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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2015 Jun 12;172(15):3817–3830. doi: 10.1111/bph.13165

Inhibition of breast cancer progression by a novel histone deacetylase inhibitor, LW479, by down-regulating EGFR expression

Jingjie Li 1,*, Tao Zhang 1,2,*, Feifei Yang 1, Yuan He 1, Fujun Dai 1, Dan Gao 1, Yihua Chen 1,✉, Mingyao Liu 1,3,✉, Zhengfang Yi 1,✉
PMCID: PMC4523338  PMID: 25884486

Abstract

Background and Purpose

Compounds targeting epigenetic events of tumours are likely to be an important addition to anticancer therapy. Histone deacetylase inhibitors (HDACI) have emerged as a promising novel class for therapeutic interventions associated with cancer, and many of them are currently in clinical investigation. Here, we assessed a novel hydroxamate-based HDACI, LW479, in breast cancer progression and explored its underlying mechanism(s).

Experimental Approach

LW479 was identified using the HDACI screening kit. Western blot and flow cytometry were used to analyse the biological effects of LW479 as a novel HDACI. The effects of LW479 were assessed in mouse models of spontaneous and experimental breast cancer. Co-immunoprecipitation, immunofluorescent staining and chromatin immunoprecipitation assays along with immunohistochemical analysis, were used to elucidate the molecular basis of the actions of LW479.

Key Results

LW479 was identified as a novel HDACI and showed marked cytotoxicity and induced apoptosis, as well as cell cycle arrest, in a panel of breast cancer cell lines. Intraperitoneal injections of LW479 markedly suppressed breast tumour growth and pulmonary metastasis in nude mice. LW479 also decreased levels of EGF receptors (EGFR) by blocking the binding of the transcription factor Sp1 and HDAC1 to the EGFR promoter region.

Conclusions and Implications

Our data have elucidated the mechanisms underlying the inhibition by LW479 of tumour growth and metastasis, in models of breast cancer with aberrant EGFR expression. LW479 could be a candidate drug for breast cancer prevention.

Tables of Links

TARGETS
Enzymesa
Akt
FAK, focal adhesion kinase
HDAC1, histone deacetylase 1
>JNK
Catalytic receptorsb
EGFR, EGF receptor
LIGANDS
Butyrate
Panobinostat
SAHA (suberoylanilide hydroxamic acid), vorinostat
Trichostatin A
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,bAlexander et al., 2013a,b).

Introduction

Breast cancer is a serious health problem among women worldwide. It is estimated that 1.67 million women worldwide suffered from breast cancer in 2012, making breast cancer as the most common cancer type diagnosed in women. In addition, breast cancer is the most common cause of cancer death among women (http://globocan.iarc.fr/Default.aspx). Classically, breast cancer is considered to initiate from genetic defects that lead to the hyperactivation of oncogenes and hypoactivation of tumour suppressor genes. To date, accumulating evidence has indicated that epigenetic modifications also play important roles in breast cancer (Huang et al., 2011).

Histone acetylation is the most widely studied among epigenetic modifications, apart from DNA methylation, in cancer development (Marks et al., 2001; Esteller, 2007). The reciprocal activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) are critically involved in chromatin modification. HATs add acetyl groups to the N-terminal lysine residues of core histones, whereas HDACs remove acetyl groups from histones. Research on the association between HDACs and cancer development was started with the discovery that HDACs were the target of sodium butyrate, trichostatin A and trapoxin, which induced G1 and G2 phase cell cycle arrest and differentiation in certain mammalian cell lines (Ginsburg et al., 1973; Altenburg et al., 1976). Subsequent studies demonstrated that other compounds, initially identified as TGFβ mimetics or anti-tumour antibiotics, also exhibited potent inhibitory effect against HDACs (Su et al., 2000; Glaser et al., 2002). In recent years, many studies have suggested the involvement of HDACs in cancer initiation and progression. Several HDACs induced angiogenic stimulators and thus increased angiogenesis (Deroanne et al., 2002; Rossig et al., 2002; Sasakawa et al., 2003). In addition, HDACs regulated the expression of metastasis-associated genes (Xu et al., 2007). It is now widely accepted that HDACs are one of the more promising targets in cancer therapy.

Recently, low MW compunds targeting HDACs have received much attention as anticancer agents and some of them have demonstrated favourable anticancer activities in both preclinical and clinical settings (Bots and Johnstone, 2009; Khan and La Thangue, 2012). Aberrant activation of HDACs in breast cancer samples has been described (Byler et al., 2014), and histone deacetylase inhibitors (HDACIs) as anticancer agents have shown therapeutic potential in breast cancer (Bots and Johnstone, 2009). HDACIs can overcome oestrogen receptor modulator/aromatase inhibitor resistance in oestrogen receptor (ER)-positive breast cancer (Munster et al., 2011) and sensitize ER-negative breast cancer to hormone therapy (Jang et al., 2004). Recently, Tate et al. (2012) demonstrated that the HDACI panobinostat (LBH589) selectively targeted triple-negative breast cancer (TNBC) cells in vitro and in vivo.

The human EGF receptor (EGFR/ErbB1) contains the first receptor tyrosine kinase to be identified and cloned and is a member of the ErbB family of receptor tyrosine knases. Upon ligand binding, the EGFR forms kinase-active homodimesr or heterodimers and induces its downstream signalling pathways (Avraham and Yarden, 2011). The EGFR plays pleiotropic roles in human cancer and overexpression of EGFR is observed in a wide range of solid tumours including breast cancer (Wheeler et al., 2010). Aberrant EGFR in breast cancer correlates with aggressive clinical behaviour (Baselga, 2002) and increased EGFR was shown to be a predictor of ER-positive breast cancer risk (Pitteri et al., 2010). Furthermore, EGFR dysregulation is more frequently detected in triple-negative breast cancer and inflammatory breast cancer, two subtypes of breast cancer with highly aggressive phenotypes (Masuda et al., 2012). The 5′-regulatory region of the EGFR gene contains a GC-rich, TATA-less, CAAT-less, promoter and thus many transcription initiation sites are locates in the promoter region. A variety of DNA-binding factors including the transcription factor Sp1 have been identified as interacting with the EGFR promoter region. In addition, several Sp1 binding sites have been discovered and Sp1 is deemed necessary for basal transcription of the EGFR gene (Nishi et al., 2002; Brandt et al., 2006).

