Pasquale De Luca

Concordant morphologic and gene expression data show that a vaccine halts HER-2/neu preneoplastic lesions

While much experimental data shows that vaccination efficiently inhibits a subsequent challenge by a transplantable tumor, its ability to inhibit the progress of autochthonous preneoplastic lesions is virtually unknown. In this article, we show that a combined DNA and cell vaccine persistently inhibits such lesions in a murine HER-2/neu mammary carcinogenesis model. At 10 weeks of age, all of the ten mammary gland samples from HER-2/neu-transgenic mice displayed foci of hyperplasia that progressed to invasive tumors. Vaccination with plasmids coding for the transmembrane and extracellular domain of rat p185neu followed by a boost with rp185neu+ allogeneic cells secreting IFN-gamma kept 48% of mice tumor free. At 22 weeks, their mammary glands were indistinguishable from those of 10-week-old untreated mice. Furthermore, the transcription patterns of the two sets of glands coincided. Of the 12,000 genes analyzed, 17 were differentially expressed and related to the antibody response. The use of B cell knockout mice as well as the concordance of morphologic and gene expression data demonstrated that the Ab response is the main mechanism facilitating tumor growth arrest. This finding suggests that a new way can be found to secure the immunologic control of the progression of HER-2/neu preneoplastic lesions.

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.

Gene expression profiling at early organogenesis reveals both common and diverse mechanisms in foregut patterning

The thyroid and lungs originate as neighboring bud shaped outgrowths from the midline of the embryonic foregut. When and how organ specific programs regulate development into structures of distinct shapes, positions and functions is incompletely understood. To characterize, at least in part, the genetic basis of these events, we have employed laser capture microdissection and microarray analysis to define gene expression in the mouse thyroid and lung primordia at E10.5. By comparing the transcriptome of each bud to that of the whole embryo as well as to each other, we broadly describe the genes that are preferentially expressed in each developing organ as well as those with an enriched expression common to both. The results thus obtained provide a valuable resource for further analysis of genes previously unrecognized to participate in thyroid and lung morphogenesis and to discover organ specific as well as common developmental mechanisms. As an initial step in this direction we describe a regulatory pathway involving the anti-apoptotic gene Bcl2 that controls cell survival in early thyroid development.

Human monocyte-derived dendritic cells exposed to hyperthermia show a distinct gene expression profile and selective upregulation of IGFBP6

Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.Fever plays a role in activating innate immunity while its relevance in activating adaptive immunity is less clear. Even brief exposure to elevated temperatures significantly impacts on the immunostimulatory capacity of dendritic cells (DCs), but the consequences on immune response remain unclear. To address this issue, we analyzed the gene expression profiles of normal human monocyte-derived DCs from nine healthy adults subjected either to fever-like thermal conditions (39°C) or to normal temperature (37°C) for 180 minutes. Exposure of DCs to 39°C caused upregulation of 43 genes and downregulation of 24 genes. Functionally, the up/downregulated genes are involved in post-translational modification, protein folding, cell death and survival, and cellular movement. Notably, when compared to monocytes, DCs differentially upregulated transcription of the secreted protein IGFBP-6, not previously known to be specifically linked to hyperthermia. Exposure of DCs to 39°C induced apoptosis/necrosis and resulted in accumulation of IGFBP-6 in the conditioned medium at 48 h. IGFBP-6 may have a functional role in the hyperthermic response as it induced chemotaxis of monocytes and T lymphocytes, but not of B lymphocytes. Thus, temperature regulates complex biological DC functions that most likely contribute to their ability to induce an efficient adaptive immune response.

The paired box transcription factor Pax8 is essential for function and survival of adult thyroid cells.

The transcription factor Pax8 is already known to be essential at very early stages of mouse thyroid gland development, before the onset of thyroid hormone production. In this paper we show, using a conditional inactivation strategy, that the removal of the Pax8 protein late in gland development results in severe hypothyroidism, consequent to a reduced gland size and a deranged differentiation. These results demonstrate that Pax8 is also an essential player in controlling survival and differentiation of adult thyroid follicular cells.

Cross-species toxicogenomic analyses and phenotypic anchoring in response to groundwater low-level pollution

BackgroundComparison of toxicogenomic data facilitates the identification of deregulated gene patterns and maximizes health risk prediction in human.ResultsHere, we performed phenotypic anchoring on the effects of acute exposure to low-grade polluted groundwater using mouse and zebrafish. Also, we evaluated two windows of chronic exposure in mouse, starting in utero and at the end of lactation. Bioinformatic analysis of livers microarray data showed that the number of deregulated biofunctions and pathways is higher after acute exposure, compared to the chronic one. It also revealed specific profiles of altered gene expression in all treatments, pointing to stress response/mitochondrial pathways as major players of environmental toxicity. Of note, dysfunction of steroid hormones was also predicted by bioinformatic analysis and verified in both models by traditional approaches, serum estrogens measurement and vitellogenin mRNA determination in mice and zebrafish, respectively.ConclusionsIn our report, phenotypic anchoring in two vertebrate model organisms highlights the toxicity of low-grade pollution, with varying susceptibility based on exposure window. The overlay of zebrafish and mice deregulated pathways, more than single genes, is useful in risk identification from chemicals implicated in the observed effects.

