Parental Imprinting Regulates Insulin-Like Growth Factor Signaling: a Rosetta Stone for Understanding the Biology of Pluripotent Stem Cells, Aging and Cancerogenesis

REVIEW Parental imprinting regulates insulin-like growth factor signaling: a Rosetta Stone for understanding the biology of pluripotent stem cells, aging and cancerogenesis

MZ Ratajczak1,3, D-M Shin2, G Schneider1, J Ratajczak1,3 and M Kucia1,3

In recent years, solid evidence has accumulated that insulin-like growth factor-1 (IGF-1) and 2 (IGF-2) regulate many biological processes in normal and malignant cells. Recently, more light has been shed on the epigenetic mechanisms regulating expression of genes involved in IGF signaling (IFS) and it has become evident that these mechanisms are crucial for initiation of embryogenesis, maintaining the quiescence of pluripotent stem cells deposited in adult tissues (for example, very-small embryonic- like stem cells), the aging process, and the malignant transformation of cells. The expression of several genes involved in IFS is regulated at the epigenetic level by imprinting/methylation within differentially methylated regions (DMRs), which regulate their expression from paternal or maternal chromosomes. The most important role in the regulation of IFS gene expression is played by the Igf-2-H19 locus, which encodes the autocrine/paracrine mitogen IGF-2 and the H19 gene, which gives rise to a non-coding RNA precursor of several microRNAs that negatively affect cell proliferation. Among these, miR-675 has recently been demonstrated to downregulate expression of the IGF-1 receptor. The proper imprinting of DMRs at the Igf-2-H19 locus, with methylation of the paternal chromosome and a lack of methylation on the maternal chromosome, regulates expression of these genes so that Igf-2 is transcribed only from the paternal chromosome and H19 (including miR-675) only from the maternal chromosome. In this review, we will discuss the relevance of (i) proper somatic imprinting, (ii) erasure of imprinting and (iii) loss of imprinting within the DMRs at the Igf-2-H19 locus to the expression of genes involved in IFS, and the consequences of these alternative patterns of imprinting for stem cell biology.

Leukemia (2013) 27, 773–779; doi:10.1038/leu.2012.322 Keywords: imprinting; Igf2-H19; PGCs; VSELs; longevity; cancerogenesis

INTRODUCTION her resources as possible toward future births (without Among the 3.0–3.5 Â 104 genes in the mammalian genome, there compromising the health of the fetus she is currently carrying) are B80 genes that are paternally imprinted and expressed from by epigenetic modulation of genes bearing maternal imprinting 1 the maternally or paternally derived chromosome only. This marks. pattern of expression regulates the appropriate dosage and level Evidence has accumulated that among the imprinted genes, the of expression of these genes in mammalian cells.1 The expression most important is insulin-like growth factor 2 (Igf-2)-H19. This of imprinted genes is regulated by the imposition of epigenetic tandem gene is imprinted both in mice and humans and regulates marks by DNA methylation within differentially methylated IGF-2 and insulin-like growth factor-1 (IGF-1) signaling, which 4 regions (DMRs), which are regulatory CpG-rich cis-elements at affects many vital aspects of cell biology. In particular, while the the gene locus.2 Most imprinted genes are methylated in mouse Igf-2 locus encodes IGF-2, which is an autocrine/paracrine on maternally derived chromosomes, and only four, Igf-2-H19, mitogen, transcription of H19 gives rise to a non-coding RNA RasGrf1, Dlk1/Dio3 and Zdbf2, are methylated on paternally (non-coding RNA) that is a precursor of several microRNAs that derived chromosomes.3 negatively affect cell proliferation. For example, it has recently According to the parent–offspring conﬂict theory, while been demonstrated that miR-675 is involved in downregulation of paternally expressed imprinted genes enhance embryo growth expression of the IGF-1 receptor (IGF-1R).5 The foregoing indicates and size of the offspring, the maternally expressed genes inhibit the dual role of this ‘yin-yang locus’, which involves opposite cell proliferation and somewhat negatively affect its size.1 Based functional effects of Igf-2 and H19 genes on cell proliferation, on this, during pregnancy, the father, through proper expression and suggests its important role in initiation and regulation of of paternally imprinted genes, contributes to body size and embryonic development.1 Furthermore, recent evidence indicates muscle mass of the developing fetus and ‘wants’ the mother to that erasure of imprinting (hypomethylation) of the DMRs at the devote as much of her resources as possible towards the growth Igf-2-H19 locus on both chromosomes, which leads to of his child. By contrast, the mother wants to conserve as much of downregulation of Igf-2 and upregulation of H19 expression, has

