Tumor cell proliferation requires both growth signals and sufficient cellular bioenergetics.The AMP-activated kinase (AMPK) pathway appears dominant over the oncogenic signaling pathway suppressing cell proliferation. This study investigated the preclinical efficacy of targeting the tumor bioenergetic pathway using a glycolysis inhibitor 2-deoxy glucose (2DG) and AMPK agonists, AICAR and metformin. We evaluated the in vitro anti-tumor activity of 2DG, metformin or AICAR alone, and 2DG in combination either with metformin or AICAR. We examined in vivo efficacy using xenograft mouse models. 2DG alone was not sufficient to promote tumor cell death, reflecting the limited efficacy demonstrated in clinical trials. A combined use of 2DG and AICAR also failed to induce cell death. However, 2DG and metformin led to significant cell death associated with decrease in cellular ATP, prolonged activation of AMPK, and sustained autophagy. Gene expression analysis and functional assays revealed that the selective AMPK agonist AICAR augments mitochondrial energy transduction (OXPHOS) while metformin compromises OXPHOS. Importantly, forced energy restoration with methylpyruvate reversed the cell death induced by 2DG and metformin, suggesting a critical role of energetic deprivation in the underlying mechanism of cell death. The combination of 2DG and metformin inhibited tumor growth in mouse xenograft models. Deprivation of tumor bioenergetics by dual inhibition of energy pathways might be an effective novel therapeutic approach for a broad spectrum of human tumors.
Tumor bioenergetics; Targeted therapy; Cancer energy metabolic pathway
Lung cancer is the leading cause of cancer death, developing over prolonged periods through genetic and epigenetic changes induced and exacerbated by tobacco exposure. Many epigenetic changes including DNA methylation and histone methylation and acetylation are reversible, and agents that can modulate these aberrations are a potentially effective approach to cancer chemoprevention. Combined epigenetic-targeting agents have gained interest for their potential to increase efficacy and lower toxicity. The present study applied recently developed statistical methods to validate the combined effects of the demethylating agent 5-aza-2-deoxycytidine (5-AZA-CdR, or AZA, or decitabine) and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, or vorinostat). This validation compared AZA alone with SAHA alone and with their combinations (at later or earlier time points and in varying doses) for inhibiting the growth of cell lines of an in vitro lung carcinogenesis system. This system comprises isogenic premalignant and malignant cells that are immortalized (earlier premalignant), transformed (later premalignant), and tumorigenic human bronchial epithelial (HBE) cells (immortalized BEAS-2B and its derivatives 1799 [immortalized], 1198 [transformed], and 1170-I [tumorigenic]). AZA alone and SAHA alone produced a limited (< 50%) inhibition of cell growth, whereas combined AZA and SAHA inhibited cell growth more than did either agent alone, reaching 90% inhibition under some conditions. Results of drug-interaction analyses in the Emax model and semiparametric model supported the conclusion that the drug combinations exert synergistic effects, i.e., beyond additivity in the Loewe model. The present results demonstrate the applicability of our novel statistical methodology for quantitatively assessing drug synergy across a wide range of doses of agents with complex dose-response profiles, a methodology with great potential for advancing the development of chemopreventive combinations.
lung cancer; epigenetics; vorinostat; decitabine; premalignant; epithelial cells
Retinoids have shown antiproliferative and chemopreventive activity. We analyzed data from a randomized, placebo-controlled chemoprevention trial to determine whether a 3-month treatment with either 9-cis-retinoic acid (RA) or 13-cis-RA and α-tocopherol reduced Ki-67, a proliferation biomarker, in the bronchial epithelium.
Former smokers (n = 225) were randomly assigned to receive 3 months of daily oral 9-cis-RA (100 mg), 13-cis-RA (1 mg/kg) and α-tocopherol (1200 IU), or placebo. Bronchoscopic biopsy specimens obtained before and after treatment were immunohistochemically assessed for changes in the Ki-67 proliferative index (i.e., percentage of cells with Ki-67–positive nuclear staining) in the basal and parabasal layers of the bronchial epithelium. Per-subject and per–biopsy site analyses were conducted. Multicovariable analyses, including a mixed-effects model and a generalized estimating equations model, were used to investigate the treatment effect (Ki-67 labeling index and percentage of bronchial epithelial biopsy sites with a Ki-67 index ≥ 5%) with adjustment for multiple covariates, such as smoking history and metaplasia. Coefficient estimates and 95% confidence intervals (CIs) were obtained from the models. All statistical tests were two-sided.
