LBH589 | Cyp17 Signal

The histone deacetylase inhibitor LBH589 inhibits undifferentiated pleomorphic sarcoma growth via downregulation of FOS-like antigen 1†

Yoshinobu Saitoh1, Costansia Bureta1, Hiromi Sasaki1, Satoshi Nagano1, Shingo Maeda2, Tatsuhiko

Furukawa3, 4, Noboru Taniguchi1, Takao Setoguchi2*

1 Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan.
2 Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan.
3 Center for the Research of Advanced Diagnosis and Therapy of Cancer, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8520, Japan.
4 Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

†This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/mc.22922]

Additional Supporting Information may be found in the online version of this article.
Received 1 May 2018; Revised 27 August 2018; Accepted 5 October 2018
Molecular Carcinogenesis This article is protected by copyright. All rights reserved
DOI 10.1002/mc.22922

Department of Medical Joint Materials, Graduate School of Medical and Dental Sciences, Kagoshima

University, Kagoshima 890-8520, Japan; E-mail: [email protected]

Undifferentiated pleomorphic sarcoma (UPS) is the second most frequent soft tissue sarcoma. Because of its resistance to chemotherapy, UPS patients are treated with surgical resection and
complementary radiotherapy. However, since standard chemotherapy has not been established, unresectable or metastatic cases result in a poor prognosis. Therefore, the identification of a more effective therapy for
UPS patients is needed.

The development and progression of malignant tumors involve epigenetic alterations, and histone deacetylases (HDAC) have become a promising chemotherapeutic target. In this study, we investigated the potential effects and mechanisms of an HDAC inhibitor, LBH589, in UPS cells. We confirmed that LBH589 exhibits potent antitumor activities in four human UPS cell lines (GBS-1, TNMY- 1, Nara-F, and Nara-H) and IC50 values ranged from 7 to 13 nM. A mouse xenograft model showed that LBH589 treatment effectively suppressed tumor growth. FACS analysis showed that LBH589 induced apoptosis and G2/M cell cycle arrest. Among apoptosis-related proteins, the expressions of Bcl-2 and Bcl- xL were decreased and the expression of Bak and Bim increased. Among cell cycle-related proteins, reductions of CDK1, p-CDK1, cyclin B1, Aurora A, and Aurora B were observed after LBH589 treatment. RNA microarray identified the FOS-like antigen 1 (FOSL1) gene as a downregulated gene in response to LBH589 in UPS cells. While knockdown of FOSL1 decreased UPS cell proliferation, overexpression
induced cell proliferation. Our results show that LBH589 could be a promising chemotherapeutic agent in
the treatment of UPS and downregulation of the FOSL1 gene could be the new molecular target of UPS treatment. This article is protected by copyright. All rights reserved

Keywords: undifferentiated pleomorphic sarcoma (UPS), histone deacetylase inhibitor (HDACi), FOSL1

(FOS-like antigen 1), p21

to surgical resection and complementary radiotherapy. No standard chemotherapy has been established, and

thus unresectable or metastatic cases result in a poor prognosis [3]. Therefore, there is an urgent need to

develop a more effective therapy for UPS patients.

Recent studies have demonstrated that the development and progression of malignant tumors involves not

only genetic mutations but also epigenetic mutations [4]. Epigenetic changes are regulated by histone

acetylation, methylation, phosphorylation, ubiquitination, and sumoylation and markedly alter gene

expression [5]. The acetylation of histone is tightly regulated by histone acetyltransferases (HATs) and

histone deacetylases (HDACs). HATs neutralize the positive charge of histones by transferring an acetyl

group to the lysine residue of the histone tail, which reduces the affinity of histone and DNA and facilitates

the binding of transcription factors. Conversely, HDACs deacetylate the histone tail lysine residues to

promote chromatin condensation. HDACs are classified into four classes (class I, II, III, and IV) according
to their function, localization and homology with yeast genes [6]. The expression of HDACs are increased in several malignant tumors such as breast [7], colon [8], prostate [9], hepatocellular carcinoma [10] and

others, and overexpression of HDACs are correlated with poor outcome in some malignant tumors [11].

