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.

Prognostic factors in patients with advanced non-small cell lung cancer : data from the phase III FLEX study

The FLEX study demonstrated that the addition of cetuximab to chemotherapy significantly improved overall survival in the first-line treatment of patients with advanced non-small cell lung cancer (NSCLC). In the FLEX intention to treat (ITT) population, we investigated the prognostic significance of particular baseline characteristics. Individual patient data from the treatment arms of the ITT population of the FLEX study were combined. Univariable and multivariable Cox regression models were used to investigate variables with potential prognostic value. The ITT population comprised 1125 patients. In the univariable analysis, longer median survival times were apparent for females compared with males (12.7 vs 9.3 months); patients with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 compared with 1 compared with 2 (13.5 vs 10.6 vs 5.9 months); never smokers compared with former smokers compared with current smokers (14.6 vs 11.1 vs 9.0); Asians compared with Caucasians (19.5 vs 9.6 months); patients with adenocarcinoma compared with squamous cell carcinoma (12.4 vs 9.3 months) and those with metastases to one site compared with two sites compared with three or more sites (12.4 months vs 9.8 months vs 6.4 months). Age (<65 vs ≥65 years), tumor stage (IIIB with pleural effusion vs IV) and percentage of tumor cells expressing EGFR (<40% vs ≥40%) were not identified as possible prognostic factors in relation to survival time. In multivariable analysis, a stepwise selection procedure identified age (<65 vs ≥65 years), gender, ECOG PS, smoking status, region, tumor histology, and number of organs involved as independent factors of prognostic value. In summary, in patients with advanced NSCLC enrolled in the FLEX study, and consistent with previous analyses, particular patient and disease characteristics at baseline were shown to be independent factors of prognostic value. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. © 2012 Elsevier Ireland Ltd.

EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer : analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 First-Line ErbituX in lung cancer (FLEX) study showed that the addition of cetuximab to first-line chemotherapy significantly improved overall survival compared with chemotherapy alone (hazard ratio [HR] 0·871, 95% CI 0·762-0·996; p=0·044) in patients with advanced non-small-cell lung cancer (NSCLC). To define patients benefiting most from cetuximab, we studied the association of tumour EGFR expression level with clinical outcome in FLEX study patients. Methods: We used prospectively collected tumour EGFR expression data to generate an immunohistochemistry score for FLEX study patients on a continuous scale of 0-300. We used response data to select an outcome-based discriminatory threshold immunohistochemistry score for EGFR expression of 200. Treatment outcome was analysed in patients with low (immunohistochemistry score <200) and high (≥200) tumour EGFR expression. The primary endpoint in the FLEX study was overall survival. We analysed patients from the FLEX intention-to-treat (ITT) population. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Tumour EGFR immunohistochemistry data were available for 1121 of 1125 (99·6%) patients from the FLEX study ITT population. High EGFR expression was scored for 345 (31%) evaluable patients and low for 776 (69%) patients. For patients in the high EGFR expression group, overall survival was longer in the chemotherapy plus cetuximab group than in the chemotherapy alone group (median 12·0 months [95% CI 10·2-15·2] vs 9·6 months [7·6-10·6]; HR 0·73, 0·58-0·93; p=0·011), with no meaningful increase in side-effects. We recorded no corresponding survival benefit for patients in the low EGFR expression group (median 9·8 months [8·9-12·2] vs 10·3 months [9·2-11·5]; HR 0·99, 0·84-1·16; p=0·88). A treatment interaction test assessing the difference in the HRs for overall survival between the EGFR expression groups suggested a predictive value for EGFR expression (p=0·044). Interpretation: High EGFR expression is a tumour biomarker that can predict survival benefit from the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC. Assessment of EGFR expression could offer a personalised treatment approach in this setting. Funding: Merck KGaA. © 2012 Elsevier Ltd.

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.

