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17 pages, 1093 KiB

Open AccessArticle

Chromatin-Independent Interplay of NFATc1 and EZH2 in Pancreatic Cancer

by Shilpa Patil, Teresa Forster, Kristina Reutlinger, Waltraut Kopp, Lennart Versemann, Jessica Spitalieri, Jochen Gaedcke, Philipp Ströbel, Shiv K. Singh, Volker Ellenrieder, Albrecht Neesse and Elisabeth Hessmann

Cells 2021, 10(12), 3463; https://doi.org/10.3390/cells10123463 - 08 Dec 2021

Cited by 5 | Viewed by 2798

Abstract

Background: The Nuclear Factor of Activated T-cells 1 (NFATc1) transcription factor and the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) significantly contribute to the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). Herein, we aimed at dissecting the mechanistic background of their interplay in [...] Read more.

Background: The Nuclear Factor of Activated T-cells 1 (NFATc1) transcription factor and the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) significantly contribute to the aggressive phenotype of pancreatic ductal adenocarcinoma (PDAC). Herein, we aimed at dissecting the mechanistic background of their interplay in PDAC progression. Methods: NFATc1 and EZH2 mRNA and protein expression and complex formation were determined in transgenic PDAC models and human PDAC specimens. NFATc1 binding on the Ezh2 gene and the consequences of perturbed NFATc1 expression on Ezh2 transcription were explored by Chromatin Immunoprecipitation (ChIP) and upon transgenic or siRNA-mediated interference with NFATc1 expression, respectively. Integrative analyses of RNA- and ChIP-seq data was performed to explore NFATc1-/EZH2-dependent gene signatures. Results: NFATc1 targets the Ezh2 gene for transcriptional activation and biochemically interacts with the methyltransferase in murine and human PDAC. Surprisingly, our genome-wide binding and expression analyses do not link the protein complex to joint gene regulation. In contrast, our findings provide evidence for chromatin-independent functions of the NFATc1:EZH2 complex and reveal posttranslational EZH2 phosphorylation at serine 21 as a prerequisite for robust complex formation. Conclusion: Our findings disclose a previously unknown NFATc1-EZH2 axis operational in the pancreas and provide mechanistic insights into the conditions fostering NFATc1:EZH2 complex formation in PDAC. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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16 pages, 2066 KiB

Open AccessArticle

Plasma Metabolome Profiling Identifies Metabolic Subtypes of Pancreatic Ductal Adenocarcinoma

by Ujjwal Mukund Mahajan, Ahmed Alnatsha, Qi Li, Bettina Oehrle, Frank-Ulrich Weiss, Matthias Sendler, Marius Distler, Waldemar Uhl, Tim Fahlbusch, Elisabetta Goni, Georg Beyer, Ansgar Chromik, Markus Bahra, Fritz Klein, Christian Pilarsky, Robert Grützmann, Markus M. Lerch, Kirsten Lauber, Nicole Christiansen, Beate Kamlage, Ivonne Regel and Julia Mayerle Show full author list Hide full author list

Cells 2021, 10(7), 1821; https://doi.org/10.3390/cells10071821 - 19 Jul 2021

Cited by 9 | Viewed by 3747

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Developing biomarkers for early detection and chemotherapeutic response prediction is crucial to improve the dismal prognosis of PDAC patients. However, molecular cancer signatures based on transcriptome analysis do not reflect intratumoral heterogeneity. To [...] Read more.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Developing biomarkers for early detection and chemotherapeutic response prediction is crucial to improve the dismal prognosis of PDAC patients. However, molecular cancer signatures based on transcriptome analysis do not reflect intratumoral heterogeneity. To explore a more accurate stratification of PDAC phenotypes in an easily accessible matrix, plasma metabolome analysis using MxP^® Global Profiling and MxP^® Lipidomics was performed in 361 PDAC patients. We identified three metabolic PDAC subtypes associated with distinct complex lipid patterns. Subtype 1 was associated with reduced ceramide levels and a strong enrichment of triacylglycerols. Subtype 2 demonstrated increased abundance of ceramides, sphingomyelin and other complex sphingolipids, whereas subtype 3 showed decreased levels of sphingolipid metabolites in plasma. Pathway enrichment analysis revealed that sphingolipid-related pathways differ most among subtypes. Weighted correlation network analysis (WGCNA) implied PDAC subtypes differed in their metabolic programs. Interestingly, a reduced expression among related pathway genes in tumor tissue was associated with the lowest survival rate. However, our metabolic PDAC subtypes did not show any correlation to the described molecular PDAC subtypes. Our findings pave the way for further studies investigating sphingolipids metabolisms in PDAC. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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14 pages, 1428 KiB

