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ABSTRACT: Abstract
Background
Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), increases survival in patients with advanced EGFR-positive non-small cell lung cancer when administrated in combination with chemotherapy. In this study, we investigated the role of complement activation in the antitumor mechanism of this therapeutic drug.
Results
EGFR-expressing lung cancer cell lines were able to bind cetuximab and initiate complement activation by the classical pathway, irrespective of the mutational status of EGFR. This activation led to deposition of complement components and increase in complement-mediated cell death. The influence of complement activation on the activity of cetuximab in vivo was evaluated in xenografts of A549 lung cancer cells on nude mice. A549 cells express wild-type EGFR and have a KRAS mutation. Cetuximab activity against A549 xenografts was highly dependent on complement activation, since complement depletion completely abrogated the antitumor efficacy of cetuximab. Moreover, cetuximab activity was significantly higher on A549 cells in which a complement inhibitor, factor H, was genetically downregulated.
Conclusions
We demonstrate for the first time that the in vivo antitumor activity of cetuximab can be associated with a complement-mediated immune response. These results may have important implications for the development of new cetuximab-based therapeutic strategies and for the identification of markers that predict clinical response.
Molecular Cancer. 01/2010;
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ABSTRACT: Abstract Background Different isoforms of VEGF-A (mainly VEGF121, VEGF165 and VEGF189) have been shown to display particular angiogenic properties in the generation of a functional tumor vasculature. Recently, a novel class of VEGF-A isoforms, designated as VEGFxxxb, generated through alternative splicing, have been described. Previous studies have suggested that these isoforms may inhibit angiogenesis. In the present work we have produced recombinant VEGF121/165b proteins in the yeast Pichia pastoris and constructed vectors to overexpress these isoforms and assess their angiogenic potential. Results Recombinant VEGF121/165b proteins generated either in yeasts or mammalian cells activated VEGFR2 and its downstream effector ERK1/2, although to a lesser extent than VEGF165. Furthermore, treatment of endothelial cells with VEGF121/165b increased cell proliferation compared to untreated cells, although such stimulation was lower than that induced by VEGF165. Moreover, in vivo angiogenesis assays confirmed angiogenesis stimulation by VEGF121/165b isoforms. A549 and PC-3 cells overexpressing VEGF121b or VEGF165b (or carrying the PCDNA3.1 empty vector, as control) and xenotransplanted into nude mice showed increased tumor volume and angiogenesis compared to controls. To assess whether the VEGFxxxb isoforms are differentially expressed in tumors compared to healthy tissues, immunohistochemical analysis was conducted on a breast cancer tissue microarray. A significant increase (p < 0.05) in both VEGFxxxb and total VEGF-A protein expression in infiltrating ductal carcinomas compared to normal breasts was observed. A positive significant correlation (r = 0.404, p = 0.033) between VEGFxxxb and total VEGF-A was found. Conclusions Our results demonstrate that VEGF121/165b are not anti-angiogenic, but weakly angiogenic isoforms of VEGF-A. In addition, VEGFxxxb isoforms are up-regulated in breast cancer in comparison with non malignant breast tissues. These results are to be taken into account when considering a possible use of VEGF121/165b-based therapies in patients.
Molecular Cancer. 01/2010;
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Ruben Pio,
David Blanco,
Maria Pajares,
Elena Aibar,
Olga Durany,
Teresa Ezponda,
Jackeline Agorreta,
Javier Gomez-Roman,
Miguel Anton,
Angel Rubio,
Maria Lozano,
Jose López-Picazo,
Francesc Subirada,
Tamara Maes, Luis Montuenga
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ABSTRACT: Abstract
Background
Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events.
Results
The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6 , VEGF , and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1 , FHL-1 , MLPH , and SUSD2. None of these splicing isoforms had been previously associated with lung cancer.
Conclusions
This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies.
BMC Genomics. 01/2010;
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Paul A Nguewa,
Jackeline Agorreta,
David Blanco,
Maria Dolores Lozano,
Javier Gomez-Roman,
Blas A Sanchez,
Iñaki Valles,
Maria J Pajares,
Ruben Pio,
Maria Jose Rodriguez, Luis M Montuenga,
Alfonso Calvo
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ABSTRACT: The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven.
We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |Delta Ct| (= |Ct Normal-Ct tumor|) +/- SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |Delta Ct| (0.70 +/- 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.
Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.
BMC Molecular Biology 12/2008; 9:103. · 2.86 Impact Factor
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Paul Nguewa,
Jackeline Agorreta,
David Blanco,
Maria Lozano,
Javier Gomez-Roman,
Blas Sanchez,
Iñaki Valles,
Maria Pajares,
Ruben Pio,
Maria Rodriguez, Luis Montuenga,
Alfonso Calvo
[show abstract]
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ABSTRACT: Abstract
Background
The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven.