In our present study, we demonstrated that LW479, a novel HDACI, inhibited breast cancer progression in mouse models in vitro and in vivo. Further mechanistic studies revealed that LW479 suppressed EGFR expression in breast cancer cells and that the low EGFR level may be ascribed to the disassociation of Sp1 and HDAC1 from the EGFR promoter. The ability of LW479 to block breast cancer growth and metastasis supports LW479 as a novel candidate for breast cancer therapy.

Methods

Synthesis, characterization and preparation of LW-479 and suberoylanilide hydroxamic acid (SAHA)

6-{2-[2-(3-Bromophenyl)-4-oxo-3-thiazolidinyl]phenoxy}-N-hydroxylhexanamide (LW479) and SAHA were synthesized in the Shanghai Key Laboratory of Regulatory Biology, East China Normal University. The synthetic route and purity analysis of LW479 are shown in Supporting Information Figs. S1–S3. SAHA was synthesized as described previously (Gediya et al., 2005). Compounds were dissolved in DMSO and stored at −20°C as small aliquots.

Cell line and cell culture

Human breast cancer cell lines MDA-MB231 (hereafter designated as MDA-231), BT549, HCC1937 and MDA-MB468 (hereafter designated as MDA-468) were obtained from the ATCC and maintained in the complete growth media recommended. MDA-231 human breast cancer cells transfected with a reporter gene encoding luciferase (MDA-231-Luc) were a kind gift from Dr Qian Zhao at Shanghai Jiaotong University and grown in MEM (Gibco, Gaithersburg, MD, USA) with 1% non-essential amino acids and 10% FBS.

HDACI identification

HDACI activity assay was performed using the HDACI screening kit. Candidate HDACIs or SAHA were incubated with HeLa nuclear extracts or MDA-231 cell lysates and HDAC fluorometric substrate containing an acetylated lysine side chain at 37°C for 1 h. The lysine developer was then added to stop the reaction and the fluorescence was measured, using 350–380nm excitation and 440–460 nm emission. Three independent experiments were carried out in triplicate.

Western blotting

Western blotting was performed as described previously (Dong et al., 2010). Cells were harvested with RIPA buffer containing 150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris and cocktails of protease and phosphatase inhibitors. Heat-denatured protein was then analysed by 8–15% SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% BSA, incubated with specific primary antibodies and exposed to HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Sigma, St. Louis, MO, USA). The chemiluminescence signals emanating from the membranes were detected by ECL reagent. Three independent experiments were carried out in triplicate.

MTS cell viability assay

MTS assay was performed according to the manufacturer’s instruction (Promega, Madison, WI, USA). Breast cancer cells (5 × 103) were seeded in 96-well plates and, after incubation for 12 h; different concentrations of LW479 were added and incubated for another 48 h. Aqueous One Solution (20μL) was subsequently added and the absorption at 490 nm was measured by a microplate spectrophotometer. Three independent experiments were carried out in triplicate.

Cell cycle analysis

MDA-231 cells were plated in 6 cm dishes, treated with indicated concentrations of LW479 for 24 h and trypsinized. Cells were collected by centrifugation and then fixed in ice-cold 70% ethanol at 4°C for 24 h. After several PBS washes, fixed cells were resuspended in PBS containing RNase and PI solution and incubated at 37°C for 30 min in the dark. Cell cycle distribution was analysed by flow cytometry (BD Biosciences, Pasadena, CA, USA). Three independent experiments were carried out in triplicate.

Cell apoptosis detection

MDA-231 cells were cultured in 6 cm dishes in the presence of different concentrations of LW479 for 48 h. Cells were washed in PBS and resuspended in binding buffer (BD Biosciences) containing FITC-conjugated Annexin V and PI. Cell apoptosis was determined by a flow cytometer (BD Biosciences). Three independent experiments were carried out in triplicate.

Transwell invasion assay

Transwell invasion assay was performed using a modified Boyden chamber system (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. After starvation overnight, MDA-231 cells were resuspended at 5 × 104 cells in 100 μL serum-free medium with or without indicated concentrations of LW479 and added to each transwell insert, precoated with 1 mg·mL−1 Matrigel. Serum-free medium (0.5 mL) or the same volume containing EGF (50 ng·mL−1) was placed in each bottom well. Twelve hours post-seeding, non-invaded cells on the upper side of the transwell were removed using cotton swabs. Invaded cells on the undersurface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images were acquired using an inverted microscope (Olympus, Tokyo, Japan) and cells were counted manually. Three independent experiments were carried out in triplicate.

Wound-healing migration assay

Wound-healing migration assay was performed by seeding cells in a 6-well plate as previously reported with modifications (Zhang et al., 2012). When the cells were fully confluent, cells were starved overnight and a ‘wound’ was made by a 100 μL pipette tip. Fresh serum-free medium or that containing EGF (50 ng·mL−1) and different concentrations of LW479 were added. Cells were allowed to migrate for 12 h and fixed with 4% paraformaldehyde. Images were taken by an inverted microscope (Olympus) and migrated cells were counted manually. Three independent experiments were carried out in triplicate.

Immunofluorescence (IF) assay

MDA-231 cells were seeded on gelatin-coated coverslips and treated with different concentrations of LW-479 for 24 h before stimulation with EGF (50 ng·mL−1) for 30 min. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked with 0.5% BSA for 30 min. Target proteins in cells were visualized by incubation with the corresponding antibodies overnight at 4°C, followed by incubation with FITC-conjugated secondary antibodies for 1 h. Rhodamine-conjugated phalloidin and 4′, 6-diamidino-2-phenylindole were subsequently used to localize F-actin and nucleus. Images were acquired with a confocal microscope (Leica, Wetzlar, Germany). Three independent experiments were carried out in triplicate.

RT-PCR

RNA samples from cells were prepared using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocols. Total RNA (1μg) was converted to cDNA using oligo dT primer. The relative expression of EGFR was analysed by RT-PCR with GAPDH as an internal control. The primer sequences used for PCR for EGFR were 5′-CCTGGTCTGGAAGTACGCAG-3′ and 5′-CTTCGCATGAAGAGGCCGAT-3′. PCR products were separated on 1.2% agarose gel and then stained with ethidium bromide. Three independent experiments were carried out in triplicate.

Haematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis

Primary tumours, lungs or other organs were freshly excised from mice and fixed in 4% paraformaldehyde overnight prior to paraffin embedding. Sections (4 μm) were then deparaffinized for H&E and IHC staining. For IHC, deparaffinized slides were subjected to epitope retrieval in sodium citrate solution and blocked in 3% H2O2, then probed with specific antibodies (EGFR and acetyl-H3) at 4°C overnight. Immunodetection was visualized by a diaminobenzidine detection kit.

