Hydroxamic acid and benzoic acid-based Stat3 inhibitors ... · inhibitors) (Roche, Indianapolis, IN) for lysis. To shear the DNA, the nuclear lysates were sonicated (Omni International,

1

Hydroxamic acid and benzoic acid-based Stat3 inhibitors suppress human glioma and breast cancer

phenotypes in vitro and in vivo

Peibin Yue1,2 Francisco Lopez-Tapia1,2,4, David Paladino1,2, Yifei Li3, Chih-Hong Chen3, Tyvette

Hilliard1,2, Yuan Chen3, Marcus A. Tius1,2,4, and James Turkson1,2,*

1Natural Products and Experimental Therapeutics and 2Cancer Biology Programs, University of Hawaii

Cancer Center, University of Hawaii, Manoa, Honolulu, HI, USA 96813, 3Department of Molecular

Medicine, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA, 4Department

of Chemistry, University of Hawaii, Manoa, Honolulu, HI, USA 96822

Running Title: Hydroxamic acid and benzoic acid-based Stat3 inhibitors Keywords: STAT, signal transducer and activator of transcription; SH5-07, SH4-54, small-molecule inhibitors; glioblastoma, breast cancer, antitumor cell effects, and tumor growth inhibition This work was supported by NIH/NCI R01 CA161931 (JT) and GM086171 (YC) and The University of Hawaii start-up funds (JT). *Address correspondence to: James Turkson Professor and Program Director Natural Products & Experimental Therapeutics Program University of Hawaii Cancer Center 701 Ilalo Street, Suite 344 Honolulu, HI 96817 Tel. 808-440-5207 Fax 808-587-0742 Email: [email protected]

The authors declare no potential conflicts of interest

Word count: 5,953

Figures: 7

on April 1, 2020. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 18, 2015; DOI: 10.1158/0008-5472.CAN-14-3558

http://cancerres.aacrjournals.org/

2

Abstract

STAT3 offers an attractive target for cancer therapy but small molecule inhibitors with appealing

pharmacologic properties have been elusive. Here we report hydroxamic acid-based and benzoic acid-

based inhibitors (SH5-07 and SH4-54, respectively) with robust bioactivity. Both inhibitors blocked

STAT3 DNA binding activity in vitro and in human glioma, breast, and prostate cancer cells and in v-Src-

transformed murine fibroblasts. STAT3-dependent gene transcription was blocked along with Bcl-2, Bcl-

xL, Mcl-1, Cyclin D1, c-Myc and Survivin expression. Nuclear magnetic resonance analysis of STAT3-

inhibitor complexes defined interactions with the SH2 and DNA binding domains of STAT3. Ectopic

expression of the SH2 domain in cells was sufficient to counter the STAT3 inhibitory effects of SH4-54.

Neither compound appreciably affected STAT1 or STAT5 DNA binding activities, STAT3-independent

gene transcription or activation of a panel of oncogenic kinases in malignant cells.

Each compound decreased the proliferation and viability of glioma, breast and prostate cancer cells and v-

Src-transformed murine fibroblasts harboring constitutively active STAT3. Further, in mouse xenograft

models of glioma and breast cancer, administration of SH5-07 or SH4-54 effectively inhibited tumor

growth. Our results offer preclinical proof of concept for SH5-07 and SH4-54 as candidates fof further

development as cancer therapeutics.




3

Introduction

The signal transducer and activator of transcription (STAT) proteins mediate cytokine and growth factor

responses, including promoting cell growth and differentiation, and immune responses [1,2]. Ligand

binding to the receptor promotes STAT activation by inducing the critical tyrosine (Tyr) phosphorylation

by growth factor receptor Tyr kinases, JAKs or Src. Phosphorylation in turn drives STAT:STAT

dimerization through a reciprocal phospho-Tyr-Src Homology (SH)2 domain interaction. STAT:STAT

dimers translocate to the nucleus and bind specific DNA-response elements in target gene promoters to

mediate transcription, thereby regulating fundamental cellular processes.

Aberrant-activation of Stat3, however occurs in malignant transformation and is implicated in glioma,

breast, prostate, ovarian, and many other cancers [3-5]. Mechanisms to induce tumorigenesis and tumor

progression include dysregulation of gene expression that leads to uncontrolled growth and survival of

tumor cells, enhanced tumor angiogenesis, and tumor metastasis [1,3,6-8]. Stat3 activity further represses

tumor immune surveillance [5,9]. Moreover, Stat3 cross-talks with nuclear factor (NF)-κB [10] and

regulates mitochondrial functions to drive malignant transformation in specific contexts [11]. The Stat3

signaling pathway is considered an attractive target for the discovery of novel anticancer drugs.