1Stem Cell Biology Program at the James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA; 2Department of Medicine, Graduate School, University of Ulsan, Seoul, Korea and 3Department of Physiology, Pomeranian Medical University, Szczecin, Poland. Correspondence: Professor MZ Ratajczak, Stem Cell Biology Program at the James Graham Brown Cancer Center, University of Louisville, 500 S. Floyd Street, Room 107, Louisville, KY 40202, USA. E-mail: [email protected] Received 2 October 2012; revised 23 October 2012; accepted 26 October 2012; accepted article preview online 8 November 2012; advance online publication, 23 November 2012 Igf-2-H19 as a master regulatory tandem gene MZ Ratajczak et al 774 an important role in regulating the quiescence of pluripotent stem chromosome depict methylation, and open lollypops on the cells residing in adult tissues and thus may be involved in the maternal chromosome indicate lack of methylation. If the DMR is regulation of life span.6–9 On the other hand, loss of imprinting methylated, it cannot bind the regulatory DNA-binding zinc finger (hypermethylation) of DMRs at this locus on both chromosomes insulator protein, CTCF, which establishes a functional boundary results in Igf-2 overexpression and H19 downregulation and is a between the Igf-2 and H19 transcription regions.11–12 The binding phenomenon observed in several malignancies.10 of CTCF has immediate consequences for expression of these loci. In this review, we will discuss the biological consequences of As expression of both Igf-2 and H19 is regulated by a 30-distal changes in imprinting at the Igf-2-H19 locus. We envision that the enhancer (shown as a red box), the presence of CTCF bound to the changes in expression of genes encoded at this locus are a kind of DMR at the maternal locus prevents transcription of Igf-2, and in ‘genetic Rosetta Stone’ that allows one to better understand this situation, only H19 is transcribed to RNA. In contrast, the development, aging and cancerogenesis. presence of a methylated DMR on the paternal chromosome prevents binding of CTCF, and in this situation, the 30-distal enhancer promotes transcription of mRNA from the Igf-2 locus. PROPER IMPRINTING OF THE IGF-2-H19 LOCUS AND INITIATION This ensures a proper balance in expression of both genes: Igf-2 OF EMBRYOGENESIS from the paternal and H19 from the maternal chromosome The tandem Igf-2-H19 locus is located on chromosome 11p15 in (Figure 1a). Overall, four CTCF-binding sites have been identified humans and chromosome 7 in mice and, as mentioned above, is so far in the murine Igf-2-H19 DMR and seven in its human paternally imprinted both in humans and in mice. This preserva- counterpart.13–14 Interestingly, the human Igf-2-H19 DMR is not tion across species suggests the importance of its involvement in able to function when introduced as a transgene into the murine mammalian development. genome, which suggests some species differences that tune the Figure 1a is a schematically simplified structure for this locus, regulation of this DMR.15 showing that the regulatory DMR is methylated on the paternal To explain the biological consequences of the gene expression chromosome and erased on the maternal chromosome. Accordingly, encoded by this locus, the IGF-2 protein product of the Igf-2 gene the filled lollypops at the DMR regulatory region of the paternal stimulates cells in both an autocrine and paracrine way after

