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We have investigated alterations of microRNA expression profiles in the apparently healthy lung of mice and rats as an early response to exposure to cigarette smoke, either mainstream (MCS) or environmental, and/or to treatment with chemopreventive agents. Further on, we evaluated microRNA alterations at a later stage, when lung tumors were detectable in MCS-exposed mice. Lung samples were available from previous studies, in which strain H mice had been exposed to MCS for 4 months, starting immediately after birth, and then kept in filtered air for an additional 3 months. Some samples were from MCS-exposed mice treated either with N-acetyl-l-cysteine during pregnancy or with phenethyl isothiocyanate after weaning. The analysis of 576 mouse microRNAs showed that MCS strongly dysregulated microRNA expression and that both chemopreventive agents efficiently attenuated this trend, especially in noncancer tissue. MicroRNA expression was affected by histopathology, with specific signatures related to occurrence of pneumonia, adenoma, or bronchoalveolar carcinoma. Within pairs of samples from individual mice, microRNA analysis discriminated adenomatous tissue and especially carcinomatous tissue from the surrounding normal appearing tissue. A series of microRNA alterations characterized the sequential stages of pulmonary carcinogenesis. The involved functions included oncogene activation, inhibition of oncosuppressor genes, recruitment of undifferentiated stem cells, inflammation, inhibition of gap-junctional intercellular communications, angiogenesis, invasiveness, and metastatization. Thus, microRNA expression profiles in lung are dysregulated by MCS along all steps of the carcinogenesis process and depend on the interplay among exposure to noxious agents, treatment with dietary and pharmacological agents, and occurrence of pulmonary diseases.

The International Society for Eye Research--XIXth Biennial Meeting, held in Montreal, included topics covering new therapeutic developments in the field of eye disease research, including ocular oncology. This conference report highlights selected presentations on uveal melanoma and retinoblastoma.

Background: Several secondary metabolites from herbal nutrient products act as weak estrogens (phytoestrogens), competing with endogenous estrogen for binding to the estrogen receptors and inhibiting steroid converting enzymes. However, it is still unclear whether these compounds elicit estrogen dependent transcription of genes at physiological concentrations. Methods: We compare the effects of physiological concentrations (100 nM) of the two phytoestrogens Enterolactone and Quercetin and the suspected phytoestrogen Curcumin on gene expression in the breast cancer cell line MCF7 with the effects elicited by 17-β-estradiol (E2). Results: All three phytocompounds have weak effects on gene transcription; most of the E2 genes respond to the phytoestrogens in the same direction though to a much lesser extent and in the order Curcumin > Quercetin > Enterolactone. Gene regulation induced by these compounds was low for genes strongly induced by E2 and similar to the latter for genes only weakly regulated by the classic estrogen. Of interest with regard to the treatment of menopausal symptoms, the survival factor Birc5/survivin and the oncogene MYBL1 are strongly induced by E2 but only marginally by phytoestrogens. Conclusion: This approach demonstrates estrogenic effects of putative phytoestrogens at physiological concentrations and shows, for the first time, estrogenic effects of Curcumin.

Although microRNAs (miRNA) have extensively been investigated in cancer research, less attention has been paid to their regulation by carcinogens and/or protective factors in early stages of the carcinogenesis process. The present study was designed to evaluate the modulation of mRNA expression as related to exposure of neonatal mice to environmental cigarette smoke (ECS) and to treatment with chemopreventive agents. Exposure to ECS started immediately after birth and for 2 weeks after weaning. Thereafter, groups of mice received daily either budesonide (BUD) or phenethyl isothiocyanate (PEITC) with the diet. The expression of 576 miRNAs was evaluated by miRNA microarray in liver and lung. In sham-exposed mice, the expression of miRNAs tended to be higher in liver than in lung. ECS downregulated the expression of a number of miRNAs in lung, whereas mixed alterations were observed in liver. PEITC and BUD did not substantially affect the physiological situation in lung, whereas both agents caused intense variations in liver, reflecting the occurrence of damage mechanisms, such as inflammation, DNA and protein damage, cellular stress, proliferation and apoptosis. PEITC and BUD protected the lung from ECS-induced alterations of miRNA expression but exhibited some adverse effects in liver.

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse-chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH4Cl, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated apo-RBP retention by resident ER proteins.

Angiogenesis is a highly regulated physiological process that has been studied in considerable detail given its importance in several chronic pathologies. Many endogenous factors and hormones intervene in the regulation of angiogensis and classical as well as targeted drugs have been developed for its control. Angiogenesis inhibition has come off the bench and entered into clinical application for cancer therapy, particularly for metastatic disease. While the clinical benefit is currently in terms of months, preclinical data suggest that novel drugs and drug combinations could lead to substantial improvement. The many targets of endogenous angiogenesis inhibitors reflect the complexity of the process; in contrast, current clinical therapies mainly target the vascular endothelial growth factor system. Cancer chemopreventive compounds can retard tumor insurgence and delay or prevent metastasis and many of these molecules hinder angiogenesis, a mechanism that we termed angioprevention. Angiopreventive drugs appear to prevalently act through the inhibition of the pro-inflammatory and anti-apoptotic player NFkappaB, thus contrasting inflammation dependent angiogenesis. Relatively little is known concerning the effects of these angiogenesis inhibitors on gene expression of endothelial cells, the main target of many of these molecules. Here we provide an exhaustive list of anti-angiogenic molecules, and summarize their effects, where known, on the transcriptome and functional genomics of endothelial cells. The regulation of specific genes can be crucial to preventive or therapeutic intervention. Further, novel targets might help to circumvent resistance to anti-angiogenic therapy. The studies we review are relevant not only to cancer but also to other chronic degenerative diseases involving endothelial cells, such as cardiovascular disorders, diabetes, rheumatoid arthritis and retinopaties, as well as vessel aging.

