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Shokry D, Khan MW, Powell C, Johnson S, Rennels BC, Boyd RI, Sun Z, Fazal Z, Freemantle SJ, Parker MH, Vieson MD, Samuelson JP, Spinella MJ, Singh R. Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B. Cell Commun Signal 2024; 22:528. [PMID: 39482699 PMCID: PMC11529429 DOI: 10.1186/s12964-024-01912-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2024] [Accepted: 10/27/2024] [Indexed: 11/03/2024] Open
Abstract
Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.
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Affiliation(s)
- Doha Shokry
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
- Department of Anatomy and Embryology, Alexandria University, Alexandria, Egypt
| | - Mehwish W Khan
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Christine Powell
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Samantha Johnson
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Brayden C Rennels
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Raya I Boyd
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Zhengyang Sun
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Zeeshan Fazal
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Sarah J Freemantle
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA
| | - Maryanna H Parker
- Department of Pathobiology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA
| | - Miranda D Vieson
- Department of Pathobiology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA
- Department of Veterinary Clinical Medicine, University of Illinois Urbana-Champaign, Urbana, IL, 61802, USA
| | - Jonathan P Samuelson
- Department of Pathobiology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA
| | - Michael J Spinella
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA.
- Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, 61802, USA.
- Cancer Center of Illinois, University of Illinois Urbana-Champaign, Urbana, IL, 61802, USA.
| | - Ratnakar Singh
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL, 61802, USA.
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Shokry D, Khan MW, Powell C, Johnson S, Rennels BC, Boyd RI, Sun Z, Fazal Z, Freemantle SJ, Parker MH, Vieson MD, Samuelson JP, Spinella MJ, Singh R. Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B. RESEARCH SQUARE 2024:rs.3.rs-4986186. [PMID: 39483904 PMCID: PMC11527238 DOI: 10.21203/rs.3.rs-4986186/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/03/2024]
Abstract
Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.
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Nicu AT, Ionel IP, Stoica I, Burlibasa L, Jinga V. Recent Advancements in Research on DNA Methylation and Testicular Germ Cell Tumors: Unveiling the Intricate Relationship. Biomedicines 2024; 12:1041. [PMID: 38791003 PMCID: PMC11117643 DOI: 10.3390/biomedicines12051041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2024] [Revised: 04/30/2024] [Accepted: 05/02/2024] [Indexed: 05/26/2024] Open
Abstract
Testicular germ cell tumors (TGCTs) are the most common type of testicular cancer, with a particularly high incidence in the 15-45-year age category. Although highly treatable, resistance to therapy sometimes occurs, with devastating consequences for the patients. Additionally, the young age at diagnosis and the treatment itself pose a great threat to patients' fertility. Despite extensive research concerning genetic and environmental risk factors, little is known about TGCT etiology. However, epigenetics has recently come into the spotlight as a major factor in TGCT initiation, progression, and even resistance to treatment. As such, recent studies have been focusing on epigenetic mechanisms, which have revealed their potential in the development of novel, non-invasive biomarkers. As the most studied epigenetic mechanism, DNA methylation was the first revelation in this particular field, and it continues to be a main target of investigations as research into its association with TGCT has contributed to a better understanding of this type of cancer and constantly reveals novel aspects that can be exploited through clinical applications. In addition to biomarker development, DNA methylation holds potential for developing novel treatments based on DNA methyltransferase inhibitors (DNMTis) and may even be of interest for fertility management in cancer survivors. This manuscript is structured as a literature review, which comprehensively explores the pivotal role of DNA methylation in the pathogenesis, progression, and treatment resistance of TGCTs.
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Affiliation(s)
- Alina-Teodora Nicu
- Genetics Department, Faculty of Biology, University of Bucharest, 030018 Bucharest, Romania; (A.-T.N.); (I.S.)
| | - Ileana Paula Ionel
- Department of Specific Disciplines, Faculty of Midwifery and Nursing, University of Medicine and Pharmacy “Carol Davila”, 050474 Bucharest, Romania
| | - Ileana Stoica
- Genetics Department, Faculty of Biology, University of Bucharest, 030018 Bucharest, Romania; (A.-T.N.); (I.S.)
| | - Liliana Burlibasa
- Genetics Department, Faculty of Biology, University of Bucharest, 030018 Bucharest, Romania; (A.-T.N.); (I.S.)
