Background
The neural transcription factor SOX11 is a novel diagnostic antigen for mantle cell lymphoma (MCL) [
1]. However, the prognostic relevance of nuclear expression of SOX11 in MCL remains unclear since it has been associated with both improved and reduced survival [
2,
3]. Our recent investigations demonstrated that nuclear staining of SOX11 is also seen in Burkitt Lymphoma (BL) and precursor B and T cell lymphoblastic neoplasia [
4], indicating a more widespread role in lymphoproliferative diseases than initially anticipated as also confirmed by others [
5]. Furthermore, analysis of solid tumors revealed a strong nuclear expression of SOX11 in epithelial ovarian cancer (EOC), which correlated with a prolonged recurrence-free survival [
6], suggesting a functional role for SOX11 in regulation of tumor growth. Abundant
SOX11 expression has been described in both the fetal central nervous system (CNS) and CNS-derived malignancies, such as medulloblastoma [
7] and malignant glioma [
8]. Furthermore, overexpression of SOX11 has been shown to prevent tumorigenesis in human glioma initiating cells [
9]. However, our previous study on EOC demonstrated that SOX11 also might be involved in growth regulation of malignancies not related to the CNS [
6].
SOX11 belongs to a group of 20 transcription factors within the high-mobility group (HMG) box protein super family, which are characterized by high sequence homology within their DNA-binding HMG domain [
10]. It has been shown that this HMG domain serves two functions, i.e. DNA binding as well as partner selection, which may permit selective recruitment of SOX proteins to specific promoters and transcription factors [
11‐
13]. To date, the main function of SOX11 in non-malignant tissues has been its involvement in neural development [
14,
15] and organogenesis [
13] during fetal development. Recent data also suggest an important role for SOX11 as a transcriptional regulator in adult immature neurons [
16].
The correlation between SOX11 and differences in survival in MCL [
2,
3] and EOC [
6] lead us to further investigate the mechanisms underlying its expression. In the present study, we used functional and epigenetic analyses of B cell malignancies to demonstrate a regulatory mechanism of
SOX11 expression on tumor cell growth. In conclusion, we provide the first evidence of a growth regulatory role for SOX11 outside the CNS. Furthermore, this protein not only has a tumor suppressor function but is also epigenetically silenced through DNA methylation in a subset of B cell malignancies.
Discussion
Recent data have suggested a functional role for the developmentally associated transcription factor SOX11 outside the CNS [
2,
3,
6], and this marker has been shown to be expressed in specific subtypes of B cell lymphomas [
1,
4,
18], as well as in solid tumors [
6]. Furthermore, the potential regulatory role of SOX11, indicated by its association with clinical features, such as survival [
2,
3,
6], require further investigations to understand the underlying biology and potential clinical application of this marker.
Gene expression can be regulated by epigenetic mechanisms, such as DNA methylation of CpG islands in the 5' promoter region [
20,
21]. Methylation-mediated silencing of various genes, often tumor suppressor genes, is a well studied phenomenon in many cancers [
22], since such methylation provides a growth advantage for the malignant cells. An increasing number of hypermethylated genes have been reported in lymphomas [
23‐
30], where they have been shown to be involved in various cellular functions, such as cell cycle control [
23], cytokine signaling [
27], DNA repair and apoptosis [
28].
Analysis of the
SOX11 promoter identified the presence of CpG islands, and bisulfite sequencing demonstrated a strong correlation between promoter methylation status and
SOX11 mRNA and protein levels in both B cell lymphoma cell lines and primary tumors. Thus, as previously reported, data from cell lines can represent the methylation status of primary malignant tissue well [
31]. However, as our experiments illustrate, and also reported by others, the magnitude of methylation is more pronounced in cell lines, since they often display either a full methylation or no methylation at all [
32]. Altogether, it is clear that the absence of SOX11 expression is tightly coupled to a methylated promoter in primary tumor samples, however no specific determinative CpG position could be identified as most alleles were either fully methylated or unmethylated.
