University of Groningen
MicroRNA expression and functional analysis in Hodgkin lymphoma
Yuan, Ye
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1
MicroRNA expression and functional
analysis in Hodgkin lymphoma
ISBN (printed): 978-94-034-1397-6
ISBN (digital): 978-94-034-1396-9
The studies described in this thesis were financially supported by:
Graduate School of medical university
Publication of this thesis was financially supported by:
University of Groningen
Graduate School of medical university
MicroRNA expression and functional analysis in Hodgkin lymphoma
©copyright 2019 Ye Yuan
All rights reserved.
Cover design: Ye Yuan
Layout: Ye Yuan
Printed by: Gildeprint
MicroRNA expression and
functional analysis in Hodgkin
lymphoma
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus prof. E. Sterken
and in accordance with the decision by the College of Deans. This thesis will be defended in public on Wednesday 13 February 2019 at 12.45 hours
by
Ye Yuan
born on 25 September 1987 in Heilongjiang, China
2
ISBN (printed): 978-94-034-1397-6
ISBN (digital): 978-94-034-1396-9
The studies described in this thesis were financially supported by:
Graduate School of medical university
Publication of this thesis was financially supported by:
University of Groningen
Graduate School of medical university
MicroRNA expression and functional analysis in Hodgkin lymphoma
©copyright 2019 Ye Yuan
All rights reserved.
Cover design: Ye Yuan
Layout: Ye Yuan
Printed by: Gildeprint
3
MicroRNA expression and
functional analysis in Hodgkin
lymphoma
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus prof. E. Sterken
and in accordance with the decision by the College of Deans. This thesis will be defended in public on Wednesday 13 February 2019 at 12.45 hours
by
Ye Yuan
born on 25 September 1987 in Heilongjiang, China
Supervisor
Prof. J.H.M. van den Berg
Co-supervisor
Dr. J.L. KluiverAssessment Committee
Prof. G. de Haan Prof. E. Vellenga Prof. D. de Jong4 5
Supervisor
Prof. J.H.M. van den Berg
Co-supervisor
Dr. J.L. KluiverAssessment Committee
Prof. G. de Haan Prof. E. Vellenga Prof. D. de JongParanymphs
Fubiao Niu Wangzhao SongContents
Chapter 1 Introduction and scope of the thesisChapter 2 MicroRNA-24-3p is overexpressed in Hodgkin lymphoma and protects
Hodgkin and Reed-Sternberg cells from apoptosis
The American Journal of Pathology, February 16, 2017
Chapter 3 Setting up a high throughput screen in Hodgkin lymphoma
Part A Feasibility testing of high-throughput screen in Hodgkin lymphoma cell lines using an empty vector barcoded approach
Part B A pilot high-throughput screen to elucidate the role of microRNAs in Hodgkin lymphoma cell growth
Chapter 4 High-throughput microRNA gain-of-function screening in Hodgkin
lymphoma Work in progress
Chapter 5 MicroRNA high throughput loss-of-function screening reveals an
oncogenic role for miR-21-5p in Hodgkin lymphoma
Cellular Physiology and Biochemistry, Epub September 5, 2018
Chapter 6 Summary, discussion and future perspectives Appendix Nederlandse samenvatting
Publications Acknowledgements Biography
6
Paranymphs
Fubiao Niu Wangzhao Song 7Contents
Chapter 1 Introduction and scope of the thesis
Chapter 2 MicroRNA-24-3p is overexpressed in Hodgkin lymphoma and protects
Hodgkin and Reed-Sternberg cells from apoptosis
The American Journal of Pathology, February 16, 2017
Chapter 3 Setting up a high throughput screen in Hodgkin lymphoma
Part A Feasibility testing of high-throughput screen in Hodgkin lymphoma cell lines using an empty vector barcoded approach
Part B A pilot high-throughput screen to elucidate the role of microRNAs in Hodgkin lymphoma cell growth
Chapter 4 High-throughput microRNA gain-of-function screening in Hodgkin
lymphoma Work in progress
Chapter 5 MicroRNA high throughput loss-of-function screening reveals an
oncogenic role for miR-21-5p in Hodgkin lymphoma
Cellular Physiology and Biochemistry, Epub September 5, 2018
Chapter 6 Summary, discussion and future perspectives Appendix Nederlandse samenvatting
Publications Acknowledgements Biography 9 21 53 55 77 97 121 151 173 179 181 185
CHAPTER 1
Introduction
and scope of the thesis
8 9
CHAPTER 1
Introduction
and scope of the thesis
CHAPTER 1
Introduction
1. Micro (mi) RNAs
MiRNAs are small single-stranded non-coding RNA molecules consisting of about 22 nucleotides. They have a function in post-transcriptional regulation of gene expression [1] and thereby play important roles in biological processes such as development, proliferation and apoptosis. Early studies have linked the function of specific miRNAs to self-renewal and differentiation of embryonic stem cells (ESC) [2].
Most primary (pri-)miRNA transcripts are transcribed by RNA polymerase II (Pol II) [3]. They are transcribed from their own host genes or co-transcribed with non-coding or protein-coding genes [4]. In the case of co-transcription, miRNAs are frequently located in the introns and in some cases in exons [5]. Pri-miRNA transcripts contain one or multiple stem-loop like structures of about 70 nucleotides each. These hairpin-like structures are recognized by Digeorge syndrome critical region 8 (DGCR8) in the nucleus [6]. DGCR8 associates with Drosha and this protein complex cleaves the flanking regions from the pri-miRNA resulting in a stem-loop fragment referred to as the precursor (pre-)miRNA. The pre-miRNA is exported from the nucleus to the cytoplasm by Exportin-5. The RNase III enzyme Dicer, cleaves the loop structure of the pre-miRNA hairpin [7]. One strand of the resulting ~22 nucleotides duplex is incorporated into the RNA-induced silencing complex (RISC), whereas the other strand is degraded (Figure 1). RISC contains several proteins such as Dicer, Ago, TRBP, PACT and Gemin3. There are four Ago proteins, of which Ago1 and Ago2 are the most abundantly expressed members in human. The Ago proteins contain four domains, i.e. the N-terminal, PAZ, middle and PIWI domain. The PAZ domain binds to the 3-end of the miRNA, while the other three domains form a unique structure, creating grooves for target mRNA and miRNA interactions [8, 9].
More than two thousand miRNAs have been discovered in humans and they are thought to regulate more than half of all protein coding genes. A single miRNA can regulate up to hundreds of genes and vice versa, each gene can be regulated by multiple miRNAs [10]. The most commonly used feature for miRNA - target gene predictions is a strong base pairing between the miRNA seed region, i.e. nucleotides 2 to 7 or 8, and its corresponding antisense region in the 3’-UTR of a mRNA [11]. The remaining nucleotides of the miRNA usually show a lower degree of complementarity to the target gene sequence. Various algorithms for miRNA target predictions have been established over the past decade [12, 13]. However, different miRNA target gene prediction algorithms frequently provide different target gene lists. Cross checking multiple algorithms may give an additional layer of confidence for true interactions. However, none of the algorithms take into consideration whether both the miRNA and predicted target gene are co-expressed in the cell type of interest. In addition, most
algorithms do not consider non-canonical miRNA binding sites such as binding to the coding region or the 5’-UTR or imperfect seed binding. Thus, although target prediction programs can help to identify miRNA-target gene interactions, they have considerable limitations compared to genome-wide target gene identification experiments in the cell type of interest.
The first human disease that was reported to be associated with miRNAs was chronic lymphocytic leukemia [14]. After that, many miRNAs have been linked to specific types of cancer and other diseases [15, 16].
Figure 1. A schematic representation of the miRNA biogenesis pathway. MiRNAs are transcribed as pri-miRNA transcripts, which are processed by the Drosha-DGCR8 complex to miRNAs. The pre-miRNAs are exported to the cytoplasm by Exportin-5 and processed by Dicer to a miRNA duplex. The mature miRNA is incorporated into the RISC and this complex binds to the 3’-UTR of its target genes and inhibits translation or induces RNA degradation [17].
