Part 1: The PIWI-piRNA Pathway Is an Immune-Like Surveillance Process That Controls Genome Integrity by Silencing Transposable Elements

PiRNAs [P-element-induced wimpy testis (PIWI)-interacting RNAs] represent the most frequent but the least well-investigated subtype of small ncRNAs and are characterized by their interaction with PIWI proteins, a subclass of the Argonaute family. PiRNAs and PIWI proteins maintain integrity of the genomic structure and regulate gene expression in germline and somatic cells. The PIWI-piRNA pathway primarily constitutes a conserved immune-like surveillance process that recognizes self and nonself. This axis controls genome integrity of germline cells and nonaging somatic cells by silencing and suppressing propagation of transposable elements through epigenetic and posttranscriptional mechanisms. However, mounting evidences indicate that the PIWI-piRNA pathway has broader implications in both germinal and somatic cells in various physiological and pathological processes. It modulates mRNAs levels of expression, stability, turnover, and translation and interacts directly with many transcription factors and signaling pathways molecules. PIWI proteins and piRNAs play pivotal roles in germline stem cell maintenance and self-renewal, fertilization and development, genes and proteins expression, genome rearrangement, and homeostasis

Part 2: Deregulated Expressions of PIWI Proteins and piRNAs as New Candidate Biomarkers and Potential Therapeutic Tools in Cancer

Epigenetic abnormalities are early events in carcinogenesis and associate heterogeneity of DNA methylation, modifications of histones, and deregulation of noncoding RNAs. Aberrant expressions of PIWI proteins and piRNAs were recently observed in numerous subtypes of malignant tumors and were implicated in occurrence of most cancer hallmarks such as cell proliferation, genomic stability, apoptosis inhibition, invasion, and metastatic spread. However, this pathway is a new emerging research field, and further investigations are necessary to elucidate their oncogenic or tumor-suppressing status. Since the aberrant expression of this pathway may induce stemness, analysis of relationship between PIWI proteins, piRNAs, and cancer stem cells may open new avenues in cancer research. The objective of this review is to provide a broad overview of the emerging implication of PIWI proteins and piRNAs in carcinogenesis and their potential clinical interest as diagnostic and prognostic biomarkers and therapeutic tools

Significance of Tumor Microenvironment Scoring and Immune Biomarkers in Patient Stratification and Cancer Outcomes

Tumors appear as heterogeneous tissues that consist of tumor cells surrounding by a tumor microenvironment (TME). TME is a complex network composed of extracellular matrix (ECM), stromal cells, and immune/inflammatory cells that drive cancer cells fate from invasion to intravasation and metastasis. The stromal-inflammatory interface represents a dynamic space, in which exchange of numerous molecular information is associated with the transition into tumorigenic microenvironment. Recruitment, activation, and reprogramming of stromal and immune/inflammatory cells in the extracellular space are the consequences of a reciprocal interaction between TME and cancer cells. Recent data suggest that cancer development is influenced by TME and controlled by the host’s immune system, underlying the importance of TME components and immune biomarkers in the determination of prognosis and response to therapy. The immune classification has prognostic value and may be a useful supplement to the histopathological, molecular, and TNM classifications. Nevertheless, the complexity of quantitative immunohistochemistry and the variable assay protocols, stromal and immune cell types analyzed underscore the need to harmonize the quantified methods. It is therefore important to incorporate TME and immune scoring in determinations of cancer prognosis and to make sure they become a routine part of the histopathological diagnostic and prognostic assessment of patients

Single-cell transcriptomics reveals shared immunosuppressive landscapes of mouse and human neuroblastoma

