• Users Online: 98
  • Home
  • Print this page
  • Email this page
Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 45  |  Issue : 2  |  Page : 105-110

β-catenin mutations in acute myeloid leukemia


1 Vocational School of Health Services; Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara, Turkey
2 Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara, Turkey
3 Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara; Departmentof Medical Biology, Faculty of Medicine, Usak University, Usak, Turkey, Turkey
4 Department of Hematology, Faculty of Medicine, Ankara University, Ankara, Turkey

Date of Submission24-Nov-2019
Date of Acceptance19-Apr-2020
Date of Web Publication29-Dec-2020

Correspondence Address:
Buket A Gunes
Department of Medical Biology, Faculty of Medicine, Vocational School of Health Services, Ankara University, Ankara 06290
Turkey
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_54_19

Rights and Permissions
  Abstract 


Background Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder characterized by uncontrolled proliferation and impaired differentiation of normal hematopoietic stem or progenitor cells. Various pathways like RAF/MEK/ERK, PI3K/AKT, receptor tyrosine kinases, members of RAS family, and Wnt/β-catenin are disrupted in AML. Stabilization of β-catenin, the key point of activated Wnt/β-catenin pathway, has been shown in AML in various studies. One of the mechanisms that may lead to the β-catenin stabilization is the mutations in exon 3 at its N-terminal domain, where β-catenin is phosphorylated particularly during the degradation process, and these mutations have been investigated in chronic myeloid leukemia and many other cancer types.
Aim β-Catenin gene exon 3 mutations were analyzed in this study with the aim of determining the relationship between mutations and molecular biology of AML.
Patients and methods In this study, we examined β-catenin gene mutations in AML cell line U937 and 31 untreated patients with AML by using the DNA sequence analysis for the first time in a Turkish population.
Results No β-catenin gene exon 3 mutations were detected in patients with AML and the cell line.
Conclusion β-catenin mutations have been detected in various types of cancer; however, the authors did not find any mutation in this gene, which may be responsible for the activation of Wnt signaling in AML. Further research is required to understand the mechanisms apart from mutations that may induce β-catenin stabilization in AML.

Keywords: acute myeloid leukemia, Wnt signaling pathway, β-catenin


How to cite this article:
Gunes BA, Ozkan T, Gurel AK, Hekmatshoar Y, Beksac M, Sunguroglu A. β-catenin mutations in acute myeloid leukemia. Egypt J Haematol 2020;45:105-10

How to cite this URL:
Gunes BA, Ozkan T, Gurel AK, Hekmatshoar Y, Beksac M, Sunguroglu A. β-catenin mutations in acute myeloid leukemia. Egypt J Haematol [serial online] 2020 [cited 2021 Jul 31];45:105-10. Available from: http://www.ehj.eg.net/text.asp?2020/45/2/105/305402




  Introduction Top


Acute myeloid leukemia (AML) is a disease characterized by the accumulation of a great number of immature myeloid cells (myeloblasts) in bone marrow/peripheral blood as a result of a number of genetic changes in hematopoietic stem cells (HSCs) or hematopoietic progenitor cells. It is seen in patients younger than 65 years old, with an annual incidence of 3–8/100 000 [1-4]. The most basic genetic occurrences in the development of AML are changes in myeloid transcription factors, mutations in signaling pathways, and abnormal signal stimulation. These mechanisms are independent of each other, and they cause reduction in apoptosis, increase in the self-renewal ability of HSCs, and blockage of differentiation potential of AML cells [5]. Although standard chemotherapy treatments based on cytosine arabinoside and anthracycline derivative daunorubicin or idarubicin agents provide complete remissions (CRs) in ∼75% of patients with de novo AML (<60 years of age), relapse develops in many patients [2],[4],[6]. Relapse and the resistance to existing therapies are among the main problems in the treatment of AML disease [4],[7]. Although CR is possible with chemotherapy, this clinical attitude has created the impression that the presence of an underlying leukemic cell population can cause the relapse development in AML [6]. In the past two decades, transplantation studies based on mice models demonstrated the presence of leukemic stem cells (LSCs) or cancer stem cells, which occur as a result of the malign transformation of HSCs or hematopoietic progenitor cells, which lead to relapse of AML disease [6],[8],[9]. The most basic characteristic of HSC is self-renewal. These cells can generate multilineage progenitor cells, which can differentiate to cells in the hematopoietic system. Normal HSC biology is the basic subject of various studies, which pioneer the explanation of specific pathways and molecules that play a role in stem cell organization. One of the signaling pathways that have a key role in the self-renewal and differentiation characteristics of HSCs is the Wnt/β-catenin signaling pathway [10],[11].