Open AccessArticle

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models

by Rosa Lynn Schmitz, Julia Weissbach, Jan Kleilein, Jessica Bell, Stefan Hüttelmaier, Fabrice Viol, Till Clauditz, Patricia Grabowski, Helmut Laumen, Jonas Rosendahl, Patrick Michl, Jörg Schrader and Sebastian Krug

Cells 2021, 10(6), 1408; https://doi.org/10.3390/cells10061408 - 06 Jun 2021

Cited by 12 | Viewed by 3548

Abstract

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. [...] Read more.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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14 pages, 1626 KiB

Open AccessArticle

RNA Extraction from Endoscopic Ultrasound-Acquired Tissue of Pancreatic Cancer Is Feasible and Allows Investigation of Molecular Features

by Livia Archibugi, Veronica Ruta, Valentina Panzeri, Miriam Redegalli, Sabrina Gloria Giulia Testoni, Maria Chiara Petrone, Gemma Rossi, Massimo Falconi, Michele Reni, Claudio Doglioni, Claudio Sette, Paolo Giorgio Arcidiacono and Gabriele Capurso

Cells 2020, 9(12), 2561; https://doi.org/10.3390/cells9122561 - 30 Nov 2020

Cited by 11 | Viewed by 2779

Abstract

Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority [...] Read more.

Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB; the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality (RNA integrity index; RIN) were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL; p < 0.0001) and RNALater (0.19 ng/uL; p < 0.0001). The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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15 pages, 3211 KiB

Open AccessArticle

Maintenance Therapy for ATM-Deficient Pancreatic Cancer by Multiple DNA Damage Response Interferences after Platinum-Based Chemotherapy

by Elodie Roger, Johann Gout, Frank Arnold, Alica K. Beutel, Martin Müller, Alireza Abaei, Thomas F. E. Barth, Volker Rasche, Thomas Seufferlein, Lukas Perkhofer and Alexander Kleger

Cells 2020, 9(9), 2110; https://doi.org/10.3390/cells9092110 - 16 Sep 2020

Cited by 19 | Viewed by 3257

Abstract

Personalized medicine in treating pancreatic ductal adenocarcinoma (PDAC) is still in its infancy, albeit PDAC-related deaths are projected to rise over the next decade. Only recently, maintenance therapy with the PARP inhibitor olaparib showed improved progression-free survival in germline BRCA1/2-mutated PDAC patients [...] Read more.

Personalized medicine in treating pancreatic ductal adenocarcinoma (PDAC) is still in its infancy, albeit PDAC-related deaths are projected to rise over the next decade. Only recently, maintenance therapy with the PARP inhibitor olaparib showed improved progression-free survival in germline BRCA1/2-mutated PDAC patients after platinum-based induction for the first time. Transferability of such a concept to other DNA damage response (DDR) genes remains unclear. Here, we conducted a placebo-controlled, three-armed preclinical trial to evaluate the efficacy of multi-DDR interference (mDDRi) as maintenance therapy vs. continuous FOLFIRINOX treatment, implemented with orthotopically transplanted ATM-deficient PDAC cell lines. Kaplan–Meier analysis, cross-sectional imaging, histology, and in vitro analysis served as analytical readouts. Median overall survival was significantly longer in the mDDRi maintenance arm compared to the maintained FOLFIRINOX treatment. This survival benefit was mirrored in the highest DNA-damage load, accompanied by superior disease control and reduced metastatic burden. In vitro analysis suggests FOLFIRINOX-driven selection of invasive subclones, erased by subsequent mDDRi treatment. Collectively, this preclinical trial substantiates mDDRi in a maintenance setting as a novel therapeutic option and extends the concept to non-germline BRCA1/2-mutant PDAC. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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Review

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28 pages, 2897 KiB

Open AccessReview

Nuclear Dynamics and Chromatin Structure: Implications for Pancreatic Cancer

by Luis F. Flores, Brooke R. Tader, Ezequiel J. Tolosa, Ashley N. Sigafoos, David L. Marks and Martin E. Fernandez-Zapico

Cells 2021, 10(10), 2624; https://doi.org/10.3390/cells10102624 - 01 Oct 2021

Cited by 6 | Viewed by 4533

Abstract

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often [...] Read more.

