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Germ line cell-derived pluripotent stem cells (GPSCs) are similar to embryonic stem (ES) cells in that they can proliferate intensively and differentiate into a variety of cell types. Previous studies have revealed some inherent differences in gene expression between undifferentiated mouse ES cells and GPSCs. Our aims were to generate functional hepatocytes from mouse GPSCs in vitro and to investigate whether the differences in gene expression may impact on the hepatocyte differentiation capacity of the GPSCs compared with ES cells. Mouse GPSCs and ES cells were induced to differentiate into hepatocytes through embryoid body formation, with very high efficiency. These hepatocytes were characterized at cellular, molecular, and functional levels. The GPSC-derived hepatocytes expressed hepatic markers and were metabolically active as shown by albumin and haptoglobin secretion, urea synthesis, glycogen storage, and indocyanine green uptake. We also performed an unprecedented DNA microarray analysis comparing different stages of hepatocyte differentiation. Gene expression profiling demonstrated a strong similarity between GPSC and ES cells at different stages of induced hepatic differentiation. Moreover, Pearson correlation analysis of the microarray datasets suggested that, at late hepatic differentiation stages, the in vitro-derived cells were closer to fetal mouse primary hepatocytes than to those obtained from neonates. We have shown for the first time that adult GPSCs can be induced to differentiate into functional hepatocytes in vitro. These GPSC-derived hepatocytes offer great potential for cell replacement therapy for a wide variety of liver diseases.

Tumor onset and progression require the accumulation of many genetic and epigenetic lesions. In some cases, however, cancer cells rely on only one of these lesions to maintain their malignant properties, and this dependence results in tumor regression upon oncogene inactivation ("oncogene addiction"). Determining which nodes of the many networks operative in the transformed phenotype specifically mediate this response to oncogene neutralization is crucial to identifying the vulnerabilities of cancer. Using the Met receptor as the major model system, we combined multiplex phosphoproteomics, genome-wide expression profiling, and functional assays in various cancer cells addicted to oncogenic receptor tyrosine kinases. We found that Met blockade affected a limited subset of Met downstream signals: Little or no effect was observed for several pathways downstream of Met; instead, only a restricted and pathway-specific signature of transducers and transcriptional effectors downstream of Ras or phosphoinositide 3-kinase (PI3K) was inactivated. An analogous signature was also generated by inhibition of epidermal growth factor receptor in a different cellular context, suggesting a stereotyped response that likely is independent of receptor type or tissue origin. Biologically, Met inhibition led to cell-cycle arrest. Inhibition of Ras-dependent signals and PI3K-dependent signals also resulted in cell-cycle arrest, whereas cells in which Met was inhibited proliferated when Ras or PI3K signaling was active. These findings uncover "dominant" and "recessive" nodes among the numerous oncogenic networks regulated by receptor tyrosine kinases and active in cancer, with the Ras and PI3K pathways as determinants of therapeutic response.

Acquisition of independence from anchorage to the extracellular matrix is a critical event for onset and progression of solid cancers. To identify and characterize new genes conferring anchorage independence, we transduced MCF10A human normal breast cells with a retroviral cDNA expression library and selected them by growth in suspension. Microarray analysis targeted on library-derived transcripts revealed robust and reproducible enrichment, after selection, of cDNAs encoding the scaffolding adaptor Gab2. Gab2 was confirmed to strongly promote anchorage-independent growth when overexpressed. Interestingly, downregulation by RNA interference of endogenous Gab2 in neoplastic cells did not affect their adherent growth, but abrogated their growth in soft agar. Gab2-driven anchorage independence was found to specifically involve activation of the Src-Stat3 signaling axis. A transcriptional 'signature' of 205 genes was obtained from GAB2-transduced, anchorage-independent MCF10A cells, and found to contain two main functional modules, controlling proliferation and cell adhesion/migration/invasion, respectively. Extensive validation on breast cancer data sets showed that the GAB2 signature provides a robust prognostic classifier for breast cancer metastatic relapse, largely independent from existing clinical and genomic indicators and from estrogen receptor status. This work highlights a pivotal role for GAB2 and its transcriptional targets in anchorage-independent growth and breast cancer metastatic progression.

Providing clinical predictions for cancer patients by analyzing their genetic make-up is a difficult and very important issue. With the goal of identifying genes more correlated with the prognosis of breast cancer, we used data mining techniques to study the gene expression values of breast cancer patients with known clinical outcome. Focus of our work was the creation of a classification model to be used in the clinical practice to support therapy prescription. We randomly subdivided a gene expression dataset of 311 samples into a training set to learn the model and a test set to validate the model and assess its performance. We evaluated several learning algorithms in their not weighted and weighted form, which we defined to take into account the different clinical importance of false positive and false negative classifications. Based on our results, these last, especially when used in their combined form, appear to provide better results.

International audience; The different behaviors of colonies of two cell lines, ARO (tyroid carcinoma-derived cells) and MLP-29 (Mouse Liver Progenitor cells), in response to Hepatocyte Growth Factor (HGF) are described deducing suitable Cellular Potts Models (CPM). It is shown how increased motility and decreased adhesiveness are responsible for cell-cell dissociation and tissue invasion in the ARO cells. On the other hand, it is shown that, in addition to the biological mechanisms above, it is necessary to include directional persistence in cell motility and HGF diffusion to describe the scattering and the branching processes characteristic of MLP-29 cells

Sporadic colorectal cancer is a major cause of death worldwide. Development takes place in a sequential manner from benign adenomas leading to carcinomas. In 90% of tumours bearing a Ras mutation it is Ki-Ras that is mutated. We have developed a model cell system to study oncogenic Ras mutations in colorectal cancer cell lines. In this analysis two Caco-2 derived cell lines expressing Ha-RasV12 (Caco-H) and Ki-RasV12 (Caco-K), respectively, have been used in large-scale microarray profiling against a Caco-2 control. This was carried out using an Illumina microarray containing 24,000 genes. Genes have been identified as differentially expressed in each isoform as well as commonly regulated. In addition the Caco-H cell line has a strong epithelial–mesenchymal phenotype that is reflected in many of its differentially expressed genes. These include the known EMT markers Vimentin, E-cadherin and Slug. Other genes of interest include several members of the Claudin family, Forkhead transcription factors and GATA-factors. The Caco-K cell line shows strong downregulation of the Dickkopf transcriptional repressor implicating it in WNT signalling. Pathway and functional analysis has also been carried out for the differentially expressed genes for both cell lines using Ingenuity software. This genome wide microarray analysis has provided a molecular signature for EMT in a Caco-H colon cancer cell line. It has also revealed a number of key genes for Caco-K expression and identified novel markers for Ras expression that have been verified by PCR analysis.