Proper Somatic Imprint

CTCF Igf2 H19 M

Igf2 H19 Igf2 H19 P DMR Enhancer

Erasure of Imprint

CTCF Igf2 H19 M H19 CTCF Igf2 Igf2 H19 P Enhancer

Loss of Imprint

Igf2 H19 M DMR Igf2 H19 Igf2 H19 P DMR Enhancer Figure 1. Changes in the methylation state of DMRs and their impact on insulin-like growth factor (Igf)-2 and H19 expression. (a) Normal somatic imprinting at the Igf-2 and H19-coding regions are separated by a differentially methylated region (DMR) that is methylated (as shown by ﬁlled lollypops) on the paternal chromosome (P) and unmethylated (open lollypops) on the maternal chromosome (M). Expression of both genes is regulated by a 30- distal enhancer depicted in green. Methylation of the DMR on the paternal chromosome (P) prevents binding of the CTCF insulator protein and allows activation of the Igf-2 promoter by the distal enhancer and transcription of Igf-2 mRNA from the paternal chromosome (P) (red arrow). By contrast, as the DMR is unmethylated on the maternal chromosome (M), it binds CTCF, and this prevents activation of the Igf-2 promoter by the distal enhancer. As a result, only the H19 ncRNA is transcribed from the maternal chromosome (M) (red arrow). As the end result, the cell has a balanced expression of Igf-2 and H19 from both the chromosomes. (b) Erasure of imprinting at the Igf- 2-H19 locus as seen in PGCs and very-small embryonic-like stem cells (VSELs) residing post-developmentally in adult tissues. DMRs on both the paternal and maternal chromosomes are engaged by the CTCF insulator protein, and thus only the H19 ncRNA is transcribed (red arrows) from the maternal (M) and paternal (P) chromosomes, contributing to the quiescent state of these cells (lacking autocrine IGF-2). (c) Loss of imprinting at the Igf-2-H19 locus as seen in tumor cells from several types of cancer (e.g., rhabdomyosarcoma, nephroblastoma and gastrointestinal tumors). As both DMRs are methylated, the insulator protein CTCF cannot bind to the DNA and the distal enhancer stimulates transcription of mRNA for IGF-2 from both maternal (M) and paternal (P) chromosomes (red arrows). Cells that have this epigenetic change are under autocrine IGF-2 stimulation.