There are 19 proteins of Wnt gene family defined in humans so far, which are similar to each other in terms of basic structure, and these are signaling pathway stimulants that contain cysteine-rich domains and are released to the extracellular area in the glycoprotein structure [12-14]. These stimulants bind to the receptors on cell surface and stimulate the signaling pathways that control the determination of the proliferation, migration, polarity, gene expression, and overall fate of the cell. Wnt signaling is required during the embryogenesis process and is active in various adult tissues such as lymphoid, colon, skin, hair follicles, and bones [14]. Wnt signaling can be categorized into canonical and noncanonical pathways [15]. Canonical pathway has been defined as β-catenin dependent, whereas noncanonical pathway has been defined as Wnt-calcium (Wnt-Ca2+) and Wnt-planar cell polarity (Wnt-PCP), independent of β-catenin [14],[16]. The fundamental role of canonical or Wnt/β-catenin pathway has been revealed in various studies for two decades [17]. It is a well-known fact that Wnt/β-catenin pathway controls the proliferation of hematopoietic cells, as well as their survival and differentiation [11],[18]. Persistent activation of Wnt signaling results in a neoplastic transformation of myeloid and lymphoid lineages. Physiologically, this pathway is regulated very tightly [18]. In the absence of Wnt stimulus, β-catenin, who cytoplasmic levels are usually kept quite low, is degraded by a protein complex called destruction complex. This complex comprises tumor suppressors adenomatous polyposis coli and scaffold protein Axin, casein kinase 1α acting as kinase, and glycogen synthase kinase 3β (GSK-3β) [19]. The last two components ensure β-catenin to be phosphorylated from serine and threonine residues in N-terminal region. Phosphorylated β-catenin is recognized by β-transducin, as a part of ubiquitin ligase complex. It leads to the polyubiquitination and proteasomal degradation of β-catenin. When Wnt ligand binds to Frizzled (Fzd) receptors with low-density lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors, Wnt signaling pathway is activated, and disheveled (Dvl) phosphorylation is stimulated [19],[20]. Dvl leads to inhibition of the destruction complex formation [21]. Binding of Dvl to Fzd allows the release of DIX domains of Dvl, as the binding site for Axin; thus, Axin binds to the intracellular tail of LRP. After binding to receptor complex, Axin can not only bind to β-catenin but can also inhibit GSK-3 activity. This effect of the pathway prevents the β-catenin phosphorylation, and stabilized β-catenin then accumulates in the cytoplasm and translocates to the nucleus where it binds to the transcription factor family members T-cell factor/lymphoid enhancer factor, and it regulates the transcription of Wnt target genes. In this manner, β-catenin intracellular levels are regulated with canonical Wnt pathway [20-22].

In a number of studies, aberrant activation of Wnt/β-catenin signaling pathway has been indicated, where the stabilization of β-catenin was discussed in-depth, which is thought to be the key point of this pathway [23]. Recent studies suggested that various possible factors including mutations in β-catenin gene, activating mutations of FMS-like tyrosine kinase-3 (FLT3) (Flt-3 ITD (Internal Tandem Duplication)), abnormalities in β-catenin destruction complex (Axin, APC, GSK-3β), overexpression of Wnt ligands, deregulation of Wnt pathway inhibitors (WIF (Wnt inhibitor factor), SFRP (secreted Frizzled-related protein and DKK (Dickkopf)) and the inhibition of regulatory pathways lead to β-catenin upregulation [5],[21]. We illustrate these mechanisms in [Figure 1].
Figure 1 Basic mechanisms for β-catenin stabilization. (a) In the presence of Flt-3 ITD (Internal Tandem Duplication) mutations induce Frizzled-4 expression and increase β-catenin nuclear localization. (b) When Wnt binds to Fzd receptors with LRP5/6 co-receptors, Wnt pathway is activated leading to the stabilization of β-catenin. (c, d, and e) Mutations in Axin, adenomatous polyposis coli, and β-catenin genes can impair the downregulation of β-catenin. (f) Akt-dependent phosphorylation increased nuclear β-catenin. (g, h, and i) Epigenetic inactivation of WIF, SFRP, and DKK leads to the stabilization of β-catenin. DKK, Dickkopf; SFRP, secreted Frizzled-related protein; WIF, Wnt inhibitor factor.