Invasive growth is a complex biological program triggered by hepatocyte growth factor (HGF) through its tyrosine kinase receptor encoded by the Met proto-oncogene. The program involves-besides proliferation-cell dissociation, motility and invasiveness, controlled by intracellular signals impinging on PI3K and on the small G-proteins of the Rac/Rho family. The mechanism(s) unbalancing Rac/Rho activation are still not completely clarified. Here, we describe a functional link between HGF and Arhgap12, a gene encoding a previously uncharacterized protein of the RhoGAP family. We identified Arhgap12 as a transcriptional target of HGF, through a novel gene trapping strategy. We found that Arhgap12 mRNA and protein are robustly suppressed by HGF treatment, but not by serum. Arhgap12 displayed GTPase activating protein (GAP) activity towards Rac1 and, upon overexpression, impaired cell scattering, invasion and adhesion to fibronectin in response to HGF. Consistently, Arhgap12 silencing by RNA interference selectively increased the scatter and adhesion responses. These data show that HGF-driven invasive growth involves transcriptional regulation of a Rac1-specific GAP.

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.

KRAS mutations are present at a high frequency in human cancers. The development of therapies targeting mutated KRAS requires cellular and animal preclinical models. We exploited adeno-associated virus–mediated homologous recombination to insert the Kras G12D allele in the genome of mouse somatic cells. Heterozygous mutant cells displayed a constitutively active Kras protein, marked morphologic changes, increased proliferation and motility but were not transformed. On the contrary, mouse cells in which we overexpressed the corresponding Kras cDNA were readily transformed. The levels of Kras activation in knock-in cells were comparable with those present in human cancer cells carrying the corresponding mutation. Kras-mutated cells were compared with their wild-type counterparts by gene expression profiling, leading to the definition of a “mutated Kras-KI signature” of 345 genes. This signature was capable of classifying mouse and human cancers according to their KRAS mutational status, with an accuracy similar to or better than published Ras signatures. The isogenic cells that we have developed recapitulate the oncogenic activation of KRAS occurring in cancer and represent new models for studying Kras-mediated transformation. Our results have implications for the identification of human tumors in which the oncogenic KRAS transcriptional response is activated and suggest new strategies to build mouse models of tumor progression. [Cancer Res 2007;67(18):8468–76]

Colorectal Cancer (CRC) is characterized by wide genetic, biological and clinical heterogeneity. Oncogenic events, such as KRAS, NRAS and BRAF mutations, are known negative biomarker of response to EGFR targeted therapies. In addition to mutational analysis, transcriptional profiling has been recently exploited to identify distinct CRC molecular subtypes, associated with biological and clinical features such as cell of origin, microsatellite instability, prognosis and response to treatments. Detailed analysis of the relationships between the subtypes and clinical/biological features requires a large panel of preclinical models closely recapitulating the molecular heterogeneity of CRC. To this aim, we assembled a large collection of 151 CRC cell lines. For each line, we: (i) verified genetic identity by short tandem repeat (STR) analysis; (ii) assessed microsatellite instability (MSI) status; (iii) sequenced mutational hotspots in the KRAS, BRAF, NRAS and PIK3CA genes; (iv) performed microarray-based global mRNA expression profiling; (v) evaluated sensitivity to the EGFR-targeting drug cetuximab. STR analysis revealed that some cell lines previously thought to be unrelated are indeed derived from the same individual. This result was confirmed by cell line hierarchical clustering based on mRNA expression profiles. Overall, the mutational landscape of the compendium was concordant with what observed in patients (mutation rates: KRAS = 47%; BRAF = 17%; NRAS = 0.7%; PIK3CA = 18%). Similarly, sensitivity to cetuximab was confined to lines without RAS or BRAF mutations. Gene expression profiling was exploited to assign cell lines to molecular subtypes, according to five different published transcriptional classifiers. We found that all molecular subtypes previously identified in CRC patients were robustly maintained in the lines. Moreover, significant overlaps were detected between individual subtypes across distinct classifiers. Subtype-specific molecular and pharmacological associations previously defined in CRC samples were largely recapitulated in the compendium. In particular, MSI+ cells were significantly enriched in inflammatory and goblet subtypes and less prevalent in the Transit Amplifying (TA) and Stem groups. Cell lines carrying BRAF mutations clustered in the inflammatory subtype, while RAS mutations were equally distributed among all subtypes. Notably, in TA lines without RAS or BRAF mutations, the fraction of cetuximab-sensitive cells was strongly enriched (56%), confirming clinical data and indicating that addiction to the EGFR pathway is an intrinsic feature of this CRC subtype. In conclusion, our results describe a powerful preclinical resource reflecting the molecular and functional heterogeneity of CRC, which can be used to explore multidimensional information of potential clinical relevance. Citation Format: Gabriele Picco, Mariangela Russo, Carlotta Cancelliere, Michela Buscarino, Claudio Isella, Simona Lamba, Barbara Martinoglio, Federica Di Nicolantonio, Alberto Bardelli, Enzo Medico. Colorectal cancer cell lines recapitulate molecular and pharmacological features of clinical samples. [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 583. doi:10.1158/1538-7445.AM2015-583

Recent studies have identified a stem/serrated/mesenchymal (SSM) molecular subtype of colorectal cancer (CRC) that is associated with poor prognosis. We noted that genes upregulated in this subtype are also prominently expressed by stromal cells. This led us to hypothesize that the SSM transcripts could derive from the tumor microenvironment, rather than being intrinsic to cancer cells. To test this hypothesis, we analyzed microarray and RNAseq expression data from patient-derived xenografts (PDXs) of CRC, where human cancer cells are supported by murine stroma. Species-specific expression analysis revealed that mRNA levels of SSM genes are mostly due to stromal expression. Considering only genes exclusively expressed by stromal cells in PDXs, We then built three expression signatures specifically reporting the abundance of cancer-associated fibroblasts (CAFs), leucocytes or endothelial cells. In human CRC samples, all stromal signatures were strongly associated with the SSM subtype. A high CAF signature was associated with poor prognosis in untreated CRC patients, while in rectal cancer high stromal signatures jointly predicted radioresistance. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stroma. Citation Format: Claudio Isella, Andrea Terrasi, Sara E. Bellomo, Consalvo Petti, Andrea Muratore, Alfredo Mellano, Mark De Ridder, Paola Cassoni, Guy Storme, Andrea Bertotti, Enzo Medico. Stromal contribution to the colorectal cancer transcriptome. [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 4760. doi:10.1158/1538-7445.AM2015-4760