Leukemia (2013) 773 – 779 & 2013 Macmillan Publishers Limited Igf-2-H19 as a master regulatory tandem gene MZ Ratajczak et al 775 binding to IGF-1R and with lower affinity to the insulin receptor.16 (PGCs).23 Figure 1b depicts the consequences of this erasure of Cells also express the high-affinity-binding IGF-2 receptor (IGF-2R); methylation within the DMRs at the Igf-2-H19 locus, which leads to however, this is a non-signaling receptor that simply binds IGF-2 downregulation of growth-promoting IGF-2 from both paternal and prevents its signaling through IGF-1R and insulin receptor.17 and maternal chromosomes, and overexpression from these On other hand H19, as mentioned above, transcribes a long, 2.3-kb, chromosomes of proliferation-limiting H19 ncRNA. This is an ncRNA that is evolutionarily conserved at the nucleotide level in important regulatory mechanism that keeps PGCs quiescent and humans and rodents and not translated to protein. Instead, it is prevents them from teratoma formation. A similar phenomenon processed into small microRNAs18 of which miR-675 as mentioned also occurs in very-small embryonic-like stem cells (VSELs), which above negatively regulates the expression of IGF-1R.5 In addition, share several markers with migrating PGCs24–25 and are deposited in situ hybridization of the H19 ncRNA revealed that it is detectable during development in developing organs, including adult bone in cytoplasmic ribonucleoprotein particles, which suggests that the marrow (BM).26–28 Figure 2a shows a representative hypomethyla- H19-derived microRNAs are involved in ribosomal function and tion state of the DMR for the Igf-2-H19 locus in murine BM-derived translation. However, the loss of H19 is not lethal in mice, and such VSELs and normal (B50%) methylation observed in hematopoie- animals display an organ overgrowth phenotype similar to babies tic stem cells (HSCs). These changes in methylation of DMRs at this with Beckwith–Wiedemann syndrome.19 On the other hand, locus result, as shown in Figure 2b, in downregulation of IGF-2 overexpression of H19 is a dominant lethal mutation and mouse mRNA and upregulation of H19 ncRNA in VSELs. The biological embryos overexpressing H19 die after embryonic day 14. This may significance of this epigenetic modification in PGCs and VSELs will reflect its overall suppressive role in early stages of development be discussed below. involving suppression of IGF-1R expression,17 as well as negative regulation of other yet-to-be identified targets. The close coupling of Igf-2 and H19 expression is explained by Erasure of Igf-2-H19 imprinting in PGCs the fact that these two genes share the same 30-gene enhancer As the precursor cells for gametes (oocytes or sperm), PGCs are (shown in Figure 1a as red boxes), and it has been reported that the most important cell population, because they transfer parental deletion of this 30-enhancer results in downregulation of both Igf-2 DNA and mitochondria to the next generation. PGCs become and H19 expression.20 However, there are also some indications specified as the first population of stem cells during embryogen- that the 30-enhancer has a more robust effect on expression of esis in the proximal part of the epiblast, which forms all three H19 than Igf-2, which could be explained by the fact that (i) H19 layers of the trilaminar germ disc of the embryo proper in a has a stronger promoter than Igf-2 and/or (ii) the H19 gene is process called gastrulation.29 After being specified, PGCs migrate physically closer to the 30-enhancer than Igf-2 (Figure 1).20 to the extra-embryonic tissues, enter through the primitive streak Interestingly, it has recently been postulated that the H19 locus the embryo proper, and migrate to the genital ridges.30 As has is also a source of antisense RNA (H91 RNA), which regulates been demonstrated, during this migration process PGCs erase the expression of Igf-2 by interacting with a novel promoter for this methylation at several maternally and paternally imprinted loci, gene.21 This latter effect adds more complexity to the regulation of the Igf-2-H19 locus, but as it has been described so far only in myoblasts, its biological significance is still awaiting further VSEL HSC validation in stem cells. Evidence has accumulated that the dual yin-yang role of this master locus is relevant to several biological functions, including normal fetal development, as the properly balanced expression of Igf-2 and H19 is required for initiation of embryogenesis.22 Accordingly, imprinting at the Igf-2-H19 locus is one of the major factors preventing parthenogenetic development in mammals, and the biological importance of this locus is demonstrated by the creation of viable bimaternal mice derived 22 from two female sets of chromosomes. These mice are created 8 % methylation 53 % methylation by fusion of two haploid nuclei, one from a non-growing and the other from a fully growing oocyte, into a diploid bimaternal H19 IGF-2 zygote. As female chromosomes have unmethylated DMRs at the 12.5 1.0 Igf-2-H19 locus (Figure 1a), which leads to overexpression of inhibitory H19 ncRNA in this ‘zygote-like’ totipotent cell, the crucial step in creating bimaternal mice is an appropriate genetic 10.0 0.8 modulation of the Igf-2-H19 locus from one of the maternally derived chromosomes, which promotes expression of IGF-2 and 7.5 0.6 thus properly balanced dosage of IGF-2/H19.22 In sum, proper imprinting of the Igf-2-H19 locus is required for 5.0 0.4 balanced expression of both genes in normal embryonic development and is maintained later on in all somatic cells of 2.5 0.2

the growing embryo and postnatal infant. Thereafter, in all the Fold difference (mRNA) Fold difference (mRNA) adult tissues except rare population of developmental early cells 0.0 0.0 that will be discussed below, the somatic cells express proper VSEL HSC VSEL HSC somatic imprinting, as depicted in Figure 1a. Figure 2. The methylation state of the DMR at the Igf-2-H19 locus in murine very-small embryonic-like stem cells (VSELs). (a) Bisulfite ERASURE OF IMPRINTING AT IGF-2-H19 LOCI REGULATES THE sequencing profiles of DNA methylation of DMRs at the insulin-like QUIESCENCE OF PLURIPOTENT STEM CELLS RESIDING IN growth factor (Igf)-2-H19 locus in VSELs and hematopoietic stem cells (HSCs. The percentage of methylated CpG sites is indicated as a ADULT TISSUES percentage (%). (b) Real time–polymerase chain reaction (RQ-PCR) In contrast to somatic cells, imprinting within DMRs at Igf-2-H19 analysis of Igf-2 and H19 RNA expression in purified, double-sorted loci is erased during early embryogenesis in primordial germ cells murine VSELs and HSCs. Representative results are shown.

