Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer

BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced/absent in human NSCLC protein samples (P <.0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P =.004) and in male patients (P <.05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P <.001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. © 2011 American Cancer Society.

COX-derived prostanoid pathways in gastrointestinal cancer development and progression : novel targets for prevention and intervention

Arachidonic acid metabolism through cyclooxygenase (COX) pathways leads to the generation of biologically active eicosanoids. Eicosanoid expression levels vary during development and progression of gastrointestinal (GI) malignancies. COX-2 is the major COX-isoform responsible for G.I. cancer development/progression. COX-2 expression increases during progression from a normal to cancerous state. Evidence from observational studies has demonstrated that chronic NSAID use reduces the risk of cancer development, while both incidence and risk of death due to G.I. cancers were significantly reduced by daily aspirin intake. A number of randomized controlled trials (APC trial, Prevention of Sporadic Adenomatous Polyps trial, APPROVe trial) have also shown a significant protective effect in patients receiving selective COX-2 inhibitors. However, chronic use of selective COX-2 inhibitors at high doses was associated with increased cardiovascular risk, while NSAIDs have also been associated with increased risk. More recently, downstream effectors of COX-signaling have been investigated in cancer development/progression. PGE 2, which binds to both EP and PPAR receptors, is the major prostanoid implicated in the carcinogenesis of G.I. cancers. The role of TXA 2 in G.I. cancers has also been examined, although further studies are required to uncover its role in carcinogenesis. Other prostanoids investigated include PGD 2 and its metabolite 15d-PGJ2, PGF 1α and PGI 2. Targeting these prostanoids in G.I. cancers has the promise of avoiding cardiovascular toxicity associated with chronic selective COX-2 inhibition, while maintaining anti-tumor reactivity.A progressive sequence from normal to pre-malignant to a malignant state has been identified in G.I. cancers. In this review, we will discuss the role of the COX-derived prostanoids in G.I. cancer development and progression. Targeting these downstream prostanoids for chemoprevention and/or treatment of G.I. cancers will also be discussed. Finally, we will highlight the latest pre-clinical technologies as well as avenues for future investigation in this highly topical research field. © 2011 Elsevier B.V.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI2/TXA2 ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer. © 2010 Elsevier B.V. All rights reserved.

A biological staging model for operable non-small cell lung cancer

Background Currently the best prognostic index for operable non-small cell lung cancer (NSCLC) is the TNM staging system. Molecular biology holds the promise of predicting outcome for the individual patient and identifying novel therapeutic targets. Angiogenesis, matrix metalloproteinases (MMP)-2 and -9, and the erb/HER type I tyrosine kinase receptors are all implicated in the pathogenesis of NSCLC. Methods A retrospective analysis of 167 patients with resected stage I-IIIa NSCLC and >60 days postoperative survival with a minimum follow up of 2 years was undertaken. Immunohistochemical analysis was performed on paraffin embedded sections for the microvessel marker CD34, MMP-2 and MMP-9, EGFR, and c-erbB-2 to evaluate the relationships between and impact on survival of these molecular markers. Results Tumour cell MMP-9 (HR 1.91 (1.23-2.97)), a high microvessel count (HR 1.97 (1.28-3.03)), and stage (stage II HR 1.44 (0.87-2.40), stage IIIa HR 2.21 (1.31-3.74)) were independent prognostic factors. Patients with a high microvessel count and tumour cell MMP-9 expression had a worse outcome than cases with only one (HR 1.68 (1.04-2.73)) or neither (HR 4.43 (2.29-8.57)) of these markers. EGFR expression correlated with tumour cell MMP-9 expression (p<0.001). Immunoreactivity for both of these factors within the same tumour was associated with a poor prognosis (HR 2.22 (1.45-3.41)). Conclusion Angiogenesis, EGFR, and MMP-9 expression provide prognostic information independent of TNM stage, allowing a more accurate outcome prediction for the individual patient. The development of novel anti-angiogenic agents, EGFR targeted therapies, and MMP inhibitors suggests that target specific adjuvant treatments may become a therapeutic option in patients with resected NSCLC.

