Frequent downregulation of BTB and CNC homology
2 expression in Epstein–Barr virus-positive diffuse
large B-cell lymphoma
Mai Noujima-Harada,1 Katsuyoshi Takata,1
Tomoko Miyata-Takata,1 Hiroaki Sakurai,2 Kazuhiko Igarashi,3
4
1
1
Etsuro Ito, Keina Nagakita, Kohei Taniguchi, Nobuhiko Ohnishi,1 Shizuma Omote,1 Tetsuya Tabata,1Yasuharu Sato1 and Tadashi Yoshino1
1Department of Pathology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama; 2Department of Cancer
Cell Biology, Graduate School of Medicine and Pharmaceutical Sciences, Toyama University, Toyama; 3Department of Biochemistry, Tohoku University
Graduate School of Medicine, Sendai; 4Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan

Key words
BACH2, diffuse large B-cell lymphoma, Epstein–Barr virus,
NFjB, TAK1
Correspondence
Katsuyoshi Takata, Department of Pathology, Okayama
University Graduate School of Medicine, Dentistry andPharmaceutical Sciences, 2-5-1 Shikata-cho, kita-ku,
Okayama City, Okayama 700-8558, Japan.
Tel: +81-86-235-7150; Fax: +81-86-235-7156;
E-mail: katsuyoshi.t@h5.dion.ne.jp
Funding Information
This work was supported by a grant from the Japan
Society for the Promotion Science (JSPS no. 24790350 and
15K19053).
Received December 15, 2016; Revised February 17, 2017;
Accepted February 20, 2017
Cancer Sci 108 (2017) 1071–1079
doi: 10.1111/cas.13213

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell lymphoma subtype, and the Epstein–Barr virus (EBV)-positive subtype of DLBCL is known toshow a more aggressive clinical behavior than the EBV-negative one. BTB and
CNC homology 2 (BACH2) has been highlighted as a tumor suppressor inhematopoietic malignancies; however, the role of BACH2 in EBV-positive DLBCL isunclear. In the present study, BACH2 expression and its significance were studied
in 23 EBV-positive and 43 EBV-negative patient samples. Immunohistochemistry
revealed BACH2 downregulation in EBV-positive cases (P < 0.0001), although biallelic deletion of BACH2 was not detected by FISH. Next, we analyzed the contribution of BACH2 negativity to aggressiveness in EBV-positive B-cell lymphomasusing FL-18 (EBV-negative) and FL-18-EB cells (FL-18 sister cell line, EBV-positive).
In BACH2-transfected FL-18-EB cells, downregulation of phosphorylated transforming growth factor-b-activated kinase 1 (pTAK1) and suppression in p65
nuclear fractions were observed by Western blot analysis contrary to non-transfected FL-18-EB cells. In patient samples, pTAK1 expression and significant nuclear
p65, p50, and p52 localization were detected immunohistochemically in BACH2negative DLBCL (P < 0.0001, P = 0.006, and P = 0.001, respectively), suggestingthat BACH2 downregulation contributes to constitutive activation of the nuclear
factor-jB pathway through TAK1 phosphorylation in BACH2-negative DLBCL(most EBV-positive cases). Although further molecular and pathological studies
are warranted to clarify the detailed mechanisms, downregulation of BACH2 may
contribute to constitutive activation of the nuclear factor-jB pathway throughTAK1 activation.

T

he Epstein–Barr virus (EBV), a linear double-stranded
DNA virus and member of the Herpesviridae family, wasinitially discovered from a Burkitt lymphoma-derived cell line
in 1964.(1) Epstein–Barr virus was subsequently found to induce
the proliferation of human B cells(2) and is currently considered
to contribute to the oncogenesis of several types of B-cell lymphoma, including Burkitt lymphoma and diffuse large B-cell
lymphoma (DLBCL).(3) Of these, DLBCL is the most common
subtype accounting for 40% of all non-Hodgkin lymphoma
cases. EBV-positive DLBCL accounts for 8–10% of DLBCL
cases in Asian countries and is frequently detected in immunocompetent people aged 50 years or older.(4) Compared with
EBV-negative DLBCLs, EBV-positive DLBCLs show anaggressive clinical behavior and were accordingly designated asa separate category in the 2008 WHO classification.
Immunophenotypically, most EBV-positive DLBCLs are of the
non-germinal center B cell type;(5) in such cases, nuclear factorjB (NFjB) activation through TNFAIP3, CARD11, and othergene mutations has been reported to play a pathogenic role.(6,7)