On-going discovery campaigns for novel therapeutic modulators of Stat3 signaling [5,12-19] have largely

focused on targeting the critical dimerization step to develop Stat3 inhibitors [5,12,13,15,19-23]. The

Stat3 dimerization inhibitor, BP-1-102 emerged as an important lead compound that induced antitumor

cell effects in vitro at 10-20 μM and antitumor effects in pre-clinical models of breast and non-small cell

lung cancers [15]. Towards further improving the potency of the salicylic acid, BP-1-102 [15], we have

synthesized and evaluated the hydroxamic acid, SH5-07 and benzoic acid, SH4-54, analogs, which show

improved in vitro inhibitory activities at 1-8 μM. Structural data suggests these agents interact with the




4

Stat3 SH2 and DNA-binding domains. Further, both agents inhibit in vivo growth of human glioma and

breast cancer xenografts that harbor aberrantly-active Stat3.

Materials and Methods

Chemical synthesis of SH4-54 and SH5-07-Synthesis and detailed characterization of agents are described

in Supplementary Materials, “Methods”.

Cells and reagents-Normal mouse fibroblasts (NIH3T3), counterparts transformed by v-Src (NIH3T3/v-

Src) or overexpressing the human epidermal growth factor (EGF) receptor (NIH3T3/hEGFR), and the

human breast (MDA-MB-231 and MCF-7), pancreatic (Panc-1) and prostate (DU145) cancer cells have

all been reported [15,21,24,25] [26,27]. Stat3 null mouse embryonic fibroblast line (MEF/ST3KO) and

ovarian cancer cells (A2780S) were kind gifts of Drs. Valeria Poli, University of Turin, Italy and Jin

Cheng, Moffitt Cancer Center, Tampa, FL, respectively. The human glioma lines, U251MG, U373MG

and U87MG (Sigma-Aldrich Corporation, St. Louis, MO), and SF-295 (Division of Cancer Treatment

and Diagnosis Tumor Repository of the National Cancer Institute, Frederick, MD) were obtained from the

designated sources and cultured in Roswell Park Memorial Institute medium-1640 supplemented with 1%

nonessential amino acids (Corning Inc., Corning, NY) and containing 10% heat-inactivated fetal bovine

serum (FBS). All other cells were grown in Dulbecco's modified Eagle's medium plus 10% heat-

inactivated FBS. Except where designated, all antibodies were purchased from Cell Signaling

Technologies (Danvers, MA).

Plasmids and molecular cloning-The Stat3-dependent luciferase reporter, pLucTKS3, and the Stat3-

independent reporter, pLucSRE, have been previously reported [28,29]. The pLucTKS3 reporter contains

seven copies of the Stat3-specific binding sequence in the C-reactive protein gene promoter driving firefly

luciferase expression, while the Stat3-independent, pLucSRE reporter is driven by the serum response




5

element (SRE) of the c-fos promoter. More details of the reporters and the Stat3 SH2 and DNA-binding

domain constructs are provided in Supplementary Materials, “Methods”.

Transient transfection of expression vectors and luciferase reporter plasmids and reporter assay-

Transient transfection using Lipofectamine 3000 (Life Technologies, Grand Island, NY) and luciferase

assays were performed as previously reported [28,29]. Details are provided in Supplementary Materials,

“Methods”.

Nuclear extract preparation and gel shift assays-Nuclear extract preparation and DNA-

binding/electrophoretic mobility shift assay (EMSA) were performed as previously described [24,29].

Details are provided in Supplementary Materials, “Methods”.

Surface plasmon resonance analysis-Studies were performed as previously reported [14,15]. Purified

Stat3 (50 μg/ml) was injected onto the HisCap Sensor Chip for immobilization. Various concentrations of

agents in running buffer (1X PBS, 0.5% DMSO) were passed over the chip to produce response signals.

The association and dissociation rate constants were calculated using the Qdat software. The ratio of the

association and dissociation rate constants was determined as the binding affinity (KD).

Nuclear magnetic resonance (NMR) studies of Stat3-compound interactions- NMR studies were

performed using the human Stat3β protein encompassing residues 127-711 in solution with agents and are

described in detail in Supplementary Materials, “Methods”.

Immunoprecipitation and SDS-PAGE/Western blotting analysis-These studies were performed as

previously described [29,30].