peptide, short chain fatty acid, and benzamide. LBH589, a derivative of hydroxamic acid, is a pan-HDAC

inhibitor that potently inhibits class I, II, and IV HDACs. LBH589 has been approved by the FDA as a

therapeutic agent for refractory multiple myeloma [14]. HDACi induce growth inhibition, differentiation,

and apoptosis in vitro and suppress tumor progression in vivo [15]. However, the precise underlying

mechanism of these drugs has not been fully elucidated.

In this study, we investigated the antitumor effect of LBH589 against UPS cells in vitro and in

vivo, and examined its molecular mechanism using RNA microarray and found that FOS-like antigen 1

(FOSL1) is involved in the antitumor effect of LBH589.

MATERIALS AND METHODS

Drugs
LBH589 was purchased from Selleckchem (Houston, TX, USA).
Cell culture

The UPS cell lines GBS-1, TNMY-1, Nara-F, and Nara-H were kindly provided by Kobe University [16-

cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum,

penicillin (100 U/mL) and streptomycin (100 μg/mL). Cell lines were cultured in a humidified incubator with 5% CO2 at 37ºC.
Patient specimens

8 human UPS biopsy specimens were obtained from primary lesions. Biopsies were performed for

diagnostic purposes. Cases in which chemotherapy was performed before surgery were excluded. Normal

muscle tissue was obtained from adjacent tissue. The Institutional Review Board of our University

approved the study protocol (Permission Number: S29010).

Small interfering RNA (siRNA) transfection

The siRNAs for FOSL control siRNA were purchased from Dharmacon (CO, USA). UPS cells were

cultured in 6-well plates in antibiotic-free medium until they reached 80% confluence and were then

transfected with siRNAs or control siRNA at a final concentration of 10 nM using Lipofectamine RNAimax

(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After 48 h, cells were harvested for subsequent analysis.
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) for cell lines

reference gene for normalization.

Western blotting

Cells were scraped and lysed in M-PER lysis buffer (Life Technologies, Carlsbad, CA) supplemented with

aprotinin and phenylmethylsulfonyl fluoride. Equal amounts of total protein extracts were fractionated by

15% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (BIO-RAD). The membrane

was blocked with skimmed milk and incubated with primary antibodies overnight at 4ºC, and then

incubated with secondary antibody. Signals were analyzed using the LAS4000 mini-image analyzer

(Fujifilm, Tokyo, Japan) and the ECL Western Blotting detection reagent (GE Healthcare, Buckinghamshire,

UK). The following antibodies were used: HDAC1 (#5356, CST, Danvers, MA, USA), HDAC2 (#2540,

CST), HDAC3 (#3949, CST), HDAC8 (sc-11405, Santa Cruz Biotechnology, Santa Cruz, CA, USA),

Histone H3 (#4499, CST), Acetyl-Histone H3 (Lys9) (#9649, CST), Bcl-2 (#2870, CST), Bcl-xL (#2764, CST), Bak (#12105, CST), Bim (#2933, CST), Bad (#9239, CST), cleaved PARP (#9541, CST), γH2AX
(#2577, CST), FRA-1 (#5281, CST), p21 (#2947, CST), p53 (sc-126, Santa Cruz Biotechnology), Acetyl-

p53 (Lys382) (#2525, CST), CDK1 (#28439, CST), Phospho-CDK1 (Thr161) (#9114, CST), Aurora A

Cell proliferation assay

Cell proliferation assays were performed using WST-1 (Roche) according to the standard protocol of

manufacturer. Cells were seeded in 96-well plates at 1×103 cells/well in 100 μl media and incubated

overnight. Cells were then treated with vehicle (0.1% DMSO) or various concentrations of LBH589 for 24

h to 72 h. After treatment, 10 μl of WST-1 was added and cells were incubated for 2 h at 37ºC. The

absorbance was analyzed on a microplate reader (BIORAD).