| | - Viorel Jinga
- Department of Urology, Faculty of Medicine, University of Medicine and Pharmacy “Carol Davila”, 050474 Bucharest, Romania;
- The Academy of Romanian Scientists, 050044 Bucharest, Romania
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Vitillo L, Anjum F, Hewitt Z, Stavish D, Laing O, Baker D, Barbaric I, Coffey P. The isochromosome 20q abnormality of pluripotent cells interrupts germ layer differentiation. Stem Cell Reports 2023; 18:782-797. [PMID: 36801002 PMCID: PMC10031278 DOI: 10.1016/j.stemcr.2023.01.007] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2022] [Revised: 01/17/2023] [Accepted: 01/18/2023] [Indexed: 02/18/2023] Open
Abstract
Chromosome 20 abnormalities are some of the most frequent genomic changes acquired by human pluripotent stem cell (hPSC) cultures worldwide. Yet their effects on differentiation remain largely unexplored. We investigated a recurrent abnormality also found on amniocentesis, the isochromosome 20q (iso20q), during a clinical retinal pigment epithelium differentiation. Here we show that the iso20q abnormality interrupts spontaneous embryonic lineage specification. Isogenic lines revealed that under conditions that promote the spontaneous differentiation of wild-type hPSCs, the iso20q variants fail to differentiate into primitive germ layers and to downregulate pluripotency networks, resulting in apoptosis. Instead, iso20q cells are highly biased for extra-embryonic/amnion differentiation following inhibition of DNMT3B methylation or BMP2 treatment. Finally, directed differentiation protocols can overcome the iso20q block. Our findings reveal in iso20q a chromosomal abnormality that impairs the developmental competency of hPSCs toward germ layers but not amnion, which models embryonic developmental bottlenecks in the presence of aberrations.
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Affiliation(s)
- Loriana Vitillo
- Rescue, Repair and Regeneration, Institute of Ophthalmology, University College London, EC1V 9EL London, UK.
| | - Fabiha Anjum
- Rescue, Repair and Regeneration, Institute of Ophthalmology, University College London, EC1V 9EL London, UK
| | - Zoe Hewitt
- Centre for Stem Cell Biology, School of Biosciences, University of Sheffield, S10 2TN Sheffield, UK
| | - Dylan Stavish
- Centre for Stem Cell Biology, School of Biosciences, University of Sheffield, S10 2TN Sheffield, UK
| | - Owen Laing
- Centre for Stem Cell Biology, School of Biosciences, University of Sheffield, S10 2TN Sheffield, UK
| | - Duncan Baker
- Sheffield Diagnostic Genetic Services, Sheffield Children's Hospital, Sheffield, UK
| | - Ivana Barbaric
- Centre for Stem Cell Biology, School of Biosciences, University of Sheffield, S10 2TN Sheffield, UK
| | - Pete Coffey
- Rescue, Repair and Regeneration, Institute of Ophthalmology, University College London, EC1V 9EL London, UK; Centre for Stem Cell Biology and Engineering, University of California, Santa Barbara, Santa Barbara, CA, USA; NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust, UCL Institute of Ophthalmology, London, UK
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Singh R, Fazal Z, Bikorimana E, Boyd RI, Yerby C, Tomlin M, Baldwin H, Shokry D, Corbet AK, Shahid K, Hattab A, Freemantle SJ, Spinella MJ. Reciprocal epigenetic remodeling controls testicular cancer hypersensitivity to hypomethylating agents and chemotherapy. Mol Oncol 2021; 16:683-698. [PMID: 34482638 PMCID: PMC8807365 DOI: 10.1002/1878-0261.13096] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2021] [Revised: 07/25/2021] [Accepted: 09/02/2021] [Indexed: 12/12/2022] Open
Abstract
Testicular germ cell tumors (TGCTs) are aggressive but sensitive to cisplatin-based chemotherapy. Alternative therapies are needed for tumors refractory to cisplatin with hypomethylating agents providing one possibility. The mechanisms of cisplatin hypersensitivity and resistance in TGCTs remain poorly understood. Recently, it has been shown that TGCTs, even those resistant to cisplatin, are hypersensitive to very low doses of hypomethylating agents including 5-aza deoxy-cytosine (5-aza) and guadecitabine. We undertook a pharmacogenomic approach in order to better understand mechanisms of TGCT hypomethylating agent hypersensitivity by generating a panel of acquired 5-aza-resistant TGCT cells and contrasting these to previously generated acquired isogenic cisplatin-resistant cells from the same parent. Interestingly, there was a reciprocal relationship between cisplatin and 5-aza sensitivity, with cisplatin resistance associated with increased sensitivity to 5-aza and 5-aza resistance associated with increased sensitivity to cisplatin. Unbiased transcriptome analysis revealed 5-aza-resistant cells strongly downregulated polycomb target gene expression, the exact opposite of the finding for cisplatin-resistant cells, which upregulated polycomb target genes. This was associated with a dramatic increase in H3K27me3 and decrease in DNMT3B levels in 5-aza-resistant cells, the exact opposite changes seen in cisplatin-resistant cells. Evidence is presented that reciprocal regulation of polycomb and DNMT3B may be initiated by changes in DNMT3B levels as DNMT3B knockdown alone in parental cells resulted in increased expression of H3K27me3, EZH2, and BMI1, conferred 5-aza resistance and cisplatin sensitization, and mediated genome-wide repression of polycomb target gene expression. Finally, genome-wide analysis revealed that 5-aza-resistant, cisplatin-resistant, and DNMT3B-knockdown cells alter the expression of a common set of polycomb target genes. This study highlights that reciprocal epigenetic changes mediated by DNMT3B and polycomb may be a key driver of the unique cisplatin and 5-aza hypersensitivity of TGCTs and suggests that distinct epigenetic vulnerabilities may exist for pharmacological targeting of TGCTs.