6

Chromatin-Immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) studies-

ChIP assay was performed as previously reported [31], with minor modification. Briefly, 1 X 107 cells in

culture were treated with 5 or 8 µM SH4-54 for 3 h and then fixed with 1% formaldehyde for 7 min at

room temperature. Cells were then treated with glycine (0.125 M, 5 min) at room temperature for cross-

linking, washed with ice-cold PBS and lysed with ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM

NaCl, 3 mM MgCl2, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) and centrifuged. Nuclear

pellet was then resuspended in buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS and protease

inhibitors) (Roche, Indianapolis, IN) for lysis. To shear the DNA, the nuclear lysates were sonicated

(Omni International, Kennesaw, GA) at 30% power for 3 pulses for 10 s intervals on ice. Samples were

pre-cleared with protein A/G agarose beads (Santa Cruz) for 1 h at 4 oC, with rocking and incubated with

anti-Stat3 (C20X) or anti-Stat5 (C-17) antibodies or with normal rabbit IgG overnight at 4 oC overnight,

with rocking for immunoprecipitation. Immunecomplexes were collected with 20 µl protein A/G agarose

bead, washed multiple times with wash buffer A (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM

Tris-HCl, pH 8.0) and two times with wash buffer B (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500

mM NaCl, 20 mM Tris-HCl, pH 8.0) and eluted with freshly prepared elution buffer (1% SDS, 100 mM

NaHCO3). Cross-links were reversed by heating at 65 oC in the presence of NaCl followed by proteinase

K treatment (20 µl, 20 mg/ml) (Pierce) overnight. The DNA was recovered and purified using ChIP spin

columns (Zymo Research Corp, Irvine, CA). The purified chromatin immunoprecipitated DNA was used

as a template for PCR amplification of the promoters for inducible nitric oxide synthase (iNOS), Survivin,

Bcl-2 and β-casein genes. The PCR products were resolved on 2% agarose gel. ChIP results were

analyzed by triplicate qPCR of the IP samples and corresponding 1% input samples using primers

flanking the Stat3 binding region of each indicated gene promoter. Quantification was performed using

the ΔCt method, appropriate due to the >90% PCR efficiency of each primer set. The percent input

enrichment was determined by normalizing the Ct value for each sample to its corresponding 1% input Ct




7

value. These absolute values were plotted as in the figures. Details and the PCR oligonucleotide primers

are described in Supplementary Materials, “Methods”.

Cell viability assays-CyQuant cell proliferation assay to evaluate compounds was performed, as

previously reported [14,15] and following the manufacturer’s (Invitrogen Corp/Life Technologies Corp,

Carlsbad, CA) instructions.

Soft-agar colony formation and clonogenic survival assays-These studies were performed as previously

reported [15,28,29]. Details are provided in Supplementary Materials, “Methods”.

Cell cycle profile and Annexin V binding with Flow cytometry analyses- Cells were treated with 0-8 µM

agent for 24-48 h. For cell cycle profile analysis, cells were harvested and fixed with 70% ice-cold

ethanol and stained with propidium iodide (PI). For apoptosis analysis, cells were collected and stained

with FITC-Annexin V using Apoptosis Detection Kit (BD Biosciences, San Jose, CA). Both the DNA

content of cells and the Annexin V-positive cells were analyzed by FACScan flow cytometer (BD

Biosciences). Cell cycle phase distribution was analyzed using the Cell-Fit program. Data acquisition was

gated to exclude cell doublets.

Wound healing assay for migration-Studies were performed as previously reported [15]. Details are

provided in Supplementary Materials, “Methods”.

Mice and in vivo tumor studies- All animal experiments were performed under a protocol approved by the

Institutional Animal Care and Use Committee. Four-to-five week-old female athymic nude mice were

purchased from Jackson Laboratory and maintained in the institutional animal facilities approved by the

American Association for Accreditation of Laboratory Animal Care. Mice were injected subcutaneously




8

in the left flank area with U251MG cells (1 x 107) in 200 µL of PBS/Matrigel matrix (1:1, BD

Biosciences), or MDA-MB-231 cells (5 x 106) in 100 µL of PBS. Mice with tumors of 90-150 mm3

(MDA-MB-231) or 150 mm3 (U251MG) were grouped for identical mean tumor sizes, administered 3, 5

or 6 mg/kg SH5-07 or SH4-54 via oral gavage daily or tail vein injection every 2 or 3 days, and

monitored every 3-7 days. Tumor sizes were measured with calipers and converted to tumor volume, V,

as follows: V=0.52 x a2 x b, where a, smallest superficial diameter, and b, largest superficial diameter. For

each treatment group, the tumor volumes for each set of measurements were statistically analyzed relative

to the control (1% DMSO-treated) group.

