Metabolic Footprinting of a Clear Cell Renal Cell Carcinoma in Vitro Model for Human Kidney Cancer Detection.

A protocol for harvesting and extracting extracellular metabolites from an in vitro model of human renal cell lines was developed to profile the exometabolome by means of a discovery-based metabolomics approach using ultraperformance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. Metabolic footprints provided by conditioned media (CM) samples ( n = 66) of two clear cell Renal Cell Carcinoma (ccRCC) cell lines with different genetic backgrounds and a nontumor renal cell line, were compared with the human serum metabolic profile of a pilot cohort ( n = 10) comprised of stage IV ccRCC patients and healthy individuals. Using a cross-validated orthogonal projection to latent structures-discriminant analysis model, a panel of 21 discriminant features selected by iterative multivariate classification, allowed differentiating control from tumor cell lines with 100% specificity, sensitivity, and accuracy. Isoleucine/leucine, phenylalanine, N-lactoyl-leucine, and N-acetyl-phenylalanine, and cysteinegluthatione disulfide (CYSSG) were identified by chemical standards, and hydroxyprolyl-valine was identified with MS and MS/MS experiments. A subset of 9 discriminant features, including the identified metabolites except for CYSSG, produced a fingerprint of classification value that enabled discerning ccRCC patients from healthy individuals. To our knowledge, this is the first time that N-lactoyl-leucine is associated with ccRCC. Results from this study provide a proof of concept that CM can be used as a serum proxy to obtain disease-related metabolic signatures.

The neural cell adhesion molecule is involved in the metastatic capacity in a murine model of lung cancer.

Neural cell adhesion molecule (NCAM) is involved in cell growth, migration, and differentiation. Its expression and/or polysialylation appear to be deregulated in many different cancer types. We employed the lung tumor cell line LP07, syngeneic in BALB/c mice to investigate the role of NCAM in malignant progression. LP07 cells express the three main NCAM isoforms, all of them polysialylated. This cells line, pretreated with an anti-NCAM antibody and inoculated intravenously (i.v.) into syngeneic mice, developed less and smaller lung metastases. In vitro studies showed that NCAM bound antibody inhibited cell growth, mainly due to an increase in apoptosis, associated with a decrease of cyclin D1 and enhanced expression of active caspase 3 and caspase 9. Anti-NCAM-treated LP07 cells showed impairment in their ability to migrate and adhere to several extracellular matrix components. Secreted uPA activity was also reduced. NCAM-140 knocked-down by siRNA in LP07 cells pretreated or not with anti-NCAM showed an impaired metastasizing ability upon i.v. inoculation into mice. These results suggest that anti-NCAM treatment could be mimicking homophilic trans-interactions and NCAM-140 knocked-down impairs heterophilic interactions, both leading to inhibition of metastatic dissemination. The involvement of NCAM in lung tumor progression was confirmed in human NSCLC tumors. Sixty percent of the cases expressed NCAM at tumor cell level. A multivariate analysis indicated that NCAM expression was associated with a shorter overall survival in this homogeneous series of Stages I and II NSCLC patients. NCAM may be able to modulate mechanisms involved in lung carcinoma progression and represents an attractive target to control metastatic progression.

Fibronectin is distinctly downregulated in murine mammary adenocarcinoma cells with high metastatic potential.

In the present work we used a murine mammary cancer model of two related adenocarcinomas with different lung metastasizing abilities, to compare their global gene expression profiles. Clontech Atlas mouse cDNA microarrays of primary cultured tumor cells were employed to identify genes that are modulated in the more metastatic variant MM3 relative to its parental tumor M3. A total of 88 from 1,176 genes were differentially expressed in MM3 primary cultures, most of them (n=86) were upregulated. Genes were grouped according to their functions as associated with signal transduction and transcription regulation (e.g. Stat1 and Zfp 92), with cell adhesion and motility (cadherin 1, fibronectin), with invasion and angiogenesis (uPA, 72 kDa MMP2), with the regulation of cell proliferation and cell death (cyclins G and A2, TNF), and also included growth factors and receptors, oncogenes and tumor suppressors genes (p107, TGFbeta2, TBR-I, PDGFR). Only 2 genes, TTF1 and fibronectin (FN), showed a significant downregulation. Notably FN expression, loss of which has been associated with a malignant phenotype, was reduced about 19-fold in the more metastatic MM3 cells. Previously known differences in expression patterns associated with the metastatic capacity of MM3 and M3 adenocarcinomas, including downregulation of FN or upregulated expression of TGFbeta and proteases, were confirmed by the array data. The fact that FN was one of the only two genes significantly down-regulated out of the 1,176 genes analyzed stresses the hypothesis that FN may behave as an important metastasis suppressor gene in mammary cancer.