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Affiliation(s)
- Ratnakar Singh
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Zeeshan Fazal
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Emmanuel Bikorimana
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Raya I Boyd
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Cliff Yerby
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Megan Tomlin
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Hannah Baldwin
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Doha Shokry
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Andrea K Corbet
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Khadeeja Shahid
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Aleyah Hattab
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Sarah J Freemantle
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA
| | - Michael J Spinella
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, IL, USA.,Carle Illinois College of Medicine and Cancer Center of Illinois, University of Illinois at Urbana-Champaign, IL, USA
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Between a Rock and a Hard Place: An Epigenetic-Centric View of Testicular Germ Cell Tumors. Cancers (Basel) 2021; 13:cancers13071506. [PMID: 33805941 PMCID: PMC8036638 DOI: 10.3390/cancers13071506] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2021] [Revised: 03/16/2021] [Accepted: 03/22/2021] [Indexed: 02/07/2023] Open
Abstract
Simple Summary This minireview focuses on the role of epigenetics in testicular cancer. A working model is developed that postulates that epigenetic features that drive testicular cancer malignancy also enable these tumors to be cured at a high rate with chemotherapy. Chemoresistance may occur by epigenetic uncoupling of malignancy and chemosensitivity, a scenario that may be amenable to epigenetic-based therapies. Abstract Compared to many common solid tumors, the main genetic drivers of most testicular germ cell tumors (TGCTs) are unknown. Decades of focus on genomic alterations in TGCTs including awareness of a near universal increase in copies of chromosome 12p have failed to uncover exceptional driver genes, especially in genes that can be targeted therapeutically. Thus far, TGCT patients have missed out on the benefits of targeted therapies available to treat most other malignancies. In the past decade there has been a greater appreciation that epigenetics may play an especially prominent role in TGCT etiology, progression, and hypersensitivity to conventional chemotherapy. While genetics undoubtedly plays a role in TGCT biology, this mini-review will focus on the epigenetic “states” or features of testicular cancer, with an emphasis on DNA methylation, histone modifications, and miRNAs associated with TGCT susceptibility, initiation, progression, and response to chemotherapy. In addition, we comment on the current status of epigenetic-based therapy and epigenetic biomarker development for TGCTs. Finally, we suggest a unifying “rock and a hard place” or “differentiate or die” model where the tumorigenicity and curability of TGCTs are both dependent on common but still ill-defined epigenetic states.
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Ari F, Napieralski R, Akgun O, Magdolen V, Ulukaya E. Epigenetic modulators combination with chemotherapy in breast cancer cells. Cell Biochem Funct 2021; 39:571-583. [PMID: 33608886 DOI: 10.1002/cbf.3626] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 02/02/2021] [Accepted: 02/05/2021] [Indexed: 12/15/2022]
Abstract
Despite the concerning adverse effects on tumour development, epigenetic drugs are very promising in cancer treatment. The aim of this study was to compare the differential effects of standard chemotherapy regimens (FEC: 5-fluorouracil plus epirubicine plus cyclophosphamide) in combination with epigenetic modulators (decitabine, valproic acid): (a) on gene methylation levels of selected tumour biomarkers (LINE-1, uPA, PAI-1, DAPK); (b) their expression status (uPA and PAI-1); (c) differentiation status (5meC and H3K27me3). Furthermore, cell survival as well as changes concerning the invasion capacity were monitored in cell culture models of breast cancer (MCF-7, MDA-MB-231). A significant overall decrease of cell survival was observed in the FEC-containing combination therapies for both cell lines. Methylation results showed a general tendency towards increased demethylation of the uPA and PAI-1 gene promoters for the MCF-7 cells, as well as the proapoptotic DAPK gene in the treatment regimens for both cell lines. The uPA and PAI-1 antigen levels were mainly increased in the supernatant of FEC-only treated MDA-MB-231 cells. DAC-only treatment induced an increase of secreted uPA protein in MCF-7 cell culture, while most of the VPA-containing regimens also induced uPA and PAI-1 expression in MCF-7 cell fractions. Epigenetically active substances can also induce a re-differentiation in tumour cells, as shown by 5meC, H3K27me3 applying ICC. SIGNIFICANCE OF THE STUDY: Epigenetic modulators especially in the highly undifferentiated and highly malignant MDA-MB-231 tumour cells significantly reduced tumour malignancy thus; further clinical studies applying specific combination therapies with epigenetic modulators may be warranted.