MMP-9 and the epidermal growth factor signal pathway in non-small cell lung cancer

Background Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type-IV collagen. Enhanced expression has been related to tumour progression in a number of systems. The control of MMP expression is complex, but recently epidermal growth actor receptor (EGFR) activity has been implicated in up-regulation of MMP-9 in tumour cells in vitro. Aims To evaluate interrelations between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of 152 patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Minimum follow-up was 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-9 and EGFR. Results: MMP-9 was expressed in the tumour cells of 79/152 (52%) cases. EGFR expression was found in 86/152 (57%) cases [membranous 51/152 (34%), cytoplasmic 35/152 (23%)]. MMP-9 expression was associated with poor outcome (p=0.04). Membranous, cytoplasmic and overall EGFR expression were not associated with outcome (p=0.29, p=0.85 and p=0.41 respectively). There was a strong correlation between MMP-9 expression and EGFR expression (p=0.001) and EGFR membranous expression (p=0.01) but not with cytoplasmic EGFR expression (p=0.28). Co-expression of MMP-9 and EGFR (36%) conferred a worse prognosis (p=0.003). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (p=0.008). Conclusions Our results show that MMP-9 and EGFR are co-expressed in NSCLC. This finding suggests the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific upregulation of MMP-9. The co-expression of these markers also confers a poor prognosis.

Expression and prognostic influence of MMP-2, MMP-9 and TIMP-2 in non-small cell lung cancer

Background Matrix metalloproteinases (MMPs) are a family of endopeptidases that digest the extracellular matrix (ECM). Overexpression of different MMPs has been shown to promote tumour cell invasion in vitro. Tissue inhibitors of matrix metalloproteinases (TIMPs) are specific inhibitors of MMPs that also possess growth-promoting properties. Aims To analyse the expression profile of MMP-2, MMP-9 and TIMP-2 in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Patient follow up was a minimum of 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-2, MMP-9 and TIMP-2. Results The results of the immunohistochemistry are set out below. marker tumour expression log-rank survival stromal expression log-rank survival MMP-2 9/72 (13%) p=0.10 34/72 (47%) p=0.34 MMP-9 79/152 (52%) p=0.04* 69/152 (45%) p=0.84 TIMP-2 28/90 (31%) p=0.04* 66/90 (73%) p=0.90 Two or more 16/59 (27%) p=0.007* There were no associations between expression and clinicopathological findings for any tumour marker. There was co-expression of MMP-2 and MMP-9 in tumour cells (p=0.01). Conclusions MMP-2, MMP-9 and TIMP-2 are expressed in NSCLC. MMP-9 and TIMP-2 tumour expression correlate with a poor outcome (both p=0.04) and are potential prognostic markers for NSCLC. Cumulative expression of two or more MMPs/TIMPs may also have increased prognostic significance. Proteases and their inhibitors are novel targets for therapeutic intervention and should be evaluated in NSCLC.

Angiogenesis and non-small cell lung cancer

Lung cancer is the commonest cause of cancer death in the western world. Recent evidence suggests that angiogenesis is related to poor prognosis in many solid tumours including non-small cell lung cancer. Angiogenesis is controlled by a complex interaction between growth and apoptotic factors, proteases and adhesion molecules. The angiogenic process may prove a target for novel therapies such as matrix metalloproteinase inhibitors, growth factor antisense RNA, growth factor receptor antagonists and naturally occurring antiangiogenic peptides. These agents may be used alone or in combination with traditional chemotherapy, radiotherapy and surgery. (C) 2000 Elsevier Science Ireland Ltd.

Herceptest: HER2 expression and gene amplification in non-small cell lung cancer

HER2 is an erbB/HER type I tyrosine kinase receptor that is frequently over-expressed in malignant epithelial tumours. Herceptin, a humanised mouse monoclonal antibody to HER2, is proven therapeutically in the management of metastatic breast cancer, significantly prolonging survival when combined with cytotoxic chemotherapeutic agents. Immunohistochemical studies suggest that non-small-cell lung cancer (NSCLC) tumours may over-express HER2. Our aim was to evaluate HER2 gene amplification and semi-quantitative immuno-expression in NSCLC. A total of 344 NSCLC cases were immunostained for HER2 expression in 2 centres using the HercepTest. Fluorescence in situ hybridisation (FISH) analysis for HER2 gene amplification was performed on most positive cases and a subset of negative cases. Fifteen cases (4.3%) demonstrated 2+ or 3+ membranous HER2 immuno-expression. There was no correlation between immuno-expression and tumour histology or grade. Tumours from higher-stage disease were more often HercepTest-positive (p < 0.001). All 4 HercepTest 3 + cases demonstrated gene amplification. One of the 5 2+ cases tested for gene amplification showed areas of borderline amplification and areas of polyploidy. None of the 19 HercepTest-negative cases demonstrated gene amplification or polyploidy (p < 0.001). Gene amplification was demonstrated in all HercepTest 3+ scoring NSCLC cases. Unlike breast cancer, gene amplification and HER2 protein over-expression assessed by the HercepTest appeared to be uncommon in NSCLC. Herceptin may therefore target only a small proportion of NSCLC tumours and be of limited clinical value in this disease, particularly in the adjuvant setting. © 2001 Wiley-Liss, Inc.