& 2013 Macmillan Publishers Limited Leukemia (2013) 773 – 779 Igf-2-H19 as a master regulatory tandem gene MZ Ratajczak et al 776 including paternal imprinting at the Igf-2-H19 locus. This tissues during early embryogenesis and serve as a back-up mechanism of erasure of imprinted marks has several important population of precursor stem cells for more differentiated tissue- consequences. First, after the erasure of imprinting, PGCs (i) are committed stem cells.37 Careful molecular analysis of VSELs has quiescent and unable to proliferate in vitro, (ii) do not form revealed that their quiescence in adult BM and premature teratomas, (iii) do not complement blastocyst development and (iv) depletion from the tissues is controlled by epigenetic changes are not capable of performing as DNA donors in therapeutic cloning to imprinted genes, including the Igf-2-H19 locus, which is erased using their harvested nuclei. However, all these limitations in the in murine VSELs24–25 similarly as seen in PGCs (Figure 1b and pluripotency of PGCs are reversed when their imprinting is Figure 2). reestablished, as seen, for example, during ex vivo generation of In addition to erasure at the Igf-2-H19 locus, murine VSELs also embryonic germ cells from PGCs.31–32 These embryonic germ cells modify expression of other imprinted genes, but not all these that recover proper somatic imprinting behave as embryonic stem epigenetic changes are identical to those seen in PGCs.24–25 For cells (ESCs) in all of the assays listed above.29 example, we observed that murine BM-sorted VSELs, like PGCs, During normal development, the proper somatic pattern of erase the paternally methylated imprints (for example, DMRs at imprinting in germline cells is established when PGCs, after the Igf-2-H19 and RasGrf1 loci), while in contrast to PGCs, colonization of the genital ridges, differentiate into the precursors hypermethylate the maternally methylated imprints (for of gametes.33 However, this occurs both in female germline example, DMRs at Igf-2R). Thus, the changes in expression of (paternal imprinting) and male germline (maternal imprinting) these genes in mouse additionally impairs IGF signaling (IFS), cells, first after a meiotic division in which the precursors of because hypermethylation of the DMR at the Igf-2R locus leads to gametes containing diploid chromosomes give rise to progeny that overexpression of IGF-2R, which, as mentioned above, is a non- possess the haploid number of chromosomes and are not able to signaling receptor that binds IGF-2 and prevents its interaction proliferate. However, when haploid male and female gametes fuse with the signaling receptors IGF-1R and insulin receptor. On the during fertilization, the chromosomes in the diploid zygote have other hand, erasure of the DMR at the RasGrf1 locus in mice leads proper complementary imprinting, including at the Igf-2-H19 loci. to downregulation of RasGRF1, which is a small guanosine triphosphate (GTP) exchange factor for Ras involved in proper signal transduction from activated IGF-1R and Ins-R. It is important Erasure of Igf-2-H19 imprinting in VSELs to point out that in contrast to the Igf-2-H19 locus, which is Modification of genomic imprinting also has a crucial role in imprinted both in mice and humans,38 Igf-2R and RasGrf1 loci are maintaining the pool of pluripotent stem cells residing in adult imprinted in murine but not in human cells, even when IGF-2R is tissues. Specifically, our group demonstrated that adult murine highly expressed by human VSELs. Based on this difference, tissues harbor a population of pluripotent Oct4 þ SSEA-1 þ Sca-1 þ murine VSELs regulate more genes involved in IFS by imprinting Lin–CD45– cells,26–27 and a corresponding population of Oct- than their human counterparts (Figure 3). 4 þ SSEA-4 þ CD133 þ Lin–CD45– cells has also been identified in The epigenetic modification of these imprinted loci (including humans.34–36 We hypothesize that VSELs, are deposited in adult Igf-2-H19) explains why VSELs, like PGCs, despite expressing

