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Thyroid cancer is the most common endocrine malignancy and several genetic events have been described to promote the development of thyroid carcinogenesis. Besides the effects of specific mutations on thyroid cancer development, the molecular mechanisms controlling tumorigenesis, tumor behavior, and drug resistance are still largely unknown. Cancer organoids have been proposed as a powerful tool to study aspects related to tumor development and progression and appear promising to test individual responses to therapies. Here, using mESC-derived thyroid organoids, we developed a BrafV637E-inducible model able to recapitulate the features of papillary thyroid cancer in vitro. Overexpression of BrafV637Erapidly leads to MAPK activation, cell dedifferentiation, and disruption of follicular organization. BrafV637E-expressing organoids show a transcriptomic signature for p53, focal adhesion, ECM-receptor interactions, EMT, and inflammatory signaling pathways. Finally, PTC-like thyroid organoids were used for drug screening assays. The combination of MAPK and PI3K inhibitors reversedBrafoncogene-promoted cell dedifferentiation while restoring thyroid follicle organization and functionin vitro. Our results demonstrate that pluripotent stem cells-derived thyroid cancer organoids can mimic tumor development and features while providing an efficient tool for testing novel targeted therapies.

Supplementary Figure 1 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Supplementary Materials and Methods from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Supplementary Figure 3 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Supplementary Figure 4 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Supplementary Table 3 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Supplementary Figure 2 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Supplementary Table 1 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Cell lines are crucial to elucidate mechanisms of tumorigenesis and serve as tools for cancer treatment screenings. Therefore, careful validation of whether these models have conserved properties of in vivo tumors is highly important. Thyrocyte-derived tumors are very interesting for cancer biology studies because from one cell type, at least five histologically characterized different benign and malignant tumor types can arise. To investigate whether thyroid tumor–derived cell lines are representative in vitro models, characteristics of eight of those cell lines were investigated with microarrays, differentiation markers, and karyotyping. Our results indicate that these cell lines derived from differentiated and undifferentiated tumor types have evolved in vitro into similar phenotypes with gene expression profiles the closest to in vivo undifferentiated tumors. Accordingly, the absence of expression of most thyrocyte-specific genes, the nonresponsiveness to thyrotropin, as well as their large number of chromosomal abnormalities, suggest that these cell lines have acquired characteristics of fully dedifferentiated cells. They represent the outcome of an adaptation and evolution in vitro, which questions the reliability of these cell lines as models for differentiated tumors. However, they may represent useful models for undifferentiated cancers, and by their comparison with differentiated cells, can help to define the genes involved in the differentiation/dedifferentiation process. The use of any cell line as a model for a cancer therefore requires prior careful and thorough validation for the investigated property. [Cancer Res 2007;67(17):8113–20]

Supplementary Table 2 from Human Thyroid Tumor Cell Lines Derived from Different Tumor Types Present a Common Dedifferentiated Phenotype

Simple Summary Cancer heterogeneity, which occurs in most cancer patients, has been known and studied by experienced pathologists since the early 19th century. Intratumoral heterogeneity between cancer cells can arise from complex genetic, epigenetic, and metabolic modifications under the influence of the tumor microenvironment and confers considerable adaptability to tumors. Despite major advances in cancer therapy, tumoral heterogeneity remains a major obstacle to the successful treatment of cancer. In this review, we highlight the role of intratumoral heterogeneity, focusing on the clinical and biological implications of this phenomenon. Abstract Though heterogeneity of cancers is recognized and has been much discussed in recent years, the concept often remains overlooked in different routine examinations. Indeed, in clinical or biological articles, reviews, and textbooks, cancers and cancer cells are generally presented as evolving distinct entities rather than as an independent heterogeneous cooperative cell population with its self-oriented biology. There are, therefore, conceptual gaps which can mislead the interpretations/diagnostic and therapeutic approaches. In this short review, we wish to summarize and discuss various aspects of this dynamic evolving heterogeneity and its biological, pathological, clinical, diagnostic, and therapeutic implications, using thyroid carcinoma as an illustrative example.