Colorectal cancer (CRC) is the third world leading cause of cancer death. Cetuximab and panitumumab, two monoclonal antibodies that bind the extracellular domain of epidermal growth factor receptor (EGFR), are effective only in the subset of RAS/BRAF wild-type metastatic colorectal cancers (mCRC). However, not all the RAS/BRAF wild-type patients benefit from anti-EGFR treatments. We hypothesized that primary resistance might be driven by overexpression of different tyrosine kinase (TK) genes and consequent activation of parallel pathways. Starting from a cetuximab screening of a panel of 151 CRC cell lines, we identified 41/151 CRC RAS/BRAF wild-type cells for which the mechanism of primary resistance to cetuximab is unaccounted for. Then, transcriptional outlier analysis identifies a subset of RAS/BRAF wild type cells, resistant to EGFR blockade, functionally and pharmacologically addicted to kinase genes including ALK, FGFR2, KIT, NTRK1/2 and RET, all of them target of drugs undergoing clinical testing or approved. Outlier expression was confirmed by RNAseq analysis. For ALK, NTRK1 and FGFR2 we found that overexpression is associated to molecular alterations, such as gene translocation (ALK and NTRK1) or gene amplification (FGFR2), described also in cancer patients. An immunohistochemistry (IHC) based screen on 220 CRC FFPE samples identified one NTRK1 outlier positive sample, with genetic rearrangement (detected by FISH). In order to demonstrate that outlier kinase genes were functionally relevant in the corresponding cell models and cell-specific functional dependencies, we used two complementary approaches: reverse genetics and pharmacological inhibition. siRNA-mediated candidate-specific gene suppression reduced, in all cases, protein expression of the ‘outlier’ TK resulted in significant impairment of cell growth, which was often accompanied by downstream signaling inhibition and apoptosis. Pharmacological inhibition with specific kinase-targeted agents was cell specific and paralleled the expression profiles of individual TKs. Nonetheless, in KIT overexpressing cells the reverse genetic experiment revealed functional dependency, despite relative kinase inhibitor was not effective. In conclusion our data suggest that overexpression of TK outliers drives primary resistance to EGFR blockade, and could be used to identify patients unlikely to respond to cetuximab or panitumumab. Moreover, the approach described here can be used to pinpoint colorectal cancers with exquisite dependencies to individual kinases for which clinically approved drugs are already available. Citation Format: Mariangela Russo, Gabriele Picco, Carlotta Cancelliere, Giorgio Corti, Emanuele Valtorta, Silvio Veronese, Marco Beccuti, Francesca Cordero, Federica Di Nicolantonio, Enzo Medico, Alberto Bardelli. The molecular landscape of colorectal cancer cell lines unveils clinically actionable targets. [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 916. doi:10.1158/1538-7445.AM2015-916

Background Data clustering analysis has been extensively applied to extract information from gene expression profiles obtained with DNA microarrays. To this aim, existing clustering approaches, mainly developed in computer science, have been adapted to microarray data analysis. However, previous studies revealed that microarray datasets have very diverse structures, some of which may not be correctly captured by current clustering methods. We therefore approached the problem from a new starting point, and developed a clustering algorithm designed to capture dataset-specific structures at the beginning of the process. Results The clustering algorithm is named Fuzzy clustering by Local Approximation of MEmbership (FLAME). Distinctive elements of FLAME are: (i) definition of the neighborhood of each object (gene or sample) and identification of objects with "archetypal" features named Cluster Supporting Objects, around which to construct the clusters; (ii) assignment to each object of a fuzzy membership vector approximated from the memberships of its neighboring objects, by an iterative converging process in which membership spreads from the Cluster Supporting Objects through their neighbors. Comparative analysis with K-means, hierarchical, fuzzy C-means and fuzzy self-organizing maps (SOM) showed that data partitions generated by FLAME are not superimposable to those of other methods and, although different types of datasets are better partitioned by different algorithms, FLAME displays the best overall performance. FLAME is implemented, together with all the above-mentioned algorithms, in a C++ software with graphical interface for Linux and Windows, capable of handling very large datasets, named Gene Expression Data Analysis Studio (GEDAS), freely available under GNU General Public License. Conclusion The FLAME algorithm has intrinsic advantages, such as the ability to capture non-linear relationships and non-globular clusters, the automated definition of the number of clusters, and the identification of cluster outliers, i.e. genes that are not assigned to any cluster. As a result, clusters are more internally homogeneous and more diverse from each other, and provide better partitioning of biological functions. The clustering algorithm can be easily extended to applications different from gene expression analysis.

Objective Mesenchymal stem cells (MSCs) are multipotent cells that can self-renew, proliferate, and exhibit elevated cellular plasticity. To investigate their possible neural fate, we studied human mesenchymal stem cells (hMSCs) in different cell culture conditions from morphological, immunochemical, gene expression, and physiological points of view. Materials and Methods We tested hMSCs in three previously reported experimental conditions made of α-modified minimum essential medium (α-MEM)/1 mM β-mercaptoethanol (βME), 10 μM α-MEM/retinoic acid (RA) or α-MEM/2% dimethylsulfoxide (DMSO) + 200 μM β-hydroxyanisole (BHA), respectively, and in a new experimental condition with neural progenitor maintenance medium (NPMM). Results hMSCs were isolated from bone marrow and expanded for several passages. In βME, cells became immunoreactive for neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), Nestin, and glial fibrillary acidic protein (GFAP). In experimental conditions with RA and DMSO/BHA, hMSCs were NeuN and NSE-positive while in NPMM they were positive for GFAP and NSE. Untreated hMSCs showed a weak mRNA expression for microtubule-associated protein, NSE, and neurofilament protein-medium and GFAP, which strongly increased in NPMM-treated hMSCs. In the electrophysiological study, NPMM-differentiated hMSCs expressed two delayed rectifier K + currents related to two ether-a-go-go K + channels (eag1, eag2), which are fundamental for setting the negative resting potentials required for neuronal survival and basal cell activity. The two K + channels were absent in undifferentiated hMSCs. These data were confirmed by real-time polymerase chain reaction. Conclusion In our new culture condition, hMSCs acquired new morphological characteristics, neural markers, and electrophysiological properties, which are suggestive of neural differentiation. This might lead to clinical use of hMSCs in neural degenerative diseases.

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here, we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably, 1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression, HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters, a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions.

