bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non- small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-NO, 1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P = 0.04) and both were inversely associated with microvessel density (P < 0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P < 0.01). A significant co-expression of c-erbB-2 with TP was noted (P = 0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P < 0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P = 0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c- erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c- erbB-2-related immune response remains to be further investigated.

Potential role of bcl-2 as a suppressor of tumour angiogenesis in non- small-cell lung cancer

It has been reported that genes regulating apoptosis may play a role in tumoral angiogenesis. This study examined the relationship between tumour vascularization, a measure of tumour angiogenesis, and bcl-2 and p53 expression in operable non-small-cell lung cancer (NSCLC). The relationship between bcl-2, p53 and tumour vascularization and epidermal-growth-factor- receptor(EGFR) and c-erbB-2 expression was also studied. Tissue sections from resected tumour specimens of 107 NSCLC patients were evaluated immunohistochemically for vascular grade and bcl-2, p53, EGFR and c-erbB-2 expression. bcl-2 expression was found in 20/107 (19%) cases and was associated with squamous-cell histology (p = 0.03). A strong inverse relationship was found between bcl-2 expression and vascular grade (p = 0.005). All c-erbB-2-positive cases were negative for bcl-2 expression (p = 0.01). Overall no association was found between c-erbB-2 expression and vascular grade. However, in bcl-2-negative cases positive c-erbB-2 expression correlated with low angiogenesis (p = 0.05). No relationship was found between p53 and EGFR expression and bcl-2, c-erbB-2 or vascular grade. The improved prognosis reported in bcl-2-positive NSCLC may be related to low tumour vascularization. The results suggest that the anti-apoptotic gene bcl- 2 plays a role in regulating tumour angiogenesis. Since normal lung epithelium expresses bcl-2, a sequence of tumour progression involving loss of bcl-2, then activation of c-erbB-2 or increase in tumour vascularization is proposed.

Histone deacetylase inhibitors target diabetes via chromatin remodeling or as chemical chaperones?

Globally, obesity and diabetes (particularly type 2 diabetes) represents a major challenge to world health. Despite decades of intense research efforts, the genetic basis involved in diabetes pathogenesis & conditions associated with obesity are still poorly understood. Recent advances have led to exciting new developments implicating epigenetics as an important mechanism underpinning diabetes and obesity related disease. One epigenetic mechanism known as the "histone code" describes the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as lysine acetyltransferases or KATs and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. Some of the known inhibitors of HDACs (HDACi) have also been shown to act as "chemical chaperones" to alleviate diabetic symptoms. In this review, we discuss the available evidence concerning the roles of HDACs in regulating chaperone function and how this may have implications in the management of diabetes. © 2009 Bentham Science Publishers Ltd.

Oxidative stress induced lung cancer and COPD : opportunities for epigenetic therapy

Reactive oxygen species (ROS) form as a natural by-product of the normal metabolism of oxygen and play important roles within the cell. Under normal circumstances the cell is able to maintain an adequate homeostasis between the formation of ROS and its removal through particular enzymatic pathways or via antioxidants. If however, this balance is disturbed a situation called oxidative stress occurs. Critically, oxidative stress plays important roles in the pathogenesis of many diseases, including cancer. Epigenetics is a process where gene expression is regulated by heritable mechanisms that do not cause any direct changes to the DNA sequence itself, and disruption of epigenetic mechanisms has important implications in disease. Evidence is emerging that histone deacetylases (HDACs) play decisive roles in regulating important cellular oxidative stress pathways including those involved with sensing oxidative stress and those involved with regulating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by HDACs may play critical roles in cancer progression. In this review we discuss the current evidence linking epigenetics and oxidative stress and cancer, using chronic obstructive pulmonary disease and non-small cell lung cancer to illustrate the importance of epigenetics on these pathways within these disease settings. © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Expression and prognostic significance of a panel of tissue hypoxia markers in head-and-neck squamous cell carcinomas

