J.R. Delgado

The impact of PKR activation: from neurodegeneration to cancer

An inverse association between cancer and neurodegeneration is plausible because these biological processes share several genes and signaling pathways. Whereas uncontrolled cell proliferation and decreased apoptotic cell death governs cancer, excessive apoptosis contributes to neurodegeneration. Protein kinase R (PKR), an interferon‐inducible double‐stranded RNA protein kinase, is involved in both diseases. PKR activation blocks global protein synthesis through eIF2α phosphorylation, leading to cell death in response to a variety of cellular stresses. However, PKR also has the dual role of activating the nuclear factor κ‐B pathway, promoting cell proliferation. Whereas PKR is recognized for its negative effects on neurodegenerative diseases, in part, inducing high level of apoptosis, the role of PKR activation in cancer remains controversial. In general, PKR is considered to have a tumor suppressor function, and some clinical data show a correlation between suppressed or inactivated PKR and a poor prognosis for several cancers. However, other studies show high PKR expression and activation levels in various cancers, suggesting that PKR might contribute to neoplastic progression. Understanding the cellular factors and signals involved in the regulation of PKR in these age‐related diseases is relevant and may have important clinical implications. The present review highlights the current knowledge on the role of PKR in neurodegeneration and cancer, with special emphasis on its regulation and clinical implications.—Marchal, J. A., Lopez, G. J., Peran, M., Comino, A., Delgado, J. R., García‐García, J. A., Conde, V., Aranda, F. M., Rivas, C., Esteban, M., Garcia, M. A. The impact of PKR activation: from neurodegeneration to cancer. FASEB J. 28, 1965–1974 (2014). www.fasebj.org

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

BackgroundTumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients.MethodsPatients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique).ResultsOur results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively.ConclusionSilencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

The potential role of the glycoprotein osteoactivin/glycoprotein nonmetastatic melanoma protein B in pancreatic cancer.

Objectives Pancreatic ductal adenocarcinoma is still one of the deadliest solid cancers so the finding of new therapeutic approaches and novel targets are of utmost importance. Glycoprotein nonmetastatic melanoma protein B (GPNMB), initially termed glycoprotein nonmetastatic gene B and also named osteoactivin (OA), is a type 1 transmembrane protein that has been recently found to play a role in cancer cell proliferation, angiogenesis, and invasion. Due to its potential responsibility in cancer aggressiveness, the main objective of this work was to assess the role of GPNMB/OA in human pancreatic cancer. Methods Using the human pancreatic cancer cell line Panc-1 in vitro, the effects of GPNMB on growth, proliferation, and invasion were tested by BrdU uptake, cell cycle and Annexin V-FITC analysis, RT-PCR, protein expression, and invasion chamber assays. Results Our results showed that GPNMB/OA protein expression prevents cells from apoptosis-enhancing proliferation and represents a novel modulator of the invasion and metastasis in pancreatic cancer cells. Conclusions Due to its main membrane localization in cancer cells and its role in the aggressiveness of pancreatic cancer, GPNMB/OA could represent a novel targeted therapy for pancreatic cancer being attractive for antibody-based therapies.

Interplay Between Gemcitabine and Erlotinib Over Pancreatic Adenocarcinoma Cells.

