pneumonia, large intestine inflammation, and type I
diabetes [4]. Furthermore, primary non-response as well as
acquired resistance to immune-checkpoint blockers remaina challenge. Thus, the need for the development of novel
treatment approaches remain.
Histone deacetylases (HDACs) remove acetylgroups from lysine residues on histones. Removing theacetyl group alters chromatin structure by facilitating
chromatin condensation to promote transcriptional
repression [5]. Aberrant HDAC expression, dis-regulationof HDAC activity or imbalances between HDACs andhistone acetyltransferases are likely involved in thedevelopment and progression of several malignant tumors[6]. Melanomas contain high levels of HDAC-1, 2 andmelanoma cells have been shown to overexpress HDAC1, -2, -3, compared to non-malignant cells [6]. HDAC
inhibitors are pharmacologic compounds that interferewith the deacetylation reaction mediated by HDACs.
HDAC inhibitors have potent anti-proliferative effects
on melanoma cells [7–9] and thus represent promisingtherapeutic agents for malignant melanoma [10].
Transducin-like Enhancer of Split (TLE) family
members are transcriptional co-factors that play criticalroles in cell proliferation and differentiation [11]. Recently,
we reported that TLE3, a TLE family member, inducescell proliferation and suppresses cell differentiation

skeletal muscle stem cells. Skeletal muscle stem cells, likemelanocytes, are also mesenchymal lineage cells [12]. TLE
proteins do not bind DNA directly but are instead recruited
to chromatin by other transcription factors where they
then reduce the activity of a target transcriptional factor[11]. TLE proteins consist of a five-domain structure [13]:Q domain; a glycine/proline rich (GP) domain; a CcN
domain; a serine/proline rich (SP) domain; and a WD40
domain. TLE proteins usually work as transcriptional corepressors by interacting with and recruiting HDACs [14].
The GP domain is especially essential for interaction of
TLE proteins with HDACs [15–18].
Here, we examined the relationship between
expression of TLE3 and malignant melanoma, as well as
the effect of TLE3 on cell proliferation using melanoma
cells.

RESULTSTLE3 expression levels are increased in humanmalignant melanomas
We first examined the mRNA levels of TLE3
in human melanoma patients by analyzing an NCBI
Gene Expression Omnibus (GEO) dataset of melanoma
microarray profiles [19]. The expression of TLE3 in

Figure 1: The expression levels of TLE3 are increased in human malignant melanoma. The expression of TLE3 in normalskin, benign nevi, and malignant melanoma of patients (GSE3189 dataset) [19]. Expression levels of TLE3 are presented as boxplots
and means were compared using unpaired ANOVA with Tukey-Kramer post-hoc test and Wilcoxon’s signed rank test (A). HMV-II cells
were stained with TLE3 antibody, rhodamine phalloidin (phalloidin), or DAPI (B). Skin from 12-week-old C57BL/6J male mice was
immunostained with anti-Tle3 antibody. The boxed areas in the left panel are shown as magnified images of hair follicles in the right panel.
Scale bars indicate 500 μm (left panel) and 100 μm (right panel) respectively. Representative images of several sections are shown (C).
B16 cells were stained with Tle3 antibody, phalloidin, or DAPI (D). Representative images are several experimental repeats shown. Scalebar corresponds to 100 μm (B and D).
www.oncotarget.com

405

Oncotargetbenign skin nevi was higher than in normal skin. The
expression of TLE3 was further increased in malignant
samples compared to benign skin nevi (Figure 1A),
suggesting that the expression of TLE3 is involvedin the progression of melanoma. We then confirmed
whether TLE3 was expressed in an additional melanoma
cell type. Immunofluorescence imaging revealed that
TLE3 was also expressed in HMV-II human melanoma
cells (Figure 1B). next, we examined Tle3 expression inmurine melanocytes. Tle3 was highly expressed in hairfollicles melanocytes, which contain distinct melanin
granules (Figure 1C and Supplementary Figure 1). We
also confirmed that Tle3 was expressed within the nucleiof B16 murine melanoma cells (Figure 1D).

showed that CyclinD1 expression correlated with theoverexpression of Tle3 (Figure 2E). Consistent with thechanges in mRNA expression, overexpression of TLE3
also increased the protein levels of CyclinD1 (Figure2F). An in vitro cell proliferation assay also demonstrated
that overexpression of Tle3 increased the proliferationof B16 cells (Figure 2G). To determine if Tle3 affects
proliferation in vivo, we subcutaneously injected micewith B16 cells over-expressing Tle3 and assessed thedevelopment of tumors. The size of tumors formed from
B16 cells over-expressing Tle3 was larger than fromcontrol cell tumors (Figure 3). These data indicate thatTle3 stimulates cell proliferation of B16 in vivo andin vitro.

