Epigenetic dysregulation of secreted Frizzled-related proteins in multiple myeloma

Cancer Letters 281 (2009) 24–31

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Epigenetic dysregulation of secreted Frizzled-related proteins in multiple myeloma E. Jost a,*,1, D. Gezer a,1, S. Wilop a, H. Suzuki b, J.G. Herman c, R. Osieka a, O. Galm a a b c

Medizinische Klinik IV, Universitaetsklinikum Aachen, RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany First Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA

a r t i c l e

i n f o

Article history: Received 24 July 2008 Received in revised form 9 January 2009 Accepted 2 February 2009

Keywords: Wnt pathway SFRP Epigenetics Multiple myeloma DNA methylation

a b s t r a c t We analysed the clinical impact of epigenetic dysregulation of the Wnt pathway in malignant plasma cell disorders. In multiple myeloma (MM) cell lines, aberrant promoter hypermethylation of the secreted Frizzled-related protein (SFRP) genes was a common event, and hypermethylation of SFRP1,-2 and -5 was associated with transcriptional silencing. Among 76 primary patient samples, the frequency of aberrant methylation was 35.5% for SFRP1, 52.6% for SFRP2, 1.3% for SFRP4 and 6.9% for SFRP5. Hypermethylation of SFRP1 and -2 genes was detected in monoclonal gammopathy of undetermined significance and all MM stages including plasma cell leukaemia (PCL), while SFRP5 methylation was restricted to advanced MM stages and PCL. Our data indicate that epigenetic silencing of Wnt antagonists is an early event in MM pathogenesis and that SFRP5 hypermethylation may play a role in disease progression. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Multiple myeloma (MM) is a B-cell neoplasm characterised by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM [1]. There is increasing evidence that in addition to genetic abnormalities, epigenetic processes play a major role in carcinogenesis [2,3]. Aberrant DNA methylation is the best studied epigenetic mechanism and was shown to influence fundamental cellular pathways in the pathogenesis of haematopoietic malignancies [4]. The importance of the Wnt signalling pathway in the development of many organ systems has been established [5]. Wnt signalling is a key factor in the regulation of cell proliferation as well as haematopoietic stem cell maintenance and B-cell differentiation [6–8]. A large number of

* Corresponding author. Tel.: +49 (0) 241 80 89806; fax: +49 (0) 241 80 82449. E-mail address: [email protected] (E. Jost). 1 These two authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.02.002

Wnt signalling abnormalities have been detected in solid tumours as well as haematopoietic malignancies, and constitutive activation of the Wnt pathway has been found in acute myelogenous leukaemia (AML) and acute lymphoblastic leukaemia (ALL) [9–11]. Wnt signalling comprises the canonical pathway with b-catenin as a key component and the non-canonical pathways involving calcium ions and planar cell polarity [12–14]. In the absence of Wnt ligands, b-catenin is phosphorylated by glycogen synthase kinase-3b (GSK3b) leading to ubiquitination and rapid degradation of the protein [15]. Binding of Wnt ligands to the Frizzled receptor leads to inhibition of GSK3b by activation of the adaptor protein Dishevelled. This results in the stabilisation of b-catenin and its translocation into the nucleus, where it binds to a family of transcription factors such as lymphoid enhancing factor-1 and T-cell factor [16]. High levels of b-catenin have been shown to block lymphoid, myeloid and erythroid cell differentiation accompanied by a considerable increase of haematopoietic stem cells and loss of repopulating activity [17,18]. Overexpression of b-catenin leads to increased proliferation of malignant plasma cells [10]. In consequence, the inactiva-