Die europaeische Richtlinie 2008/50/EG enthaelt als zentrales neues Element Regelungen zur Senkung der Luftbelastung durch die Feinstaubfraktion PM2.5. Die neuen Regelungen werden vorgestellt; sie folgen einerseits dem klassischen Ansatz allgemein verbindlicher Grenz- und Zielwerte, fuehren jedoch andererseits ein neues Element ein: die Senkung der durchschnittlichen urbanen Exposition in den Mitgliedstaaten auch bei Einhaltung der Ziel- und Grenzwerte. Der Beitrag untersucht die Frage, ob die neuen Regelungen zusaetzliche Anforderungen an den Immissionsschutz in Deutschland stellen werden. Dazu wird zunaechst die Korrelation der Feinstaubfraktionen PM10 und PM2.5 bestimmt, die insbesondere an verkehrsbezogenen Messpunkten sehr eng ist. Die gute Korrelation der Jahresmittel beider Feinstaubfraktionen ermoeglicht es, das dichte Messnetz fuer die Feinstaubfraktion PM10 auch fuer die Prognose von Ueberschreitungen der Ziel- und Grenzwerte fuer die Feinstaubfraktion PM2.5 heranzuziehen. Danach wird bis 2020 der Grenzwert fuer das Tagesmittel der Feinstaubfraktion PM10 weiterhin die strengste Regelungsgroesse darstellen. Langjaehrige Zeitreihen aus dem Westen Deutschlands zeigen einen fallenden Trend der PM2.5-Belastung, der derzeit groesser ist als die zu erwartende durchschnittliche nationale Einsparverpflichtung von 15 Prozent bis 2020. (A)

Interferon (IFN)-alpha is a cytokine with marked therapeutic activity in transplantable tumor models, that is in part due to angiogenesis inhibition. Aim of this study was to investigate the effects of IFN-alpha during the early phases of tumor development in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. To provide sustained IFN-alpha production, TRAMP mice were injected intraperitoneally with lentiviral vectors. IFN-alpha administration resulted in rapid and protracted upregulation of IFN-alpha-regulated genes associated with antiangiogenic and antiproliferative functions in the prostate of TRAMP mice, including guanylate-binding protein 1 (GBP-1), IFI204 and CXCL10-11. These transcriptional changes were accompanied by effects on the tumor vasculature, including significant reduction of intraductal microvessel density and increased pericyte coverage, and marked reduction of tumor cell proliferation, without induction of tumor necrosis. Intriguingly, GBP-1 and myxovirus resistance A, two IFN-regulated proteins, were found expressed in approximately 40% of human prostate cancer samples analyzed, suggesting expression of endogenous IFN-alpha. Overall, these findings demonstrate that IFN-alpha is able to counteract the angiogenic switch and impairs tumor cell proliferation in preinvasive lesions. Since the angiogenic switch also marks progression of human prostatic cancer, these results highlight the potential of angiogenesis inhibitors for the development of chemoprevention strategies in high-risk individuals.

The cytokine C-X-C motif chemokine 12 (CXCL12) is synthesised by metastasis target tissues and has been shown to attract tumour cells that express the receptor, C-X-C chemokine receptor type 4 (CXCR4). However, epigenetic silencing of CXCL12 has recently been reported to increase the metastatic potential of breast cancer cells and the reintroduction of the cytokine gene into MDA-MB-231 breast carcinoma cells decreases the number of metastases formed in vivo. We therefore wished to know whether CXCL12 expression correlates with relapse-free and overall survival in human breast cancer patients. The expression of C-X-C motif chemokine 12 (CXCL12) and C-X-C chemokine receptor type 4 (CXCR4) was analysed in 100 archival breast cancer samples by immunohistochemistry and in two breast cancer microarray datasets of 408 cases. Data were analysed by univariate and multivariate COX regression analyses. CXCL12 and CXCR4 are expressed by epithelial tumour cells and by stromal and endothelial cells. Microarray gene expression analysis and immunohistochemistry revealed that expression of CXCL12 but not of CXCR4 significantly correlates with disease-free and overall survival in oestrogen receptor-positive and -negative cancers. The expression of the oestrogen receptor α and that of CXCL12 do not correlate. CXCL12 is a strong, independent prognostic marker. We propose that saturation of the receptor through autocrine CXCL12 production reduces chemotaxis towards CXCL12-releasing metastasis target tissues.

We have investigated the effects of cigarette smoke exposure in three different strains of mice. DBA/2 and C57BL/6J are susceptible to smoke and develop different lung changes in response to chronic exposure, whereas ICR mice are resistant to smoke and do not develop emphysema. The present study was carried out to determine early changes in the gene expression profile of mice exposed to cigarette smoke with either a susceptible or resistant phenotype. The three strains of mice were exposed to smoke from three cigarettes per day, 5 days/wk, for 4 wk. Microarray analysis was carried out on total RNA extracted from the lung using the Affymetrix platform. Cigarette smoke modulates several clusters of genes (i.e., proemphysematous, acute phase response, and cell adhesion) in smoke-sensitive DBA/2 or C57BL/6J strains, but the same genes are not altered by smoke in ICR resistant mice. Only a few genes were commonly modulated by smoke in the three strains of mice. This pattern of gene expression suggests that the response to smoke is strain-dependent and may involve different molecular signaling pathways. Real-time quantitative PCR was used to verify the pattern of modulation of selected genes and their potential biological relevance. We conclude that gene expression response to smoke is highly dependent on the mouse genetic background. We speculate that the definition of gene clusters associated, to various degrees, with mouse susceptibility or resistance to smoke may be instrumental in defining the molecular basis of the individual response to smoke-induced lung injury in humans.

We have recently described a novel nuclear antigen, AF-2, which is related to cell cycle dependent alterations of chromatin structure. We show by two parameter flow cytometry on a cell by cell basis that the antigen is accessible to specific monoclonal antibodies only in mitotic and postmitotic early G1-phase cells. The evaluation of nuclease susceptibility and AF-2 antigen accessibility reveals different subcompartments of the G1 -phase of the cell cycle with distinct chromatin conformations. Digestion with DNase I seems to alter the chromatin structure according to concentration and this is reflected by an increase of the antigen accessibility. Chromatin in the more condensed early G1-phase is specifically digested by lower concentrations of the enzyme than chromatin in later stages of interphase. Chromatin from cells in the late-G1, S-, and G2-phases shows a higher relative resistance to DNase I and a reduced accessibility of the AF-2 antigen to monoclonal antibodies. Nuclease S1 has a similar effect on chromatin topology, as revealed by the reaction with anti-AF-2 antibodies, without digestion of detectable amounts of DNA. The antigen becomes available to the antibodies in almost all cells by digestion with high concentrations of DNase I or Nuclease S1.