The function of the thyroid gland is to capture iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans which is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, there is a need for new therapeutic approaches that involve replacement of thyroid tissue responsive to changing demands for thyroid hormone. Here we report the first transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone to athyreotic mice as a proof of concept for future therapeutic development.

Our expanding knowledge of the interactions between tumor cells and their microenvironment has helped to revolutionize cancer treatments, including the more recent development of immunotherapies. Immune cells are an important component of the tumor microenvironment that influence progression and treatment responses, particularly to the new immunotherapies. Technological advances that help to decipher the complexity and diversity of the tumor immune microenvironment (TIME) are increasingly used in translational research and biomarker studies. Current techniques that facilitate TIME evaluation include flow cytometry, multiplex bead-based immunoassays, chromogenic immunohistochemistry (IHC), fluorescent multiplex IHC, immunofluorescence, and spatial transcriptomics. This article offers an overview of our representative data, discusses the application of each approach to studies of the TIME, including their advantages and challenges, and reviews the potential clinical applications. Flow cytometry and chromogenic and fluorescent multiplex IHC were used to immune profile a HER2+ breast cancer, illustrating some points. Spatial transcriptomic analysis of a luminal B breast tumor demonstrated that important additional insight can be gained from this new technique. Finally, the development of a multiplex panel to identify proliferating B cells, Tfh, and Tfr cells on the same tissue section demonstrates their co-localization in tertiary lymphoid structures.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover heterogeneity in the thyrocyte population and molecularly characterize the non‐thyrocyte cells surrounding the follicle, we developed a single‐cell transcriptome atlas of the region containing the zebrafish thyroid gland. The 6249‐cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells, and fibroblasts. Further, the thyrocytes show expression heterogeneity, including bimodal expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9‐based pax2a knock‐in line that monitors pax2a expression in the thyrocytes. A population of pax2a‐low mature thyrocytes interspersed in individual follicles can be distinguished. We corroborate heterogeneity within the thyrocyte population using RNA sequencing of pax2a‐high and pax2a‐low thyrocytes, which demonstrates 20% differential expression in transcriptome between the two subpopulations. Our results identify and validate transcriptional differences within the presumed homogenous thyrocyte population.

The vast majority of thyroid cancers originate from follicular cells. We outline outstanding issues at each step along the path of cancer patient care, from prevention to post-treatment follow-up and highlight how emerging technologies will help address them in the coming years. Three directions will dominate the coming technological landscape. Genomics will reveal tumoral evolutionary history and shed light on how these cancers arise from the normal epithelium and the genomics alteration driving their progression. Transcriptomics will gain cellular and spatial resolution providing a full account of intra-tumor heterogeneity and opening a window on the microenvironment supporting thyroid tumor growth. Artificial intelligence will set morphological analysis on an objective quantitative ground laying the foundations of a systematic thyroid tumor classification system. It will also integrate into unified representations the molecular and morphological perspectives on thyroid cancer.

The advent of next generation sequencing technologies has boosted the interest in exploring the role of fusion genes in the development and progression of solid tumors. In breast cancer, most of the detected gene fusions seem to be "passenger" events while the presence of recurrent and driver fusions is still under study. We performed RNA sequencing in 55 well-characterized breast cancer samples and 10 adjacent normal breast tissues, complemented by an analysis of SNP array data. We explored the presence of fusion genes and defined their association with breast cancer subtypes, clinical-pathologic characteristics and copy number aberrations. Overall, 370 fusions were detected across the majority of the samples. HER2+ samples had significantly more fusions than triple negative and luminal subtypes. The number of fusions was correlated with histological grade, Ki67 and tumor size. Clusters of fusion genes were observed across the genome and a significant correlation of fusions with copy number aberrations and more specifically amplifications was also revealed. Despite the large number of fusion events, only a few were recurrent, while recurrent individual genes forming fusions with different partners were also detected including the estrogen receptor 1 gene in the previously detected ESR1-CCDC170 fusion. Overall we detected novel gene fusion events while we confirmed previously reported fusions. Genomic hotspots of fusion genes, differences between subtypes and small number of recurrent fusions are the most relevant characteristics of these events in breast cancer. Further investigation is necessary to comprehend the biological significance of these fusions.