Prognostic significance of platelet and microvessel counts in operable non-small cell lung cancer

Background: Microvessel density, an indirect measure of angiogenesis, has been shown to be an independent prognostic marker in many solid tumours including non-small cell lung cancer (NSCLC). Platelets transport and release angiogenic growth factors. Platelets are increasingly likely to adhere to tumour microvessels due to raised expression of platelet-binding proteins and stasis in blood-flow. Increased vascular permeability in tumour microvessels facilitates platelet extravasation into the extracellular matrix. Adherence and extravasation both lead to platelet activation and release of growth factors capable of instigating the angiogenic process. Methods: A total of 181 patients were identified who underwent resection of stage I-IIIa NSCLC with a post-operative survival >60 days. Patients were followed-up for a minimum of 24 months. Sections from the tumour periphery were stained for the endothelial marker CD34 (Novocastra NCL-END) using standard ABC immunohistochemistry. Chalkley counting was used to assess microvessel density. Results: A pre-operative platelet count greater than the median and above the normal range (>400) was associated with a poor outcome (P = 0.01 and P = 0.04, respectively). Tumours with an above median and high Chalkley count (upper tertile) had a worse prognosis (P = 0.007 and P = 0.0006, respectively). There was no association between platelet count and Chalkley count. Conclusions: Platelet and microvessel counts are both potential prognostic markers for NSCLC. The role of platelets in the angiogenic process needs to be further investigated. (C) 2000 Elsevier Science Ireland Ltd.

Platinum-based chemotherapy in metastatic breast cancer : current status

Cisplatin and carboplatin are active in previously untreated patients with metastatic breast cancer (MBC) with mean response rates (RRs) of 50 and 32%, respectively. In pretreated patients the RR to cisplatin/carboplatin monotherapy declines markedly to <10%. Cisplatin and carboplatin have been combined with many other cytotoxics. In first-line setting high activity has been observed in combination with taxanes or vinorelbine (RRs consistently ∼60%). It appears that these newer combinations are superior to older regimens with etoposide (RRs 30 to 50%) or 5-fluorouracil (RRs 40 to 60%). Cisplatin-/carboplatin-based regimens with infusional 5-FU and epirubicin/paclitaxel/vinorelbine achieve high RRs of around 60 to 80%. However these regimens are difficult to administer in all patients because they require central venous access for continuous 5-FU infusion. In pretreated MBC the combinations of cisplatin-taxane/vinorelbine/gemcitabine or carboplatin-docetaxel/vinorelbine yield RRs of 40 to 50%, which are higher than those achieved with platinum-etoposide/5-FU. In locally advanced disease cisplatin-based regimens achieve very high RRs (>80%). This would suggest that in chemotherapy-naïve patients platinum-based therapy might have an important role to play. Additionally the synergy demonstrated between platinum compounds, taxanes and herceptin, in preclinical and clinical studies is of immense importance and the results of the two ongoing Breast Cancer International Research Group randomized phase III studies are eagerly awaited. These studies may help clarify the role of platinum compounds in the treatment of metastatic and possibly early breast cancer. © 2003 Elsevier Ltd. All rights reserved.

Platinum-based chemotherapy in metastatic breast cancer : the Leicester (UK) experience