Objectives Pancreatic ductal adenocarcinoma remains as a chemoresistant disease with the poorest prognosis. Gemcitabine has been the standard treatment during the last decade. Erlotinib, a tyrosine kinase inhibitor, in combination with gemcitabine produces a small increase in survival. However, these results remain insufficient. The aim of this study was to investigate the molecular interplay in vitro between them regarding their effects over cytotoxicity, proliferation, apoptosis, and invasion. Methods Using the human pancreatic cancer cell lines Panc-1 and BxPC-3 in vitro, the effects of gemcitabine and erlotinib therapy on growth, proliferation, and invasion were tested by cytotoxicity, cell cycle, and Annexin V-Fluorescein Isothiocyanate analysis, reverse transcription polymerase chain reaction, protein expression, and Chip assays. Results Therapy decreased cell proliferation causing G0/G1 phase cell cycle arrest with induction of apoptosis in the Panc-1 cell line. This blockade was associated with increased p27 expression. Besides, treatments enhanced the nuclear factor-&kgr;B (NF-&kgr;B) pathway and the binding of NF-&kgr;B to the promoters of genes related to the proliferation and the evasion of apoptosis. Conclusions Our data suggest that, although gemcitabine and erlotinib exert antiproliferative effects over pancreatic cancer cell lines, the gemcitabine-induced activation of NF-&kgr;B expression and its DNA-binding activities are important drawbacks of this treatment against pancreatic cancer.

Consolidation treatment with yttrium-90 ibritumomab tiuxetan after new induction regimen in advanced stage follicular lymphoma: update results from the Spanish Lymphoma Oncology Group trial after a median follow-up of 8.5-years

Mariano Provencio, Fernando Franco, Jos e G omez-Codina, Cristina Quero Blanco, Marta Llanos, Francisco Garcia-Arroyo, Luis de la Cruz, Josep Gum a, Juan Ram on Delgado, Ruth Alvarez, Jos e Ignacio Chac on, Ana Royuela and Antonio Rueda Hospital Puerta de Hierro Majadahonda, Madrid, Spain; Department of Medical Oncology, Hospital Puerta de Hierro, Majadahonda, Spain; Department of Medical Oncology, Hospital La Fe, Valencia, Spain; Hospital Clinico de Malaga, Malaga, Spain; Department of Medical Oncology, Hospital Universitario de Canaras, Tenerife, Spain; Department of Medical Oncology, Hospital de Pontevedra, Pontevedra, Spain; Department of Medical Oncology, Hospital Universitario Virgen Macarena, Sevilla, Spain; Department of Medical Oncology, Hospital Universitario de Reus, Reus, Spain; Department of Medical Oncology, Hospital Virgen de las Nieves, Granada, Spain; Department of Medical Oncology, Hospital Universitario Virgen de la Salud, Toledo, Spain; Department of Biostatistic, Hospital Universitario Puerta del Hierro Majadahonda, Majadahonda, Spain; Department of Medical Oncology, Hospital Costa del Sol, Marbella, Spain

Abstract 1828: Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume

Most preclinical oncology studies (xenograft, PDX, GEMMS) involve monitoring tumour growth rates, measuring them with callipers, and calculating the volume. Volume is calculated from the width and the length to estimate a 3D volume and is directly used to assess treatment efficacy. Although this technique is useful, it is unable to accurately assess non-uniformly shaped or very small tumours and introduces a systematic bias by assuming that tumours present with spheroid shape. Furthermore callipers do not inform of the tumour condition, which is dependent upon a visual estimation. Here we describe the development and validation of a 3D scanner as an alternative method to callipers to monitor tumour progression in rodents. The resulting 3D scanner solution made up of hardware and software, has the potential to impact on the 3Rs guiding principles underpinning the humane use of animals in oncology research. The 3Rs benefits identified are primarily through reduction of animals via improved data accuracy allowing reduction in group sizes or the ability to include irregularly shaped tumours to test. In addition the scanner system described will make it possible to record tumour measurements in a rapid, minimally invasive, morphology-independent, and human-bias-free way, removing interoperator variability. This photo-based technique captures external symptoms of redness, paleness, ulceration of tumours, etc., which could ultimately be used to detect early toxicities of compounds or determine scales of animal welfare. We describe the development and early validation of the scanner system within our laboratories. Using the 3D scanner alongside tumour callipers to monitor tumour growth of Oncology tumour studies we demonstrated that we can accurately measure tumour size parameters (length, width and volume) in multiple mouse strains and across a range of tumour models. 3D scanning tumour data is comparable to tumour measures generated from tumour callipers If successful the introduction of this system to replace tumour callipers could have a large impact for groups running oncology in-vivo tumour studies. Citation Format: Zena Wilson, Juan Delgado, Michael Davies, Rebecca Whiteley, Jennifer Hare, Amar Rahi, Stephen Marshall, Andrew Smith, Stephen Atkinson, Jarno Ralli, Adeala Zabair, Adeala Zabair, Jane Kendrew. Are callipers obsolute? A novel 3D scanning technology to measure subcutaneous tumor volume [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1828. doi:10.1158/1538-7445.AM2017-1828