This abstract was withdrawn by the authors.

The thyroid gland regulates growth and metabolism via production of thyroid hormone in follicles composed of thyrocytes. So far, thyrocytes have been assumed to be a homogenous population. To uncover genetic heterogeneity in the thyrocyte population, and molecularly characterize the non-thyrocyte cells surrounding the follicle, we developed a single-cell transcriptome atlas of the zebrafish thyroid gland. The 6249-cell atlas includes profiles of thyrocytes, blood vessels, lymphatic vessels, immune cells and fibroblasts. Further, the thyrocytes could be split into two sub-populations with unique transcriptional signature, including differential expression of the transcription factor pax2a. To validate thyrocyte heterogeneity, we generated a CRISPR/Cas9-based pax2a knock-in line, which demonstrated specific pax2a expression in the thyrocytes. However, a population of pax2a-low mature thyrocytes interspersed within individual follicles could be distinguished, corroborating heterogeneity within the thyrocyte population. Our results identify and validate transcriptional differences within the nominally homogenous thyrocyte population., info:eu-repo/semantics/published

C. Maenhaut and V. Detours contributed equally, info:eu-repo/semantics/published

Background: The early molecular events in human thyrocytes after 131I exposure have not yet been unravelled. Therefore, we investigated the role of TSH in the 131I-induced DNA damage response and gene expression in primary cultured human thyrocytes. Methods: Following exposure of thyrocytes, in the presence or absence of TSH, to 131I (β radiation), γradiation (3 Gy), and hydrogen peroxide (H2O2), we assessed DNA damage, proliferation, and cell-cycle status. We conducted RNA sequencing to profile gene expression after each type of exposure and evaluated the influence of TSH on each transcriptomic response. Results: Overall, the thyrocyte responses following exposure to β or γradiation and to H2O2 were similar. However, TSH increased 131I-induced DNA damage, an effect partially diminished after iodide uptake inhibition. Specifically, TSH increased the number of DNA double-strand breaks in nonexposed thyrocytes and thus predisposed them to greater damage following 131I exposure. This effect most likely occurred via Gα?q cascade and a rise in intracellular reactive oxygen species (ROS) levels. β and γradiation prolonged thyroid cell-cycle arrest to a similar extent without sign of apoptosis. The gene expression profiles of thyrocytes exposed to β/γradiation or H2O2 were overlapping. Modulations in genes involved in inflammatory response, apoptosis, and proliferation were observed. TSH increased the number and intensity of modulation of differentially expressed genes after 131I exposure. Conclusions: TSH specifically increased 131I-induced DNA damage probably via a rise in ROS levels and produced a more prominent transcriptomic response after exposure to 131I., SCOPUS: ar.j, info:eu-repo/semantics/published

As in many cancer types, miRNA expression profiles and functions have become an important field of research on non-medullary thyroid carcinomas, the most common endocrine cancers. This could lead to the establishment of new diagnostic tests and new cancer therapies. However, different studies showed important variations in their research strategies and results. In addition, the action of miRNAs is poorly considered as a whole because of the use of underlying dogmatic truncated concepts. These lead to discrepancies and limits rarely considered. Recently, this field has been enlarged by new miRNA functional and expression studies. Moreover, studies using next generation sequencing give a new view of general miRNA differential expression profiles of papillary thyroid carcinoma. We analyzed in detail this literature from both physiological and differential expression points of view. Based on explicit examples, we reviewed the progresses but also the discrepancies and limits trying to provide a critical approach of where this literature may lead. We also provide recommendations for future studies. The conclusions of this systematic analysis could be extended to other cancer types., SCOPUS: re.j, Hydra, info:eu-repo/semantics/published

Spatial transcriptomics measures mRNA at hundreds of 100 micrometer-diameter spots evenly spread across 6.5×6.9 mm2 histological slices. Gene expression within each spot is commonly normalized by total read counts. However we show that the transcriptional output of individual spots reflects the number of cells they contain, hence total read counts per spot reflect relevant biology. Although per-spot read-count normalization reveals important enrichment trends, it may heavily distort cell-type-related absolute local expression and conceal important biological information.