In addition to investigating the cause of differential
SOX11 expression, we also explored the relationship between SOX11 and cellular growth, as a correlation with improved survival has been reported [
1,
3]. SOX11 function in the CNS has previously been assessed, using siRNA in a mouse neuroblastoma cell line and in cultured mouse dorsal root ganglia neurons, where
SOX11 was shown to modulate the levels of several other unrelated mRNAs involved in cell survival and death, suggesting an anti-apoptotic role [
33]. In contrast, SOX11 was recently shown to prevent gliomagenesis
in vivo by induced neuronal differentiation and abolished expression of oncogenic
plagl1 [
9]. Recent clinical studies have shown both a positive and negative correlation of SOX11 to survival and further studies are consequently needed to fully explore the clinical implications of this marker [
2,
3,
6]. In the present study, transient knock-down experiments confirm a growth regulatory role for SOX11 in B cell malignancies, as decreased levels result in increased proliferation in several
in vitro models of MCL. To further clarify if SOX11 is the limiting factor in a signaling cascade or if SOX11 possibly exhibits a master regulatory property, we overexpressed
SOX11 in different B cell lymphoma cells lines with variable degree of wild-type
SOX11 expression. Overexpression was achieved in all cell lines, independent of the original
SOX11 status, and resulted in an increase in SOX11 protein. Of note, all cell lines were functionally affected and their growth rates were significantly reduced. The direct effect on proliferation upon increasing SOX11 levels confirms that SOX11 is a master growth regulator.
It is known since previously that proliferation in MCL is partly driven by an overexpression of
CCND1, which leads to an increased ability to pass through the G1/S cell cycle checkpoint [
34]. Using global gene expression analysis we now show that the
CCND1-related Rb-E2F pathway [
19,
35] is affected by the increased level of SOX11. Among others, up-regulation of the CDKN2A locus, coding for p16
INK4A and p14
ARF, is seen already at 24 h of ectopic SOX11-overexpression. The CDKN2A locus encode both p16
INK4A and p14
ARF, but the proteins have no sequence homology due to alternative reading frames [
36]. The precise mechanisms of CDKN2A regulation and induction is unknown, but it has been shown that p16
INK4A and p14
ARF levels responds to, (i) external stress signals, (ii) Jun, Ets and Id families of transcription factors and (iii) hyperproliferative signals from for example Ras, Myc or deregulated E2F, as reviewed by Lowe and Sherr [
37]. Thus, it remains to be determined how SOX11 induces changes to the Rb-E2F signaling pathway although it is of major biological interest that this MCL-associated protein affects the same signaling pathway as
CCND1.
In agreement with the observed decrease in proliferation both p16
INK4A and p14
ARF possess anti-proliferative functions [
36,
38] and constitute two of the three pathways that control the G1/S transition of the cell cycle and are thus targeted in many tumors [
39]. E2F1 is down-regulated at 48 h and is one of the down-stream targets of p14
ARF. It has been suggested that E2F1 is the limiting factor for cell cycle transition [
36], and the decrease may thus directly contribute to the observed growth reduction.
Furthermore, TGF-β is up-regulated at 24 h and induces expression of down-stream genes, including TGFβR1, SMAD2/3 and TAB1/2 at 48 h. Of note, we have previously demonstrated that MCL cell lines are responsive to TGF-β-mediated decrease in proliferation when the corresponding receptor is available [
40]. Thus, the up-regulation of TGF-βR seen at 48 h emphasizes the need for a functional receptor to achieve TGF-β-mediated growth reduction. Consequently, it is likely that the anti-proliferative activity of TGF-β together with reduced levels of E2F1 is directly involved in growth reduction induced by ectopic SOX11-overexpression. The anti-proliferative effect of TGF-β is widely known and its dual role as both tumor suppressor and pro-metastatic mediator makes it an interesting target for intervention [
41‐
43].