In per-subject analyses, Ki-67 labeling in the basal layer was not changed by any treatment; the percentage of subjects with a high Ki-67 labeling in the parabasal layer dropped statistically significantly after treatment with 13-cis-RA and α-tocopherol treatment (P = .04) compared with placebo, but the drop was not statistically significant after 9-cis-RA treatment (P = .17). A similar effect was observed in the parabasal layer in a per-site analysis; the percentage of sites with high Ki-67 labeling dropped statistically significantly after 9-cis-RA treatment (coefficient estimate = −0.72, 95% CI = −1.24 to −0.20; P = .007) compared with placebo, and after 13-cis-RA and α-tocopherol treatment (coefficient estimate = −0.66, 95% CI = −1.15 to −0.17; P = .008).
In per-subject analyses, treatment with 13-cis-RA and α-tocopherol, compared with placebo, was statistically significantly associated with reduced bronchial epithelial cell proliferation; treatment with 9-cis-RA was not. In per-site analyses, statistically significant associations were obtained with both treatments.
Lung cancer is the leading cause of cancer-related mortality worldwide. Early detection or prevention strategies are urgently needed to increase survival. Hyperplasia is the first morphologic change that occurs in the bronchial epithelium during lung cancer development, followed by squamous metaplasia, dysplasia, carcinoma in situ, and invasive tumor. The current study was designed to determine the molecular mechanisms that control bronchial epithelium hyperplasia. Using primary normal human tracheobronchial epithelial (NHTBE) cells cultured using the 3-dimensional organotypic method, we found that the epidermal growth factor receptor (EGFR) ligands EGF, transforming growth factor-alpha, and amphiregulin induced hyperplasia, as determined by cell proliferation and multilayered epithelium formation. We also found that EGF induced increased cyclin D1 expression, which plays a critical role in bronchial hyperplasia; this overexpression was mediated by activating the mitogen-activated protein kinase pathway but not the phosphoinositide 3-kinase/Akt signaling pathway. Erlotinib, an EGFR tyrosine kinase inhibitor, and U0126, a MEK inhibitor, completely inhibited EGF-induced hyperplasia. Furthermore, a promoter analysis revealed that the activator protein-1 transcription factor regulates EGF-induced cyclin D1 overexpression. Activator protein-1 depletion using siRNA targeting its c-Jun component completely abrogated EGF-induced cyclin D1 expression. In conclusion, we demonstrated that bronchial hyperplasia can be modeled in vitro using primary NHTBE cells maintained in a 3-dimensional (3-D) organotypic culture. EGFR and MEK inhibitors completely blocked EGF-induced bronchial hyperplasia, suggesting that they have a chemopreventive role.
NHBE; bronchial hyperplasia; dysplasia; erlotinib; MEK inhibitor
Agents can enter clinical development for cancer prevention either initially or after previous development for a different indication, such as arthritis, with both approaches consuming many years of development before an agent is fully evaluated for cancer prevention. We propose the following, third approach: Reverse migration, that is, importing agents, targets and study designs to personalize interventions, and concepts developed in advanced cancer to the setting of cancer prevention. Importing these “ready-made” features from therapy will allow reverse migration to streamline preventive-agent development. We recently reported the Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial of personalized lung-cancer therapy and now propose the reverse-migration development of personalized lung-cancer prevention based on the BATTLE model.
chemoprevention; personalized; targeted; lung cancer
To compare the characteristics of HER receptors and their ligands deregulation between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).
Fifty five NSCLC primary tumors (PT) and corresponding brain metastases (BM) specimens were examined for the immunohistochemical expression of EGFR, phosphorylated (p)–EGFR, Her2, Her3, and p-Her3, and their ligands EGF, TGF-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, and neuregulins-1 and -2. Analysis of EGFR copy number using fluorescent in situ hybridization and mutation by PCR-based sequencing was also performed.
Metastases showed significantly higher immunohistochemical expression of EGF (membrane, BM 66.0 vs. PT 48.5; P=0.027; and nucleus, BM 92.2 vs. 67.4; P=0.008), amphiregulin (nucleus, BM 53.7 vs. PT 33.7; P=0.019), p-EGFR (membrane, BM 161.5 vs. PT 76.0; P<0.0001; and cytoplasm, BM 101.5 vs. PT 55.9; P=0.014), and p-Her3 (membrane, BM 25.0 vs. PT 3.7; P=0.001) than primary tumors (PT) did. Primary tumors showed significantly higher expression of cytoplasmic TGF–α (PT 149.8 vs. BM 111.3; P=0.008) and neuregulin-1 (PT 158.5 vs. BM 122.8; P=0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time points.
Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.
Interindividual variation in genetic background may influence the response to chemotherapy and overall survival for patients with advanced-stage non–small cell lung cancer (NSCLC).