This study investigated the prevalence of TIL subpopulations, TLS, PD-1 and PD-L1 in tumors from TNBC patients harboring wild-type or mutated BRCA1 or BRCA2 germline genes. This TNBC cohort included 85% TIL-positive (≥10%) tumors with 21% classified as TILhi (≥50%). Interestingly, the BRCAmut group had a significantly higher incidence of TILpos tumors compared to the BRCAwt group (P = 0.037). T cells were dominant in the infiltrate but no statistically significant differences were detected between BRCAwt and BRCAmut for CD3+, CD4+ and CD8+ T cells or CD20+ B cells. TLS were detected in 74% of tumors but again no significant differences between the BRCA groups. PD-1 expression was observed in 33% and PD-L1 in 53% (any cell, cut-off ≥1%) tumors for the entire TNBC cohort. PD-1 expression correlated with PD-L1 and both with TIL and TLS but was not associated with BRCA mutational status. Our analyses reveal that BRCAwt and BRCAmut TNBC are similar except for a significant increase of TILpos tumors in the BRCAmut group. While BRCA gene mutations may not directly drive immune infiltration, the greater number of TILpos tumors could signal greater immunogenicity in this group.

We studied the mechanism that may explain the relative resistance of thyrocytes to H2O2 compared to other cell types. Ability to degrade H2O2, glutathione peroxidase (GPx) activity, heme oxygenase-1 (HO-1) expression, cell survival and capacity to repair DNA damage after H2O2 exposure or irradiation were measured in human thyrocytes in primary culture and compared to the values obtained in human T-cells and different cell lines. Compared to other cell types, thyrocytes presented a low mortality rate after H2O2 exposure, rapidly degraded extracellular H2O2 and presented a high basal seleno-dependent GPx activity. Only in thyrocytes, H2O2 up-regulated GPx activity and expression of HO-1 mRNA. These effects were not reproduced by irradiation. DNA damage caused by H2O2 was more slowly repaired than that caused by irradiation and not repaired at all in T-cells. Our study demonstrates that the thyrocyte has specific protective mechanisms against H2O2 and its mutagenic effects., SCOPUS: ar.j, info:eu-repo/semantics/published

The cerebral cortex underwent rapid expansion and increased complexity during recent hominid evolution. Gene duplications constitute a major evolutionary force, but their impact on human brain development remains unclear. Using tailored RNA sequencing (RNA-seq), we profiled the spatial and temporal expression of hominid-specific duplicated (HS) genes in the human fetal cortex and identified a repertoire of 35 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among them NOTCH2NL, human-specific paralogs of the NOTCH2 receptor, stood out for their ability to promote cortical progenitor maintenance. NOTCH2NL promote the clonal expansion of human cortical progenitors, ultimately leading to higher neuronal output. At the molecular level, NOTCH2NL function by activating the Notch pathway through inhibition of cis Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes active during human corticogenesis and reveals how human-specific NOTCH paralogs may have contributed to the expansion of the human cortex. Human-specific NOTCH2NL expands cortical progenitors and neuronal output and thus may have contributed to the expansion of the human cortex., SCOPUS: ar.j, info:eu-repo/semantics/published