Purpose: To investigate the expression pattern of hypoxia-induced proteins identified as being involved in malignant progression of head-and-neck squamous cell carcinoma (HNSCC) and to determine their relationship to tumor pO 2 and prognosis. Methods and Materials: We performed immunohistochemical staining of hypoxia-induced proteins (carbonic anhydrase IX [CA IX], BNIP3L, connective tissue growth factor, osteopontin, ephrin A1, hypoxia inducible gene-2, dihydrofolate reductase, galectin-1, IκB kinase β, and lysyl oxidase) on tumor tissue arrays of 101 HNSCC patients with pretreatment pO 2 measurements. Analysis of variance and Fisher's exact tests were used to evaluate the relationship between marker expression, tumor pO 2, and CA IX staining. Cox proportional hazard model and log-rank tests were used to determine the relationship between markers and prognosis. Results: Osteopontin expression correlated with tumor pO 2 (Eppendorf measurements) (p = 0.04). However, there was a strong correlation between lysyl oxidase, ephrin A1, and galectin-1 and CA IX staining. These markers also predicted for cancer-specific survival and overall survival on univariate analysis. A hypoxia score of 0-5 was assigned to each patient, on the basis of the presence of strong staining for these markers, whereby a higher score signifies increased marker expression. On multivariate analysis, increasing hypoxia score was an independent prognostic factor for cancer-specific survival (p = 0.015) and was borderline significant for overall survival (p = 0.057) when adjusted for other independent predictors of outcomes (hemoglobin and age). Conclusions: We identified a panel of hypoxia-related tissue markers that correlates with treatment outcomes in HNSCC. Validation of these markers will be needed to determine their utility in identifying patients for hypoxia-targeted therapy. © 2007 Elsevier Inc. All rights reserved.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

The role of epigenetics in resistance to cisplatin chemotherapy in lung cancer

Non-small cell lung cancer (NSCLC) is the most common cause of cancer related death in the world. Cisplatin and carboplatin are the most commonly used cytotoxic chemotherapeutic agents to treat the disease. These agents, usually combined with drugs such as gemcitabine or pemetrexed, induce objective tumor responses in only 20-30% of patients. Aberrant epigenetic regulation of gene expression is a frequent event in NSCLC. In this article we review the emerging evidence that epigenetics and the cellular machinery involved with this type of regulation may be key elements in the development of cisplatin resistance in NSCLC. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

Molecular biomarkers in non-small-cell lung cancer : a retrospective analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 FLEX study showed that the addition of cetuximab to cisplatin and vinorelbine significantly improved overall survival, compared with cisplatin and vinorelbine alone, in the first-line treatment of EGFR-expressing, advanced non-small-cell lung cancer (NSCLC). We investigated whether candidate biomarkers were predictive for the efficacy of chemotherapy plus cetuximab in this setting. Methods: Genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour tissue of patients enrolled in the FLEX study was screened for KRAS codon 12 and 13 and EGFR kinase domain mutations with PCR-based assays. In FFPE tissue sections, EGFR copy number was assessed by dual-colour fluorescence in-situ hybridisation and PTEN expression by immunohistochemistry. Treatment outcome was investigated according to biomarker status in all available samples from patients in the intention-to-treat population. The primary endpoint in the FLEX study was overall survival. The FLEX study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT00148798. Findings: KRAS mutations were detected in 75 of 395 (19%) tumours and activating EGFR mutations in 64 of 436 (15%). EGFR copy number was scored as increased in 102 of 279 (37%) tumours and PTEN expression as negative in 107 of 303 (35%). Comparisons of treatment outcome between the two groups (chemotherapy plus cetuximab vs chemotherapy alone) according to biomarker status provided no indication that these biomarkers were of predictive value. Activating EGFR mutations were identified as indicators of good prognosis, with patients in both treatment groups whose tumours carried such mutations having improved survival compared with those whose tumours did not (chemotherapy plus cetuximab: median 17·5 months [95% CI 11·7-23·4] vs 8·5 months [7·1-10·8], hazard ratio [HR] 0·52 [0·32-0·84], p=0·0063; chemotherapy alone: 23·8 months [15·2-not reached] vs 10·0 months [8·7-11·0], HR 0·35 [0·21-0·59], p<0·0001). Expression of PTEN seemed to be a potential indicator of good prognosis, with patients whose tumours expressed PTEN having improved survival compared with those whose tumours did not, although this finding was not significant (chemotherapy plus cetuximab: median 11·4 months [8·6-13·6] vs 6·8 months [5·9-12·7], HR 0·80 [0·55-1·16], p=0·24; chemotherapy alone: 11·0 months [9·2-12·6] vs 9·3 months [7·6-11·9], HR 0·77 [0·54-1·10], p=0·16). Interpretation: The efficacy of chemotherapy plus cetuximab in the first-line treatment of advanced NSCLC seems to be independent of each of the biomarkers assessed. Funding: Merck KGaA. © 2011 Elsevier Ltd.