BACKGROUND High-risk neuroblastoma is a pediatric cancer with still a dismal prognosis, despite multimodal and intensive therapies. Tumor microenvironment represents a key component of the tumor ecosystem the complexity of which has to be accurately understood to define selective targeting opportunities, including immune-based therapies. METHODS We combined various approaches including single-cell transcriptomics to dissect the tumor microenvironment of both a transgenic mouse neuroblastoma model and a cohort of 10 biopsies from neuroblastoma patients, either at diagnosis or at relapse. Features of related cells were validated by multicolor flow cytometry and functional assays. RESULTS We show that the immune microenvironment of MYCN-driven mouse neuroblastoma is characterized by a low content of T cells, several phenotypes of macrophages and a population of cells expressing signatures of myeloid-derived suppressor cells (MDSCs) that are molecularly distinct from the various macrophage subsets. We document two cancer-associated fibroblasts (CAFs) subsets, one of which corresponding to CAF-S1, known to have immunosuppressive functions. Our data unravel a complex content in myeloid cells in patient tumors and further document a striking correspondence of the microenvironment populations between both mouse and human tumors. We show that mouse intratumor T cells exhibit increased expression of inhibitory receptors at the protein level. Consistently, T cells from patients are characterized by features of exhaustion, expressing inhibitory receptors and showing low expression of effector cytokines. We further functionally demonstrate that MDSCs isolated from mouse neuroblastoma have immunosuppressive properties, impairing the proliferation of T lymphocytes. CONCLUSIONS Our study demonstrates that neuroblastoma tumors have an immunocompromised microenvironment characterized by dysfunctional T cells and accumulation of immunosuppressive cells. Our work provides a new and precious data resource to better understand the neuroblastoma ecosystem and suggest novel therapeutic strategies, targeting both tumor cells and components of the microenvironment