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BACKGROUND Adjuvant chemotherapy with anthracyclines improves disease-free and overall survival compared with non-anthracycline-based adjuvant chemotherapy regimens in the treatment of early breast cancer. The role of HER2 status as a marker of anthracycline responsiveness has been explored by subset analyses within randomized clinical trials, with inconsistent results. We performed a pooled analysis of the interaction between HER2 status and the efficacy of adjuvant anthracyclines based on the published subset data. METHODS We searched literature databases to identify randomized trials that compared anthracycline-based with non-anthracycline-based adjuvant chemotherapy regimens in the treatment of early breast cancer and reported efficacy data according to HER2 status. Log hazard ratios (HRs) for disease-free and overall survival were pooled across the studies according to HER2 status by inverse variance weighting. A pooled test for treatment by HER2 status interaction was performed by weighted linear meta-regression. All statistical tests were two-sided. RESULTS Eight studies (with 6564 randomly assigned patients, of whom 5354 had HER2 status information available) were eligible for this analysis. In HER2-positive disease (n = 1536 patients), anthracyclines were superior to non-anthracycline-based regimens in terms of disease-free (pooled HR of relapse = 0.71; 95% confidence interval [CI] = 0.61 to 0.83; P < .001) and overall (pooled HR of death from any cause = 0.73; 95% CI = 0.62 to 0.85; P < .001) survival. In HER2-negative disease (n = 3818 patients), anthracyclines did not improve disease-free (HR = 1.00; 95% CI = 0.90 to 1.11; P = .75) or overall (HR = 1.03; 95% CI = 0.92 to 1.16; P = .60) survival. The test for treatment by HER2 status interaction yielded statistically significant results: for disease-free survival, the chi-square statistic for interaction was 13.7 (P < .001), and for overall survival, it was 12.6 (P < .001). CONCLUSIONS The added benefits of adjuvant chemotherapy with anthracyclines appear to be confined to women who have HER2 overexpressed or amplified breast tumors.

The possibility of predicting clinical outcome of cancer patients through the analysis of gene expression profiles in the primary tumor is a kind of ideological revolution as the multistep carcinogenesis model postulates that the proportion of cells within the primary tumor that actually acquire metastasis driving mutation(s) is small; too small to leave its imprint on the gene expression profile. The data collected to date have brought a new paradigm to reality in the metastasis field: metastasis must at least in part rely on mutations and/or gene regulation events present in the majority of cells which constitute the primary tumor mass. By analyses of differential expression of primary tumors versus metastases or by functional analyses of putative metastasis genes in experimental metastasis, many metastasis-associated gene expression events have been identified that correlate with the development of metastases. Among genes "favoring" metastasis, we find many molecules that are expressed not by the tumor cell itself but by the cells of the microenvironment, as well as genes over-expressed in the primary tumor that have a principle role in mediating tumor-host interactions. Here we review these concepts and advance hypotheses on how gene expression of the primary tumor and the microenvironment can favor the spread of the metastasis seeds and how this knowledge can provide tools to secondary prevention.

Dissemination of metastatic cells probably occurs long before diagnosis of the primary tumor. Metastasis during early phases of carcinogenesis in high risk patients is therefore a potential prevention target. The plant polyphenol Curcumin has been proposed for dietary prevention of cancer. We therefore examined its effects on the human breast cancer cell line MDA-MB-231 in vitroand in a mouse metastasis model. Curcumin strongly induces apoptosis in MDA-MB-231 cells in correlation with reduced activation of the survival pathway NFkappaB, as a consequence of diminished IotakappaB and p65 phosphorylation. Curcumin also reduces the expression of major matrix metalloproteinases (MMPs) due to reduced NFkappa B activity and transcriptional downregulation of AP-1. NFkappa B/p65 silencing is sufficient to downregulate c-jun and MMP expression. Reduced NFkappa B/AP-1 activity and MMP expression lead to diminished invasion through a reconstituted basement membrane and to a significantly lower number of lung metastases in immunodeficient mice after intercardiac injection of 231 cells (p=0.0035). 68% of Curcumin treated but only 17% of untreated animals showed no or very few lung metastases, most likely as a consequence of down-regulation of NFkappa B/AP-1 dependent MMP expression and direct apoptotic effects on circulating tumor cells but not on established metastases. Dietary chemoprevention of metastases appears therefore feasible.

The dietary antioxidant Curcumin has been proposed for cancer chemoprevention since it induces apoptosis and inhibits the formation of breast cancer metastases. Curcumin acts through the inhibition of phosphorylation of the inhibitor of kappa B (IkappaB), which in turn reduces the nuclear translocation of nuclear factor kappa B (NFkappaB), an inflammation- and cell survival-related transcription factor. However, it is not clear whether the strong antimetastatic effect can exclusively be explained by inhibition of NFkappaB. Here, we addressed the effects of Curcumin (IC(50) = 17 muM) in MDA-MB-231 breast cancer cells using microarray gene expression analyses. Among the 62 genes whose expression was significantly altered, we found the two inflammatory cytokines CXCL1 and -2 (Groalpha and -beta) that were downregulated. Further validation of the microarray results by quantitative real-time reverse transcription-polymerase chain reaction, western blots and enzyme-linked immunosorbent assay revealed that Curcumin impairs transcription of CXCL1 and -2 >24 h and reduces the corresponding proteins. Using small interfering RNA techniques, we elucidated the underlying molecular mechanism revealing that reduction of CXCL1 and -2 messenger RNA levels is NFkappaB dependent and requires intact IkappaBalpha expression. Moreover, CXCL1 and -2 silencing leads to downregulation of several metastasis-promoting genes among which we found the cytokine receptor CXCR4. We therefore suggest that the decrease of CXCL1 and -2 mediated by Curcumin is involved in the inhibition of metastasis.