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Affiliation(s)
- Ferda Ari
- Science and Art Faculty, Department of Biology, Bursa Uludag University, Bursa, Turkey
| | - Rudolf Napieralski
- Department of Obstetrics and Gynecology, Clinical Research Unit, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.,Therawis Diagnostics GmbH, Munich, Germany
| | - Oguzhan Akgun
- Science and Art Faculty, Department of Biology, Bursa Uludag University, Bursa, Turkey
| | - Viktor Magdolen
- Department of Obstetrics and Gynecology, Clinical Research Unit, Klinikum rechts der Isar, Technische Universität München, Munich, Germany
| | - Engin Ulukaya
- Faculty of Medicine, Department of Clinical Biochemistry, Istinye University, Istanbul, Turkey
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Albany C, Fazal Z, Singh R, Bikorimana E, Adra N, Hanna NH, Einhorn LH, Perkins SM, Sandusky GE, Christensen BC, Keer H, Fang F, Nephew KP, Spinella MJ. A phase 1 study of combined guadecitabine and cisplatin in platinum refractory germ cell cancer. Cancer Med 2020; 10:156-163. [PMID: 33135391 PMCID: PMC7826483 DOI: 10.1002/cam4.3583] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2020] [Revised: 10/07/2020] [Accepted: 10/13/2020] [Indexed: 02/06/2023] Open
Abstract
Purpose Germ cell tumors (GCTs) are cured with therapy based on cisplatin, although a clinically significant number of patients are refractory and die of progressive disease. Based on preclinical studies indicating that refractory testicular GCTs are hypersensitive to hypomethylating agents (HMAs), we conducted a phase I trial combining the next‐generation HMA guadecitabine (SGI‐110) with cisplatin in recurrent, cisplatin‐resistant GCT patients. Methods Patients with metastatic GCTs were treated for five consecutive days with guadecitabine followed by cisplatin on day 8, for a 28‐day cycle for up to six cycles. The primary endpoint was safety and toxicity including dose‐limiting toxicity (DLT) and maximum tolerated dose (MTD). Results The number of patients enrolled was 14. The majority of patients were heavily pretreated. MTD was determined to be 30 mg/m2 guadecitabine followed by 100 mg/m2 cisplatin. The major DLTs were neutropenia and thrombocytopenia. Three patients had partial responses by RECIST criteria, two of these patients, including one with primary mediastinal disease, completed the study and qualified as complete responses by serum tumor marker criteria with sustained remissions of 5 and 13 months and survival of 16 and 26 months, respectively. The overall response rate was 23%. Three patients also had stable disease indicating a clinical benefit rate of 46%. Conclusions The combination of guadecitabine and cisplatin was tolerable and demonstrated activity in patients with platinum refractory germ cell cancer.
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Affiliation(s)
- Costantine Albany
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Zeeshan Fazal
- Department of Comparative Biosciences and the Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Ratnakar Singh
- Department of Comparative Biosciences and the Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Emmanuel Bikorimana
- Department of Comparative Biosciences and the Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Nabil Adra
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Nasser H Hanna
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Lawrence H Einhorn
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Susan M Perkins
- Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN, USA
| | - George E Sandusky
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Brock C Christensen
- Department of Epidemiology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA
| | - Harold Keer
- Astex Pharmaceuticals, Inc, Pleasanton, CA, USA
| | - Fang Fang
- Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN, USA
| | - Kenneth P Nephew
- Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN, USA
| | - Michael J Spinella
- Department of Comparative Biosciences and the Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
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9
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Fazal Z, Singh R, Fang F, Bikorimana E, Baldwin H, Corbet A, Tomlin M, Yerby C, Adra N, Albany C, Lee S, Freemantle SJ, Nephew KP, Christensen BC, Spinella MJ. Hypermethylation and global remodelling of DNA methylation is associated with acquired cisplatin resistance in testicular germ cell tumours. Epigenetics 2020; 16:1071-1084. [PMID: 33126827 DOI: 10.1080/15592294.2020.1834926] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Testicular germ cell tumours (TGCTs) respond well to cisplatin-based therapy. However, cisplatin resistance and poor outcomes do occur. It has been suggested that a shift towards DNA hypermethylation mediates cisplatin resistance in TGCT cells, although there is little direct evidence to support this claim. Here we utilized a series of isogenic cisplatin-resistant cell models and observed a strong association between cisplatin resistance in TGCT cells and a net increase in global CpG and non-CpG DNA methylation spanning regulatory, intergenic, genic and repeat elements. Hypermethylated loci were significantly enriched for repressive DNA segments, CTCF and RAD21 sites and lamina associated domains, suggesting that global nuclear reorganization of chromatin structure occurred in resistant cells. Hypomethylated CpG loci were significantly enriched for EZH2 and SUZ12 binding and H3K27me3 sites. Integrative transcriptome and methylome analyses showed a strong negative correlation between gene promoter and CpG island methylation and gene expression in resistant cells and a weaker positive correlation between gene body methylation and gene expression. A bidirectional shift between gene promoter and gene body DNA methylation occurred within multiple genes that was associated with upregulation of polycomb targets and downregulation of tumour suppressor genes. These data support the hypothesis that global remodelling of DNA methylation is a key factor in mediating cisplatin hypersensitivity and chemoresistance of TGCTs and furthers the rationale for hypomethylation therapy for refractory TGCT patients.