Cell cycle and apoptosis assay

UPS cells were seeded in 6-well plates and treated with either DMSO (0.1%) or LBH589 (10 nM or 50

nM). After 24 h, cells were trypsinized and collected. Cells were fixed in 70% ethanol for 24 h at -20ºC,

rehydrated by ice-cold PBS, and stained by Guava cell cycle reagent (Millipore, Billerica, MA, USA). Cell

cycle analysis was then performed. For apoptosis analysis, the Annexin A5 FITC/7-AAD Kit (Beckman Coulter, CA, USA) was used according to the manufacturer’s instructions. Cells were sorted using a
CytoFLEX (Beckman Coulter) and data were analyzed using the CytExpert software (Beckman Coulter).

Lentiviral infection

GBS-1 cells were treated with 100 nM LBH589 for 0 h, 12 h or 24 h. Total mRNA was extracted using the

High Pure RNA Isolation Kit (Roche). The mRNA samples were assayed on a SurePrint G3 Human GE v3

8×60k Microarray (Agilent Technologies, CA, USA) and data were analyzed by Simple Array Analyzer for

human ver 1.1 (iAnalyze, Kagoshima, Japan). All normalized and non-normalized microarray data have

been submitted to the GEO database (accession number GSE104256).

Xenograft model

The animal experiment protocol was approved by the Institutional Animal Care and Use Committee,

Graduate School of Medical and Dental Sciences, Kagoshima University (Permission Number: MD16065).

GBS-1 cells (3×106) were suspended in 100 μl of Matrigel (BD Bioscience, NJ, USA) and subcutaneously

inoculated into the lower flanks of 5-week-old BALB/c Slc-nu/nu mice (Japan SLC, Inc., Shizuoka, Japan)

as previously reported [19]. The mice were randomly allocated to control, LBH589 5 mg/kg, and LBH589
10 mg/kg groups (n=6, each group). Treatment was started after 1 week of inoculation, and drugs were administered intraperitoneally three times per a week. Tumor volumes were estimated using the following

formula: tumor volume (mm3) = length (mm)×width2 (mm) /2. All surgeries were performed under general

(Takara Bio Inc. Shiga, Japan) according to the manufacturer’s instructions.

Statistical analysis

All experiments were performed at least three times. The results are presented as the mean ± SD. Statistical

analysis were performed using student’s t-test between two parametric groups. ANOVA was used for

comparison between multiple groups, and post hoc analysis by Dunnett method was performed for those

with significant difference. All analyses were performed using the Excel Statistics 2012 (SSRI, Tokyo,

Japan) software. p < 0.05 was considered significant.

I HDACs in 8 UPS clinical samples using RT-PCR and human UPS cell lines (GBS-1, TNMY-1, Nara-F,

and Nara-H) using RT-PCR and western blot. As shown in Fig. 1A, in UPS clinical samples, the expressions

of HDAC1, HDAC2, and HDAC3 mRNA levels were significantly increased compared with adjacent non-

neoplastic muscle tissues (p < 0.05), but there was no statistical difference in HDAC8 mRNA levels. Except

for GBS-1, TNMY-1, Nara-F in HDAC 1, and GBS-1 in HDAC 3, the expression of Class 1 HDACs was

significantly elevated in all the cell lines as compared with the control (p < 0.01) (Fig. 1B). Western blot

showed that all class 1 HDACs were increased in UPS cell lines compared with UBE6T15 cells, except for

HDAC8 in Nara-F cells (Fig. 1C). Together our data showed that class I HDACs are highly expressed in

UPS cells and tissues.