Aims: After failure of anthracycline- and taxane-based chemotherapy in metastatic breast cancer, treatment options until recently were limited. Until the introduction of capecitabine and vinorelbine, no standard regimen was available. We conducted a retrospective study to determine the efficacy and toxicity of platinum-based chemotherapy in metastatic breast cancer. Materials and methods: Forty-two women with metastatic breast cancer previously treated with anthracyclines (93%) and/or taxanes (36%) received mitomycin-vinblastine-cisplatin (MVP) (n = 23), or cisplatin-etoposide (PE) (n = 19), as first-, second- and third-line treatment at a tertiary referral centre between 1997 and 2002. Chemotherapy was given every 3 weeks as follows: mitomycin-C (8 mg/m 2) (cycles 1, 2, 4, 6), vinblastine (6 mg/m 2), and cisplatin (50 mg/m 2) all on day 1; and cisplatin (75 mg/m 2) and etoposide (100 mg/m 2) on day 1 and (100 mg/m 2) orally twice a day on days 2-3. Results: The response rate for 40 evaluable patients (MVP: n = 23; PE: n = 17) was 18% (95% confidence interval [CI]: 9-32%). The response rate to MVP was 13% (95% CI: 5-32%, one complete and two partial responses) and to PE 24% (10-47%, four partial responses). Disease stabilised in 43% (26-63%) and 47% (26-69%) of women treated with MVP and PE, respectively. After a median follow-up of 18 months, 37 women (MVP: n = 19; PE: n = 18) died from their disease. Median (range) progression-free survival and overall survival were 6 months (0.4-18.7) and 9.9 months (1.3-40.8), respectively. Median progression-free survival for the MVP and PE groups was 5.5 and 6.2 months (Log-rank, P = 0.82), and median overall survival was 10.2 and 9.4 months (Log-rank, P = 0.46), respectively. The main toxicity was myelosuppression. Grades 3-4 neutropenia was more common in women treated with PE than in women treated with MVP (74% vs 30%; P = 0.012), but the incidence of neutropenic sepsis, relative to the number of chemotherapy cycles, was low (7% overall). The toxicity-related hospitalisation rate was 1.2 admissions per six cycles of chemotherapy. No treatment-related deaths occurred. MVP and PE chemotherapy have modest activity and are safe in women with metastatic breast cancer. © 2005 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Analysis of acute-phase proteins, AHSG, C3, CLI, HP and SAA, reveals distinctive expression patterns associated with breast, colorectal and lung cancer

Early detection, clinical management and disease recurrence monitoring are critical areas in cancer treatment in which specific biomarker panels are likely to be very important in each of these key areas. We have previously demonstrated that levels of alpha-2-heremans-schmid-glycoprotein (AHSG), complement component C3 (C3), clusterin (CLI), haptoglobin (HP) and serum amyloid A (SAA) are significantly altered in serum from patients with squamous cell carcinoma of the lung. Here, we report the abundance levels for these proteins in serum samples from patients with advanced breast cancer, colorectal cancer (CRC) and lung cancer compared to healthy controls (age and gender matched) using commercially available enzyme-linked immunosorbent assay kits. Logistic regression (LR) models were fitted to the resulting data, and the classification ability of the proteins was evaluated using receiver-operating characteristic curve and leave-one-out cross-validation (LOOCV). The most accurate individual candidate biomarkers were C3 for breast cancer [area under the curve (AUC) = 0.89, LOOCV = 73%], CLI for CRC (AUC = 0.98, LOOCV = 90%), HP for small cell lung carcinoma (AUC = 0.97, LOOCV = 88%), C3 for lung adenocarcinoma (AUC = 0.94, LOOCV = 89%) and HP for squamous cell carcinoma of the lung (AUC = 0.94, LOOCV = 87%). The best dual combination of biomarkers using LR analysis were found to be AHSG + C3 (AUC = 0.91, LOOCV = 83%) for breast cancer, CLI + HP (AUC = 0.98, LOOCV = 92%) for CRC, C3 + SAA (AUC = 0.97, LOOCV = 91%) for small cell lung carcinoma and HP + SAA for both adenocarcinoma (AUC = 0.98, LOOCV = 96%) and squamous cell carcinoma of the lung (AUC = 0.98, LOOCV = 84%). The high AUC values reported here indicated that these candidate biomarkers have the potential to discriminate accurately between control and cancer groups both individually and in combination with other proteins. Copyright © 2011 UICC.

Daxx regulates mitotic progression and prostate cancer predisposition

Mitotic progression of mammalian cells is tightly regulated by the E3 ubiquitin ligase anaphase promoting complex (APC)/C. Deregulation of APC/C is frequently observed in cancer cells and is suggested to contribute to chromosome instability and cancer predisposition. In this study, we identified Daxx as a novel APC/C inhibitor frequently overexpressed in prostate cancer. Daxx interacts with the APC/C coactivators Cdc20 and Cdh1 in vivo, with the binding of Cdc20 dependent on the consensus destruction boxes near the N-terminal of the Daxx protein. Ectopic expression of Daxx, but not the D-box deleted mutant (DaxxΔD-box), inhibited the degradation of APC/Cdc20 and APC/Cdh1 substrates, leading to a transient delay in mitotic progression. Daxx is frequently upregulated in prostate cancer tissues; the expression level positively correlated with the Gleason score and disease metastasis (P = 0.027 and 0.032, respectively). Furthermore, ectopic expression of Daxx in a non-malignant prostate epithelial cell line induced polyploidy under mitotic stress. Our data suggest that Daxx may function as a novel APC/C inhibitor, which promotes chromosome instability during prostate cancer development.