Figure 3. Epigenetic changes that affect insulin factor signaling (IFS) in murine Oct-4 þ SSEA-1 þ Sca-1 þ Lin-CD45- (a) and human Oct-4 þ SSEA- 4 þ CD133 þ Lin-CD45- (b) very-small embryonic-like stem cells (VSELs). VSELs are deposited in adult tissues as a back-up population for tissue- committed stem cells. Erasure of imprinting at the insulin-like growth factor (Igf)-2-H19 locus results in a decrease in autocrine IGF-2 secretion and, via miR675, a decrease in IGF-2 and IGF-1 signaling through IGF-1R. At the same time, overexpression of non-signaling IGF-2R prevents the interaction of paracrine-secreted IGF-2 with IGF-1R and Ins-R. Of note, because of erasure of imprinting of the DMR at the RasGrf1 locus, murine VSELs lack RasGRF1, which is an important GTP exchange factor involved in IGF-1R and INS-R signaling (a). By contrast, RasGrf1 is not an imprinted gene in human cells (b). This balance in expression from the Igf-2-H19 locus can be perturbed by chronic elevation of IGF-1 and insulin levels, as seen, for example, in a chronic increase in calorie uptake that, over time, may lead to (i) depletion of VSELs from the tissues, which may lead to accelerated aging, and/or (ii) chronic activation of VSELs, which may result in their malignant transformation. As also demonstrated, at least for murine VSELs, imprinting at the Igf-2-H19 locus is at least partially reversed over time to the normal somatic imprinting pattern, which also makes VSELs more susceptible to IFS with increasing age.