Evaluation of The Roles of lncRNAs in Breast Carcinomas

Le cancer du sein représente à l’échelle mondiale le deuxième cancer le plus fréquent et la première des tumeurs malignes de la femme. Actuellement, seuls certains biomarqueurs (RO, RP, récepteur HER2, index Ki67) et la signature transcriptomique PAM50 sont pris en compte dans la classification morphologique et l’orientation thérapeutique. Les analyses transcriptomiques à haut débit ont révélé que plus de 80% du génome humain est transcrit en ARN. Parmi les ARNs non codants, les transcrits dont la longueur est supérieure à 200 nt sont arbitrairement qualifiés de longs ARNs non codants (lncRNAs). Les lncRNAs jouent un rôle crucial dans le maintien de l’homéostasie cellulaire et présentent des profils d’expression anormaux dans diverses pathologies, dont le cancer. L’objectif principal de mon projet de thèse a consisté à analyser l’expression des lncRNAs, leur fonctionnalité et leur rôle dans l’oncogénèse mammaire. La première partie s’est focalisée sur l’étude des gènes ANRIL (ainsi que 10 gènes de la même voie de signalisation) et MALAT1, deux lncRNAs dont les mécanismes d’action et la signification clinique au cours de la cancérogénèse mammaire sont encore controversés. ANRIL et MALAT1 sont respectivement surexprimés dans 20% et 14% des tumeurs de notre série, confirmant leurs rôles pro-oncogénique dans la cancérogénèse mammaire. La surexpression de MALAT1 se traduit en RNA-FISH par la présence de volumineux speckles intranucléaires. La complexité de leur dérégulation est liée à la présence d’isoformes et de réseaux d’interactions avec les mRNAs et les miRNAs. Concernant les sous-unités appartenant aux complexes Polycomb PRC2 et PRC1qui interagissent avec ANRIL, EZH2 (PRC2) est normalement ciblé par 3 miRNAs onco-suppresseurs (miR-26A1, miR-125B et miR-214) qui sont sous-exprimés dans notre série de CCIs. Les 2 oncomiRs miR-181B1 and miR-181A2 qui ciblent et inactivent CBX7 (PRC1), apparaissent surexprimés dans notre série, en relation avec l’activation de l’oncogène HMGA1. Concernant MALAT1, le complexe de biogénèse des miRNAs Drosha-DGCR8-Microprocesseur régule l’expression d’un variant d’épissage ∆-MALAT1 et ce dernier est impliqué dans l’activation de la voie PI3K/Akt. Des corrélations significatives sont observées entre MALAT1 et des gènes impliqués dans l’épissage alternatif, le cycle cellulaire, l’apoptose, la réparation de l’ADN et la migration cellulaire. Les profils transcriptomiques aberrants de ces 2 lncRNAs semblent caractéristiques des carcinomes mammaires. Ainsi, ANRIL (i) présente une association positive inattendue avec le cluster p16-CDKN2A/p15-CDKN2B/p14-ARF dans notre série, alors que cette association apparait négative dans les carcinomes de prostate et (ii) inactive épigénétiquement les miRNAs onco-suppresseurs miR-99a/miR-449a dans les carcinomes gastriques et non dans notre série de CCIs. D’un point de vue clinique, deux signatures pronostiques indépendantes ont pu être identifiées, l’une intégrant les 2 partenaires protéiques d’ANRIL appartenant aux complexes Polycomb (surexpression d’EZH2 / sous-expression de CBX7), et l’autre représentée par la sous-expression de Δ-MALAT1, observée dans 20% des tumeurs de notre série. La présence de variants d’épissage alternatifs, de réseaux d’interactions multiples et d’une spécificité d’organe devra être prise en compte lors de l’évaluation des thérapies épigénétiques ciblant ANRIL (inhibiteurs des bromodomaines et des oncoMIRs) ou MALAT1 (ASOs) dans les cancers du sein. La deuxième partie du projet de thèse a consisté en l’analyse du transcriptome non codant des carcinomes mammaires par une stratégie pangénomique, afin d’identifier de nouveaux types de lncRNAs, tels les nouveaux lncRNAs antisens, circulaires et associés à des séquences ultra-conservées ou induisant des résistances médicamenteuses.Breast cancer is the second most common cancer and the first malignancy of women. Currently, only few biomarkers (ER, PR, receptor HER2, index Ki67) and transcriptomic signature PAM50 are included in the morphological classification and therapeutic orientation. Transcriptome genome-wide analyses unexpectedly revealed that over 80% of the DNA is transcribed into RNA. Among these noncoding RNAs, transcripts longer than 200 nt are arbitrarily qualified as long noncoding RNAs (lncRNAs). LncRNAs play a crucial role in maintenance of cellular homeostasis and present abnormal expression patterns in various diseases, including cancer. The main objective of my project was to analyze expression of lncRNAs, their functionality and their roles in breast oncogenesis. The first part focused on the study of ANRIL and MALAT1 genes, two lncRNAs whose mechanisms of action and clinical significance in breast carcinogenesis are still controversial. ANRIL and MALAT1 respectively overexpressed in 20% and 14% of tumors in our series, confirming their pro-oncogenic roles in mammary carcinogenesis. MALAT1 overexpression results in RNA-FISH by presence of huge intranuclear speckles. Complexity of their deregulation is associated with presence of various isoforms and interaction networks with miRNAs, mRNAs and other lncRNAs. Concerning PRC2/PRC1 polycomb sub-units interacting with ANRIL, EZH2 (PRC2) is normally targeted by 3 onco-suppressor miRNAs (miR-26A1, miR-125B and miR-214) that are under-expressed in our series of CCIs. The 2 oncomiRs miR-181B1 and miR-181A2 that normally target and inactivate CBX7 (PRC1) appear overexpressed in our series of CCIs, resulting from activation of the oncogene HMGA1. Concerning MALAT1, the miRNAs biogenesis complex Drosha-DGCR8-Microprocessor regulates expression levels of the splicing variant Δ-MALAT1 and the latter is involved in activation of PI3K/Akt pathway. Significant correlations were observed between MALAT1 and genes involved in alternative splicing, cell cycle, apoptosis, DNA repair and migration. Aberrant transcriptomic profiles of these two lncRNAs seem characteristics of mammary carcinomas. Thus, ANRIL (i) presents an unexpected positive association with the p16-CDKN2A/p15-CDKN2B/p14-ARF cluster in our series of CCIs, whereas this association appears negative in prostate carcinomas and (ii) epigenetically inactivates onco-suppressor miRNAs miR99a/miR-449a in gastric carcinomas, but not in our series. From a clinic point of view, two independent prognostic signatures were identified, one incorporating two protein partners of ANRIL belonging to the polycomb complexes (EZH2 overexpression / CBX7 under-expression) and the other represented by under-expression of the variant Δ-MALAT1 observed in 20% of tumors in our series. The presence of alternative splice variants, multiple interactions with mRNAs and miRNAs and organ specificity should be considered when evaluating epigenetic antitumoral drugs designed to target ANRIL (bromodomains and oncoMIRs inhibitors) and MALAT1 (ASOs) in breast cancers. The second part of the project involved analysis of non-coding transcriptome of mammary carcinomas to identify new types of lncRNAs, including new antisens lncRNAs, circular lncRNAs, induced lncRNAs, noncoding ultraconserved transcripts and lncRNAs associated with resistance to systemic treatments. The preliminary analysis performed on a small cohort of breast cancers (n=8) will allow the implementation of the main (n=40) which will enhance robustness of identified signatures