Background Obesity and the insulin resistance syndrome are risk factors for breast cancer and might also affect breast cancer progression. The anti-diabetic drug Metformin (METF) reduces the breast cancer risk in diabetic women. Insulin like growth factor 1 (IGF1) and insulin are involved in breast cancer tumorigenesis and progression. We tested the effect of METF on the IGF1/insulin pathway and its involvement in breast cancer progression. Methods We developed a prognostic signature based on IGF1/insulin pathway genes using the Stockholm breast cancer microarray dataset of 149 cases for training and primary validation and the Uppsala dataset of 249 for external validation. The effect of METF on the prognostic gene set identified was tested in vitro on a panel of breast cancer cell lines. METF effects on proliferation and glucose metabolism were analyzed in vitro and in vivo. The insulin receptor substrate 2 (IRS2) was silenced by transfection with shRNA-lentiviral vectors. Xenograft growth, in the presence and absence of METF, was studied and 18FDG-uptake was measured in vitro and in vivo. Results A 15-gene signature (Insulin sensitivity score, ISS) was developed and predicted breast cancer metastasis with an accuracy similar to the Recurrence Score. ISS genes were expressed at variable levels in a breast cancer cell line panel and showed variable responsiveness to METF. The high expression correlation among the ISS genes observed in untreated breast cancer cell lines was lost upon treatment with METF. METF reduced breast cancer cell growth in vitro with IC50 values ranging from 1mM to 25mM. Growth of MDA-MB-231 cells and hyper-invasive subpopulations derived therefrom was reduced in vivo by oral administration of METF to xenografted nude mice. Response to METF in terms of IC50 values correlated with basal expression of the 15 ISS genes with the strongest inverse correlation observed for IRS2. Stable silencing of IRS2 reduced the MDA-231 cell responsiveness to METF in vitro. Discussion METF acts on the insulin/IGF1 axis by disturbing a network of breast cancer progression related genes and appears to depend in its action on the expression of IRS2 that inversely correlates with the sensitivity of cell lines to the drug. The disruption of the ISS gene network is expected to correlate with an effect on breast cancer growth and progression and in fact, mouse xenografts show reduced growth upon treatment with METF. IRS2 appears to be a major mediator of METF effects. Citation Format: Alessia I. Esposito, Adriana Amaro, Giovanna Angelini, Laura Emionite, Alessandra Gennari, Stefano Indraccolo, Davide Maggi, Cecilia Marini, Barabara Salani, Gianmario Sambuceti, Maria Pia Sormani, Ulrich Pfeffer. Metformin affects breast cancer cell growth and disturbs an IGF1/insulin related gene network that correlates with breast cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1182. doi:10.1158/1538-7445.AM2015-1182

Kaposi's sarcoma (KS) is characterized by an abnormal growth of blood vessels. KS was found mainly in older men of Mediterranean or African origin (classic KS) or in patients after organ transplantation (iatrogenic KS). However, in the early 1980s, an aggressive epidemic form, linked to AIDS, was noticed and was one of the first clues to the existence of HIV-1 pandemy. The link between KS occurrence and HIV has raised multiple hypotheses. The drastic reduction of KS after the introduction of HAART, suggests HIV as a powerful co-factor for KS progression. We and others have contributed to the elucidation of KS cell nature and the possible involvement of extracellular HIV Tat. Tat is proangiogenic and is a true promoter of KS lesions acting as a VEGFR2 ligand both on KS and endothelial cells, in addition Tat is able to bind and activate chemokine receptors on monocytes and granulocytes causing a pro-inflammatory status. Evaluation of the effects of extracellular Tat on KS cells by microarray analysis after 24 h of incubation shows an interesting clustering of gene products involved in signal transduction, especially GTP-ase, Kinase and cAMP activity, confirming that Tat acts extracellularly by ways that are probably unrelated to its nuclear activity. KS occurrence is reduced by HAART but still present and in Africa is one of the most frequent oncologic disease. To find suitable drugs with low toxic impact on KS patients, we have tested several drugs and gene therapy approaches in in vivo models. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

SummaryOur recent studies on breast carcinoma cell lines with differing tumorigenicity / invasiveness (MCF-7< MDA-MB-468< MDAMB-231< MDA-MB-435) had shown significantly decreasing expression levels of MMPs-1,-2,-3,-8,-9,-10,-11 and –13 with increasing cell density while the levels of TIMP-1 and –2 increased. This correlated well with a lower invasiveness of confluent cells. In the present study, we extend our in vitro studies on three-dimensional cultures of breast cancer cell lines MCF-7 and MDAMB-435 and the transcriptional control of MMP and TIMP-expression in two-dimensional cultures of MDA-MB-231 and –435 cells. The tumor spheroid model showed that MMP expression and proteolytic activity were considerably higher in loosely structured tumor groups as compared to densely growing “compact” cell complexes. These data suggested that cell density regulates MMP and TIMP transcription and therefore, we tested whether AP-1, NFκB and CRE are involved in this process. Gene silencing of c-jun in sparse cultures had an inhibitory effect on MMP-3,–9 and –13 expression, on proteolytic activity as well as on the invasive potential of the cells, thus confirming a role for AP-1. TIMP-1, and –2 expression was up-regulated as compared to control cells. Consistent with this, overexpression of c-jun and c-fos in confluent breast cancer cell lines leads to up-regulation of MMP expression, proteolytic activity and invasion as well as down-regulation of TIMP-1. In summary, we provide evidence that cell density influences the invasive potential of tumor cells via regulation of MMPs and TIMPs by AP-1, NFκB and CRE transcription factors. Overexpression of MMPs in sparse cultures could help explain early dissemination of potentially metastatic cells.

The anti-oxidants N-acetyl-l-cysteine (NAC) and (-)-epigallocatechin-3-gallate (EGCG) inhibit tumor vascularization by reducing endothelial cell migration and invasion in a similar, non additive and non synergistic manner but do not alter the growth of human umbilical vein endothelial cells. Here we address the effects of the two chemopreventive drugs on endothelial cell signaling by means of expression profiling and real-time PCR validation. We identify a series of angiogenesis related genes that are similarly regulated by the two drugs. Anti-oxidant treated endothelial cells show gene expression profiles compatible with a less activated, less apoptosis prone and less migratory phenotype. The anti-oxidants affect expression of several components of the TNFalpha response pathway including downstream genes that are regulated in the opposite direction in the absence of the inflammatory cytokine. The interference with the TNFalpha pathway is reflected by reduced NFkappaB activation in anti-oxidants treated cells but the compounds are not able to contrast TNFalpha mediated activation of NFkappaB. The chemopreventive action of these compounds thus relies on a reduction of basal levels of endothelial cell activation. Down-regulation of the TNFalpha responsive pro-metastatic, pro-inflammatory genes, urokinase plasminogen activator and selectin E, further implies anti-metastatic effects for these drugs.