Overexpression of Tle3 increases theproliferation of B16 melanoma cells

Knockdown of Tle3 (TLE3) in melanoma cells
decreases proliferation

A characteristic feature of melanoma is rapid cellproliferation [2]. Tle3 has been shown to stimulate cellproliferation in skeletal muscle satellite cells [12]. We
hypothesized that Tle3 may also play role in proliferationof melanoma cells. Overexpression of Tle3 in B16
melanoma cells stimulated mRNA expression of cell
cycle related genes such as CyclinD1,CyclinD2, andCyclinA2 (Figure 2B-2D). Immunofluorescence staining

We next examined the effect of Tle3 reduction on
the proliferation of B16 cells. In contrast to the effect ofTle3 overexpression, siRNA-mediated knockdown of Tle3
led to a reduction in the protein levels of CyclinD1 as well
as a reduction in the number of CyclinD1 positive cells(Figure 4A and 4B). An in vitro cell proliferation assay
also demonstrated that Tle3 knockdown cells proliferated
at a reduced rate compared to Control siRNA cells

Figure 2: Overexpression of Tle3 increases proliferation in B16 melanoma cells. (A-F) B16 cells stably expressing Myc-tagged

Tle3 or empty vector were generated after positive selection with G418. The messenger RNA levels of Tle3 (A), CyclinD1 (B), CyclinD2(C), or CyclinA2 (D) were determined by qPCR on day 2. B16 cells with high expression of Tle3 co-expressed CyclinD1 in the nuclei.
Scale bar corresponds to 40 μm (E). Protein levels of CyclinD1, Myc-tagged Tle3, or β-actin were determined by western-blot analysis
on day 2 (F). Overexpression of Tle3 increased the proliferation of B16 cells assessed by water-soluble tetrazolium salt (WST) -8 assay.
Proliferation was quantified on day 2 by spectrophotometric absorbance measurement at 450 nm (G). Data are expressed as the mean ± SD
(n = 3). **, p < 0.01 versus control (A-D, G). Representative images were shown (E and F).
www.oncotarget.com

406

Oncotarget

Figure 3: Overexpression of Myc-Tle3 in subcutaneously injected B16 melanoma cells increases tumor size in vivo.

BALB/cA Jcl-nu/nu mice (n=5) were injected subcutaneously with 1 × 105 control B16 cells (left side; arrow heads) or cells stably
expressed Myc-tagged Tle3 (right side; arrows). Representative photograph of a mouse (A) and resected tumors (B) 3 weeks after injectionof with B16 cells. Scale bar corresponds to 10 mm (B). Resected tumors were immunostained with anti-Tle3 antibody. Scale bars indicate
25 μm (C). Representative images of several sections are shown. The volume of resected tumors was quantified. The data are expressed as
the mean ± SD (n = 10). *, p < 0.05 versus control (D).

Figure 4: Knockdown of Tle3 (TLE3) in melanoma cells decreases proliferation. (A-C) B16 cells were transfected withscramble siRNA, or siRNA against murine Tle3 (siTle3-1, siTle3-2). Protein levels of Tle3, cyclinD1, or β-actin were assessed bywestern blotting analysis (A). The numbers of cyclinD1 positive cells were decreased in the Tle3 knockdown B16 cells (B). In cells Tle3
knockdown cells, proliferation ability on day 2 and day 3 was decreased in comparison to scramble siRNA cells (C). (D-F) HMV-II cells
were transfected with scrambled siRNA or siRNA against human TLE3 (siTLE3-1, siTLE3-2). Protein levels of TLE3, CYCLIN A2, or
β-ACTIN were assessed by western blotting analysis (D). The numbers of KI67 positive cells were decreased in the TLE3 knockdown
HMV-II cells (E). In TLE3 knockdown HMV-II cells, proliferation on day 4 was decreased in comparison to scrambled siRNA cells (F).
Scale bar corresponds to 100 mm (B and E). **, p < 0.01 versus scramble (C and F).
www.oncotarget.com

407

Oncotarget

(Figure 4C). In human HMV-II melanoma cells, siRNAknockdown of TLE3 expression resulted in a reductionof CYCLIN A2 protein levels (Figure 4D). siRNAmediated knockdown of TLE3 led to a reduction in thenumber of KI67-positive cells (Figure 4E) as well as adecrease in proliferation (Figure 4F) compared to Control
siRNA cells. Moreover, the size of tumors derived fromthe subcutaneous injection of B16 cells in which Tle3 hadstably been knocked-down by shRNA were smaller thanthat of control tumors (Figure 5). These data indicate thatTle3 is required, at least in part, for proliferation in the
B16 mouse melanoma model.

M344 (Supplementary Figure 2A and 2B). TSA, Apicidin,
and M344 were also able to suppress proliferation in
HMV-II cells (Supplementary Figure 2C), and COLO679
cells (Supplementary Figure 2D). TSA and Apicidin also
eliminated the positive effect of Tle3 overexpression on
B16 cell proliferation (Figure 6A and Supplementary
Figure 2E), suggesting that Tle3 functions via a HDACdependent mechanism. Tle3 (1-140), a truncated mutant
of Tle3 lacking the HDAC-binding GP domain, failed toincrease the expression of CyclinD1, and proliferation(Figure 6B-6D). Moreover, the size of tumors derived fromthe subcutaneous injection of B16 stably overexpressing
Myc tagged-Tle3 (1-140) cells were not larger than that ofcontrol tumors (Figure 6E and 6F). Altogether, these datasuggest that HDACs are required for the TLE3’s ability toincrease proliferation of melanoma.