Since migratory thrombophlebitis was noted by Trousseau during diagnosis of his own pancreatic cancer in 1865, blood clotting disorders have been regularly reported as a sign of occult (as in without readily detectable symptoms) malignancies. No direct link between tumorigenesis and blood clotting had been found, until now. In a mouse model for liver cancer, the human oncogene MET that triggers tumour development is shown to regulate genes responsible for haemostasis. Mice in which MET is active develop venous thromboses similar to those seen in human occult malignancies and overt cancers. The close relationship between activation of blood coagulation and cancer is an old enigma. In 1865, migrans trombophlebitis (‘a condition of the blood that predisposes it to spontaneous coagulation’) was described as a forewarning of occult malignancy (Trousseau's sign1). This pioneering observation emphasized the existence of haemostasis disorders associated with cancer onset; this phenomenon has since been extensively reported in clinical and epidemiological studies2,3,4, but has so far resisted a mechanistic explanation. Here we report a mouse model of sporadic tumorigenesis based on genetic manipulation of somatic cells. Targeting the activated, human MET oncogene to adult liver caused slowly progressing hepatocarcinogenesis. This was preceded and accompanied by a syndrome manifesting first with blood hypercoagulation (venous thromboses), and then evolving towards fatal internal haemorrhages. The pathogenesis of this syndrome is driven by the transcriptional response to the oncogene, including prominent upregulation of plasminogen activator inhibitor type 1 (PAI-1) and cyclooxygenase-2 (COX-2) genes. In vivo analysis showed that both proteins support the thrombohaemorrhagic phenotype, thus providing direct genetic evidence for the long-sought-after link between oncogene activation and haemostasis.

Papillary thyroid carcinomas are characterized by rearrangements of the RET receptor tyrosine kinase generating RET/ PTC oncogenes. Here we show that osteopontin ( OPN), a secreted glycoprotein, is a major RET/ PTC- induced transcriptional target in PC Cl 3 thyroid follicular cells. OPN upregulation depended on the integrity of the RET/ PTC kinase and tyrosines Y1015 and Y1062, two major RET/ PTC autophosphorylation sites. RET/ PT C also induced a strong overexpression of CD44, a cell surface signalling receptor for OPN. Upregulation of CD44 was dependent on RET/ PTC Y1062, as well. Constitutive OPN overexpression or treatment with exogenous recombinant OPN sharply increased proliferation, Matrigel invasion and spreading in collagen gels of RET/ PTC- transformed PC Cl 3 cells. These effects were impaired by the treatment of PC Cl 3- RET/ PTC cells with OPN- and CD44- blocking antibodies. Thus, RET/ PTC signalling triggers an autocrine loop involving OPN and CD44 that sustains proliferation and invasion of transfomed PC Cl 3 thyrocytes.

Insulin receptor isoform A (IR-A) is a fetal insulin receptor isoform that is overexpressed in cancer. We investigated whether insulin-like growth factor (IGF)-II may elicit a different gene expression response from insulin in cells expressing only IR-A and lacking IGF-I receptor (R−/IR-A cells). Cells were stimulated with either insulin or IGF-II (at 0.5, 3, and 8 h), and global gene expression was studied by microarray technology. Results were validated by quantitative real-time PCR. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 transcripts were differentially regulated. of these 45 genes, 18 were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both ligands but with a significant difference at at least one time point. IGF-II was a more potent regulator than insulin for these genes. In conclusion, insulin and IGF-II, acting via the same receptor (IR-A), may differentially affect gene expression in cells. These findings provide a molecular basis in clarifying the biological role of IR-A in embryonic/fetal life and in cancer.

The human insulin receptor (IR) exists in two isoforms (IR-A and IR-B). IR-A is a short isoform, generated by the skipping of exon 11, a small exon encoding for 12 amino acid residues at the carboxyl terminus of the IR alpha-subunit. Recently, we found that IR-A is the predominant isoform in fetal tissues and malignant cells and binds with a high affinity not only insulin but also insulin-like growth factor-II (IGF-II). To investigate whether the activation of IR-A by the two ligands differentially activate post-receptor molecular mechanisms, we studied gene expression in response to IR-A activation by either insulin or IGF-II, using microarray technology. To avoid the interfering effect of the IGF-IR, IGF-II binding to the IR-A was studied in IGF-IR-deficient murine fibroblasts (R- cells) transfected with the human IR-A cDNA (R-/IR-A cells). Gene expression was studied at 0.5, 3, and 8 h. We found that 214 transcripts were similarly regulated by insulin and IGF-II, whereas 45 genes were differentially transcribed. Eighteen of these differentially regulated genes were responsive to only one of the two ligands (12 to insulin and 6 to IGF-II). Twenty-seven transcripts were regulated by both insulin and IGF-II, but a significant difference between the two ligands was present at least in one time point. Interestingly, IGF-II was a more potent and/or persistent regulator than insulin for these genes. Results were validated by measuring the expression of 12 genes by quantitative real-time reverse transcriptase-PCR. In conclusion, we show that insulin and IGF-II, acting via the same receptor, may differentially affect gene expression in cells. These studies provide a molecular basis for understanding some of the biological differences between the two ligands and may help to clarify the biological role of IR-A in embryonic/fetal growth and the selective biological advantage that malignant cells producing IGF-II may acquire via IR-A overexpression.

Anaplastic large cell lymphoma (ALCL) represents a category of T-cell Non-Hodgkin Lymphomas characterized by marked cellular pleomorphism, and expression of CD30. Two systemic forms of ALCL are defined by the presence or absence of chromosomal translocations involving the Anaplastic Lymphoma Kinase (ALK) gene at 2p23 locus. Among several pathways triggered by ALK signaling, it has been widely shown that the constitutive activation of STAT3 is strictly required for ALK-mediated transformation and survival. To discover essential mediators of STAT3 oncogenic activity that may represent feasible targets for ALCL therapies, we performed gene and miRNA expression profiling experiments in association with functional validation approaches. The transcriptome of STAT3 was analysed in ALK positive ALCL cell lines using a tightly controlled time course experimental condition, in which the STAT3 signalling was abrogated by an inducible short hairpin RNA (shRNA). Gene expression profiling analysis identified a selected number of genes (1730) specifically modulated by STAT3 silencing. A significant overrepresentation of putative STAT3 binding sites was found in regulatory regions of early down-regulated genes. Functional studies using a shRNA lentiviral library established that Interferon Regulatory Factor-4 (IRF4) targeting specifically affect cell viability of ALK+ ALCL cells. In contrast, forced expression of IRF4 partially rescued STAT3 knock-down sustaining the survival of ALK+ cells. In a parallel experiment, genome-wide miRNA expression profiling identified 48 miRNAs concordantly modulated by the inducible knock down of ALK and STAT3. Among these, we demonstrated that expression of miR-17∼92 cluster significantly reverted STAT3 deprivation, sustaining both proliferation and survival of ALCL cells. In conclusion, genes and miRNA expression profiling associated to functional screenings allowed the identification of new biologically relevant targets for ALK+ALCL. We speculate that IRF4 and miR-17∼92 cluster are involved in the lymphomagenesis of STAT3+ ALCL, and that their inhibition might represent an alternative avenue to interfere with ALK signaling in Anaplastic Large Cell Lymphomas. Citation Format: Elisa Spaccarotella, Aldi Pupuleku, Elisa Pellegrino, Cecilia Bandini, Manuela Ferracin, Daniela Cantarella, Andrea Rinaldi, Paolo Provero, Ferdinando Di Cunto, Enzo Medico, Francesco Bertoni, Giorgio Inghirami, Roberto Piva. STAT3 network dissection in ALK positive Anaplastic Large Cell Lymphomas. [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 2219. doi:10.1158/1538-7445.AM2014-2219