170PMODIFICATION OF METHYLATION LEVEL DNA PROMOTER IN SERUM OF A PANEL OF GENE BEFORE AND AFTER TREATMENT AND IMPACT IN PROGNOSTIC FACTOR IN PATIENTS WITH BREAST CANCER

ABSTRACT Aim: Alterations of genetic and epigenetic feature can provide important insghts into the natural history of breast cancer. Although DNA methylation analysis is a rapidly developing field, a reproducible epigenetic blood-based assay for diagnosis and follow up of breast cancer has yet to be successfully developed into a routine clinical test. The aim of ths study was to review DNA methylation of promoter methylation of 14.3.3 sigma and ESR1 in circulating DNA of breast cancer patients and to highlight the value of those novel biomarkers in prediction of therapeutic outcom Methods: Plasma was sampled prospectively from 110 patients diagnosed of breast cancer. A PCR quantitative technique was used to analyzed the utility of circulating DNA with CpG island hypermethylation of ESR1, 14.3.3 sigma, APC and Rar Beta genes promoter regions as breast cancer biomarkers Results: The cutt-off points for the genes methylated promoters were established from the ROC curves, selecting values that gave the maximal likelihood ratio. Mean serum values of methylated genes before treatment was for ESR1:0.009 microg/ml, 14.3.3 sigma: 0.047 microg/ml, RarBeta: 0.0001 microg/ml and APC: 0.012 microg/ml. Mean values of methylated genes after treatment was for ESR: 0.003 microg/ml, 14.3.3 sigma: 0.038 microg/ml, RarBeta: 0 microg/ml and APC:0.001microg/ml. In the light of the discriminatory power of the ESR1 and 14.3.3 sigma and the finding that serum levels of methylated gene promoter changed after breast cancer treatment, post treatment modifications in these two promoters were analyzed. We observed lower methylated ESR1, 14.3.3 sigma and APC values after surgery respect pretreatment levels, but without an overall statistically significant difference. With a median follow up of 8 years we found that patients with a significant decrease of sera methylated levels of these genes after surgery had better interval free progression and overall survival respect patients without this observation. Conclusions: These findings cast some doubts on the utility for early cancer diagnosis of highly sensitive techniques to identify hypermethylation os specific gene promoters in DNA extracted from serum. Althought numerous issues remain to be resolved the quantitative measurement of circulating methylated DNA is a promising tool for cancer prognostic assessment. Disclosure: All authors have declared no conflicts of interest.

Abstract A47: Study of promoter methylation pattern of 14-3-3 sigma gene in serum of patients with breast cancer: A potential biomarker for diagnostic anf prognostic factor.