Three-dimensional top culture assay

Three-dimensional top culture assay was conducted as previously reported (Lee et al., 2007). Growth factor-reduced Matrigel was thawed at 4°C overnight. Matrigel solution (80 μL per well) was added to 48-well plates and left at 37°C for 30 min to solidify. A total of 1.5 × 104 MDA-231 cells were resuspended in 100 μL serum-free medium followed by seeding on solidified Matrigel. Fifteen minutes post cell attachment, 100 μL serum-free media containing 10% Matrigel indicated concentrations of LW-479 and EGF was added on top of the plated culture. The on-top Matrigel medium was replaced every 2 days. All experiments were performed in triplicate and repeated at least three times.

Co-immunoprecipitation

Co-immunoprecipitation was performed as previously reported (Dai et al., 2012). MDA-231 cells were treated with or without LW479 for 24 h and nuclear lysates were prepared. Equal amount of nuclear extracts was incubated with anti-Sp1 and anti-HDAC1 antibodies for 12 h at 4°C. The immunoprecipitated pellets were then incubated with protein A/G agarose beads followed by five washes with wash buffer.

Chromatin immunoprecipitation (ChIP) assay

ChIP assay was performed as previously described (Dong et al., 2010). MDA-231 cells were cross-linked in 1% formaldehyde in PBS for 10 min, followed by adding glycine to quench unreacted formaldehyde. Cell lysates were then collected with cold lysis buffer for ChIP and sonicated to obtain chromatin with an average fragment size of 500 bp. The chromatin samples were precleared with protein A/G agarose/salmon sperm DNA beads for 1 h and then immunoprecipitated with antibodies against IgG, HDAC1 and Sp1. After 12 h of incubation, the samples were incubated with protein A/G agarose beads for 2 h. After five sequential washes, cross-links were eluted with elution buffer plus proteinase K and reversed at 65°C for 12 h. DNA was extracted with phenol-chloroform. Immunoprecipitated DNA was analysed by real-time PCR.

siRNA-mediated knockdown

MDA-231 cells were seeded in a 6-well plate 24 h before transfection. siRNAs targeting HDAC1 or non-specific control (siControl) were transfected using lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA, USA). The sequences of siRNAs against HDAC1 were 5′-TAAGGTTCTCAAACAGTCG-3′ and 5′-AAGCCGGUCAUGUCCAAAGUA-3′ (Mottet et al., 2007; Pulukuri et al., 2007).

Luciferase reporter gene assay

The EGFR promoter–luciferase construct was transiently transfected into MDA-231 cells. Briefly, MDA-231 cells were seeded in 24-well plates and cultured for 24 h. Cells were co-transfected with the EGFR promoter–luciferase construct and Renilla luciferase, according to the manufacturer’s instructions (Promega). The cells were then exposed to LW479 for 24 h. Luciferase activity was measured and normalized against Renilla luciferase activity.

EMSA

EMSA was performed using Odyssey Infrared Sp1 EMSA Kit (LI-COR Biosciences, Lincoln, NE, USA) following the manufacturer’s protocol. In brief, nuclear extracts from MDA-231 cells were prepared and incubated with Sp1 IRDye™ 700 infrared dye-labelled oligonucleotides (LI-COR Biosciences). The protein–DNA complex was applied to 6.6% native polyacrylamide gels. The gels were visualized with Odyssey infrared system.

Animals

All animal care and experimental studies were conducted according to the guidelines and approval of the Animal Investigation Committee of the Institute of Biomedical Sciences and School of Life Sciences, East China Normal University. Studies with animals are described in compliance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010).

BALB/c athymic nude mice and BALB/c mice were bred and housed at the animal centre in East China Normal University [21°C, 55% humidity, on a 12 h light/dark cycle (SPF)]. The total numbers used in each experiment were female BALB/c athymic nude mice in the spontaneous metastasis model, 32; female BALB/c athymic nude mice in the experimental metastasis model, 28; female BALB/c mice in toxicity study, 15.

Xenograft animal studies

Xenograft animal studies were performed as described previously (Zhang et al., 2014).

Spontaneous metastasis mouse model

Female nude mice, 6–8 weeks of age, received orthotropic inoculations in the no. 4 inguinal mammary fat pad with 50 μL of MDA-231-Luc cell suspension containing 1 × 106 cells. Treatment began 1 week after tumour cell injection when palpable tumours had formed. Mice were allocated into four groups for treatment based on the in vivo imaging results on treatment day 0 using the Xenogen In Vivo Imaging System (IVIS; Xenogen Corporation, Danville, CA, USA). Tumour growth was measured weekly by monitoring tumour length (L) and width (W). Tumour volume was calculated from L × W2 × 0.52. Each group contained eight mice. When the study was terminated, various organs were collected and imaged by IVIS to detect distant metastasis.

Experimental metastasis mouse model

A total of 1 × 106 MDA-231-Luc cells in 100 μL PBS were injected into the nude mice at the age of 6–8 weeks through the tail vein. Mice given tumour cells on day 0 were assigned into four groups for treatment with or without i.p. injections of indicated doses of LW-479 or SAHA (n = 7 per group). Real-time whole body images were acquired using the Xenogen IVIS. Lung metastasis was expressed in units of photons s-1 cm-2 sr-1.

Toxicity study of LW479

For potential toxicity testing, female BALB/c mice (6–8 weeks of age) were randomly assigned to three groups (n = 5 per group) and received i.p. injections of DMSO or LW479 (40 mg·kg−1) or SAHA (40 mg·kg−1) once a day. Body weight was monitored every 6 days. On day 30, mice were killed and major organs were freshly dissected. Histological examinations were carried out using H&E staining.

Data analysis

Results are expressed as mean ± SE. Groupwise comparisons were determined by Student’s t-test. All experiments were performed at least three times, except for the whole animal experiments. P-values less than 0.05 were considered statistically significant.

Materials

Growth factor-reduced Matrigel was purchased from BD Biosciences. HDACi screening kit was obtained from BioVision (Milpitas, CA, USA).

Antibodies for Western blot, immunofluorescence and IHC included phospho-EGFR/EGFR, phospho-FAK/FAK, phospho-JNK/JNK, phospho-AKT/AKT, acetyl-histone H3, histone H3, acetyl-histone H4, HDAC1, HDAC2, HDAC3, HDAC4 and HDAC6 (Cell Signaling Technology, Danvers, MA, USA); Sp1 and acetyl-lysine (Millipore); Paxillin (BD Transduction Laboratories, Pasadena, CA, USA); and β-actin (Sigma).