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tion of Wnt antagonists may contribute to the pathogenesis of MM. A loss of tumour suppressor genes function may result from mutations, chromosomal deletions or epigenetic dysregulation [4]. The best studied epigenetic mechanism for silencing of cancer-related genes is hypermethylation of CpG islands in the promoter region of genes. CpG island hypermethylation of tumour suppressor genes has been described in almost all solid tumours and haematopoietic malignancies [19,20]. Epigenetic silencing of negative regulators of the Wnt signalling pathway may affect the Wnt inhibitory factor1 (WIF1), the four mammalian homologues of Dickkopf (DKK) and the secreted Frizzled-related proteins (SFRPs) [9,21–23]. Wnt5a, a negative regulator of the non-canonical Wnt pathway, is also frequently epigenetically silenced in haematopoietic malignancies [24,25]. Promoter hypermethylation of the WIF1 gene has been described in about half of the patients with acute promyelocytic leukaemia (APL) [21]. Myeloid leukaemias with an AML1/ETO or CBFb/MYH11 fusion transcript, the so-called core binding factor (CBF) leukaemias, showed preferential hypermethylation of DKK1 [26] or SFRP2 [27]. Downregulation of WIF1, DKKs and SFRPs by promoter hypermethylation has also been observed in ALL and was associated with poorer survival [22]. The family of SFRPs belongs to a group of proteins antagonizing the Wnt signalling pathway by interaction with the Frizzled receptor. SFRP function in normal and malignant B-cell differentiation has not yet been systematically investigated. Four of the five known SFRP genes have been found to be epigenetically silenced in association with CpG island hypermethylation in cancer cells, as was shown particularly for SFRP1 and -2 in colorectal cancer [28]. SFRP3 has no promoter-associated CpG island and a correlation between DNA methylation and decrease of expression of the gene has not been established in malignant diseases [29]. In MM, a large number of tumour suppressor genes, such as p16, p73, death-associated protein kinase 1 (DAPK1), E-cadherin (E-cad) and suppressor of cytokine signalling 1 (SOCS1), are silenced through DNA methylation [30,31]. In contrast to genetic alterations, epigenetic disturbances are reversible, and a role of the DNA demethylating agents 5-azacytidine (AZA) and 5-aza-20 -deoxycytidine (DAC) in the treatment of haematopoietic malignancies has been established [32–35]. In vitro experiments provide evidence for a possible synergism between AZA with bortezomib or doxorubicin in the treatment of MM [36]. In this study, we have analysed the occurrence and the possible impact of epigenetic dysregulation of the SFRP genes in MM cell lines and samples from patients with malignant plasma cell disorders. Furthermore, correlations between epigenetic dysregulation of SFRP genes and clinical parameters were investigated. 2. Materials and methods 2.1. Human tissue samples After informed consent was given, bone marrow (BM) and peripheral blood (PB) specimens were obtained at

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the time of diagnosis or at relapse during routine clinical assessment of 76 patients with plasma cell disorders, who presented at the University Hospital Aachen, Germany, between 1995 and 2007. The collection of patient samples for the analysis of genetic and epigenetic changes was approved by the local ethics committee. Three patients presented with monoclonal gammopathy of undetermined significance (MGUS), 66 with MM and seven with plasma cell leukaemia (PCL) at the time of sample collection. 51 samples were obtained from therapy-naïve patients at diagnosis, and 25 patients had received treatment before sampling but were in relapse at the time of collection. MM diagnosis and staging were made in accordance with the Salmon and Durie criteria [37]. The main clinical and laboratory features of the patient cohort are summarised in the table. Healthy volunteers provided control PB samples and served as controls. Mononuclear cells from PB and BM were separated by density gradient centrifugation and frozen at 80 °C prior to further analysis. 2.2. Cell culture We obtained the MM cell lines U266, OPM-2, RPMI8226 and LP-1 from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). XG1 cells were kindly provided by P. C. Heinrich (RWTH Aachen). All cell lines except for LP-1 and XG1 were routinely cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10–20% foetal calf serum (Gemini Bio-Products, Woodland, CA). LP-1 and XG1 cells were routinely cultured in Iscove’s modified Dulbecco’s medium (Life Technologies, Rockville, MD) with 10% and 20% FCS, respectively. For XG1 cells, medium was supplemented with interleukin-6 at a final concentration of 10 U/ml. For gene expression and demethylation studies, cell lines were incubated with or without a final concentration of 1.0 lM DAC (Sigma, St. Louis, MO) for 96 h, harvested and subjected to RNA and DNA extraction. 2.3. Real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR) Total RNA was extracted from cell lines using a commercially available kit (Qiagen, Hilden, Germany). Reverse transcription reactions were performed with 1 lg total RNA in a final volume of 20 ll, and the concentration of reagents were 10 lM for random hexamers, 0.5 mM for dNTPs and 5.5 mM for magnesium chloride. Before the addition of 50 U superscript enzyme (Invitrogen, Carlsbad, CA), 2 ll of the mixture were separated to serve as a negative control. Real-time RT-PCR was performed in a Perkin–Elmer 7700 thermocycler using standard PCR conditions and the commercially available universal master mix (Applied Biosystems, Foster City, NJ). The quality of the cDNA was assessed by the housekeeping gene GAPDH using commercially available probe and primers (Applied Biosystems, Foster City, NJ). Primers for SFRP1 were 5-AGA TGC TTA AGT GTG ACA AGT TCC C-30 (forward) and 50 -TGG CCT CAG ATT TCA ACT CGT-30 (reverse) and the Taqman probe was 50 -FAM-ACC GAA GCC TCC AAG