In addition, several groups also described NFjB activation
through latent infection membrane protein 1 (LMP1) in EBVpositive lymphoma cells.(8,9)
BTB and CNC homology 2 (BACH2) has been highlighted
as a transcriptional factor in the regulation of plasma cell differentiation. Specifically, BACH2 inhibits plasma cell differentiation by repressing PRDM1 expression, thus allowing B cells
to undergo class switch recombination and somatic hypermutation.(10,11) Sakane-Ishikawa et al.(12) reported that patients withhigher levels of BACH2 expression had a better prognosis in
DLBCL, although there seemed to be little consensus regarding the significance of BACH2 as a prognostic factor.(13) As
BACH2 was reported to mediate the negative selection of preB cells by p53 upregulation, the tumor-suppressive function of
BACH2 has been highlighted.(14) However, the role of BACH2
expression and its tumor-suppressive function in EBV-positive
DLBCL is still unclear.
In the present study, we examined BACH2 expression in
EBV-positive DLBCLs and found it to be downregulated in© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.
This is an open access article under the terms of the Creative Commons Attrib
ution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used forcommercial purposes.

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Original Article
BACH2 downregulation in EBV + DLBCL

78.3% of cases. In addition, we sought to investigate how
BACH2 negativity contributes to aggressiveness in EBV-positive DLBCL using B-cell lymphoma-derived EBV-positive and-negative cell lines.
Materials and Methods
Patient selection. Formalin-fixed, paraffin-embedded tissues(FFPET) were obtained from 23 patients with EBV-positive
DLBCL who were diagnosed from 2008 to 2011 and 43
patients with EBV-negative DLBCL who were diagnosed
from 2004 to 2012 at Okayama University Graduate Schoolof Medicine (Okayama, Japan). Three hematopathologists
diagnosed the cases according to the criteria described in
the 2008 WHO classification. The study protocol wasapproved by the Institutional Review Board of Okayama
University (IRB No. 493, November 29, 2011). All study
procedures were undertaken in accordance with the guidelines of the Declaration of Helsinki.
Immunohistochemical analysis and in situ hybridization. All
immunohistochemical analyses of FFPET were carried out using
an automated immunostainer (Bond-max; Leica Microsystems,
Wetzlar, Germany). The following primary antibodies and dilutions were used: CD20 (L26, 1:200), CD3 (PS-1, 1:50), CD10
(56C6, 1:50), CD5 (4C7, 1:100), Ki-67 (MIB-1, 1:5000), LMP1
(1:10) (all Leica Microsystems); multiple myeloma oncogene 1(MUM1) (MUM1p, 1:50), NFjB p65 (1:1000) (both from Dako,
Glostrup, Denmark); BCL-6 (D-8, 1:100) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); germinal center B-cell
expressed transcript 1 (GCET1) (RAM341, 1:100), NFjB p105/
p50 (E381, 1:250) (Abcam, Cambridge, UK); NFjB2 p100/p52(18D10, 1:100) (Cell Signaling Technology, Danvers, MA,USA); forkhead box protein P1 (FOXP1) (JC12, 1:500), andBACH2 (1:400) (both from Life Span Biosciences, Seattle, WA,
USA). In situ hybridization with EBV-encoded small RNA
probes (Leica Microsystems) was used to detect EBV. A sample
was scored as positive if >30% of the lymphoma cells were positively stained.
Fluorescence in situ hybridization for BACH2. The FFPET
were subjected to FISH using a spectrum red-labeled BACH2
probe and spectrum green-labeled centromeric probe for chromosome 6 (CEP6) (Vysis/Abbott Molecular Laboratories, Des
Plaines, IL, USA) according to the manufacturer’s instructions.
To identify the BACH2 gene, we prepared specific probes