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Affiliation(s)
- Zeeshan Fazal
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Ratnakar Singh
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Fang Fang
- Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN, USA
| | - Emmanuel Bikorimana
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Hannah Baldwin
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Andrea Corbet
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Megan Tomlin
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Cliff Yerby
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Nabil Adra
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Costantine Albany
- Division of Hematology/Oncology, Indiana University Melvin and Bren Simon Comprehensive Cancer Center, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Sarah Lee
- Department of Epidemiology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA
| | - Sarah J Freemantle
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Kenneth P Nephew
- Medical Sciences Program, Indiana University School of Medicine, Bloomington, IN, USA
| | - Brock C Christensen
- Department of Epidemiology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA
| | - Michael J Spinella
- Department of Comparative Biosciences, The University of Illinois at Urbana-Champaign, Urbana, IL, USA.,Carle Illinois College of Medicine and Cancer Center of Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA
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Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors. Cancers (Basel) 2020; 12:cancers12102903. [PMID: 33050470 PMCID: PMC7601457 DOI: 10.3390/cancers12102903] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Revised: 10/06/2020] [Accepted: 10/08/2020] [Indexed: 12/16/2022] Open
Abstract
Simple Summary There is a need for novel treatment options for patients with testicular germ cell tumors, especially for those that are resistant to standard chemotherapy, who show poor prognosis. In this work, we test two compounds that inhibit epigenetic enzymes called histone deacetylases—belinostat and panobinostat. We show that these enzymes are expressed at different levels in different germ cell tumor subtypes (seminomas and non-seminomas) and that both drugs are effective in reducing tumor cell viability, by decreasing cell proliferation and increasing cell death. These results are promising and should prompt further works with these compounds, envisioning the improvement of care of germ cell tumor patients. Abstract Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents.
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Cisplatin Resistance in Testicular Germ Cell Tumors: Current Challenges from Various Perspectives. Cancers (Basel) 2020; 12:cancers12061601. [PMID: 32560427 PMCID: PMC7352163 DOI: 10.3390/cancers12061601] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2020] [Revised: 06/13/2020] [Accepted: 06/16/2020] [Indexed: 02/07/2023] Open
Abstract
Testicular germ cell tumors share a marked sensitivity to cisplatin, contributing to their overall good prognosis. However, a subset of patients develop resistance to platinum-based treatments, by still-elusive mechanisms, experiencing poor quality of life due to multiple (often ineffective) interventions and, eventually, dying from disease. Currently, there is a lack of defined treatment opportunities for these patients that tackle the mechanism(s) underlying the emergence of resistance. Herein, we aim to provide a multifaceted overview of cisplatin resistance in testicular germ cell tumors, from the clinical perspective, to the pathobiology (including mechanisms contributing to induction of the resistant phenotype), to experimental models available for studying this occurrence. We provide a systematic summary of pre-target, on-target, post-target, and off-target mechanisms putatively involved in cisplatin resistance, providing data from preclinical studies and from those attempting validation in clinical samples, including those exploring specific alterations as therapeutic targets, some of them included in ongoing clinical trials. We briefly discuss the specificities of resistance related to teratoma (differentiated) phenotype, including the phenomena of growing teratoma syndrome and development of somatic-type malignancy. Cisplatin resistance is most likely multifactorial, and a combination of therapeutic strategies will most likely produce the best clinical benefit.
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12
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Singh R, Fazal Z, Freemantle SJ, Spinella MJ. Mechanisms of cisplatin sensitivity and resistance in testicular germ cell tumors. CANCER DRUG RESISTANCE (ALHAMBRA, CALIF.) 2019; 2:580-594. [PMID: 31538140 PMCID: PMC6752046 DOI: 10.20517/cdr.2019.19] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Testicular germ cell tumors (TGCTs) are a cancer pharmacology success story with a majority of patients cured even in the highly advanced and metastatic setting. Successful treatment of TGCTs is primarily due to the exquisite responsiveness of this solid tumor to cisplatin-based therapy. However, a significant percentage of patients are, or become, refractory to cisplatin and die from progressive disease. Mechanisms for both clinical hypersensitivity and resistance have largely remained a mystery despite the promise of applying lessons to the majority of solid tumors that are not curable in the metastatic setting. Recently, this promise has been heightened by the realization that distinct (and perhaps pharmacologically replicable) epigenetic states, rather than fixed genetic alterations, may play dominant roles in not only TGCT etiology and progression but also their curability with conventional chemotherapies. In this review, it discusses potential mechanisms of TGCT cisplatin sensitivity and resistance to conventional chemotherapeutics.