Summary Little is known about how RNA editing operates in cancer. Transcriptome analysis of 68 normal and cancerous breast tissues revealed that the editing enzyme ADAR acts uniformly, on the same loci, across tissues. In controlled ADAR expression experiments, the editing frequency increased at all loci with ADAR expression levels according to the logistic model. Loci-specific “editabilities,” i.e., propensities to be edited by ADAR, were quantifiable by fitting the logistic function to dose-response data. The editing frequency was increased in tumor cells in comparison to normal controls. Type I interferon response and ADAR DNA copy number together explained 53% of ADAR expression variance in breast cancers. ADAR silencing using small hairpin RNA lentivirus transduction in breast cancer cell lines led to less cell proliferation and more apoptosis. A-to-I editing is a pervasive, yet reproducible, source of variation that is globally controlled by 1q amplification and inflammation, both of which are highly prevalent among human cancers., Graphical Abstract, Highlights • A-to-I editing is a major source of mRNA variability in breast and other cancers • RNA editing is globally controlled by tumor interferon and ADAR copy number • Both these factors are highly prevalent among human cancers • RNA editing sites might represent a new class of therapeutic targets, Fumagalli et al. identify the principles governing A-to-I editing in breast and potentially all types of cancer, demonstrating that A-to-I editing is a pervasive source of transcriptome variation that is mainly controlled by two factors, 1q amplification and inflammation, both of which are highly prevalent among human cancers.

Transcriptome profiling has helped characterise nodal spread. The interpretation of these data, however, is not without ambiguities., C. Maenhaut and V. Detours contributed equally, info:eu-repo/semantics/published

The contribution of intratumor heterogeneity to thyroid metastatic cancers is still unknown. The clonal relationships between the primary thyroid tumors and lymph nodes (LN) or distant metastases are also poorly understood. The objective of this study was to determine the phylogenetic relationships between matched primary thyroid tumors and metastases. We searched for non-synonymous single-nucleotide variants (nsSNVs), gene fusions, alternative transcripts, and loss of heterozygosity (LOH) by paired-end massively parallel sequencing of cDNA (RNA-Seq) in a patient diagnosed with an aggressive papillary thyroid cancer (PTC). Seven tumor samples from a stage IVc PTC patient were analyzed by RNA-Seq: two areas from the primary tumor, four areas from two LN metastases, and one area from a pleural metastasis (PLM). A large panel of other thyroid tumors was used for Sanger sequencing screening. We identified seven new nsSNVs. Some of these were early events clonally present in both the primary PTC and the three matched metastases. Other nsSNVs were private to the primary tumor, the LN metastases and/or the PLM. Three new gene fusions were identified. A novel cancer-specific KAZN alternative transcript was detected in this aggressive PTC and in dozens of additional thyroid tumors. The PLM harbored an exclusive whole-chromosome 19 LOH. We have presented the first, to our knowledge, deep sequencing study comparing the mutational spectra in a PTC and both LN and distant metastases. This study has yielded novel findings concerning intra-tumor heterogeneity, clonal evolution and metastases dissemination in thyroid cancer.

SummaryThe human cerebral cortex has undergone rapid expansion and increased complexity during recent evolution. Hominid-specific gene duplications represent a major driving force of evolution, but their impact on human brain evolution remains unclear. Using tailored RNA sequencing (RNAseq), we profiled the spatial and temporal expression of Hominid-specific duplicated (HS) genes in the human fetal cortex, leading to the identification of a repertoire of 36 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among these we focused on NOTCH2NL, previously uncharacterized HS paralogs of NOTCH2. NOTCH2NL promote the clonal expansion of human cortical progenitors by increasing self-renewal, ultimately leading to higher neuronal output. NOTCH2NL function by activating the Notch pathway, through inhibition of Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes linking genomic evolution to human brain development, and reveals how hominin-specific NOTCH paralogs may have contributed to the expansion of the human cortex.