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CCC CAA G-TAMRA-30 (GenBank accession number NM 003012). For SFRP2, primers were 50 -ATC CCC CTC GCT AGC AGC-30 (forward) and 50 -AAG CGT TTC CAT TAT GTC GTT GT-30 (reverse) and the probe was 50 -FAM-CTC CTG CCA GCC ACC GAG GAA-TAMRA-30 (GenBank accession number NM 003013). Primers for SFRP5 were 5-GTG CAG TTC GGG CAC CTG-30 (forward) and 50 -CAC ATC TGC TCC ATG AGG CC-30 (reverse) and the Taqman probe was 50 FAM-CAG TGA CCA AGA TCT GCG CCC AGT GT-TAMRA-30 (GenBank accession number NM 003015). The final concentration was 800 mM for the primers and 200 mM for the probes. Results were calculated using the threshold cycle (Ct) method, and expression levels of SFRP1, -2 and -5 were normalised to GAPDH expression levels (deltaCt). 2.4. Sodium bisulfite treatment and methylation-specific polymerase chain reaction (MSP) Genomic DNA was isolated from cell lines and primary tissues using standard methods (Qiagen, Hilden, Germany). A purification of plasma cells prior to further analysis was not performed owing to the high sensitivity of the MSP technique [38]. Approximately 1 lg DNA was sodium bisulfite-modified and subjected to MSP with primers specifically recognizing the unmethylated or the methylated sequence of SFRP1, -2, -4 and -5, respectively. MSP primers for the SFRP genes and reaction conditions have been published previously [28]. Normal DNA from PB was treated in vitro with SssI methyltransferase (New England Biolabs, Beverly, MA) in order to generate in vitro methylated DNA (IVD) that served as a positive control for methylated alleles [39]. PCR products were separated on 2.5% agarose gels and visualised by ethidium bromide staining. 2.5. Statistical methods Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the

log-rank test. Survival was calculated from the date of diagnosis until the patients’ death or last visit. Correlations between variables were analysed using the Fisher’s exact two-sided test, the two-sided Student’s t-test. All calculations were performed using the SAS statistical software (version 9.1.3, SAS, Cary, NC). 3. Results 3.1. Methylation status of the SFRP genes promoter regions in MM cell lines We first analysed the methylation status of the SFRP1, -2, -4 and -5 promoter regions by MSP in MM cell lines (Fig. 1). Methylation was found for all four SFRP genes in LP-1, XG1 and OPM-2 cells as well as for SFRP1, -4 and -5 in U266 cells. RPMI8226 cells were unmethylated for all four genes. 3.2. Expression of SFRP genes in MM cell lines We next examined the expression of SFRP1 and SFRP2 in the MM cell lines U266, OPM-2 and RPMI8226 as well as the expression of SFRP5 in the MM cell lines U266 and LP-1 by real-time RT-PCR. As shown in Fig. 2a, hypermethylation of SFRP1 was associated with transcriptional silencing in U266 and OPM-2 cells. In both cell lines, treatment with the demethylating agent DAC for 96 h at a 1.0 lM concentration induced reexpression of SFRP1. Fig. 2b shows reexpression of SFRP2 after DAC exposure in OPM-2 cells with hypermethylation for SFRP2, while no change in expression is found in the unmethylated cell lines RPMI8226 and U266. Reexpression of SFRP5 was observed after incubation with DAC in the methylated cell lines U266 and LP-1 (Fig. 2c). Thus, there was an association between hypermethylation of SFRP1, -2 and -5 and transcriptional silencing as well as gene reexpression after DAC exposure. 3.3. Methylation of SFRP genes in primary patient samples We then investigated the methylation status of the SFRP promoter regions in PB and BM from normal healthy donors and in 76 primary samples from patients with plasma cell disorders. Representative MSP results are shown in Fig. 3. No aberrant methylation was observed previously in 7 BM samples from patients without evidence for haematological malignancy [27]. The frequency of aberrant methylation among primary patient samples was 35.5% (27/76) for SFRP1, 52.6% (40/76) for SFRP2, 1.3% (1/76) for SFRP4 and 6.9% (5/76) for SFRP5 (Fig. 4). Hypermethylation of at least one SFRP gene occurred in 63.2% (48/76) of the patients. For the entire patient cohort, aberrant methylation of the SFRP1 promoter was associated with concomitant promoter methylation of SFRP2 (p = 0.0016). Promoter hypermethylation of SFRP1 or -2 was found in

Fig. 1. MSP analysis of the four SFRP genes in MM cell lines and normal PB. In vitro methylated DNA (IVD) and water served as controls. Lane U, amplified product with primers recognising unmethylated SFRP1, -2, -4 and -5 sequences. Lane M, amplified product recognising methylated SFRP1, -2, -4 and -5 sequences.