A metastatic neuroendocrine anaplastic small cell tumor in a patient with multiple endocrine neoplasia type 1 syndrome : assessment of disease status and response to doxorubicin, cyclophosphamide, etoposide chemotherapy through scintigraphic imaging with 111In-pentetreotide

Extrapulmonary small cell and small cell neuroendocrine tumors of unknown primary site are, in general, aggressive neoplasms with a short median survival. Like small cell lung cancer (SCLC), they often are responsive to chemotherapy and radiotherapy. Small cell lung cancer and well differentiated neuroendocrine carcinomas of the gastrointestinal tract and pancreas tend to express somatostatin receptors. These tumors may be localized in patients by scintigraphic imaging using radiolabeled somatostatin analogues. A patient with an anaplastic neuroendocrine small cell tumor arising on a background of multiple endocrine neoplasia type 1 syndrome is reported. The patient had a known large pancreatic gastrinoma and previously treated parathyroid adenopathy. At presentation, there was small cell cancer throughout the liver and skeleton. Imaging with a radiolabeled somatostatin analogue, 111In- pentetreotide (Mallinckrodt Medical B. V., Petten, Holland), revealed all sites of disease detected by routine biochemical and radiologic methods. After six cycles of chemotherapy with doxorubicin, cyclophosphamide, and etoposide, there was almost complete clearance of the metastatic disease. 111In-pentetreotide scintigraphy revealed uptake consistent with small areas of residual disease in the liver, the abdomen (in mesenteric lymph nodes), and posterior thorax (in a rib). The primary gastrinoma present before the onset of the anaplastic small cell cancer showed no evidence of response to the treatment. The patient remained well for 1 year and then relapsed with brain, lung, liver, and skeletal metastases. Despite an initial response to salvage radiotherapy and chemotherapy with carboplatin and dacarbazine, the patient died 6 months later.

Somatostatin receptor expression in lung cancer

Experimental evidence suggests that somatostatin analogues may have a role to play in the management of lung tumours. We evaluated membrane preparations of nine small cell lung cancer (SCLC) cell lines and of tumour samples from 3 patients with non-small cell lung cancer (NSCLC), 1 patient with an atypical carcinoid and another with a bronchial carcinoid for the presence of specific binding sites for RC-160, a potent growth inhibitory octapeptide analogue of somatostatin. Specific binding was noted on six of nine SCLC lines. Radio-receptor assay on the cell line NCI H 69 showed evidence of two specific binding sites for RC-160, one with high affinity and the other with low affinity. Binding sites were also found on all five tumour samples. Scatchard analysis indicated the presence of a single class of receptors with high affinity in each case. Histological assessment of the resected specimens before binding assay showed them to be comprised of tumour cells and necrotic tissue, stroma and/or inflammatory cells. Therefore, the specific binding of RC-160 may be to tissues other than the tumour cells. In 3 patients, from whom the tumour samples were obtained, radiolabelled somatostatin analogue scintigraphy using [111In] pentetreotide was performed prior to surgery. In all cases, the radiolabel localised the disease. This study demonstrates the presence of specific binding sites for RC-160 in SCLC. Furthermore, the detection of specific binding in vitro and in vivo in NSCLC and intrapulmonary carcinoids demonstrates that these tumours contain cells which express specific binding sites for somatostatin. These results suggest that RC-160 may have a role toplay as a therapeutic agent in lung cancer.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.

