Mutation and expression alterations of histone methylation-related NSD2, KDM2B and SETMAR genes in colon cancers
A B S T R A C T
Epigenetic dysregulation is a hallmark of cancers, and examples of its cancer-associated expression and mutation alterations are rapidly growing. Histone methylation, a process by which methyl groups are transferred to amino acids of histone proteins, is crucial for the epigenetic gene regulation. NSD2 (nuclear receptor-binding SET domain protein 2) and SETMAR are epigenetic regulators for histone methylation. KDM2B, also known as FBXL10, is a histone demethylase that targets histone methylation processes. They are known to be altered in many cancers, but somatic frameshift mutation and expression of these genes remain undetermined in many other subsets of cancers, including high microsatellite instability (MSI-H) colon cancer (CC). In this study, we analyzed mononucleotide repeats in coding sequences of NSD2, KDM2B and SETMAR genes, and found frame- shift mutations in 10 %, 2 % and 1 % of CCs with MSI-H, respectively. Of note, there was no frameshift mutation of these genes in microsatellite stable (MSS) CCs. In addition, we discovered that 2 and 2 of 16 CRCs (12.5 % and 12.5 %) harbored intratumoral heterogeneity (ITH) of the NSD2 and KDM2B frameshift mutations, respectively. In the immunohistochemistry for NSD2, intensity of NSD2 immunostaining in MSI-H CC is decreased compared to that in MSS. These results suggest that NSD2 might be altered at multiple levels (frameshift mutation, mutational ITH and expression) in MSI-H CCs, and could be related to MSI-H cancer pathogenesis.
1.Introduction
Coordinated epigenetic gene regulation is important in many bio- logical phenotypes, and alterations of this process frequently result in pathologic changes [1]. EXpressional and mutational alterations in genes involved in the epigenetic regulation are important in cancer pathogenesis [2]. For example, epigenetic regulators for histone modi- fication and chromatin remodeling, including subunits of SWI/SNF complex and p300 are overexpressed in human cancers [3]. Also, so- matic mutations in epigenetic regulation machineries such as DNMT3A and EZH2 genes are frequent in many cancers [4]. However, alterations in genes involved in epigenetic regulation in cancers remain incom- pletely defined in many other cancers.Histone methylation is a process by which methyl groups are trans- ferred to amino acids of histone proteins that make up nucleosomes, which the DNA double heliX wraps around to form chromosomes [5]. It can either increase or decrease transcription of genes, depending on which amino acids in the histones are methylated, and how many methyl groups are attached. NSD2 (nuclear receptor-binding SET domain protein 2), also known as MMSET and WHSC1, is an epigenetic regulator for histone methylation in histone 3 lysine 36 (H3K36) [6,7]. NSD2 is overexpressed, amplified or somatically mutated in multiple types of cancer, suggesting its critical role in cancer. The most well-known genetic alteration of NSD2 has been t(4;14) translocation in multiple myeloma, which confers a poor prognosis [6]. NSD2 protein is overexpressed in many human cancers, including colon (CC), prostate and lung cancers, and its overexpression is to associate with cancer aggressiveness [7], suggesting NSD2 is a potential oncogene and is implicated as a therapeutic cancer target [8]. KDM2B, also known as FBXL10, is a histone demethylase that targets histone methylations in H3K36me2 and H3K4me3 [9]. KDM2B has been known to promote oncogenic activity by upregulating cell proliferation, differentiation and migration [10]. However, KDM2B exhibits opposing roles, i.e., antago- nizing tumor development, depending on the cellular context [10].
SETMAR is a DNA sequence-specific histone-methylase that plays a role in DNA repair [11]. Knockdown of SETMAR gene induces apoptosis [11], suggesting its roles in antagonizing tumor development. For the therapeutic application, it is important to know alterations for the tar- gets [2], but somatic mutation and expression status of these genes remain undetermined in many other subsets of cancers.
DNA mismatch repair (MMR) is a cellular mechanism for correcting erroneous bases by MMR-specific proteins, alterations of which would result in microsatellite instability (MSI) and mutator phenotypes [12]. The mutator phenotype is characterized by mutation accumulation in repetitive DNA sequences (frequently mono- or dinucleotide repeats). In coding DNA sequences, the MSI produces frameshift mutations within the affected genes that would truncate protein synthesis [12]. The CC is the most common cancers with high MSI (MSI-H) phenotype [12]. It is believed that MSI is random, but there is evidence suggesting that MSI targets include a growing list of cancer genes [13,14]. In the genome database, we observed that the NSD2, KDM2B and SETMAR genes possess mononucleotide repeats that could be mutation targets. In the present study, we identified NSD2, KDM2B and SETMAR frameshift mutations, or loss of expression in CC with MSI-H.