Results

LW-479 is identified as a HDACI

LW479 was discovered using the HDACI drug-screening kit in our internal chemical library. The chemical structure of LW479 is shown in Figure 1A, with the details of the synthetic route and identification provided in Supporting Information Figs. S1–S3. As shown in Figure 1B, LW479 was a potent inhibitor of HDAC enzyme activity, in HeLa cell nuclear extracts and MDA-231 cell lysates. Treatment with 10 μM of LW479 resulted in marked increases in histone H3 and H4 acetylation, the characteristic indicators of HDAC inhibition, in a panel of breast cancer cell lines (Figure 1C and D and Supporting Information Fig. S4A). In parallel, we used total histone H3 as a loading control. The HDAC inhibition could also be observed by the varying degrees of reduction of individual HDACs (HDAC1-HDAC4, HDAC6) (Figure 1C). HDACIs are known for their ability to selectively block proliferation, induce apoptosis and cell cycle arrest in cancer cells (Minucci and Pelicci, 2006). The effect of LW479 was thus tested on these aspects. MTS assay (Figure 1E) showed LW479-inhibited proliferation of all four types of breast cancer cells in a dose-dependent manner. The anti-proliferation characteristic of LW479 was similar to that of SAHA (Supporting Information Fig. S5). Annexin V/PI staining in Figure 1F indicated enhancement of apoptosis by LW479. In addition, we performed annexin V/PI assays and caspase activity assays and found that LW479 (10 μM) induced cell apoptosis and, using Western blots, up-regulated the expression of cleaved caspase 3 and down-regulated the expression of caspase 3 (Supporting Information Fig. S4B and C). Furthermore, flow cytometry analysis showed that LW479 induced G2/M phase arrest in MDA-231 cells (Figure 1G). Results from all of these assays suggest that LW479 acts as a HDACI.

Figure 1.

Figure 1

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LW479 is identified as a HDACI. (A) Chemical structure of LW479. (B) HeLa cell nuclear extracts and MDA-231 cell lysates were treated with 10 μM LW479 or SAHA followed by co-incubation with HDAC fluorometric substrate at 37°C for 1 h. The lysine developer was added to stop the reaction, and the fluorescence units was measured at Ex/Em 350–380/440–460 nm. SAHA was used as a positive control. (C) MDA-231 cells were exposed to 10 μM LW479 (LW) or SAHA (SA) for 24 h and then whole cell lysates were probed using indicated antibodies by Western blot; total histone 3 as loading control. (D) Acetylated histone 3 (Ac-H3) protein level was induced by LW479 in various breast cancer cell lines. (E) Different breast cancer cell lines were treated with increasing concentrations of LW479 for 48 h and MTS assay was conducted. (F) MDA-231 cells were left untreated or treated with LW479 at the indicated doses for 48 h. Apoptotic cells were labelled with annexin V and PI and analysed by flow cytometry. (G) MDA-231 cells were treated with different concentrations of LW479 and incubated for 24 h. Cell population distribution was determined following PI staining and further analysed by flow cytometry. ***P < 0.001 versus control.

LW479 silences EGFR expression in breast cancer cells through disrupting Sp1 and HDAC1 binding to EGFR promoter

Aberrant EGFR expression has been commonly observed in breast cancers (Pitteri et al., 2010) and the EGFR profiles have prognostic value (Baselga, 2002). As shown in Figure 2A, treatment of four breast cancer cell lines with LW479 (1–20 μM) decreased the levels of EGFR protein, in a dose-dependent fashion. Because the EGFR protein expression was so markedly reduced by LW479, we queried whether LW479 treatment was attenuating EGFR at the mRNA level. MDA-231 cells were treated with increasing concentrations of LW479 and conventional RT-PCR revealed the suppression of EGFR mRNA (Figure 2B). These data suggested that the diminished EGFR protein expression may be, in part, caused by a decrease in mRNA levels. A earlier study demonstrated abnormal expression of individual HDACs in tumour samples and overexpression of HDAC1 is seen in breast carcinoma (Bolden et al., 2006). To examine the role of HDAC1 in EGFR expression, endogenous HDAC1 was knocked down in MDA-231 cells by treatment with specific siRNAs directed against HDAC1 (Figure 2C). In HDAC1 knockdown cells, a diminished level of EGFR expression was observed when compared with cells transfected with control siRNA (Figure 2C). Basal EGFR gene transcription is modulated by the transcription factor Sp1 (Brandt et al., 2006) and post-translational modifications such as acetylation are known to influence the transcriptional activity of Sp1 (Tan and Khachigian, 2009). Aberrant recruitment of HDAC1 to promoters through interacting with Sp1 has shown to regulate gene transcription (Varshochi et al., 2005). To determine whether Sp1 was acetylated or bound to HDAC1 and what effect LW479 had on these interactions, cell nuclear lysates were prepared and then immunoprecipitated with anti-Sp1 or anti-HDAC1 antibodies followed by Western blot analysis with antibodies against acetyl-lysine or Sp1. As shown in Figure 2D, one band located at ∼90 kD could be recognized by anti-acetyl-lysine antibody in anti-Sp1 immunoprecipitants in the LW479 (20 μM) treated group. After normalization for the amount of protein present, we found a decreased binding of HDAC1 to Sp1, in the presence of LW479. The IF assay also showed a reduced co-localization between HDAC1 and Sp1 in nuclei of cells treated with LW479 (Figure 2E). To assess if the effect of LW479 on the interaction between HDAC1 and Sp1 modulated their binding to the EGFR promoter, we performed quantitative ChIP assays. There are four Sp1 recognition sites in the EGFR promoter region (−329/−320, −310/−301, −221/−212 and −99/−90) (Liu et al., 2005). We designed primers to cover the regions −329/−320 and −310/−301 in the EGFR promoter. A high level of HDAC1 was observed at EGFR promoters in untreated cells, whereas LW479 induced the dissociation of HDAC1 from the EGFR promoter (Figure 2F). Similar results was obtained when the recruitment of Sp1was tested (Figure 2F). Moreover, knockdown of Sp1 caused a decreased level of EGFR mRNA and protein in MDA-231 cells (Supporting Information Fig. S6A and B). Overexpression of Sp1 in cells treated with LW479 attenuated the reduction of EGFR mRNA and protein expression (Supporting Information Fig. S6C and D). In addition, in order to determine that the reduced level of EGFR was due to transcriptional inhibition, cells were transiently transfected with the EGFR promoter–luciferase construct and treated with or without LW479 for 24 h. EGFR promoter activity was drastically reduced by LW479 (Supporting Information Fig. S6E), suggesting that the transcriptional inhibition leads to the decreased EGFR expression. Using EMSA assay, we demonstrated that LW479 or knockdown of Sp1 suppressed the endogenous Sp1 binding activity (Supporting Information Fig. S6F). These data indicated that the diminished interaction between HDAC1 and Sp1 induced by LW479 contributes importantly to the LW479-induced suppression of EGFR.

