Identification and functional characterization of epigenetic determinants of pancreatic CSCs

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-11-2015Human tumours including pancreatic cancer display significant functional heterogeneity in their respective cancer cell populations. Cancer stem cells (CSCs) represent a subpopulation of cells distinguishable from the bulk of the tumour by their exclusive ability to drive tumourigenesis based on their unlimited self-renewal capacity and their ability to give rise to more differentiated progenies (non-CSCs). Since CSCs and non-CSCs share an identical genetic background, the purpose of our study is to identify and functionally characterize the epigenetic signature of pancreatic CSCs in order to better understand the differences between these highly tumourigenic cells and their less tumourigenic progenies. Using high-throughput methylation analysis, we discovered that CSCs (i.e autofluorescent-positive cells) have a higher level of global methylation compared to their negative counterparts, regardless of the heterogeneity or polyclonality of the CSC populations present in the tumours analysed. Moreover, we found that CSCs express higher level of DNMT methylentrasferase proteins, with DNMT1 showing the most consistent up-regulation, which makes this protein a potentially attractive target for chemotherapy and chemoprevention. Specifically, targeting DNMT1 in CSCs using the DNMT inhibitor Zebularine significantly reduced their sphere formation capacity and in vivo tumourigenic potential. This inhibitory effect on CSCs, is mediated, in part, through epigenetic regulation of microRNAs, specifically, the cluster miR-17-92. Furthermore, we discovered that CSCs and non-CSCs have different expression level of proteins involved in DNA demethylation, with the TET2 protein being expressed at low levels in CSCs. Subsequent loss-of-function experiments in more differentiated cells resulted in their increased self-renewal, higher migration and in vivo tumourigenicity. Together our findings indicate that DNA methylation and demethylation play a crucial role in CSC biology and at the same time highlight the realistic potential for developing modulators that could modulate the epigenetic plasticity of CSCs offering new therapies to improve the poor outcome of patients with pancreatic ductal adenocarcinomaLos tumores humanos, incluyendo el adenocarcinoma ductal de páncreas (PDAC, del inglés pancreatic ductal adenocarcinoma), son muy heterogéneos respecto a las poblaciones de células cancerosas que contienen. Las células madre tumorales (CSCs, del inglés cancer stem cells) forman una subpoblación de células cancerosas caracterizada por su alta capacidad tumorigénica, autorenovación y su habilidad para repoblar el tumor. Dado que las CSCs y las células no-CSCs comparten el mismo perfil genético, el propósito de este estudio es identificar y caracterizar la firma epigenética de las CSCs pancreáticas que pueda explicar las diferencias funcionales entre estas células altamente tumorigénicas y sus progenies. Usando un análisis de metilación de alto rendimiento, hemos descubierto que las CSCs (identificadas como células autofluorescentes) tienen un nivel de metilación global más alto que las células diferenciadas, sin que influya el grado de heterogeneidad de los tumores analizados. Además, hemos encontrado que las CSCs expresan un mayor nivel de las metiltransferasas DNMT, sobre todo DNMT1, lo que convierte estas proteínas en una diana especialmente interesante para quimioterapia y quimioprevención. Específicamente, la inhibición de DNMT1 con zebularine redujo significativamente la capacidad de formar esferas y su potencial tumorigénico in vivo. Este efecto está parcialmente mediado por la regulación epigenética de los microRNAs de la familia miR-17-92. Además, hemos descubierto que las CSCs y las células tumorales diferenciadas expresan diferentes niveles de proteínas encargadas de demetilar el ADN, siendo la proteína TET2 la menos expresada en CSCs. Los experimentos de pérdida de función en células diferenciadas resultaron en una mayor capacidad de autorenovación, migración y tumorigénesis in vivo. En resumen, nuestros resultados indican que la metilación-demetilación del ADN tiene un papel crucial en la biología de las CSCs. Este hecho pone de relevancia que el desarrollo de moduladores de la plasticidad epigenética de las CSCs podría utilizarse como quimioterapia, potencialmente suponiendo una importante mejora de la situación de los pacientes con adenocarcinoma de páncrea