TLE3 induces proliferation of B16 cells via
HDACs
TLE3 usually requires interaction with HDACs via
the TLE3 GP domain to function as a co-repressor for the
repression of several different transcription factors [15].
Furthermore, several studies have demonstrated that HDAC
inhibitors repress proliferation of malignant melanoma[7–9]. We thus examined whether HDACs are involved inaugmenting the effects of Tle3 on the proliferation of B16
cells. Using a GST pulldown assay, we first confirmed thatTle3 does indeed bind directly to Hdac1 (Supplementary
Figure 3). Consistent with a role for HDACs in melanoma
proliferation, treatment of B16 cells with trichostatin
A (TSA), an HDAC inhibitor, resulted in a reductionof proliferation (Figure 6A). This effect could also be
observed with two other HDAC inhibitors – Apicidin and

DISCUSSIONThe Groucho/TLE proteins are a family oftranscriptional co-factors implicated in the regulation ofcell proliferation, differentiation and cell-fate events in
multiple tissues. In mesenchymal tissues, we and others
have previously demonstrated that under physiologicalconditions TLE3 regulates proliferation and differentiationof adipocytes [20, 21], osteoblasts [21] and skeletal
muscle cells [12]. Here in this study, we have extended
our understanding of TLE3 function by exploring its rolein the pathological proliferation of neural crest-derived
melanocyte cancer cells. More specifically, we show that

Figure 5: Knockdown of Tle3 in subcutaneously injected B16 melanoma cells decreases tumor size in vivo. B16 cells,

stably expressing control shRNA (Control) or shRNA against Tle3 (sh-Tle3) were generated by blasticidin selection. Protein levels of Tle3
were decreased in cells stably expressing sh-Tle3 (A). BALB/cA Jcl-nu/nu mice (n=5) were injected subcutaneously with 1 × 105 control
cells (left side; arrow heads) or sh-Tle3 (right side; arrows). Representative pictures of subcutaneous tumors (B) and resected tumors(C) 3 weeks after injection of B16 cells are shown. Scale bar corresponds to 10 mm (C). Resected tumors were immunostained with anti
-Tle3 antibody. Scale bars indicate 25 μm. Representative images of several sections are shown (D). The volume of resected tumors were
quantified (E). The data are expressed as the mean ± SD (n = 10). *, p < 0.05 versus control (E).
www.oncotarget.com

408

Oncotarget

the TLE3 is enriched during the malignant alteration ofmelanocytes in human patient tumor samples, and wefurther present evidence that Tle3 promotes proliferationin murine B16 and human HMV-II melanoma cellsby an HDAC activity-dependent mechanism. Finally,
we demonstrated the clinical relevancy of Tle3 as atherapeutic target by showing that Tle3 knockdown led toa significant reduction in tumor size in vivo.
Consistent with a role for TLE3 in melanoma
development, our analysis of gene expression data frompatient samples showed that TLE3 mRNA was highly
enriched in tumor samples compared to normal skinor benign skin nevi samples. A correlation between
elevated TLE family expression and tumorigenesis
was first observed in cervical squamous metaplasias
and carcinomas [22]. Since then, TLE2 and TLE3 havealso been shown to be induced during the malignant
progression of meningiomas [23]. More recently, analternatively spliced isoform of TLE3 was found to besignificantly enriched in prostate tumors [24]. Taken
together, dysregulation of TLE3 expression appears
to have a major impact on tumorigenesis. Our dataalso demonstrates that Tle3 has a positive effect on the
proliferation of melanoma cells. Overexpression of

Figure 6: HDACs are involved in the enhancement of the proliferation of B16 cells by Tle3. (A-C) B16 cells were transiently

transfected with empty vector (Mock) or Myc-tagged Tle3 and then treated with DMSO, or the indicated concentration of trichostatin A.
Cell proliferation was evaluated on day 2 by water-soluble tetrazolium salt (WST) assay and absorbance measurement at 450 nm (A).
Schematic of the C-terminally truncated forms of the Myc-tagged Tle3 plasmids used in these experiments. Q; glutamine rich domain,
GP; glycine/proline rich domain, CcN; CcN domain, SP; serine/proline rich domain, WD, tryptophan/aspartic acid repeat domain (B).
C-terminally truncated forms of Tle3 were transfected in B16 cells and proliferation ability measured on day 2 by WST assay (C). The
data are expressed as the mean ± SD (n = 3). **, p < 0.01 versus Mock transfection (A and C). B16 cells were transfected with empty vector(Mock), Myc-tagged Tle3 (1-782), or Myc-tagged Tle3 (1-140). Protein levels of cyclinD1, Myc (tagged) or β-actin were assessed bywestern blotting analysis on day 2 (D). (E and F) B16 cells stably expressing Myc-tagged Tle3 (1-782), Myc-tagged Tle3 (1-140), or empty
vector were generated after positive selection with G418. BALB/cA Jcl-nu/nu mice (n=5) were injected subcutaneously with 1 × 105 mock
B16 cells (a), cells stably expressing Myc-tagged Tle3 (1-782) (b), or cells stably expressing Myc-tagged Tle3 (1-140) (c). Representative
photograph of a mouse 3 weeks after injection with B16 cells (E). The volume of resected tumors was quantified. The data are expressedas the mean ± SD (n = 5). *, p < 0.05 (F).
www.oncotarget.com