We examined the effects of fenretinide [N-(4-hydroxyphenyl)retinamide; (4HPR)] on highly angiogenic Kaposi's sarcoma tumors in vivo and investigated the mechanisms involved for potential clinical applications.(CD-1)BR nude mice bearing KS-Imm cell tumors were randomized to receive 4HPR or vehicle until sacrifice. In vitro, KS-Imm and endothelial cells were treated with 4HPR to study the effects on proliferation, apoptosis, migration, and invasion; in vivo angiogenesis was evaluated in the Matrigel model. Angiogenesis-related and retinoid receptor molecules were examined at the mRNA and protein expression levels.In vivo, 4HPR significantly (P0.001) reduced growth of detectable Kaposi's sarcoma (KS) xenografts and inhibited angiogenesis in the Matrigel plug assay (P0.04). In vitro, 4HPR affected KS-Imm and endothelial cell growth and KS-Imm migration and invasion. 4HPR invasion inhibition was associated with decreased release of matrix metalloprotease-2 and rapid reduction of vascular endothelial growth factor (VEGF) expression by KS cells and of vascular endothelial growth factor receptor 2 (VEGFR2) by KS and endothelial cells. Finally, 4HPR repression of angiogenesis was associated with a 4HPR-induced increase in retinoic acid receptor beta expression.These data indicate that 4HPR inhibits KS tumor growth in vivo through a mechanism involving the modulation of angiogenesis-associated growth factors and their receptors on both tumor and endothelial cells. In addition, 4HPR inhibited invasion by decreasing of matrix metalloprotease-2 activity. Our results justify further studies to evaluate the utility of 4HPR as a chemopreventive or therapeutic agent in KS, a malignancy associated with immune suppression that has a high risk of recurrence with highly active antiretroviral therapy failure.

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.

The 1785 nucleotides of the coding region of the estrogen receptor alpha (ER-alpha) are dispersed over a region of more than 300,000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-alpha splice sites by Hamming clustering, a self learning method trained on more than 28,000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mini-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor (5' splice site) and exon 5 acceptor sites (3' splice site) is responsible for skipping of the exon in between.

Somatostatin was reported to inhibit Kaposi's sarcoma (KS) cell (KS-Imm) xenografts through an antiangiogenic activity. Here, we show that somatostatin blocks growth of established KS-Imm tumors with the same efficacy as adriamycin, a clinically effective cytotoxic drug. Whereas KS-Imm cells do not express somatostatin receptors (SSTRs), endothelial cells express several SSTRs, in particular SSTR3. We investigated the molecular mechanisms and receptor specificity of somatostatin inhibition of angiogenesis. Somatostatin significantly inhibited angiogenesis in vivo in the matrigel sponge assay; this inhibition was mimicked by the SSTR3 agonist L-796778 and reversed by the SSTR3 antagonist BN81658, demonstrating involvement of SSTR3. In vitro experiments showed that somatostatin directly affected different endothelial cell line proliferation through a block of growth-factor-stimulated MAPK and endothelial nitric oxide (NO) synthase (eNOS) activities. BN81658 reversed somatostatin inhibition of cell proliferation, NO production, and MAPK activity, indicating that SSTR3 activation is required for the effects of somatostatin in vitro. Finally in vivo angiogenesis assays demonstrated that eNOS inhibition was a prerequisite for the antiangiogenic effects of somatostatin, because high concentrations of sodium nitroprusside, an NO donor, abolished the somatostatin effects. In conclusion, we demonstrate that somatostatin is a powerful antitumor agent in vivo that inhibits tumor angiogenesis through SSTR3-mediated inhibition of both eNOS and MAPK activities.

The 1785 nucleotides of the coding region of the estrogen receptor \alpha (ER-\alpha) are dispersed over a region of more than 300.000 nucleotides in the primary transcript. Splicing of this precursor RNA frequently leads to variants lacking one or more exons that have been associated to breast cancer progression. The most frequent splice variant lacks exon 4 and is expressed in the human mammary carcinoma cell line MCF-7 at a level similar to that of the full-length messenger. The in silico analysis of ER-\alpha splice sites by Hamming clustering, a self learning method trained on more than 28.000 experimentally proved splice sites, reveals high relevance for the 5' and 3' splice sites of exon 4. The splicing analysis of transfected mini-gene constructs containing drastically shortened introns excludes that weak splice sites, intron or exon lengths or splice enhancers are responsible for exon skipping. Exon 6 is never skipped in MCF-7 cells but is spliced out from mine-gene derived primary transcripts if inserted between exons 3 and 5 instead of exon 4. As a consequence, it appears that a particular splice site affinity of exon 3 donor and exon 5 acceptor sites is responsible for skipping of the exon in between.

Kaposis Sarcoma (KS) is a highly angiogenic neoplasm associated with infection by the human γherpesvirus, HHV-8 or Kaposis sarcoma herpes virus (KSHV). When in 1872 the Hungarian scientist Moritz Kaposi described the sarcoma, which was later named after him, he was dealing with a rare dermatologic disease. Today, KS is a more common pathology due to its high incidence in AIDS, in immunosuppressed transplantation patients and, in its endemic form, in Africa. The introduction of highly active antiretroviral therapy (HAART) has led to a drastic reduction of KS incidence in HIVinfected patients, but in some cases KS resists the treatment. KS is more common in men than in women. The observation of spontaneous remissions during pregnancy stimulated investigations into the potential antiKS activity of the pregnancy hormone human chorionic gonadotropin (hCG). The variable effect in clinical trials using urinary preparations of the hormone (uhCG) has led to the hypothesis that contaminating moieties present in these preparations may account for the antiKS effect observed in vitro. While the discrepancy between laboratory tests and clinical trials remains a mystery, little is known about potential antiKS mechanisms of the hormone itself and/or other active moieties present in uhCG.

The first exon of the human androgen receptor (AR) contains a translated CAG (poly-glutamine) repeat. The repeat length is polymorphic in the normal population ranging from 8 to 35 repeats. Expansions to over 40 repeats lead to spinal bulbar muscular atrophy (SBMA), a late onset neurodegenerative disease. The repeat is located between the two parts of a bipartite amino-terminal transactivation function and the repeat length, also within in the normal range, is inversely correlated to the transactivation power of the receptor. P160 type co-activators bind more strongly to shorter repeats. A correlation between AR CAG repeat length and total risk, age at diagnosis, recurrence after surgery and aggressive growth has been reported for tumors of classical androgen target tissues. In the prostate, where androgens exert a mitogenic effect, the cancer risk increases with decreasing AR-CAG repeat length. In contrast, in the breast, where the hormone probably acts as anti-mitogen, a higher risk and earlier onset of breast cancer has been reported for carriers of BRCA1 mutations who also have long CAG repeats in the receptor gene. Somatic alterations during carcinogenesis appear to be frequent in endometrial and in colon cancer. In the endometrium the AR CAG repeat prevalently undergoes expansions consistent with the putative protective function of androgens in this tissue. Frequent repeat reductions during colon carcinogenesis would be consistent with a mitogenic effect of androgens. Analysis of AR protein expression by Western blot reveals expression of the AR in healthy and neoplastic colon tissues. Normal mucosa of the colon expresses both AR-isoforms of 110 and 87 kDa, while the tumor samples have lost the expression of the 110-kDa isoform. The 87-kDa isoform is devoid of the amino-terminal portion of the receptor molecule that also contains the poly-glutamine tract. The temporal and causal relation between isoform switch and somatic repeat reductions during colon carcinogenesis is as yet unclear, but the two events could both enhance p160 mediated androgen signaling. The recent finding that smad3 interacts with the AR in a way similar to p160 links the AR to TGFbeta signaling. Interruption of this signaling pathway is a frequent event in colon carcinogenesis.