Epidermal growth factor receptors and cyclooxygenase-2 in the pathogenesis of non-small cell lung cancer : potential targets for chemoprevention and systemic therapy

The epidermal growth factor receptor (EGFR) is part of a family of plasma membrane receptor tyrosine kinases that control many important cellular functions, from growth and proliferation to cell death. Cyclooxygenase (COX)-2 is an enzyme which catalyses the conversion of arachidonic acid to prostagladins and thromboxane. It is induced by various inflammatory stimuli, including the pro-inflammatory cytokines, Interleukin (IL)-1β, Tumour Necrosis Factor (TNF)-α and IL-2. Both EGFR and COX-2 are over-expressed in non-small cell lung cancer (NSCLC) and have been implicated in the early stages of tumourigenesis. This paper considers their roles in the development and progression of lung cancer, their potential interactions, and reviews the recent progress in cancer therapies that are directed toward these targets. An increasing body of evidence suggests that selective inhibitors of both EGFR and COX-2 are potential therapeutic agents for the treatment of NSCLC, in the adjuvant, metastatic and chemopreventative settings. © 2002 Elsevier Science 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.

Phospho-Akt expression is associated with a favorable outcome in non-small cell lung cancer

Akt, a Serine/Threonine protein kinase, mediates growth factor-associated cell survival. Constitutive activation of Akt (phosphorylated Akt, P-Akt) has been observed in several human cancers, including lung cancer and may be associated with poor prognosis and chemotherapy and radiotherapy resistance. The clinical relevance of P-Akt in non-small cell lung cancer (NSCLC) is not well described. In the present study, we examined 82 surgically resected snap-frozen and paraffin-embedded stage I to IIIA NSCLC samples for P-Akt and Akt by Western blotting and for P-Akt by immunohistochemistry. P-Akt protein levels above the median, measured using reproducible semiquantitative band densitometry, correlated with a favorable outcome (P = 0.007). Multivariate analysis identified P-Akt as a significant independent favorable prognostic factor (P = 0.004). Although associated with a favorable prognosis, high P-Akt levels correlated with high tumor grade (P = 0.02). Adenocarcinomas were associated with low P-Akt levels (P = 0.039). Akt was not associated with either outcome or clinicopathologic variables. Cytoplasmic (CP-Akt) and nuclear (NP-Akt) P-Akt tumor cell staining was detected in 96% and 42% of cases, respectively. Both CP-Akt and NP-Akt correlated with well-differentiated tumors (P = 0.008 and 0.017, respectively). NP-Akt also correlated with nodal metastases (P = 0.022) and squamous histology (P = 0.037). These results suggest P-Akt expression is a favorable prognostic factor in NSCLC. Immunolocalization of P-Akt, however, may be relevant as NP-Akt was associated with nodal metastases, a known poor prognostic feature in this disease. P-Akt may be a potential novel therapeutic target for the management of NSCLC. © 2005 American Association for Cancer Research.

Coexpression of epidermal growth factor receptor with related factors is associated with a poor prognosis in non-small-cell lung cancer

The epidermal growth factor receptor (EGFR) is commonly expressed in non-small-cell lung cancer (NSCLC) and promotes a host of mechanisms involved in tumorigenesis. However, EGFR expression does not reliably predict prognosis or response to EGFR-targeted therapies. The data from two previous studies of a series of 181 consecutive surgically resected stage I-IIIA NSCLC patients who had survived in excess of 60 days were explored. Of these patients, tissue was available for evaluation of EGFR in 179 patients, carbonic anhydrase (CA) IX in 177 patients and matrix metalloproteinase-9 (MMP-9) in 169 patients. We have previously reported an association between EGFR expression and MMP-9 expression. We have also reported that MMP-9 (P=0.001) and perinuclear (p)CA IX (P=0.03) but not EGFR expression were associated with a poor prognosis. Perinuclear CA IX expression was also associated with EGFR expression (P<0.001). Multivariate analysis demonstrated that coexpression of MMP-9 with EGFR conferred a worse prognosis than the expression of MMP-9 alone (P<0.001) and coexpression of EGFR and pCA IX conferred a worse prognosis than pCA IX alone (P=0.05). A model was then developed where the study population was divided into three groups: group 1 had expression of EGFR without coexpression of MMP-9 or pCA IX (number=21); group 2 had no expression of EGFR (number=75); and group 3 had coexpression of EGFR with pCA IX or MMP-9 or both (number=70). Group 3 had a worse prognosis than either groups 1 or 2 (P=0.0003 and 0.027, respectively) and group 1 had a better prognosis than group 2 (P=0.036). These data identify two cohorts of EGFR-positive patients with diametrically opposite prognoses. The group expressing either EGFR and or both MMP-9 and pCA IX may identify a group of patients with activated EGFR, which is of clinical relevance with the advent of EGFR-targeted therapies. © 2004 Cancer Research UK.