409

Oncotarget

Tle3 led to an increase in the proliferation of B16 cells
in vitro and in vivo whereas Tle3 knockdown decreased
proliferation. This data is in agreement with our previous
study in skeletal muscle where TLE3 expression promoted
the expansion and proliferation of satellite cells [12]. TLE
family proteins play multiple and sometimes opposing
roles on cell proliferation. Although several studies
have shown a correlation in TLE protein expression andtumor progression, few have demonstrated causality by
manipulating TLE levels. Forced expression of TLE1
in embryonic fibroblasts promotes cell growth andtransformation [25] and similarly, TLE1 transgenic micedevelop lung tumors with increased alveolar epithelial
proliferation [26]. In acute myeloid lymphoma, loss of
TLE1 or TLE4 increases cell proliferation suggesting that
under some conditions TLE proteins may act as tumor
suppressors [27]. Our data not only shows an enrichment
of TLE3 in melanoma, but also shows that knockdown of
TLE3 can reduce cell proliferation and tumor growth thus
identifying a potential therapeutic target.
The mechanism by which TLE3 affects melanoma
proliferation remains unknown although our data strongly
suggests that it involves the modulation of HDAC activity.
TLE3 does not have DNA binding domain and is thus
not expected to bind DNA on its own. Instead, TLE3,
along with HDACs, are recruited to DNA by multiple
transcription factors where they subsequently reduce theactivity of several targets [11]. Our data demonstrates
that TLE3 can indeed bind to HDAC1 and moreover
inhibition of HDAC activity prevented the promotion ofcell proliferation by TLE3. HDAC inhibitors have beenshown to have anti-proliferative effects on malignant
melanoma cells [7–9]. HDAC inhibitors induce theexpression of cyclin dependent kinase inhibitors (CK1)
p21 by increasing the acetylation of chromatin at thegene promoter region. High levels of p21 are associatedwith a G1/S phase cell cycle arrest due to the inhibitionof CDK2 by p21 [8, 28]. In preliminary experiments, wehave observed that overexpression of Tle3 significantly
reduced the expression of CK1 (p21) (Data not shown),suggesting that Tle3 along with HDACs may interact
with and inhibit the transcription factor(s) that regulate
the expression of CK1 (p21). Our data does not howeverdetermine which specific class of HDACs are involved inthese mechanisms (Supplementary Figure 2). In addition,
since TSA treatment reduced the expression of TLE3 (datanot shown) it is also possible that the suppressive effectof TSA on the proliferation of melanoma is due to direct
HDAC repression of TLE3 expression. HDAC inhibitors
also induce apoptosis in several kinds of malignant
tumor cells [2]. Our preliminary data showed that overexpression of Tle3 reduced the number of apoptotic
B16 cells induced by TSA treatment (data not shown),
indicating that the reduction of apoptosis by Tle3 may
be involved in the regulation of B16 tumor size by Tle3.

www.oncotarget.com

Further experiments are needed to elucidate the exact roleof TLE3 in the pathophysiology of melanoma.
Canonical Wnt (Wnt-β-catenin) signaling is
mediated through β-catenin. Wnt-β-catenin is activatedby the binding of secreted canonical Wnts, such as Wnt1
and Wnt3a to Lrp and Frizzled coreceptor complexes tostabilize intracellular pools of β-catenin and activate Tcf/
Lef dependent transcription [29]. Although it is clear thatβ-catenin is critical during the early stages of melanocytetransformation [30], conflicting studies on the role ofβ-catenin in melanoma proliferation and metastasis havebeen published. Malignant melanoma patients with highlevels of β-catenin have better prognosis [31, 32]. Wnt3aor small-molecule activators of β-catenin signaling
reduce the proliferation in B16 melanoma cells and otherhuman melanoma cell lines in vitro. B16 melanoma
cells overexpressing Wnt3a also have decreased tumor
size and metastasis when cells are implanted into mice
[32, 24]. TLE family members, including TLE3, act astranscriptional co-repressors of canonical Wnt-β-cateninsignaling via binding to the downstream effectors TCF/LEF
and subsequently inhibiting Wnt target gene transcription[11, 33–35]. Indeed, in our in vitro experimental model,
overexpression of Wnt3a decreased the proliferation of
B16 cells and overexpression of Tle3 also repressed Wntβ-catenin signaling induced by Wnt3a or a constitutively
active form of β-catenin (Supplementary Figure 4). These
data suggest that the suppression of Wnt-β-catenin signaling
by Tle3 might be involved in the mechanism underlying theproliferative effect of Tle3 in B16 cells.
In conclusion, the reduction of TLE3 levels
may provide a novel and beneficial method to control
melanoma. Needless to say, it is important to examine therole of Tle3 not only in cell proliferation but also in cell
apoptosis, invasion and distal metastasis to understand thefull scope of melanoma especially in vivo.

MATERIALS AND METHODS
Microarray data mining
To examine TLE3 expression in patient melanoma
samples, dataset GSE3189 [19] was downloaded from the
NCBI Gene Expression Omnibus (GEO) and analyzed.

Mice
12 week-old male C57BL/6 and 12 week-old
male BALB/cA Jcl-nu/nu mice were purchased from
CLEA Japan Inc. (Tokyo, Japan). All mice were usedin accordance with guidelines from the Kyushu Dental
University Animal Care and Use Committee. All
experiments were carried out with the approval of theAnimal Use and Care Committee of the Kyushu Dental
University (Approval number #17-23).

410

Oncotarget

B16 cells, transfection, and selection of G418 or
blasticidin resistant clones

aaggccaaccgtgaaaagat; reverse, gtggtacgaccagaggcatac).
SYBR green-based quantitative real-time PCR was
performed using PowerUp SYBR Green MasterMix (Thermo Fisher Scientific, Waltham, MA) with
QuantStudio 3 system (Thermo Fisher Scientific). Values
were normalized to β-actin using the 2-ΔΔCt method [38].