Uveal melanoma is the most frequent primary tumor of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight UV-exposure on the etiology of uveal melanoma is matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma. We also analyzed the frequency of other recently discovered uveal melanoma specific mutations in the genes GNAQ, GNA11, BAP1, SF3B1, and EIFAX1, and we analyzed the association of these mutation with histopathological and clinical features including progression free survival. Finally, we analyzed TERT expression in these tumors. The mutation frequencies observed confirmed the prevalence of GNAQ mutations in cases with disomy of chromosome 3 that have a better prognosis whereas GNA11 was more frequent in monosomic cases. Together these two mutations account for more than 85% of the cases. BAP1 mutations are associated with chromosome 3 monosomy but not with progression free survival. SF3B1 and EIF1AX mutations occur in 10.3 and 22.5% of uveal melanomas, in disomic cases only. We detected a TERT mutation in only one case of a 57-year old white male with clinical and histo-pathological features typical for uveal melanoma. The tumor showed mutations in GNA11 and EIF1AX. No mutations were detected in GNAQ, BAP1, and SF3B1. Both copies of chromosome 3 were retained. Several tumors among which the one carrying the TERT promoter mutation showed elevated TERT expression. These data indicate that TERT mutations are rare in uveal melanoma, consistent with a reduced etiological influence of sunlight. No conclusion can be drawn on the potential influence of TERT mutations on tumor progression. Citation Format: Adriana Agnese AMARO, Marina Gualco, Maria Dono, Ulrich Pfeffer, Irena Maric, Carlo Mosci. TERT promoter mutations are rare in uveal melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3420. doi:10.1158/1538-7445.AM2014-3420

The region on chromosome 7q21-22 is frequently altered in several human neoplasias such as uterine leiomyoma, myeloid leukemia and breast cancer. The same region has also been linked to split hand/split foot malformation type 1 and to involutional osteoporosis. Our analysis of genes that map to this region has led to the identification of the so far unknown first exon of the homeobox gene DLX6, a mammalian homologue of the Drosophila distal-less gene. Distal-less is a downstream target of the trithorax transcription factors. Translocations involving the mammalian homologue of trithorax, ALL-1, leading to its constitutive activation cause leukemia. We describe here that the first exons of human and mouse DLX6 genes contain a multiple trinucleotide repeat region. We have analyzed the CAG repeat length in 90 subjects and were able to identify five alleles with 11 to 20 CAG repeats.

In the framework of new European directives in the field of ambient air quality assessment specific requirements for measurement methods for benzene will be defined and have to be met in the future. In a residential area in the vicinity of a cokery a comprehensive monitoring programme for BTX compounds (benzene, toluene, xylenes) was carried out with diffusive samplers. Measurement results from 20 sites show a distinct spatial distribution of benzene concentrations which is caused by the emissions of the plant. Comparisons with two pumped measurement methods reveal excellent agreement in the case of benzene. An assessment of measurement uncertainty for the diffusive method is presented in terms of standard deviations from parallel measurements (precision) and by comparison with a reference method according to national and international standards. Data quality objectives required by an upcoming European directive for benzene can be met by diffusive sampling.

Many groups have examined of androgen the effects on normal and neoplastic colon tissues, but no clear picture has hitherto emerged. In particular, the presence and the function of the androgen receptor (AR) has only partially been investigated in the past. The present study reports analysis of expression of the AR gene as messenger RNA and as protein in surgical samples of neoplastic colon mucosa and of corresponding healthy surrounding tissue. Specific binding for DHT, demonstrating the presence of AR, was observed in almost all the samples (2 samples out of 12 were negative). No significant difference was observed between healthy and neoplastic mucosa, or between male and female patients. A further characterization of AR was performed with Western blot, using 2 different primary antibodies. Both AR isoforms, AR-B and AR-A, were detected in healthy mucosa, while only AR-A, resolving at 87 kDa, was observed in neoplastic mucosa. RT-PCR analysis revealed the transcript for AR in both healthy and neoplastic mucosa in 10 samples; no message was detectable in 2 samples (negative also for binding); 2 additional samples presented AR mRNA only in healthy colon mucosa, 2 others only in neoplastic mucosa. In addition, a variant AR messenger RNA, probabily derived from alternative splicing, was observed. We found that AR is expressed both in healthy and in neoplastic colon mucosa, either as mRNA or as protein. Neoplastic colon tissue shows a characteristic loss of expression of the AR-B isoform, while AR-A expression is maintained. These findings underscore the possible role of androgen and its receptor in colon carcinogenesis.

Publisher Summary This chapter describes different methods employed for the expression analysis of various retinoid receptors. Different one-tube reverse transcription–polymerase chain reaction (RT–PCR) that assembles the reverse transcription reaction and the cyclic amplification reaction in a single tube are described. One of the protocols described exploits the reverse transcriptase activity of thermostable polymerases. In the presence of Mn 2+ ions the enzyme isolated from Thermus thermophilus (Tth polymerase) uses RNA as a template for DNA polymerization. In the second step of the one-tube reaction, Mn 2+ ions are sequestered by EGTA, a chelator with a high affinity for Mn 2+ as compared to Mg 2+ ; the latter ion and PCR primers are added to the diluted reaction mix and amplification of the cDNA produced can be performed in the same tube. Depending on the expression level of the receptors in the sample analyzed and on the amount of RNA used for the reaction, the cycle number of the first round of amplification can be adjusted and different amounts of the first round of amplification can be used as a template for the second round to calibrate the sensitivity of the system.