using the BAC clones RP11-16C2, RP11-402C18, and RP11147G14, which cover approximately 500 kb of the BACH2
gene, and incubated samples with these probes at 37°C for
approximately 48 h in a Hybridizer (Dako). Cells with two
CEP6 signals were scored, and the signal ratio of BACH2 to
CEP6 was calculated. BACH2 biallelic and monoallelicdeletions were defined as having signal ratios of 20–60% and60–80%, respectively, as previously described.(15)Cell lines, RNA extraction, and RT-PCR. We prepared humannon-Hodgkin’s lymphoma cell lines (FL-18, FL-218, and FL318) and an EBV-positive sister cell line (FL-18-EB),(16,17) all
provided by Dr. Ohno at Kyoto University (Kyoto, Japan).
RNA was extracted from cultured cells using the miRNeasy
Mini kit (Qiagen, Hilden, Germany), and cDNA was produced
using SuperScript VILO MasterMix (Life Technologies, Palo
Alto, CA, USA). BACH2 and b-actin were amplified as previously described;(18) the primers are listed in Table S1.
Transfection assay. FL-18-EB cells were transfected with apIRES2-EGFP plasmid containing a BACH2 sequence using
the Neon transfection system (Life Technologies). The© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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optimum transfection conditions (pulse voltage, 1100 V;
pulse width, 30 ms; once) were set using the control plasmid pmaxGFP (Amaxa Bioscience, Basel, Switzerland).
Transfected cells were grown in RPMI 1640 medium containing 10% (v/v) FBS at 37°C in an atmosphere with 5%
CO2.
Western blot analysis. Whole cell lysates and nuclear andcytoplasmic fractions were resolved by SDS-PAGE and transferred onto nitrocellulose membranes using a Trans-Blot Turbo
Blotting System (Bio-Rad, Hercules, CA, USA). The nuclear
and cytoplasmic fractions were separated using a nuclear/cytosol fractionation kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. Antibody reactions werecarried out as previously described;(19) the primary antibodies
and conditions are listed in Table S2 (phosphorylated transforming growth factor-b-activated kinase 1 [pTAK1] was theresource from the previous report.(20)). ImageJ (NIH, Bethesda,
MD, USA) was used to quantify the protein expression and theratio to control was calculated.
Statistical analysis. STATCEl3 software (OMS, Saitama, Japan)
was used to undertake v2-test and t-test analyses. Kaplan–
Meier plots were made using SPSS version 14.0 (IBM, Chicago,
Table 1. Clinicopathological features of patients with diffuse large
B-cell lymphoma (DLBCL), grouped according to Epstein–Barr virus(EBV) positivity
EBV-positive
DLBCL (n = 23)
Clinical features
Median age,77 (2–88)
years (range)
Male : female
1.6:1.0
Clinical stage
I–II (%)
14/23 (61)
III–IV (%)
9/23 (39)Median LDH,
290 (141–1255)
IU/L (range)
Median sIL2R,
1932 (340–11000)
U/mL (range)
Anemia8/11 (73)Three-year
38.9
survival rate, %Immunohistochemical
featuresCD5-positive (%)
0/15 (0)Median49.95 (20.5–76.1)
Ki-67 labelling
index (range)
ABC type (%)
15/18 (83)
GCB type (%)
3/18 (17)Treatment
R-CHOP or
8/13 (62)
R-CHOP-likeregimen (%)
CHOP or CHOP-like
2/13 (15)regimen (%)
Radiotherapy (%)
0/13 (0)Other (%)
3/13 (23)EBV-negative
DLBCL (n = 43)

71 (30–91)

P-value

0.66

1.0:1.0

0.50

24/40 (60)
16/40 (40)243.5 (158–970)

0.84
0.31

1483.5 (165–25400)

0.63

13/31 (42)
72.2

0.16
0.04

5/43 (12)60 (27.9–83.7)

0.40
0.08

22/43 (51)21/43 (49)0.04

23/32 (72)

6/32 (19)
1/32 (3)2/32 (6)

Bold indicates statistical significance (P < 0.05). ABC, activated B-celllike; CHOP, cyclophosphamide, doxorubicin, vincristine, and prednisolone; GCB, germinal centre B-cell-like; LDH, lactate dehydrogenase; R, rituximab; sIL2R, soluble interleukin-2 receptor.