LBH589 increases histone acetylation and suppresses cell proliferation in vitro

We next examined the effect of LBH589 on the acetylation of histone H3 in UPS cell lines
using western blot. In all cell lines, LBH589 treatment resulted in increased levels of acetylation of histone 3 at lysine 9 (H3K9Ac), but slight impact on the steady state expression of histone H3 (Fig. 2A). To examine

the effect of LBH589 on the proliferation of UPS cell lines, we performed cell proliferation assays using

LBH589 induces apoptosis in UPS cells

To determine whether effect of LBH589 involved apoptosis, we performed FACS analysis and

western blot. After 24 h of treatment with DMSO or 100 nM LBH589, both early and late apoptotic cells

were increased after LBH589 treatment in all four cell lines (Fig. 3A). Furthermore, western blot showed

that the anti-apoptotic proteins Bcl-2 and Bcl-xL decreased and the pro-apoptotic proteins Bak, Bim, and

Bad increased in response to LBH589 (Fig. 3B). An increase in cleaved PARP indicating the induction of

apoptosis and γH2AX indicating DNA damage were observed in all four cell lines in response to LBH589.

LBH589 induces G2/M cell cycle arrest

We next performed cell cycle analysis using FACS and western blot for cell cycle-related

proteins. Treatment of UPS cells with LBH589 increased the G2/M population (Fig. 3C) and altered

expression of several cell cycle-related proteins. Among the proteins affecting G2/M progression, the
expressions of CDK1, cyclin B1, Phosphorylated CDK1, Aurora A, and Aurora B were decreased in response to LBH589 treatment in UPS cell lines (Fig. 3D). These findings suggested that LBH589 causes

G2/M arrest by down-regulating several proteins regulating the G2/M phase.

treatment significantly diminished the growth of tumors compared with the control group. After 21 days of

treatment, the mean estimated tumor volumes in control, LBH589 5 mg/kg, and LBH589 10 mg/kg groups

were 2924±777 mm3, 1249±484 mm3 and 929±603 mm3, respectively (Fig. 4B). In the LBH589

administration group, the tumor growth was significantly suppressed as compared with the control (p <

0.01). LBH589 treatment did not cause significant differences in body weight compared with the controls

(Fig. 4C). LBH589 treatment groups showed decreased tumor weight compared with the control (p < 0.01),

but there was no significant difference between low dose and high dose groups (Fig. 4E). We also performed

hematoxylin and eosin staining and TUNEL staining on tumor sections from each treatment group.

Apoptosis was observed in the LBH589 treatment groups, while little apoptosis was observed in the control

group (Fig. 4F). These findings suggest that LBH589 treatment exhibits an antitumor effect in vivo

involving the induction of apoptosis.
LBH589 treatment reduces the expression of FOSL1
To identify target genes of the LBH589, we performed RNA microarray analysis in GBS-1 cells

treated with 100 nM of LBH589 for 12 h and 24 h. We selected the top 30 genes (cut-off value: 100) whose

To assess whether FOSL1 is a target gene of the LBH589, we first examined the expression of

the FOSL1 gene and FRA-1 protein in the 4 UPS cell lines. FOSL1 mRNA was significantly elevated in all

UPS cell lines (p < 0.01) (Fig. 5A) and FRA1 protein was elevated in GBS-1, TNMY-1, and Nara-H cell

lines compared with the UBE6T15 which is a mesenchymal cell line (Fig. 5B). FRA-1 levels were

considerably decreased in all UPS cells after LBH589 treatment (Fig. 5C). Knockdown of the FOSL1 gene

significantly suppressed the proliferation of all 4 UPS cell lines compared with control siRNA-transfected

cells (p < 0.01) (Fig. 5D, E). These results were based on the use of a cocktail of four siRNAs for FOSL1;

to rule out any off-target effect, we next knocked down the FOSL1 gene using four independent siRNAs in

GBS-1 cells (Fig. 5F). WST-1 assays showed that each siRNA also reduced proliferation of GBS-1 cells

according to its knockdown efficiency (Fig. 5G). Furthermore, we used lentiviral particles to overexpress

the FOSL1 gene in UPS cells and performed WST-1 assay. Overexpression of FOSL1 increased the
proliferation of UPS cells (Fig. 5H, I). Taken together, these findings show that FOSL1 is involved in the proliferation of UPS cells and that its downregulation may be one of the antitumor mechanisms of LBH589.