1
11
Introduction and scope of the thesis
10
Introduction
1. Micro (mi) RNAs
MiRNAs are small single-stranded non-coding RNA molecules consisting of about 22 nucleotides. They have a function in post-transcriptional regulation of gene expression [1] and thereby play important roles in biological processes such as development, proliferation and apoptosis. Early studies have linked the function of specific miRNAs to self-renewal and differentiation of embryonic stem cells (ESC) [2].
Most primary (pri-)miRNA transcripts are transcribed by RNA polymerase II (Pol II) [3]. They are transcribed from their own host genes or co-transcribed with non-coding or protein-coding genes [4]. In the case of co-transcription, miRNAs are frequently located in the introns and in some cases in exons [5]. Pri-miRNA transcripts contain one or multiple stem-loop like structures of about 70 nucleotides each. These hairpin-like structures are recognized by Digeorge syndrome critical region 8 (DGCR8) in the nucleus [6]. DGCR8 associates with Drosha and this protein complex cleaves the flanking regions from the pri-miRNA resulting in a stem-loop fragment referred to as the precursor (pre-)miRNA. The pre-miRNA is exported from the nucleus to the cytoplasm by Exportin-5. The RNase III enzyme Dicer, cleaves the loop structure of the pre-miRNA hairpin [7]. One strand of the resulting ~22 nucleotides duplex is incorporated into the RNA-induced silencing complex (RISC), whereas the other strand is degraded (Figure 1). RISC contains several proteins such as Dicer, Ago, TRBP, PACT and Gemin3. There are four Ago proteins, of which Ago1 and Ago2 are the most abundantly expressed members in human. The Ago proteins contain four domains, i.e. the N-terminal, PAZ, middle and PIWI domain. The PAZ domain binds to the 3-end of the miRNA, while the other three domains form a unique structure, creating grooves for target mRNA and miRNA interactions [8, 9].
More than two thousand miRNAs have been discovered in humans and they are thought to regulate more than half of all protein coding genes. A single miRNA can regulate up to hundreds of genes and vice versa, each gene can be regulated by multiple miRNAs [10]. The most commonly used feature for miRNA - target gene predictions is a strong base pairing between the miRNA seed region, i.e. nucleotides 2 to 7 or 8, and its corresponding antisense region in the 3’-UTR of a mRNA [11]. The remaining nucleotides of the miRNA usually show a lower degree of complementarity to the target gene sequence. Various algorithms for miRNA target predictions have been established over the past decade [12, 13]. However, different miRNA target gene prediction algorithms frequently provide different target gene lists. Cross checking multiple algorithms may give an additional layer of confidence for true interactions. However, none of the algorithms take into consideration whether both the miRNA and predicted target gene are co-expressed in the cell type of interest. In addition, most
11
algorithms do not consider non-canonical miRNA binding sites such as binding to the coding region or the 5’-UTR or imperfect seed binding. Thus, although target prediction programs can help to identify miRNA-target gene interactions, they have considerable limitations compared to genome-wide target gene identification experiments in the cell type of interest.
The first human disease that was reported to be associated with miRNAs was chronic lymphocytic leukemia [14]. After that, many miRNAs have been linked to specific types of cancer and other diseases [15, 16].
Figure 1. A schematic representation of the miRNA biogenesis pathway. MiRNAs are transcribed as pri-miRNA transcripts, which are processed by the Drosha-DGCR8 complex to miRNAs. The pre-miRNAs are exported to the cytoplasm by Exportin-5 and processed by Dicer to a miRNA duplex. The mature miRNA is incorporated into the RISC and this complex binds to the 3’-UTR of its target genes and inhibits translation or induces RNA degradation [17].
CHAPTER 1
2. Hodgkin lymphoma (HL)
HL is one of the most common B-cell lymphoma subtypes. Based on differences in histology and phenotype of the tumor cells, HL can be divided into classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant HL (NLPHL). The cHL variant is further classified into nodular sclerosis (NS), mixed cellularity (MC), lymphocyte-rich (LR) and lymphocyte-depleted (LD) subtypes. The malignant cells comprise less than 1% of the total tumor mass [18]. In cHL, the malignant cells are referred to as Hodgkin (H) and Reed-Sternberg (RS) cells (commonly referred to as HRS cells), and in NLPHL they are called lymphocyte-predominant (LP) cells [19]. The tumor cells in cHL present as mono-nucleated Hodgkin (H) or multi-nucleated Reed-Sternberg (RS) cells. HRS cells intermingled in an abundant reactive infiltrate consisting of small lymphocytes and various inflammatory cells are required for HL diagnosis. Presence of immunoglobulin (Ig) gene rearrangements indicated that HRS cells are derived from B cells. The presence of somatic mutations in all B-cell derived cases and crippling somatic hypermutations in a proportion of the cases indicated that HRS cells most likely originate from pre-apoptotic germinal center (GC) B cells [20]. Despite their B-cell origin, HRS cells usually lack expression of common B-cell markers such as Ig, CD19, CD20, CD22, and CD79a and B-cell transcription factors such as Bob-1, Oct-2, PU.1, and Pax5 [21, 22]. HRS cells always express CD30, a member of the tumor necrosis factor (TNF) receptor superfamily [23]. In addition, HRS cells retain the antigen presenting phenotype that is observed in normal B cells [24, 25]. In contrast, the LP
cells of NLPHL do express most common B-cell markers [26].
HL patients are treated with a combination of chemotherapy and radiotherapy. In general, the adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy regimen is successful in the majority of early stage HL patients. The 5-year survival rate is about 90% based on the SEER database of the National Cancer Institute, including more than 8,000 HL patients diagnosed between 1988 and 2001. For advanced stage patients, chemotherapy with a regimen of bleomycin, etoposide, adriamycin, cyclophosphamide, oncovin, procarbazine and prednisone (BEACOPP) gives better survival than ABVD, with a 5-years survival of about 80% for stage III and 65% for stage IV patients. However, toxicity is significantly higher for BEACOPP-treated as compared to ABVD-BEACOPP-treated patients [27].
3. HL and miRNAs
Many researchers have shown that miRNAs play an important role in the pathogenesis of B-cell lymphoma [14, 28-30]. The first report on a miRNA in HL indicated a high expression of the B cell integration cluster (BIC) gene in HL [31]. MiR-155 was derived from the highly conserved stem-loop region located in the third exon of the human non-coding BIC gene [32]. In many types of lymphoma, miR-155 is expressed at a high
level and is assumed to play a crucial oncogenic role in the pathogenesis [33]. One of the identified target genes relevant for B-cell lymphoma is the growth inhibitor TBRG1 also known as NIAM [34]. In Burkitt lymphoma, miR-155 levels are low or absent and may act as a tumor suppressor miRNA [35]. This contradictory function in Burkitt lymphoma has been explained by the finding that miR-155 regulates the expression of activation-induced cytidine deaminase (AID), which regulates class switching and somatic hypermutation [36]. In Burkitt lymphoma-initiating cells low expression of miR-155 may lead to high AID levels, which may facilitate formation of Myc-Ig translocations that are characteristic of Burkitt lymphoma [37].
Identification of differentially expressed miRNAs in HL as compared to their normal counterparts, the GC-B cells, may improve our understanding of the HL pathogenesis. Small RNA cloning and subsequent sequencing analysis of 250 cancer samples including 4 cHL cell lines and various normal B-cell subsets revealed a high expression of miR-16, miR-21, miR-29b, miR-142, miR-155 in HL cells [38]. Gibcus et al. used microarrays to define the miRNA profile of HL cell lines and identified the miR-17~92 cluster, miR-16, miR-21, miR-24, and miR-155 as HL-specific miRNAs [39]. Navarro et al. identified 25 miRNAs differentially expressed between total cHL tissue samples and reactive lymph node tissue [40]. Comparison of microdissected HRS cells from nine
cHL patients to CD77+ GC-B cells revealed 12 over- and 3 under-expressed miRNAs
[41]. Semra et al. analyzed the expression of 377 miRNAs by qPCR in 32 total cHL tissues and 60 control tissues with reactive lymphadenopathy and identified 13 miRNAs with decreased and 11 miRNAs with increased expression [42]. Van
Eijndhoven et al. detected miRNAs inplasma of cHL patients and found higher levels
of miR-24-3p, miR-127-3p, miR-21-5p, miR-155-5p, and let-7a-5p in circulating extra cellular vesicles (EV) of cHL patients compared with EV fractions from healthy subjects [43]. Another study identified 25, 30a/d, 26b, 182, 186, miR-140* and miR-125a as up-regulated and miR-23a, miR-122, miR-93 and miR-144 as down-regulated in plasma from HL patients compared to healthy adults [44].
