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Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC.Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays.Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival.Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Supplementary Table S7. Upstream regulators identified by the Upstream Regulator Analysis in HBCx22 TamR and HBCx34 TamR xenografts.

Bracoviruses and ichnoviruses are endogenous viruses of parasitic wasps that produce particles containing virulence genes expressed in host tissues and necessary for parasitism success. In the case of bracoviruses the particles are produced by conserved genes of nudiviral origin integrated permanently in the wasp genome, whereas the virulence genes can strikingly differ depending on the wasp lineage. To date most data obtained on bracoviruses concerned species from the braconid subfamily of Microgastrinae. To gain a broader view on the diversity of virulence genes we sequenced the genome packaged in the particles of Chelonus inanitus bracovirus (CiBV) produced by a wasp belonging to a different subfamily: the Cheloninae. These are egg-larval parasitoids, which means that they oviposit into the host egg and the wasp larvae then develop within the larval stages of the host. We found that most of CiBV virulence genes belong to families that are specific to Cheloninae. As other bracoviruses and ichnoviruses however, CiBV encode v-ank genes encoding truncated versions of the immune cactus/IκB factor, which suggests these proteins might play a key role in host–parasite interactions involving domesticated endogenous viruses. We found that the structures of CiBV V-ANKs are different from those previously reported. Phylogenetic analysis supports the hypothesis that they may originate from a cactus/IκB immune gene from the wasp genome acquired by the bracovirus. However, their evolutionary history is different from that shared by other V-ANKs, whose common origin probably reflects horizontal gene transfer events of virus sequences between braconid and ichneumonid wasps.

Reverse-phase protein array (RPPA) technology uses panels of high-specificity antibodies to measure proteins and protein post-translational modifications in cells and tissues. The approach offers sensitive and precise quantification of large numbers of samples and has thus found applications in the analysis of clinical and pre-clinical samples. For effective integration into drug development and clinical practice, robust assays with consistent results are essential. Leveraging a collaborative RPPA model, we set out to assess the variability between three different RPPA platforms using distinct instrument set-ups and workflows. Employing multiple RPPA-based approaches operated across distinct laboratories, we characterised a range of human breast cancer cells and their protein-level responses to two clinically relevant cancer drugs. We integrated multi-platform RPPA data and used unsupervised learning to identify protein expression and phosphorylation signatures that were not dependent on RPPA platform and analysis workflow. Our findings indicate that proteomic analyses of cancer cell lines using different RPPA platforms can identify concordant profiles of response to pharmacological inhibition, including when using different antibodies to measure the same target antigens. These results highlight the robustness and the reproducibility of RPPA technology and its capacity to identify protein markers of disease or response to therapy.

Additional file of Identification of differentially expressed genes in chickens differing in muscle glycogen content and meat quality

Clinical-grade mesenchymal stromal cells (MSCs) can be expanded from bone marrow and adipose tissue to treat inflammatory diseases and degenerative disorders. However, the influence of their tissue of origin on their functional properties, including their immunosuppressive activity, remains unsolved. In this study, we produced paired bone marrow-derived mesenchymal stromal cell (BM-MSC) and adipose-derived stromal cell (ASC) batches from 14 healthy donors. We then compared them using transcriptomic, phenotypic, and functional analyses and validated our results on purified native MSCs to infer which differences were really endowed by tissue of origin. Cultured MSCs segregated together owing to their tissue of origin based on their gene expression profile analyzed using differential expression and weighted gene coexpression network analysis. This translated into distinct immune-related gene signatures, phenotypes, and functional cell interactions. Importantly, sorted native BM-MSCs and ASCs essentially displayed the same distinctive patterns than their in vitro-expanded counterparts. As a whole, ASCs exhibited an immune profile consistent with a stronger inhibition of immune response and a lower immunogenicity, supporting the use of adipose tissue as a valuable source for clinical applications.

