets, we studied copy number and methylation profiles of SCLC. We found disruption of the E2F/Rb pathway was a prominent feature deregulated in 96% of the SCLC samples investigated and was strongly associated with increased expression of EZH2, an oncogene and core member of the polycomb repressive complex 2 (PRC2). Through its catalytic role in the PRC2 complex, EZH2 normally functions to epigenetically silence genes during development, however, it aberrantly silences genes in human cancers. We provide evidence to support that EZH2 is functionally active in SCLC tumours, exerts pro-tumourigenic functions in vitro, and is associated with aberrant methylation profiles of PRC2 target genes indicative of a "stem-cell like" hypermethylator profile in SCLC tumours. Furthermore, lentiviral-mediated knockdown of EZH2 demonstrated a significant reduction in the growth of SCLC cell lines, suggesting EZH2 has a key role in driving SCLC biology. In conclusion, our data confirm the role of EZH2 as a critical oncogene in SCLC, and lend support to the prioritization of EZH2 as a potential therapeutic target in clinical disease.
Identification of a homozygous deletion in cancer cells provides strong evidence for the location of a tumor suppressor gene (TSG). We analyzed the 2p24 homozygous deletion of a non-small-cell lung cancer (NSCLC) cell line, NCI-H2882, and found that the deletion size was 3.7 Mbp. Since RhoB, which has been suggested to be a candidate TSG, was located in this region, we analyzed RhoB for alterations in NSCLC. Although we found no mutations in 48 cell lines including 20 NSCLCs, a loss of heterozygosity (LOH) analysis in 128 primary NSCLCs showed that 25 of 62 informative samples had LOH at the RhoB locus. Northern blot analysis of 28 cell lines (including 15 NSCLCs) indicated that RhoB expression was downregulated in 27. We analyzed RhoB expression in 112 primary NSCLCs with immunohistochemistry and found no or a weak RhoB expression in 33 (42%) of 78 adenocarcinomas, whereas we found it in 29 (94%) of 31 squamous cell carcinomas. No or a weak expression of RhoB was more frequently observed in poorly- or moderately-differentiated adenocarcinomas than in well-differentiated ones (p = 0.0014). Furthermore, no or a weak expression of RhoB indicated a tendency to poor patient prognosis. Although hypermethylation was not found at the promoter region, the RhoB expression in NSCLC cell lines was induced by histone deacetylase inhibition, suggesting that RhoB downregulation may be due to histone modification. The present study demonstrates that RhoB expression is frequently downregulated in NSCLCs by multiple mechanisms, suggesting that RhoB is a candidate TSG for NSCLC.
Telomeres maintain genomic integrity in normal cells, and their progressive shortening during successive cell divisions induces chromosomal instability. In the large majority of cancer cells, telomere length is maintained by telomerase. Thus, telomere length and telomerase activity are crucial for cancer initiation and the survival of tumors. Several pathways that regulate telomere length have been identified, and genome-scale studies have helped in mapping genes that are involved in telomere length control. Additionally, genomic screening for recurrent human telomerase gene hTERT promoter mutations and mutations in genes involved in the alternative lengthening of telomeres pathway, such as ATRX and DAXX, has elucidated how these genomic changes contribute to the activation of telomere maintenance mechanisms in cancer cells. Attempts have also been made to develop telomere length- and telomerase-based diagnostic tools and anticancer therapeutics. Recent efforts have revealed key aspects of telomerase assembly, intracellular trafficking and recruitment to telomeres for completing DNA synthesis, which may provide novel targets for the development of anticancer agents. Here, we summarize telomere organization and function and its role in oncogenesis. We also highlight genomic mutations that lead to reactivation of telomerase, and mechanisms of telomerase reconstitution and trafficking that shed light on its function in cancer initiation and tumor development. Additionally, recent advances in the clinical development of telomerase inhibitors, as well as potential novel targets, will be summarized.
Genomic amplification of regions on chromosome arm 5p has been observed frequently in small cell lung cancer (SCLC), implying the presence of multiple oncogenes on this arm. Although conventional comparative genomic hybridization (CGH) detects gross chromosomal copy number changes, gene discovery requires a higher-resolution approach in order to identify regions of alteration precisely. To identify candidate genes on this chromosome arm, we developed a high-resolution, 10-clone-per-megabase bacterial artificial chromosome CGH array for 5p and examined a panel of 15 SCLC cell lines. Utilization of this CGH array has allowed the fine-mapping of breakpoints to regions as small as 200 kb in a single experiment. In addition to reporting our observations of aberrations at the well-characterized SKP2 and TERT loci, we describe the identification of microdeletions that have escaped detection by conventional screens and the identification TRIO and ANKH as novel putative oncogenes.