'Reduced malignancy as a mechanism for longevity in mice with adenylyl cyclase type 5 disruption'.

Disruption of adenylyl cyclase type 5 (AC5) knockout (KO) is a novel model for longevity. Because malignancy is a major cause of death and reduced lifespan in mice, the goal of this investigation was to examine the role of AC5KO in protecting against cancer. There have been numerous discoveries in genetically engineered mice over the past several decades, but few have been translated to the bedside. One major reason is that it is difficult to alter a gene in patients, but rather a pharmacological approach is more appropriate. The current investigation employs a parallel construction to examine the extent to which inhibiting AC5, either in a genetic knockout (KO) or by a specific pharmacological inhibitor protects against cancer. This study is unique, not only because a combined genetic and pharmacological approach is rare, but also there are no prior studies on the extent to which AC5 affects cancer. We found that AC5KO delayed age-related tumor incidence significantly, as well as protecting against mammary tumor development in AC5KO × MMTV-HER-2 neu mice, and B16F10 melanoma tumor growth, which can explain why AC5KO is a model of longevity. In addition, a Food and Drug Administration approved antiviral agent, adenine 9-ß-D-arabinofuranoside (Vidarabine or AraAde), which specifically inhibits AC5, reduces LP07 lung and B16F10 melanoma tumor growth in syngeneic mice. Thus, inhibition of AC5 is a previously unreported mechanism for prevention of cancers associated with aging and that can be targeted by an available pharmacologic inhibitor, with potential consequent extension of lifespan.

Clinical relevance of galectin-1 expression in non-small cell lung cancer patients.

BACKGROUND: Identification of biomarkers in lung cancer, a leading cause of cancer-related mortality, has a meaningful clinical relevance in the quest of novel prognostic factors and therapeutic targets. The glycan-binding protein galectin-1 (Gal-1) modulates tumor progression by mediating cell-cell and cell-extracellular matrix interactions, as well as angiogenesis and tumor immune-escape. Previous works reported the expression of Gal-1 in lung cancer, although its clinical significance remains uncertain. OBJECTIVE: To assess the clinicopathologic relevance and prognostic value of Gal-1 expression in a cohort of 103 Stage I-III non-small cell lung cancer (NSCLC) patients. METHODS: Gal-1 expression was determined by immunohistochemistry in tumor tissue samples. The percentage of immunoreactive tumor cells and stroma, as well as the presence of blood vessels with positively stained endothelium in the tumor and surrounding normal tissue, were recorded. Results were correlated with the clinicopathologic factors of the patients (Spearman's rank correlation coefficient, chi-square test) and overall survival by univariate (Kaplan Meier) and multivariate analyses (Cox regression hazard model). RESULTS: We did not observe significant associations between Gal-1 expression and relevant clinicopathologic features at diagnosis of NSCLC. However, Kaplan Meier analysis revealed a significant association between Gal-1 expression and overall survival, when Gal-1 expression was analyzed on tumor cells alone ("tumor cell percentage") or when an integrated score accounting for tumor cell as well as stromal expression of Gal-1 ("total score") was assessed. Patients showing high Gal-1 expression evidenced a poorer clinical outcome. Furthermore, "total score" remained significantly associated with survival by multivariate Cox regression analysis in the whole cohort of patients, even when controlling for the classical predictors and prognostic factors of NSCLC. CONCLUSION: We conclude that Gal-1 expression may be a useful biomarker for better prediction of the clinical outcome and management of NSCLC patients.

Modulation of pancreatic tumor potential by overexpression of protein kinase C ß1.

OBJECTIVE: This study aimed to investigate whether the overexpression of protein kinase C ß1 (PKCß1) is able to modulate the malignant phenotype displayed by the human ductal pancreatic carcinoma cell line PANC1. METHODS: PKCß1 overexpression was achieved using a stable transfection approach. PANC1-PKCß1 and control cells were analyzed both in vitro and in vivo. RESULTS: PANC1-PKCß1 cells displayed a lower growth capacity associated with the down-regulation of the MEK/ERK pathway and cyclin expression. Furthermore, PKCß1 overexpression was associated with an enhancement of cell adhesion to fibronectin and with reduced migratory and invasive phenotypes. In agreement with these results, PANC1-PKCß1 cells showed an impaired ability to secrete proteolytic enzymes. We also found that PKCß1 overexpressing cells were more resistant to cell death induced by serum deprivation, an event associated with G0/G1 arrest and the modulation of PI3K/Akt and NF-κB pathways. Most notably, the overexpression of PKCß1 completely abolished the ability of PANC1 cells to induce tumors in nude mice. CONCLUSIONS: Our results established an important role for PKCß1 in PANC1 cells suggesting it would act as a suppressor of tumorigenic behavior in pancreatic cancer.