Click here to view


Exon 3 mutations, in N-terminal region [Serine (Ser) 33, Ser 37, Ser 45 and Threonine (Thr) 41st position], where β-catenin is phosphorylated particularly during degradation, were studied within chronic myeloid leukemia (CML) and many other types of cancer [10],[24]. In this study, the β-catenin gene exon 3 mutations of Turkish population were analyzed, and their effects on AML were investigated.


  Patients and methods Top


Primary cells and cell lines

Bone marrow/blood samples were collected from 31 untreated patients (age average 54) with newly diagnosed AML from Ankara University Faculty of Medicine, Department of Hematology. The Ankara University Faculty of Medicine’s Institutional Ethics Committee approved the study (approval no: 19-956-16), and a written informed consent was obtained from each patient. Mononuclear cells of patients with AML were obtained by Ficoll–Hypaque density gradient centrifugation (PAA/Austria). CD34+ cells from the mononuclear cells of each patient were isolated by using EasySep CD34+ isolation kit (Stem Cell Technologies, Vancouver, Canada), according to the manufacturer’s protocol. CD34+ cell purity was assessed by flow cytometry (FACS), and the purity of FACS-isolated cells was greater than 90%. U937 AML cell line was also included in the study. U937 cells were cultured with RPMI 1640 medium, comprising of 10% fetal bovine serum (Sigma, St. Louis, Missouri, USA), 100 units/ml penicillin, 100 g/ml streptomycin (Gibco, Co Dublin, Ireland), and 2 mmol/l l-glutamine (Gibco).

RNA isolation and cDNA synthesis

Total RNA from U937 cell line and CD34+ cells was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA concentration was verified by using the absorbance ratio of OD260/OD280 nm. cDNA was generated from 1 μg RNA by reverse transcriptase (Transcriptor High Fidelity cDNA Synthesis Kit; Roche, Germany).

Sequence analysis

The sequence reactions were run and analyzed on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Exon 3 region was amplified with PCR for 227 bp product of β-catenin gene using the following primers: forward:5′-GCTGATTTGATGGAGTTGGA-3′, and reverse: 5′-GCTACTTGTTCTTGAGTGAA-3′ [10]. PCR reactions were performed with 10× PCR buffer, 25 mmol/l magnesium chloride (SolisBiodyne, Tartu, Estonia), 2.5 mmol/l of each deoxyribonucleoside triphosphate (dNTP) (Sigma, St. Louis, Missouri, USA), 4 µl cDNA, 0.4 pmol of each primer (Alfa DNA, Montreal, Canada), and 0.5 mU Taq polymerase (SolisBiodyne, Estonia) in a final volume of 50 ml. The amplified PCR products were visualized for detecting product size and nonspecific binding by gel electrophoresis in 2% agarose gel. After amplification, PCR products were purified with Promega A9281 Wizard SV Gel ve PCR Clean-Up System (Promega, Madison, Wisconsin, USA) according to the manufacturer’s protocol and were then sequenced using Big Dye Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Foster City, California, USA). Bidirectional sequencing was carried out in an ABI 310 Genetic Analyzer (Applied Biosystems). The sequence analysis results were evaluated using Chromas program. Obtained sequences were analyzed and aligned with β-catenin (CTNNB1) reference sequence from the Gen Bank database accession number, NG_013302.


  Results and discussion Top


It is a known fact that Wnt/β-catenin pathway has an essential role in the regulation of apoptosis, as well as the proliferation and differentiation of HSCs. Although Wnt/β-catenin signaling is critical during the embryonic development, it should be downregulated in differentiated cells [21]. Recently, it has been suggested that the deregulation of this pathway leads to various malignancies, including AML.

It is generally accepted that a small subpopulation of AML cells, known as LSCs, are capable of self-renewal. LSCs are analyzed as potential treatment targets, considering their vital role in chemotherapeutic drug resistance and relapse of disease. Signaling pathways, controlling the development and survival of these stem cells, have come to the forefront in recent years [25]. One of these pathways, β-catenin, plays an important role in self-renewal characteristics of HSCs, and it is deregulated in LSCs according to recent studies [24]. Several studies demonstrated that β-catenin is overexpressed via Wnt-dependent and independent mechanisms [5],[21],[23]. Increased β-catenin expression level by Wnt-dependent/independent mechanisms leads to β-catenin entry to the nucleus, which affects the expression levels of Wnt target genes. In a clinical study on patients in various phases of CML, β-catenin gene activating mutations (exon 3) were analyzed. According to this study, β-catenin is not activated through these mutations [26]. Coluccia et al. [27] declared that the β-catenin mutations were not detected or were very rare in CML; conversely, these mutations were widely observed in patients with solid tumors. As similar to the results of these studies, Sercan et al. analyzed the β-catenin N-terminal mutations in Turkish population, and mutations were not detected in patients with CML in chronic phase. Thus, they suggested β-catenin mutation was not the underlying mechanism that leads to Wnt signaling activation within CML [10].Groen et al. [28] concluded that β-catenin mutations play a role in the abnormalities of T-cell differentiation, by analyzing the β-catenin exon 3 mutations in T-lineage lymphomas. Based on this study, Ng et al. [29] analyzed β-catenin gene exon 2 and 3 hot-spot regions of patients with T-ALL in Turkish population. In contrast to the study by Groen and colleagues it has been found that the β-catenin mutation is detected in only 1 patient with T-ALL and that abnormal Wnt activation is independent of β-catenin mutations in ALL.