Statistical analysis-Statistical analysis was performed on mean values using Prism GraphPad Software,

Inc. (La Jolla, CA). The significance of differences between groups was determined by the paired t-test at

p <0.05*, <0.01**, and < 0.001***.

Results

Compounds preferentially inhibit Stat3:Stat3 DNA-binding activity. SH4-54 and SH5-07 (Fig. 1A)

are benzoic and hydroxamic acid analogs, respectively, of BP-1-102 [15]. Pre-incubation of NIH3T3/v-

Src nuclear extracts of equal total protein containing constitutively-active Stat3 with 0-10 µM SH5-07 or

SH4-54 for 30 min at room temperature, prior to incubation with the radiolabeled high-affinity sis-

inducible element (hSIE) probe that binds Stat3 and Stat1 and subjecting to EMSA analysis [12,13,15]

dose-dependently inhibited Stat3 activity (Fig. 1B), with IC50, 3.9 ± 0.6 μM and 4.7 ± 0.5 μM,

respectively. These potencies are improved over that of BP-1-102, of IC50, 6.8 μM [15] (Fig. S1A).

Similar studies using EGF-stimulated NIH3T3/hEGFR nuclear extracts containing active Stat1, Stat3 and

Stat5 show agents preferentially inhibited Stat3:Stat3 DNA-binding activity, ahead of inhibiting

Stat1:Stat3 activity, with minimal effects on Stat1:Stat1 activity (Fig. 1C). Parallel EMSA analysis using

nuclear extracts from EGF-stimulated NIH3T3/hEGFR cells and the radiolabeled mammary gland factor




9

element (MGFe) probe that binds Stat1 and Stat5 showed no inhibition of Stat1:Stat1 or Stat5:Stat5

activity (Fig. 1D). Supershift study with anti-Stat3 antibody (α-ST3) identifies Stat3-DNA complex (Fig.

S1B).

Inhibition of intracellular Stat3 activation. EMSA analysis [14,15] of nuclear extracts from human

glioma (U251MG, U373MG, U87MG and SF295), breast (MDA-MB-231), and prostate cancer (DU145)

cells, or NIH3T3/v-Src cells [4,5,8] and treated with 0-10 µM SH5-07 or SH4-54 for 1 h (Fig. 2A, B)

shows inhibition of constitutively-active Stat3 in the U251MG and U87MG cells at 1 µM and higher,

U373MG and SF295 lines at 1-3 µM and higher, and in MDA-MB-231 and DU145 cells at 3-5 µM and

higher (Fig. 2A, B). These compare more favorably over the potencies BP-1-102 of 10-20 µM [15].

Immunoblots confirmed pY705Stat3 reduction by agents at 3-10 µM (Fig. 2C, D), compared to 10-20 µM

activity for BP-1-102 [15]. Inhibition of Stat3 DNA-binding activity (Fig. 2B) or pY705Stat3 (Fig. 2D)

occurs early (0.5-1 h) and is sustained for 3-6 h, followed by an apparent weakening at 6 h or later. By

contrast, BP-1-102 inhibited pY705Stat3 at 15 µM and 24 h, with little effect at 1 h (Fig. S2A). Assuming

the possibility of diminishing intracellular inhibitor levels, a time-course study for the effects of 5 μM

SH5-07 with or without a second treatment at time 5 h after the first treatment for additional 1 h (6#) and

4 h (9#) showed a sustained pY705Stat3 inhibition for up to 9 h (Fig. 2E, lanes 5 and 6). Both inhibitors

had little effect on pS727-Stat3 (Fig. S2B).

The observation that intracellular constitutive Stat3 DNA-binding activity decreased (Fig. 2A) with no

corresponding change in pY705Stat3 at 1-3 µM, except at higher, 5-10 µM (Fig. 2C, S2C), suggests that

inhibition is strongest on Stat3 DNA-binding activity, compared to pY705Stat3 (Fig. 2A vs. C, and Fig.

S2C). This apparent lack of correlation within the context of constitutively-active Stat3 suggests the

disruption of the pre-existing Stat3:Stat3 dimers, which directly leads to lower DNA-binding activity

(Fig. 1B) [12-15,32], has a non-linear relationship with the turnover of the disrupted pStat3 molecules.