2.Materials and methods
In the present study, formalin-fiXed and paraffin embedded (FFPE) tissues of 168 CCs from Korean patients were used. Briefly, they con- sisted of 100 CCs with MSI-H and 68 CCs with microsatellite-stable (MSS) phenotype. For the evaluation of the MSI status of each cancer, we adopted five mononucleotide repeats (BAT25, BAT26, NR-21, NR-24 and MONO-27) that were known to be frequently mutated in MSI-H cancers [15]. Of the 168 CCs, multi-region sampling per CC was done in 39 CCs (16 MSI-H and 23 MSS CCs), while single region sampling was done in 129 CCs (84 MSI-H and 45 MSS CCs). Malignant cells and normal cells were separately collected from hematoXylin-eosin slides using a 30G1/2 hypodermic needle by microdissection as described in earlier studies [16,17]. The tumor cell fraction was estimated to be approXi- mately 70–80 % under microscope by a pathologist. The microdissected cell number in each case was similar among the cancers. DNA extraction was performed by a modified single-step DNA extraction method using proteinase K [16,17]. Research approval was obtained from the insti-
tutional review board of Catholic University of Korea. All FFPE samples were made anonymous and waived the need for written informed consent.
Since frameshift mutations of genes in MSI-H frequently accompany expressional alteration of the affected proteins [18], we analyzed the NSD2 protein expression status in the CCs by immunohistochemistry using anti-NSD2 antibody (Atlas Antibodies, Stockholm, Sweden; dilu- tion 1/50). Of the 3 genes analyzed in this study, NSD2 was the one with most frequent mutation. The immunohistochemistry procedures have been described in our earlier studies [18]. Briefly, sections from FFPE tissues of CCs were studied using ImmPRESS System (Vector Labora- tories, Burlingame, CA, USA). After deparaffinization, heat-induced epitope retrieval was conducted by immersing the slides in Coplin jars filled with 10 mmol/L citrate buffer (pH 6.0) and boiling the buffer for 30 min in a pressure cooker (Nordic Ware, Minneapolis, MN) inside a microwave oven at 700 W; the jars were then cooled for 20 min. We used diaminobenzidine (brown) as chromogen for the immunohistochemistry reactions and counterstained with hematoXylin (blue). The staining intensity was graded as follows: 0, negative; 1+, weak staining in cytosol or nucleus; 2+, moderate; and 3+, intense. The extent was graded as follows: 0, 0–10 % positivity of cells; 1, 10–39 %; 2, 40–69 %; 3, > 70 %. The intensity and the extent were multiplied for the immunohistochemistry score (IS), which consisted of IS 0 or1 as negative, 2 or 3 or 4 as and 6 or 9 as . Negative control of the immunostaining was the replacement of primary antibody with the blocking reagent.
3.Results
In the present study, we analyzed the nucleotide repeats in the NSD2, KDM2B and SETMAR coding sequences by PCR-based SSCP analysis for the detection of frameshift mutations. The PCR-SSCP for NSD2, KDM2B and SETMAR revealed aberrant migrating bands in 10, 2 and 1 CCs, respectively, which were subsequently confirmed as frameshift muta- tions (deletion or duplication mutation in the repeats) by DNA sequencing (Table 1 and Fig. 1). They were interpreted somatic as there was no such mutations in matched normal tissues. All of cancers with the mutations were MSI-H cases, but there was none in the MSS cases (sig- nificant difference, p 0.006). The DNA sequencing exhibits both wild- type and mutation sequences, indicating they are heterozygous muta-tions (Figs. 1 and 2). There was no significant difference of 5-year sur- vival between patients with and without the mutations (p > 0.05).We further analyzed the mutations in multi-regional areas of 16 CCs(96 areas, 4–7 areas per case). Two of the CCs (12.5 %) showed differ- ence in the NSD2 c.4028dupC (1 mutant and 5 wild-type areas in one CC and 2 mutants; 4 wild-type areas in the other CC), indicating there wasintratumoral heterogeneity (ITH) of the frameshift mutation (Fig. 2). Also, 2 (12.5 %) and 0 of the 16 CCs exhibited regional difference inKDM2B (c.77delA) and SETMAR mutations, respectively. Also, there was significant difference neither in patients’ survival nor clinical out- comes between ITH and non-ITH cases (p > 0.05). sequenced by Sanger DNA sequencing of both forward and reverse strands to confirm the mutated sequences (3730 DNA Analyzer, Applied Biosystem, Carlsbad, CA, USA).