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IL, USA).
significant.

P-values <0.05

wereconsidered

statisticallyResults
Significant downregulation of BACH2 expression in EBV-positive DLBCL. The clinicopathological features of 23 cases of

EBV-positive DLBCL and 43 cases of EBV-negative DLBCL
are summarized in Table 1. The histological features andimmunohistochemical findings of representative cases areshown in Figure 1. The EBV-positive cases had a significantly
poorer prognosis than EBV-negative cases (P = 0.04; Table 1,
Fig. S1). In addition, 15 of the 18 cases (83%) of EBV-positive DLBCL showed an activated B-cell-like (ABC) phenotype
according to Choi’s criteria (P = 0.04; Table 1, Fig. 2). There

were no other significant differences in clinicopathologicalcharacteristics between EBV-positive and -negative cases of
DLBCL.
Immunohistochemically, 38 of 43 (88.4%) EBV-negative
DLBCL cases were BACH2-positive, compared to only 5 of
23 (21.7%) EBV-positive cases (P < 0.0001; Table 2a). Whenthe analysis was restricted to ABC cases, EBV-positive
DLBCL cases also showed a significantly lower BACH2
expression rate relative to EBV-negative cases (P < 0.0001;
Table 2b), suggesting that the downregulation of BACH2
expression is associated with the presence of EBV. When compared with ABC-type (22 cases) or germinal center B-cell-type(21 cases) EBV-negative DLBCL cases, EBV-positive cases
showed a significantly lower rate of BACH2 positivity(P < 0.0001 and P < 0.0001, respectively; Table S3).

Fig 1. Histologicalfeaturesandimmunohistochemical findings of representative cases ofdiffuse large B-cell lymphoma (DLBCL). (a)Immunohistochemical analysis of Epstein–Barr virus(EBV)-positive DLBCL, activated B-cell-like (ABC)type reveals that the sample is CD20-positive, CD3negative, germinal center B-cell expressed transcript
1 (GCET1)-positive, multiple myeloma oncogene 1(MUM1)-positive,
CD10-negative,
EBV-encoded
small RNA in situ hybridization (EBER (ISH))-positive,
and BACH2-negative with a Ki-67 labeling index of
62.8%. (b) Immunohistochemical analysis of EBVnegative DLBCL, ABC type indicates that the sample
is CD20-positive, CD3-negative, GCET1-positive,
MUM1-positive, CD10-negative, EBER(ISH)-negative,
and BACH2-positive with a Ki-67 labeling index of
81.2%.
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Original Article
BACH2 downregulation in EBV + DLBCL

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Fig. 2. Distribution of Epstein–Barr virus (EBV)negative (a) and EBV-positive (b) diffuse large B-cell
lymphoma (DLBCL) molecular phenotypes. ?,Positive; ⇢, negative. ABC, activated B-cell-like;
FOXP1, forkhead box protein P1; GCB, germinal
center B-cell-like; GCET1, germinal center B-cell
expressed transcript 1; MUM1, multiple myeloma
oncogene 1.

In accordance with a previous report,(21) we categorized ourdata into negative, weakly positive, or strongly positive BACH2
expression (Table S4). In total, BACH2 expression was negative, weakly positive, and strongly positive in 34.8%, 15.2%,
and 50% of cases, respectively. In EBV-negative cases, BACH2
negative, weakly positive, and strongly positive expression constituted 11.6%, 23.3%, and 65.1%. In EBV-positive cases, allfive BACH2-positive cases were strongly positive, and in EBVnegative cases, 10 cases of 38 BACH2-positive cases were weakand 28 cases of BACH2-positive cases were strongly positive.
There was no significant difference between EBV status and thestaining intensity of BACH2 (P = 0.247).