B16 cells (RCB1283) (MTA; RM87746), HMVII cells (RCB0777) (MTA; RM87747), or COLO679
cells (RCB0989) (MTA; RM87746) were purchasedfrom RIKEN BRC (RIKEN, Ibaragi, Japan). B16 cells
were maintained in DMEM supplemented with 10%
fetal bovine serum (FBS) and 2 mM L-Glutamine [36].
HMV-II cells were maintained in DMEM/Ham’s F-12
supplemented with10% FBS. COLO679 were maintainedin RPMI Medium1640 supplemented with 20% FBS. B16
cells were transfected with Myc-tagged murine Tle3 (1782), Myc-taged murine Tle3 (1- 140) [12], Myc-pcDEF3
empty vector (Control), shRNA against murine Tle3(#1792), or shRNA against LacZ (Control) [29]. Cells
transfected with Myc-pcDEF3 or Myc-pcDEF3 empty
vector were treated with G418 (Roche, Basel, Switzerland)for 2 weeks until G418-resistant clones emerged. Cells
transfected with shRNA against murine Tle3 (#1792)or LacZ (control), were treated with blasticidin (Wako,
Osaka, Japan) to obtain blasticidin resistant clones.

Immunohistochemistry analysis
Freshly isolated skin from C57BL/6 mice wereimmediately fixed in 4% paraformaldehyde in PBS andsubsequently embedded in paraffin. Vertical sections, 6 to
8 μm thick, were deparaffinized in xylene and rehydrated
with a graded series of ethanol concentrations. Sections
were incubated at 4°C overnight with polyclonal antiTLE3 antibody (Proteintech, Chicago, IL, USA), or normal
Rabbit IgG (MBL, Aichi, Japan). After washing, sections
were incubated for 1h with peroxidase-labeled secondary
antibodies (Histofine Simple Stain)(Nichirei Biosciences,
Tokyo, Japan). Diaminobenzidine (Histofine DAB-3S kit)
(Nichirei Biosciences) served as the peroxidase substrate.
ABZ-9000 (Keyence, Tokyo, Japan) microscope was used
for these analyses.

HDAC inhibitor treatment

Immunocytochemistry analysis

B16 cells, HMV-II cells, or COLO678 cells
were treated with trichostatin A (TSA) (Sigma Aldrich
Chemicals, St. Louis, MO, USA), Apicidin (BioVison,
Milpitas, CA, USA), M344 (BioVison, Milpitas, CA,USA), Sodium 4-Phenylbutyrate (BioVison, Milpitas,
CA, USA), Splitomicin (BioVison, Milpitas, CA, USA),or Valproic Acid (VPA) (BioVison, Milpitas, CA, USA) at
the indicated concentrations for 12 hours.

B16 cells were incubated with primary antibodies at
4°C overnight following blocking/permeabilization with
PBS containing 0.3% Triton X100 and 5% goat serum for
20 minutes at room temperature. The following antibodies
were used for immunocytochemistry: polyclonal anti-TLE3
antibody (Proteintech), and CyclinD1 mouse monoclonal
antibody (72-13G)(Santa Cruz, Santa Cruz, CA, USA).
anti-Ki67 rabbit monoclonal antibody (ab92742, Abcam,
Cambridge, UK). The target proteins were visualized using
an Alexa 488- or Alexa 594-conjugated secondary antibody(Invitrogen, Carlsbad, CA, USA). ABZ-9000 (Keyence)microscope was used for these analyses. To visualize thecell nuclei, the cells were mounted with Hard Set Mounting
Medium with DAPI (Vector laboratories, Burlingame,
CA, USA) and to visualize the cellular skeleton, the cells
were stained with Rhodamine Phalloidin (Thermo Fisher
Scientific).

B16 melanoma mouse model
Mice were injected subcutaneously with 1 × 105
B16 cells in a 100 μL volume [37]. Tumor diameters were
measured with calipers. Body weight and physiologic
status were monitored daily.

RNA isolation and quantitative real-time PCR
Total RNA was isolated from cells using
FastGeneTM RNA Basic Kit (Nippon Genetics, Tokyo,
Japan) and then reverse-transcribed into cDNA using
ReverTra Ace (Toyobo, Osaka, Japan). The cDNA wasamplified by PCR using specific primers for murine
Tle3 (primer sequences: forward, agtctcgcctccattcctg;
reverse, catctgcccatcagcactc), murine Cyclin A2(primer sequences: forward, cttggctgcaccaacagtaa;
reverse, caaactcagttctcccaaaaaca), murine Cyclin D1(primer sequences: forward, tttctttccagagtcatcaagtgt;
reverse, tgactccagaagggcttcaa), murine Cyclin D2(primer sequences: forward, tcccgactcctaagacccatc;
reverse, taccagttcccactccagca), and β-actin (forward,

www.oncotarget.com

Western blot analysis
The following antibodies were used for Western blot
analysis: anti-TLE3 antibody (Proteintech), anti-cyclinD1
mouse monoclonal antibody (72-13G)(Santa Cruz), antiMyc-tag polyclonal antibody (MLB), CyclinA2 rabbit
polyclonal antibody (GTX103042, GeneTex, Irvine, CA),and anti-β-actin mouse monoclonal antibody (Sigma
Aldrich Chemicals, St. Louis, MO, USA). The target
proteins were detected using an anti-mouse or anti-rabbit
IgG antibody conjugated with horseradish peroxidase

411

Oncotarget

(Cell signaling, Beverly, MA, USA) and visualized by
ImmunoStar LD (Wako).