For part of the differentially expressed miRNAs a functional role in the pathogenesis of HL has been linked to specific cellular pathways. Navarro et al. showed that increased miR-135a induced downregulation of JAK2 and as a consequence led to reduced Bcl-xL expression in HL [45]. The miR-17/106b seed family targets CDKN1A encoding for the P21 protein. High miR-17/106b levels in HRS cells result in a decrease of the P21 protein levels and this enabled cell cycle progression [46]. The high miR-9 levels in HRS cells resulted in reduced levels of the miR-9 target plasma cell differentiation gene PRDM1, which might explain the differentiation block observed in HRS cells [47]. The interaction between EBV-miR-BHRF1-2 and PRDM1 may be one of the mechanisms by which Epstein-Barr virus (EBV) miRNA EBV-miR-BHRF1-2 promotes EBV lymphomagenesis [48]. In addition, miR-9 targets DICER1 and HuR, a regulator of cytokine expression and inhibition of miR-9 resulted in higher cytokine production
1
13
Introduction and scope of the thesis
12
2. Hodgkin lymphoma (HL)
HL is one of the most common B-cell lymphoma subtypes. Based on differences in histology and phenotype of the tumor cells, HL can be divided into classical Hodgkin lymphoma (cHL) and nodular lymphocyte-predominant HL (NLPHL). The cHL variant is further classified into nodular sclerosis (NS), mixed cellularity (MC), lymphocyte-rich (LR) and lymphocyte-depleted (LD) subtypes. The malignant cells comprise less than 1% of the total tumor mass [18]. In cHL, the malignant cells are referred to as Hodgkin (H) and Reed-Sternberg (RS) cells (commonly referred to as HRS cells), and in NLPHL they are called lymphocyte-predominant (LP) cells [19]. The tumor cells in cHL present as mono-nucleated Hodgkin (H) or multi-nucleated Reed-Sternberg (RS) cells. HRS cells intermingled in an abundant reactive infiltrate consisting of small lymphocytes and various inflammatory cells are required for HL diagnosis. Presence of immunoglobulin (Ig) gene rearrangements indicated that HRS cells are derived from B cells. The presence of somatic mutations in all B-cell derived cases and crippling somatic hypermutations in a proportion of the cases indicated that HRS cells most likely originate from pre-apoptotic germinal center (GC) B cells [20]. Despite their B-cell origin, HRS cells usually lack expression of common B-cell markers such as Ig, CD19, CD20, CD22, and CD79a and B-cell transcription factors such as Bob-1, Oct-2, PU.1, and Pax5 [21, 22]. HRS cells always express CD30, a member of the tumor necrosis factor (TNF) receptor superfamily [23]. In addition, HRS cells retain the antigen presenting phenotype that is observed in normal B cells [24, 25]. In contrast, the LP cells of NLPHL do express most common B-cell markers [26].
HL patients are treated with a combination of chemotherapy and radiotherapy. In general, the adriamycin, bleomycin, vinblastine and dacarbazine (ABVD) chemotherapy regimen is successful in the majority of early stage HL patients. The 5-year survival rate is about 90% based on the SEER database of the National Cancer Institute, including more than 8,000 HL patients diagnosed between 1988 and 2001. For advanced stage patients, chemotherapy with a regimen of bleomycin, etoposide, adriamycin, cyclophosphamide, oncovin, procarbazine and prednisone (BEACOPP) gives better survival than ABVD, with a 5-years survival of about 80% for stage III and 65% for stage IV patients. However, toxicity is significantly higher for BEACOPP-treated as compared to ABVD-BEACOPP-treated patients [27].
3. HL and miRNAs
Many researchers have shown that miRNAs play an important role in the pathogenesis of B-cell lymphoma [14, 28-30]. The first report on a miRNA in HL indicated a high expression of the B cell integration cluster (BIC) gene in HL [31]. MiR-155 was derived from the highly conserved stem-loop region located in the third exon of the human non-coding BIC gene [32]. In many types of lymphoma, miR-155 is expressed at a high
13
level and is assumed to play a crucial oncogenic role in the pathogenesis [33]. One of the identified target genes relevant for B-cell lymphoma is the growth inhibitor TBRG1 also known as NIAM [34]. In Burkitt lymphoma, miR-155 levels are low or absent and may act as a tumor suppressor miRNA [35]. This contradictory function in Burkitt lymphoma has been explained by the finding that miR-155 regulates the expression of activation-induced cytidine deaminase (AID), which regulates class switching and somatic hypermutation [36]. In Burkitt lymphoma-initiating cells low expression of miR-155 may lead to high AID levels, which may facilitate formation of Myc-Ig translocations that are characteristic of Burkitt lymphoma [37].
Identification of differentially expressed miRNAs in HL as compared to their normal counterparts, the GC-B cells, may improve our understanding of the HL pathogenesis. Small RNA cloning and subsequent sequencing analysis of 250 cancer samples including 4 cHL cell lines and various normal B-cell subsets revealed a high expression of miR-16, miR-21, miR-29b, miR-142, miR-155 in HL cells [38]. Gibcus et al. used microarrays to define the miRNA profile of HL cell lines and identified the miR-17~92 cluster, miR-16, miR-21, miR-24, and miR-155 as HL-specific miRNAs [39]. Navarro et al. identified 25 miRNAs differentially expressed between total cHL tissue samples and reactive lymph node tissue [40]. Comparison of microdissected HRS cells from nine cHL patients to CD77+ GC-B cells revealed 12 over- and 3 under-expressed miRNAs
[41]. Semra et al. analyzed the expression of 377 miRNAs by qPCR in 32 total cHL tissues and 60 control tissues with reactive lymphadenopathy and identified 13 miRNAs with decreased and 11 miRNAs with increased expression [42]. Van Eijndhoven et al. detected miRNAs inplasma of cHL patients and found higher levels of miR-24-3p, miR-127-3p, miR-21-5p, miR-155-5p, and let-7a-5p in circulating extra cellular vesicles (EV) of cHL patients compared with EV fractions from healthy subjects [43]. Another study identified 25, 30a/d, 26b, 182, 186, miR-140* and miR-125a as up-regulated and miR-23a, miR-122, miR-93 and miR-144 as down-regulated in plasma from HL patients compared to healthy adults [44].
For part of the differentially expressed miRNAs a functional role in the pathogenesis of HL has been linked to specific cellular pathways. Navarro et al. showed that increased miR-135a induced downregulation of JAK2 and as a consequence led to reduced Bcl-xL expression in HL [45]. The miR-17/106b seed family targets CDKN1A encoding for the P21 protein. High miR-17/106b levels in HRS cells result in a decrease of the P21 protein levels and this enabled cell cycle progression [46]. The high miR-9 levels in HRS cells resulted in reduced levels of the miR-9 target plasma cell differentiation gene PRDM1, which might explain the differentiation block observed in HRS cells [47]. The interaction between EBV-miR-BHRF1-2 and PRDM1 may be one of the mechanisms by which Epstein-Barr virus (EBV) miRNA EBV-miR-BHRF1-2 promotes EBV lymphomagenesis [48]. In addition, miR-9 targets DICER1 and HuR, a regulator of cytokine expression and inhibition of miR-9 resulted in higher cytokine production
CHAPTER 1
levels [49]. DICER1 has been indicated as a candidate predisposing gene in familial HL [50]. In summary, it is evident that miRNAs are involved in the pathogenesis of HL and deregulated miRNAs have an effect on different cellular pathways (Figure 2).