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Affiliation(s)
- Ratnakar Singh
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Zeeshan Fazal
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Sarah J Freemantle
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Michael J Spinella
- Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.,The Carle Illinois College of Medicine , University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.,The Cancer Center of Illinois, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
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13
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Oing C, Skowron MA, Bokemeyer C, Nettersheim D. Epigenetic treatment combinations to effectively target cisplatin-resistant germ cell tumors: past, present, and future considerations. Andrology 2019; 7:487-497. [PMID: 30924611 DOI: 10.1111/andr.12611] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2019] [Revised: 02/18/2019] [Accepted: 02/24/2019] [Indexed: 12/20/2022]
Abstract
BACKGROUND Type II germ cell tumors represent the most common solid malignancy in men aged 15-45 years. Despite high cure rates of >90% over all stages, 10-15% of advanced patients develop treatment resistance and potentially succumb to their disease. Treatment of refractory germ cell tumors remains unsatisfactory, and new approaches are needed to further improve outcomes. OBJECTIVES With this narrative review, we highlight epigenetic mechanisms related to resistance to standard systemic treatment, which may act as promising targets for novel combined epigenetic treatment approaches. MATERIALS AND METHODS A comprehensive literature search of PubMed and MEDLINE was conducted to identify original and review articles on resistance mechanisms and/or epigenetic treatment of germ cell tumors in vitro and in vivo. Review articles were hand-searched to identify additional articles. RESULTS Distinct epigenetic phenomena have been linked to chemotherapy resistance in germ cell tumors, among which DNA hypermethylation, histone acetylation, and bromodomain proteins appear as promising targets for therapeutic exploitation. Inhibitors of key regulators, for example DNA methyltransferases (e.g. decitabine, guadecitabine), histone deacetylases (e.g. romidepsin), and bromodomain proteins (e.g. JQ1) decreased cell viability, triggered apoptosis, and growth arrest. Additionally, these epigenetic drugs induced differentiation and led to loss of pluripotency and re-sensitization towards cisplatin in cell lines and animal models. DISCUSSION Epigenetic treatments hold promise to (i) reduce the treatment burden of and (ii) overcome resistance to standard cisplatin-based chemotherapy. Combined approaches may enhance activity, while the ideal target and treatment combination of epigenetic drugs, either with another epigenetic agent or conventional cytotoxic agents need to be defined. CONCLUSION Epigenetic (combination) treatment for germ cell tumors should be further explored in pre-clinical and clinical research for its potential to further improve germ cell tumor treatment.
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Affiliation(s)
- C Oing
- Department of Oncology, Hematology and Bone Marrow Transplantation with Division of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.,Laboratory of Radiobiology and Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - M A Skowron
- Department of Urology, Urological Research Lab, Translational Urooncology, University Medical School Duesseldorf, Duesseldorf, Germany
| | - C Bokemeyer
- Department of Oncology, Hematology and Bone Marrow Transplantation with Division of Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - D Nettersheim
- Department of Urology, Urological Research Lab, Translational Urooncology, University Medical School Duesseldorf, Duesseldorf, Germany
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14
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Sellers ZP, Schneider G, Maj M, Ratajczak MZ. Analysis of the Paternally-Imprinted DLK1-MEG3 and IGF2-H19 Tandem Gene Loci in NT2 Embryonal Carcinoma Cells Identifies DLK1 as a Potential Therapeutic Target. Stem Cell Rev Rep 2018; 14:823-836. [PMID: 29980981 DOI: 10.1007/s12015-018-9838-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
The paternally-imprinted genes insulin-like growth factor 2 (IGF2), H19, delta-like homologue 1 (DLK1), and maternally-expressed gene 3 (MEG3) are expressed from the tandem gene loci IGF2-H19 and DLK1-MEG3, which play crucial roles in initiating embryogenesis and development. The erasure of imprinting (EOI) at differentially methylated regions (DMRs) which regulate the expression of these genes maintains the developmental quiescence of primordial germ cells (PGCs) migrating through the embryo proper during embryogenesis and prevents them from forming teratomas. To address the potential involvement of the IGF2-H19 and DLK1-MEG3 loci in the pathogenesis of embryonal carcinoma (EC), we investigated their genomic imprinting at DMRs in the human PGC-derived EC cell line NTera-2 (NT2). We observed EOI at the IGF2-H19 locus and, somewhat to our surprise, a loss of imprinting (LOI) at the DLK1-MEG3 locus. As a result, NT2 cells express imprinted gene ratios from these loci such that there are i) low levels of the proliferation-promoting IGF2 relative to ii) high levels of the proliferation-inhibiting long noncoding RNA (lncRNA) H19 and iii) high levels of proliferation-promoting DLK1 relative to iv) low levels of the proliferation-inhibiting lncRNA MEG3. Consistent with this pattern of expression, the knockdown of DLK1 mRNA by shRNA resulted in decreased in vitro cell proliferation and in vivo tumor growth as well as decreased in vivo organ seeding by NT2 cells. Furthermore, treatment of NT2 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaD) inhibited their proliferation. This inhibition was accompanied by changes in expression of both tandem gene sets: a decrease in the expression of DLK1 and upregulation of the proliferation-inhibiting lncRNA MEG3, and at the same time upregulation of IGF2 and downregulation of the lncRNA H19. These results suggest that the DLK1-MEG3 locus, and not the IGF2-H19 locus, drives the tumorigenicity of NT2 cells. Based on these results, we identified DLK1 as a novel treatment target for EC that could be downregulated by 5-azaD.