Leukemia (2013) 773 – 779 & 2013 Macmillan Publishers Limited Igf-2-H19 as a master regulatory tandem gene MZ Ratajczak et al 777 several markers of pluripotency such as (i) an open chromatin VSELs and a novel view of aging structure at the promoters for Oct-4 and Nanog, (ii) bivalent IFS negatively correlates with life span in different species, domains at developmentally important homeobox-domain-con- including mice and humans.44–48 We have already reported that taining genes, (iii) reactivation of the X chromosome in female VSELs can be specified into HSCs and have proposed that in one VSELs and (iv) in vitro differentiation into cells from all three germ marrow (BM) they correspond to the most primitive precursors for layers, do not complement blastocyst development after injection HSCs.49–50 If this is also true for VSELs residing in other organs (for into the pre-implantation blastocyst and do not grow teratomas in example, liver, skeletal muscles and epidermis), they could also be 24–28,39 immunodeficient mice. a potential back-up population for other types of tissue- The fact that erasure of imprinting at the Igf-2-H19 locus may committed stem cells, though more evidence is needed. have a crucial role in keeping VSELs quiescent in adult tissues has Nevertheless, as the erasure of Igf-2-H19 imprinting in VSELs important practical implications. Specifically, we envision that negatively affects IFS signaling, it potentially maintains their reestablishment of proper expression of the IGF-2/H19 ratio in quiescent state and protects them from premature depletion from VSELs will be crucial for effective expansion of these cells ex vivo the tissues. Based on this, we proposed a novel hypothesis that for potential application in regenerative medicine. Supporting the relates aging, longevity and IFS to the abundance and function of feasibility of this goal, the IGF-2/H19 ratio is also perturbed in pluripotent VSELs deposited in adult tissues. A decrease in the parthenogenetic stem cells, and it has recently been demon- number of these cells should negatively affect pools of tissue- strated in two independent reports that downregulation of committed stem cells in various organs and have an impact on H19 ncRNA in these cells significantly improves their ex vivo tissue rejuvenation and life span.47,51–52 In support of this 40 expansion. expectation, we observed a significantly higher number of VSELs and HSCs in the BM of long-living murine strains (for example, Laron dwarfs and Ames dwarfs) whose longevity is explained by LOSS OF IMPRINTING AT IGF-2-H19 LOCI AND MALIGNANT low levels of circulating IGF-1 and decreased IFS.7 It is known that TRANSFORMATION IFS involves TORC1 (TOR (target of rapamycin) complex 1)— A growing body of evidence suggests that cancer originates in the ribosomal protein S6K (S6 kinase), and that this TORC1–S6K axis stem/progenitor cell compartment as a result of mutations controls several basic cellular processes, including transcription, 41 translation, protein and lipid synthesis, cell growth/size and cell accumulating over a lifetime. These mutations are maintained 53 in stem cell compartments, and self-renewing stem cells may be metabolism that affect aging. It explains why inhibition of TORC1/S6K pathway by rapamycin has been shown to efficiently subjected to additional mutations and epigenetic changes so 53 that the genome is destabilized and uncontrolled neoplastic extend life span in several experimental animal models in vivo. proliferation is initiated. Interestingly, during the 19th and early Similar effect on TORC1/S6K signaling is achieved in VSELs by 20th centuries, several investigators proposed that cancer erasure of imprinting at Igf-2-H19 locus. develops in populations of embryonic-like cells that are left in a By contrast, compared with normally aging littermates, the number dormant state in developing organs during embryogenesis.41 This of VSELs and HSCs is significantly reduced in short-living mouse strains (for example, growth hormone-overexpressing transgenic mice) with ‘embryonic rest hypothesis of cancer origin’ suggested that adult 8–9 tissues contain embryonic remnants that normally lie dormant, high levels of circulating IGF-1 and thus elevated IFS. but that can be activated to become cancerous. Based on the presence of PGCs and VSELs in adult tissues, it is tempting to VSELs and their potential involvement in cancerogenesis hypothesize that these cells could be the missing link that Elevated IFS is also well known to be involved in development of reconciles this past theory of cancer origin with current theories malignancies.54 Specifically, both obesity and high-caloric uptake, envisioning cancer as a stem cell disorder. However, this which are associated with highly active IFS, are risk factors for hypothesis needs more experimental corroboration. cancer development. Experimental animals with high levels of Nevertheless, hypermethylation of the Igf-2-H19 locus on both circulating IGF-1 are not only short-lived but also have a high chromosomes, which is called loss of imprinting (in contrast to incidence of cancer.48 On the other hand, long-living animals, such erasure of imprinting), results in Igf-2 overexpression (Figure 1c) as the Laron dwarf and Ames dwarf mice mentioned above, with and is observed as an epigenetic change in several malignancies low levels of circulating IGF-1, have a much lower incidence of (for example, rhabdomyosarcoma and nephroblastoma) where tumor development.48 Importantly, this animal data also correlates overexpressed IGF-2 acts as an autocrine growth factor for tumor very well with the human Laron dwarf mutation, where affected cells. The best example of this mechanism is Beckwith– individuals have a very-low level of circulating IGF-1 and at the Wiedemann syndrome, which is associated with the development 55–56 10 same time are highly prone to cancer development. of several pediatric sarcomas. A similar loss of imprinting, Based on the observations that predisposition to malignancies in however, has been also reported in pediatric sarcomas developing 41 42–43 mice correlates with VSEL numbers in their tissues. To explain this, independently as part of Beckwith–Wiedemann syndrome. we envision that chronic stimulation of VSELs by IFS may potentially activate these cells in an uncontrolled way and promote their malignant transformation. Therefore, it is also likely that some human VSELS—IMPRINTING OF THE IGF-2-H19 LOCUS, IFS AND THE tumors may originate in VSELs, and IFS may have an important IMPLICATIONS FOR AGING AND CANCEROGENESIS promoting role.57 Again, we envision two possible mechanisms. First, The above-mentioned changes in expression of imprinted genes VSELs exposed to constant high circulating levels of IGF-1 could in VSELs and, in particular, the common epigenetic change, transform into neoplastic cells and second, as will be discussed erasure of imprinting at the Igf-2-H19 locus, observed in both below, they could transform because of a loss of imprinting at the Igf- murine and human VSELs, leads to significant attenuation of IFS 2-H19 locus, which would expose them to the autocrine IGF-2 loop (Figure 3) in these cells.24–25 As a result, due to epigenetic changes and restore normal expression of IGF-1R. Currently, we are testing in imprinted genes, VSELs are protected from autocrine and this hypothesis in appropriate animal models. paracrine IFS, which would otherwise lead to their premature depletion from adult tissues, as well as potentially trigger uncontrolled proliferation leading to teratoma formation. This CONCLUSIONS attenuation of IFS in VSELs may have important implications, both Evidence has accumulated that the imprinted Igf-2-H19 tandem for aging and cancerogenesis. gene has a pleiotropic role in several biological processes,