Evaluation du rôle des longs ARN non codants dans les carcinomes mammaires infiltrants

Breast cancer is the second most common cancer and the first malignancy of women. Currently, only few biomarkers (ER, PR, receptor HER2, index Ki67) and transcriptomic signature PAM50 are included in the morphological classification and therapeutic orientation. Transcriptome genome-wide analyses unexpectedly revealed that over 80% of the DNA is transcribed into RNA. Among these noncoding RNAs, transcripts longer than 200 nt are arbitrarily qualified as long noncoding RNAs (lncRNAs). LncRNAs play a crucial role in maintenance of cellular homeostasis and present abnormal expression patterns in various diseases, including cancer. The main objective of my project was to analyze expression of lncRNAs, their functionality and their roles in breast oncogenesis. The first part focused on the study of ANRIL and MALAT1 genes, two lncRNAs whose mechanisms of action and clinical significance in breast carcinogenesis are still controversial. ANRIL and MALAT1 respectively overexpressed in 20% and 14% of tumors in our series, confirming their pro-oncogenic roles in mammary carcinogenesis. MALAT1 overexpression results in RNA-FISH by presence of huge intranuclear speckles. Complexity of their deregulation is associated with presence of various isoforms and interaction networks with miRNAs, mRNAs and other lncRNAs. Concerning PRC2/PRC1 polycomb sub-units interacting with ANRIL, EZH2 (PRC2) is normally targeted by 3 onco-suppressor miRNAs (miR-26A1, miR-125B and miR-214) that are under-expressed in our series of CCIs. The 2 oncomiRs miR-181B1 and miR-181A2 that normally target and inactivate CBX7 (PRC1) appear overexpressed in our series of CCIs, resulting from activation of the oncogene HMGA1. Concerning MALAT1, the miRNAs biogenesis complex Drosha-DGCR8-Microprocessor regulates expression levels of the splicing variant Δ-MALAT1 and the latter is involved in activation of PI3K/Akt pathway. Significant correlations were observed between MALAT1 and genes involved in alternative splicing, cell cycle, apoptosis, DNA repair and migration. Aberrant transcriptomic profiles of these two lncRNAs seem characteristics of mammary carcinomas. Thus, ANRIL (i) presents an unexpected positive association with the p16-CDKN2A/p15-CDKN2B/p14-ARF cluster in our series of CCIs, whereas this association appears negative in prostate carcinomas and (ii) epigenetically inactivates onco-suppressor miRNAs miR99a/miR-449a in gastric carcinomas, but not in our series. From a clinic point of view, two independent prognostic signatures were identified, one incorporating two protein partners of ANRIL belonging to the polycomb complexes (EZH2 overexpression / CBX7 under-expression) and the other represented by under-expression of the variant Δ-MALAT1 observed in 20% of tumors in our series. The presence of alternative splice variants, multiple interactions with mRNAs and miRNAs and organ specificity should be considered when evaluating epigenetic antitumoral drugs designed to target ANRIL (bromodomains and oncoMIRs inhibitors) and MALAT1 (ASOs) in breast cancers. The second part of the project involved analysis of non-coding transcriptome of mammary carcinomas to identify new types of lncRNAs, including new antisens lncRNAs, circular lncRNAs, induced lncRNAs, noncoding ultraconserved transcripts and lncRNAs associated with resistance to systemic treatments. The preliminary analysis performed on a small cohort of breast cancers (n=8) will allow the implementation of the main (n=40) which will enhance robustness of identified signatures.Le cancer du sein représente à l’échelle mondiale le deuxième cancer le plus fréquent et la première des tumeurs malignes de la femme. Actuellement, seuls certains biomarqueurs (RO, RP, récepteur