Methods
Cultivation of cell lines
Nineteen lymphoma cell lines were used to study
SOX11 including eight MCL, four follicular lymphoma (FL), three diffuse large B cell lymphoma (DLBCL), three Burkitt lymphoma (BL) and one acute monocytic leukemia (MONO-L), as shown in Table
1. Most cell lines were provided and authenticated by DSMZ (Table
1). All cell lines were cultured in RPMI-1640 medium (HyClone, Sout Logan, UT) supplemented with 10% (v/v) fetal bovine serum (Invitrogen Gibco, Carlsbad, CA, USA) and 2 mM L-Glutamine (Sigma-Aldrich, St. Louis, MO, USA), hereafter referred to as R10 medium, except ULA which was cultured in 45% optiMEM (HyClone), 45% IMDM (HyClone) supplemented with 10% (v/v) fetal bovine serum (Invitrogen).
Collection and purification of primary samples
Lymphocytes were isolated from four MCLs, five FLs and one DLBCL through density centrifugation, as previously described [
44]. All five FL samples and two of the MCL samples (MCL1 and MCL6) were purified by positive selection, using a CD19 specific antibody (clone HD37, DAKO, Glostrup, Denmark) coupled to Dynabeads Pan Mouse IgG magnetic beads (Invitrogen Dynal), according to the protocol of the manufacturer. Flow cytometry was used to determine the purity of MCL 3 and 4 and the DLBCL. Patient material information is shown in Additional File
1: Table S1.
DNA methylation analysis
MethPrimer
http://www.urogene.org/methprimer/index1.html[
45] was used to analyze the 2000 bp region directly upstream of the
SOX11 transcription start site (the
SOX11 promoter region) for the presence of CpG islands. Using the MethPrimer default algorithm, three CpG islands were identified as >200 bp regions with G and C contents >50% and Observed/Expected CpG-rates of >0.6. One additional CpG island was detected when the region size constraint was lowered to 100 bp without altering the other criteria (Figure
1). The methylation status of the 5'-promoter region was determined by sodium bisulfite sequencing [
46] of the 213 bp CpG island located -435 to -222 bp upstream of the
SOX11 transcription start site. Briefly, total genomic DNA was extracted from five million cells per cell line or primary samples, using QIAamp DNA MINI kit (QIAgen) according to the protocol of the manufacturer. To convert unmethylated cytosine to uracil, we performed bisulfite conversion of 0.5 - 1 μg of DNA with EpiTect Bisulfite Kit (QIAgen). The CpG island was amplified from bisulfite converted DNA, using primers 5'-AGA GAG ATT TTA ATT TTT TGT AGA AGG A-3'and 5'-CCC CCT TCC AAA CTA CAC AC-3' and Platinum Taq DNA polymerase (Invitrogen). PCR products were both directly sequenced as well as ligated into the vector pCR.21-TOPO (Invitrogen) for clonal analysis. Sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany and GATC Biotech (Konstanz, Germany). Quality control of methylation data was performed in a standardized manner, using the BiQ Analyzer software [
47],
http://biq-analyzer.bioinf.mpi-inf.mpg.de/index.php. Images of CpG methylation for figures
3A-C were constructed using the BDPC web server [
48], using output files from BiQ Analyzer. All amplicons included in the study had, (i) bisulfite conversion rates above 95% for unmethylated non-CpG C:s to T:s, and (ii) sequence similarity above 90% compared to the original genomic sequence.
Nucleofection of cell lines with SOX11-specific siRNA or overexpression plasmid
The Amaxa protocol
http://www.lonzabio.com/protocols.html for nucleofection of suspension cell lines was followed, using program 0-017 and Cell Line Nucleofector Solution T (Amaxa Biosystems, Cologne, Germany). For the knock-down experiments, 5 × 10
6 cells were mixed with 50 pmol of siRNA (Ambion, Austin, TX, USA) in each reaction and a scrambled sequence and GFP-producing plasmid were used as controls. The sequences of the siRNAs in the pool targeting the
SOX11 gene can be found in Additional File: Table S3. For the overexpression experiments, 5 × 10
6 cells were mixed with 2 μg of OmicsLink™Expression Clone for SOX11 (EX-M0425-M60, the sequence can be found in the Additional File
1: Table S2) in each reaction and a GFP control vector was used as a control (both from GeneCopoeia, Germantown, MD, USA).