Metformin, an anti-diabetic drug commonly used for type 2 diabetes therapy, is associated with a decreased risk for certain cancer. Due to extensive reports of its in vitro and in vivo anti-tumor activity, we and others have focused on the anti-angiogenic potential as a possible mechanism through which metformin could affect tumor progression, and to propose its use as an angiopreventive drug. Metformin was able to interfere with endothelial cell proliferation, apoptosis and invasion in vitro. Gene arrays analyses were performed to verify the effect of metformin on endothelial gene expression profiles and the matrigel sponge assay to test its effect in vivo. We show that metformin inhibited endothelial cell proliferation by effecting cell cycle regulation rather than inducing apoptosis. In vitro, metformin reduces endothelial cell invasion and represses their ability to organize into capillary-like networks. These effects are partially reverted by compound C, a specific inhibitor of AMPK, confirming that metformin could interfere with angiogenesis responses by activating AMPK signaling. Gene expression profiles of endothelial cells treated with metformin showed down-regulation of several angiogenesis-related genes but up-regulation of chemokine pathways. In vivo, metformin inhibited angiogenesis induced by VEGF. Considering the strict correlation between type II diabetes and obesity, we evaluated the ability of Metformin to modulate endothelial precursor cells in an obese murine model and we reported that Metformin decreases the number of adipose tissue progenitor endothelial cells in vivo only in obese mice. These data suggest that metformin has anti-angiogenic effect associated with down-regulation of specific molecule although it also induces some inflammatory mediators, and that it may be most effective in the context of obesity. Citation Format: Katiuscia Dallaglio, Antonino Bruno, Anna Rita Cantelmo, Ulrich Pfeffer, Francesco Bertolini, Douglas M. Noonan, Adriana Albini. Metformin as a potent antiangiogenic factor: From diabetes to cancer angioprevention. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A23.

11063 Background: Tumor biomarkers can help to identify pts with early-stage NSCLC with high risk of relapse and poor prognosis. The aim of this study was to investigate the prognostic value of 7 biomarkers involved in DNA synthesis and repair. Methods: Tumour tissues from 82 radically resected, stage I-III NSCLC pts were consecutively collected to investigate the following biomarkers: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunit 1 (RRM1), ribonucleotide reductase subunit 2 (RRM2), subunit p53R2, thymidylate synthase (TS) and class III beta-tubulin (TUBB3) using immunohistochemistry (IHC) and quantitative real time-polymerase chain reaction (qRT-PCR). Expression levels of these genes were also investigated in a large publicly available NSCLC microarray dataset (Director Challenge Consortium, DCC). Results: RRM2 expression (p=0.031), TS expression (p=0.023), and pathologic stage (p

In America and Western Europe prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the pro-inflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. Here we analyse the effects of Curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that Curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase, IKKβ, leading to stabilization of the inhibitor of NFκB, IκBα, in PC3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of Curcumin with the synthetic IKKβ inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with Curcumin as wells as siRNA based knock-down of CXCL1 and -2 induce apoptosis, inhibit proliferation, and down-regulate several important metastasis-promoting factors like COX2, SPARC, and EFEMP. In an orthotopic mouse model of haematogenous metastasis, treatment with Curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive pro-inflammatory and pro-metastatic feed-back loop between NFκB and CXCL1/-2. Curcumin disrupts this feed-back loop by the inhibition of NFκB signalling leading to reduced metastasis formation in vivo. Citation Format: Peter H. Killian, Emanuel Kronski, Simonetta Astigiano, Ottavia Barbieri, Christian P. Sommerhoff, Andreas G. Nerlich, Ulrich Pfeffer, Beatrice E. Bachmeier. Curcumin inhibits prostate cancer metastasis in vivo by targeting the inflammatory cytokines CXCL1 and -2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2590. doi:10.1158/1538-7445.AM2013-2590

We report that growth of LNCaP human prostate cancer cells is significantly stimulated (up to 120% above control) by physiological estradiol (E2) concentrations. This growth increase appears to be comparable to that induced by either testosterone or dihydrotestosterone, as also reported by others. This paper presents novel illustrative evidence for estrogen-binding proteins and messenger RNA transcripts in LNCaP cells. In fact, 1) the reverse transcriptase-polymerase chain reaction system documented normal messenger RNA for estrogen receptors (ER); 2) the radioligand binding assay allowed the detection of high affinity, reduced capacity binding sites in both soluble and nuclear cell fractions; and 3) the immunocytochemical analysis showed a consistently intensive staining for both ER and progesterone receptors. Compared to other human estrogen-responsive mammary cancer cells, MCF7 and ZR75-1, ER expression in LNCaP cells was not significantly lower, as shown by levels of the ER transcripts, number of sites per cell, or femtomoles per mg DNA as well as the percentage and intensity of immunocytochemical staining. A relative estimate of ER expression obtained by matching LNCaP with another human prostate cancer cell line, PC3, always displayed significantly and consistently higher levels in LNCaP cells. The detection of relatively high type I ER content in either cell compartment of LNCaP cells was paralleled by a highly intensive staining for progesterone receptors. In addition, evidence that the synthetic androgen R1881 did not compete for type I binding of E2 and that any E2-induced growth was completely reversed by the pure antiestrogen ICI-182,780, but unaffected by the antiandrogen Casodex, clearly suggests that the biological response of LNCaP cells to E2 is mediated via its own receptor.

Background Biomarkers can help in identifying patients (pts) with early-stage NSCLC with high risk of relapse and poor prognosis. The aim of this study was to investigate the relationship between the levels of expression of 7 biomarkers, various clinicopathological characteristics and their prognostic significance. Methods Tumor tissue from 82 radically resected stage I-III NSCLC pts were consecutively collected to investigate the mRNA expression and protein levels of the following biomarkers using quantitative reverse transcriptase real-time PCR (qRT-PCR) and immunohistochemistry (IHC) with a tissue microarray technique: excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase subunit 1 (RRM1), RRM2, p53R2, thymidylate synthase (TS) and class III beta-tubulin (β-Tub-III). Results On a univariate analysis, p53R2 expression was significantly higher in adenocarcinoma (ADK) compared to squamous cell carcinoma (SSC) samples (p = 0.002) and in stage I compared to stage II-III (p ≤ 0.001). ERCC1 expression was significantly higher in females compared to males (p = 0.03), and β-Tub-III expression was significantly higher in ADK than in SSC (p = 0.03). Pts with lower RRM2 expression survived longer than pts with higher RRM2 expression (p = 0.069). The multivariate analysis confirmed RRM2 as an independent prognostic marker of shorter survival (p= 0.031). The comparison between survival curves with qRT-PCR and IHC showed similar results with a trend towards longer survival among ERCC1 negative pts, BRCA1 negative pts, p53R2 positive pts and among pts with low levels of RRM1 and RRM2, although the difference was not statistically significant with both methods. qRT-PCR and IHC have shown that β-Tub-III and TS had no significant impact on survival. Conclusions This is the first study that identifies RRM2 expression as a negative prognostic factor in resected stage I-III NSCLC. Moreover, we have demonstrated the differential expression of p53R2 and β-Tub-III in ADK compared to SSC and higher expression of p53R2 in pts with stage I compared to stage II-III NSCLC. Disclosure All authors have declared no conflicts of interest.