& 2013 Macmillan Publishers Limited Leukemia (2013) 773 – 779 Igf-2-H19 as a master regulatory tandem gene MZ Ratajczak et al 778 including quiescence of VSELs deposited in adult organs. It is dwarf mice—novel view on Igf-1, stem cells and aging. Leukemia 2011; 25: important to mention that we have observed that with increasing 729–733. age, the DMRs at the Igf-2-H19 locus become gradually 8 Kucia M, Shin DM, Liu R, Ratajczak J, Bryndza E, Masternak MM et al. Reduced methylated, and thus VSELs become more sensitive to IFS over number of VSELs in the bone marrow of growth hormone transgenic mice time.6–8 This phenomenon may contribute to their age-related indicates that chronically elevated Igf1 level accelerates age-dependent exhaus- depletion, as well as render them more sensitive to IFS and put tion of pluripotent stem cell pool: a novel view on aging. Leukemia 2011; 25: them at risk of malignant transformation. Thus, modification of 1370–1374. expression at the Igf-2-H19 locus may have an important role in 9 Kucia M, Masternak M, Liu R, Shin DM, Ratajczak J, Mierzejewska K et al. The negative effect of prolonged somatotrophic/insulin signaling on an adult bone inhibiting aging processes and preventing cancerogenesis. marrow-residing population of pluripotent very small embryonic-like stem cells Furthermore, we envision that proper methylation of the DMR at (VSELs). Age (Dordr) 2012; DOI: 10.1007/s11357-011-9364-8. 24–25 this locus, which is erased in VSELs, will be crucial for 10 Feinberg AP. Phenotypic plasticity and the epigenetics of human disease. Nature development of ex vivo strategies for expansion of these cells for 2007; 447: 433–440. the purposes of regenerative medicine.57 11 Thorvaldsen JL, Duran KL, Bartolomei MS. Deletion of the H19 differentially In addition to imprinting, expression at the Igf-2-H19 locus is methylated domain results in loss of imprinted expression of H19 and Igf2. Genes tightly regulated by the CTCF protein, which is involved in the Dev 1998; 12: 3693–3702. balanced expression of IGF-2 and H19 from the paternal and 12 Srivastava M, Hsieh S, Grinberg A, Williams-Simons L, Huang S-P, Pfeifer K. H19 maternal chromosomes. Of note, an interesting mechanism has and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19. Genes Dev 2000; 14: 1186–1195. been described in which elevated level of IGF-2 in senescent 13 Schoenherr CJ, Levorse JM, Tilghman SM. CTCF maintains differential methylation human epithelial cells is the result of a reduction in CTCF at the Igf2/H19 locus. Nat Genet 2003; 33: 66–69. expression, which controls the Igf-2-H19 locus. As reported, a 14 Takai D, Gonzales FA, Tsai YC, Thayer MJ, Jones PA. Large scale mapping of decrease in the intracellular CTCF level, leading to lower methylcytosines in CTCF-binding sites in the human H19 promoter and occupancy of DMRs by CTCF within the Igf-H19 locus, resulted in aberrant hypomethylation in human bladder cancer. Hum Mol Genet 2001; 10: a 10-fold increase in intracellular Igf-2 expression.58 Therefore, 2619–2626. modulation of CTCF expression could also be an option for 15 Jones BK, Levorse J, Tilghman SM. A human H19 transgene exhibits impaired regulating IFS in VSELs. Furthermore, it is likely that, in addition to paternal-specific imprint acquisition and maintenance in mice. Hum Mol Genet CTCF, other proteins are also involved in regulation of this locus 2002; 11: 411–418. 16 Brooks AJ, Waters MJ. The growth hormone receptor: mechanism of activation that still await identification. and clinical implications. Nat Rev Endocrinol 2010; 6: 515–525. 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