E. Aranda

Nitric Oxide and Cancer: The Emerging Role of S-Nitrosylation

Nitric oxide (NO˙) is a short-lived, endogenously produced gas that is highly diffusible across cell membranes and acts as a signaling molecule in the body. The redox state and chemistry of NO˙ facilitate its interaction with various proteins thus regulating various intracellular and intercellular events. One of the key mechanisms by which NO˙ regulates the function of various target proteins is through the coupling of a nitroso moiety from NO-derived metabolites to a reactive cysteine leading to the formation of a S-nitrosothiol (SNO), a process commonly known as S-nitrosylation. S-nitrosylation signaling events within the cell have led to the discovery of many other physiological functions of NO˙ in many other types of cells including cancer cells. Only recently are the diverse roles of S-nitrosylation in cancer beginning to be understood. In the present review we discuss the recent evidence for the diverse roles of NO˙/SNO-related mechanisms in cancer biology and therapy, including the participation of NO˙ in the pathogenesis of cancer, its duality in protecting against or inducing cancer cell death and the contribution of NO˙ to metastatic processes. In addition, NO˙ can be therapeutically used in the reversal of tumor cell resistance to cytotoxic drugs and as a sensitizing agent to chemo- and radiotherapy. Finally, recent studies providing evidence for NO-related mechanisms of epigenetic gene expression regulation will also be discussed. Undoubtedly, new exciting results will contribute to this rapidly expanding area of cancer research.

Phase II open-label study of erlotinib in combination with gemcitabine in unresectable and/or metastatic adenocarcinoma of the pancreas: relationship between skin rash and survival (Pantar study)

BACKGROUND Skin rash is an adverse event which might be associated with longer survival in patients treated with epidermal growth factor receptor tyrosine kinase inhibitors. The aim of this nonrandomised phase II clinical trial is to prospectively evaluate the relationship between skin rash and overall survival (OS) in advanced/metastatic pancreatic cancer treated with erlotinib plus gemcitabine. PATIENTS AND METHODS Patients were given gemcitabine (1000 mg/m2/week, 3 weeks every 4 weeks) plus erlotinib (100 mg/day orally continuously) until disease progression/unacceptable toxicity. The primary end point was OS. RESULTS A total of 153 eligible patients were enrolled (grade≥2 rash, 25%; grade<2 rash, 75%). OS was longer in patients with grade≥2 rash versus grade<2 (11 versus 5 months; P<0.001). Progression-free survival was longer in patients with grade≥2 rash versus grade<2 (6 versus 3 months; P<0.001) and shorter in those without rash versus grade 1 (2 versus 4 months; P=0.005) or grade≥2 (2 versus 6 months; P<0.001). Patients with grade≥2 rash showed higher rates of overall response (21% versus 7%; P<0.05) and disease control (84% versus 43%; P<0.05) versus grade<2. CONCLUSIONS This study prospectively confirms the relationship between rash and longer OS in unresectable locally advanced/metastatic pancreatic cancer treated with erlotinib plus gemcitabine.

CoCl2, a Mimic of Hypoxia, Induces Formation of Polyploid Giant Cells with Stem Characteristics in Colon Cancer

The induction of polyploidy is considered the reproductive end of cells, but there is evidence that polyploid giant cancer cells (PGCCs) contribute to cell repopulation during tumor relapse. However, the role of these cells in the development, progression and response to therapy in colon cancer remains undefined. Therefore, the main objective of this study was to investigate the generation of PGCCs in colon cancer cells and identify mechanisms of formation. Treatment of HCT-116 and Caco-2 colon cancer cells with the hypoxia mimic CoCl2 induced the formation of cells with larger cell and nuclear size (PGCCs), while the cells with normal morphology were selectively eliminated. Cytometric analysis showed that CoCl2 treatment induced G2 cell cycle arrest and the generation of a polyploid cell subpopulation with increased cellular DNA content. Polyploidy of hypoxia-induced PGCCs was confirmed by FISH analysis. Furthermore, CoCl2 treatment effectively induced the stabilization of HIF-1α, the differential expression of a truncated form of p53 (p47) and decreased levels of cyclin D1, indicating molecular mechanisms associated with cell cycle arrest at G2. Generation of PGCCs also contributed to expansion of a cell subpopulation with cancer stem cells (CSCs) characteristics, as indicated by colonosphere formation assays, and enhanced chemoresistance to 5-fluorouracil and oxaliplatin. In conclusion, the pharmacological induction of hypoxia in colon cancer cells causes the formation of PGCCs, the expansion of a cell subpopulation with CSC characteristics and chemoresistance. The molecular mechanisms involved, including the stabilization of HIF-1 α, the involvement of p53/p47 isoform and cell cycle arrest at G2, suggest novel targets to prevent tumor relapse and treatment failure in colon cancer.