To identify genetic variants associated with poor overall survival in these patients, we conducted a genome-wide scan of 307 260 single-nucleotide polymorphisms (SNPs) in 327 advanced-stage NSCLC patients who received platinum-based chemotherapy with or without radiation at the University of Texas MD Anderson Cancer Center (the discovery population). A fast-track replication was performed for 315 patients from the Mayo Clinic followed by a second validation at the University of Pittsburgh in 420 patients enrolled in the Spanish Lung Cancer Group PLATAX clinical trial. A pooled analysis combining the Mayo Clinic and PLATAX populations or all three populations was also used to validate the results. We assessed the association of each SNP with overall survival by multivariable Cox proportional hazard regression analysis. All statistical tests were two-sided.
SNP rs1878022 in the chemokine-like receptor 1 (CMKLR1) was statistically significantly associated with poor overall survival in the MD Anderson discovery population (hazard ratio [HR] of death = 1.59, 95% confidence interval [CI] = 1.32 to 1.92, P = 1.42 × 10−6), in the PLATAX clinical trial (HR of death = 1.23, 95% CI = 1.00 to 1.51, P = .05), in the pooled Mayo Clinic and PLATAX validation (HR of death = 1.22, 95% CI = 1.06 to 1.40, P = .005), and in pooled analysis of all three populations (HR of death = 1.33, 95% CI = 1.19 to 1.48, P = 5.13 × 10−7). Carrying a variant genotype of rs10937823 was associated with decreased overall survival (HR of death = 1.82, 95% CI = 1.42 to 2.33, P = 1.73 × 10−6) in the pooled MD Anderson and Mayo Clinic populations but not in the PLATAX trial patient population (HR of death = 0.96, 95% CI = 0.69 to 1.35).
These results have the potential to contribute to the future development of personalized chemotherapy treatments for individual NSCLC patients.
Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of NSCLCs, in particular lung adenocarcinomas.
To better characterize TITF-1 abnormality: patterns in NSCLC, we studied TITF-1’s gene copy number using fluorescent in situ hybridization (FISH) and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprised of surgically resected NSCLC (N=321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCCs). TITF-1 copy number and protein expression were correlated with patients’ clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.
We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (p<0.001), never smokers (p=0.004), presence of EGFR mutations (p=0.05) and better overall survival (all stages, p=0.0478. stages I and II, p=0.002). TITF-1 copy number gain (CBG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (p=0.004) and presence of KRAS mutations (p=0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (p=0.039).
Our integrative study demonstrates that the protein versus genomic expression patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.
NSCLC; TITF-1; gene copy gain; lineage-specific oncogenes
Despite continual efforts to develop a prognostic model of gastric cancer by using clinical and pathological parameters, a clinical test that can discriminate patients with good outcomes from those with poor outcomes after gastric cancer surgery has not been established. We aim to develop practical biomarker-based risk score that can predict relapse of gastric cancer after surgical treatment.
Using microarray technologies, we generated and analyzed gene expression profiling data from 65 gastric cancer patients to identify biomarker genes associated with relapse. The association of expression patterns of identified genes with relapse and overall survival was validated in independent gastric cancer patients.
We uncovered two subgroups of gastric cancer that were strongly associated with the prognosis. For the easy translation of our findings into practice, we developed a scoring system based on the expression of six genes that predicted the likelihood of relapse after curative resection. In multivariate analysis, the risk score was an independent predictor of relapse in a cohort of 96 patients. We were able to validate the robustness of the 6-gene signature in an additional independent cohort.
The risk score derived from the 6-gene set successfully prognosticated the relapse of gastric cancer patients after gastrectomy.
Identification of effective markers for outcome is expected to improve the clinical management of non-small cell lung cancer (NSCLC). Here, we assessed in NSCLC the prognostic efficacy of genes, which we had previously found to be differentially expressed in an in vitro model of human lung carcinogenesis.
Prediction algorithms and risk-score models were applied to the expression of the genes in publicly available NSCLC expression datasets. The prognostic capacity of the immunohistochemical expression of proteins encoded by these genes was also tested using formalin-fixed paraffin-embedded (FFPE) tissue specimens from 156 lung adenocarcinomas and 79 squamous cell carcinomas (SCCs).
The survival of all-stages (p<0.001, HR=2.0) or stage-I (p<0.001, HR=2.84) adenocarcinoma patients that expressed the five-gene in vitro lung carcinogenesis model (FILM) signature was significantly poorer than that of patients who did not. No survival differences were observed between SCCs predicted to express or lack FILM signature. Moreover, all stages (p<0.001, HR=1.95) or stage-I (p=0.001, HR=2.6) adenocarcinoma patients predicted to be at high risk by FILM transcript exhibited significantly worse survival than patients at low risk. Furthermore, the corresponding protein signature was associated with poor survival (all stages, p<0.001, HR=3.6; stage-I, p<0.001, HR=3.5; stage-IB, p<0.001, HR=4.6) and mortality risk (all stages, p=0.001, HR=4.0; stage-I, p=0.01, HR=3.4; stage-IB, p<0.001, HR=7.2) in lung adenocarcinoma patients.