Context Although 60% of papillary thyroid carcinomas are BRAFV600E mutant (PTCV600E), the increased aggressiveness of these cancers is still debated. Objective For PTCV600E we aimed to further characterize the extent of the stroma and its activation, the three-dimensional (3D) tumor-stroma interface, and the proliferation rates of tumor and stromal fibroblasts. Design We analyzed exomes, transcriptomes, and images of 364 papillary thyroid carcinoma (PTCs) from The Cancer Genome Atlas (TCGA), including 211 PTCV600E; stained 22 independent PTCs for BRAFV600E and Ki67; sequenced the exomes and stained BRAFV600E in 5 primary tumor blocks and 4 nodal metastases from one patient with PTCV600E; and reconstructed the 3D volumes of one tumor and one metastatic block at histological resolution. Results In TCGA, BRAFV600E was associated with higher expression of proliferation markers and lower expression of thyroid differentiation markers, independently of tumor purity. Moreover, PTCV600E, in line with their overall lower purity, also had higher expression of fibroblast- and T cell-associated genes and presented more fibrosis. Tumor cells that appeared disconnected on two-dimensional histological slices were revealed to be part of a unique tumor component in the 3D reconstructed microvolumes, and they formed a surprisingly complex connected space, infiltrating a proliferative stroma. Finally, in our PTC set, both stromal fibroblasts and tumor cells presented higher proliferation rates in PTCV600E. Conclusions Our results support the increased aggressiveness associated with BRAFV600E in PTC and shed light on the important role of the stroma in tumor expansion. The greater and more active fibrotic component predicts better efficiency of combined targeted treatments, as previously proposed for melanomaV600E.

Background:Papillary thyroid cancer (PTC) incidence increased dramatically in children after the Chernobyl accident, providing a unique opportunity to investigate the molecular features of radiation-induced thyroid cancer. In contrast to the previous studies that included age-related confounding factors, we investigated mRNA expression in PTC and in the normal contralateral tissues of patients exposed and non-exposed to the Chernobyl fallout, using age- and ethnicity-matched non-irradiated cohorts.Methods:Forty-five patients were analysed by full-genome mRNA microarrays. Twenty-two patients have been exposed to the Chernobyl fallout; 23 others were age-matched and resident in the same regions of Ukraine, but were born after 1 March 1987, that is, were not exposed to (131)I.Results:A gene expression signature of 793 probes corresponding to 403 genes that permitted differentiation between normal tissues from patients exposed and from those who were not exposed to radiation was identified. The differences were confirmed by quantitative RT-PCR. Many deregulated pathways in the exposed normal tissues are related to cell proliferation.Conclusion:Our results suggest that a higher proliferation rate in normal thyroid could be related to radiation-induced cancer either as a predisposition or as a consequence of radiation. The signature allows the identification of radiation-induced thyroid cancers.British Journal of Cancer advance online publication, 24 July 2012; doi:10.1038/bjc.2012.302 www.bjcancer.com., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Differentiation is central to development, while dedifferentiation is central to cancer progression. Hence, a quantitative assessment of differentiation would be most useful. We propose an unbiased method to derive organ-specific differentiation indices from gene expression data and demonstrate its usefulness in thyroid cancer diagnosis. We derived a list of thyroid-specific genes by selecting automatically those genes that are expressed at higher level in the thyroid than in any other organ in a normal tissue's genome-wide gene expression compendium. The thyroid index of a tissue was defined as the median expression of these thyroid-specific genes in that tissue. As expected, the thyroid index was inversely correlated with meta-PCNA, a proliferation metagene, across a wide range of thyroid tumors. By contrast, the two indices were positively correlated in a time course of thyroid-stimulating hormone (TSH) activation of primary thyrocytes. Thus, the thyroid index captures biological information not integrated by proliferation rates. The differential diagnostic of follicular thyroid adenomas and follicular thyroid carcinoma is a notorious challenge for pathologists. The thyroid index discriminated them as accurately as did machine-learning classifiers trained on the genome-wide cancer data. Hence, although it was established exclusively from normal tissue data, the thyroid index integrates the relevant diagnostic information contained in tumoral transcriptomes. Similar results were obtained for the classification of the follicular vs classical variants of papillary thyroid cancers, that is, tumors dedifferentiating along a different route. The automated procedures demonstrated in the thyroid are applicable to other organs.