This study's objectives were to determine whether tumor response measured by computed tomography (CT) and evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) correlated with overall survival (OS) in patients with non-small-cell lung cancer (NSCLC) after neoadjuvant chemotherapy and surgical resection.
We measured primary tumor size on CT before and after neoadjuvant chemotherapy in 160 NSCLC patients who underwent surgical resection. The relationship between CT-measured response (RECIST) and histopathologic response (≤ 10% viable tumor) and OS were assessed by Kaplan-Meier survival, univariable, and multivariable Cox proportional hazards regression.
There was a statistically significant association between CT-measured response (RECIST) and OS (p = 0.03). However, histopathologic response was a stronger predictor of OS (p = 0.002), with a more pronounced separation of the survival curves when compared with CT-measured response. In multivariable Cox regression analysis, only pathologic stage and histopathologic response were significant predictors of OS. A 41% overall discordance rate was noted between CT RECIST response and histopathologic response. CT RECIST classified as nonresponders a subset of patients with histopathologic response (8 out of 30 points, 27%) who demonstrated prolonged survival after neoadjuvant chemotherapy.
We were unable to show that CT RECIST is a reliable predictor of OS in patients with NSCLC undergoing surgical resection after neoadjuvant chemotherapy. The failure of CT RECIST to predict long-term outcome may be because of the inability of CT imaging to consistently identify patients with histopathologic response. CT RECIST may have only a limited role as an efficacy endpoint after neoadjuvant chemotherapy in patients with resectable NSCLC.
This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited.The reproducibility of mass spectrometry (MS) data collected using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) has been questioned. This investigation was designed to test the reproducibility of SELDI data collected over time by multiple users and instruments. Five laboratories prepared arrays once every week for six weeks. Spectra were collected on separate instruments in the individual laboratories. Additionally, all of the arrays produced each week were rescanned on a single instrument in one laboratory. Lab-to-lab and array-to-array variability in alignment parameters were larger than the variability attributable to running samples during different weeks. The coefficient of variance (CV) in spectrum intensity ranged from 25% at baseline, to 80% in the matrix noise region, to about 50% during the exponential drop from the maximum matrix noise. Before normalization, the median CV of the peak heights was 72% and reduced to about 20% after normalization. Additionally, for the spectra from a common instrument, the CV ranged from 5% at baseline, to 50% in the matrix noise region, to 20% during the drop from the maximum matrix noise. Normalization reduced the variability in peak heights to about 18%. With proper processing methods, SELDI instruments produce spectra containing large numbers of reproducibly located peaks, with consistent heights.
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.
Despite recent advances in cancer therapy, the overall 5-year survival rate of patients with lung cancer remains low. The aim of our study was to search for novel markers for early diagnosis in patients with lung cancer.
Complementary DNA microarray analysis was performed in primary lung adenocarcinomas and cell lines to search for differentially expressed genes, followed by in vivo and in vitro tumorigenic assays to characterize the oncogenic potential of the candidate genes. Gene body methylation was analyzed by 450K methylation array, bisulfite sequencing, and quantitative methylation-specific polymerase chain reaction assays. In silico analysis of The Cancer Genome Atlas data set was also performed.
Inositol-trisphosphate 3-kinase A gene (ITPKA), a kinase with limited tissue distribution, was identified as a potential oncogene. We showed that ITPKA expression is up-regulated in many forms of cancers, including lung and breast cancers, and that overexpressed ITPKA contributes to tumorigenesis. We also demonstrated that ITPKA expression is regulated by epigenetic DNA methylation of ITPKA gene body through modulation of the binding of SP1 transcription factor to the ITPKA promoter. ITPKA gene body displayed low or absent levels of methylation in most normal tissue but was significantly methylated in malignant tumors. In lung cancer, ITPKA gene body methylation first appeared at the in situ carcinoma stage and progressively increased after invasion.
ITPKA is a potential oncogene that it is overexpressed in most tumors, and its overexpression promotes tumorigenesis. ITPKA gene body methylation regulates its expression and thus serves as a novel and potential biomarker for early cancer detection.