Biallelic BACH2 deletion was not detected in EBV-positive
DLBCL cases and human B-cell lymphoma cell lines. We used

FISH for the 18 EBV-positive DLBCL cases that were
immunohistochemically negative for BACH2. Of these, 15cases (83.3%) had no evidence of BACH2 gene deletion(Fig. 3a). The other three cases (16.7%) showed monoallelicdeletion; no cases had biallelic deletion. Notably, BACH2 geneloss was not detected in any EBV-negative case (Fig. S2). Further studies using human B-cell lymphoma-derived cell lines(FL-18, FL-218, FL-318, and the FL-18 sister cell line FL-18EB) were carried out. Through RT-PCR analysis, BACH2mRNA was detected in FL-18, FL-218, and FL-318 but not in
the EBV-infected line, FL-18-EB (Fig. 3b). An immunohistochemical study yielded consistent results (Fig. 3c). Notably,biallelic BACH2 deletion was not detected in either FL-18-EB
or FL-18 using FISH (Fig. 3d), indicating that BACH2 expression was downregulated in the former despite the lack of genedeletion. This result was concordant with the findings frompatient sample analyses.
Transfection of the BACH2 gene into FL-18-EB inactivated theTAK1–NFjB pathway. We transfected the BACH2 gene into FL-

18-EB cells to analyze the effect of BACH2 on the expressionof other genes. First, we confirmed the overexpression of© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

BACH2 in FL-18 cells using RT-PCR (Fig. 4a), and we examined the NFjB pathway protein expression by Western blot
analysis. From real-time PCR analysis, NFKB2 (encodes p52)
and RELA (encodes p65), the products of which comprise the
NFjB pathway, were significantly downregulated in the
BACH2-transfected FL-18-EB cells (Fig. S3). Then we particularly focused on the expression of TAK1, a representative
NFjB pathway molecule in EBV-positive B cells.(22) FL-18EB cells contained pTAK1, an active form of TAK1, whereas
this form was not detected in FL-18 cells. Notably, the transfection of BACH2 into FL-18-EB cells repressed the activationof TAK1, as shown by the reduced levels of pTAK1 (Fig. 4b).
No significant difference was observed in the expression of
total TAK1. Moreover, p65 expression of the nuclear fractions
was suppressed in BACH2-transfected FL-18-EB cells,

Table 2. Differential BTB and CNC homology 2 (BACH2) expression
in Epstein–Barr virus (EBV)-positive and EBV-negative diffuse large Bcell lymphoma (DLBCL) and EBV-positive and EBV-negative DLBCL,
activated B-cell-like (ABC) type (b)BACH2 expression
P-valueDiagnosis(a)EBV-positive DLBCL (n = 23)
EBV-negative DLBCL (n = 43)(b)
EBV-positive DLBCL, ABC type (n = 15)
EBV-negative DLBCL, ABC type (n = 22)

Positive

Negative

5
38

18
5

<0.0001

3
20

12
2

<0.0001

According to the criteria reported by Kikuchi et al.,(21) all five BACH2positive cases of EBV-positive DLBCL were strongly positive for BACH2.
Among 38 BACH2-positive cases of EBV-negative DLBCL, 10 cases wereweakly positive and 28 cases were strongly positive for BACH2.

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suggesting that BACH2 represses the TAK1–NFjB pathway(Fig. 4c,d).
Phosphorylated TAK1 expression and p65, p50, and p52 nuclear
localization in BACH2-negative DLBCL. Finally, we immunohisto-

chemically evaluated the localization of p65, which was significantly downregulated in BACH2-transfected FL-18-EB cells,
by real-time PCR, in 36 cases of BACH2-positive DLBCL and14 cases of BACH2-negative DLBCL that were available forfurther examination and successfully stained. Among the
BACH2-positive DLBCLs, only 2 of 36 cases (5.6%) showed
nuclear p65 localization, whereas 10 of 14 cases (71.4%) of
BACH2-negative DLBCL showed nuclear p65 localization(P < 0.0001; Table 3, Fig. 4e,f). The BACH2-negative cases
were found to express pTAK1, whereas BACH2-positive cases
did not (Fig. 4e,f). Furthermore, BACH2-positve DLBCL
showed significant cytoplasmic staining of p50 and p52(P = 0.006 and P = 0.001, respectively; Fig. 4g,h, Table 3),although the total number of examined cases was limited due
to shortage of samples. In addition, significant nuclear localization of p65, p50, and p52 was observed in EBV-positive cases(P < 0.0001, P < 0.01, and P < 0.01, respectively; Table S5).
Discussion