Transcription profiling of laser microdissected microsporocytes in an Arabidopsis mutant (Atmcc1) with enhanced histone acetylation

In this work, pollen mother cells (PMCs) isolated in Arabidopsis thaliana by laser-capture microdissection (LCM) were subjected to transcription profiling by microarray (LMM). PMCs covering all meiotic stages, from leptotene to tetrad, were collected in an Atmcc1 characterized by overexpression of a GCN5-like histone acetylase (AtMCC1). A total of 150 genes showed differential expression in Atmcc1 PMCs as compared to the wild type. Histone hyperacetylation affected the transcription of genes belonging to categories such as the meiotic and mitotic cycle, the ubiquitinproteasome-system, and the chromatin structure. We also discuss the putative role of ASK1 and RAD51C upregulation in the meiotic defects observed in Atmcc1. PMCLMM experiments allowed identification of candidate AtMCC1 targets with known and potential function in meiosis, providing data for further investigation on plant meiosis.

Nitric Oxide regulates mouth development in amphioxus

The development of the mouth in animals has fascinated researchers for decades, and a recent study proposed the modern view of recurrent evolution of protostomy and deuterostomy. Here we expanded our knowledge about conserved traits of mouth formation in chordates, testing the hypothesis that nitric oxide (NO) is a potential regulator of this process. In the present work we show for the first time that NO is an essential cell signaling molecule for cephalochordate mouth formation, as previously shown for vertebrates, indicating its conserved ancestral role in chordates. The experimental decrease of NO during early amphioxus Branchiostoma lanceolatum development impaired the formation of the mouth and gill slits, demonstrating that it is a prerequisite in pharyngeal morphogenesis. Our results represent the first step in the understanding of NO physiology in non-vertebrate chordates, opening new evolutionary perspectives into the ancestral importance of NO homeostasis and acquisition of novel biological roles during evolution.

Abstract 2936: Molecular profiling of primary malignant pleuralmesothelioma histotypes cell lines reveals relationship between GDF15overexpression and cisplatin resistance both in in vitro and in vivo models

Malignant pleural mesothelioma (MPM) development is mainly correlated with exposure to asbestos and appears to be a trend toward an increase in its incidence in the years to come. Although novel therapeutic strategies are under investigation, alone or in combination with conventional therapy, MPM remains a cancer with high medical need. With the aim to identify putative novel therapeutic targets/biomarkers expressed in specific MPM histotypes we investigated 9 different primary MPM cell lines at molecular, biochemical and pharmacological levels. A transcriptomic analysis has been conducted comparing the gene expression in epitheliod (E-MM473), sarcomatoid (S-MM432) and biphasic (B-MM487) primary cell lines. We found more than 1000 genes differentially regulated and statistically validated (p 90%) and E-MM473/B-MM487 cell survival (>50%). GDF15 belongs to the TGF-β superfamily and its role in different pathologies including cancer is controversial (Breit et al., 2011). Its overexpression has been associated to tumor chemoresistance (Chung-Ying et al.,2007) including Cisplatin (Cis) (Li et al., 2015; Yang et al. 2014). To this end, we investigated if GDF15 protein levels in MPM cells exposed to Cis correlate with its citotoxicity. Interestingly GDF15 protein level, which is high in E-MM473, medium in B-M M432 and low in S-MM487, is inversely correlated with the cells sensitivity to the treatment (i.e. E-MM473 are 2-5 times more resistant) and GDF15 silencing increases Cis-induced cytotoxicity. To assess the relationship between GDF15 levels/ Cis resistance, efficacy of Cis (4mg/kg; q7dx3w;i.v.) on stably expressing luciferase E-MM473(highGDF15) and B-MM487 (lowGDF15), orthotopically xenografted in nude mice, were investigated. While, E-MM473 showed a poor response to the Cis (TGI 58%), an impressive tumor response (TGI 95%) was observed in treated B-MM487. Our results suggest and support the need of further clinical investigation to correlate GDF15 plasma level to the chemotherapy response in MPM patients. Citation Format: Claudio Pisano, Gianluca Carletta, Fulvio D’Angelo, Antonietta Esposito, Erminia Bianchino, Assunta Riccio, Michela Festa, Francesco Cardile, Emanuele Carchia, Walter D’Acunto, Giacomo Signorino, Mario B. Guglielmi, Pasquale De Luca. Molecular profiling of primary malignant pleuralmesothelioma histotypes cell lines reveals relationship between GDF15overexpression and cisplatin resistance both in in vitro and in vivo models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2936.