10

This likely reflects differences in kinetics and highlights the complexity of the events involved in the

Stat3 activation and inactivation. We observe a similar non-agreement between the pStat3 and Stat3

DNA-binding activity changes caused by the over-expression of 1 µg exogenous Stat3 SH2 domain,

which suppressed DNA-binding activity, with no corresponding pStat3 change (Fig. S2D, lane 4). The

over-expression of higher, 2 or 4 µg Stat3 SH2 domain, however, caused concurrent reductions in both

pStat3 and Stat3 DNA-binding activity (Fig. S2D, lanes 5 and 6), consistent with previous report that a

peptide derived from the SH2 domain inhibited Stat3 signaling [33] and that the SH2 domain expressed

alone is functional. Moreover, dimerization disruptors could inhibit DNA-binding activity and function,

without necessarily inhibiting pY705Stat3. Nuclear extracts containing constitutively-active Stat3 and

pre-incubated with 5 µM SH4-54, SH5-07 or BP-1-102 [15] had lower Stat3 activity in the cell-free

EMSA analysis (Fig. S2E(i)), while the corresponding pY705Stat3 immunoblots were unchanged (Fig.

S2E(ii)), presumably due to the absence of intracellular events to promote pY705Stat3 turnover.

Therefore, despite being a direct measure of the effects of dimerization disruptors on Stat3 activation,

DNA-binding activity inhibition may not necessarily correlate with the pY705Stat3 inhibition.

Altogether, these data highlight complexities in the intracellular Stat3 signaling induction and turnover

that impact the measured activities of dimerization disruptors or SH2 domain antagonists.

Luciferase reporter studies showed v-Src induces Stat3-dependent pLucTKS3 luciferase reporter activity

[20,21,29] by 25 to 34-fold, which was suppressed by 3-8 µM SH5-07 (Fig. 2F(i), lanes 2 vs. 1, and lanes

3 and 4 vs. 2), while similar treatment did not inhibit v-Src-induced, Stat3-independent pLucSRE reporter

activity [20,21,28,29] (Fig. 2F(ii)). Agents had no significant effects on pY1068EGFR, pY416Src,

pJAK2, pShc, pErk1/2MAPK, and pS473Akt levels (Fig.S3A-D) at concentrations that inhibit Stat3 activity

(Fig. 2A-F). We note the agreement between the inhibitory potencies in both the cell-free Stat3 DNA-

binding assay (IC50, 3.9-4.7 μM; Fig. 1B) and the intracellular constitutively-active Stat3 studies (1-8 µM;




11

Fig. 2A-F). These potencies are an improvement over the 10-20 µM activities for BP-1-102 [15] (Fig.

S2A).

Agents bind Stat3, disrupt Stat3 association with growth factor receptor, and thereby inhibit Stat3

phosphorylation. Stat3 dimerization disruptors suppress pY705Stat3 [13-16,20,21,34-36]. STATs are

recruited to the receptor phospho-Tyr motif for close proximity to Tyr kinases. We focused on Stat3

recruitment and assessed the effect of agents on EGFR:Stat3 association. In co-immunoprecipitation

studies, EGF stimulation of NIH3T3/hEGFR fibroblasts induced 1.38-fold increase in Stat3:EGFR

complexation (Fig. 3A, lanes 3 vs 2, Stat3), which was associated with pY705Stat3 induction (Fig. 3B,

lanes 2 vs 1, pY705Stat3). Twenty-four hour prior treatment of fibroblasts with agents decreased EGF-

stimulated Stat3:EGFR co-immunoprecipitation (Fig. 3A, lanes 4 and 5 vs 3, Stat3), in parallel with

decreased EGF-induced pY705Stat3 (Fig. 3B, lanes 3 and 4 vs 2, pY705Stat3), without inhibiting

pY1068EGFR or pERK1/2 induction (Fig. 3C). Moreover, prior SH5-07 treatment of fibroblasts had little

effect on EGF-stimulated pY1068EGFR induction, or pY705Stat3, except at 24 h (Fig. S4). By contrast,

1-h prior treatment of MDA-MB-231 cells with agents inhibited pre-existing and/or interleukin-6 (IL-6)-

stimulated pY705Stat3 induction (Fig. 3D, lanes 3 vs 2 and 1, and lanes 5 vs 6). We note the differences

in the time-to-inhibition between the EGF-stimulated pY705Stat3 in mouse fibroblasts, IL-6-stimulated

pY705Stat3 in human breast cancer cells, and pre-existing pY705Stat3 in tumor cells, which altogether

indicate signal-type (constitutive, EGF- or IL-6-stimulated) and cell-type (normal mouse fibroblasts

versus human tumor cells) contexts of pY705Stat3 suppression. Therefore, agents inhibit ligand-

stimulated de novo Stat3 induction. Furthermore, disruption of Stat3:receptor interaction represents one of

the pY705Stat3 inhibition mechanisms.