HER2, index Ki67) et la signature transcriptomique PAM50 sont pris en compte dans la classification morphologique et l’orientation thérapeutique. Les analyses transcriptomiques à haut débit ont révélé que plus de 80% du génome humain est transcrit en ARN. Parmi les ARNs non codants, les transcrits dont la longueur est supérieure à 200 nt sont arbitrairement qualifiés de longs ARNs non codants (lncRNAs). Les lncRNAs jouent un rôle crucial dans le maintien de l’homéostasie cellulaire et présentent des profils d’expression anormaux dans diverses pathologies, dont le cancer. L’objectif principal de mon projet de thèse a consisté à analyser l’expression des lncRNAs, leur fonctionnalité et leur rôle dans l’oncogénèse mammaire. La première partie s’est focalisée sur l’étude des gènes ANRIL (ainsi que 10 gènes de la même voie de signalisation) et MALAT1, deux lncRNAs dont les mécanismes d’action et la signification clinique au cours de la cancérogénèse mammaire sont encore controversés. ANRIL et MALAT1 sont respectivement surexprimés dans 20% et 14% des tumeurs de notre série, confirmant leurs rôles pro-oncogénique dans la cancérogénèse mammaire. La surexpression de MALAT1 se traduit en RNA-FISH par la présence de volumineux speckles intranucléaires. La complexité de leur dérégulation est liée à la présence d’isoformes et de réseaux d’interactions avec les mRNAs et les miRNAs. Concernant les sous-unités appartenant aux complexes Polycomb PRC2 et PRC1qui interagissent avec ANRIL, EZH2 (PRC2) est normalement ciblé par 3 miRNAs onco-suppresseurs (miR-26A1, miR-125B et miR-214) qui sont sous-exprimés dans notre série de CCIs. Les 2 oncomiRs miR-181B1 and miR-181A2 qui ciblent et inactivent CBX7 (PRC1), apparaissent surexprimés dans notre série, en relation avec l’activation de l’oncogène HMGA1. Concernant MALAT1, le complexe de biogénèse des miRNAs Drosha-DGCR8-Microprocesseur régule l’expression d’un variant d’épissage ∆-MALAT1 et ce dernier est impliqué dans l’activation de la voie PI3K/Akt. Des corrélations significatives sont observées entre MALAT1 et des gènes impliqués dans l’épissage alternatif, le cycle cellulaire, l’apoptose, la réparation de l’ADN et la migration cellulaire. Les profils transcriptomiques aberrants de ces 2 lncRNAs semblent caractéristiques des carcinomes mammaires. Ainsi, ANRIL (i) présente une association positive inattendue avec le cluster p16-CDKN2A/p15-CDKN2B/p14-ARF dans notre série, alors que cette association apparait négative dans les carcinomes de prostate et (ii) inactive épigénétiquement les miRNAs onco-suppresseurs miR-99a/miR-449a dans les carcinomes gastriques et non dans notre série de CCIs. D’un point de vue clinique, deux signatures pronostiques indépendantes ont pu être identifiées, l’une intégrant les 2 partenaires protéiques d’ANRIL appartenant aux complexes Polycomb (surexpression d’EZH2 / sous-expression de CBX7), et l’autre représentée par la sous-expression de Δ-MALAT1, observée dans 20% des tumeurs de notre série. La présence de variants d’épissage alternatifs, de réseaux d’interactions multiples et d’une spécificité d’organe devra être prise en compte lors de l’évaluation des thérapies épigénétiques ciblant ANRIL (inhibiteurs des bromodomaines et des oncoMIRs) ou MALAT1 (ASOs) dans les cancers du sein. La deuxième partie du projet de thèse a consisté en l’analyse du transcriptome non codant des carcinomes mammaires par une stratégie pangénomique, afin d’identifier de nouveaux types de lncRNAs, tels les nouveaux lncRNAs antisens, circulaires et associés à des séquences ultra-conservées ou induisant des résistances médicamenteuses

Long Noncoding RNAs as New Architects in Cancer Epigenetics, Prognostic Biomarkers, and Potential Therapeutic Targets

International audienc

High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism.