W. A. and S. K. performed the literature review, prepared
the initial and final versions of the article, and submitted
the document.

Expression plasmids, shRNA, and siRNA

CONFLICTS OF INTEREST

Myc-tagged murine TLE3 expression plasmid [12],C-terminally truncated forms of myc-tagged Tle3 plasmids[12], murine Wnt3a [32] and constitutively active form ofmurine β-catenin [39] were previously described. Super TOP
flash luciferase reporter vector [40] was kindly provided by
Dr. Randall Moon. Short hairpin RNA against murine Tle3
constructs (shTLE3) was designed using BLOCK-IT RNAi
designer tool (Invitrogen), sense and antisense oligos wereannealed and cloned into pcDNA 6.2-GW/miR [29]. The
following shRNA oligos were used: control shRNA, TG
CTGAAATCGCTGATTTGTGTAGTCGTTTTGGCCACTGACTGACGACTACACATCAGCGATTT;shRNA
against Tle3, TGCTGTGCTGAGGCTGTCTTTCTCTT
GTTTTGGCCACT GACTGACAAGAGAAACAGCCTCAGCA. Only sense strands are shown [21, 29]. siRNA-1
against murine Tle3 (Stealth siRNA, MSS238514)(Thermo
Fisher Scientific), siRNA-2 against murine Tle3 (StealthsiRNA, MSS2385136), siRNA-1 against human TLE3
(Stealth siRNA, HSS186348), or siRNA-2 against human
TLE3 (Stealth siRNA, HSS110791) were transfected into
B16 cells or HMV-II cells using Lipofectamine RNAiMAX
Transfection Reagent (Thermo Fisher Scientific) according to
the manufacturer’s Protocol.

The authors declare that they have no conflicts ofinterest.

FUNDING
This study was supported by KAKEN (Y. S.-T.,
K. M. and H. H.). This work was also supported by the
Fukuoka Foundation for Sound Health (to Y. S.-T.).

REFERENCES
1. Garbe C, Leiter U. Melanoma epidemiology
andtrends.
Clin
Dermatol.
2009;
27:3–9.
https://doi.org/10.1016/j.clindermatol.2008.09.001.
2. Garmpis N, Damaskos C, Garmpi A, Dimitroulis D, Spartalis
E, Margonis GA, Schizas D, Deskou I, Doula C, Magkouti E,
Andreatos N, Antoniou EA, Nonni A, et al. Targeting histone
deacetylases in malignant melanoma: a future therapeutic
agent or just great expectations? Anticancer Res. 2017;37:5355–62. https://doi.org/10.21873/anticanres.11961.
3. Larkin J, Hodi FS, Wolchok JD. Combined nivolumab
and ipilimumab or monotherapy in untreatedmelanoma. N Engl J Med. 2015; 373:1270–1
https://doi.org/10.1056/NEJMc1509660.

Cell proliferation assay

4. Horvat TZ, Adel NG, Dang TO, Momtaz P, Postow MA,
Callahan MK, Carvajal RD, Dickson MA, D’Angelo SP, Woo
KM, Panageas KS, Wolchok JD, Chapman PB. Immunerelated adverse events, need for systemic immunosuppression,and effects on survival and time to treatment failure in patientswith melanoma treated with ipilimumab at Memorial Sloan
Kettering Cancer Center. J Clin Oncol. 2015; 33:3193–8.
https://doi.org/10.1200/JCO.2015.60.8448.

The proliferation of cells was assessed using a Cell
Counting kit-8 (Dojindo, Kumamoto, Japan), according to
the manufacturer’s Protocol [34].

Luciferase assays
Luciferase assays were performed using Super
TOPflash-luciferase reporter vector or phRL-SV40(Promega, Madison, WI) with the Dual-Glo Luciferase
Assay System (Promega)

5. Peterson CL, Laniel MA. Histones and histone
modifications. Curr Biol. 2004; 14: R546-51.
https://doi.org/10.1016/j.cub.2004.07.007.

Statistical analysis

6. Hornig E, Heppt MV, Graf SA, Ruzicka T, Berking
C. Inhibition of histone deacetylases in melanoma-a
perspective from bench to bedside. Exp Dermatol. 2016;
25:831–8. https://doi.org/10.1111/exd.13089.

Comparisons were made using an unpaired ANOVA
with Tukey-Kramer post-hoc test and Wilcoxon’s signed
rank test. The results are shown as the mean ± S.D. The
statistical significance is indicated as follows: **, p < 0.01
and *, p < 0.05.

7. Murakami T, Sato A, Chun NA, Hara M, Naito Y,
Kobayashi Y, Kano Y, Ohtsuki M, Furukawa Y, Kobayashi
E. Transcriptional modulation using HDACi depsipeptide
promotes immune cell-mediated tumor destruction ofmurine B16 melanoma. J Invest Dermatol. 2008; 128:1506–
16. https://doi.org/10.1038/sj.jid.5701216.

Author contributions
M. O., T. Y., C. N., T. N., Y. T.-S., W. A., M. U., T.
M., K. W., H. H., and S. K. performed the experiments.
C. N., W. A., T. M., K. M., T. S., H. H., S. W., and S. K.
reviewed the intermediate draft. S. K. designed the study.
www.oncotarget.com

8. Boyle GM, Martyn AC, Parsons PG. Histone deacetylase
inhibitors and malignant melanoma. Pigment Cell Res. 2005;
18:160–6. https://doi.org/10.1111/j.1600-0749.2005.00228.x.