Simultaneous inhibition of EGFR/VEGFR and cyclooxygenase-2 targets stemness-related pathways in colorectal cancer cells.

Despite the demonstrated benefits of anti-EGFR/VEGF targeted therapies in metastatic colorectal cancer (mCRC), many patients initially respond, but then show evidence of disease progression. New therapeutic strategies are needed to make the action of available drugs more efficient. Our study aimed to explore whether simultaneous targeting of EGFR/VEGF and cyclooxygenase-2 (COX-2) may aid the treatment and management of mCRC patients. The dual tyrosine kinase inhibitor AEE788 and celecoxib were used to inhibit EGFR/VEGFR and COX-2, respectively, in colorectal cancer cells. COX-2 inhibition with celecoxib augmented the antitumoral and antiangiogenic efficacy of AEE788, as indicated by the inhibition of cell proliferation, induction of apoptosis and G1 cell cycle arrest, down-regulation of VEGF production by cancer cells and reduction of cell migration. These effects were related with a blockade in the EGFR/VEGFR signaling axis. Notably, the combined AEE788/celecoxib treatment prevented β-catenin nuclear accumulation in tumor cells. This effect was associated with a significant downregulation of FOXM1 protein levels and an impairment in the interaction of this transcription factor with β-catenin, which is required for its nuclear localization. Furthermore, the combined treatment also reduced the expression of the stem cell markers Oct 3/4, Nanog, Sox-2 and Snail in cancer cells, and contributed to the diminution of the CSC subpopulation, as indicated by colonosphere formation assays. In conclusion, the combined treatment of AEE788 and celecoxib not only demonstrated enhanced anti-tumoral efficacy in colorectal cancer cells, but also reduced colon CSCs subpopulation by targeting stemness-related pathways. Therefore, the simultaneous targeting of EGFR/VEGF and COX-2 may aid in blocking mCRC progression and improve the efficacy of existing therapies in colorectal cancer.

Altered S-nitrosothiol homeostasis provides a survival advantage to breast cancer cells in HER2 tumors and reduces their sensitivity to trastuzumab

The monoclonal antibody trastuzumab against HER2/neu, which is overexpressed in 15-20% of breast cancers, has clinical efficacy but many patients do not respond to initial treatment or develop resistance during treatment. Nitric oxide (NO) regulates cell signaling by targeting specific cysteine residues in proteins, forming S-nitrosothiols (SNO) in a process known as S-nitrosylation. We previously reported that molecular characteristics in breast cancer may dictate the tumor response to impaired SNO homeostasis. In the present study, we explored the role of SNO homeostasis in HER2 breast tumors. The antiproliferative action of trastuzumab in HER2-overexpressing BT-474 and SKBR-3 cells was suppressed when S-nitrosoglutathione reductase (GSNOR/ADH5) activity, which plays a key role in SNO homeostasis, was specifically inhibited with the pyrrole derivative compound N6022. Moreover, GSNOR inhibition restored the activation of survival signaling pathways involved in the resistance to anti-HER2 therapies (AKT, Src and c-Abl kinases and TrkA/NRTK1, TrkB/NRTK2, EphA1 and EphA3 receptors) and reduced the apoptotic effect of trastuzumab. Accordingly, GSNOR inhibition augmented the S-nitrosylation of apoptosis-related proteins, including Apaf-1, pSer73/63 c-Jun, calcineurin subunit α and HSF1. In agreement with in vitro data, immunohistochemical analyses of 51 breast tumors showed that HER2 expression was associated with lower expression of GSNOR protein. Moreover, gene expression analysis confirmed that high ADH5/GSNOR gene expression was associated with high patient survival rates in HER2 tumors. In conclusion, our data provide evidence of molecular mechanisms contributing to the progression of HER2+ breast cancers and could facilitate the development of therapeutic options to counteract resistance to anti-HER2 therapies.