Pharmacological targeting of the receptor ALK inhibits tumorigenicity and overcomes chemoresistance in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients

SCIRT lncRNA Restrains Tumorigenesis by Opposing Transcriptional Programs of Tumor-Initiating Cells

In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet, how transcriptional regulation of cell-cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle gene expression and increase in self-renewal gene expression are coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long noncoding RNA (lncRNA) that we named Stem Cell Inhibitory RNA Transcript (SCIRT), which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell-cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell-cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing toward repression. These data suggest that the interaction of an lncRNA with EZH2 can alter the affinity of EZH2 for its protein-binding partners to regulate cancer cell state transitions. Significance: These findings show that a novel lncRNA SCIRT counteracts breast tumorigenesis by opposing transcriptional networks associated with cell cycle and self-renewal.</p

DNMT1 Inhibition Reprograms Pancreatic Cancer Stem Cells via Upregulation of the miR-17-92 Cluster.

Pancreatic ductal adenocarcinoma (PDAC) and other carcinomas are hierarchically organized, with cancer stem cells (CSC) residing at the top of the hierarchy, where they drive tumor progression, metastasis, and chemoresistance. As CSC and non-CSC share an identical genetic background, we hypothesize that differences in epigenetics account for the striking functional differences between these two cell populations. Epigenetic mechanisms, such as DNA methylation, play an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the role of DNA methylation in pancreatic CSC is obscure. In this study, we investigated the genome-wide DNA methylation profile of PDAC CSC, and we determined the importance of DNA methyltransferases for CSC maintenance and tumorigenicity. Using high-throughput methylation analysis, we discovered that sorted CSCs have a higher level of DNA methylation, regardless of the heterogeneity or polyclonality of the CSC populations present in the tumors analyzed. Mechanistically, CSC expressed higher DNMT1 levels than non-CSC. Pharmacologic or genetic targeting of DNMT1 in CSCs reduced their self-renewal and in vivo tumorigenic potential, defining DNMT1 as a candidate CSC therapeutic target. The inhibitory effect we observed was mediated in part through epigenetic reactivation of previously silenced miRNAs, in particular the miR-17-92 cluster. Together, our findings indicate that DNA methylation plays an important role in CSC biology and also provide a rationale to develop epigenetic modulators to target CSC plasticity and improve the poor outcome of PDAC patients. Cancer Res; 76(15); 4546-58. ©2016 AACR

SCIRT lncRNA restrains tumorigenesis by opposing transcriptional programs of tumor-initiating cells

In many tumors, cells transition reversibly between slow-proliferating tumor-initiating cells (TIC) and their differentiated, faster-growing progeny. Yet how transcriptional regulation of cell cycle and self-renewal genes is orchestrated during these conversions remains unclear. In this study, we show that as breast TIC form, a decrease in cell-cycle and increase in self-renewal gene expression is coregulated by SOX2 and EZH2, which colocalize at CpG islands. This pattern was negatively controlled by a novel long non-coding RNA (lncRNA) that we name SCIRT, which was markedly upregulated in tumorspheres but colocalized with and counteracted EZH2 and SOX2 during cell cycle and self-renewal regulation to restrain tumorigenesis. SCIRT specifically interacted with EZH2 to increase EZH2 affinity to FOXM1 without binding the latter. In this manner, SCIRT induced transcription at cell cycle gene promoters by recruiting FOXM1 through EZH2 to antagonize EZH2-mediated effects at target genes. Conversely, on stemness genes, FOXM1 was absent and SCIRT antagonized EZH2 and SOX2 activity, balancing towards repression. These data suggest that the interaction of a lncRNA with EZH2 can alter the affinity of EZH2 for its protein binding partners to regulate cancer cell state transitions