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Affiliation(s)
- Zachariah Payne Sellers
- Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY, 40202, USA
| | - Gabriela Schneider
- Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY, 40202, USA
| | - Magdalena Maj
- Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY, 40202, USA
| | - Mariusz Z Ratajczak
- Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, 500 South Floyd Street, Louisville, KY, 40202, USA.
- Medical University of Warsaw and Center for Preclinical Research and Technology, Warsaw, Poland.
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15
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Marques-Magalhães Â, Graça I, Henrique R, Jerónimo C. Targeting DNA Methyltranferases in Urological Tumors. Front Pharmacol 2018; 9:366. [PMID: 29706891 PMCID: PMC5909196 DOI: 10.3389/fphar.2018.00366] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2017] [Accepted: 03/28/2018] [Indexed: 12/14/2022] Open
Abstract
Urological cancers are a heterogeneous group of malignancies accounting for a considerable proportion of cancer-related morbidity and mortality worldwide. Aberrant epigenetic traits, especially altered DNA methylation patterns constitute a hallmark of these tumors. Nonetheless, these alterations are reversible, and several efforts have been carried out to design and test several epigenetic compounds that might reprogram tumor cell phenotype back to a normal state. Indeed, several DNMT inhibitors are currently under evaluation for therapeutic efficacy in clinical trials. This review highlights the critical role of DNA methylation in urological cancers and summarizes the available data on pre-clinical assays and clinical trials with DNMT inhibitors in bladder, kidney, prostate, and testicular germ cell cancers.
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Affiliation(s)
- Ângela Marques-Magalhães
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal
| | - Inês Graça
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal
| | - Rui Henrique
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal.,Department of Pathology, Portuguese Oncology Institute of Porto, Porto, Portugal.,Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
| | - Carmen Jerónimo
- Cancer Biology and Epigenetics Group - Research Center, Portuguese Oncology Institute of Porto, Porto, Portugal.,Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
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Sonnenburg D, Spinella MJ, Albany C. Epigenetic Targeting of Platinum Resistant Testicular Cancer. Curr Cancer Drug Targets 2017; 16:789-795. [PMID: 26694252 DOI: 10.2174/1568009616666151222150359] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2015] [Revised: 12/11/2015] [Accepted: 12/18/2015] [Indexed: 02/08/2023]
Abstract
The involvement of epigenetic aberrations in the development and progression of tumors is now well established. However, little is known of the epigenetic alterations in testicular cancer and particularly in platinum refractory germ cell tumors. Germ cell derived testicular cancers, as compared to somatic tumors, appear to have a unique epigenetic profile that features more extensive DNA hypomethylation. Emerging data from clinical specimens suggest that epigenetic aberrations, especially DNA hypermethylation, can contribute to chemotherapy resistance and poor clinical outcomes in testicular germ cell tumors. Recent data indicate that testicular cancer cells, even those resistant to platinum, are highly sensitive to low doses of demethylating agents. Based on these promising preclinical studies, we suggest that DNA methylation inhibitors in combination with chemotherapeutic agents may offer a path to overcome acquired drug resistance in testicular cancer, laying the foundation and rationale for testing this class of epigenetic drugs in the clinical setting. In this mini-review we provide a brief overview of the promise of DNA methylation therapy to treat patients with refractory cancer of the testes.
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Affiliation(s)
| | | | - Costantine Albany
- Department of Medicine, Indiana University Melvin and Bren Simon Cancer Center, 535 Barnhull Drive RT-473, Indianapolis, IN 46202, USA.