TGF-ß autocrine pathway and MAPK signaling promote cell invasiveness and in vivo mammary adenocarcinoma tumor progression.

Breast cancer progression and metastasis have been linked to abnormal signaling by transforming growth factor-ß (TGF-ß) cytokines. In early-stage breast cancers, TGF-ß exhibits tumor suppressor activity by repressing cell proliferation and inducing cell death, whereas in advanced-stage tumors, TGF-ß promotes invasion and metastatic dissemination. The molecular mechanisms underlying pro-oncogenic activities of TGF-ß are not fully understood. The present study validates the role of TGF-ß signaling in cancer progression and explores mediators of pro-oncogenic TGF-ß activities using the LM3 mammary adenocarcinoma cell line, derived from a spontaneous murine mammary adenocarcinoma. Expression of kinase-inactive TGF-ß receptors decreased both basal and TGF-ß-induced invasion. Analysis of signal transduction mediators showed that p38MAPK and MEK contribute to TGF-ß stimulation of cell motility and invasion. TGF-ß disrupted the epithelial actin structures supporting cell-cell adhesions, and increased linear actin filaments. Moreover, MEK and p38MAPK pathways showed opposite effects on actin remodeling in response to TGF-ß. Blockade of Raf-MEK signaling enhanced TGF-ß induction of actin stress-fibers whereas p38MAPK inhibitors blocked this effect. A novel observation was made that TGF-ß rapidly activates the actin nucleation Arp2/3 complex. In addition, TGF-ß stimulated matrix metalloproteinase MMP-9 secretion via a MAPK-independent pathway. Experiments using syngeneic mice showed that kinase-inactive TGF-ß receptors inhibit the first stages of LM3 tumor growth in vivo. Our studies demonstrate that autocrine TGF-ß signaling contributes to the invasive behavior of mammary carcinoma cells. Moreover, we show that both MAPK-dependent and -independent pathways are necessary for TGF-ß-induced effects. Therefore, MEK-ERK and p38 MAPK pathways are potential venues for therapeutic intervention in pro-oncogenic TGF-ß signaling.

Association between mast cells of different phenotypes and angiogenesis in colorectal cancer.

It is known that mast cells proliferate in solid tumours and increase tumour angiogenesis. Nevertheless, there is no consensus regarding their role in colorectal cancer (CRC). In this study, we aimed to clarify the relationship of mast cells positive for tryptase (MCts) and tryptase-chymase (MCtcs) with microvessel density (MVD) in the intratumoral zone and the invasive edge of 80 CRC patient tumours. We evaluated these parameters and associated their expression with clinicopathological parameters, including survival rate. Tumour sections from each patient were immunostained for tryptase to evaluate MCts, chymase to evaluate MCtcs, and CD34 to evaluate microvessel counts under x100 microscopy. The number of MCs of both phenotypes and the MVD counts were higher in the invasive edge than in the intratumoral zone (p<0.001). MCt numbers were higher than those of MCtcs in all Astler-Coller stages in both regions. A positive correlation between MVD and MCts or MCtcs was observed (Pearson's test p<0.001). Neither the number of MCs nor MVD was associated with overall survival (log rank test). However, only 8.3% of patients with low numbers of MCtcs in the invasive edge succumbed to the disease, compared to 32% with high numbers of MCtcs. Our results indicate that angiogenesis and MC hyperplasia are events which appear early during CRC development. The correlation of MC phenotypes with MVD is in agreement with the role attributed to MCs, that of angiogenesis enhancement. Collectively, these findings suggest that screening during the early malignization of CRC can provide valuable clinical information.

Involvement of TGF-beta(s)/T(beta)Rs system in tumor progression of murine mammary adenocarcinomas.

We studied the expression of TGF-beta/T(beta)R system and its biological role in tumor development, in M3 and MM3 murine mammary adenocarcinomas with different metastasizing capability and in LM3 and LMM3 derived cell lines. All the studied cells secreted TGF-beta(s) and expressed T(beta)Rs. While the proliferation of the poorly metastatic M3 cells was significantly inhibited by 4 ng/ml TGF-beta(s), the highly metastatic MM3 cells were only slightly inhibited in response to the highest dose used. LM3 and LMM3 cells, highly invasive and metastatic, were totally refractory to TGF-beta antiproliferative effect. The role of TGF-beta in modulating key proteolytic cascades in tumor progression was also studied. TGF-beta(s) enhanced metalloproteinases production in all the studied cells while induced a stimulatory net effect on plasmin system activity only in the more metastatic cells. Our results in this murine mammary tumor lineage support the concept that dissociation of TGF-beta regulated growth control versus proteolytic enzyme pathways promotes tumor dissemination.