Figure 2.

Figure 2

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LW479 silences EGFR expression in breast cancer cells through disrupting HDAC1 and Sp1 binding to the EGFR promoter. (A) A panel of breast cancer cell lines were incubated with or without LW479 for 24 h; whole cell lysates were analysed by immunoblotting for EGFR and β-actin. (B) MDA-231 cells were treated with LW479 for 24 h and then analysed for EGFR mRNA expression by RT-PCR. (C) MDA-231 cells were transfected with siRNAs targeting HDAC1 (siHDAC1 1# and 2#) or non-specific control (siCtrl). Western blot analysis was used to detect the specific knockdown of HDAC1. HDAC1 knockdown repressed EGFR expression. (D) MDA-231 cells were pre-incubated with LW479 and nuclear extracts were prepared. The immunoprecipitation was performed using anti-Sp1 and anti-HDAC1 antibodies. The immunoprecipitated pellets were analysed by immunoblotting with anti-acetyl-lysine and anti-Sp1 antibodies. (E) MDA-231 cells treated with or without LW479 were stained for HDAC1 (green) and Sp1 (red). (F) MDA-231 cells were incubated with LW479 and the proteins were cross-linked with DNA and analysed by ChIP assay with anti-HDAC1 and anti-Sp1 antibodies.

LW479 blocks EGF/EGFR signalling pathway and EGF-stimulated motility

We next examined whether the downstream targets of the EGF/EGFR signalling cascade were also inhibited by LW479. Although the phosphorylation of EGFR, FAK, Akt and JNK was clearly increased by stimulation of the cells by upon EGF stimulation, LW479 repressed their activation in a dose-dependent manner (Figure 3A), and this blockade of the EGF/EGFR signalling pathway by LW479 could be an indirect consequence of inhibiting EGFR transcription. Previous studies have demonstrated that dysregulated EGFR and EGF-induced signalling promote tumour cell motility, invasion and metastasis (Lu et al., 2001). As we had found that LW479 down-regulated EGFR at protein and mRNA level (Figure 2A and B), we tested the effects of LW479 on EGF-directed migration and invasion. As shown in Figure 3B, LW479-treated cancer cells were unable to respond to EGF stimulus to migrate and invade (Figure 3B). The activation of cancer cell focal adhesions by soluble growth factors plays pivotal roles in cell motility (Turner, 2000). Our data showed that LW479 impaired EGF-induced formation of focal adhesion, as can be seen from the reduced focal adhesion adaptor protein paxillin (Figure 3C).

Figure 3.

Figure 3

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LW479 blocks EGF/EGFR signalling pathway and EGF-stimulated motility. (A) MDA-231 cells were pretreated with the indicated concentrations of LW479 for 24 h and then stimulated with EGF (50 ng·μL−1) for 30 min. Whole cell extracts were prepared and analysed by Western blotting, using the relevant antibodies. (B) Confluent MDA-231 cells in 6-well plates were scratched to create a wound and starved in serum-free medium overnight followed by exposure to EGF and different concentrations of LW479. Images were taken after 12 h of incubation at 37°C (upper right). Cell migration was quantified manually (upper left). MDA-231 cells (1 × 105) were resuspended in serum-free medium and seeded into the upper side of the transwell inserts precoated with Matrigel. Increasing concentrations of LW479 and EGF (50 ng·μL−1) were added in the bottom well. Images were obtained after 12 h of incubation (lower right). Inhibition of cell invasion by LW479 was expressed as % untreated control. (C) MDA-231 cells were treated with LW479 followed by 30 min of stimulation with EGF. Cells were fixed, permeabilized and incubated with anti-Paxillin antibody (green) and phalloidin (red). Cell nuclei were stained with DAPI. (D) MDA-231 cells were seeded onto solidified Matrigel. Serum-free medium (0.1mL) containing 10% Matrigel plus different concentrations of LW479 and EGF was added. After 4 days, culture cells were photographed using an inverted microscope. Arrow heads indicated invasive structures. **P < 0.01; ***P < 0.001 versus control.

Three-dimensional culture systems provide structural and functional aspects of cancer progression. Unlike monolayer cultures, mammary epithelial cells grown in three-dimensional systems reproduce the mammary glandular structure and many other features of breast epithelium in vivo (Lee et al., 2007). Moreover, malignant breast epithelial cells invade the surrounding matrix in three-dimensional cultures and the invasive stellate structure can be easily observed by microscopy (Lu et al., 2009). In our three-dimensional cultures, using growth factor-reduced Matrigel, LW479 inhibit EGF-induced breast cancer cell invasion, as few cells escaped from the acini and invaded the surrounding Matrigel (Figure 3D). These data demonstrated that LW479 impaired EGF-induced formation of focal adhesion and invasive structures, in three-dimensional cultures.

LW479 is effective in preventing breast tumour growth and spontaneous metastasis

To test the suppressive effects of LW-479 on breast cancer progression, we used a model in which MDA-231 cells encoding a luciferase reporter gene were injected into the mammary fat pad of nude mice. Bioluminescent images from this model on day 0 and day 35 are shown in Figure 4A. Mice treated with LW479 (40 mg·kg−1) showed a marked reduction in tumour volume (Figure 4B). On day 35, the major organs of all groups of mice were removed and imaged for tumour presence. As shown in Figure 4C, only three mice in LW479 (40 mg·kg−1) group were found to bear lung metastasis and no other distant metastasis were observed. In contrast, the untreated group progressed in a more aggressive manner: all mice developed lung metastases, five of the eight mice had liver metastases, four had spleen metastases and two had kidney metastases. Histological examination of these mice showed that the decreased tumour growth was accompanied by reduced expression of EGFR (Figure 4D, upper row). We also measured histone acetylation, within the tumour. In accord with the in vitro data, treatment with LW479 (40 mg·kg−1) for 35 days significantly increased acetylation of histone H3 (Figure 4D, lower row).