We present a method for fast and efficient trapping of genes whose transcription is regulated by exogenous stimuli. We constructed a promoterless retroviral vector transducing a green fluorescent protein-nitroreductase (GFNR) fusion protein downstream from a splice acceptor site. Flow cytometric analysis of the infected population allows identification and sorting of cells in which the trap is integrated downstream from an active promoter. Conversely, the nitroreductase (NTR) moiety allows pharmacological selection against constitutive GFNR expression. Using hepatocyte growth factor (HGF) stimulation of liver cells combined with either positive or negative selection, we recovered cell populations carrying traps in induced or suppressed genes, respectively. Several distinct responsive clones were isolated, and regulated expression of the trapped gene was confirmed at the RNA level. Positive and negative selection can be calibrated to recover traps in genes showing different levels of basal expression or transcriptional regulation. The flexibility and efficiency of the GFNR-based trap screening procedure make it suitable for wide surveys of transcriptionally regulated genes.

Anaplastic large cell lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that includes two entities ALK+ and ALK- ALCL based on chromosomal translocations of the anaplastic lymphoma kinase (ALK). ALK- ALCL have very poor clinical outcome with conventional chemotherapy. Lack of representative cell lines and/or models has compromised the development of successful therapy. Toward this end, we report the generation and characterization of a group of novel ALCL tumor grafts. We implanted subcutaneously (s.c.) tumor tissue fragments, fresh or viable cryopreserved, from 11 cases of primary systemic ALCL (3 ALK+ and 8 ALK-) in six- to eight-week old severe immunocombined immunodeficiency NOD Cg-Prkdcscid/Il2rgtm1wjl/SzJ (NSG) mice. A single leukaemic ALCL sample was injected i.v. or s.c. Six cases led to successful lymphoma growth within 6-12 weeks as subcutaneous solid spheroid masses constituted by a uniform population of large pleomorphic cells CD30-positive. All tumor grafts were re-implanted in recipient animals, in same cases up to 21 consecutive implants with an analogous time growth rate. Two cases, in the same way of the original human neoplasia, were early associated with concomitant enlargement of multiple organs. All tumor graft lines eventually showed disseminate disease with involvement of distant lymph nodes and of spleen (with homig in peri-arteriolar spaces), liver (initially observed within the sinusoidal spaces) and kidney. The morphological and immunohistochemical (IHC) evaluation (expression of CD30, cytotoxic proteins and ALK) of the serially propagated ALCL tumor grafts demonstrated a high concordance between primary and derived animal tumors which showed highly stable profile along different passages from the early generation (T1) until the final ones (up to T16). The clonal relationship with primary and corresponding tumor grafts was confirmed by gene TCR rearrangement analyses and by ALK FISH studies. In addition we interrogated the model using gene expression profiling and single nucleotide polymorphism which established a close relationship among primary and derived tumors. Massive parallel sequencing on mRNA transcripts defines the presence of two novel chimeras involving NF1-MAZ and TRAF1-ALK1, which confirmes the evidence of ALK signaling and pinpoint a role of RAS in ALK- ALCL. The tumor grafts displayed, also, therapeutic responses with conventional therapy (CHOP) which reflected that seen in the donor patients: initial partial responses followed invariably by clinical relapses. To overcome this refractory we assayed the sensitivity of two tumorgraft lines, generated from ALK+ ALCL, to a new selective ALK inhibitor (CEP28122) that led a rapid and stable response. This study demonstrated the key role of these libraries of tumor grafts, especially in developing and testing new therapeutic regimens in refractory ALCL patients. Citation Format: Fabrizio Tabbo’, Antonella Barreca, Rodolfo Machiorlatti, Katia Messana, Indira Landra, Francesco Abate, Michela Boi, Maria Todaro, Cristina Abele, Domenico Novero, Alberto Zamo’, Marco Chilosi, Maurilio Ponzoni, Mangeng Cheng, Bruce Ruggeri, Pierpaolo Piccaluga, Stefano Pileri, Enrico Tiacci, Brunangelo Falini, Lenny Shultz, Roberto Piva, Enzo Medico, Francesco Bertoni, Raul Rabadan, Giorgio Inghirami. Humanized NOD/Scid/IL2g-/- tumor grafts recapitulate primary anaplastic large cell lymphoma. [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 3853. doi:10.1158/1538-7445.AM2013-3853

nef is a human immunodeficiency virus (HIV) gene encoding a 27-kDa myristoylated protein with structural features of a signal transducing molecule, but whose functions are largely unknown. We studied the interactions of Nef with the signal transduction pathways triggered by the platelet-derived growth factor (PDGF) receptor. The association of phosphatidylinositol (PI) 3-kinase with the activated receptor was severely impaired by nef expression. Conversely, PDGF-induced receptor tyrosine phosphorylation, binding to phospholipase C-gamma and to Ras-GAP were not modified. Microtubule-associated protein kinase activation and intracellular calcium influx in response to PDGF were either unaffected or only slightly enhanced. Nef significantly reduced the proliferative response to the growth factor, while the chemotactic response was unchanged. These data show that Nef affects selectively the PI 3-kinase signaling pathway and suggest that this interference results in some of the HIV adverse effects on host cell functions.