Changes in nuclear shape have been extensively associated with the dynamics and functionality of cancer cells. In most normal cells, nuclei have a regular ellipsoid shape and minimal variation in nuclear size; however, an irregular nuclear contour and abnormal nuclear size is often observed in cancer, including pancreatic cancer. Furthermore, alterations in nuclear morphology have become the ‘gold standard’ for tumor staging and grading. Beyond the utility of altered nuclear morphology as a diagnostic tool in cancer, the implications of altered nuclear structure for the biology and behavior of cancer cells are profound as changes in nuclear morphology could impact cellular responses to physical strain, adaptation during migration, chromatin organization, and gene expression. Here, we aim to highlight and discuss the factors that regulate nuclear dynamics and their implications for pancreatic cancer biology. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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18 pages, 1027 KiB

Open AccessReview

Store-Operated Calcium Entry: Shaping the Transcriptional and Epigenetic Landscape in Pancreatic Cancer

by Ana P. Kutschat, Steven A. Johnsen and Feda H. Hamdan

Cells 2021, 10(5), 966; https://doi.org/10.3390/cells10050966 - 21 Apr 2021

Cited by 4 | Viewed by 3556

Abstract

Pancreatic ductal adenocarcinoma (PDAC) displays a particularly poor prognosis and low survival rate, mainly due to late diagnosis and high incidence of chemotherapy resistance. Genomic aberrations, together with changes in the epigenomic profile, elicit a shift in cellular signaling response and a transcriptional [...] Read more.

Pancreatic ductal adenocarcinoma (PDAC) displays a particularly poor prognosis and low survival rate, mainly due to late diagnosis and high incidence of chemotherapy resistance. Genomic aberrations, together with changes in the epigenomic profile, elicit a shift in cellular signaling response and a transcriptional reprograming in pancreatic tumors. This endows them with malignant attributes that enable them to not only overcome chemotherapeutic challenges, but to also attain diverse oncogenic properties. In fact, certain genetic amplifications elicit a rewiring of calcium signaling, which can confer ER stress resistance to tumors while also aberrantly activating known drivers of oncogenic programs such as NFAT. While calcium is a well-known second messenger, the transcriptional programs driven by aberrant calcium signaling remain largely undescribed in pancreatic cancer. In this review, we focus on calcium-dependent signaling and its role in epigenetic programs and transcriptional regulation. We also briefly discuss genetic aberration events, exemplifying how genetic alterations can rewire cellular signaling cascades, including calcium-dependent ones. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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13 pages, 992 KiB

Open AccessReview

Coming in the Air: Hypoxia Meets Epigenetics in Pancreatic Cancer

by Claudia Geismann and Alexander Arlt

Cells 2020, 9(11), 2353; https://doi.org/10.3390/cells9112353 - 25 Oct 2020

Cited by 19 | Viewed by 2901

Abstract

With a five-year survival rate under 9%, pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest tumors. Although the treatment options are slightly improving, PDAC is the second leading cause of cancer related death in 2020 in the US. In addition to a [...] Read more.

With a five-year survival rate under 9%, pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest tumors. Although the treatment options are slightly improving, PDAC is the second leading cause of cancer related death in 2020 in the US. In addition to a pronounced desmoplastic stroma reaction, pancreatic cancer is characterized by one of the lowest levels of oxygen availability within the tumor mass and these hypoxic conditions are known to contribute to tumor development and progression. In this context, the major hypoxia associated transcription factor family, HIF, regulates hundreds of genes involved in angiogenesis, metabolism, migration, invasion, immune escape and therapy resistance. Current research implications show, that hypoxia also modulates diverse areas of epigenetic mechanisms like non-coding RNAs, histone modifications or DNA methylation, which cooperate with the hypoxia-induced transcription factors as well as directly regulate the hypoxic response pathways. In this review, we will focus on hypoxia-mediated epigenetic alterations and their impact on pancreatic cancer. Full article

(This article belongs to the Special Issue Genome Dynamics in Pancreatic Cancer Biology and Therapy)

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