Figure 4.

Figure 4

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LW479 prevents breast tumour growth and spontaneous metastasis. (A) MDA-231-Luc cells were injected into the mammary fat pad of female nude mice, resulting in a primary breast tumour. Real-time images on treatment day 0 and day 35 were acquired using the Xenogen IVIS. (B) Primary tumour was monitored weekly by a digital calliper. (C) Ex vivo bioluminescence images were obtained in each group to determine the effects of LW479 against distant metastasis. Metastasis incidence in distant organ was quantified. (D) Primary tumours were removed at the end of the experiment, fixed and paraffin embedded. Four micrometre Sections (4μm) were analysed by IHC using anti-EGFR and anti-acetyl-H3 antibodies. *P < 0.05 versus control.

LW479 attenuates breast cancer metastasis to the lung

To assess the anti-metastatic effects of LW479, we used a model with MDA-231-Luc cells injected i.v. in female nude mice. As shown in Figure 5A and B, treatment with LW479 (40 mg·kg−1) markedly decreased lung metastases, achieving about 90% inhibition relative to the untreated control group, on day 32.

Figure 5.

Figure 5

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LW479 attenuates breast cancer metastasis to the lung. (A) MDA-231-Luc cells were inoculated i.v. into nude mice. Mice were assigned to four groups and received treatment. Lung colonization was assessed by Xenogen IVIS on day 0 and day 32. (B) Normalized bioluminescence quantification on day 0 and day 32. *P < 0.05 versus control; **P < 0.01.

Toxicity test of LW479

To investigate the systemic toxicity of LW479, female BALB/c mice were given DMSO (vehicle), LW479 (40 mg·kg−1) or SAHA (40 mg·kg−1) for 30 days. Body weight was measured every 6 days over the treatment period. No loss of weight was observed in LW479-treated group compared with untreated or DMSO-treated group (Supporting Information Fig. S7). To further assess the toxicity of LW479, all mice were killed on day 30 and the major organs were fixed and paraffin embedded for H&E staining. Histological analysis revealed that LW479 showed no obvious damage to major organs including brain, heart, lung, liver, spleen and kidney (Figure 6). Our results indicated that LW479 caused no systemic toxicity to mice at the test concentrations.

Figure 6.

Figure 6

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LW479 shows no systemic toxicity in mice. Major organs from the different groups of animals were stained with H&E.

Discussion

Numerous studies have revealed HDACs as excellent targets for cancer therapy and more than 20 different HDACIs are currently in clinical trials (http://clinicaltrials.gov/). In this report, we have identified a novel HDACI, LW479, and investigated the molecular mechanisms underlying the anti-breast cancer effects of this compound.

We demonstrated that LW479 inhibited HDAC activity using a HDACI screening kit. We further found that LW479 induced substantial increases in HDAC-related biomarkers, such as histone H3 and H4 acetylation, and abolished the expression of individual HDACs. HDACIs have been reported to induce apoptosis and cell cycle arrest in cancer cells. We demonstrated that LW479 sensitized breast cancer cells to apoptosis and caused G2/M phase cell cycle arrest. These results suggested that LW479 acts as a novel pan-HDACI.

EGFR is a member of the ErbB family that is frequently dysregulated in a variety of human epithelial cancers, including NSCLC, colorectal cancer, pancreatic cancer, brain cancer and breast cancer (Wheeler et al., 2010). Many reports have described the importance of the EGFR in breast cancer (Baselga, 2002; Pitteri et al., 2010; Masuda et al., 2012). In our studies, we found that LW479 decreased the protein and mRNA levels of EGFR in breast cancer cells and the altered expression of EGFR protein may be a consequence of the reduced mRNA level. The regulation of EGFR by HDACIs has been reported in other studies. For instance, SAHA attenuated EGFR expression in ER-negative breast cancer cells (Zhou et al., 2009) and diminished expression of EGFR was also found in colorectal cancer cells after SAHA treatment (Chou et al., 2011). However, exactly how HDACIs affect the EGFR level in distinct cell types has ont been defined. The transcription factor Sp1 that regulates basal transcription of the EGFR gene is ubiquitously expressed and the effects of HDAC inhibition on Sp1 may be involved.

Although EGFR was known to be down-regulated by HDACIs in breast cancer cells, the mechanism involved in this down-regulation was not investigated. As noted earlier, the universally expressed transcription factor Sp1 is a key regulator for EGFR transcription. Sp1 recruits HDAC1 to the promoter of regulated genes and mounting evidence indicates that the post-translational modifications (phosphorylation, acetylation, sumoylation, etc.) can influence the transcription activity of Sp1. Like many other non-histone cellular proteins, Sp1 is the target of acetylation events by HDACs (Schrump, 2009). Our results revealed that Sp1 was, constitutively, at low levels of acetylation in MDA-231 cells and this acetylation was increased by LW479 treatment. In addition, the interaction between Sp1 and HDAC1 was disrupted. LW479 also reduced the binding of Sp1 as well as that of HDAC1 to the EGFR promoter, leading to the transcriptional suppression of EGFR (Supporting Information Fig. S9). These data together indicated that suppression of EGFR by LW479 may involve the inhibition of HDAC1, which in turn up-regulates the acetylation level of Sp1. Acetylation has shown to be involved in protein–protein interaction (Arnesen, 2011). Acetylated Sp1 may be unfavourable for its interaction with HDAC1 in breast cancer cells. Previous reports have correlated transcriptional activity with increased acetylation of Sp1 (Torigoe et al., 2005). However, studies by Waby et al. (2010) suggested that exposure to butyrate (a known HDACI) elevated p21 expression by increasing acetylation of Sp1, which prevented Sp1 binding to the p21 promoter. ChIP analysis indicated that Sp1 acetylation induced by trichostatin A, another HDACI, inhibited its DNA binding at promoters of 12(S)-lipoxygenase, leading to a diminished level of this enzyme (Chen et al., 2008). To date, there still remains a controversy on the relationship between acetylated Sp1 and its transcriptional potency. Our results would support the proposition ‘the more acetylation, the less transcriptional activation’, as LW479 exposure increased both the dissociation of Sp1 from the EGFR promoter and the acetylation of Sp1. The constitutively low-acetylated Sp1 we observed in MDA-231 cells might therefore play a role in the transcription of EGFR in breast cancer.