Figure 2. Overview of miRNAs and target genes involved in HL pathogenesis. MiRNAs with aberrant expression in HRS cells include miR-9, miR-17 seed family, miR-96, miR-155, miR-182 and miR-183, which are all increased in HL and play roles in inhibition of cell differentiation and cell apoptosis, production of cytokines and inducing proliferation. Moreover, miR-135a is downregulated in HL and this leads to inhibition of apoptosis due to derepression of JAK2 [34, 45-47, 49, 51, 52]. Adapted from [53].
4. Cancer stem cells
Cancer stem cells (CSCs) are a subpopulation of cancer cells that share characteristics with normal stem cells including the potential to self-renew and differentiate [54]. Self-renewal is defined as a cell division that enables a stem cell to produce another stem cell with the same characteristics (Figure 3). The second daughter cell can subsequently differentiate into a tissue-specific more differentiated cell [55]. Aldehyde dehydrogenase (ALDH), a cytosolic enzyme responsible for oxidizing a variety of intracellular aldehydes to carboxylic acids [56], is a commonly used stem cell marker [57]. Its expression is enriched in hematopoietic stem cells and progenitor cells with enhanced self-renewal capacity [57].
Most cancer cells have a limited ability to proliferate, whereas, a small subset of the cancer cells is able to proliferate extensively. Dick and colleagues showed that a small subset of human acute myeloid leukemia (AML) cells was phenotypically similar to normal hematopoietic stem cells and these cells could transfer AML when transplanted
into immune-deficient mice [58]. Other AML cells were unable to induce leukemia upon transplantation [59]. To date, the existence of CSCs has been proven in leukemia, glioblastoma [60], gastric cancer [61], breast cancer [62], prostate cancer [63], lung cancer [64] and colon cancer [65]. CSCs are generated upon genetic aberrations that are associated with oncogenesis and play essential roles in tumor formation and treatment resistance [66]. In general, CSCs are more difficult to eradicate and become resistant to therapy causing local relapse or upon migration distant metastasis [67].
Figure 3. Model of cancer cell division and cancer stem cell (CSC) division. A cancer cell (A, blue) can divide symmetrically into two daughter cancer cells. The CSC (B, red) can divide symmetrically into CSCs and asymmetrically into stem cells and cancer cells. CSCs proliferate faster than normal cancer cells.
5. Cancer stem cells in Hodgkin lymphoma
The presence of CSCs in HL cell lines might interfere with high throughput screening studies as performed in this thesis. A few studies reported presence of a side population (SP) or cancer initiating cells in HL cell lines [68-71]. Jones and colleagues showed that L428 and KM-H2 contain a subpopulation of cells that express the immunoglobulin light chain, the memory B-cell antigen CD27, and the stem cell marker ALDH. [68] The ALDH+ subpopulation of cells was responsible for the generation and maintenance of the dominant multinucleated RS cell population. In addition, they
showed that ALDH+ cells had a better colony forming ability than the ALDH- HL cells.
They also showed a small population of clonotypic B cells in HL patients by sequencing of the Ig complementary determining region 3 (CDR3) PCR products of circulating
light-chain restricted CD27 ALDHhigh B cells sorted from 2 newly diagnosed HL patients.
The sequence of these cells was identical to those of the HRS cells of the same patients. These data supported presence of clonotypic B cells or CSCs in HL patients. In another study, Schafer et al showed a SP in L428 and HDLM2 HL cell lines, but not in L1236 and L540 cell lines. The SP cells showed increased resistance to gemcitabine,
1
15
Introduction and scope of the thesis
14
levels [49]. DICER1 has been indicated as a candidate predisposing gene in familial HL [50]. In summary, it is evident that miRNAs are involved in the pathogenesis of HL and deregulated miRNAs have an effect on different cellular pathways (Figure 2).
Figure 2. Overview of miRNAs and target genes involved in HL pathogenesis. MiRNAs with aberrant expression in HRS cells include miR-9, miR-17 seed family, miR-96, miR-155, miR-182 and miR-183, which are all increased in HL and play roles in inhibition of cell differentiation and cell apoptosis, production of cytokines and inducing proliferation. Moreover, miR-135a is downregulated in HL and this leads to inhibition of apoptosis due to derepression of JAK2 [34, 45-47, 49, 51, 52]. Adapted from [53].
4. Cancer stem cells
Cancer stem cells (CSCs) are a subpopulation of cancer cells that share characteristics with normal stem cells including the potential to self-renew and differentiate [54]. Self-renewal is defined as a cell division that enables a stem cell to produce another stem cell with the same characteristics (Figure 3). The second daughter cell can subsequently differentiate into a tissue-specific more differentiated cell [55]. Aldehyde dehydrogenase (ALDH), a cytosolic enzyme responsible for oxidizing a variety of intracellular aldehydes to carboxylic acids [56], is a commonly used stem cell marker [57]. Its expression is enriched in hematopoietic stem cells and progenitor cells with enhanced self-renewal capacity [57].
Most cancer cells have a limited ability to proliferate, whereas, a small subset of the cancer cells is able to proliferate extensively. Dick and colleagues showed that a small subset of human acute myeloid leukemia (AML) cells was phenotypically similar to normal hematopoietic stem cells and these cells could transfer AML when transplanted
15
into immune-deficient mice [58]. Other AML cells were unable to induce leukemia upon transplantation [59]. To date, the existence of CSCs has been proven in leukemia, glioblastoma [60], gastric cancer [61], breast cancer [62], prostate cancer [63], lung cancer [64] and colon cancer [65]. CSCs are generated upon genetic aberrations that are associated with oncogenesis and play essential roles in tumor formation and treatment resistance [66]. In general, CSCs are more difficult to eradicate and become resistant to therapy causing local relapse or upon migration distant metastasis [67].
Figure 3. Model of cancer cell division and cancer stem cell (CSC) division. A cancer cell (A, blue) can divide symmetrically into two daughter cancer cells. The CSC (B, red) can divide symmetrically into CSCs and asymmetrically into stem cells and cancer cells. CSCs proliferate faster than normal cancer cells.
5. Cancer stem cells in Hodgkin lymphoma
The presence of CSCs in HL cell lines might interfere with high throughput screening studies as performed in this thesis. A few studies reported presence of a side population (SP) or cancer initiating cells in HL cell lines [68-71]. Jones and colleagues showed that L428 and KM-H2 contain a subpopulation of cells that express the immunoglobulin light chain, the memory B-cell antigen CD27, and the stem cell marker ALDH. [68] The ALDH+ subpopulation of cells was responsible for the generation and maintenance of the dominant multinucleated RS cell population. In addition, they showed that ALDH+ cells had a better colony forming ability than the ALDH- HL cells.
They also showed a small population of clonotypic B cells in HL patients by sequencing of the Ig complementary determining region 3 (CDR3) PCR products of circulating light-chain restricted CD27 ALDHhigh B cells sorted from 2 newly diagnosed HL patients.
The sequence of these cells was identical to those of the HRS cells of the same patients. These data supported presence of clonotypic B cells or CSCs in HL patients. In another study, Schafer et al showed a SP in L428 and HDLM2 HL cell lines, but not in L1236 and L540 cell lines. The SP cells showed increased resistance to gemcitabine,
CHAPTER 1
a common drug for the treatment of refractory HL and increased cell viability compared to non-SP cells [69]. The third study on SP cells compared single nucleated H cells to multinucleated RS cells in L1236 and L428 HL cell lines [70]. Cultures of H cells yielded both H and RS cells, whereas RS cell cultures yielded only RS cells and no cells with the H cell phenotype. The proliferative capacity of H cells in NOD/SCID mice was substantial, whereas RS cells did not proliferate. This study supported the presence of a CSC population within the single nucleated H cell population of the HL cell lines. Nakashima and colleagues identified SP cells in KM-H2 and L428 HL cell lines. These SP cells consisted almost entirely of distinct small mononuclear cells, whereas the non-SP cells were a mixture of relatively large cells with H or RS cell-like morphology [71].