Our findings highlight a gene and corresponding protein signature with effective capacity for identification of stage-I lung adenocarcinoma patients with poor prognosis that are likely to benefit from adjuvant therapy.
Lung adenocarcinoma; NSCLC; gene signature; prognosis
Patients with oral preneoplastic lesion (OPL) have high risk of developing oral cancer. Although certain risk factors such as smoking status and histology are known, our ability to predict oral cancer risk remains poor. The study objective was to determine the value of gene expression profiling in predicting oral cancer development. Gene expression profile was measured in 86 of 162 OPL patients who were enrolled in a clinical chemoprevention trial that used the incidence of oral cancer development as a prespecified endpoint. The median follow-up time was 6.08 years and 35 of the 86 patients developed oral cancer over the course. Gene expression profiles were associated with oral cancer-free survival and used to develope multivariate predictive models for oral cancer prediction. We developed a 29-transcript predictive model which showed marked improvement in terms of prediction accuracy (with 8% predicting error rate) over the models using previously known clinico-pathological risk factors. Based on the gene expression profile data, we also identified 2182 transcripts significantly associated with oral cancer risk associated genes (P-value<0.01, single variate Cox proportional hazards model). Functional pathway analysis revealed proteasome machinery, MYC, and ribosomes components as the top gene sets associated with oral cancer risk. In multiple independent datasets, the expression profiles of the genes can differentiate head and neck cancer from normal mucosa. Our results show that gene expression profiles may improve the prediction of oral cancer risk in OPL patients and the significant genes identified may serve as potential targets for oral cancer chemoprevention.
gene expression profiling; oral cancer; oral leukoplakia; biomarker
Leukoplakia is the most common premalignant lesion of the oral cavity. Epidermal growth factor receptor (EGFR) abnormalities are associated with oral tumorigenesis and progression. We hypothesized that EGFR expression and gene copy number changes are predictors of the risk of an oral premalignant lesion (OPL) for progressing to oral squamous cell carcinoma (OSCC). A formalin-fixed, paraffin-embedded OPL biopsy specimen was collected from each of 162 patients in a randomized controlled clinical trial. We assessed EGFR expression by immunohistochemistry with two methods: a semi-quantitative analysis (145 evaluable specimens) and an automated quantitative analysis (127 evaluable specimens). EGFR gene copy number was assessed by fluorescence in situ hybridization (FISH) in a subset of 49 OPLs with high EGFR expression defined by the semi-quantitative analysis. We analyzed EGFR abnormalities for associations with OSCC development. High EGFR expression occurred in 103 (71%) of the 145 OPLs and was associated with a nonsignificantly higher risk of OSCC (P = 0.10). Twenty (41%) of 49 OPLs assessed by FISH had an increased EGFR gene copy number (FISH-positive). Patients with FISH-positive lesions had a significantly higher incidence of OSCC than did patients with FISH-negative (a normal copy number) lesions (P = 0.0007). Of note, 10 of 11 OSCCs that developed at the site of the examined OPL were in the FISH-positive group, leaving only one FISH-negative OPL that did so (P < 0.0001). Our data indicate that an increased EGFR gene copy number is common in and associated with OSCC development in patients with OPLs expressing high EGFR, particularly OSCC developing at the site of a high-expression OPL; they also suggest that EGFR inhibitors may prevent oral cancer in patients with OPLs having an increased EGFR gene copy number.
epithelial growth factor receptor; oral cancer; oral leukoplakia; immunohistochemistry; fluorescence in situ hybridization; biomarker
Tumor hypoxia regulates many cytokines and angiogenic factors (CAFs) and is associated with worse prognosis in head and neck squamous cell cancer (HNSCC). Serum CAF profiling may provide information regarding the biology of the host and tumor, prognosis, and response to therapy. We investigated 38 CAFs in HNSCC patients receiving induction therapy on a Phase II trial of carboplatin, paclitaxel, and cetuximab. CAFs were measured by multiplex bead assay and enzyme-linked immunosorbent assay in 32 patients. Baseline and post-induction CAF levels were correlated with disease progression (PD) and human papilloma virus (HPV) status by Wilcoxon rank sum test. Baseline levels of 8 hypoxia-regulated CAFs (the “high-risk signature” including vascular endothelial growth factor, interleukins-4 and -8, osteopontin, growth-related oncogene-α (Gro-α), eotaxin, granulocyte-colony stimulating factor, and stromal cell derived factor-1α) were associated with subsequent PD. Elevation in ≥6/8 factors was strongly associated with shorter time to progression (p=0.001) and was 73% specific and 100% sensitive for PD. Rising Gro-α from baseline to week six was also associated with PD. Progression free and overall survival were shorter in patients with HPV-negative tumors (p=0.012 and 0.046, respectively), but no individual CAF was associated with HPV-status. However, among 14 HPV-negative patients, the high-risk CAF signature was seen in all 6 patients with PD, but only 2/14 without PD. In conclusion, serum CAF profiling, particularly in HPV-negative patients, may be useful for identifying those at highest risk for recurrence.