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Fig. 2. Relative changes in SFRP1 (Fig. 2a), -2 (Fig. 2b) and -5 (Fig. 2c) expression levels before (light grey) and after (dark grey) treatment of MM cell lines with the demethylating agent DAC for 96 h at a 1 lM concentration (DAC). Expression levels of SFRP1, -2 and -5 were determined by real-time RT-PCR and normalised to the housekeeping gene GAPDH. Error bars indicate standard deviation.

MGUS, MM and PCL. In contrast, SFRP5 hypermethylation was only detected in stage III MM or in PCL, and the association between aberrant methylation of SFRP5 and PCL was statistically significant (p = 0.0046). Concomitant hypermethylation of all four SFRP genes was found in one PCL patient. There were no correlations between the methylation status of any SFRP gene with the clinical parameters age and gender, white blood cell count, haemoglobin, platelet count, creatinine or calcium. However, we found a correlation between elevated lactate dehydrogenase (LDH) and hypermethylation of SFRP5 (p = 0.014). Furthermore, there was no impact of the SFRP methylation patterns on overall survival for the cohort of 51 therapy-naïve patients at diagnosis (See Table 1).

4. Discussion Wnt signalling plays an important role in stem cell selfrenewal as well as in differentiation and proliferation of haematopoietic progenitor cells. Aberrant activation of the Wnt pathway has been demonstrated to contribute to the development of lymphoid malignancies, but in contrast to solid tumours, no mutations in genes of the Wnt

pathway such as b-catenin have been described in haematopoietic malignancies until now. In AML, epigenetic silencing of negative regulators of Wnt signalling has been found for WIF1 preferentially in APL and for DKK1 and SFRP2 in CBF leukaemia [26,27]. In addition, SFRP gene silencing in association with promoter hypermethylation has been described in ALL [22]. However, despite a growing knowledge about the Wnt pathway [40], the role of SFRP proteins in normal and malignant haematopoiesis is still poorly defined. In patients suffering from MM, DNA hypermethylation of Wnt antagonists has very recently been described Chim et al. [41] who detected hypermethylation of WIF1, DKK3, APC as well as SFRP1, -2, -4 and -5. In MM cell lines, reexpression of the silenced genes could be observed after exposure to DAC, and the addition of recombinant SFRP1 to the culture medium resulted in downregulation of Wnt signalling.

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Table 1 Characteristics of the patient cohort. Number of patients, n Age (median and range, years) Gender Male Female Disease stage (Salmon and Durie) MGUS Stage I Stage II Stage III PCL Paraprotein isotype IgA IgG IgM Light chain Non secretory Laboratory parameters (median and range) WBC (109/l) Haemoglobin (g/l) Platelet count (109/l) LDH (U/l) creatinine (mg/dl) serum calcium (mmol/l)

76 65 (40–94) 45 31 3 7 8 51 7 15 48 1 11 1 5.8 (0.9–63.4) 114 (57–192) 224 (22–665) 173 (79–908) 1.1 (0.5–4.4) 2.3 (1.9–4.4)

In our study, we confirm frequent epigenetic dysregulation of the Wnt antagonists SFRP1, -2, -4 and -5 in MM. We have demonstrated that aberrant CpG island methylation of SFRP1 and -2 near the transcriptional start site is associated with gene silencing in MM cell lines. In vitro treatment with the demethylating agent DAC resulted in reexpression of SFRP1, -2 and -5. In comparison to Chim et al., we confirm in our larger patient cohort aberrant CpG island methylation for SFRP1, -2, -4 and -5 in malignant plasma cell disorders, but at a higher frequency for SFRP1. Especially concomitant hypermethylation of SFRP1 and -2 is a common event in MM. Aberrant methylation of at least one of the four SFRP genes was detected in 63.2% of the primary patient samples. In addition, we found aberrant hypermethylation of SFRP1 and -2 in all