Hypoxia-inducible factor-1α in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathways

Hypoxia-inducible factor (HIF)-1α is the regulatory subunit of HIF-1 that is stabilized under hypoxic conditions. Under different circumstances, HIF-1α may promote both tumorigenesis and apoptosis. There is conflicting data on the importance of HIF-1α as a prognostic factor. This study evaluated HIF-1α expression in 172 consecutive patients with stage I-IIIA non small cell lung cancer (NSCLC) using standard immunohistochemical techniques. The extent of HIF-1α nuclear immunostaining was determined using light microscopy and the results were analyzed using the median (5%) as a low cut-point and 60% as a high positive cut-point. Using the low cut-point, positive associations were found with epidermal growth factor receptor (EGFR; p = 0.01), matrix metalloproteinase (MMP)-9 (p = 0.003), membranous (p < 0.001) and perinuclear (p = 0.004) carbonic anhydrase (CA) IX, pS3 (p = 0.008), T-stage (p = 0.042), tumor necrosis (TN; p < 0.001) and squamous histology (p < 0.001). No significant association was found with Bcl-2 or either N- or overall TMN stage or prognosis. When the high positive cut-point was used, HIF-1α was associated with a poor prognosis (p = 0.034). In conclusion, the associations with EGFR, MMP-9, p53 and CA IX suggest that these factors may either regulate or be regulated by HIF-1α. The association with TN and squamous-type histology, which is relatively more necrotic than other NSCLC types, reflects the role of hypoxia in the regulation of HIF-1α. The prognostic data may reflect a change in the behavior of HIF-1α in increasingly hypoxic environments. © 2004 Wiley-Liss, Inc.

Tumour necrosis is an independent prognostic marker in non-small cell lung cancer : correlation with biological variables

Background Tumour necrosis (TN) is recognized to be a consequence of chronic cellular hypoxia. TN and hypoxia correlate with poor prognosis in solid tumours. Methods In a retrospective study the prognostic implications of the extent of TN was evaluated in non-small cell lung cancer (NSCLC) and correlated with clinicopathological variables and expression of epidermal growth factor receptor, Bcl-2, p53 and matrix metalloproteinase-9 (MMP-9). Tissue specimens from 178 surgically resected cases of stage I-IIIA NSCLC with curative intent were studied. The specimens were routinely processed, formalin-fixed and paraffin-embedded. TN was graded as extensive or either limited or absent by two independent observers; disagreements were resolved using a double-headed microscope. The degree of reproducibility was estimated by re-interpreting 40 randomly selected cases after a 4 month interval. Results Reproducibility was attained in 36/40 cases, Kappa score=0.8 P<0.001. TN correlated with T-stage (P=0.001), platelet count (P=0.004) and p53 expression (P=0.031). Near significant associations of TN with N-stage (P=0.063) and MMP-9 expression (P=0.058) were seen. No association was found with angiogenesis (P=0.98). On univariate (P=0.0016) and multivariate analysis (P=0.023) TN was prognostic. Conclusion These results indicate that extensive TN reflects an aggressive tumour phenotype in NSCLC and may improve the predictive power of the TMN staging system. The lack of association between TN and angiogenesis may be important although these variables were not evaluated on serial sections. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Delineating breast cancer cell interactions with engineered bone microenvironments