Next, we examined the mechanism of FOSL1 regulation by LBH589. Previous studies showed that the

signal transducers of the Wnt/β-catenin pathway, including GSK3β, phosphorylated GSK3β, and β-catenin.

Although expression of β-catenin was decreased only in Nara-H cells after treatment with LBH589, other

cell lines showed no change of β-catenin expression (Fig. 5K). We thus concluded that the MAPK and the

Wnt/β-catenin pathways are not involved in the control of FOSL1 by LBH589 in UPS cells.

Knockdown of FOSL1 induces the upregulation of p21

Previous studies reported that HDACi increase the expression of p21, but the effect of HDACi

on p53, a well-known regulator of p21 [23], is controversial [24]. Therefore, we first examined the changes

in the expression of p21 and p53 in response to LBH589 treatment. As shown in Fig. 6A and 6B, 50nM of

LBH589 significantly increased the expression of CDKN1A mRNA (p < 0.01) and p21 protein and

decreased the expression of p53 protein in GBS-1, TNMY-1, and Nara-F cell lines. Notably, the Nara-H

cell line does not express p53 protein. Several studies reported that HDACi cause acetylation and activation of p53 [25], and thus we next investigated the effect of LBH589 on acetylation of p53. However, we found
that LBH589 had no effect on the acetylation of p53 in UPS cells (Fig. 6B).

We next evaluated the effect of FOSL1 on cell proliferation-related proteins and found that

(Fig. 6F).

DISCUSSION

Previous studies have shown that the aberrant activation of HDACs is critical in oncogenesis,

and therefore HDACi have been promising targets for anticancer therapy [26]. HDACi induce cell cycle

arrest, apoptosis, senescence and differentiation in cancer cells at concentrations that do not affect normal

cells[27,28]. Because the biological effects and treatment outcomes vary with tumors and HDACs [20], the

detailed underlying mechanisms have been unknown. In this study, we examined the antitumor effects and

mechanism of the LBH589 against UPS. LBH589 has been approved by the FDA for the treatment of

multiple myeloma. Maiso et al. reported that the IC50 values of LBH589 for multiple myeloma cell lines

are 5.7 nM (MM1S), 6.5 nM (MM1R), 8.1 nM (U266), 24 nM (U266LR7), and 45.5 nM (U266DOX4),

respectively [29]. In this study, the IC50 values of LBH589 for UPS cells are 10 nM (GBS-1), 9 nM (TNMY- 1), 6 nM (Nara-F), and 13 nM (Nara-H), respectively. Our findings showed that LBH589 provide equivalent
therapeutic effects in the treatment of UPS in vitro compared with multiple myeloma cells.

To evaluate the effect of LBH589 in vivo, Ocio et al. generated a disseminated myeloma

mg/kg and control groups [30]. In our study, both 5 mg/kg and 10 mg/kg of LBH589 significantly

suppressed tumor growth, suggesting that LBH589 also has high antitumor activity against UPS in vivo.

HDACi have been reported to cause G1/S [31] or G2/M cell cycle arrest [32]. In this study, we

found that LBH589 caused G2/M arrest in UPS cells. To investigate the mechanism of G2/M cell cycle

arrest, we performed western blot for cell cycle-related proteins and demonstrated that CDK1 and cyclin

B1, which regulate G2/M phase progression [33], were decreased after LBH589 treatment. To further

investigate the molecular mechanism, we focused on the Aurora kinase family (Aurora A, Aurora B, and

Aurora C). Aurora kinases are serine-threonine kinases that play a critical role in chromosome maturation

and separation and bipolar spindle assembly during G2/M transition of mitosis. The Aurora A kinase

functions upstream of CDK1 [34] and is required for initial activation of CDK1 and mitotic commitment.

