Monocarboxylate transporters 1-4 in NSCLC: MCT1 is an independent prognostic marker for survival.

INTRODUCTION: Monocarboxylate transporters (MCTs) 1-4 are lactate transporters crucial for cancers cells adaption to upregulated glycolysis. Herein, we aimed to explore their prognostic impact on disease-specific survival (DSS) in both cancer and tumor stromal cells in NSCLC. METHODS: Tissue micro arrays (TMAs) were constructed, representing both cancer and stromal tumor tissue from 335 unselected patients diagnosed with stage I-IIIA NSCLC. Immunohistochemistry was used to evaluate the expression of MCT1-4. RESULTS: In univariate analyses; ↓ MCT1 (P = 0.021) and ↑ MCT4 (P = 0.027) expression in cancer cells, and ↑ MCT1 (P = 0.003), ↓ MCT2 (P = 0.006), ↓ MCT3 (P = 0.020) expression in stromal cells correlated significantly with a poor DSS. In multivariate analyses; ↓ MCT1 expression in cancer cells (HR: 1.9, CI 95%: 1.3-2.8, P = 0.001), ↓ MCT2 (HR: 2.4, CI 95%: 1.5-3.9, P<0.001), ↓ MCT3 (HR: 1.9, CI 95%: 1.1-3.5, P = 0.031) and ↑ MCT1 expression in stromal cells (HR: 1.7, CI 95%: 1.1-2.7, P = 0.016) were significant independent poor prognostic markers for DSS. CONCLUSIONS: We provide novel information of MCT1 as a candidate marker for prognostic stratification in NSCLC. Interestingly, MCT1 shows diverging, independent prognostic impact in the cancer cell and stromal cell compartments.

Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth.

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1ß, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.

Expression of TWEAK/Fn14 in neuroblastoma: implications in tumorigenesis.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) family of cytokines, acts on responsive cells via binding to a cell surface receptor called Fn14. TWEAK binding to an Fn14 receptor or constitutive Fn14 overexpression has been shown to activate nuclear factor κB signaling which is important in tumorigenesis and cancer therapy resistance. In the present study, we demonstrate that TWEAK and Fn14 are expressed in neuroblastoma cell lines and primary tumors, and both are observed at increased levels in high-stage tumors. The treatment of neuroblastoma cell lines with recombinant TWEAK in vitro causes increased survival, and this effect is partially due to the activation of NF-κB signaling. Moreover, TWEAK induces the release of matrix metalloprotease-9 (MMP-9) in neuroblastoma cells, suggesting that TWEAK may play a role in the invasive phase of neuroblastoma tumorigenesis. TWEAK-induced cell survival was significantly reduced by silencing the TWEAK and Fn14 gene functions by siRNA. Thus, the expression of TWEAK and Fn14 in neuroblastoma suggests that TWEAK functions as an important regulator of primary neuroblastoma growth, invasion and survival and that the therapeutic intervention of the TWEAK/Fn14 pathway may be an important clinical strategy in neuroblastoma therapy.

In NSCLC, VEGF-A response to hypoxia may differ between squamous cell and adenocarcinoma histology.

AIM: To investigate if hypoxia induces vascular endothelial growth factor (VEGF)-A and VEGF-C secretion in non-small cell lung cancer (NSCLC) cells and if the secretion is cell type-dependent. MATERIALS AND METHODS: Adenocarcinoma (AC) (H522, PAC) and squamous cell carcinoma (SCC) (H520) cell lines were exposed to hypoxia and normoxia. Supernatants were analysed with enzyme-linked immunosorbent assay (ELISA). Tissue microarrays, from 304 patients diagnosed with stage I-IIIA NSCLC, were immunohistochemically-stained and scored for VEGF-A and VEGF-C. RESULTS: In vitro, VEGF-A expression in hypoxic AC cells was significantly higher than that in normoxic cells (H522: p=0.004, PAC; p=0.007). In contrast, hypoxia led to significantly reduced VEGF-A production in the SCC cell line compared to normoxic cells (p=0.005). CONCLUSION: In vitro, AC and SCC exhibit different VEGF-A responses to hypoxia. Hypoxia mediates a pro-angiogenic response in AC, but apparently not in SCC.

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced.

BACKGROUND: Cancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers. METHODS: CAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining. RESULTS: Exposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, ß1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed. CONCLUSIONS: Our data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.

Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy.