Surface plasmon resonance (SPR) analysis of Stat3:agent interactions shows SH4-54 and SH5-07 bound

Stat3, with affinities (KD) of 2.4 µM (Fig. 4A and data not shown). We performed NMR analysis of




12

Stat3:compound interactions. 1D 1H NMR spectra of 200 µM compounds (from DMSO stock) in aqueous

solution suggested 20 μM effective concentration (Fig. S5A, and data not shown), suggesting Stat3 could

not be fully saturated by the compounds in NMR studies. Given Stat3 protein concentration of 20 µM and

the 20 µM maximal compound detected, the sample reached approximately 1:1 stoichiometry. The %

saturation, based on KD of 2.4 µM, is approximately 70%. Solubility issues prevented BP-1-102 analysis

in a similar study. NMR chemical shift perturbation (CSP) indicates that compounds interact with Stat3 in

a specific manner. The overlay of the 13C-1H HMQC spectra of Stat3, free and in complex with the

representative compound, SH4-54, is shown (Fig. 4B, left). All 35 peaks from Ile residues of Stat3 have

been assigned (Namanja et al, manuscript submitted), providing probes for every structured domain. In

addition, site-directed mutations have been made to replace Leu residues in the DNA-binding domain

(DB), one at a time, in order to identify their resonances. Binding of compounds caused selective changes

in line-widths at Ile residues 597, 386 and 439 and CSP of Leu411 (Fig. 4B, left). The line broadening

effects observed by the Ile signals indicate that the compounds bind to both the Stat3 SH2 and DB

domains (Fig. 4B, left). Residues Leu411, Ile386 and Ile439 form a hydrophobic pocket that is likely

involved in compound binding. The peak from I364, which is next to the other perturbed residues in the

DB domain, also showed CSP. The ribbon representation of Stat3 structure in complex with DNA and

with the locations of these residues is shown (Fig. 4B, right).

NMR data identify a novel putative binding to the DB domain. We probed the specificity of the

Stat3:compound interaction and the significance of the DB domain. The most likely non-specific event

that induces CSP is the alkylation of the Cys residues that do not form disulfide bonds in the DB domain,

one of which is at the DB interface. NMR analyses of 20 µM Stat3 sample with and without 100 µM

NEM (Fig. S5B) showed the CSP due to alkylation is different from that due to interaction with

compounds. The CSP at residues I364 and L411 upon compound interaction did not occur upon

alkylation by NEM, suggesting the Stat3:compound interaction is specific. Unfortunately, the poor




13

solubility and moderately weaker activity of BP-1-102 prevented the collection of 2D 1H-13C HMQC

spectra for comparison. Moreover, EMSA analysis shows transiently-expressed exogenous Stat3 SH2

domain rescued Stat3 activity from compound effects, compared to the moderate rescue by the DB

domain (Fig. 4C), indicating that the expressed SH2 domain is functional (Fig. S2D) and represents the

primary target site for the compounds, while the DB domain interaction only moderately contributes to

the overall compound effect against Stat3. We note that the expressed SH2 domain functions as a

negative regulator of Stat3 signaling (Fig. S2D, lanes 4-6). However, the exogenously expressed Stat3

SH2 domain, when concurrently present, binds compound, consistent with the previous report that the

SH2 domain and its peptide, SPI, both sufficiently interacted with the dimerization disruptor, S3I-201 and

other Stat3 SH2 domain-binding peptides [33]. The easier accessibility to the exogenously expressed SH2

domain favors interaction with the compound more than the occluded SH2 domain in the Stat3:Stat3

dimer.