Scope of the thesis
HL is a characteristic disease with a minority of tumor cells that have lost most of the common B-cell markers. The identification of differentially expressed miRNAs in HL and functional follow-up studies will improve our understanding of the pathogenesis of HL. Although some studies showed a pathogenic role of specific miRNAs in HL, the role of most of the aberrantly expressed miRNAs is still unknown.
The aim of this thesis was to investigate the expression and function of miRNAs and
unravel their underlying pathogenic mechanisms in cHL. In chapter 2, small RNA
sequencing and Ago2-IP was performed on HL cell lines to determine differentially expressed known and novel miRNAs and to characterize the miRNA-targetome in HL cell lines. We selected miR-24 for further characterization of its role in the growth of
HRS cells. In chapter 3, we explored the feasibility of a high throughput screen in HL.
In the first part (chapter 3A), we used a barcoded empty vector library screen to
determine whether a next generation sequencing (NGS) based high throughput screen
can be applied in HL cell lines. In the second part (chapter 3B), we performed a pilot
experiment with a pool of miRNA overexpression and inhibition constructs to test the actual feasibility of the NGS-based approach to identify miRNAs that regulate growth
of cHL cell lines and to set up the pipeline for data analysis. In chapter 4, after
optimization of the experimental conditions and the pipeline for data analysis, we performed a high-throughput miRNA overexpression screen to determine the effect on HL cell growth. The effect of selected miRNAs was validated by GFP competition
assays. In chapter 5, we performed a high-throughput screen in HL cell lines with a
pool of miRNA/gene inhibition (n=249) constructs. We selected miR-21 for further
evaluation of its role in HL cell growth and the underlying mechanisms. In chapter 6,
we summarized and discuss the results presented in this thesis and present future perspectives.
References
1. Bartel, D.P., MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004. 116(2): p. 281-97.
2. Kanellopoulou, C., et al., Dicer-deficient mouse embryonic stem cells are defective in
differentiation and centromeric silencing. Genes Dev, 2005. 19(4): p. 489-501.
3. Lee, Y., et al., MicroRNA genes are transcribed by RNA polymerase II. EMBO J, 2004. 23(20): p. 4051-60.
4. Kim, V.N. and J.W. Nam, Genomics of microRNA. Trends Genet, 2006. 22(3): p. 165-73. 5. Rodriguez, A., et al., Identification of mammalian microRNA host genes and transcription
units. Genome Res, 2004. 14(10A): p. 1902-10.
6. Gregory, R.I., T.P. Chendrimada, and R. Shiekhattar, MicroRNA biogenesis: isolation and
characterization of the microprocessor complex. Methods Mol Biol, 2006. 342: p. 33-47.
7. Lund, E. and J.E. Dahlberg, Substrate selectivity of exportin 5 and Dicer in the biogenesis
of microRNAs. Cold Spring Harb Symp Quant Biol, 2006. 71: p. 59-66.
8. Song, J.J., et al., Crystal structure of Argonaute and its implications for RISC slicer activity. Science, 2004. 305(5689): p. 1434-7.
9. Ma, J.B., et al., Structural basis for 5'-end-specific recognition of guide RNA by the A.
fulgidus Piwi protein. Nature, 2005. 434(7033): p. 666-70.
10. Lim, L.P., et al., Microarray analysis shows that some microRNAs downregulate large
numbers of target mRNAs. Nature, 2005. 433(7027): p. 769-73.
11. Lewis, B.P., et al., Prediction of mammalian microRNA targets. Cell, 2003. 115(7): p. 787-98.
12. John, B., C. Sander, and D.S. Marks, Prediction of human microRNA targets. Methods Mol Biol, 2006. 342: p. 101-13.
13. Ritchie, W., J.E. Rasko, and S. Flamant, MicroRNA target prediction and validation. Adv Exp Med Biol, 2013. 774: p. 39-53.
14. Calin, G.A., et al., Frequent deletions and down-regulation of micro- RNA genes miR15 and
miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A, 2002. 99(24):
p. 15524-9.
15. He, L., et al., A microRNA polycistron as a potential human oncogene. Nature, 2005. 435(7043): p. 828-33.
16. Mraz, M., et al., MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease
subtypes. Leuk Lymphoma, 2009. 50(3): p. 506-9.
17. Kroesen, B.J., et al., Immuno-miRs: critical regulators of T-cell development, function and
ageing. Immunology, 2015. 144(1): p. 1-10.
18. Kuppers, R., The biology of Hodgkin's lymphoma. Nat Rev Cancer, 2009. 9(1): p. 15-27. 19. Drakos, E., et al., Nodular lymphocyte predominant Hodgkin lymphoma with clusters of LP
Cells, acute inflammation, and fibrosis: a syncytial variant. Am J Surg Pathol, 2009. 33(11):
p. 1725-31.
20. Kuppers, R., et al., Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from
histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A, 1994.
91(23): p. 10962-6.
21. Watanabe, K., et al., Varied B-cell immunophenotypes of Hodgkin/Reed-Sternberg cells in
classic Hodgkin's disease. Histopathology, 2000. 36(4): p. 353-61.
22. Hertel, C.B., et al., Loss of B cell identity correlates with loss of B cell-specific transcription
factors in Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma. Oncogene, 2002.
21(32): p. 4908-20.
23. Smith, C.A., et al., CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose
ligand defines an emerging family of cytokines with homology to TNF. Cell, 1993. 73(7): p.
1349-60.
24. Delabie, J., et al., The antigen-presenting cell function of Reed-Sternberg cells. Leuk Lymphoma, 1995. 18(1-2): p. 35-40.
25. Schwering, I., et al., Loss of the B-lineage-specific gene expression program in Hodgkin
1
17
Introduction and scope of the thesis
16
a common drug for the treatment of refractory HL and increased cell viability compared to non-SP cells [69]. The third study on SP cells compared single nucleated H cells to multinucleated RS cells in L1236 and L428 HL cell lines [70]. Cultures of H cells yielded both H and RS cells, whereas RS cell cultures yielded only RS cells and no cells with the H cell phenotype. The proliferative capacity of H cells in NOD/SCID mice was substantial, whereas RS cells did not proliferate. This study supported the presence of a CSC population within the single nucleated H cell population of the HL cell lines. Nakashima and colleagues identified SP cells in KM-H2 and L428 HL cell lines. These SP cells consisted almost entirely of distinct small mononuclear cells, whereas the non-SP cells were a mixture of relatively large cells with H or RS cell-like morphology [71].
Scope of the thesis
HL is a characteristic disease with a minority of tumor cells that have lost most of the common B-cell markers. The identification of differentially expressed miRNAs in HL and functional follow-up studies will improve our understanding of the pathogenesis of HL. Although some studies showed a pathogenic role of specific miRNAs in HL, the role of most of the aberrantly expressed miRNAs is still unknown.
The aim of this thesis was to investigate the expression and function of miRNAs and unravel their underlying pathogenic mechanisms in cHL. In chapter 2, small RNA
sequencing and Ago2-IP was performed on HL cell lines to determine differentially expressed known and novel miRNAs and to characterize the miRNA-targetome in HL cell lines. We selected miR-24 for further characterization of its role in the growth of HRS cells. In chapter 3, we explored the feasibility of a high throughput screen in HL.
In the first part (chapter 3A), we used a barcoded empty vector library screen to
determine whether a next generation sequencing (NGS) based high throughput screen can be applied in HL cell lines. In the second part (chapter 3B), we performed a pilot
experiment with a pool of miRNA overexpression and inhibition constructs to test the actual feasibility of the NGS-based approach to identify miRNAs that regulate growth of cHL cell lines and to set up the pipeline for data analysis. In chapter 4, after
optimization of the experimental conditions and the pipeline for data analysis, we performed a high-throughput miRNA overexpression screen to determine the effect on HL cell growth. The effect of selected miRNAs was validated by GFP competition assays. In chapter 5, we performed a high-throughput screen in HL cell lines with a
pool of miRNA/gene inhibition (n=249) constructs. We selected miR-21 for further evaluation of its role in HL cell growth and the underlying mechanisms. In chapter 6,
we summarized and discuss the results presented in this thesis and present future perspectives.