stages of plasma cell disorders ranging from MGUS to PCL, while SFRP5 hypermethylation was limited to advanced MM or PCL. CpG island hypermethylation for SFRP1 and -2 was found in MM, but also in MGUS and PCL suggesting that epigenetic dysregulation of these genes is an early event in the pathogenesis of malignant plasma cell disorders. This early detection of promoter hypermethylation in MM has also been described for other cancer-related genes such as p15 and p16 [30,42]. In contrast, hypermethylation of SFRP5 was found to occur only in stage III MM and especially in PCL. These data indicate that SFRP5 may be involved in the progression of MM or be related to aggressive forms of plasma cell disorders. Additionally, SFRP5 hypermethylation correlated with higher levels of LDH. These findings are in accordance with the well-described adverse prognostic impact of LDH elevation in MM patients [43–45]. The lack of association between hypermethylation of SFRP5 and a worse prognosis in our cohort may be related to the small number of patients in our cohort. An association between PCL and epigenetic dysregulation has also been described for E-cad, DAPK1 and p16 [30]. This pattern of distribution of hypermethylation illustrates a differential role of Wnt antagonists in normal and malignant plasma cell development. A specific role of various SFRP genes and SFRP hypermethylation has also been described in AML [27] as well as in precancerous lesions in the pathogenesis of colorectal cancer [28]. In contrast to MM, a possible prognostic role was found for SFRP2 hypermethylation in specific AML subgroups [27]. Epigenetic dysregulation of the Wnt pathway may be involved in the pathogenesis of plasma cell disorders and also in the maintenance of tumour stem cells [46]. As cancer stem cells are thought to be more resistant to most chemotherapeutic agents, epigenetic silencing of Wnt antagonists may play a role in relapse of MM after successful treatment with conventional chemotherapeutic agents or autologous stem cell transplantation. Since epigenetic

Fig. 3. Representative SFRP1, -2, -4 and -5 MSP analysis of MM patient samples. In vitro methylated DNA (IVD) and water served as controls. Lane U, amplified product with primers recognising unmethylated SFRP1, -2, -4 and -5 sequences. Lane M, amplified product recognising methylated SFRP1, -2, -4 and -5 sequences.

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Fig. 4. Methylation profile of the SFRP genes in the patient cohort. Grey boxes indicate patient samples with methylation signal by MSP. Grey grid squares indicate methylated sites, open grid squares indicate unmethylated sites.

changes are pharmacologically reversible, therapeutic approaches with demethylating agents may contribute to reactivation of SFRP1 and SFRP2 function in patients with methylation-associated silencing of those genes. Other genes have been shown to be reactivated by demethylating agents [47]. Thus, the addition of demethylating drugs may be a novel promising strategy for the treatment of MM or PCL [48]. Furthermore, the development of Wnt pathway antagonists [49–51] may be of potential therapeutic value in malignant plasma cell disorders. Additionally, the follow-up of CpG island hypermethylation of SFRP1 and SFRP2 could serve for monitoring the in vivo effects of demethylating agents [52–54]. Synergism between conventional chemotherapeutic agents, novel drugs successfully used in MM such as bortezomib and DNA demethylating agents has been recently described in vitro [36].

In our cohort of patients suffering from MM, no impact of SFRP methylation patterns on overall survival could be observed. Since the patient group was heterogeneous for age, risk factors and treatment regimens, no definitive conclusion can be retained from these data regarding the prognostic role of SFRP promoter hypermethylation. However, analysis of the prognostic relevance of aberrant SFRP gene methylation is warranted in larger prospective trials. In ALL and AML, an association between SFRP hypermethylation or a CpG island methylator phenotype (CIMP) and survival has been described [22,27,55]. Further investigations are required in MM to study the impact on survival for hypermethylation of single genes or CIMP. In addition, the epigenetic signature of MM samples may be useful as a biomarker for further prognostic stratification in defined risk groups. Since CpG island hypermethylation can also be

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used as a marker for minimal residual disease in MM, monitoring of SFRP gene methylation may also serve for relapse prediction and risk stratification [56]. In summary, our study shows that in MM cell lines, CpG island methylation in the promoter regions of SFRP1, -2 and -5 is associated with transcriptional silencing. 63.2% of patient samples presented at least one aberrant methylation signal for SFRP genes. Hypermethylation was most frequently detected for SFRP1 and -2. For SFRP5 a correlation between promoter hypermethylation and PCL could be observed. The role of SFRPs in normal and malignant lymphopoiesis as well as maintenance of cancer stem cells needs to be investigated more in detail. The potential use of SFRP promoter hypermethylation as a diagnostic biomarker in MM requires further assessment in prospective trials. Conflicts of Interest Statement

[12] [13] [14] [15] [16]

[17]

[18]

[19]

[20]

None declared. [21]

Disclosure [22]

J.G. Herman is a paid consultant to and receives research support from OncoMethylome Sciences. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflicts of interest policies.

[23] [24]

Acknowledgements We thank Claudia Schubert, Ramona Latton, Ingeborg Wiegand, Peter Glatte and Lucia Vankann for expert technical assistance. This work was supported by a grant from the Deutsche Krebshilfe.

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