head and neck squamous cell cancer; serum markers; hypoxia
Melanoma antigens (MAGE) are frequently expressed in lung cancer and are promising targets of anticancer immunotherapy. Our preliminary data suggested that MAGE may be expressed during early lung carcinogenesis, raising the possibility of targeting MAGE as a lung cancer prevention strategy. The purpose of this study was to investigate MAGE activation patterns in the airways of chronic smokers without lung cancer. MAGE-A1, -A3 and -B2 gene expression was determined in bronchial brush cells from chronic former smokers without lung cancer by reverse transcription-PCR (RT-PCR). The results were correlated with clinical parameters. The 123 subjects had a median age of 57 years, a median of 40 pack-years smoking history, and had quit smoking for at least one year prior to enrollment. Among the subjects, 31 (25%), 38 (31%), and 46 (37%) had detectable MAGE-A1, -A3 and -B2 expression, respectively, in their bronchial brush samples. Expression of MAGE-A1 and -B2 positively correlated with pack-years smoking history (P=0.03 and 0.03, respectively). The frequency of expression did not decrease despite a prolonged smoking cessation period. In conclusion, MAGE-A1, -A3 and -B2 genes are frequently expressed in the bronchial epithelial cells of chronic smokers without lung cancer, suggesting that chronic exposure to cigarette smoke activates these genes even before the malignant transformation of bronchial cells in susceptible individuals. Once activated, the expression persists despite long-term smoking cessation. These data support the targeting of MAGE as a novel lung cancer prevention strategy.
melanoma antigens; airway; smokers; lung cancer; prevention
Squamous cell carcinoma (SCC) in the lung originates from bronchial epithelial cells that acquire increasingly abnormal phenotypes. Currently, no known biomarkers are clinically efficient for the early detection of premalignant lesions and lung cancer. We sought to identify secreted molecules produced from squamous bronchial epithelial cells cultured with organotypic culture methods. We analyzed protein expression patterns in the apical surface fluid (ASF) from aberrantly differentiated squamous metaplastic normal human tracheobronchial epithelial (NHTBE) and mucous NHTBE cells. Comparative two-dimensional PAGE analysis revealed 174 unique proteins in the ASF of squamous NHTBE cells compared to normal mucociliary differentiated NHTBE cells. Among them, 64 well-separated protein spots were identified using liquid chromatography-tandem mass spectrometry, revealing 22 different proteins in the ASF from squamous NHTBE cells. Expression of six of these proteins (SCCA1, SCCA2, S100A8, S100A9, annexin I, and annexin II) in the squamous NHTBE cells was further confirmed with immunoblot analysis. Notably, SCCA1 and SCCA2 were verified as being expressed in squamous metaplastic NHTBE cells but not in normal mucous NHTBE or normal bronchial epithelium. Moreover, SCCA1 and SCCA2 expression increased in in vitro lung carcinogenesis model cell lines with increasing malignancy. In summary, we identified proteins that are uniquely secreted from squamous metaplastic primary human bronchial epithelial cells cultured by the organotypic air-liquid interface method. These ASF proteins may be used to detect abnormal lesions in the lung without collecting invasive biopsy specimens.
bronchial epithelial cells; squamous metaplasia; secretion; biomarker; proteomics
The risk of malignant transformation of oral preneoplastic lesion (OPL) is difficult to assess. DeltaNp63 is an early oncoprotein associated with mucosal tumorigenesis. The purpose of this study was to assess deltaNp63 expression in OPL and its role as a marker of oral cancer risk.
DeltaNp63 expression was determined using immunohistochemistry in 152 OPL patients included in a clinical trial comparing retinyl palmitate alone or plus beta-carotene with low dose 13-cis retinoic acid. The associations between deltaNp63 expression as well as deltaNp63 expression with other potential risk factors for oral cancer development were analyzed.
deltaNp63 expression was positive in 41 (27%) patients, clusters of intraepithelial inflammatory cells (EIC) were noted in 37 (26%) patients, and podoplanin (previously reported), was positive in 56 (37%) patients. Significantly more patients whose lesions were deltaNp63 positive or exhibited EIC developed oral cancers. In the multicovariate analysis including age, treatment, and histologic status as cofactors, positive deltaNp63 expression was associated with an increased hazard ratio of 3.308 (95% CI, 1.663 to 6.580; P=0.0007). Patients whose lesions showed positive deltaNp63, podoplanin, and EIC had the highest oral cancer risk with a hazard ratio of 4.372 (95% CI, 1.912 to 9.992; P=0.0005) and 61% oral cancer development rate at 5 years compared to 15% of other OPL patients (P<0.0001).
deltaNp63 overepression in OPL is associated with increased oral cancer risk. Together, deltaNp63, podoplanin, and EIC may be used as biomarkers to identify OPL patients with substantially high oral cancer risk.