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Multiple mechanisms determine the sensitivity of human-induced pluripotent stem cells to the inducible caspase-9 safety switch. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2016; 3:16003. [PMID: 27626039 PMCID: PMC5008202 DOI: 10.1038/mtm.2016.3] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Revised: 01/03/2016] [Accepted: 01/04/2016] [Indexed: 12/17/2022]
Abstract
Expression of the inducible caspase-9 (iC9) suicide gene is one of the most appealing safety strategies for cell therapy and has been applied for human-induced pluripotent stem cells (hiPSC) to control the cell fate of hiPSC. iC9 can induce cell death of over 99% of iC9-transduced hiPSC (iC9-hiPSC) in less than 24 hours after exposure to chemical inducer of dimerization (CID). There is, however, a small number of resistant cells that subsequently outgrows. To ensure greater uniformity of the hiPSC response to iC9 activation, we purified a resistant population by culturing iC9-hiPSC with CID and analyzing the mechanisms by which the cells evade killing. We found that iC9-resistant hiPSC have significant heterogeneity in terms of their escape mechanisms from caspase-dependent apoptosis including reduced expression of iC9 by promoter silencing and overexpression of BCL2. As a consequence, modifying a single element alone will be insufficient to ensure sustained susceptibility of iC9 in all cells and prevent the eventual outgrowth of a resistant population. To solve this issue, we propose to isolate an iC9-sensitive population and show that this hiPSC line has sustained a uniform responsiveness to iC9-mediated growth control.
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18
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Phan NLC, Trinh NV, Pham PV. Low concentrations of 5-aza-2'-deoxycytidine induce breast cancer stem cell differentiation by triggering tumor suppressor gene expression. Onco Targets Ther 2015; 9:49-59. [PMID: 26730203 PMCID: PMC4694670 DOI: 10.2147/ott.s96291] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
Background Breast cancer stem cells (BCSCs) are considered the cause of tumor growth, multidrug resistance, metastasis, and recurrence. Therefore, differentiation therapy to reduce self-renewal of BCSCs is a promising approach. We have examined the effects of 5-aza-2′-deoxycytidine (DAC) on BCSC differentiation. Materials and methods BCSCs were treated with a range of DAC concentrations from 0.625 to 100 µM. The differentiation status of DAC-treated BCSCs was graded by changes in cell proliferation, CD44+CD24− phenotype, expression of tumor suppressor genes, including BRCA1, BRCA2, p15, p16, p53, and PTEN, and antitumor drug resistance. Results DAC treatment caused significant BCSC differentiation. BCSCs showed a 15%–23% reduction in proliferation capacity, 3.0%–21.3% decrease in the expression of BCSC marker CD44+/CD24−, activation of p53 expression, and increased p15, p16, BRCA1, and BRCA2 expression. Concentrations of DAC ranging from 0.625 to 40 µM efficiently induce cell cycle arrest in S-phase. ABCG2, highly expressed in BCSCs, also decreased with DAC exposure. Of particular note, drug-sensitivity of BCSCs to doxorubicin, verapamil, and tamoxifen also increased 1.5-, 2.0-, and 3.7-fold, respectively, after pretreatment with DAC. Conclusion DAC reduced breast cancer cell survival and induced differentiation through reexpression of tumor suppressor genes. These results indicate the potential of DAC in targeting specific chemotherapy-resistant cells within a tumor.
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Affiliation(s)
- Nhan Lu-Chinh Phan
- Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam
| | - Ngu Van Trinh
- Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam
| | - Phuc Van Pham
- Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh City, Vietnam
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Wongtrakoongate P. Epigenetic therapy of cancer stem and progenitor cells by targeting DNA methylation machineries. World J Stem Cells 2015; 7:137-148. [PMID: 25621113 PMCID: PMC4300924 DOI: 10.4252/wjsc.v7.i1.137] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2014] [Revised: 10/01/2014] [Accepted: 10/29/2014] [Indexed: 02/06/2023] Open
Abstract
Recent advances in stem cell biology have shed light on how normal stem and progenitor cells can evolve to acquire malignant characteristics during tumorigenesis. The cancer counterparts of normal stem and progenitor cells might be occurred through alterations of stem cell fates including an increase in self-renewal capability and a decrease in differentiation and/or apoptosis. This oncogenic evolution of cancer stem and progenitor cells, which often associates with aggressive phenotypes of the tumorigenic cells, is controlled in part by dysregulated epigenetic mechanisms including aberrant DNA methylation leading to abnormal epigenetic memory. Epigenetic therapy by targeting DNA methyltransferases (DNMT) 1, DNMT3A and DNMT3B via 5-Azacytidine (Aza) and 5-Aza-2’-deoxycytidine (Aza-dC) has proved to be successful toward treatment of hematologic neoplasms especially for patients with myelodysplastic syndrome. In this review, I summarize the current knowledge of mechanisms underlying the inhibition of DNA methylation by Aza and Aza-dC, and of their apoptotic- and differentiation-inducing effects on cancer stem and progenitor cells in leukemia, medulloblastoma, glioblastoma, neuroblastoma, prostate cancer, pancreatic cancer and testicular germ cell tumors. Since cancer stem and progenitor cells are implicated in cancer aggressiveness such as tumor formation, progression, metastasis and recurrence, I propose that effective therapeutic strategies might be achieved through eradication of cancer stem and progenitor cells by targeting the DNA methylation machineries to interfere their “malignant memory”.
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