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes

[Background & Aims]: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. [Methods]: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. [Results]: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. [Conclusions]: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.This work was supported by the Deutsche Forschungsgemeinschaft (grants AL 1174/4-1, AL1174/4-2, and Collaborative Research Center 1321 “Modeling and Targeting Pancreatic Cancer” to Hana Algül; SFB824 Z2 to Katja Steiger), the Deutsche Krebshilfe (grant 111646 to Hana Algül), a Ramon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain (to Bruno Sainz Jr), a Coordinated Grant from Fundación Asociación Española Contra el Cáncer (GC16173694BARB to Bruno Sainz Jr), funding from The Fero Foundation (to Bruno Sainz Jr), and a Proyecto de Investigacion de Salud, ISCIII, Spain (no. PI18/00757 to Bruno Sainz Jr). Jiaoyu Ai is supported by the “China Scholarship Council” grant program

GATA4 and GATA6 loss-of-expression is associated with extinction of the classical programme and poor outcome in pancreatic ductal adenocarcinoma

ObjectiveGATA6 is a key regulator of the classical phenotype in pancreatic ductal adenocarcinoma (PDAC). Low GATA6 expression associates with poor patient outcome. GATA4 is the second most expressed GATA factor in the pancreas. We assessed whether, and how, GATA4 contributes to PDAC phenotype and analysed the association of expression with outcome and response to chemotherapy.DesignWe analysed PDAC transcriptomic data, stratifying cases according to GATA4 and GATA6 expression and identified differentially expressed genes and pathways. The genome-wide distribution of GATA4 was assessed, as well as the effects of GATA4 knockdown. A multicentre tissue microarray study to assess GATA4 and GATA6 expression in samples (n=745) from patients with resectable was performed. GATA4 and GATA6 levels were dichotomised into high/low categorical variables; association with outcome was assessed using univariable and multivariable Cox regression models.ResultsGATA4 messenger RNA is enriched in classical, compared with basal-like tumours. We classified samples in 4 groups as high/low for GATA4 and GATA6. Reduced expression of GATA4 had a minor transcriptional impact but low expression of GATA4 enhanced the effects of GATA6 low expression. GATA4 and GATA6 display a partially overlapping genome-wide distribution, mainly at promoters. Reduced expression of both proteins in tumours was associated with the worst patient survival. GATA4 and GATA6 expression significantly decreased in metastases and negatively correlated with basal markers.ConclusionsGATA4 and GATA6 cooperate to maintain the classical phenotype. Our findings provide compelling rationale to assess their expression as biomarkers of poor prognosis and therapeutic response

The Epigenetic Landscape of Pancreatic Cancer Stem Cells

Data now indicates that in addition to genetic alterations/mutations, human cancer cells exhibit important changes in their epigenome. In the context of this review, we define the epigenome as the chemical compounds and/or proteins that can interact with nuclear DNA to direct the specific and localized activation or silencing of genes to control the production of cellular proteins (directly or indirectly) in a given cell. Our ever-growing knowledge of how the epigenome can affect cellular processes has largely changed our view of cancer being a solely genetic disease. Nowadays, cancer is largely defined and characterized by the dynamic changes in both the genome and epigenome, which function together and contribute concomitantly to cancer initiation and progression. Since epigenetic modifications are crucial processes involved in controlling cellular identity and lineage fate, perturbations in this layer of gene regulation can contribute to the acquisition of new cellular characteristics different than those that were &ldquo;initially&rdquo; intended. For example, aberrant epigenetic alterations may transform normal non-cancer cells into cancer stem cells (CSCs), endowing them with the loss of differentiation and the acquisition of stem-like characteristics. In this review, we will focus our discussion on CSCs in the context of pancreatic ductal adenocarcinoma (PDAC). We will discuss how different epigenetic modifications create a landscape that can impact CSC identity and the way this small sub-population of cells contributes to tumor initiation, progression, and resistance to therapy. Moreover, we will highlight the latest discoveries in epigenetic-based therapies as a means of targeting CSCs