Podoplanin; oral cancer; oral leukoplakia; immunohistochemistry; biomarker
Insulin-like growth factor 1 receptor (IGF-1R) signaling has been implicated in several human neoplasms. However, the role of serum levels of insulin-like growth factors (IGFs) in lung cancer risk is controversial. We assessed the role of tissue-derived IGFs in lung carcinogenesis. We found that IGF-1 and IGF-2 levels in bronchial tissue specimens containing high-grade dysplasia were significantly higher than in those containing normal epithelium, hyperplasia, and squamous metaplasia. Derivatives of human bronchial epithelial cell lines with activation mutation in KRAS (V12) or loss of p53 overexpressed IGF-1 and IGF-2. Transformed characteristics of these cells were significantly suppressed by inactivation of IGF-1R or inhibition of IGF-1 or IGF-2 expression but enhanced by ovrexpresion of insulin-like growth factor receptor (IGF-1R) or exposure to the tobacco carcinogens (TC) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP). We further determined the role of IGF-1R signaling in lung tumorigenesis by determining antitumor activities of the selective IGF-1R tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl] -1-(2-phenyl-quinolin-7-yl) –imidazo [1,5-a]pyrazin-8-ylamine (PQIP) using an in vitro progressive cell system and an in vivo mouse model with a lung-specific IGF-1 transgene after exposure to TCs, including NNK plus BaP. Our results demonstrate that airway epithelial cells produce IGFs in an autocrine or paracrine manner, and these IGFs act jointly with TCs to enhance lung carcinogenesis. Further, the use of selective IGF-1R inhibitors may be a rational approach to controlling lung cancer.
IGF; IGF-1R; lung cancer; cigarette smoking; carcinogenesis
Risk prediction models are useful in clinical decision making. We have published an internally validated prediction tool for lung cancer based on easily obtainable epidemiologic and clinical data. Because the precision of the model was modest, we now estimate the improvement obtained by adding two markers of DNA repair capacity.
Assay data (host-cell reactivation and mutagen sensitivity) were available for 725 White lung cancer cases and 615 controls, all former or current smokers, a subset of cases and controls from the previous analysis. Multivariable models were constructed from the original variables with addition of the biomarkers separately and together. Pairwise comparisons of the area under the receiver operating characteristic curves (AUC) and 3-fold cross-validations were done.
For former smokers, the AUC and 95% confidence intervals were 0.67 (0.63–0.71) for the baseline model and 0.70 (0.66–0.74) for the expanded model. For current smokers, the comparable AUC values were 0.68 (0.64–0.72) and 0.73 (0.69–0.77). For both groups, the expanded models were statistically significantly better than the baseline models (P = 0.006 and P = 0.0048, respectively), although the increases in the concordance statistics were modest. We also recomputed 1-year absolute risks of lung cancer as described previously for two different risk profiles and showed that individuals who exhibited poor repair capacity or heightened mutagen sensitivity had increased absolute risks of lung cancer.
Addition of biomarker assays improved the sensitivity of the expanded models.
Because existing risk prediction models for lung cancer were developed in white populations, they may not be appropriate for predicting risk among African-Americans. Therefore, a need exists to construct and validate a risk prediction model for lung cancer that is specific to African-Americans. We analyzed data from 491 African-Americans with lung cancer and 497 matched African-American controls to identify specific risks and incorporate them into a multivariable risk model for lung cancer and estimate the 5-year absolute risk of lung cancer. We performed internal and external validations of the risk model using data on additional cases and controls from the same ongoing multiracial/ethnic lung cancer case-control study from which the model-building data were obtained as well as data from two different lung cancer studies in metropolitan Detroit, respectively. We also compared our African-American model with our previously developed risk prediction model for whites. The final risk model included smoking-related variables [smoking status, pack-years smoked, age at smoking cessation (former smokers), and number of years since smoking cessation (former smokers)], self- reported physician diagnoses of chronic obstructive pulmonary disease or hay fever, and exposures to asbestos or wood dusts. Our risk prediction model for African-Americans exhibited good discrimination [75% (95% confidence interval, 0.67−0.82)] for our internal data and moderate discrimination [63% (95% confidence interval, 0.57−0.69)] for the external data group, which is an improvement over the Spitz model for white subjects. Existing lung cancer prediction models may not be appropriate for predicting risk for African-Americans because (a) they were developed using white populations, (b) level of risk is different for risk factors that African-American share with whites, and (c) unique group-specific risk factors exist for African-Americans. This study developed and validated a risk prediction model for lung cancer that is specific to African-Americans and thus more precise in predicting their risks. These findings highlight the importance of conducting further ethnic-specific analyses of disease risk.