The metabolism of arachidonic acid by the cyclooxygenase (COX) or lipoxygenase (LO) pathways generates eicosanoids that have been implicated in the pathogenesis of a variety of human diseases, including cancer. In this study, we examined the expression and significance of components within the 5-LO pathway in human neuroblastoma, an embryonal tumor of the sympathetic nervous system. High expression of 5-LO, 5-LO-activating protein (FLAP), leukotriene A(4) hydrolase, leukotriene C(4) synthase, and leukotriene receptors was detected in a majority of primary neuroblastoma tumors and all cell lines investigated. Expression of 5-LO and FLAP was evident in tumor cells but not in nonmalignant adrenal medulla where neuroblastomas typically arise. Moreover, neuroblastoma cells produce leukotrienes, and stimulation of neuroblastoma cells with leukotrienes increased neuroblastoma cell viability. Inhibitors of 5-LO (AA-861), FLAP (MK-886), or the leukotriene receptor antagonist montelukast inhibited neuroblastoma cell growth by induction of G(1)-cell cycle arrest and apoptosis. Similarly, specific 5-LO and leukotriene receptor silencing by small interfering RNA decreased neuroblastoma cell growth. These findings provide new insights into the pathobiology of neuroblastoma, and the use of leukotriene pathway inhibitors as a novel adjuvant therapy for children with neuroblastoma warrants further consideration.

Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides.

BACKGROUND/AIMS: Bacterial DNA and synthetic oligonucleotides containing unmethylated motifs have become the focus of many studies due to their ability to activate cells of the innate immune system through interaction with Toll-like receptor 9 (TLR9). This study was undertaken to investigate if and how CpG-oligonucleotides (CpGs) activate liver sinusoidal endothelial cells (LSECs), known to be the main site of clearance of DNA-oligonucleotides from the circulation. METHODS: Expression of TLR9 was analyzed by RT-PCR and immunohistochemistry. Production of IL-1beta and IL-6 was measured by ELISA. RESULTS: Here we show for the first time that mouse LSECs express TLR9 mRNA and protein. Moreover, our findings suggest that CpGs are first taken up by LSECs by scavenger receptor(s)-mediated endocytosis, and then join TLR9 in the lysosomal compartments. Furthermore, we found that uptake of CpGs in LSECs results in the activation of transcription factor NF-kappaB and secretion of IL-1beta and IL-6. CONCLUSIONS: The presence of functional TLR9 in LSECs emphasizes the importance of these cells in the innate defense mechanisms of the liver.

Osteoprotegerin is expressed in colon carcinoma cells.

BACKGROUND: Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor family, is produced by various cell types and tissues and plays a key role in the physiological regulation of osteoclast differentiation and activity. Also, OPG is a soluble decoy receptor for tumor necrosis factor-related apoptosis-inducing factor (TRAIL). In the present study we investigated whether the human colon cancer cell lines HT-29 and SW-480 produce and secrete OPG in vitro. MATERIALS AND METHODS: Expression of OPG mRNA was examined by RT-PCR. OPG protein was analysed by ELISA assay and immunostaining methods. The effect of OPG secretion on TRAIL-mediated apoptosis was also investigated. RESULTS: By RT-PCR, it was demonstrated that mRNA transcripts for OPG were produced by both cell lines. By ELISA analysis, OPG was detected in the culture medium; and treatment of cells with proinflammatory cytokines TNF-alpha and IL-1beta increased OPG secretion significantly. Tumor xenografts in nude mice also were shown to express OPG by immunohistochemistry. When RANKL, which selectively binds OPG, was added to cell cultures along with recombinant TRAIL, apoptosis was shown to increase significantly. CONCLUSION: These data indicate that OPG may be involved in tumorigenesis and the progression of colon cancer.

NSAIDs in neuroblastoma therapy.

Cyclooxygenases (COX) catalyse the conversion of arachidonic acid to prostaglandins. COX-2 is upregulated in several adult epithelial cancers. In neuroblastoma it has been shown that the majority of primary tumours and cell lines express high levels of COX-2, whereas normal adrenal medullas from children do not express COX-2. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs (NSAIDs), inhibitors of COX, induces caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Established neuroblastoma xenografts in nude rats treated with the dual COX-1/COX-2 inhibitor, diclofenac, or the COX-2 specific inhibitor, celecoxib significantly inhibits neuroblastoma growth in vivo. In vitro, arachidonic acid and diclofenac synergistically induces neuroblastoma cell death. This effect is further pronounced when lipoxygenases is inhibited simultaneously. Proton MR-spectroscopy (1H MRS) of neuroblastoma cells treated with COX-inhibitors demonstrates accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, 1H MRS, which can be performed with clinical MR-scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX-inhibition. Taken together, these data suggest the use of NSAIDs as a novel adjuvant therapy for children with neuroblastoma.

Synergistic induction of apoptosis in neuroblastoma cells using a combination of cytostatic drugs with interferon-gamma and TRAIL.