Despite previous reports of the aggressive behavior of EBVpositive DLBCL, no reports have addressed the tumor-suppressive factor BACH2. The findings from the present study reveal
significant downregulation of BACH2 expression in EBV-positive DLBCL and suggest its importance in EBV-positive B-cell
lymphomas. In our study, we used the FL-18 and EBV-positive FL-18-EB cell lines, established from a single patient
diagnosed with follicular lymphoma, which harbor t(14;18)
and the MYC-related translocation t(8;22)(q24;q13).(16,17)
Therefore, these paired cell lines were considered a goodmodel to analyze the impact of EBV in aggressive B-cell lymphoma. Additionally, FL-18-EB showed partial LMP1 positivity in our examination (Fig. S4).

Original Article
Noujima-Harada et al.

A member of the CNC (Cap’n’collar) group of transcription
factors, BACH2 forms heterodimers with small Maf family
proteins. These heterodimers repress the expression of target
genes by binding to a DNA element termed the Maf recognition element.(10,23) Expression of BACH2 has been describedin hematopoietic cells and nerve cells,(24,25) and the functionof BACH2 has been well investigated in B lymphocytes.
BACH2 represses plasma cell differentiation by repressing
BLIMP1 transcription, and studies of BACH2-deficient mice
have led to the description of this protein as indispensable forthe induction of class switch recombination.(11,26)
In high-grade lymphomas, a loss of heterozygosity on thelong arm of chromosome 6 is frequently detected.(27) BACH2
is accordingly among the candidate genes associated with lymphomagenesis,(28,29) and several research groups have recently
described the tumor-suppressive function of its protein product.
For example, Swaminathan et al.(14) reported that BACH2positive B cells resist MYC-induced leukemic transformation
through p53 upregulation. In chronic myeloid leukemia,
Yoshida et al.(30) reported that BACH2 phosphorylation, which
occurred downstream of BCR/ABL signaling, repressed
nuclear translocation and led to the expression of the antiapoptotic factor heme oxygenase-1. Furthermore, Chenet al.(31) reported that, in mantle cell lymphoma, BACH2
localization determined cell viability in response to oxidative
stress. In the present study, significant downregulation of
BACH2 expression was observed in EBV-positive DLBCL,
suggesting that BACH2 negativity contributes to the presenceof EBV.
In previous reports, immunohistochemical BACH2 expression was described as negative, weakly positive, and strongly
positive. The negative-case ratio of our series (34.8%) is very
similar to that of the series (35%) by Kikuchi et al.(21)
Sakane-Ishikawa et al.(12) and Ichikawa et al.(13) reported no
BACH2-negative case in their series, which seems to be different from our data. However, in our series, only 11.6% of
EBV-negative cases were completely negative for BACH2.

Fig. 3. (a) Fluorescence in situ hybridization (FISH)
analysis using a spectrum red labeled BACH2 probe
and spectrum green labeled centromeric probe forchromosome 6 (CEP6). Epstein–Barr virus (EBV)positive diffuse large B-cell lymphoma (DLBCL)
yielded a signal ratio of 90.5%. (b–d) BACH2
expression in human non-Hodgkin’s lymphoma celllines. (b) BACH2 expression was not detected by
PCR in the FL-18-EB cell line. (c) BACH2 wasexpressed in FL-18 cells but not in FL-18-EB cells. (d)FL-18 and FL-18-EB showed signal ratios of 94%
and 93%, respectively, by FISH analysis with the
BACH2 probe.
Cancer Sci | May 2017 | vol. 108 | no. 5 | 1075