Deguelin has shown promising chemopreventive and therapeutic activities in diverse types of cancers. However, the potential side effect of deguelin over a certain dose could be the substantial hurdle in the practical application of the drug. One of the successful strategies for the use of deguelin in clinical trials could be lung-specific delivery of the drug. The present study evaluates the efficacy of liposome-encapsulated deguelin with a dose of 0.4mg/kg, which is 10 times less than the dose (4mg/kg) for preventive and therapeutic activities validated in previous in vivo studies. Liposomal deguelin revealed cytotoxic activity in vitro in premalignant and malignant human bronchial epithelial (HBE) cells and non-small-cell lung cancer (NSCLC) cells through the same mechanistic pathway previously reported for deguelin, i.e., suppression of the HSP90 chaperone function and induction of apoptosis. Delivery of liposomal deguelin at a dose of 0.4mg/kg by intranasal instillation resulted in markedly increased drug partitioning to the lungs compared to that of 4mg/kg deguelin or 0.4mg/kg liposomal deguelin administered by oral gavage. Lung-specific delivery of deguelin (0.4mg/kg) via nasal or intratracheal instillation in a liposomal formulation also showed significant chemopreventive and therapeutic activities in 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK)/benzo(a)pyrene (BaP)-treated A/J mice and K-rasLAC57Bl6/129/sv F1 mice with no detectable toxicity. Our findings support the potential use of deguelin in a liposomal formulation via lung-specific delivery to improve efficacy and to reduce the potential side effects of the agent.
liposome-encapsulated deguelin; chemoprevention; therapy; lung cancer; instillation
Hypoxia-inducible factor-1 (HIF-1) has been suggested to play a major role in tumor radioresistance. However, the mechanisms through which irradiation regulates HIF-1α expression remain unclear. The purpose of this study was to investigate the mechanisms that mediate HIF-1 activation and thus radioresistance. Here we show that irradiation induces survival and angiogenic activity in a subset of radioresistant lung cancer cell lines by elevating HIF-1α protein expression. Radiation induced HIF-1α protein expression mainly through two distinct pathways, including an increase in de novo protein synthesis via activation of PI3K/Akt/mTOR and stabilization of HIF-1α protein via augmenting the interaction between heat shock protein 90 (Hsp90) and HIF-1α protein. While the PI3K/Akt/mTOR pathway was activated by irradiation in all the lung cancer cells examined, the HSP90-HIF-1α interaction was enhanced in the resistant cells only. Inhibition of Hsp90 function by 17-AAG or deguelin, a novel natural inhibitor of HSP90, suppressed increases in HIF-1α/Hsp90 interaction and HIF-1α expression in radioresistant cells. Furthermore, combined treatment of radiation with deguelin significantly decreased the survival and angiogenic potential of radioresistant lung cancer cells in vitro. We finally determined in vivo that systemic administration of deguelin resulted in profound inhibition of tumor growth and angiogenesis when combined with radiation. These results provide a strong rationale to target Hsp90 as a means to block radiation-induced HIF-1α and thus to circumvent radioresistance in lung cancer cells.
HIF-1α; Hsp90; Radiation; resistance; deguelin
To investigate whether retinyl palmitate (RP) alone or plus beta-carotene (BC) would be as effective and less toxic than low-dose 13-cis retinoic acid (13cRA) in treating oral premalignant lesions (OPLs) and reducing the risk of oral cancer.
Patients and Methods
Initially, patients were randomly assigned to receive low-dose 13cRA or BC plus RP for 3 years (plus 2 years follow-up). After other randomized trials established an adverse effect of BC on lung cancer incidence/mortality, BC was dropped (patients randomly assigned to 13cRA or RP alone). The primary end point was OPL clinical response at 3 months.
We randomly assigned 162 eligible patients. The 3-month clinical response rate of the combined BC plus RP and RP alone arm (32.5%) was not statistically equivalent to that of 13cRA (48.1%). The clinical response rate of RP alone (20.0%) was significantly lower than that of BC plus RP (42.9%; P = .03). Similar oral cancer–free survival rates were observed across all arms. There was no significant association between 3-month OPL response and subsequent oral cancer development (P = .11). Grades 2 and higher adverse events were more common in the 13cRA than other groups (P < .0001).
This large chemoprevention trial did not establish the equivalence of RP plus BC or RP alone with low-dose 13cRA in reducing the long-term risk of oral cancer. At present, 13cRA, BC plus RP, and RP alone cannot be recommended for chemoprevention, and new, better agents are needed in this setting. Our results did not establish short-term OPL response as a surrogate end point for oral cancer–free survival.