The mechanisms leading to colonization of metastatic breast cancer cells (BCa) in the skeleton are still not fully understood. Here, we demonstrate that mineralized extracellular matrices secreted by primary human osteoblasts (hOBM) modulate cellular processes associated with BCa colonization of bone. A panel of four BCa cell lines of different bone-metastatic potential (T47D, SUM1315, MDA-MB-231, and the bone-seeking subline MDA-MB-231BO) was cultured on hOBM. After 3 days, the metastatic BCa cells had undergone morphological changes on hOBM and were aligned along the hOBM's collagen type I fibrils that were decorated with bone-specific proteins. In contrast, nonmetastatic BCa cells showed a random orientation on hOBM. Atomic force microscopy-based single-cell force spectroscopy revealed that the metastatic cell lines adhered more strongly to hOBM compared with nonmetastatic cells. Function-blocking experiments indicated that β1-integrins mediated cell adhesion to hOBM. In addition, metastatic BCa cells migrated directionally and invaded hOBM, which was accompanied by enhanced MMP-2 and -9 secretion. Furthermore, we observed gene expression changes associated with osteomimickry in BCa cultured on hOBM. As such, osteopontin mRNA levels were significantly increased in SUM1315 and MDA-MB-231BO cells in a β1-integrin-dependent manner after growing for 3 days on hOBM compared with tissue culture plastic. In conclusion, our results show that extracellular matrices derived from human osteoblasts represent a powerful experimental platform to dissect mechanisms underlying critical steps in the development of bone metastases.

Fibrogenic stresses activate different mitogen-activated protein kinase pathways in renal epithelial, endothelial or fibroblast cell populations

Fibrogenic stresses promote progression of renal tubulointerstitial fibrosis, disparately affecting survival, proliferation and trans-differentiation of intrinsic renal cell populations through ill-defined biomolecular pathways. We investigated the effect of fibrogenic stresses on the activation of cell-specific mitogen-activated protein kinase (MAPK) in renal fibroblast, epithelial and endothelial cell populations. The relative outcomes (cell death, proliferation, trans-differentiation) associated with activation or inhibition of extracellular-regulated protein kinase (ERK) or stress activated/c-Jun N terminal kinase (JNK) were analysed in each renal cell population after challenge with oxidative stress (1 mmol/L H2O2), transforming growth factor-beta1 (TGF-beta1, 10 ng/mL) or tumour necrosis factor-alpha (TNF-alpha, 50 ng/mL) over 0-20 h. Apoptosis increased significantly in all cell types after oxidative stress (P < 0.05). In fibroblasts, oxidative stress caused the activation of ERK (pERK) but not JNK (pJNK). Inhibition of ERK by PD98059 supported its role in a fibroblast death pathway. In epithelial and endothelial cells, oxidative stress-induced apoptosis was preceded by early induction of pERK, but its inhibition did not support a pro-apoptotic role. Early ERK activity may be conducive to their survival or promote the trans-differentiation of epithelial cells. In epithelial and endothelial cells, oxidative stress induced pJNK acutely. Pretreatment with SP600125 (JNK inhibitor) verified its pro-apoptotic activity only in epithelial cells. Transforming growth factor-beta1 did not significantly alter mitosis or apoptosis in any of the cell types, nor did it alter MAPK activity. Tumor necrosis factor-alpha caused increased apoptosis with no associated change in MAPK activity. Our results demonstrate renal cell-specific differences in the activation of ERK and JNK following fibrotic insult, which may be useful for targeting excessive fibroblast proliferation in chronic fibrosis.

Selective regulation of p38β protein and signaling by integrin-linked kinase mediates bladder cancer cell migration

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK–p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK–p38β signaling complexes, playing a key role in bladder cancer cell migration.

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results Serum TXB2 levels were correlated with VEGF (p<0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p<0.0001) and microvessel density (CD-31; p<0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p<0.0001) and female patients (p<0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p<0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.