Our in vivo studies showed that LW479 was effective in breast cancer xenograft models. We observed a diminished expression of EGFR and an increased level of acetylated histone H3 in primary tumour tissues following treatment with LW479 (40 mg·kg−1), indicating that the actions of LW479 in xenograft models was target specific. Although LW479 was effective in suppresiing breast tumour growth, the results are not particularly impressive as the inhibition rate was only about 40%. One possible reason may lie in the daily dose of LW479. An earlier study showed that SAHA as well as other novel hydroxamate-based HDACIs exhibited potent anti-breast tumour activity at a daily dose of 90 mg·kg−1 (Zhang et al., 2011) while the maximum dose of LW479 we used was 40 mg·kg−1. However, this dose of LW479 showed significant inhibition of tumour metastasis in both spontaneous and experimental metastasis models (Figures 4 and 5). Therefore, LW479 may be a potential anti-metastatic agent. The epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose their epithelial characteristics and acquire mesenchymal-like phenotypes, endows cells with invasive properties. Accumulating evidence has indicated that the EMT is a relevant and important process in tumour metastasis (Ombrato and Malanchi, 2014). We found that LW479 up-regulated epithelial markers and down-regulated mesenchymal markers in highly mesenchymal-like MDA-231 cells, suggesting that LW479 could reverse the EMT and act as a metastasis inhibitor (Supporting Information Fig. S8). In addition, preclinical studies using cell-based assay have revealed that EGFR inhibitors in combination with cytotoxic chemotherapies resulted in little effect (Corkery et al., 2009) and a phase II clinical trial in TNBC patients indicated that compounds targeting EGFR showed no obvious benefit in survival (Carey et al., 2012). Lee et al. (2012) emphasized that sequential, rather than concurrent, application of EGFR inhibitors and DNA-damaging agents sensitized breast cancer cells to death. Besides, in clinical settings, HDACIs have shown only moderate effects as single-agent anticancer therapies (Bots and Johnstone, 2009). Considering all these aspects, the therapeutic potential of LW479 may be best fulfilled through appropriate combinations with other chemotherapeutic agents.

In conclusion, our studies suggested that LW479 possesses the potential for inhibiting breast cancer progression and that it acts by diminishing levels of EGFR. However, it appears that EGFR is not the only target gene of LW479 in breast cancer cells. Further investigations are needed to identify other genes and proteins interacting with LW479, which will give us a better understanding of the molecular basis of breast cancer inhibition by LW479.

Acknowledgments

This study was partially supported by Major State Basic Research Development Program of China (2015CB910400, 2012CB910401), National Natural Science Foundation of China (81472788, 81272463, 81202407, 81330049), National Major Scientific and Technological Special Project for ‘Significant New Drugs Development’ (2013ZX09507001) and Innovation Program of Shanghai Municipal Education Commission (13zz034).

Glossary

EGFR

EGF receptor

HATs

histone acetyltransferases

HDACI

histone deacetylase inhibitor

HDACs

histone deacetylases

IVIS

In Vivo Imaging System

SAHA

suberoylanilide hydroxamic acid

Author contributions

J. L., T. Z. and F. Y. performed the research. Y. C., M. L., Z. Y. and J. L. designed the research study. F. D., F. Y. and Y. C. contributed essential reagents or tools. J. L., T. Z., Y. H., D. G., Y. C. and Z. Y. analysed the data. J. L., Y. C., M. L. and Z. Y. wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher's web-site:

Figure S1 The synthesis of LW479. Regents and conditions: (A) 3-bromobenzaldehyde, EtOH, 0oC then to room temperature; (B) NaBH4, 0oC then to room temperature; (C) pimelic acid anhydride, 1,4-dioxane, reflux; (D) MeOH, Cat.SOCl2, reflux; (E) NH2OH.HCl, MeOH, KOH. The intermediate 2-(3-bromophenyl)-3-(2-hydroxyphenyl)-4-thiazolidinone (2) was constructed from o-aminophenol (1), 3-bromobenzaldehyde and thioglycolic acid, which was alkylated with 6-bromine caproic acid methyl ester to yield ester compound (3), and the ester groups were treated with NH2OH●HCl in methanol to get the compound LW479. All starting reagents and materials were purchased from commercial sources and used without further purification. All reactions were monitored by TLC and UV spectra, column chromatography was performed on silica gel, NMR spectra was recorded on a Bruker 300 or 500 MHz instrument and obtained as CDCl3 DMSO-d6 solutions (reported in p.p.m.), using DMSO-d6 as the reference standard (2.50 p.p.m.). Mass spectral data (ESI) were gathered on VG ZAB-HS or VG-7070 instrument. HPLC (Agilent Technologies 1200 Series) was utilized for purification, injection volume was 10 L, flow rate of 1.5 mL/min, solvent A: H2O; solvent B: MeOH; gradient of 40–90% B (0–10 min), 90% B (10–15 min), 90–40% B (15–20 min). Compound purity was determined by HPLC with a confirming purity of ≥98% for the testing compounds.

Figure S2 1HMR spectrum of LW479. 1H NMR (DMSO, 300 MHz): δ 10.39 (brs, 1H), 8.69 (brs, 1H), 7.63 (s, 1H), 7.41 (dd, J = 9.0, 9.0 Hz, 2H), 7.24 (d, J = 7.8 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 7.8 Hz, 2H), 6.82 (dd, J = 7.5, 7.5 Hz, 1H), 6.16 (s, 1H), 4.03 (d, J = 15.6 Hz, 1H), 3.96-3.91(m, 2H), 3.81 (d, J = 15.6 Hz, 1H), 2.03-1.98 (m, 2H), 1.79-1.75 (m, 2H), 1.64-1.57 (m, 2H), 1.50-1.42 (m, 2H).

Figure S3 13C NMR spectrum of LW479. 13C NMR (DMSO, 125 MHz) δ 170.41, 169.04, 154.17, 142.67, 131.47, 130.59, 130.02, 129.33, 126.52, 125.58, 121.56, 120.24, 113.15, 67.79, 62.60, 32.29, 32.06, 28.42, 25.16, 24.91.

Figure S4 LW479 induces histone acetylation and breast cancer cell apoptosis. (A) LW479 up-regulated histone H3 acetylation in a dose-dependent manner. (B, C) LW479 (10μM)-induced cell apoptosis and the expression of cleaved caspase 3.

Figure S5 Cytotoxicity of SAHA. MDA-231 cells were seeded in 96-well plate and treated with different concentrations of SAHA for 48 h. Aqueous One Solution (20 μL) was subsequently added and the absorption at 490 nm was measured by a microplate spectrophotometer.