Randomized study of weekly irinotecan plus high-dose 5-fluorouracil (FUIRI) versus biweekly irinotecan plus 5-fluorouracil/leucovorin (FOLFIRI) as first-line chemotherapy for patients with metastatic colorectal cancer: a Spanish Cooperative Group for the Treatment of Digestive Tumors Study

BACKGROUND Irinotecan plus infusional 5-fluorouracil/leucovorin (FOLFIRI) is accepted as a reference treatment for the first-line treatment of patients with metastatic colorectal cancer (MCRC). The aim of this study was to demonstrate that a regimen without leucovorin (LV) (FUIRI) is not inferior to the standard FOLFIRI (response rate). PATIENTS AND METHODS Chemotherapy-naive patients with MCRC were randomized to receive either irinotecan (180 mg/m(2) on day 1) + 5-fluorouracil (5-FU) (400 mg/m(2) bolus and 600 mg/m(2) 22-h infusion) + LV (200 mg/m(2) on days 1-2) (FOLFIRI) every 2 weeks or irinotecan (80 mg/m(2)) + 5-FU (2.250 mg/m(2) 48-h infusion) (FUIRI) weekly. RESULTS In all, 346 patients were included, 173 in each arm. In the intention-to-treat analysis, the response rates for FOLFIRI and FUIRI were 57% [95% confidence interval (CI) 49% to 64%] and 51% (95% CI 43% to 59%), respectively (P = 0.2809). No statistically significant differences were observed between FOLFIRI and FUIRI regarding median progression-free survival (8.3 versus 8.4 months; P = 0.4339) nor median overall survival (21.6 versus 19.2 months; log-rank test P = 0.2941). Grade 3/4 neutropenia was significantly more frequent on FOLFIRI arm (27% versus 9%), while the proportion of diarrhea was higher on FUIRI arm (21% versus 42%). CONCLUSION FUIRI represents a valid alternative without LV to the FOLFIRI regimen as MCRC first-line treatment.

Genetic variants in the renin-angiotensin system predict response to bevacizumab in cancer patients.

Currently, there are no predictive biomarkers for anti‐angiogenic strategies in cancer, but response to anti‐angiogenic drugs is associated with development of hypertension secondary to treatment. Therefore, this study explored the clinical relevance of genetic polymorphisms in some components of the renin–angiotensin system (RAS).

Integrative multi-platform meta-analysis of gene expression profiles in pancreatic ductal adenocarcinoma patients for identifying novel diagnostic biomarkers

Applying differentially expressed genes (DEGs) to identify feasible biomarkers in diseases can be a hard task when working with heterogeneous datasets. Expression data are strongly influenced by technology, sample preparation processes, and/or labeling methods. The proliferation of different microarray platforms for measuring gene expression increases the need to develop models able to compare their results, especially when different technologies can lead to signal values that vary greatly. Integrative meta-analysis can significantly improve the reliability and robustness of DEG detection. The objective of this work was to develop an integrative approach for identifying potential cancer biomarkers by integrating gene expression data from two different platforms. Pancreatic ductal adenocarcinoma (PDAC), where there is an urgent need to find new biomarkers due its late diagnosis, is an ideal candidate for testing this technology. Expression data from two different datasets, namely Affymetrix and Illumina (18 and 36 PDAC patients, respectively), as well as from 18 healthy controls, was used for this study. A meta-analysis based on an empirical Bayesian methodology (ComBat) was then proposed to integrate these datasets. DEGs were finally identified from the integrated data by using the statistical programming language R. After our integrative meta-analysis, 5 genes were commonly identified within the individual analyses of the independent datasets. Also, 28 novel genes that were not reported by the individual analyses (‘gained’ genes) were also discovered. Several of these gained genes have been already related to other gastroenterological tumors. The proposed integrative meta-analysis has revealed novel DEGs that may play an important role in PDAC and could be potential biomarkers for diagnosing the disease.