We also analyzed intratumoral hetero-geneity (ITH) of the NSD2, KDM2B and SETMAR frameshift mutations, We next analyzed protein expression status of NDS2 protein (Fig. 3).In normal epithelial cells of colonic mucosa, NDS2 was negatively expressed (IS 0) in immunohistochemistry (Fig. 3A and D). Inflamma- tory cells in the mucosa were positive for the immunostaining (Fig. 3A, B and D). In the cancers, the MSS (88.9 %, 40/45) and MSI-H (80.0 %, 80/100) CCs exhibited positive immunostaining (+ or ++, Fig. 3B–E) with no statistically different prevalence (Fisher’s exact test, p 0.141) (Table 2). However, intensity of the positive immunostaining was different between the MSI-H (++ in 58 CCs, + in 22 CCs) and MSS (++ in 38 CCs, + in 2 CCs) (Fisher’s exact test, p = 0.002) (Table 2). In were either – (n 1) or (n 9) (Fig. 3F). The negative control using blocking reagent instead of the primary antibody showed no immu- nostainings in the tissues. There was no significant difference in pa-tients’ survival nor clinical outcomes between negative (n 25) and positive (n 120) CCs (p > 0.05). The positive immunostaining was mainly detected in the nuclei (Fig. 3), where NDS2 has been known to perform its activities [6–8]. The immunostaining was seen not only innuclei, but also in cytosol in some CCs (Fig. 3B and C). The negative control using blocking reagent instead of the primary antibody showed no immunostainings in the tissues.
4.Discussion
Epigenetic dysregulation is well-known in cancers, and some are being used as targets for cancer therapy [2,8]. For example, alterations of NSD genes are known in many cancers and NSD-targeting cancer therapies are currently being developed [8], but their alterations remain unknown in many subtypes of cancers. The aim of this study was to address whether genetic and expressional alterations of NSD2, KDM2B and SETMAR genes involved in histone methylation/demethylation, an important epigenetic gene regulation, in MSI-H CC. In the present study, we discovered that the frameshift mutations of NSD2, KDM2B and SETMAR were frequent in MSI-H CC (10.0 %, 2 % and 1 %, respectively); the NSD2 and KDM2B frameshift mutations showed ITH (12.5 % and 12.5 %, respectively); and intensity of NSD2 immunostaining in MSI-H CC was decreased compared to that in MSS, suggesting that NSD2 might be altered in MSI-H CCs. In many cases, a cancer gene usually shows not only high incidence of genetic alterations but also functional implications for tumorigenesis [19]. Functions of NSD2 alterations are currently known to be related to cancer-promoting activities in many cancers [6–8]. Increased NSD2 level induces H3K36 di-methylation, chromatin structure, which sub- sequently leads to cellular proliferation and cancer development in multiple myeloma [6,8]. Furthermore, NSD2 is overexpressed in different cancers and is related to their aggressiveness or prognosis [7]. A frameshift mutation is usually considered a loss-of-function mutation and the NSD2 frameshift mutations identified in the present study would result in premature stops and would produce truncated NSD2 proteins, which might lead the wild-type NSD2 to an inactive form. The MSI-H CCs exhibited decreased intensity of NSD2 expression and increased NSD2 frameshift mutation compared to the MSS CCs. These results suggest that NSD2 might be less activated in
MSI-H CCs than in MSS CCs, which is somewhat different from the over-activated NSD2 in other cancers [6–8]. However, it is unknown whether
altered NSD2 has a causative role in pathogenesis, or they simply reflect the phenomenon that the MSI-H CC has increased mutation frequency. The Human Pro- tein Atlas database (https://drive.google.com/file/d/1M87mhyd D-1m8iUKZ9w4NRR5YkrvgtfNR/view?usp sharing) data show that that low NSD2 mRNA expression is associated with worse prognosis of patients with CC, which is not matched to our data with no significant difference in patients’ survival between negative and positive expression in CCs. On the other hand, it appears to be in agreement with our data that show low NSD2 expression in some CCs.
The anti-NSD2 antibody adopted in this study was made using a synthetic peptide of amino acids 3–146 (NP_001035889.1). Based on the nucleotide changes of NSD2 by the frameshift mutations (p. Pro1343GlnfsX49), the mutants could be detected by the immunohis-
tochemistry. However, the NSD2 expression was either negative or mild positive ( ) in all CCs with the frameshift mutations (Table 2). This observation may suggest that the mutants could possibly be degraded by nonsense-mediated mRNA decay that is frequently present in MSI-H cancers [20]. In addition, overall decreased intensity of the positive immunostaining in MSI-H compared to MSS CCs (Table 2) may suggest that NSD2 expression is weakly activated in the MSH-H CCs.Our data indicate that the regional differences in NSD2 and KDN2B mutations are common in MSI-H CC (Fig. 2). Cancer ITH is known to play an important role in driving disease selection and adaptation in response to selection pressures in a cancer, and is known to be associated with poor prognosis and clinical outcomes [21]. The ITH in our study could ameliorate the phenotypic differences of the frameshift mutation and further functional and clinical implication of the ITH remains to be UNC8153 defined.