INTRODUCTION: RNA editing is a post-transcriptional modification that changes double stranded RNA sequences (dsRNA). The most common way of editing in humans is the Adenosine-to-Inosine type, which is catalyzed by members of the ADAR enzymes family. A-to-I RNA editing has been shown to be essential for normal tissue development but also plays a crucial role in many cancers. We have notably shown that in breast cancer the editing frequency is increased in tumors as compared to normal tissue and correlates with ADAR expression. We also showed that type I IFN response and ADAR DNA copy number together explain 53% of ADAR expression in breast cancers. Moreover, the implication of ADAR-mediated RNA editing in preventing the sensing of endogenous dsRNA leading to inhibition of the immune response has been recently reported. ADAR-mediated RNA editing may play a crucial role in tumor progression by modulating immune response through the dsRNA sensing pathway. Here, we aim to assess the influence of ADAR-mediated RNA editing in the sensing of endogenous dsRNA and the immune response in breast cancer. MATERIAL & METHODS: ADAR expression was modulated in MDAMB231 breast cancer cell line using shRNA lentiviral transduction and lentiviral plasmid transduction for ADAR inhibition and ADAR wild-type and editing-deficient mutant overexpression respectively. ADAR expression was assessed by RTqPCR/Western blotting. dsRNA level was evaluated by immunofluorescence using the dsRNA-specific J2 antibody (Scicons). RESULTS: Poly I:C transfection, mimicking dsRNAs, as well as IFN Type I exposure induced the immune response in BC cell lines, in particular it increases the expression of RIG-I, MDA5 and IRF7 involved in the dsRNA sensing pathway. DNMTi treatment, known as an inducer of dsRNA level, also activated cytosolic dsRNA response in these cells in a dose dependent manner. In order to assess the role of ADAR-mediated RNA editing in the activation of the dsRNA sensing pathway, we exposed MDAMB231 cell lines in which ADAR expression was modulated to DNMTi. A wide range of doses of DNMTi, from 0.3 to 50μM, and time of exposure, from 3h to 72h, were tested. The cytosolic dsRNA pathway was induced in a dose dependent manner after DNMTi exposure. However, no effect of ADAR modulation on the activation of the pathway was observed. No difference in dsRNA levels was observed after activation of the immune pathway by DNMTi or IFN exposure, nor after ADAR modulation. CONCLUSION & PERSPECTIVES: dsRNA pathway activation by DNMTi, poly I:C transfection or IFN Type I treatment is not dependent of ADAR in MDAMB231 cell lines. Moreover, in contrast to data in colorectal cell lines, our data suggest that DNMTi exposure does not increase cytosolic dsRNA level. Citation Format: Floriane Dupont, Florian Clatot, David Venet, Véronique Kruys, Debora Fumagalli, Vincent Detours, Françoise Rothé, Christos Sotiriou. Functional relevance of A-to-I RNA editing on the immune response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3801.

The purpose of this paper is to correlate the molecular phenotype of papillary thyroid carcinoma (PTC) to their biological pathology. We hybridized 26 PTC on microarrays and showed that nearly 44% of the transcriptome was regulated in these tumors. We then combined our data set with two published PTC microarray studies to produce a platform- and study-independent list of PTC-associated genes. We further confirmed the mRNA regulation of 15 genes from this list by quantitative reverse transcription-PCR. Analysis of this list with statistical tools led to several conclusions: (1) there is a change in cell population with an increased expression of genes involved in the immune response, reflecting lymphocyte infiltration in the tumor compared to the normal tissue. (2) The c-jun N-terminal kinase pathway is activated by overexpression of its components. (3) The activation of ERKK1/2 by genetic alterations is supplemented by activation of the epidermal growth factor but not of the insulin-like growth factor signaling pathway. (4) There is a downregulation of immediate early genes. (5) We observed an overexpression of many proteases in accordance with tumor remodeling, and suggested a probable role of S100 proteins and annexin A2 in this process. (6) Numerous overexpressed genes favor the hypothesis of a collective migration mode of tumor cells.