Cha et al. reported that LBH589 causes G2/M arrest via reduced expression of Aurora A and Aurora B in
renal cell carcinoma [35]. We showed that LBH589 also decreases the expression of Aurora A and Aurora B in UPS cells. Our results suggest that decreased Aurora A and Aurora B may play an important role in

the G2/M arrest of UPS cells caused by LBH589.

encoded by the FOSL1 gene, associates with members of the Jun family to form AP-1 transcription

complexes [37]. The AP-1 complex regulates a variety of cellular processes, such as proliferation,

differentiation, apoptosis, cell invasion, and cell adhesion [37,38]. The FOSL1 gene is overexpressed in

breast [39], esophageal [40], lung [41], colon [42], and brain tumors [43] and is related to the poor prognosis

of these cancers, and therefore considered to be an oncogene. We found that UPS cell proliferation was

decreased by knockdown of the FOSL1 gene and enhanced by FOSL1 gene overexpression. These findings

show that the FOSL1 gene is related to the proliferation ability of UPS cells.

The effects of HDACi on p53 expression vary according to reports, but the increase of p21

expression in response to HDACi has been well established [44]. In our study, we observed reduction of

p53 and increase in p21 in UPS cells after LBH589 treatment. Importantly we observed these results in the

Nara-H cell line, which is deficient in p53. These findings thus suggest that the increase of p21 by the LBH589 HDACi occurs in a p53-independent manner in UPS cells. We also found that knockdown of the
FOSL1 gene by RNAi increased not only p21 protein, but also CDKN1A mRNA expression, indicating that

FOSL1 transcriptionally suppresses the expression of p21. Galvagni et al. reported that FOSL1 negatively

In conclusion, our findings show that LBH589 could be a promising chemotherapeutic agent

in the treatment of UPS and downregulation of the FOSL1 gene could be the molecular target of UPS

treatment.

ACKNKOWLEDGEMENT

We are grateful to Hui Gao for her excellent technical assistance. This work was supported by the Facility

of Laboratory Animal Science Research Support Center Institute for Research Promotion Kagoshima

University. We also wish to thank the Joint Research Laboratory, Kagoshima University Graduate School

of Medical and Dental Sciences, for the use of their facilities. We thank Kobe University for providing the

UPS cell lines. We thank Gabrielle White Wolf, PhD, from Edanz Group (www.edanzediting.com/ac) for

editing a draft of this manuscript.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

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Figure legends

Fig. 1 The expression of class 1 HDACs in the UPS tissues and cell lines.

(A) Comparison of the mRNA expression of class 1 HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) in

UPS tissues and adjacent non-neoplastic tissues (n=8) by RT-PCR. Data are mean ± S.D. of triplicate samples. *p < 0.05.
(B) Comparison of the mRNA expression of class 1 HDACs in UPS cell lines (GBS-1, TNMY-1, Nara-F,

and Nara-H) and UBE6T15, a mesenchymal stem cell line, by RT-PCR. Data represent means ± SD. **p <

0.01.

from a single experiment representative of three similar results.

Fig. 2 The effect of class 1 HDACs and LBH589 on the acetylation of histone H3 and cell proliferation in

vitro

(A) UPS cells were treated with 10 nM or 50 nM LBH589 or DMSO for 24 h, and western blot analysis

was performed for the expression of histone H3 and H3K9Ac.

(B) GBS-1, TNMY-1, Nara-F, and Nara-H cells were treated with 1 nM, 10 nM, 100 nM LBH589 or DMSO and at 24 h, 48 h, 72 h, WST-1 assays were performed and absorbance was read at 450 nm. Data represent
means ± SD (n= 3 experiments) **p < 0.01.