Lipoxygenase metabolism : roles in tumor progression and survival

The metabolism of arachidonic acid through lipoxygenase pathways leads to the generation of various biologically active eicosanoids. The expression of these enzymes vary throughout the progression of various cancers, and thereby they have been shown to regulate aspects of tumor development. Substantial evidence supports a functional role for lipoxygenase-catalyzed arachidonic and linoleic acid metabolism in cancer development. Pharmacologic and natural inhibitors of lipoxygenases have been shown to suppress carcinogenesis and tumor growth in a number of experimental models. Signaling of hydro[peroxy]fatty acids following arachidonic or linoleic acid metabolism potentially effect diverse biological phenomenon regulating processes such as cell growth, cell survival, angiogenesis, cell invasion, metastatic potential and immunomodulation. However, the effects of distinct LOX isoforms differ considerably with respect to their effects on both the individual mechanisms described and the tumor being examined. 5-LOX and platelet type 12-LOX are generally considered pro-carcinogenic, with the role of 15-LOX-1 remaining controversial, while 15-LOX-2 suppresses carcinogenesis. In this review, we focus on the molecular mechanisms regulated by LOX metabolism in some of the major cancers. We discuss the effects of LOXs on tumor cell proliferation, their roles in cell cycle control and cell death induction, effects on angiogenesis, migration and the immune response, as well as the signal transduction pathways involved in these processes. Understanding the molecular mechanisms underlying the anti-tumor effect of specific, or general, LOX inhibitors may lead to the design of biologically and pharmacologically targeted therapeutic strategies inhibiting LOX isoforms and/or their biologically active metabolites, that may ultimately prove useful in the treatment of cancer, either alone or in combination with conventional therapies. © 2007 Springer Science+Business Media, LLC.

Cyclooxygenase-2 protein levels are independent of epidermal growth factor receptor expression or activation in operable non-small cell lung cancer

Both cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR) are thought to play important roles in the pathogenesis of non-small cell lung cancer (NSCLC). A number of in vitro studies have postulated a link between EGFR activation and subsequent COX-2 upregulation. The relationship between these factors has not been established in patients with NSCLC. COX-2 and EGFR expression were studied in 172 NSCLC specimens using standard immunohistochemical techniques. Western blotting was used to determine COX-2 and EGFR levels in five NSCLC cell lines. The effect of treatment with EGF on COX-2 expression in A549 cells was assessed. Results: Both EGFR and COX-2 are overexpressed in NSCLC. The predominant pattern of COX-2 and EGFR staining was cytoplasmic. Membranous EGFR staining was seen in 23.3% of cases. There was no relationship between COX-2 and EGFR expression and survival or any clinicopathological features. No correlation was seen between EGFR expression and COX-2 expression in the immunohistochemical series or in the cell lines. Treatment with EGF did not upregulate COX-2 levels in A549 cells, either in serum free or serum-supplemented conditions. Conclusions: Although COX-2 and EGFR are over-expressed in NSCLC neither was of prognostic significance in this series of cases. There is no correlation between these two factors in either tumour samples or cell lines. Although these factors show no correlation in NSCLC, they remain potential, though independent targets for treatment. © 2004 Elsevier Ireland Ltd. All rights reserved.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

Transparency tools in gene patenting for informing policy and practice

In the recent decision Association for Molecular Pathology v. Myriad Genetics1, the US Supreme Court held that naturally occurring sequences from human genomic DNA are not patentable subject matter. Only certain complementary DNAs (cDNA), modified sequences and methods to use sequences are potentially patentable. It is likely that this distinction will hold for all DNA sequences, whether animal, plant or microbial2. However, it is not clear whether this means that other naturally occurring informational molecules, such as polypeptides (proteins) or polysaccharides, will also be excluded from patents. The decision underscores a pressing need for precise analysis of patents that disclose and reference genetic sequences, especially in the claims. Similarly, data sets, standards compliance and analytical tools must be improved—in particular, data sets and analytical tools must be made openly accessible—in order to provide a basis for effective decision making and policy setting to support biological innovation. Here, we present a web-based platform that allows such data aggregation, analysis and visualization in an open, shareable facility. To demonstrate the potential for the extension of this platform to global patent jurisdictions, we discuss the results of a global survey of patent offices that shows that much progress is still needed in making these data freely available for aggregation in the first place.