The natural compound deguelin has promising preventive and therapeutic activity against diverse cancers by directly binding to heat-shock protein 90 (Hsp90) and thus suppressing its function. Potential side effects of deguelin over a certain dose, however, could be a substantial obstacle to its clinical use. To develop a derivative(s) of deguelin with reduced potential side effects, we synthesized five deguelin analogues (SH-02, SH-03, SH-09, SH-14 and SH-15) and compared them with the parent compound and each other for structural and biochemical features, solubility, and antiproliferative effects on normal, premalignant and malignant human bronchial epithelial (HBE) and non-small-cell lung cancer (NSCLC) cell lines. Four derivatives destabilized hypoxia-inducible factor-1α as potently as did deguelin. Reverse-phase protein array (RPPA) analysis in H460 NSCLC cells revealed that deguelin and the derivatives suppressed expression of a number of proteins including Hsp90 clients and proteins involved in the phosphoinositide 3 kinase (PI3K)/Akt pathway. One derivative, SH-14, showed several features of potential superiority for clinical use: the highest apoptotic activity; no detectable influence on Src/signal transducer and activator of transcription (STAT) signaling, which can promote cancer progression and is closely related to pathogenesis of Parkinson’s disease (deguelin, SH-02 and SH-03 strongly activated this signaling); better aqueous solubility; and less cytotoxicity to immortalized HBE cells (versus deguelin) at a dose (1 μM) that induced apoptotic activity in most premalignant and malignant HBE and NSCLC cell lines. These collective results suggest that the novel derivative SH-14 has strong potential for cancer chemoprevention and therapy, with equivalent efficacy and lesser toxicity (versus deguelin).
Reverse phase protein array; Hsp90; HIF-1; deguelin; lung cancer
Genetic polymorphisms contribute to interindividual variation in drug response. However, a single polymorphism is likely to exhibit a modest effect. Therefore, we applied a pathway-based approach to evaluate the cumulative effect of multiple polymorphisms on clinical outcome of patients with non-small cell lung cancer (NSCLC).
We genotyped 25 functional polymorphisms in 16 key genes involved in cisplatin metabolism and action and evaluated their associations with overall survival in 229 NSCLC patients receiving first-line cisplatin-based chemotherapy.
Several biologically plausible main effects were identified in individual analysis. More importantly, when 6 polymorphisms in nucleotide excision repair genes were analyzed jointly, a significant trend of reduced risk of death with decreasing number of putative unfavorable genotypes was observed (P for trend <0.001 and log-rank p<0.001). Survival tree analysis revealed potential higher-order gene-gene interactions and categorized subgroups with dramatically different survival experiences, based on distinct genotype profiles. The median survival time was 78.5 months for terminal node 1 in the low-risk group, 15.1 months for terminal node 10 in the medium-risk group, and 6.7 months for terminal node 9 in the high-risk group (log rank P<0.001). We also constructed a prediction hazard model. The area under the curve (AUC) increased from 0.71 (using clinical variables only) to 0.84 (using clinical, epidemiological, and genetic variations from survival tree analysis).
Our results highlight the clinical potential of taking a pathway-based approach and using survival tree analytic approach to identify subgroups of patients with distinctly differing outcomes.
The inhibitor of apoptosis protein survivin is selectively expressed in tumor cells. The tobacco component nicotine increases the transcription of the survivin gene in non-small cell lung cancer cells. However, the role of survivin expression induced by tobacco component is not clear during lung carcinogenesis. We investigated the effects of the tobacco components nicotine and its related carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on survivin expression in normal human bronchial epithelial (NHBE) cells and examined the role of survivin in the malignant transformation of normal human bronchial epithelial (HBE) cells induced by these components. We found that survivin messenger RNA (mRNA) expression was detected in 41% (7 of 17) of bronchial brush specimens from heavy smokers. Nicotine and NNK increased survivin mRNA and protein expression levels in primary cultured NHBE cells and immortalized HBE cells. Bronchial epithelium in mice administered NNK also showed increased staining for survivin. Nicotine and NNK stimulated the Akt–mammalian target of rapamycin (mTOR) pathway in NHBE cells, leading to increased de novo synthesis of survivin protein. Induced survivin expression increased the survival potential of the cells, which was blocked by transfection with survivin-specific small interfering RNA (siRNA). siRNA-induced down-regulation of survivin expression also suppressed the tumorigenic potential of premalignant and malignant HBE cells exposed to the tobacco components. These findings suggest that NNK and nicotine induce survivin protein synthesis in NHBE cells by activating the Akt–mTOR pathway and thus blockade of the pathway effectively inhibits the tobacco-induced malignant transformation of HBE cells.