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Original Article
BACH2 downregulation in EBV + DLBCL© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Fig. 4. (a) BACH2 was detected in BACH2-transfected FL-18-EB cells (FL-18-EB [BTB and CNC homology 2 (BACH2)+]) by PCR. (b) Phosphorylatedtransforming growth factor-b-activated kinase 1 (pTAK1) expression was downregulated in FL-18-EB (BACH2+) cells as shown by Western blot
analysis. (c) p65 expression was downregulated in nuclear fractions of FL-18-EB (BACH2+) cells compared to FL-18-EB and FL-18-EB (control vector) cells. (d) The ratio of BACH2 expression level to LaminB1 is shown. (e) Immunohistochemistry in BACH2-negative diffuse large B-cell lymphoma (DLBCL) shows p65 positivity in the nucleus, pTAK1 positivity, and EBV-encoded small RNA in situ hybridization (EBER (ISH)) positivity. (f)Immunohistochemistry in BACH2-positive DLBCL shows p65 positivity in the cytoplasm, pTAK1 negativity, and EBER (ISH) negativity. (g) Immunohistochemistry in BACH2-negative DLBCL shows nuclear staining of p50 and p52. (h) Immunohistochemistry in BACH2-positive DLBCL shows cytoplasmic staining of p50 and p52.

Therefore, we suppose this difference is not crucial. We
focused on EBV status, and our proportion of EBV-positive
cases is different from other reports. We suppose that thehigher negative ratio of BACH2 was due to the larger numberof EBV-positive DLBCLs in our series. Moreover, the antiBACH2 antibody used was different among the studies, whichmight affect the results. In our cases, there was no significant
difference between EBV status and the staining level of
BACH2, indicating BACH2 positivity or negativity is more
important than BACH2 immunostaining intensity as to EBV
status.
Previous reports have not examined the mechanism underlying the downregulation of BACH2 protein expression in EBVpositive DLBCL. Notably, Takakuwa et al.(32) reported thatthe integration of EBV into BACH2 induced a loss of BACH2
expression in the Raji Burkitt lymphoma cell line. As FISH
did not detect biallelic deletion in any patient samples or celllines used in our study, viral integration may have occurred in
BACH2. Epigenetic silencing is another possible mechanism
underlying the downregulation of BACH2 expression, as waspreviously reported in gastric cancer.(33) Although we treatedFL-18-EB cells with the DNA methylation inhibitor 5-azacytidine, we were unable to recover BACH2 expression (data notshown), thus failing to support a methylation-based mechanismof reduced expression. In contrast, proteolytic underlying
mechanisms remain worth considering. Another member of the
CNC transcription factor group, Nrf2, has been widely investigated, and the role of Keap1 in Nrf2 ubiquitination and subsequent proteasomal degradation has attracted considerable
attention. Many reports have revealed an association between
abnormal Keap1–Nrf2 signaling and tumorigenesis in many
types of carcinomas.(34,35) Therefore, further evaluation of
BACH2 proteolysis may be merited.
In this report, we also attempted to elucidate the association
between BACH2 downregulation and NFjB activation. In
EBV-infected cells, LMP1, which is produced by EBV andexpressed in the cell membrane, has been reported to affectneoplastic transformation through the ligand-independent

activation of several signaling pathways that induce cell
growth.(36,37) As the NFjB pathway was among these oncogenic pathways, we focused on the association between
BACH2 and the NFjB pathway.(38) In canonical NFjB activation, tumor necrosis factor receptor-associated factor 6 ubiquitination by the C-terminal activation region 2 of LMP1
induces TAK1 binding protein 2/3 activation. Transforming
growth factor-b-activated kinase 1 forms a complex with
TAK1 binding protein 2/3 and TAK1 is subsequently activatedby phosphorylation and polyubiquitination, leading to phosphorylation of the IjB kinase complex and degradation of IjBa.
Subsequently, p50 and p65 transfers to the nucleus and induces

Table 3. Differential p65, p50, and p52 expression in BTB and CNC
homology 2 (BACH2)-positive and BACH2-negative diffuse large B-cell
lymphoma (DLBCL)
Diagnosis
p65
BACH2-positive DLBCL (n = 36)
BACH2-negative DLBCL (n = 14)
p50
BACH2-positive DLBCL (n = 17)
BACH2-negative DLBCL (n = 16)
p52
BACH2-positive DLBCL (n = 14)
BACH2-negative DLBCL (n = 16)