(A) UPS cells were treated with 100 nM LBH589 or DMSO (control) for 24 h and then FACS analysis was

performed after Annexin 5-FITC/7AAD double staining for the detection of the apoptotic rate.

(B) Western blot analysis of the expression of apoptosis-related proteins in UPS cells treated with 10 nM

or 50 nM LBH589 for 24 h.

(D) Cell cycle analysis by FACS analysis using PI staining in GBS-1, TNMY-1, Nara-F, and Nara-H cells

treated with 10 nM or 50 nM LBH589 for 24 h.

(E) GBS-1, TNMY-1, Nara-F, and Nara-H cells were treated with 10 nM or 50 nM LBH589 for 24 h, and

western blotting for cell cycle-related proteins was performed. Data shown is from a single experiment

representative of three similar results.

Fig. 4 LBH589 suppresses tumor growth in a mouse xenograft model

(A) Detailed experimental schedule for the in vivo study.
(B) Effect of LBH589 (5 or 10 mg/kg) and control (DMSO) on tumor volumes in the mouse xenograft model. Data represent means ± SD (n=6). **p < 0.05.

presented as mean ± SD (n = 6). **p < 0.01.

Fig. 5 LBH589 decreases the expression of FOSL1, which affects the proliferation of UPS cells

(A) Expression of FOSL1 mRNA in UPS cells and UBE6T15 cells by RT-PCR. Data represents mean ± SD

(n = 3 experiments). **p < 0.01.

(B) Expression of FRA-1 protein in UPS cells and UBE6T15 cells by western blot.

DMSO for 24 h.

(D) Validation of siRNA-mediated knockdown of FOSL1. Cells were transfected with control siRNA or

FOSL1 siRNA. At 48 h after transfection, western blot was performed using anti-FRA1 antibodies.

(E) WST-1 assay was performed to evaluate the effect of FOSL1 knockdown on cell proliferation. Cells

were transfected with control siRNA or FOSL1 siRNA. At 24 h and 48 h after transfection, WST-1 assays were performed and absorbance was read at 450 nm. Data represents mean ± SD (n = 3 experiments). **p
< 0.01.

(F) Western blotting was performed on GBS-1 cells transfected with four independent siRNA sequences

(H) GBS-1, TNMY-1, Nara-F, and Nara-H cells were infected with lentivirus expressing FOSL1-GFP and

GFP only as control for 48 h, and expression of FRA-1 protein was detected by western blot.

(I) WST-1 assays were performed to examine the effect of overexpression of FRA-1 on proliferation.

Absorbance was read at 450 nm at 24 h, 48 h, and 72 h. Data represent means ± SD (n= 3 experiments). *p

< 0.05. **p < 0.01.

(J) GBS-1, TNMY-1, Nara-F and Nara-H cells were treated with 10 nM and 50 nM LBH589 for 24 h and

western blot was performed on MAPK pathway-related proteins, including p-Raf, pMEK, and p-ERK.

(K) GBS-1, TNMY-1, Nara-F and Nara-H cells were treated with 10 nM and 50 nM LBH589 for 24 h and

western blot was performed on Wnt-β catenin pathway-related proteins, including GSK3β, p-GSK3β (Ser9),

and β-catenin.

Fig. 6 LBH589 treatment induces p21 expression independent of p53
(A) Expression levels of CDKN1A mRNA in GBS-1, TNMY-1, Nara-F, and Nara-H cells treated with 10nM or 50 nM LBH589 for 24 h were analyzed by RT-PCR. Data represent means ± SD (n= 3 experiments).

**p < 0.01.

(D) Western blot analysis of p21 in GBS-1, TNMY-1, Nara-F, and Nara-H cells transfected with siRNA

against FOSL1 for 48 h.

(E) Western blot for p21 expression in GBS-1 cells transfected with each independent FOSL1 siRNA.