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Additional file 4:Selected real-time RT-PCR primer sequences and accession numbers. (DOC 80 KB)

Authors’ original file for figure 1

Additional file 3:List of the common genes that were differentially expressed in the two models (Fat vs. Lean chickens and G+ vs. G- chickens generated from the F2FL population). (DOC 34 KB)

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/β) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription., Endocytosis of the type I interferon receptor (IFNAR) is required for JAK/STAT signalling. Here the authors show that the internalized IFNAR1 and IFNAR2 subunits are differentially sorted by the retromer complex at the early endosome and this controls JAK/STAT signalling and gene transcription.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a major role in DNA damage signaling and repair and is also frequently overexpressed in tumor metastasis. We used isogenic cell lines expressing different levels of DNA-PKcs to investigate the role of DNA-PKcs in metastatic development. We found that DNA-PKcs participates in melanoma primary tumor and metastasis development by stimulating angiogenesis, migration and invasion. Comparison of conditioned medium content from DNA-PKcs-proficient and deficient cells reveals that DNA-PKcs controls secretion of at least 103 proteins (including 44 metastasis-associated with FBLN1, SERPINA3, MMP8, HSPG2 and the inhibitors of matrix metalloproteinases, such as α-2M and TIMP-2). High throughput analysis of secretomes, proteomes and transcriptomes, indicate that DNA-PKcs regulates the secretion of 85 proteins without affecting their gene expression. Our data demonstrate that DNA-PKcs has a pro-metastatic activity via the modification of the tumor microenvironment. This study shows for the first time a direct link between DNA damage repair and cancer metastasis and highlights the importance of DNA-PKcs as a potential target for anti-metastatic treatment.

Virus domestication is a recurrent and beneficial process in the evolution of parasitic wasps., Relics of ancient infections are abundant in eukaryote genomes, but little is known about how they evolve when they confer a functional benefit on their host. We show here, for the first time, that the virus-like particles shown to protect Venturia canescens eggs against host immunity are derived from a nudivirus genome incorporated by the parasitic wasp into its own genetic material. Nudivirus hijacking was also at the origin of protective particles from braconid wasps. However, we show here that the viral genes produce “liposomes” that wrap and deliver V. canescens virulence proteins, whereas the particles are used as gene transfer agents in braconid wasps. Our findings indicate that virus domestication has occurred repeatedly during parasitic wasp evolution but with different evolutionary trajectories after endogenization, resulting in different virulence molecule delivery strategies.

Cervical cancer (CC) is the fourth most common cause of cancer deaths in women worldwide, for which prognostic and predictive biomarkers are largely lacking. RAIDs is a EU-funded project on cervical cancer that spans seven European countries. The main objective of the RAIDs project is to use this tumor type, which is easily accessible for repeated biopsies, to learn how to stratify patients into targeted therapies. The project includes: 1) a cognitive cohort study (BioRAIDs), one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 400 patients include Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor DNA at Erasmus MC (Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France) and immune-microenvironment analyses at CGOA (Netherlands). In addition, 20 CC cell lines have been profiled pharmacologically using a panel of drugs which potentially synergize with “standard treatment”. The present poster will mainly focus on the Reverse Phase Protein Array (RPPA) results of BioRAIDs and how these relate to the genomic profiling and to patient outcome. More than 150 cryopreserved baseline (before treatment) samples and 23 CC cell lines have been screened by RPPA. From these, whole exome sequencing is available for 92 patient samples. Stratification of patients based on proteomics and genomics has evidenced different subgroups (clusters) of patients displaying specific molecular characteristics. Genomics data and proteomics data both demonstrate that these clusters differ notably in the pathways of oxidative phosphorylation, glycolysis and DNA repair. Patient response to treatment is assessed by the presence or absence of residual tumor at six months after treatment. The correlation of proteomics and genomics data with these clinical data is ongoing and will identify putative predictive biomarkers. Correlation of protein data with response to drugs treatment in 20 CC cell lines has identified several potential biomarkers, some of which again relate to (glucose) metabolism. In conclusion, we here present new evidence for molecular subgroups of cervical cancer that could benefit from different treatment options. Notably, our data suggest that the metabolism/glycolysis pathways are a major effector in CC and constitute a potential therapeutic target in a subset of patients. This project has received funding from the European Union’s Seventh Program for research, technological development and demonstration under grant agreement No 304810. Citation Format: Leanne de Koning, Bérengère Ouine, Aurélie Cartier, Els M. Berns, Kirsten Ruigrok-Ritstier, Corine Beaufort, Balazs Balint, Attila Kereszt, Gemma Kenter, Sanne Samuels, Ekaterina S. Jordanova, Emmanuelle Jeannot, Heiko von der Leyen, Marina Popovic, Windy Luscap-Rondof, Vonick Sibut, Choumouss Kamoun, Isabel Britto, Claudia Rincon, Philippe Hupé, Maud Kamal, Suzy M. Scholl. Rational molecular assessment and innovative drug selection (RAIDs): Paving the way to personalized medicine in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2017-1746