Identification of gene expression profiling associated with erlotinib-related skin toxicity in pancreatic adenocarcinoma patients

Erlotinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that showed activity against pancreatic ductal adenocarcinoma (PDAC). The drugs most frequently reported side effect as a result of EGFR inhibition is skin rash (SR), a symptom which has been associated with a better therapeutic response to the drug. Gene expression profiling can be used as a tool to predict which patients will develop this important cutaneous manifestation. The aim of the present study was to identify which genes may influence the appearance of SR in PDAC patients. The study included 34 PDAC patients treated with erlotinib: 21 patients developed any grade of SR, while 13 patients did not (controls). Before administering any chemotherapy regimen and the development of SR, we collected RNA from peripheral blood samples of all patients and studied the differential gene expression pattern using the Illumina microarray platform HumanHT-12 v4 Expression BeadChip. Seven genes (FAM46C, IFITM3, GMPR, DENND6B, SELENBP1, NOL10, and SIAH2), involved in different pathways including regulatory, migratory, and signalling processes, were downregulated in PDAC patients with SR. Our results suggest the existence of a gene expression profiling significantly correlated with erlotinib-induced SR in PDAC that could be used as prognostic indicator in this patients.

551PRAS ANALYSIS OF THE PLANET STUDY: PHASE II TRIAL OF PANITUMUMAB (P) PLUS FOLFOX4 OR FOLFIRI IN SUBJECTS WITH WILD-TYPE (WT) KRAS COLORECTAL CANCER (CRC) AND LIVER-LIMITED DISEASE (LLD)

ABSTRACT Aim: Patients (pts) with RAS mutations other than KRAS exon 2 do not benefit from P. We explored the effect of these mutations in the efficacy of P-FOLFOX4 or P-FOLFIRI, as 1st-line treatment in WT KRAS CRC pts with LLD. Methods: Phase II, open-label, multicentre study in which WT KRAS CRC pts ≥ 18 years (y) with ≥4 liver metastases; at least 1 >10 cm; or not resectable were randomized to P-FOLFOX4 or P-FOLFIRI. Objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were analysed according to RAS status (exons 2, 3, 4 of KRAS/NRAS). Results: 77 pts were analysed (38 P-FOLFOX4 [82% male, median age: 65 y]/ 39 P-FOLFIRI [72% male, 63 y]). It was possible to determine RAS status in 83.1% of pts (82.8% WT, 17.2% mutant). Higher ORR and longer PFS and OS were observed in WT-RAS versus mutant RAS subgroups in both P-FOLFOX4 and P-FOLFIRI groups, although differences were not significant, probably due to small sample sizes (Table). There were no significant differences between P-FOLFOX4 and P-FOLFIRI within each of the RAS strata, either. Peri-operative and overall safety were similar, except for higher grade 3/4 neutropenia (39.5% vs 10.3%; p = 0.003) and neuropathy (13.2% vs 0%;p = 0.025) in the P-FOLFOX4 arm. P-FOLFOX4 % or median (95% CI) P-FOLFIRI % or median (95% CI) P value* P-FOLFOX4 vs P-FOLFIRI Total WT RAS, n 27 26 53 ORR (not confirmed) 77.8 (62.1–93.5) 73.1 (56.0–90.1) 75.5 (63.9–87.1) PFS, mo 12.8 (6.2–22.0) 14.8 (7.1–18.7) 0.675 13.4 (9.9–18.6) OS, mo 39.0 (26.4–NA) 45.8 (32.8–51.5) 0.634 44.7 (32.8–51.5) Mutant RAS, n 4 7 11 ORR (not confirmed) 50 (1.0–99.0) 57.1 (20.5–93.8) 54.6 (25.1–84.0) PFS, mo 15.4 (3.7–24.9) 12.6 (1.9–16.2) 0.699 12.6 (3.7–24.9) OS, mo 31.4 (20.6–NA) 42.4 (13.7–42.4) 0.391 31.4 (13.7–NA) P value * WT vs Mutant RAS PFS 1.000 0.238 0.403 OS 0.843 0.616 0.724 * Wilcoxon test; NA = not achieved; CI = confidence interval; mo = months. Conclusions: In pts with WT KRAS CRC and LLD, P plus chemotherapy provide a high response rate, allowing potentially curative resection. Better outcomes are observed in the WT RAS subgroup, without differences between the two regimens. Supported by Amgen S.A. Disclosure: B. Massuti Sureda: Consultant or advisory role: Amgen Honoraria. Amgen; M. Valladares-Ayerbes: Consultant or advisory role: Merck, Amgen Honoraria: Merck, Amgen Research funding: Merck, Amgen E. Aranda: Advisory Role: Roche, Merck. All other authors have declared no conflicts of interest.