Compounds induce antitumor cell effects against malignant cells harboring constitutively-active

Stat3. In agreement with Stat3’s role in tumor cell phenotype [3-5,14,15], 72-h treatment with the

compounds of cultured tumor cells harboring variable Stat3 activities (Fig. S6) inhibited viability to

different degrees (Fig. 5A), with IC50 of 1.0-2.7 µM for glioma U251MG and U87MG cells (Fig. 5A,

middle panels), which are most sensitive. Other IC50 values are 3.8-4.5 µM for breast cancer, MDA-MB-

231 (231) cells (Fig. 5A, left panels), 3.8-7.4 µM for glioma, U373MG and SF295 cells (Fig. 5A, middle

panels), 5.3-5.8 µM for DU145 prostate cancer cells (Fig. 5A, left panels), 4.1-9.2 µM for NIH3T3/v-Src

(vSrc) (Fig. 5A, left panels), and 9.6-10.3 µM for the least sensitive human pancreatic cancer, Panc-1

cells (Fig. 5A, left panels). The variable growth inhibition likely reflects the Stat3 activation levels that

show different inhibitor sensitivities (Fig. 2A-D) across the tumor cells. The 1.0-7.4 µM range that

inhibits cell viability overlaps with the 1-5 µM range that inhibits Stat3 DNA-binding activity in tumor

cells. Comparatively, the viability of MCF-7, A2780S, normal NIH3T3 (3T3) and Stat3-null mouse




14

embryonic fibroblasts (MEF/S3T3) that do not harbor aberrantly-active Stat3 (Fig. S6) is only moderately

inhibited, with IC50 values of 8.1-10.8 µM or higher (Fig. 5A, right panel). We note the general trend that

glioma and breast cancer cells harboring aberrantly-active Stat3 are more sensitive.

To validate the Stat3-dependency, the transient over-expression of the SH2 domain (Fig. 5B(ii)) rescued

MDA-MB-231 cells from the effects of SH4-54 (Fig. 5B(i), SH2), while the DB domain over-expression

only moderately rescued cells (Fig. 5B(i), DBD), consistent with the findings in Figure 4C that the SH2

domain represents the major target site for the inhibitors, and with the previous report that the Stat3 SH2

domain rescued tumor cells from BP-1-102-induced apoptosis [15]. Furthermore, siRNA knockdown of

Stat3 suppressed U251MG viability (Fig. 5C).

In colony survival assay, one-time treatment with compounds at 1-3 µM dose-dependently suppressed

U251MG and MDA-MB-231 colony formation (Fig. 5D, left two panels), with higher activity against

U251MG cells, while minimally affecting MEF/ST3KO (Fig. 5D, right panel). SH5-07 further dose-

dependently suppressed the soft-agar growth of U251MG and MDA-MB-231 cells, with a higher activity

against MDA-MB-231 cells (Fig. 5E and Fig. S7A).

Flow cytometry/cell cycle profile analysis shows sub-G0 phase population increased by 15-55% for

MDA-MB-231 cells treated with 5-8 µM SH5-07 for 24-48 h, with decreased cell numbers in G1, S and

G2/M phases (Fig. 5F(i)), and 20% for U251MG cells treated with 8 µM SH5-07 for 48 h, associated with

reduced G0/G1 phase population (Fig. 5F (ii), 8 µM), while treatment had minimal effects on

MEF/ST3KO (Fig. 5F (iii)). Annexin V binding/flow cytometry analysis showed 5.8 % and 18 %

apoptosis, respectively, for U251MG and MDA-MB-231 cells treated with 8 µM SH5-07 for 24 h (Fig.

5G(i) and (ii)) and minimal effects on similarly-treated MEF/ST3KO cells (Fig. 5G (iii)). Therefore, cell

cycle changes and apoptosis contribute to the growth inhibition of tumor cells that harbor persistently-




15

active Stat3. Further, 8 µM SH5-07 or SH4-54 treatment for 22 h suppressed MDA-MB-231, U251MG

and DU145 cells migration into the denuded area (Fig. S7B(i) and (ii), upper panels), without affecting

cell viability (Fig. S7B(i) and (ii), bottom panels).

SH5-07 inhibits the expression of known Stat3-regulated genes. Immunoblotting analysis shows

reduced Bcl-2, Bcl-xL, c-Myc, Survivin, Cyclin D1 and Mcl-1 expression in response to 24 h, 5 µM SH5-

07 treatment (Fig. 6A). Chromatin immunoprecipitation analysis with qPCR confirmed decreased

promoter occupancy by Stat3 of the iNOS, bcl-2 and survivin genes in cells treated for 3 h with 5 or 8 µM

SH4-54, and not Stat5 occupancy of β-casein gene promoter (Fig. 6B). siRNA knockdown of Stat3

suppressed iNOS, Bcl-2 and Survivin expression (Fig. 6C). Studies altogether validate the inhibition of

aberrantly-active Stat3, suppression of constitutive induction of Stat3-regulated genes [3-6,8], and the

suppression of tumor phenotype by SH5-07 and SH4-54.