Background Most patients affected by Glioblastoma multiforme (GBM, grade IV glioma) experience a recurrence of the disease because of the spreading of tumor cells beyond surgical boundaries. Unveiling mechanisms causing this process is a logic goal to impair the killing capacity of GBM cells by molecular targeting. We noticed that our long-term GBM cultures, established from different patients, may display two categories/types of growth behavior in an orthotopic xenograft model: expansion of the tumor mass and formation of tumor branches/nodules (nodular like, NL-type) or highly diffuse single tumor cell infiltration (HD-type). Methods We determined by DNA microarrays the gene expression profiles of three NL-type and three HD-type long-term GBM cultures. Subsequently, individual genes with different expression levels between the two groups were identified using Significance Analysis of Microarrays (SAM). Real time RT-PCR, immunofluorescence and immunoblot analyses, were performed for a selected subgroup of regulated gene products to confirm the results obtained by the expression analysis. Results Here, we report the identification of a set of 34 differentially expressed genes in the two types of GBM cultures. Twenty-three of these genes encode for proteins localized to the plasma membrane and 9 of these for proteins are involved in the process of cell adhesion. Conclusions This study suggests the participation in the diffuse infiltrative/invasive process of GBM cells within the CNS of a novel set of genes coding for membrane-associated proteins, which should be thus susceptible to an inhibition strategy by specific targeting. Massimiliano Monticone and Antonio Daga contributed equally to this work

Metformin (dimethylbiguanide) is used as a first-line therapy for the management of type 2 diabetes. Recently, metformin has hit the headlines as anti-cancer drug. Metformin is known to work in part through activation of AMP-activated protein kinase (AMPK). AMPK is involved in cancer cell growth and metabolism by modulation of gene expression and translation through the AMPK/ mTOR/S6K1 axis. In order for a solid tumor to grow and to disseminate, an efficient angiogenesis is required, becoming in this way one of the most successful targets not only in cancer therapy but also in prevention. Angiogenesis is the formation of new blood vessels from pre-existing ones. Some evidences have been collected that metformin activates AMPK in human umbilical vein endothelial cells (HUVEC). We aimed to better characterize the mechanism of action by which metformin can affect neo-angiogenesis. We carried out HUVEC morphogenesis assay in matrigel in presence of metformin. We found that the compound affects normal tube formation. Moreover we assessed the effect of metformin on cell viability by a MTT assay in presence of different doses of the drug. We also observed that this compound affected endothelial cell proliferation. This finding was corroborated by real time PCR data showing that in HUVEC metformin treatment induced downregulation of Cyclin D1. The lower proliferation rate was not associated to the induction of apoptotic pathway, as indicated by Annexin V/7AAD staining and FACS analysis. In order to evaluate to what extent this compound can effect endothelial cell migration and invasion of a reconstitute basement membrane in vitro, HUVEC were cultured in a Boyden chamber and showed less invasiveness in presence of metformin compare to untreated cells. Futhertrmore we investigated the effect of the drug when administered to the cells along with other antiangiogenic compounds used in chemotherapy, e.g. the synthetic triterpenoid CDDO. The combination of two drugs demonstrated synergistic effects on cells organizing in tubule-like structures in a morphogenesis assay. Finally, we examined the activity of metformin on endothelial cells by microarray gene expression profiling. Although we are still in the validation process, preliminary results suggest that metformin decrease the expression of several genes involved in biosynthetic process and in the response to stimuli, supporting its anti-proliferative effect. On the other hand it also seems to induce a pro-inflammatory setting. This result hints that the effect of the chemotherapy can be potentiated by metformin, suggesting a possible use of metformin in combination with chemotherapeutic agent in order to lower the risk of side effect following high dosage treatments. Our results indicate an effective role of metformin in inhibiting neoangiogenesis by which it may exert or, at least, support its anti-tumor effect. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5303. doi:1538-7445.AM2012-5303

The acquisition of an invasive phenotype is a pre-requisite for metastasis. We set out to develop cellular systems that can mirror transient and stbale molecular alterations that confer an invasive phenotype to breast cancer cells. We observed that it is possible to isolate invasive subpopulations from moderately invasive cancer cell lines. Enrichment of invasive sub-populations of MDA-MB-231 breast cancer cells in three successive preparative invasion assays in Matrigel covered Boyden chambers yielded a highly invasive cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype although not all phenotypic changes acquired during selection are maintained by long term cultures. Genetic analyses of these cells by cytogenetics and array based comparative genome hybridization revealed many genetic alterations including increased ploidy. The flow cytometric DNA Index (DI) changes from 1.28 to 2.28. Cells with DI 2.28 constitute 1.4% of the parental cell line. Whole genome SNP analysis shows that the two populations are genetically related excluding any cell contamination. The invasive cells proliferate and undergo apoptosis similar to the parental cells. Commitment to apoptosis is increased since invasive cells respond more strongly to curcumin or peroxide induced apoptosis. Invasive cells show relative resistance to the cytotoxic, alkylating agent Doxorubicin and increased sensitivity to the anti-mitotic drugs Vincristine and Taxol. Increased resistance to the topoisomerase II inhibitor Mitoxantrone is observed only transiently in invasive cells and lost in long term cultures. Similarly, the chemokines CXCL1 and -2 are transiently upregulated. Response to the anti-diabetic drug Metformin showed a reduced sensibility for invasive cells in terms of growth inhibition (IC50). Gene expression profiling shows complex alterations in gene expression. Many of the genes that are differentially expressed in highly versus moderately invasive cells are differentially expressed in human breast cancer cases with and without distant metastasis and correlate with disease free survival. The invasive phenotype is not related to stem cell features nor to epithelial mesenchymal transition. These cells constitute a novel model for tumor progression. Tumorigenicity and metastatic potential in vivo are currently being tested. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 496. doi:1538-7445.AM2012-496