Figure S6 LW479 inhibits EGFR expression via regulating Sp1-dependent EGFR transcription. (A, B) MDA-231 cells were transfected with Sp1 siRNAs. Forty-eight hours post-transfection, cells were harvested. RT-PCR or Western blot analysis was used for detecting Sp1 and EGFR expression. (C, D) MDA-231 cells were transfected with p3xFLAG-Sp1 vector. After 48 h, cells were left treated or untreated with LW479 (10 μM) for another 24 h. RT-PCR or Western blot analysis was used for detecting Sp1 and EGFR expression. (E) MDA-231 cells were transfected with EGFR-Luc and treated with different concentrations of LW479 for 24 h. The results were normalized to the Renilla luciferase activity. (F) LW479 dose-dependently inhibited DNA-binding activity of Sp1 in MDA-231 cells. Nuclear extract was prepared and examined by EMSA assay. ‘1#’ represents negative control; ‘2#’ represents 100-fold cold probe competition. Three independent experiments were carried out.

Figure S7 LW479 has little effect on body weight of mice.

Figure S8 LW479 affects EMT-related protein expression. MDA-231 cells were treated with different concentrations of LW479 for 48 h; cells were then harvested with RIPA. Western blot analysis was performed with specific antibodies.

Figure S9 A proposed diagram of EGFR down-regulation by LW479. In human breast cancer cells, low-acetylated Sp1 recruits HDAC1 to the EGFR promoter and transcription activation. Inhibition of HDAC activity by LW479 increases the acetylation level of Sp1 and disrupts the interaction of Sp1 with HDAC1, leading to transcription repression of EGFR.

bph0172-3817-sd1.pdf (435KB, pdf)

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Associated Data

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Supplementary Materials

Figure S1 The synthesis of LW479. Regents and conditions: (A) 3-bromobenzaldehyde, EtOH, 0oC then to room temperature; (B) NaBH4, 0oC then to room temperature; (C) pimelic acid anhydride, 1,4-dioxane, reflux; (D) MeOH, Cat.SOCl2, reflux; (E) NH2OH.HCl, MeOH, KOH. The intermediate 2-(3-bromophenyl)-3-(2-hydroxyphenyl)-4-thiazolidinone (2) was constructed from o-aminophenol (1), 3-bromobenzaldehyde and thioglycolic acid, which was alkylated with 6-bromine caproic acid methyl ester to yield ester compound (3), and the ester groups were treated with NH2OH●HCl in methanol to get the compound LW479. All starting reagents and materials were purchased from commercial sources and used without further purification. All reactions were monitored by TLC and UV spectra, column chromatography was performed on silica gel, NMR spectra was recorded on a Bruker 300 or 500 MHz instrument and obtained as CDCl3 DMSO-d6 solutions (reported in p.p.m.), using DMSO-d6 as the reference standard (2.50 p.p.m.). Mass spectral data (ESI) were gathered on VG ZAB-HS or VG-7070 instrument. HPLC (Agilent Technologies 1200 Series) was utilized for purification, injection volume was 10 L, flow rate of 1.5 mL/min, solvent A: H2O; solvent B: MeOH; gradient of 40–90% B (0–10 min), 90% B (10–15 min), 90–40% B (15–20 min). Compound purity was determined by HPLC with a confirming purity of ≥98% for the testing compounds.

Figure S2 1HMR spectrum of LW479. 1H NMR (DMSO, 300 MHz): δ 10.39 (brs, 1H), 8.69 (brs, 1H), 7.63 (s, 1H), 7.41 (dd, J = 9.0, 9.0 Hz, 2H), 7.24 (d, J = 7.8 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 7.8 Hz, 2H), 6.82 (dd, J = 7.5, 7.5 Hz, 1H), 6.16 (s, 1H), 4.03 (d, J = 15.6 Hz, 1H), 3.96-3.91(m, 2H), 3.81 (d, J = 15.6 Hz, 1H), 2.03-1.98 (m, 2H), 1.79-1.75 (m, 2H), 1.64-1.57 (m, 2H), 1.50-1.42 (m, 2H).

Figure S3 13C NMR spectrum of LW479. 13C NMR (DMSO, 125 MHz) δ 170.41, 169.04, 154.17, 142.67, 131.47, 130.59, 130.02, 129.33, 126.52, 125.58, 121.56, 120.24, 113.15, 67.79, 62.60, 32.29, 32.06, 28.42, 25.16, 24.91.

Figure S4 LW479 induces histone acetylation and breast cancer cell apoptosis. (A) LW479 up-regulated histone H3 acetylation in a dose-dependent manner. (B, C) LW479 (10μM)-induced cell apoptosis and the expression of cleaved caspase 3.

Figure S5 Cytotoxicity of SAHA. MDA-231 cells were seeded in 96-well plate and treated with different concentrations of SAHA for 48 h. Aqueous One Solution (20 μL) was subsequently added and the absorption at 490 nm was measured by a microplate spectrophotometer.

Figure S6 LW479 inhibits EGFR expression via regulating Sp1-dependent EGFR transcription. (A, B) MDA-231 cells were transfected with Sp1 siRNAs. Forty-eight hours post-transfection, cells were harvested. RT-PCR or Western blot analysis was used for detecting Sp1 and EGFR expression. (C, D) MDA-231 cells were transfected with p3xFLAG-Sp1 vector. After 48 h, cells were left treated or untreated with LW479 (10 μM) for another 24 h. RT-PCR or Western blot analysis was used for detecting Sp1 and EGFR expression. (E) MDA-231 cells were transfected with EGFR-Luc and treated with different concentrations of LW479 for 24 h. The results were normalized to the Renilla luciferase activity. (F) LW479 dose-dependently inhibited DNA-binding activity of Sp1 in MDA-231 cells. Nuclear extract was prepared and examined by EMSA assay. ‘1#’ represents negative control; ‘2#’ represents 100-fold cold probe competition. Three independent experiments were carried out.

Figure S7 LW479 has little effect on body weight of mice.

Figure S8 LW479 affects EMT-related protein expression. MDA-231 cells were treated with different concentrations of LW479 for 48 h; cells were then harvested with RIPA. Western blot analysis was performed with specific antibodies.

Figure S9 A proposed diagram of EGFR down-regulation by LW479. In human breast cancer cells, low-acetylated Sp1 recruits HDAC1 to the EGFR promoter and transcription activation. Inhibition of HDAC activity by LW479 increases the acetylation level of Sp1 and disrupts the interaction of Sp1 with HDAC1, leading to transcription repression of EGFR.

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