412

Oncotarget

9. Borriello A, Naviglio S, Bencivenga D, Caldarelli I,
Tramontano A, Speranza MC, Stampone E, Sapio L,
Negri A, Oliva A, Sinisi AA, Spina A, Della Ragione F.
Histone deacetylase inhibitors increase p27(Kip1) byaffecting its ubiquitin-dependent degradation throughSkp2 downregulation. Oxid Med Cell Longev. 2016; 2016:
2481865. https://doi.org/10.1155/2016/2481865.

of adipogenesis. Cell Metab. 2011;https://doi.org/10.1016/j.cmet.2011.02.014.

13:413–27.

21. Kokabu S, Nguyen T, Ohte S, Sato T, Katagiri T, Yoda
T, Rosen V. TLE3, transducing-like enhancer of split 3,
suppresses osteoblast differentiation of bone marrowstromal cells. Biochem Biophys Res Commun. 2013;
438:205–10. https://doi.org/10.1016/j.bbrc.2013.07.054.

10. Gatti L, Sevko A, De Cesare M, Arrighetti N, Manenti G,
Ciusani E, Verderio P, Ciniselli CM, Cominetti D, Carenini
N, Corna E, Zaffaroni N, Rodolfo M, et al. Histone
deacetylase inhibitor-temozolomide co-treatment inhibitsmelanoma growth through suppression of Chemokine (C-C
motif) ligand 2-driven signals. Oncotarget. 2014; 5:4516–
28. https://doi.org/10.18632/oncotarget.2065.

22. Liu Y, Dehni G, Purcell KJ, Sokolow J, Carcangiu ML,
Artavanis-Tsakonas S, Stifani S. Epithelial expression andchromosomal location of human TLE genes: implications
for notch signaling and neoplasia. Genomics. 1996; 31:58–
64. https://doi.org/10.1006/geno.1996.0009.
23. Cuevas IC, Slocum AL, Jun P, Costello JF, Bollen
AW, Riggins GJ, McDermott MW, Lal A. Meningiomatranscript
profilesrevealderegulated
Notchsignaling pathway. Cancer Res. 2005; 65:5070–5.
https://doi.org/10.1158/0008-5472.CAN-05-0240.

11. Jennings BH, Ish-Horowicz D. The Groucho/TLE/Grg
family of transcriptional co-repressors. Genome Biol. 2008;
9: 205. https://doi.org/10.1186/gb-2008-9-1-205.
12. Kokabu S, Nakatomi C, Matsubara T, Ono Y, Addison WN,
Lowery JW, Urata M, Hudnall AM, Hitomi S, Nakatomi M,
Sato T, Osawa K, Yoda T, et al. The transcriptional co-repressor
TLE3 regulates myogenic differentiation by repressing theactivity of the MyoD transcription factor. J Biol Chem. 2017;
292:12885–94. https://doi.org/10.1074/jbc.M116.774570.

24. Nakaya HI, Beckedorff FC, Baldini ML, Fachel AA, Reis
EM, Verjovski-Almeida S. Splice variants of TLE family
genes and up-regulation of a TLE3 isoform in prostate
tumors. Biochem Biophys Res Commun. 2007; 364:918–
23. https://doi.org/10.1016/j.bbrc.2007.10.097.
25. Sonderegger CK, Vogt PK. Binding of the corepressor
TLE1 to Qin enhances Qin-mediated transformation ofchicken embryo fibroblasts. Oncogene. 2003; 22:1749–57.
https://doi.org/10.1038/sj.onc.1206308.

13. Stifani S, Blaumueller CM, Redhead NJ, Hill RE, ArtavanisTsakonas S. Human homologs of a Drosophila Enhancer ofsplit gene product define a novel family of nuclear proteins.
Nat Genet. 1992; 2: 343. https://doi.org/10.1038/ng1292-343a.

26. Allen T, van Tuyl M, Iyengar P, Jothy S, Post M, Tsao
MS, Lobe CG. Grg1 acts as a lung-specific oncogene in atransgenic mouse model. Cancer Res. 2006; 66:1294–301.
https://doi.org/10.1158/0008-5472.CAN-05-1634.

14. Gasperowicz M, Otto F. Mammalian Groucho homologs:
redundancy or specificity? J Cell Biochem. 2005; 95:670–
87. https://doi.org/10.1002/jcb.20476.
15. Chen G, Nguyen PH, Courey AJ. A role for Groucho
tetramerization in transcriptional repression. Mol Cell Biol.
1998; 18:7259–68.
16. Pinto M, Lobe CG. Products of the grg (Groucho-related gene)family can dimerize through the amino-terminal Q domain. J
Biol Chem. 1996; 271:33026–31.

27. Dayyani F, Wang J, Yeh JR, Ahn EY, Tobey E, Zhang DE,
Bernstein ID, Peterson RT, Sweetser DA. Loss of TLE1
and TLE4 from the del(9q) commonly deleted region in
AML cooperates with AML1-ETO to affect myeloid cellproliferation and survival. Blood. 2008; 111:4338–47.
https://doi.org/10.1182/blood-2007-07-103291.

17. Grbavec D, Lo R, Liu Y, Stifani S. Transducin-like
Enhancer of split 2, a mammalian homologue of Drosophila
Groucho, acts as a transcriptional repressor, interacts with
Hairy/Enhancer of split proteins, and is expressed during
neuronal development. Eur J Biochem. 1998; 258:339–49.