RNA isolation and Real Time-qPCR analysis of wt and SOX11-knocked/overexpressed cell lines
The relative quantity (RQ) of SOX11 mRNA in various wt cell lines was identified using Real Time-quantitative PCR (RT-qPCR). The cells were lysed and cDNA synthesis performed using the Fast SYBR Green Cells-to-CT kit (Applied Biosystems), according to the protocol of the manufacturer. Briefly, 104 cells were washed in PBS, lysed and treated with DNase. Lysates were reversely-transcribed and cDNA amplified in three technical replicates with the following primer specific either for SOX11 or the house-keeping gene GAPDH (250 nM, MWG, High-Point, NC, USA); GAPDH: 5'-TGGTATCGTGGAAGGACTC-3' and 5'-AGTAGAGGCAGGGATGATG-3', SOX11-t: 5'-GGTGGATAAGGATTTGGATTCG-3' and 5'-GCTCCGGCGTGCAGTAGT-3'. q-PCR conditions were as follows: enzyme activation 20 seconds at 95°C, PCR cycle denaturation for 3 seconds at 95°C and anneal/elongation 30 seconds at 60°C run on a Fast 7500 real-time qPCR system (Applied Biosystems). All samples were run in triplicates. In the reverse-transcription, a control sample was run containing lysate but no reverse transcriptase (RT), to check for background amplification of genomic SOX11 and GAPDH. A ΔCT > 4 for GAPDH (+RT) and GAPDH (-RT) was achieved for all cell lines. Similarly, the ΔCT for SOX11 (+RT) and SOX11 (-RT) was used as a qualitative control to determine if SOX11 was expressed or not. Generally, all samples with a ΔCT (SOX11+RT, SOX11-RT) < |2| was considered negative and the RQ was set to 0.01 for those samples. Finally, RQ is calculated as 2-(ΔΔCT(SOX11-GAPDH)) comparing each cell line to GRANTA-519. All the error bars related to qPCR data have been calculated using the standard error (SE) with a 95% confidence level.
For the over-expression experiments the Fast SYBR Green Cells-to-CT kit (Applied Biosystems) was used for lysis of 0.5-1.0*105 cells and subsequent cDNA synthesis as described above. The SOX11-t and GAPDH primer sets were used for amplification
In the knock-down experiments RNA isolation was carried out, using Trizol (Invitrogen) as previously described [
44]. The cDNA synthesis was performed, as outlined in the RevertAid™First Strand cDNA Synthesis kit-protocol (Fermentas). 1 μg of RNA was mixed with 0.2 μg random hexamer primers, and a reverse transcriptase was added to produce cDNA. Samples for RT-qPCR were prepared following the iQ™SYBR Green Supermix protocol (Bio-Rad, Hercules, CA, USA). The concentration of cDNA was 1.25-2.5 μg/l. The primers were as above but primers for Eg5 were included and a different set of
SOX11 primers were used as follows:
SOX11-u: 5'-CCAGGACAGAACCACCTGAT-3' and 5'-CCCCACAAACCACTCAGACT-3', Eg5: 5'-GTTTGGCCATACGCAAAGAT-3' and 5' - GAGGATTGGCTGACAAGAGC-3'. The RT-qPCR was run in triplicate, using a 2-Step Amplification and melt-curve program (Bio-Rad) with GAPDH as the endogenous control.
Protein purification and quantification
0.5-2 × 106 cells were harvested, washed and placed in 200 μl lysis-buffer (1% NP40/Protease Inhibitor cocktail (Roche, Basel, Switzerland) in PBS) and incubated on ice for 30 min. Centrifugation (16,000 × g at 4°C for 30 min) was used to remove cell debris. Protein concentrations were determined using the BCA Kit for Protein Determination (Sigma-Aldrich) with BSA as a standard (0.08 - 0.4 mg/ml). The samples were mixed with BCA working reagent, incubated at 37°C for 30 min, and absorbance measured at 562 nm.