Increasing epidemiological and animal experimental data provide substantial support for the role of aryl hydrocarbon receptor (AhR) in mammary tumorigenesis. The effects of AhR have been clearly demonstrated in rodent models of breast carcinogenesis and in several established human breast cancer cell lines following exposure to AhR ligands or AhR overexpression. However, relatively little is known about the role of AhR in human breast cancers. AhR has always been considered to be a regulator of toxic and carcinogenic responses to environmental contaminants such as TCDD (dioxin) and benzo[a]pyrene (BaP). The aim of this study was to identify the type of breast tumors (ERα-positive or ERα-negative) that express AHR and how AhR affects human tumorigenesis. The levels of AHR, AHR nuclear translocator (ARNT) and AHR repressor (AHRR) mRNA expression were analyzed in a cohort of 439 breast tumors, demonstrating a weak association between high AHR expression and age greater than fifty years and ERα-negative status, and HR-/ERBB2 breast cancer subtypes. AHRR mRNA expression was associated with metastasis-free survival, while AHR mRNA expression was not. Immunohistochemistry revealed the presence of AhR protein in both tumor cells (nucleus and/or cytoplasm) and the tumor microenvironment (including endothelial cells and lymphocytes). High AHR expression was correlated with high expression of several genes involved in signaling pathways related to inflammation (IL1B, IL6, TNF, IL8 and CXCR4), metabolism (IDO1 and TDO2 from the kynurenine pathway), invasion (MMP1, MMP2 and PLAU), and IGF signaling (IGF2R, IGF1R and TGFB1). Two well-known ligands for AHR (TCDD and BaP) induced mRNA expression of IL1B and IL6 in an ERα-negative breast tumor cell line. The breast cancer ER status likely influences AhR activity involved in these signaling pathways. The mechanisms involved in AhR activation and target gene expression in breast cancers are also discussed

Expression, Localization and Prognosis Association of MEP50 in Breast Cancer

Breast cancer is composed of distinct subgroups, triple-negative breast cancer (TNBC), human epidermal growth factor receptor-2 (HER2), luminal A, and luminal B, which are associated with different prognosis. MEP50 is the main partner of the arginine methyltransferase PRMT5 required for its enzymatic activity. Here, we examined MEP50 expression in the different breast cancer subgroups from the transcriptomic data obtained on human breast cancer samples and on normal breast tissues in two cohorts (Curie, n = 141; The Cancer Genome Atlas—TCGA, n = 788). We observed higher levels of MEP50 mRNA in TNBC (Curie, n = 41; TCGA, n = 106) compared to the other breast cancer subgroups and normal breast tissues. Using an online KM-plotter database, which allows survival analyses in a larger number of breast cancer patients, we found that high MEP50 mRNA levels were associated with a more favorable recurrence-free survival (RFS) in TNBC (n = 953, p = 1.2 × 10−4) and luminal B (n = 1353, p = 0.013) tumors, whereas high PRMT5 mRNA levels were associated with worse RFS in these two subgroups (TNBC: n = 442, p = 1.0 × 10−4; luminal B: n = 566, p = 6.8 × 10−3). We next determined the expression and the subcellular localization of MEP50 protein by immunohistochemistry (IHC) in our Curie cohort of breast cancer (n = 94) and normal tissues (n = 7) using a validated MEP50 antibody. MEP50 was more expressed in breast tumors compared to normal breast tissues (p = 0.02). MEP50 was more localized to the cytosol in breast cancer cells compared to normal breast tissue (p = 4 × 10−4), and was more found at the plasma membrane in normal tissues compared to breast tumors (p = 0.01). We also evaluated PRMT5 activity by IHC in our Curie cohort using a validated antibody (H4R3me2s) detecting histone H4 symmetrically dimethylated on Arg3. High levels of H4R3me2s were found in normal breast tissues, whereas the lowest levels of H4R3me2s were observed in TNBC and HER2 breast cancer subgroups. Altogether, our study reports the expression of the PRMT5 cofactor (MEP50) and substrate (H4R3me2s) in breast cancer and highlights the association of PRMT5 and MEP50 mRNA with prognosis in luminal B and TNBC breast cancer subgroups and certain TNBC subtypes