Background: Papillary Thyroid Cancer (PTC) is the most prevalent type of endocrine cancer. Its incidence has rapidly increased in recent decades but little is known regarding its complete microRNA transcriptome (miRNome). In addition, there is a need for molecular biomarkers allowing improved PTC diagnosis. Methods: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deepsequencing of 59 normal samples, 495 PTC, and 8 LNM). Results: We performed small RNA deep-sequencing of 3 PTC, their matching normal tissues and lymph node metastases (LNM). We designed a new bioinformatics framework to handle each aspect of the miRNome: whole expression profiles, isomiRs distribution, non-templated additions distributions, RNA-editing or mutation. Results were validated experimentally by qRT-PCR on normal samples, tumors and LNM from 14 independent patients and in silico using the dataset from The Cancer Genome Atlas (small RNA deep-sequencing of 59 normal samples, 495 PTC, and 8 LNM). We confirmed already described up-regulations of microRNAs in PTC, such as miR-146b-5p or miR-222-3p, but we also identified down-regulated microRNAs, such as miR-7-5p or miR-30c-2-3p. We showed that these down-regulations are linked to the tumorigenesis process of thyrocytes. We selected the 14 most down-regulated microRNAs in PTC and we showed that they are potential biomarkers of PTC samples. Nevertheless, they can distinguish histological classical variants and follicular variants of PTC in the TCGA dataset. In addition, 12 of the 14 down-regulated microRNAs are significantly less expressed in aggressive PTC compared to non-aggressive PTC. We showed that the associated aggressive expression profile is mainly due to the presence of the BRAF V600E mutation. In general, primary tumors and LNM presented similar microRNA expression profiles but specific variations like the down-regulation of miR-7-2-3p and miR-30c-2-3p in LNM were observed. Investigations of the 5p-to-3p arm expression ratios, non-templated additions or isomiRs distributions revealed no major implication in PTC tumorigenesis process or LNM appearance. Conclusions: Our results showed that down-regulated microRNAs can be used as new potential common biomarkers of PTC and to distinguish main subtypes of PTC. MicroRNA expressions can be linked to the development of LNM of PTC. The bioinformatics framework that we have developed can be used as a starting point for the global analysis of any microRNA deep-sequencing data in an unbiased way., SCOPUS: ar.j, info:eu-repo/semantics/published

Tomasetti and Vogelstein (Science 347, 78–81, 2015) claimed that “primary prevention measures are not likely to be very effective” for many cancers because they arise mostly from random mutations fixed during stem cell division, independently of specific genetic or environmental factors. We demonstrate that their calculation for hepatocellular carcinomas overlooked a major subset of tumors proven to be preventable through vaccination. The problem, which is not limited to hepatocellular carcinoma, arises from the general reliance of their analysis on average USA incidences and the omission of incidences in specific risk groups.

Background: Studies evaluating the presence of viral sequences in breast cancer (BC), including various strains of human papillomavirus and human herpes virus, have yielded conflicting results. Most were based on RT-PCR and in situ hybridization. Methods: In this report we searched for expressed viral sequences in 58 BC transcriptomes using five distinct in silico methods. In addition, we complemented our RNA sequencing results with exome sequencing, PCR and immunohistochemistry (IHC) analyses. A control sample was used to test our in silico methods. Results: All of the computational methods correctly detected viral sequences in the control sample. We identified a small number of viral sequences belonging to human herpesvirus 4 and 6 and Merkel cell polyomavirus. The extremely low expression levels-two orders of magnitude lower than in a typical hepatitis B virus infection in hepatocellular carcinoma-did not suggest active infections. The presence of viral elements was confirmed in sample-matched exome sequences, but could not be confirmed by PCR or IHC. Conclusions: Our results show that no viral sequences are expressed in significant amounts in the BC investigated. The presence of non-transcribed viral DNA cannot be excluded., SCOPUS: ar.j, Hydra, info:eu-repo/semantics/published