Western Blot analysis of SOX11-knockdown and differential expression
Protein lysates, 3 or 7 μg for knock-down experiments, 3.5 μg for overexpression experiments and 32 μg for wild-type expression in nineteen lymphoma cell lines and fifteen primary specimens were run on NuPAGE 10% Bis-Tris gels (Invitrogen) under reducing conditions for ~45 min at 130 V. Separated proteins were blotted onto PVDF membranes, Amersham Hybond-P (GE Healthcare, Uppsala, Sweden) for 30 min (15 V) and blocked over night in 5% milk PBS. SOX11 protein expression was verified using anti-Sox-11
C-term (Figure
2,
3,
4 or Sox-11
N-term (Figure
5) antibodies, as previously described [
1,
4]. Primary antibodies Eg5 (Becton Dickinson, Franklin Lakes, NJ, USA) or GAPDH (Abcam) were used as loading control. HRP-labeled swine anti-rabbit antibody or rabbit anti-mouse antibody (DAKO) was used as secondary antibody and detection was made with SuperSignal West Femto Max Sensitivity Substrate (Pierce Biotechnology Inc., Rockford, IL), according to the protocol of the manufacturer. Blots were developed, using the SuperSignal West Femto Maximum Sensitivity Substrate (Nordic Biolabs, T228;by, Sweden) and detected with either with ECL Hyperfilm (GE Healthcare) in Kodak X-OMAT 1000 processor (Kodak Nordic AB, Upplands V228;sby, Sweden) or using a chemiluminescence scanner and CCD camera (Bio-Rad Laboratories, Hercules, California).
Assessment of proliferation
All proliferation assays were quantified using Methyl-3H-Thymidine (MTT) incorporation, as previously described [
49]. 50 000 cells were plated in triplicates for each sample. For all proliferation results, the ± 1 standard deviation (SD) is shown.
Isolation of tRNA for GeneChip Whole Transcript analysis
GRANTA-519 and JEKO-1 cells were nucleofected with SOX11-GFP or control GFP-containing vector, as described above, and 100 000 cells were washed once in PBS and lysed in 300 μl Trizol (Life Technologies, Gaithersburg, MD). The RNA was precipitated using chloroform/isopropanol extraction and dissolved in 6 μl of RNA-free H2O. The integrity and quantity of the RNA was assessed using Agilent 2100 bioanalyzer with the RNA 6000 Nano LabChip® reagent set (Agilent Technologies Inc., Santa Clara, CA, USA) and stored at -20°C. For all arrays 300 ng tRNA starting material was used for the first strand cDNA synthesis which was followed by amplification, fragmentation, labeling, hybridization, washing and scanning, all performed according to the Affymetrix standard protocol "GeneChip® Whole Transcript (WT) Sense Target Labeling Assay User manual, P/N 701880 Rev.5 (Affymetrix Inc., Santa Clara, CA). The labeled cRNA was hybridized to the Human Gene ST 1.0 arrays (Affymetrix Inc.). All arrays passed the initial quality control using assessment of hybridization, amplification controls and noise levels as defined by Affymetrix Inc.
Raw data was extracted from Human Affymetrix Gene arrays, HuGene ST 1.0, using the Command Console software package (Affymetrix). Quantile normalization using RMA and quality control was done in the Expression Console 1.0 software (Affymetrix). Normalized data was imported into Gene Spring GX 11.0 (Agilent Technologies Inc.) for fold change analysis. Fold change analyses for 24 and 48 h treatments were performed for GRANTA 519 and JEKO-1, comparing SOX11 overexpression to control vector. For each of the cell lines, genes that had a 1.2 fold change comparing SOX11 to control vector, at either 24 h or 48 h were selected. Genes that were differentially expressed in both cell lines were selected for further analysis using Ingenuity Pathway Analysis (Ingenuity Systems).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
EG performed all epigenetic experiments and wrote parts of the manuscript. SS performed siRNA/overexpression experiments and wrote parts of the manuscript. EA performed the overexpression studies. DB was involved in the design of the epigenetic analysis. MD and MJ provided patients material and clinical data. CB was involved in the design of the study, interpretation of the results and writing of the manuscript. SE was responsible for the design of the study, interpretation of data and writing of the manuscript. All authors approved of the final manuscript.