17
References
1. Bartel, D.P., MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004.
116(2): p. 281-97.
2. Kanellopoulou, C., et al., Dicer-deficient mouse embryonic stem cells are defective in
differentiation and centromeric silencing. Genes Dev, 2005. 19(4): p. 489-501.
3. Lee, Y., et al., MicroRNA genes are transcribed by RNA polymerase II. EMBO J, 2004.
23(20): p. 4051-60.
4. Kim, V.N. and J.W. Nam, Genomics of microRNA. Trends Genet, 2006. 22(3): p. 165-73.
5. Rodriguez, A., et al., Identification of mammalian microRNA host genes and transcription
units. Genome Res, 2004. 14(10A): p. 1902-10.
6. Gregory, R.I., T.P. Chendrimada, and R. Shiekhattar, MicroRNA biogenesis: isolation and
characterization of the microprocessor complex. Methods Mol Biol, 2006. 342: p. 33-47.
7. Lund, E. and J.E. Dahlberg, Substrate selectivity of exportin 5 and Dicer in the biogenesis
of microRNAs. Cold Spring Harb Symp Quant Biol, 2006. 71: p. 59-66.
8. Song, J.J., et al., Crystal structure of Argonaute and its implications for RISC slicer activity.
Science, 2004. 305(5689): p. 1434-7.
9. Ma, J.B., et al., Structural basis for 5'-end-specific recognition of guide RNA by the A.
fulgidus Piwi protein. Nature, 2005. 434(7033): p. 666-70.
10. Lim, L.P., et al., Microarray analysis shows that some microRNAs downregulate large
numbers of target mRNAs. Nature, 2005. 433(7027): p. 769-73.
11. Lewis, B.P., et al., Prediction of mammalian microRNA targets. Cell, 2003. 115(7): p.
787-98.
12. John, B., C. Sander, and D.S. Marks, Prediction of human microRNA targets. Methods Mol
Biol, 2006. 342: p. 101-13.
13. Ritchie, W., J.E. Rasko, and S. Flamant, MicroRNA target prediction and validation. Adv
Exp Med Biol, 2013. 774: p. 39-53.
14. Calin, G.A., et al., Frequent deletions and down-regulation of micro- RNA genes miR15 and
miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A, 2002. 99(24): p. 15524-9.
15. He, L., et al., A microRNA polycistron as a potential human oncogene. Nature, 2005.
435(7043): p. 828-33.
16. Mraz, M., et al., MicroRNAs in chronic lymphocytic leukemia pathogenesis and disease
subtypes. Leuk Lymphoma, 2009. 50(3): p. 506-9.
17. Kroesen, B.J., et al., Immuno-miRs: critical regulators of T-cell development, function and
ageing. Immunology, 2015. 144(1): p. 1-10.
18. Kuppers, R., The biology of Hodgkin's lymphoma. Nat Rev Cancer, 2009. 9(1): p. 15-27.
19. Drakos, E., et al., Nodular lymphocyte predominant Hodgkin lymphoma with clusters of LP
Cells, acute inflammation, and fibrosis: a syncytial variant. Am J Surg Pathol, 2009. 33(11): p. 1725-31.
20. Kuppers, R., et al., Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from
histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development. Proc Natl Acad Sci U S A, 1994. 91(23): p. 10962-6.
21. Watanabe, K., et al., Varied B-cell immunophenotypes of Hodgkin/Reed-Sternberg cells in
classic Hodgkin's disease. Histopathology, 2000. 36(4): p. 353-61.
22. Hertel, C.B., et al., Loss of B cell identity correlates with loss of B cell-specific transcription
factors in Hodgkin/Reed-Sternberg cells of classical Hodgkin lymphoma. Oncogene, 2002. 21(32): p. 4908-20.
23. Smith, C.A., et al., CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose
ligand defines an emerging family of cytokines with homology to TNF. Cell, 1993. 73(7): p. 1349-60.
24. Delabie, J., et al., The antigen-presenting cell function of Reed-Sternberg cells. Leuk
Lymphoma, 1995. 18(1-2): p. 35-40.
25. Schwering, I., et al., Loss of the B-lineage-specific gene expression program in Hodgkin
CHAPTER 1
26. Schmitz, R., et al., Pathogenesis of classical and lymphocyte-predominant Hodgkin
lymphoma. Annu Rev Pathol, 2009. 4: p. 151-74.
27. Diehl, V., et al., Standard and increased-dose BEACOPP chemotherapy compared with
COPP-ABVD for advanced Hodgkin's disease. N Engl J Med, 2003. 348(24): p. 2386-95.
28. Medina, P.P., M. Nolde, and F.J. Slack, OncomiR addiction in an in vivo model of
microRNA-21-induced pre-B-cell lymphoma. Nature, 2010. 467(7311): p. 86-90.
29. Cordeiro, A., M. Monzo, and A. Navarro, Non-Coding RNAs in Hodgkin Lymphoma. Int J Mol Sci, 2017. 18(6).
30. Sole, C., et al., miRNAs in B-cell lymphoma: Molecular mechanisms and biomarker
potential. Cancer Lett, 2017. 405: p. 79-89.
31. van den Berg, A., et al., High expression of B-cell receptor inducible gene BIC in all
subtypes of Hodgkin lymphoma. Genes Chromosomes Cancer, 2003. 37(1): p. 20-8.
32. Lagos-Quintana, M., et al., Identification of tissue-specific microRNAs from mouse. Curr Biol, 2002. 12(9): p. 735-9.
33. Kluiver, J., et al., BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal
and diffuse large B cell lymphomas. J Pathol, 2005. 207(2): p. 243-9.
34. Slezak-Prochazka, I., et al., Inhibition of the miR-155 target NIAM phenocopies the growth
promoting effect of miR-155 in B-cell lymphoma. Oncotarget, 2016. 7(3): p. 2391-400.
35. Kluiver, J., et al., Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt
lymphoma. Genes Chromosomes Cancer, 2006. 45(2): p. 147-53.
36. Muramatsu, M., et al., Class switch recombination and hypermutation require
activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell, 2000. 102(5): p.
553-63.
37. Dorsett, Y., et al., MicroRNA-155 suppresses activation-induced cytidine
deaminase-mediated Myc-Igh translocation. Immunity, 2008. 28(5): p. 630-8.
38. Landgraf, P., et al., A mammalian microRNA expression atlas based on small RNA library
sequencing. Cell, 2007. 129(7): p. 1401-14.
39. Gibcus, J.H., et al., Hodgkin lymphoma cell lines are characterized by a specific miRNA
expression profile. Neoplasia, 2009. 11(2): p. 167-76.
40. Navarro, A., et al., MicroRNA expression profiling in classic Hodgkin lymphoma. Blood, 2008. 111(5): p. 2825-32.
41. Van Vlierberghe, P., et al., Comparison of miRNA profiles of microdissected
Hodgkin/Reed-Sternberg cells and Hodgkin cell lines versus CD77+ B-cells reveals a distinct subset of
differentially expressed miRNAs. Br J Haematol, 2009. 147(5): p. 686-90.
42. Paydas, S., et al., Micro-RNA (miRNA) profile in Hodgkin lymphoma: association between
clinical and pathological variables. Med Oncol, 2016. 33(4): p. 34.
43. van Eijndhoven, M.A., et al., Plasma vesicle miRNAs for therapy response monitoring in
Hodgkin lymphoma patients. JCI Insight, 2016. 1(19): p. e89631.
44. Khare, D., et al., Plasma microRNA profiling: Exploring better biomarkers for lymphoma
surveillance. PLoS One, 2017. 12(11): p. e0187722.
45. Navarro, A., et al., Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin
lymphoma. Blood, 2009. 114(14): p. 2945-51.
46. Gibcus, J.H., et al., MiR-17/106b seed family regulates p21 in Hodgkin's lymphoma. J Pathol, 2011. 225(4): p. 609-17.