28. Vance KW, Carreira S, Brosch G, Goding CR. Tbx2
is overexpressed and plays an important role inmaintaining proliferation and suppression of senescencein melanomas. Cancer Res. 2005; 65:2260–8.
https://doi.org/10.1158/0008-5472.CAN-04-3045.

18. Brantjes H, Roose J, van De Wetering M, Clevers H. All
Tcf HMG box transcription factors interact with Grouchorelated co-repressors. Nucleic Acids Res. 2001; 29:1410–9.

29. Baron R, Kneissel M. WNT signaling in bone homeostasis
and disease: from human mutations to treatments. Nat Med.
2013; 19:179–92. https://doi.org/10.1038/nm.3074.

19. Talantov D, Mazumder A, Yu JX, Briggs T, Jiang Y,
Backus J, Atkins D, Wang Y. Novel genes associatedwith malignant melanoma but not benign melanocytic
lesions. Clin Cancer Res. 2005; 11:7234–42
https://doi.org/10.1158/1078-0432.CCR-05-0683.

30. Webster MR, Kugel CH 3rd, Weeraratna AT. The Wntsof change: How Wnts regulate phenotype switching inmelanoma. Biochim Biophys Acta. 2015; 1856:244–51.
https://doi.org/10.1016/j.bbcan.2015.10.002.
31. Bachmann IM, Straume O, Puntervoll HE, Kalvenes MB,
Akslen LA. Importance of P-cadherin, beta-catenin, and
Wnt5a/frizzled for progression of melanocytic tumors andprognosis in cutaneous melanoma. Clin Cancer Res. 2005;
11:8606–14. https://doi.org/10.1158/1078-0432.CCR-05-0011.

20. Villanueva CJ, Waki H, Godio C, Nielsen R, Chou
WL, Vargas L, Wroblewski K, Schmedt C, Chao LC,
Boyadjian R, Mandrup S, Hevener A, Saez E, Tontonoz
P. TLE3 is a dual-function transcriptional coregulator

www.oncotarget.com

413

Oncotarget

32. Chien AJ, Moore EC, Lonsdorf AS, Kulikauskas
RM, Rothberg BG, Berger AJ, Major MB, Hwang
ST, Rimm DL, Moon RT. Activated Wnt/beta-cateninsignaling in melanoma is associated with decreased
proliferation in patient tumors and a murine melanoma
model. Proc Natl Acad Sci U S A. 2009; 106:1193–8
https://doi.org/10.1073/pnas.0811902106.

actin filaments. PLoS Biol. 2018; 16: e2005345.
https://doi.org/10.1371/journal.pbio.2005345.
37. Zhu ML, Nagavalli A, Su MA. Aire deficiency promotes TRP1-specific immune rejection of melanoma. Cancer Res. 2013;
73:2104–16. https://doi.org/10.1158/0008-5472.CAN-12-3781.
38. Kokabu S, Gamer L, Cox K, Lowery J, Tsuji K, Raz R,
Economides A, Katagiri T, Rosen V. BMP3 suppresses
osteoblast differentiation of bone marrow stromal cells via
interaction with Acvr2b. Mol Endocrinol. 2012; 26:87–94.
https://doi.org/10.1210/me.2011-1168.

33. Cavallo RA, Cox RT, Moline MM, Roose J, Polevoy GA,
Clevers H, Peifer M, Bejsovec A. Drosophila Tcf andGroucho interact to repress Wingless signalling activity.
Nature. 1998; 395:604–8. https://doi.org/10.1038/26982.

39. Kokabu S, Sato T, Ohte S, Enoki Y, Okubo M,
Hayashi N, Nojima J, Tsukamoto S, Fukushima Y,
Sakata Y, Katagiri T, Rosen V, Yoda T. Expression of
TLE3 by bone marrow stromal cells is regulated bycanonical Wnt signaling. FEBS Lett. 2014; 588:614–9.
https://doi.org/10.1016/j.febslet.2013.12.031.

34. Roose J, Molenaar M, Peterson J, Hurenkamp J, Brantjes
H, Moerer P, van de Wetering M, Destree O, Clevers H.
The Xenopus Wnt effector XTcf-3 interacts with Grouchorelated transcriptional repressors. Nature. 1998; 395:608–
12. https://doi.org/10.1038/26989.
35. Daniels DL, Weis WI. Beta-catenin directly displaces
Groucho/TLE repressors from Tcf/Lef in Wnt-mediatedtranscription activation. Nat Struct Mol Biol. 2005; 12:364–
71. https://doi.org/10.1038/nsmb912.

40. Fukuda T, Kokabu S, Ohte S, Sasanuma H, Kanomata
K, Yoneyama K, Kato H, Akita M, Oda H, Katagiri
T. Canonical Wnts and BMPs cooperatively
induceosteoblastic
differentiation
througha
GSK3beta-dependentandbeta-catenin-independent
mechanism.
Differentiation.
2010;
80:46–52.
https://doi.org/10.1016/j.diff.2010.05.002.

36. Douglas RG, Nandekar P, Aktories JE, Kumar H, Weber
R, Sattler JM, Singer M, Lepper S, Sadiq SK, Wade
RC, Frischknecht F. Inter-subunit interactions drive
divergent dynamics in mammalian and Plasmodium

www.oncotarget.com

414

Oncotarget