Purpose: Patients with luminal breast cancer (LBC) often become endocrine resistant over time. We investigated the molecular changes associated with acquired hormonoresistances in patient-derived xenografts of LBC. Experimental Design: Two LBC xenografts (HBCx22 and HBCx34) were treated with different endocrine treatments (ET) to obtain xenografts with acquired resistances to tamoxifen (TamR) and ovariectomy (OvaR). PI3K pathway activation was analyzed by Western blot analysis and IHC and responses to ET combined to everolimus were investigated in vivo. Gene expression analyses were performed by RT-PCR and Affymetrix arrays. Results: HBCx22 TamR xenograft was cross-resistant to several hormonotherapies, whereas HBCx22 OvaR and HBCx34 TamR exhibited a treatment-specific resistance profile. PI3K pathway was similarly activated in parental and resistant xenografts but the addition of everolimus did not restore the response to tamoxifen in TamR xenografts. In contrast, the combination of fulvestrant and everolimus induced tumor regression in vivo in HBCx34 TamR, where we found a cross-talk between the estrogen receptor (ER) and PI3K pathways. Expression of several ER-controlled genes and ER coregulators was significantly changed in both TamR and OvaR tumors, indicating impaired ER transcriptional activity. Expression changes associated with hormonoresistance were both tumor and treatment specific and were enriched for genes involved in cell growth, cell death, and cell survival. Conclusions: PDX models of LBC with acquired resistance to endocrine therapies show a great diversity of resistance phenotype, associated with specific deregulations of ER-mediated gene transcription. These models offer a tool for developing anticancer therapies and to investigate the dynamics of resistance emerging during pharmacologic interventions. Clin Cancer Res; 20(16); 4314–25. ©2014 AACR.

Background Recent retrospective data1,2 identified major molecular alterations in cervical cancer (CC), but so far there has been no prospective assessment on patient outcome using a complete molecular profiling with quality control evaluation of treatment. The Cetuxicol (phase 2) clinical trial showed that the addition of Cetuximab over a 6 week period, did not improve DFS. PI3K pathway mutations in the tumor in the Cetuximab treatment arm led to a worse DFS3. We are lacking prognostic and predictive biomarkers for CC treatment and there is a growing need for the development of biomarkers to follow up the course of the disease. Methodology RAIDs (http://www.raids-fp7.eu) is a multidisciplinary co-operation between academic clinical centers, SMEs and translational research platforms in seven European countries. It includes: 1) a cognitive cohort study (BioRAIDs)4, one of the first prospective trials intended to define patient stratification for targeted therapies, 2) a targeted clinical trial using an HPV directed vaccine and 3) preclinical studies aiming at assessing new treatment strategies. Molecular analysis on quality controlled tumor and sera samples from 500 patients enrolled in BioRAIDs combine Next Generation Sequencing at SeqOmics (Hungary), PIK3CA mutations detection in circulating tumor (ct) DNA at ERASMUS (The Netherlands), Reverse Phase Protein array and HPV insertion sites analyses at Institut Curie (France). Bioinformatics pipelines to detect somatic mutations and clustering methods were developed in order to stratify the patients into different subtypes. Following quality control, the pharmacological profiling of a panel of 20 CC cell lines using a panel of drugs which may potentially synergize with “standard treatment” has been completed. Drugs were chosen so as to interfere with different signaling pathways. Results Molecular profiles including exome sequencing and ctDNA analyses on 48 quality controlled samples from the BioRAIDs patients and 20 CC cell lines will be presented. Preliminary results on HPV insertion sites in tumor and serum will be reported. Somatic and DNA copy number alterations from exome sequencing profiles confirm PI3K pathway mutations to be a dominant feature in CC. Stratifications of patients’ tumors based on their mutation profiles show that some cell lines cluster similarly to patients’ tumors, suggesting that they will be helpful for the prediction of response to drugs for patients within the same cluster. Conclusions The identification of predictive tumor/blood based biomarkers will permit the definition of new strategies for precision medicine in CC. A bioinformatics analysis which will assess drug responsiveness in CC cell lines in relation to specific genomic alterations is ongoing. Its results should permit to select treatments according to genetic “constellations” of the tumors. Citation Format: Suzy Scholl, Maud Kamal, Els Berns, Balzs Balint, Attila Kereszt, Leanne de Koning, Emmanuelle Jeannot, Windy Luscap-rondof, Vonick Sibut, Philippe Hupe, Gemma Kenter, Sanne Samuels, Katja Jordanova, Sandrine Blanchet, Laurence Lafanachere, Marc Billaud, RAIDs consortium. Rational molecular assessment and innovative drug selection (RAIDs): exome data from cervical cancer. [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 434.

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