A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK (Figure 1). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60 % (6/10) of the cases (Table 1). KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Abstract 2634 ALCL has been separated in two distinct subtypes based on the presence or absence of translocations involving the ALK gene. It is accepted that ALK+ALCL is a distinct subgroup which shares a unique phenotype, with well defined genetic and clinical features. Although the clinical presentations, translocations and genetic events vary between ALK+ and ALK-ALCL, the relationship between these two ALCL subtypes and whether ALK-ALCL may represent a subset of peripheral T-cell lymphomas, not otherwise specified (PTCL, NOS), remains unclear. In this regard, the WHO Classification classifies ALK-ALCL as a provisional entity. A better understanding of the underlying genetics would provide critical explanations to answer some of these questions. With the aim of identifying the genetic events underlying the pathogenesis of ALCL, we studied a series of 69 cases of ALCL (34 ALK-, 35 ALK+) with high-density genome wide SNP-based arrays. Methods. DNA was extracted from frozen biopsies. DNA profiles were obtained using the Affymetrix GeneChip Human Mapping SNP6 arrays. Differences in frequencies between subgroups were evaluated using Fisher's exact test. A subset of cases also had available gene expression profiles. Clinical data were available in half of the cases and genomic lesions were evaluated for their impact on clinical outcome with the log-rank test. Results. The most common losses were at 6q21, 17p13 (19%), 13q22.3 (15%), 3p21.31, 13q32.3 (14%), 1p13.3, 16q23.1 (WWOX) (13%), 16q23.3–24.1 (12%), 1p33 and 16q22.1 (10%). The most common gains occurred at 8q22 (20%), 1q (13%), 7q (10–15%; CDK6, 15%), 8q24 and 9p24.1 (10%). ALK-ALCL displayed a higher number of genomic aberrations in comparison with ALK+ALCL. The lesions presenting major differences included: -6q21 (35% vs 6%; P=0.002), -1p13 (26% vs 3%, P=0.001), -3q22 (26% vs 0%, P=0.001), -4q12-q26 (18% vs 0%; P=0.009), +9p21 (17% vs 0%, P=0.009), -17p13 (TP53, 26% vs 6%, P=0.019). The deletions at 6q21 targeted the gene PRDM1, coding for BLIMP1. The whole coding sequence of PRDM1 has been sequenced in 33 ALK- ALCL samples. Only one somatic mutation, inducing a stop codon, was identified, in one case bearing copy neutral loss of heterozygosity (cnLOH) spanning PRDM1 locus, suggesting a loss of functional protein in this patient. As a whole, 38% of ALK-ALCL presented loss of at least one allele of PRDM1. Only two cases were observed with complete gene loss: the ALK-case with somatic mutation plus cnLOH, and one ALK+ case with homozygous deletion. The presence of 6q21 deletion had an impact on progression free survival among all ALCL (P=0.048), likely reflecting its association with ALK-ALCL, but not when considering ALK- patients only. Xenografts derived from primary ALCL samples bearing 6q21 loss presented decreased BLIMP1 expression level. The detection of PRDM1 loss was present also in cell lines, in which also a decreased level of BLIMP1 RNA and protein was observed. Additional genes, members of PRDM1 pathway, were identified as targets of focal deletions. Conclusions. A series of recurrent lesions has been identified in ALCL. Alongside TP53 loss, inactivation of PRDM1 by genomic losses or somatic mutations was the most common detected lesion, and was more frequently inactivated in ALK-ALCL. PRDM1, encoding BLIMP1, a master regulator of T-cells differentiation, appears as a central gene in ALCL pathogenesis. Other genes, belonging to the same pathway, were found to have focal genomic aberrations in a smaller number of cases. Disclosures: No relevant conflicts of interest to declare.

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues.

The mutational landscape of cancer genomes displays a complex combination of genetic lesions affecting oncogenes and tumor suppressor genes. The construction of cellular models closely recapitulating cancer-linked genetic alterations is a prerequisite to understand their role in tumor progression and to identify genotype- specific pharmacological responses. To recapitulate point mutations affecting oncogenes we used homologous recombination to ‘knock-in’ specific nucleotide changes in the genome of human cells. We focused on the following alleles: EGFR (delE746-A750), KRAS (G13D), BRAF (V600E) and PIK3CA (E545K and H1047R), that are found in multiple human cancers. As recipient cells, a human non transformed epithelial cell line of breast origin (hTERT-HME1) has been employed. This cell line has been immortalized by hTERT and harbors a mutation on TP53 gene, a genetic event frequently observed in naturally occurring human tumors. To recapitulate the inactivation of additional tumor suppressor genes in our cellular models, we exploited shRNAs to permanently knock-down the expression of PTEN and RB1 genes, as they are frequently down regulated in human tumors. Knock-down of PTEN and RB1 was performed both in the parental hTERT-HME1 cells and in the ‘knock-in’ models, thus allowing us to combine tumor suppressor genes inactivation with mutational activation of specific oncogenes. The resulting cellular models are hereafter refereed to as combinatorial ‘matrix’. To assess how specific combinations of genetic lesions affect the tumorigenic properties of not transformed cells, we measured the ability of the ‘matrix’ to grow in soft agar. At the same time we injected our cellular models in the cleared, humanized mammary fat pad of immunocompromised mice. For the latter approach, we focused on genotypes recapitulating mutations found in breast tumors, such as PTEN or RB1 inactivation and PIK3CA or KRAS mutations, alone or in combination. In vitro and in vivo results showed that those combinations of genetic events are not sufficient to transform the normal hTERT-HME1 cells. However, preliminary data indicates that few genotypes of the ‘matrix’ acquired the ability to colonize the murine fat pads. With the rational of unveiling new pharmaco-genetic interactions involving one or multiple cancer mutations, the “matrix” was used to screen a panel of compounds used for cancer therapy, or for which an anti-proliferative activity was previously reported. This approach led to the identification of a subset of genotype-drug interactions thus underscoring how the multifactorial genomic milieu of cancer cells impact the response to therapies. Grant support: This work is part of the EU FP7 grant COLTHERES Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2011-230

804 Bioinformatic analysis of BRAFV600E vs RASG12V signatures in colon cancer cells reveals differential regulation of cellular pathways related to MSI or EMT A. Pintzas, T. Joyce, E. Oikonomou, E. Makrodouli, E. Medico, D. Cantarella, I. Bantounas. Institute of Biological Research and Biotechnology National Hellenic Research Foundation, Laboratory of Signal Mediated Gene Expression, Athens, Greece, The Oncogenomics Center Institute for Cancer Research and Treatment University of Torino School of Medicine, Laboratory of Functional Genomics, Candiolo, Italy