The preliminary studies on primary AML samples asserted that these cells express significantly high β-catenin mRNA and protein expression levels compared with normal hematopoietic progenitors, and active β-catenin was detected in most patients (∼% 60) [23]. An in-vivo study indicated that AML was developed in a mice model with activating β-catenin mutations [30]. The mutations in β-catenin’s N-terminal region, where β-catenin is phosphorylated, is one of the potential mechanisms involved in β-catenin stabilization that causes solid tumor progression [27]. These mutations block the serine/threonine phosphorylation of β-catenin, thus leading to an increase on the cellular level of stabilized β-catenin protein.

To detect whether β-catenin mutations in N-terminal region are responsible for β-catenin stabilization and Wnt pathway deregulation in AML, we analyzed β-catenin gene exon 3 mutations [Serine (Ser) 33, Ser 37, Ser 45 and Threonine (Thr) 41st position], which have never been researched within the scope of Turkish population. For this purpose, β-catenin gene was amplified with PCR ([Figure 2]), and sequence analysis was done on 31 untreated patients with AML and in U937 AML cell line.
Figure 2 PCR amplification products of the third exon of β-catenin in patients with acute myeloid leukemia. M, marker-100-bp ladder; NC, negative control.

Click here to view


PCR products were subjected to bidirectional sequencing. All the PCR samples displayed a normal sequence electropherogram, without any mutation on β-catenin exon 3 hot-spot region ([Figure 3]).
Figure 3 The sequence analysis results of the β-catenin mutations in the third exon. None of the patients with acute myeloid leukemia or the acute myeloid leukemia cell line were shown to carry mutations.

Click here to view


In accordance with the literature, our results suggested that β-catenin mutations were not responsible for Wnt activation in AML. Further studies are required to evaluate the responsible mechanisms for Wnt signal activation such as abnormalities in β-catenin destruction complex, overexpression of Wnt ligands, deregulation of Wnt pathway inhibitors such as soluble frizzled-related proteins and Dickkopf proteins, epigenetic mechanisms of gene regulation, and inhibition of other pathways in AML.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Stone RM, O’Donnell MR, Sekeres MA. Acute myeloid leukemia. Hematol Am Soc Hematol Educ Program 2004; (1):98–117.  Back to cited text no. 1
    
2.
Almeida AM, Ramos F. Acute myeloid leukemia in the older adults. Leuk Res Rep 2016; 6:1–7.  Back to cited text no. 2
    
3.
Sanz MA, Iacoboni G, Montesinos P, Venditti A. Emerging strategies for the treatment of older patients with acute myeloid leukemia. Ann Hematol 2016. 95:1583–1593.  Back to cited text no. 3
    
4.
Hackl H, Astanina K, Wieser R. Molecular and genetic alterations associated with therapy resistance and relapse of acute myeloid leukemia. J Hematol Oncol 2017. 10:51.  Back to cited text no. 4
    
5.
Mikesch JH, Steffen B, Berdel WE, Serve H, Müller-Tidow C. The emerging role of Wnt signaling in the pathogenesis of acute myeloid leukemia. Leukemia 2007. 21:1638–1647.  Back to cited text no. 5
    
6.
Wang M, Yang C, Zhang L, Schaar DG. Molecular mutations and their cooccurrences in cytogenetically normal acute myeloid leukemia. stem cells int 2017. 2017:6962379.  Back to cited text no. 6
    
7.
Saraceni F, Labopin M, Gorin N, Blaise D, Tabrizi R, Volin L et al. Matched and mismatched unrelated donor compared to autologous stem cell transplantation for acute myeloid leukemia in first complete remission: a retrospective, propensity score-weighted analysis from the ALWP of the EBMT. J Hematol Oncol 2016; 9:79.  Back to cited text no. 7
    
8.
Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3:730–737.  Back to cited text no. 8
    