47. Nie, K., et al., MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in
Hodgkin/Reed-Sternberg cells: a potential pathogenetic lesion in Hodgkin lymphomas. Am J Pathol, 2008.
173(1): p. 242-52.
48. Ma, J., et al., EBV-miR-BHRF1-2 targets PRDM1/Blimp1: potential role in EBV
lymphomagenesis. Leukemia, 2016. 30(3): p. 594-604.
49. Leucci, E., et al., Inhibition of miR-9 de-represses HuR and DICER1 and impairs Hodgkin
lymphoma tumour outgrowth in vivo. Oncogene, 2012. 31(49): p. 5081-9.
50. Bandapalli, O.R., et al., Whole genome sequencing reveals DICER1 as a candidate
predisposing gene in familial Hodgkin lymphoma. Int J Cancer, 2018.
51. Petri, S., et al., Increased siRNA duplex stability correlates with reduced off-target and
elevated on-target effects. RNA, 2011. 17(4): p. 737-49.
52. Leucci, E., et al., microRNA-9 targets the long non-coding RNA MALAT1 for degradation in
the nucleus. Sci Rep, 2013. 3: p. 2535.
53. Lawrie, C.H., MicroRNAs in Medicine. 2014: Wiley.
54. Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p. 105-11.
55. Lobo, N.A., et al., The biology of cancer stem cells. Annu Rev Cell Dev Biol, 2007. 23: p. 675-99.
56. Russo, J.E. and J. Hilton, Characterization of cytosolic aldehyde dehydrogenase from
cyclophosphamide resistant L1210 cells. Cancer Res, 1988. 48(11): p. 2963-8.
57. Ginestier, C., et al., ALDH1 is a marker of normal and malignant human mammary stem
cells and a predictor of poor clinical outcome. Cell Stem Cell, 2007. 1(5): p. 555-67.
58. Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.
59. Lapidot, T., et al., A cell initiating human acute myeloid leukaemia after transplantation into
SCID mice. Nature, 1994. 367(6464): p. 645-8.
60. Singh, S.K., et al., Identification of a cancer stem cell in human brain tumors. Cancer Res, 2003. 63(18): p. 5821-8.
61. Houghton, J., et al., Gastric cancer originating from bone marrow-derived cells. Science, 2004. 306(5701): p. 1568-71.
62. Ponti, D., et al., Isolation and in vitro propagation of tumorigenic breast cancer cells with
stem/progenitor cell properties. Cancer Res, 2005. 65(13): p. 5506-11.
63. Collins, A.T., et al., Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res, 2005. 65(23): p. 10946-51.
64. Kim, C.F., et al., Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell, 2005. 121(6): p. 823-35.
65. O'Brien, C.A., et al., A human colon cancer cell capable of initiating tumour growth in
immunodeficient mice. Nature, 2007. 445(7123): p. 106-10.
66. Greaves, M. and C.C. Maley, Clonal evolution in cancer. Nature, 2012. 481(7381): p. 306-13.
67. Jordan, C.T., M.L. Guzman, and M. Noble, Cancer stem cells. N Engl J Med, 2006. 355(12): p. 1253-61.
68. Jones, R.J., et al., Circulating clonotypic B cells in classic Hodgkin lymphoma. Blood, 2009. 113(23): p. 5920-6.
69. Shafer, J.A., et al., Antigen-specific cytotoxic T lymphocytes can target chemoresistant
side-population tumor cells in Hodgkin lymphoma. Leuk Lymphoma, 2010. 51(5): p.
870-80.
70. Ikeda, J., et al., Tumorigenic potential of mononucleated small cells of Hodgkin lymphoma
cell lines. Am J Pathol, 2010. 177(6): p. 3081-8.
71. Nakashima, M., et al., The side population, as a precursor of Hodgkin and Reed-Sternberg
cells and a target for nuclear factor-kappaB inhibitors in Hodgkin's lymphoma. Cancer Sci,
1
19
Introduction and scope of the thesis
18
26. Schmitz, R., et al., Pathogenesis of classical and lymphocyte-predominant Hodgkin
lymphoma. Annu Rev Pathol, 2009. 4: p. 151-74.
27. Diehl, V., et al., Standard and increased-dose BEACOPP chemotherapy compared with
COPP-ABVD for advanced Hodgkin's disease. N Engl J Med, 2003. 348(24): p. 2386-95.
28. Medina, P.P., M. Nolde, and F.J. Slack, OncomiR addiction in an in vivo model of
microRNA-21-induced pre-B-cell lymphoma. Nature, 2010. 467(7311): p. 86-90.
29. Cordeiro, A., M. Monzo, and A. Navarro, Non-Coding RNAs in Hodgkin Lymphoma. Int J
Mol Sci, 2017. 18(6).
30. Sole, C., et al., miRNAs in B-cell lymphoma: Molecular mechanisms and biomarker
potential. Cancer Lett, 2017. 405: p. 79-89.
31. van den Berg, A., et al., High expression of B-cell receptor inducible gene BIC in all
subtypes of Hodgkin lymphoma. Genes Chromosomes Cancer, 2003. 37(1): p. 20-8.
32. Lagos-Quintana, M., et al., Identification of tissue-specific microRNAs from mouse. Curr
Biol, 2002. 12(9): p. 735-9.
33. Kluiver, J., et al., BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal
and diffuse large B cell lymphomas. J Pathol, 2005. 207(2): p. 243-9.
34. Slezak-Prochazka, I., et al., Inhibition of the miR-155 target NIAM phenocopies the growth
promoting effect of miR-155 in B-cell lymphoma. Oncotarget, 2016. 7(3): p. 2391-400.
35. Kluiver, J., et al., Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt
lymphoma. Genes Chromosomes Cancer, 2006. 45(2): p. 147-53.
36. Muramatsu, M., et al., Class switch recombination and hypermutation require
activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell, 2000. 102(5): p. 553-63.
37. Dorsett, Y., et al., MicroRNA-155 suppresses activation-induced cytidine
deaminase-mediated Myc-Igh translocation. Immunity, 2008. 28(5): p. 630-8.
38. Landgraf, P., et al., A mammalian microRNA expression atlas based on small RNA library
sequencing. Cell, 2007. 129(7): p. 1401-14.
39. Gibcus, J.H., et al., Hodgkin lymphoma cell lines are characterized by a specific miRNA
expression profile. Neoplasia, 2009. 11(2): p. 167-76.
40. Navarro, A., et al., MicroRNA expression profiling in classic Hodgkin lymphoma. Blood,
2008. 111(5): p. 2825-32.
41. Van Vlierberghe, P., et al., Comparison of miRNA profiles of microdissected
Hodgkin/Reed-Sternberg cells and Hodgkin cell lines versus CD77+ B-cells reveals a distinct subset of differentially expressed miRNAs. Br J Haematol, 2009. 147(5): p. 686-90.
42. Paydas, S., et al., Micro-RNA (miRNA) profile in Hodgkin lymphoma: association between
clinical and pathological variables. Med Oncol, 2016. 33(4): p. 34.
43. van Eijndhoven, M.A., et al., Plasma vesicle miRNAs for therapy response monitoring in
Hodgkin lymphoma patients. JCI Insight, 2016. 1(19): p. e89631.
44. Khare, D., et al., Plasma microRNA profiling: Exploring better biomarkers for lymphoma
surveillance. PLoS One, 2017. 12(11): p. e0187722.
45. Navarro, A., et al., Regulation of JAK2 by miR-135a: prognostic impact in classic Hodgkin
lymphoma. Blood, 2009. 114(14): p. 2945-51.
46. Gibcus, J.H., et al., MiR-17/106b seed family regulates p21 in Hodgkin's lymphoma. J
Pathol, 2011. 225(4): p. 609-17.
47. Nie, K., et al., MicroRNA-mediated down-regulation of PRDM1/Blimp-1 in
Hodgkin/Reed-Sternberg cells: a potential pathogenetic lesion in Hodgkin lymphomas. Am J Pathol, 2008. 173(1): p. 242-52.