The majority of high-risk neuroblastomas lack the expression of caspase-8 due to gene silencing which suggest a mechanism for the selection of tumour cells that are refractory to multiple cytotoxic drugs including tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Inhibitors of DNA methyltransferases and IFN-gamma induce expression of caspase-8, and sensitise some neuroblastoma cells to TRAIL-mediated apoptosis. Here we demonstrate that a combination of cytostatic drugs with IFN-gamma and TRAIL synergistically induces neuroblastoma cell death, which may have implications for future therapy of children with neuroblastoma. Treatment of neuroblastoma cells with IFN-gamma induced caspase-8 expression in all cell lines investigated. In five of the neuroblastoma cell lines (SHEP-1, SK-N-AS, SK-N-FI, SH-SY-5Y and Kelly), IFN-gamma promoted TRAIL-mediated cleavage of caspase-8, initiating a caspase cascade involving caspase-7 and PARP followed by apoptosis. IFN-gamma-mediated facilitation of apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk and the caspase-8 specific inhibitor zIEDT-fmk, indicating an important role of caspase-8 in mediating sensitation by IFN-gamma in neuroblastoma cells. In three of the cell lines [SK-N-BE(2), SK-N-DZ and IMR-32] caspase-8 expression was induced by IFN-gamma, but the cells were still resistant to TRAIL-mediated apoptosis. The pattern of basal TRAIL receptor expression, decoy receptors, FLIP and FADD could not be correlated with resistance or sensitivity to TRAIL-induced apoptosis. Importantly, treatment of neuroblastoma cell lines with cytostatic drugs increased apoptosis in the TRAIL-sensitive cell lines whereas the resistant cell lines were susceptible to TRAIL-mediated apoptosis in the presence of the anticancer drugs. The mechanism of the increased susceptibility to apoptosis might results from drug-mediated up-regulation of the death receptors DR4 and DR5.

Cyclooxygenase-2 is expressed in neuroblastoma, and nonsteroidal anti-inflammatory drugs induce apoptosis and inhibit tumor growth in vivo.

Neuroblastoma is the single most common and deadly tumor of childhood and is often associated with therapy resistance. Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to prostaglandins. COX-2 is up-regulated in several adult epithelial cancers and is linked to proliferation and resistance to apoptosis. We detected COX-2 expression in neuroblastoma primary tumors and cell lines but not in normal adrenal medullas from children. Treatment of neuroblastoma cells with nonsteroidal anti-inflammatory drugs, inhibitors of COX, induced caspase-dependent apoptosis via the intrinsic mitochondrial pathway. Treatment of established neuroblastoma xenografts in nude rats with the dual COX-1/COX-2 inhibitor diclofenac or the COX-2-specific inhibitor celecoxib significantly inhibited tumor growth in vivo (P < 0.001). In vitro, arachidonic acid and diclofenac synergistically induced neuroblastoma cell death. This effect was further pronounced when lipooxygenases were simultaneously inhibited. Proton magnetic resonance spectroscopy ((1)H MRS) of neuroblastoma cells treated with COX inhibitors demonstrated accumulation of polyunsaturated fatty acids and depletion of choline compounds. Thus, (1)H MRS, which can be performed with clinical magnetic resonance scanners, is likely to provide pharmacodynamic markers of neuroblastoma response to COX inhibition. Taken together, these data suggest the use of nonsteroidal anti-inflammatory drugs as a novel adjuvant therapy for children with neuroblastoma.

Heterogeneity in gamma-glutamyltransferase mRNA expression and glycan structures. Search for tumor-specific variants in human liver metastases and colon carcinoma cells.

The enzyme gamma-glutamyltransferase (GGT) is frequently overexpressed in cancer cells and tissues and has significant utility as a cancer marker. Significant heterogeneity of the enzyme has been described due to both transcriptional and post-translational variations. For possible use in diagnosis and follow-up of patients with colorectal cancer, a search was performed for specific mRNA subtypes and glycan structures of the enzyme in liver metastases. We found no differences in the distribution of three GGT mRNA subtypes (fetal liver, HepG2, placenta) in metastatic tissue and normal liver tissue. Furthermore, the three subtypes were present in leukocytes isolated from both normal individuals and cancer patients. Two colon carcinoma cell lines (Colo 205 and HCC 2998) also displayed the three forms and no consistent changes in mRNA composition were noted after butyrate-induced differentiation of the cells. Thus, neither of the GGT mRNA subforms appear to be tumor-specific, although some qualitative and quantitative variations were noted. Two distinct glycosylation features were detected for GGT in metastatic tissue in contrast to normal liver GGT; an extreme sialic acid heterogeneity and a significant increase in beta1,6GlcNAc branching. The GGT glycans from the two colon carcinoma cell lines also possessed these features. As butyrate treatment of the cells resulted in an increased sialic acid content and a reduced beta1,6GlcNAc branching, the described carbohydrate structures appear to be part of a tumor-related pattern. We were, however, unable to identify such GGT isoforms in serum from patients with advanced colorectal cancer. This indicates that their usefulness in diagnostic use is doubtful.