Results
We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder , mean of |ΔCt| (= |Ct Normal-Ct tumor|) ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09), with no statistically significant differences between normal and malignant samples and with excellent expression stability.
Conclusion
Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.
BMC Molecular Biology. 01/2008;
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Juan P de Torres,
Gorka Bastarrika,
Juan P Wisnivesky,
Ana B Alcaide,
Arantza Campo,
Luis M Seijo,
Jesús C Pueyo,
Alberto Villanueva,
María D Lozano,
Usua Montes, Luis Montuenga,
Javier J Zulueta
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ABSTRACT: Identification of risk factors for lung cancer can help in selecting patients who may benefit the most from smoking cessation interventions, early detection, or chemoprevention.
To evaluate whether the presence of emphysema on low-radiation-dose CT (LDCT) of the chest is an independent risk factor for lung cancer.
The study used data from a prospective cohort of 1,166 former and current smokers participating in a lung cancer screening study. All individuals underwent a baseline LDCT and spirometry followed by yearly repeat LDCT studies. The incidence density of lung cancer among patients with and without emphysema on LDCT was estimated. Stratified and multiple regression analyses were used to assess whether emphysema is an independent risk factor for lung cancer after adjusting for age, gender, smoking history, and the presence of airway obstruction on spirometry.
On univariate analysis, the incidence density of lung cancer among individuals with and without emphysema on LDCT was 25.0 per 1,000 person-years and 7.5 per 1,000 person-years, respectively (risk ratio [RR], 3.33; 95% confidence interval [CI], 1.41 to 7.85). Emphysema was also associated with increased risk of lung cancer when the analysis was limited to individuals without airway obstruction on spirometry (RR, 4.33; 95% CI, 1.04 to 18.16). Multivariate analysis showed that the presence of emphysema (RR, 2.51; 95% CI, 1.01 to 6.23) on LDCT but not airway obstruction (RR, 2.10; 95% CI, 0.79 to 5.58) was associated with increased risk of lung cancer after adjusting for potential cofounders.
Results suggest that the presence of emphysema on LDCT is an independent risk factor for lung cancer.
Chest 01/2008; 132(6):1932-8. · 5.25 Impact Factor
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ABSTRACT: Hypoxia is an important phenomenon in the tumor microenvironment. Hypoxic tumors are more aggressive and resistant to anti-neoplastic treatments. HIF-1alpha plays a major role in the response of tumors to hypoxia, and it is mainly responsible for the "angiogenic switch". HIF-1alpha contributes to tumor aggressiveness, invasiveness, and resistance to radiotherapy and chemotherapy. Targeting HIF-1alpha is an attractive strategy, with the potential for disrupting multiple pathways crucial for tumor growth. We review recent findings on the potential efficacy of small molecules to downregulate HIF-1alpha. These promising drugs inhibit HIF-1alpha synthesis or transcriptional activity by blocking a variety of steps in several different signaling pathways. Blocking HIF-1alpha activity should not only downregulate tumor angiogenesis, but also interfere with glycolytic metabolism and tumor cell growth. This strategy could also improve the efficiency of established tumor therapies.
Cancer biology & therapy 11/2005; 4(10):1055-62. · 2.64 Impact Factor
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ABSTRACT: Human adrenomedullin (AM) is a 52 amino acid peptide, which shares homology with the calcitonin gene-related peptide. Overexpression of AM in the prostate carcinoma cell line PC-3 results in growth inhibition with a 20% (for human AM) and 35% (for rat AM) increase in doubling time compared to parental or mock-transfected cells. We demonstrate by gene expression profiling that AM overexpression results in the dysregulation of approximately 100 genes. Examples of such genes include many involved in the formation of the cytoskeleton, cell adhesion and the extracellular matrix, as well as regulators of the cell cycle and apoptosis, cytokines and transcription factors. Several genes related to cell growth arrest, such as GADD45, IGF-BP6 and RUNX-3, are upregulated by AM. Interestingly, interleukin-13 receptor alpha 2 (IL-13R alpha 2) transcripts were significantly increased in clones overexpressing AM, which was confirmed by semiquantitative RT-PCR analysis. In addition, PC-3 cells treated with AM showed an overexpression of IL-13R alpha 2, which was abolished when cells were preincubated with an anti-AM blocking antibody. When PC-3 cells overexpressing AM and the IL-13R alpha 2 were treated with the highly specific IL13-PE38 cytotoxin, which binds to this receptor, a concentration-dependent inhibition of protein synthesis was observed. The IC(50) (concentration of cytotoxin inhibiting protein synthesis by 50%) ranged from 1 to 4 ng/ml. This cytotoxicity was specific as it was neutralized by the excess of IL-13 and confirmed by clonogenic assays. This study describes a novel AM-induced mechanism of tumor sensitization through the upregulation of functional IL-13R alpha 2 chain, an ideal target for the highly specific recombinant chimeric cytotoxin IL13-PE38.