Background: Expression of 14-3-3 σ is a tumor suppressor gene induced in response to DNA damage, and has been implicated in G2/M cell cycle arrest by p53. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. Objective: To correlation methylation levels of promoter 14-3-3σ with association prognostic factors in breast cancer. Material and Methods: This is a prospective study we quantified methylation levels of promoter 14-3-3σ gene in 107 women with breast cancer and 108 control subjects by real time QMS-PCR SYBR green and analyzed association with prognostics factor in breast cancer. Results: Median age was 58 years (32-88); 69% were postmenopausal women.Nodal involvement N0;63%,N1;30%,N2;7%), tumor size (T1;58%,T2;35%,T3;4%,T4;4%) and grade G1; 20%, G2; 37%, G3; 30%). The methylation of 14-3-3σ were 60% of sporadic breast cancer patients and were 34% of normal breast (p=0.0047).The methylation of 14-3-3σ gene in serum was markedly related with T3-4 stage (p 0.05). Conclusions: Hypermethylation of the 14-3-3σ a promoter is an early and frequent event in breast neoplastic transformation, leading to the suggestion that silencing of 14-3-3σ may be an important event in tumor progression and particularly in breast carcinogenesis.Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G2 cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Perhaps in the detection of CpG methylation of 14-3-3σ may be used for diagnostic and prognostic purposes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A47. Citation Format: Martinez-Galan Joaquina, Juan Ramon Delgado, Blanca Torres-Torres, Rosario Del Moral, Sandra Rios, M. Isabel Nunez. Study of promoter methylation pattern of 14-3-3 sigma gene in serum of patients with breast cancer: A potential biomarker for diagnostic anf prognostic factor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A47.

Abstract A126: Change in status promoter of 5 panel gene during progression of atypical hyperplasia to breast cancer.

Background: DNA methylation is the main epigenetic modification in cancer. The pathological features of breast cancer follow a sequential progression from transition of a normal cell to benign proliferative hyperplasia, hyperplasia with atypia, in situ carcinoma and, eventually, to invasive and metastatic disease. Purpose: This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions to invasive ductal carcinoma and different methylation patron with benign breast disease. Material and Methods: We performed analysis for the methylation status of 5 promoter CpG island (ESR1, E-Cad, APC, RARB, and 14-3-3-; promoters) in a group with women with normal breast tissue n=74; other group with epithelial atypia/ atypical ductal hyperplasia (n=20), ductal carcinoma in situ (DCIS, n=14) and invasive ductal carcinoma (IDC, n=107). Results: Relative serum levels of methylated gene promoters ESR1 and 14-3-3-; showed significant differences between IDC y normal breast tissue (were significantly higher in IDC than normal breast tissue) and between normal breast tissue and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ. There are no differences between IDC and epithelial atypia/ atypical ductal hyperplasia/ ductal carcinoma in situ when 14-3-3-; and ESR1 mean values are compared. In fact, the present findings showed a frequent presence of methylated 14-3-3-; in sera from women with breast benign disease, which could be considered the result of a proliferative alteration of epithelial and stromal components of the breast. Which may indicate presence of occult benign breast disease, although other possible sources of this DNA include normal tissues, which show higher methylation values with increasing age; which might undergo lysis during tumor growth; or premalignant lesion during tumor growth; or premalignant lesion. Conclusions: These findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and breast cancer, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A126. Citation Format: Martinez-Galan Joaquina, Juan Ramon Delgado, Blanca Torres-Torres, Rosario Del Moral, Sandra Rios, M. Isabel Nunez. Change in status promoter of 5 panel gene during progression of atypical hyperplasia to breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A126.

Significance and clinical outcome of lymph node level assessment in biliary tract cancers.

274 Background: Biliary tract cancers (BTCs) are relatively rare neoplasms encompass both cholangiocarcinoma (CC).The role of routine lymphadenectomy at the time of surgical resection remains poorly defined.We sought to identify factors associated with outcome following and examine the impact of lymph node (LN) assessment on survival. Methods: 43 pts who underwent curative intent surgery between 2000-10 were identified from a database.We calculated prognostic factors and impact lymph node assessment for survival. Results: A total 43 pts were identified with no metastatic BTCs.The median age was 65 years (29–82 years); PS0:33/43 (76%); PS1:8/43 (19%) and PS2:2/43 (5%) pts.A histological diagnosis of adenocarcinoma was confirmed in 100%.Surgical resection was performed in all patients.After resection 42% (18/43) had positive nodes.Adjuvant chemotherapy had 31/43(72%),preferred with gemcitabine and a median number of cycles 6. Grade 3 or 4 toxicities rarely occurred.During median follow-up of 6.6 years tumor...