9.
Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367:645–648.  Back to cited text no. 9
    
10.
Sercan Z, Pehlivan M, Gokturk D, Sercan HO. Beta-catenin mutations are not observed in chronic myeloid leukemia. Tumori 2009; 95:836–839.  Back to cited text no. 10
    
11.
Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 2003; 423:409–414.  Back to cited text no. 11
    
12.
Ilyas M. Wnt signalling and the mechanistic basis of tumour development. J Pathol 2005; 205:130–144.  Back to cited text no. 12
    
13.
Yao H, Ashihara E, Maekawa T. Targeting the Wnt/beta-catenin signaling pathway in human cancers. Expert Opin Ther Targets 2011; 15:873–887.  Back to cited text no. 13
    
14.
Ahmadzadeh A, Norozi F, Shahrabi S, Shahjahani M, Saki N. Wnt/beta-catenin signaling in bone marrow niche. Cell Tissue Res 2016; 363:321–335.  Back to cited text no. 14
    
15.
Widelitz R. Wnt signaling through canonical and non-canonical pathways: recent progress. Growth Factor 2005; 23:111–116.  Back to cited text no. 15
    
16.
Reya T, Clevers H. Wnt signalling in stem cells and cancer. Nature 2005; 434:843–850.  Back to cited text no. 16
    
17.
MacDonald BT, Tamai K, He X. Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 2009; 17:9–26.  Back to cited text no. 17
    
18.
Pehlivan M, Çalışkan C, Yüce Z, Sercan HO. Secreted Wnt antagonists in leukemia: a road yet to be paved. Leuk Res 2018; 69:24–30.  Back to cited text no. 18
    
19.
Staal FJ, Famili F, Garcia Perez L, Pike-Overzet K. Aberrant Wnt signaling in leukemia. Cancers (Basel) 2016. 8:E78.  Back to cited text no. 19
    
20.
Duchartre Y, Kim YM, Kahn M. The Wnt signaling pathway in cancer. Crit Rev Oncol Hematol 2016; 99:141–149.  Back to cited text no. 20
    
21.
Taciak B, Pruszynska I, Kiraga L, Bialasek M, Krol M. Wnt signaling pathway in development and cancer. J Physiol Pharmacol 2018; 69:7.  Back to cited text no. 21
    
22.
Moon RT. Wnt/beta-catenin pathway. Sci STKE 2005; 2005:cm1.  Back to cited text no. 22
    
23.
Ysebaert L, Chicanne G, Demur C, De Toni F, Prade-Houdellier N, Ruidavets JB et al. Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis. Leukemia 2006. 20:1211–1216.  Back to cited text no. 23
    
24.
Polakis P. Wnt signaling and cancer. Genes Dev 2000; 14:1837–1851.  Back to cited text no. 24
    
25.
Wang Y, Krivtsov AV, Sinha AU, North TE, Goessling W, Feng Z et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science 2010. 327:1650–1653.  Back to cited text no. 25
    
26.
Chomel JC, Villalva C, Sorel N, Chazelas F, Guilhot F, Turhan AG. Evaluation of beta-catenin activating mutations in chronic myeloid leukemia. Leuk Res 2008. 32:838–839.  Back to cited text no. 26
    
27.
Coluccia AM, Vacca A, Duñach M, Mologni L, Redaelli S, Bustos VH et al. Bcr-Abl stabilizes beta-catenin in chronic myeloid leukemia through its tyrosine phosphorylation. EMBO J 2007. 26:1456–1466.  Back to cited text no. 27
    
28.
Groen RW, Oud ME, Schilder-Tol EJ, Overdijk MB, ten Berge D, Nusse R et al. Illegitimate WNT pathway activation by beta-catenin mutation or autocrine stimulation in T-cell malignancies. Cancer Res 2008. 68:6969–6977.  Back to cited text no. 28
    
29.
Ng OH, Erbilgin Y, Firtina S, Celkan T, Karakas Z, Aydogan G et al. Deregulated WNT signaling in childhood T-cell acute lymphoblastic leukemia. Blood Cancer J 2014; 4:e192.  Back to cited text no. 29
    
30.
Kode A, Manavalan JS, Mosialou I, Bhagat G, Rathinam CV, Luo N et al. Leukaemogenesis induced by an activating beta-catenin mutation in osteoblasts. Nature 2014; 506:240–244.  Back to cited text no. 30
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Patients and methods
Results and disc...
References
Article Figures

 Article Access Statistics
    Viewed464    
    Printed18    
    Emailed0    
    PDF Downloaded49    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]