International Journal of Cancer 06/2005; 114(6):870-8. · 5.44 Impact Factor
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Gorka Bastarrika,
María José García-Velloso,
Maria Dolores Lozano,
Usua Montes,
Wenceslao Torre,
Natalia Spiteri,
Arantza Campo,
Luis Seijo,
Ana Belén Alcaide,
Jesús Pueyo,
David Cano,
Isabel Vivas,
Octavio Cosín,
Pablo Domínguez,
Patricia Serra,
José A Richter, Luis Montuenga,
Javier J Zulueta
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ABSTRACT: Lung cancer screening using computed tomography (CT) is effective in detecting lung cancer in early stages. Concerns regarding false-positive rates and unnecessary invasive procedures have been raised.
To study the efficiency of a lung cancer protocol using spiral CT and F-18-fluorodeoxyglucose positron emission tomography (FDG-PET).
High-risk individuals underwent screening with annual spiral CTs. Follow-up CTs were done for noncalcified nodules of 5 mm or greater, and FDG-PET was done for nodules 10 mm or larger or smaller (> 7 mm), growing nodules.
A total of 911 individuals completed a baseline CT study and 424 had at least one annual follow-up study. Of the former, 14% had noncalcified nodules of 5 mm or larger, and 3.6% had nodules of 10 mm or larger. Eleven non-small cell lung cancers (NSCLC) and one small cell lung cancer (SCLC) were diagnosed in the baseline study (prevalence rate, 1.32%), and two NSCLCs in the annual study (incidence rate, 0.47%). All NSCLCs (92% of prevalence cancers) were diagnosed in stage I (12 stage IA, 1 stage IB). FDG-PET was helpful for the correct diagnosis in 19 of 25 indeterminate nodules. The sensitivity, specificity, positive predictive value, and negative predictive value of FDG-PET for the diagnosis of malignancy were 69, 91, 90, and 71%, respectively. However, the sensitivity and negative predictive value of the screening algorithm, which included a 3-month follow-up CT for nodules with a negative FDG-PET, was 100%.
A protocol for early lung cancer detection using spiral CT and FDG-PET is useful and may minimize unnecessary invasive procedures for benign lesions.
American Journal of Respiratory and Critical Care Medicine 06/2005; 171(12):1378-83. · 11.08 Impact Factor
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ABSTRACT: Our aim is to present initial baseline data from a screening trial on low-dose spiral computed tomography (CT). We describe enrollment criteria and a diagnostic algorithm based on initial low-dose CT findings.
From September 2000 to May 2001, 150 asymptomatic smokers (age range 40-78 years; mean 55 years) were studied using non-enhanced low-dose spiral CT of the chest. Repeated short-term high resolution CT follow-up was performed for non-calcified pulmonary nodules smaller than 10 mm in diameter. Non-calcified pulmonary nodules 10 mm or larger were considered as potentially malignant and a complementary positron emission tomography (PET) exam was recommended.
54 non-calcified pulmonary nodules were found in 34 out of 141 (24.15%) symptom-free subjects. The diameter was 5 mm or shorter in 24 participants (70.6%), 6-10 mm in 7 (20.6%) and longer than 10 mm in 3 (8.8%) individuals. One patient with a non-calcified pulmonary nodule of at least 10 mm underwent a complementary PET exam, which was positive. Biopsy of this nodule demonstrated lung cancer (squamous cell carcinoma). CT follow-up over one year was decided in the other two subjects having non-calcified pulmonary nodules longer than 10 mm, as no radiographic signs indicative of malignancy were observed in the baseline scan.
Following this low-dose CT based screening programme, detection of early-stage lung cancer in asymptomatic subjects at high risk of developing the disease is feasible. Further studies are however necessary to pursue more definitive results.
Medicina Clínica 07/2003; 121(2):41-7. · 1.38 Impact Factor
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ABSTRACT: There is increasing evidence for the important role played by regulatory peptides in the physiology of the normal and neoplastic prostate. Adrenomedullin (AM) and pro-adrenomedullin N-terminal 20 peptide (PAMP) are recently discovered regulatory peptides widely expressed in the normal prostate and in prostate carcinoma. AM is produced in secretory, stroma, and endothelial cells and in neurons of the prostate ganglia. PAMP is only produced by neuroendocrine cells. The expression of AM mRNA is regulated by androgens in the rat prostate. The number of neuroendocrine cells expressing PAMP is increased in prostate carcinoma after androgen deprivation, which shows that this peptide could regulate androgen-independent prostate tumor growth. However, the roles of AM and PAMP in the normal prostate and in prostate carcinoma are yet to be elucidated.
Microscopy Research and Technique 05/2002; 57(2):98-104. · 1.79 Impact Factor