48. Ma, J., et al., EBV-miR-BHRF1-2 targets PRDM1/Blimp1: potential role in EBV
lymphomagenesis. Leukemia, 2016. 30(3): p. 594-604.
49. Leucci, E., et al., Inhibition of miR-9 de-represses HuR and DICER1 and impairs Hodgkin
lymphoma tumour outgrowth in vivo. Oncogene, 2012. 31(49): p. 5081-9.
50. Bandapalli, O.R., et al., Whole genome sequencing reveals DICER1 as a candidate
predisposing gene in familial Hodgkin lymphoma. Int J Cancer, 2018.
51. Petri, S., et al., Increased siRNA duplex stability correlates with reduced off-target and
elevated on-target effects. RNA, 2011. 17(4): p. 737-49.
52. Leucci, E., et al., microRNA-9 targets the long non-coding RNA MALAT1 for degradation in
the nucleus. Sci Rep, 2013. 3: p. 2535.
19
53. Lawrie, C.H., MicroRNAs in Medicine. 2014: Wiley.
54. Reya, T., et al., Stem cells, cancer, and cancer stem cells. Nature, 2001. 414(6859): p.
105-11.
55. Lobo, N.A., et al., The biology of cancer stem cells. Annu Rev Cell Dev Biol, 2007. 23: p.
675-99.
56. Russo, J.E. and J. Hilton, Characterization of cytosolic aldehyde dehydrogenase from
cyclophosphamide resistant L1210 cells. Cancer Res, 1988. 48(11): p. 2963-8.
57. Ginestier, C., et al., ALDH1 is a marker of normal and malignant human mammary stem
cells and a predictor of poor clinical outcome. Cell Stem Cell, 2007. 1(5): p. 555-67.
58. Bonnet, D. and J.E. Dick, Human acute myeloid leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med, 1997. 3(7): p. 730-7.
59. Lapidot, T., et al., A cell initiating human acute myeloid leukaemia after transplantation into
SCID mice. Nature, 1994. 367(6464): p. 645-8.
60. Singh, S.K., et al., Identification of a cancer stem cell in human brain tumors. Cancer Res,
2003. 63(18): p. 5821-8.
61. Houghton, J., et al., Gastric cancer originating from bone marrow-derived cells. Science,
2004. 306(5701): p. 1568-71.
62. Ponti, D., et al., Isolation and in vitro propagation of tumorigenic breast cancer cells with
stem/progenitor cell properties. Cancer Res, 2005. 65(13): p. 5506-11.
63. Collins, A.T., et al., Prospective identification of tumorigenic prostate cancer stem cells.
Cancer Res, 2005. 65(23): p. 10946-51.
64. Kim, C.F., et al., Identification of bronchioalveolar stem cells in normal lung and lung cancer.
Cell, 2005. 121(6): p. 823-35.
65. O'Brien, C.A., et al., A human colon cancer cell capable of initiating tumour growth in
immunodeficient mice. Nature, 2007. 445(7123): p. 106-10.
66. Greaves, M. and C.C. Maley, Clonal evolution in cancer. Nature, 2012. 481(7381): p.
306-13.
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CHAPTER 2
MicroRNA-24-3p is overexpressed in Hodgkin
lymphoma and protects Hodgkin and
Reed-Sternberg cells from apoptosis
Ye Yuan*¶, Joost Kluiver*, Jasper Koerts*, Debora de Jong*, Bea Rutgers*, F. ReenyAbdul Razak*, Martijn Terpstra§, Boudewijn E. Plaat II, Ilja M. Nolte‡, Arjan Diepstra*,
Lydia Visser*, Klaas Kok§, Anke van den Berg*‡
Department of * Pathology and Medical Biology, § Genetics, ‡ Epidemiology,
II Otorhinolaryngology/Head and Neck Surgery, University of Groningen, University
Medical Center Groningen, Groningen, the Netherlands ¶ Institute of Clinical Pharmacology of the Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang Province, China
20 21
CHAPTER 2
MicroRNA-24-3p is overexpressed in Hodgkin
lymphoma and protects Hodgkin and
Reed-Sternberg cells from apoptosis
Ye Yuan*¶, Joost Kluiver*, Jasper Koerts*, Debora de Jong*, Bea Rutgers*, F. ReenyAbdul Razak*, Martijn Terpstra§, Boudewijn E. Plaat II, Ilja M. Nolte‡, Arjan Diepstra*,
Lydia Visser*, Klaas Kok§, Anke van den Berg*‡
Department of * Pathology and Medical Biology, § Genetics, ‡ Epidemiology,
II Otorhinolaryngology/Head and Neck Surgery, University of Groningen, University
Medical Center Groningen, Groningen, the Netherlands ¶ Institute of Clinical Pharmacology of the Second Affiliated Hospital, Harbin Medical University, Harbin, Heilongjiang Province, China
CHAPTER 2
Abstract
miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs miR-23a-3p, miR-24-3p, and miR-27a-3p were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three HL cell lines and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1,142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1,142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed
increased protein levels on miR-24-3p inhibition for CDKN1B/P27kip1 and MYC. In
summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs
cell growth possibly via targeting CDKN1B/P27kip1 and MYC.
Introduction
Hodgkin lymphoma (HL) is a B-cell–derived lymphoma classified into classic HL (cHL)
and nodular lymphocyte-predominant HL (NLPHL).[1]NLPHL is a more rare subtype
of HL accounting for approximately 5% of all cases.[2]CHL accounts for 95% of all HL
cases and is characterized by a minority of Hodgkin and Reed-Sternberg (HRS) tumor
cells,[3] which have lost their normal B-cell phenotype.[4] Furthermore, cHL is
subclassified according to the morphology of HRS cells and the composition of the cellular background into nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte-depleted cases.[5]
MiRNAs are short non-coding RNA molecules with unique expression patterns in different tissue and cell types.[6, 7] They inhibit gene expression by binding to complementary sequences at the 3’-untranslated region (UTR) of their target gene
transcripts.[8]One single miRNA can interact with multiple targets.[9]The first human
cancer type reported to be associated with miRNAs was chronic lymphocytic leukemia.[10] After that, many aberrant miRNA expression patterns have been linked
to specific types of cancer.[11]Depending on their set of target genes, miRNAs can act
as oncogenes or tumor suppressor genes.[12-14]
So far, multiple miRNAs are deregulated in B-cell lymphoma and for a subset of them pivotal functions have been shown in the pathogenesis.[10, 15, 16] Using small RNA sequencing, Landgraf et al generated amongst others miRNA expression profiles of
four EBV-cHL cell lines.[17]Van Vlierberghe and colleagues identified 12 up- and three
down-regulated miRNAs in microdissected HRS cells from nine cHL patients and HL
cell lines compared to CD77+ GC-B cells.[18] Gibcus et al determined the miRNA
profile of HL cell lines in comparison to GC-B cell–derived lymphoblastoid cell lines and other B-cell lymphoma cell lines and showed increased expression of the
miR-17~92 cluster, miR-16, miR-21, miR-24, and miR-155 in HL.[19]Functional studies in
HL are limited, but for some of the miRNAs their putative role has been established.
MiR-135a targets JAK2, which leads to reduced Bcl-xL levels in HL.[20] The
miR-17/106b seed family targets CDKN1A encoding for the P21 protein and inhibition of
this seed family results in a G1-phase cell cycle arrest.[21]HuR and Dicer were shown
to be targets of the oncogenic miR-9 and inhibition of miR-9 resulted in higher cytokine
production levels.[22] A significant correlation between miR-124a methylation status
and a high-risk international prognostic score was found in HL.[23]
Here, we established an HL-specific miRNA expression profile using small RNA sequencing and validated differential expression of selected miRNAs. Furthermore, we determined the effects of miR-23a/b-3p, miR-24-3p, and miR-27a/b-3p inhibition on cell growth. To identify target genes regulated by these miRNAs, Ago2 RNA immunoprecipitation (Ago2-RIP) followed by a microarray analysis was performed on