Anaplastic large cell lymphomas (ALCL) are a subset of T-cell Non-Hodgkin Lymphomas (T-NHL) in which the intracytoplasmic domain of the anaplastic lymphoma kinase (ALK) gene is frequently fused to nucleophosmin (NPM1). It has been previously shown that the transcription factor STAT3 is a major substrate of ALK enzymatic activity and it is strictly required for the growth and survival of ALK-transformed cells. With the aim to unravel the regulatory network underlying NPM-ALK-mediated transformation and tumor maintenance, we timely dissected the mRNA and miRNA expression signature of ALK-positive ALCL cell lines following ALK or STAT3 silencing. We observed that the majority of ALK-regulated genes were concordantly dependent on STAT3, including known and novel STAT3 target genes. Additional transcription factors (C/EBP beta, LEF1, and others) emerged as STAT3-dependent initiators and master regulators of ALK oncogenic properties in ALCL. However, STAT3 signaling was inconsistent and completely dispensable in non small cell cancer cell lines expressing EML4-ALK fusion proteins, suggesting a T-cell specific STAT3 regulatory network. Finally, we analyzed the gene expression profiling of 90 systemic peripheral T cell lymphomas, including a set of ALK positive and negative ALCL (n=36). We observed that ALK positive ALCL were significantly clustered in a separate subgroup, characterized by a relevant STAT3 signature. Using prediction analyses, we identified a set of genomic classifiers able to a clearly separate ALK positive and ALK negative ALCL from other T-NHL. In conclusion we proved that inference of ALK oncogenic transcriptional networks is a powerful tool to identify meaningful signaling molecules that regulate the transition into a pathologic cellular state. Our molecular classification suggests that ALCL may represent a unique entity discernible from other T-NHL, and that a restricted number of genes can be instrumental for clinical stratification and, possibly, to address the therapy of patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2161.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Lung cancer is the most common cancer in the world, and is lethal in 90% of the cases. In non-small cell lung cancer (NSCLC), deregulated receptor tyrosine kinases (RTKs) stand out among the causal dominant oncogenes. Consequently, the genomic and/or pharmacological ablation of RTK signaling has emerged as a novel tailored therapeutic strategy. Nevertheless, this approach has serious constrains, probably due to the frequent co-activation of multiple RTKs in a considerable subsets of solid tumors. It has become also evident that during TK inhibitor-based regimens, RTK switching and/or the selection of resistant clones can frequently occur representing a serious and problematic drawback. In a subset of NSCLC, the Anaplastic Lymphoma Kinase (ALK) gene has been described to be translocated and fused to EML4. This determines the ALK constitutive dimerization and autophosphorylation leading to cellular transformation. Here, we investigated the ALK oncogenic addiction of human NSCLC and studied the putative co-operative role of other kinases. We first demonstrated that the ectopic expression of EML4-ALK in ALK positive NSCLC cell lines (H2228 and H3122) resulted in the activation of multiple signaling pathways, in manner similar to that described for other known ALK fusions. Although EML4-ALK can induce transformation in lung in vitro and in vivo, ALK inhibition via shRNA or small molecule inhibitors (CEPs, Cephalon) induced only the apoptosis of H3122 cells, whereas in H2228 it caused cell growth arrest. Moreover, the treatment with ALK inhibitors led to tumor regression, but not tumor eradication, in vivo. Based on Phosphoproteomic analyses we demonstrated that the phosphorylation status of several tyrosine kinases (such as, EGFR, Met, FGFR, Jak1 or IGFR) was affected by the ALK inhibition only in H2228 cell line, but not in H3122. Notably, the combined treatment with anti-ALK ([CEP14083][1], CEP2550, [CEP28122][2]) and -EGFR inhibitors resulted in an increased cell death of H2228 cells to values similar to those observed for ALK treated H3122 cells. Finally, gene expression analyses showed that known EGFR substrates were specifically down regulated upon the combined treatment in ALK positive H2228 cells. Our findings suggest that ALK signaling is required for the maintenance of the neoplastic phenotype of some ALK positive NSCLC cells and that its abrogation could represent a novel strategy for the treatment of a well-defined subset of human lung cancer (complete ALK addiction). More importantly, we showed that the tumor survival and maintenance of ALK positive neoplastic cells might relay on the concomitant activation of multiple RTKs. These findings further support the notion that molecular and functional signatures are required for designing molecular-based “patient specific” therapeutic protocols in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-309. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP14083&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom [2]: /lookup/external-ref?link_type=GENPEPT&access_num=CEP28122&atom=%2Fcanres%2F70%2F8_Supplement%2FLB-309.atom

The occurrence of leukemia-like events consequent to vector integration in a successful human gene therapy trial has prompted a reassessment of the insertional mutagenesis risk by retroviral vectors. Recent reports, which used high-throughput sequencing and mapping of vector integration sites, have challenged the notion that retroviruses integrate randomly in the cell genome, and indicated preferential integration into active genes both for HIV- and MLV-based vectors. We used a novel approach based on genetic trapping to score several thousands integration sites at once in transduced cells. To this aim, we constructed HIV- and MLV-based promoter traps and compared the efficiency of promoter trapping using a selectable reporter PuroR-GFP fusion gene. We found that MLV trapped cellular promoters much more efficiently, and targeted more active genes, than HIV. Remarkably, one out of seven MLV integrations trapped an active cellular promoter. We obtained these findings in human and murine cells, including human cord blood CD34+ cells. When we delivered vector traps by CaPi-DNA transfection, we did not find significant differences in trapping efficiency of MLV and HIV traps, indicating that the retroviral integration machinery was responsible for the differences observed in transduction experiments. RNA analysis of transduced cells showed that HIV transcripts had a much wider size range as compared with those of MLV, which did not significantly exceed the length of the vector backbone. These results indicated that MLV integrations resulting in reporter expression occurred in proximity to the transcription start site, or within the first intron, of active genes. Interestingly, analysis of puromycin-selected cells showed strong enrichment in HIV transcripts of size similar to those of MLV, indicating that the few HIV integrations resulting in efficient reporter expression were those occurring proximal to a promoter. The average reporter expression level of these two selected cell populations remained higher for MLV traps, confirming that, on average, MLV vectors targeted genes more active than those targeted by HIV. These results provide functional evidence that MLV vectors preferentially integrate close to highly active promoters, and can efficiently exploit them for proviral expression. Such a strategy may have evolved in the parental viruses to provide a fraction of their rogeny with a supplementary cellular promoter, less susceptible to silencing by genome surveillance mechanisms than the one embedded in the LTR. The proximity and functional relationship established between MLV proviruses and flanking cellular promoters suggests that integration of wild-type MLV may easily up-regulate transcription from cellular promoters and overexpress the cognate genes, including proto-oncogenes. HIV vectors did not show the integration biases observed for MLV. Whether this behavior of HIV, together with the advanced vector engineering that fully inactivates its LTR, provide a safer integrating tool for gene therapy remains to be assessed.

The monoclonal antibody-defined CARS antigen is a new carcinoma associated marker which is expressed on a mucin-like molecule. Serum concentrations of CARS were assayed in 181 patients with carcinomas of different organs, 20 patients with non-carcinomatous malignancies, 123 patients with inflammatory diseases and 150 healthy controls. Serum levels of CARS were significantly increased in 51% of the patients with pancreatic carcinomas, in 60% of patients with biliary tract carcinomas and in about 15% of the patients with carcinomas of the digestive apparatus. Sera from patients with breast carcinomas were negative, as well as sera from patients with melanomas or sarcomas. CAR-3 values in samples from patients with chronic pancreatitis were constantly negative, as were samples from healthy donors. Significant concentrations of CAR-3 were detected in 20% of the sera from patients with acute pancreatitis and in 15% of the sera from patients with cirrhosis. Because of its high specificity for pancreatic carcinomas compared to chronic pancreatitis, CARS seems a promising marker for distinguishing between neoplastic and chronic inflammatory diseases of the pancreas, whose differential diagnosis is difficult.