Cytoplasmic

NuclearP-value

34
4

2
10

<0.00011681
80.0060147090.0010Cancer Sci | May 2017 | vol. 108 | no. 5 | 1077

Fig. 5. Schema of BTB and CNC homology 2 (BACH2) interaction in
the nuclear factor-jB (NFjB) signaling pathway. BACH2 repressed thetransforming growth factor-b-activated kinase 1 (TAK1) phosphorylation and induced the inactivation of the NFjB signaling pathway. In
Epstein–Barr virus (EBV)-positive B-cells, BACH2 downregulation contributes to the activation of the NFjB pathway through TAK1 activation. CTAR2, C-terminal activation region 2; IKK, IjB kinase; LMP1,
latent infection membrane protein 1; P, phosphorylation; TAB2/3,
TAK1 binding protein 2/3; TRAF6, tumor necrosis factor receptor associated factor 6; Ub, ubiquitination.
© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Original Article
BACH2 downregulation in EBV + DLBCLthe expression of genes related to cell proliferation andsurvival.(39)
Although the target protein of BACH2 in the NFjB pathway
has not yet been clarified, BACH2 expression led to a decreasein TAK1 phosphorylation, which occurs upstream of the NFjB
pathway. Therefore, BACH2 was suggested as a repressive
factor upstream of TAK1 phosphorylation. Immunohistochemistry of pTAK1 was not detected in BACH2-positive DLBCL
cases and it was determined that these cases showed cytoplasmic p65 expression, whereas pTAK1 positivity and nuclear
p65 expression were observed in BACH2-negative DLBCL.
Furthermore, nuclear p65 expression was repressed in the
BACH2-transfected cells by Western blot analysis.
Compagno et al.(40) and Montes-Moreno et al.(5) used antip50 and -p52 antibody to evaluate NFjB activation, and Ok
et al.(41) reported that expression of p65 showed no difference
between EBV-positive and -negative DLBCLs, contrary to theresults of p50 and p52 in Caucasian. In our present study, p50,
p52, and p65 had very similar status, and p65 frequently localized to the nucleus in EBV-positive cases (P < 0.0001;
Table S5). There are some possible factors for the discrepancy,such as differences in race and antibodies (although the antibody information was not available for the previous report).
EBV-associated lymphoproliferative disorder is more frequentin Asian than in Caucasian, that is, vast majority of patientswith chronic active EBV infection, or EBV-associated naturalkiller cell lymphoma of nasal type is almost exclusively foundin Asia. Therefore, this discrepancy could be potentiallyexplained by the difference of the population. All 66 DLBCL
cases in our series are from Japanese patients.
Figure 5 shows a schema of the relationships between
BACH2 and the NFjB pathway, showing that BACH2
repressed TAK1 phosphorylation and induced the inactivation

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Toshiharu Maeda, Tadayoshi Kunitomo, Yoshimi Bando, Yasushi Terasaki, Yoshinobu Maeda, Yumi Oshiro, Akira Hida, Eiji Ikeda, and
Aya Ishii.

Disclosure Statement
The authors have no conflict of interest.

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Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article:
Table S1. Primer sequences used to amplify BACH2 and b-actin.
Table S2. Antibodies and conditions for Western blot analysis.
Table S3. Differential BTB and CNC homology 2 (BACH2) expression in Epstein–Barr virus (EBV)-positive and EBV-negative diffuse large
B-cell lymphoma (activated B-cell-like [ABC] and germinal center B-cell-like [GCB] types).
Table S4. BTB and CNC homology 2 (BACH2) expression level based on staining intensity.
Table S5. Differential p65 expression in Epstein–Barr virus (EBV)-positive and EBV-negative diffuse large B-cell lymphoma.
Fig. S1. Kaplan–Meier curves for overall survival in Epstein–Barr virus (EBV)-positive and EBV-negative diffuse large B-cell lymphoma.
Fig. S2. Fluorescence in situ hybridization analysis using a spectrum red labeled BACH2 probe and spectrum green labeled centromeric probe forchromosome 6 (CEP6) in Epstein–Barr virus-negative diffuse large B-cell lymphoma.
Fig. S3. NFKB1, NFKB2, and RELA expression in FL-18-EB (BTB and CNC homology 2 [BACH2]+) cells analyzed by real-time PCR.
Fig. S4. Immunohistochemical analysis of latent infection membrane protein 1 (LMP1) in FL-18-EB cells.

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© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.