The Epigenetic Landscape of Pancreatic Cancer Stem Cells

© 2018 by the authorsData now indicates that in addition to genetic alterations/mutations, human cancer cells exhibit important changes in their epigenome. In the context of this review, we define the epigenome as the chemical compounds and/or proteins that can interact with nuclear DNA to direct the specific and localized activation or silencing of genes to control the production of cellular proteins (directly or indirectly) in a given cell. Our ever-growing knowledge of how the epigenome can affect cellular processes has largely changed our view of cancer being a solely genetic disease. Nowadays, cancer is largely defined and characterized by the dynamic changes in both the genome and epigenome, which function together and contribute concomitantly to cancer initiation and progression. Since epigenetic modifications are crucial processes involved in controlling cellular identity and lineage fate, perturbations in this layer of gene regulation can contribute to the acquisition of new cellular characteristics different than those that were “initially” intended. For example, aberrant epigenetic alterations may transform normal non-cancer cells into cancer stem cells (CSCs), endowing them with the loss of differentiation and the acquisition of stem-like characteristics. In this review, we will focus our discussion on CSCs in the context of pancreatic ductal adenocarcinoma (PDAC). We will discuss how different epigenetic modifications create a landscape that can impact CSC identity and the way this small sub-population of cells contributes to tumor initiation, progression, and resistance to therapy. Moreover, we will highlight the latest discoveries in epigenetic-based therapies as a means of targeting CSCs

Cancer-associated fibroblasts in bladder cancer: Origin, biology, and therapeutic opportunities

Context: Bladder cancer (BLCA) is a highly prevalent tumour and a health problem worldwide, especially among men. Recent work has highlighted the relevance of the tumour microenvironment (TME) in cancer biology with translational implications. Cancer-associated fibroblasts (CAFs) are a prominent, heterogeneous population of cells in the TME. CAFs have been associated with tumour development, progression, and poor prognosis in several neoplasms. However, their role in BLCA has not yet been exploited deeply. Objective: To review the role of CAFs in BLCA biology and provide an understanding of CAF origin, subtypes, markers, and phenotypic and functional characteristics to improve patient management. Evidence acquisition: A PubMed search was performed to review manuscripts published using the terms "cancer associated fibroblast" and "bladder cancer" or "urothelial cancer". All abstracts were reviewed, and the full content of all relevant manuscripts was analysed. In addition, selected manuscripts on CAFs in other tumours were considered. Evidence synthesis: CAFs have been studied less extensively in BLCA than in other tumours. Thanks to new techniques, such as single-cell RNA-seq and spatial transcriptomics, it is now possible to accurately map and molecularly define the phenotype of fibroblasts in normal bladder and BLCA. Bulk transcriptomic analyses have revealed the existence of subtypes among both non-muscle-invasive and muscle-invasive BLCA; these subtypes display distinct features regarding their CAF content. We provide a higher-resolution map of the phenotypic diversity of CAFs in these tumour subtypes. Preclinical studies and recent promising clinical trials leverage on this knowledge through the combined targeting of CAFs or their effectors and the immune microenvironment. Conclusions: Current knowledge of BLCA CAFs and the TME is being increasingly applied to improve BLCA therapy. There is a need to acquire a deeper understanding of CAF biology in BLCA. Patient summary: Tumour cells are surrounded by nontumoural cells that contribute to the determination of the behaviour of cancers. Among them are cancer-associated fibroblasts. The "neighbourhoods" established through these cellular interactions can now be studied with much greater resolution. Understanding these features of tumours will contribute to the designing of more effective therapies, especially in relationship to bladder cancer immunotherapy.Work in the laboratory of FXR is funded, in part, by a grant from Fundación Científica de la Asociación Española Contra el Cáncer. CNIO is supported by Ministerio de Ciencia, Innovación y Universidades as a Centro de Excelencia Severo Ochoa (CEX2019-000891-S (MCIN/AEI/ 10.13039/501100011033)