The monoclonal antibody (MAb) BD-5 reacts with an epitope (CAR-5) expressed in 83% of the gastric carcinomas and in 51% of the ductal pancreatic carcinomas. This MAb reacts also with epithelial cells of colorectal mucosa, but does not react at all with normal adult gastric mucosa or normal adult pancreas. We report the biochemical and immunochemical characterization of CAR-5-bearing molecule. The epitope was found to be carried on a mucin of more than 400 kDa with a density of 1.45 g/ml, metabolically labelled with 35S-sulfate, 3H-glucosamine, 3H-mannose and 35S-methionine. Antigenicity survived metaperiodate oxidation and alkalinization, while it was fully destroyed by pronase or papain. Trypsin, although cleaving the molecule, did not affect its antigenic activity. CAR-5 epitope is thus carried on the protein moiety of a sulfo-mucin. On the basis of its biochemical properties, the antigen was purified by a 3-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL-4B and affinity chromatography on wheat-germ agglutinin coupled to Sepharose 4B. Cross-competition experiments, together with the chemical properties displayed by the different epitopes, clearly indicate that CAR-5 is different from all previously characterized carcinoma-associated determinants. Cross-DDIRMA experiments performed with different "catcher" and "tracer" antibody combinations showed that CAR-5 epitope may be expressed on the same mucin bearing CA 19-9, MOv2, DU-PAN-2, Lewisa and Lewisb epitopes.

In colorectal cancer (CRC), WNT pathway activation by genetic rearrangements of RSPO3 is emerging as a promising target. However, its low prevalence severely limits availability of preclinical models for in-depth characterization. Using a pipeline designed to suppress stroma-derived signal, we find that RSPO3 "outlier" expression in CRC samples highlights translocation and fusion transcript expression. Outlier search in 151 CRC cell lines identified VACO6 and SNU1411 cells as carriers of, respectively, a canonical PTPRK(e1)-RSPO3(e2) fusion and a novel PTPRK(e13)-RSPO3(e2) fusion. Both lines displayed marked in vitro and in vivo sensitivity to WNT blockade by the porcupine inhibitor LGK974, associated with transcriptional and morphological evidence of WNT pathway suppression. Long-term treatment of VACO6 cells with LGK974 led to the emergence of a resistant population carrying two frameshift deletions of the WNT pathway inhibitor AXIN1, with consequent protein loss. Suppression of AXIN1 in parental VACO6 cells by RNA interference conferred marked resistance to LGK974. These results provide the first mechanism of secondary resistance to WNT pathway inhibition., info:eu-repo/semantics/published

The monoclonal antibody AR-3 reacts with an epitope (CAR-3) carried on a high-molecular-weight glycoprotein associated with carcinomas of the pancreas, stomach, colon, uterus, and ovary. This study reports the partial purification and characterization of CAR-3-bearing molecule. The antigen was quantified by a double determinant immunoradiometric assay. CAR-3 antigen was purified by a three-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL-4B, and affinity chromatography on AR-3 antibodies coupled to Sepharose 4B. Following this procedure CAR-3 antigen was purified about 400-fold with a 36% yield. Treatment of the CAR-3 antigen with 16 mM metaperiodate or with 1 N NaOH resulted in complete loss of activity. Antigenicity survived enzymatic treatments known to destroy proteins. The epitope was found to be carried on a molecule with a molecular weight of greater than 400,000 with a density of 1.45 g/ml, metabolically labeled with [35S]sulfate, [3H]glucosamine, and [35S]methionine. It is concluded that CAR-3 epitope is expressed on a carbohydrate moiety linked to a sulfo-mucin-like molecule via an O-glycosidic bond. Cross-competition experiments showed that CAR-3 epitope is strictly related or in close topographic proximity to Lewis(a) and Lewis(b) antigens. Cross-double determinant immunoradiometric assay experiments indicated that the same mucin carrying CAR-3 bears also CA 19-9, CA 125, and BW 494 epitopes.

Each cancer is a complex system with unique molecular features determining its dynamics, such as its prognosis and response to therapies. Understanding the role of these biological traits is fundamental in order to personalize cancer clinical care according to the characteristics of each patient’s disease. To achieve this, translational researchers propagate patients’ samples through in vivo and in vitro cultures to test different therapies on the same tumor and to compare their outcomes with the molecular profile of the disease. This in turn generates information that can be subsequently translated into the development of predictive biomarkers for clinical use. These large-scale experiments generate huge collections of hierarchical data (i.e. experimental trees) with relative annotations that are extremely difficult to analyze. To address such issues in data analyses, we came up with the Semalytics data framework, the core of an analytical platform that processes experimental information through Semantic Web technologies. Semalytics allows (i) the efficient exploration of experimental trees with irregular structures together with their annotations. Moreover, (ii) the platform links its data to a wider open knowledge base (i.e. Wikidata) to add an extended knowledge layer without the need to manage and curate those data locally. Altogether, Semalytics provides augmented perspectives on experimental data, allowing the generation of new hypotheses, which were not anticipated by the user a priori. In this work, we present the data core we created for Semalytics, focusing on its semantic nucleus and on how it exploits semantic reasoning and data integration to tackle issues of this kind of analyses. Finally, we describe a proof-of-concept study based on the examination of several dozen cases of metastatic colorectal cancer in order to illustrate how Semalytics can help researchers generate hypotheses about the role of genes alterations in causing resistance or sensitivity of cancer cells to specific drugs.

The hepatocyte growth factor (HGF) receptor (p190MET) is a tyrosine kinase composed of two disulfide-linked chains, alpha of 50 kDa and beta of 145 kDa. We have previously described an isoform (p140MET) containing a beta chain of 85 kDa, lacking the cytoplasmic kinase domain. The two receptor variants originate by post-translational processing of a common single-chain precursor of 170 kDa (Pr170). In the endoplasmic reticulum a fraction of Pr170 is cleaved at the cytosolic side generating an intermediate product of 120 kDa (Pr120). This molecule 1) is already detectable after 15 min of pulse labeling, 2) contains high mannose-branched oligosaccharides, and 3) accumulates upon treatments inhibiting the export from the endoplasmic reticulum. A second cleavage, occurring after 30 min of chase in the trans-Golgi network, converts the single-chain precursors Pr170 and Pr120 into the mature heterodimers p190MET and p140MET. This process is inhibited by brefeldin A treatment. Conditions leading to Pr170 accumulation in the endoplasmic reticulum, such as receptor overexpression, induce kinase activation and overproduction of Pr120. Conversely, cells expressing a kinase-defective HGF receptor lack the truncated isoform. The proteolytic cleavage of the cytoplasmic domain may thus represent a safety mechanism aimed at preventing ligand-independent intracellular activation of the HGF receptor kinase.