SH5-07 and SH4-54 inhibit growth of human breast and glioma tumor xenografts. Tail vein

injection (5-6 mg/kg every 2-3 days) or oral gavage delivery (3 mg/kg daily) of SH5-07 or SH4-54

inhibited growth of 90-150 mm3 established subcutaneous mouse xenografts of human glioma (U251MG)

(Fig. 7A) and breast (MDA-MB-231) (Fig. 7B) tumors that harbor aberrantly-active Stat3, associated

with decreased c-Myc, Mcl-1 and Cyclin D1 expression (Fig. 7C). No significant changes in body

weights (Fig. S8A), blood cell counts (Fig. S8B), or the gross anatomy of organs (Fig. S8C), or obvious

signs of toxicity, such as loss of appetite, decreased activity, or lethargy were observed.

Discussion

The prevalence of constitutively-active Stat3 in human tumors has placed an increasing importance on the

discovery of novel Stat3-inhibiting anticancer drugs [5]. The hydroxamic acid, SH5-07 and the benzoic

acid, SH4-54, analogs of BP-1-102 [15] have emerged as potent and selective inhibitors, with single-digit




16

micromolar activities against Stat3 signaling. Studies show that constitutively-active Stat3 in tumors

responds variably across tumor lines to small-molecule inhibitors. The differential sensitivities of

aberrantly-active Stat3 signaling suggest complexities in the Stat3 induction and decay mechanisms in

tumor cells. Compared to other notable agents, including, BP-1-102 [15] and S3I-1757 [19], both SH5-07

and SH4-54 possess stronger activities.

Present studies, together with published reports [13-15,17,19,23,37], demonstrate the biological

responsiveness to small-molecule Stat3 inhibitors of tumor models harboring persistently-active Stat3,

including glioma, breast, prostate, and lung cancer models. Interestingly, among the Stat3-relevant tumor

models, the human glioma tumor cells, U251MG and U87MG appear to be more sensitive than the

glioma lines, U373MG and SF295 or the breast cancer line, MDA-MB-231, prostate cancer cells, DU145,

or the v-Src-transformed mouse fibroblasts, while Panc-1 pancreatic cancer cells are least responsive.

Given the importance of constitutively-active Stat3 in promoting aggressive glioma phenotype and its

clinical relevance in predicting a poor clinical outcome, current studies together with others [37-40] raise

the potential that small-molecule Stat3-inhibiting therapeutics, such as SH5-07 and SH4-54, could be

particularly useful in anti-glioma therapy.

Aberrantly-active Stat3 dysregulates expression of critical genes that control tumor cell growth and

survival, tumor angiogenesis, and tumor cell migration, invasion and tumor metastasis

[15,20,21,23,25,26,41-44]. The inhibition of Stat3 signaling by SH5-07 and SH4-54 is associated with

decreased Bcl-2, Bcl-xL, Cyclin D1, c-Myc, Mcl-1, and Survivin expression, and antitumor responses in

human glioma and breast cancer models. Against this compelling evidence, however, studies by others

showed that the phosphatase-stable, cell-permeable SH2 domain-targeting prodrug inhibitor of Stat3, PM-

73G, did not inhibit cyclin D1, Bcl-2 or survivin expression or induce apoptosis [35,45] at the

concentrations that inhibited pY705Stat3 in tumor cells, except at 50-fold higher concentration [35]. The




17

failure in those studies to observe antitumor cell response to agents at concentrations that inhibit

pY705Stat3 contrasts with the current study and others, and likely suggests complex mechanisms and

functions of constitutive Stat3 activation in malignant progression that may differentially impact the

cellular responses to Stat3 inhibitors. It further indicates there is more to learn about the relationship

between the inhibition of Stat3 signaling by small-molecules and the overall biological outcome. By

comparison, the suppression of the known Stat3-regulated genes and antitumor cell responses to SH5-07

or SH4-54 occurred at 1-8 µM concentrations that inhibit constitutively-active Stat3, measured by

pY705Stat3, Stat3 DNA-binding activity, and Stat3-dependent luciferase reporter transcription. The

inhibitory responses are validated by the SH2 domain over-expression that countered the effects of the

small-molecules and by the siRNA knockdown of Stat3 that produced similar results.

Altogether, the present study identifies SH5-07 and SH4-54 as potent Stat3 inhibitors, which induce

antitumor cell effects in vitro and antitumor response in vivo against human glioma and breast cancer

models. The efficacy studies against subcutaneous xenografts are intended to provide proof-of-concept

for the potential efficacy against glioma and breast cancer. SH5-07 and SH4-54 are therefore potential

candidates for further development for clinical application, particularly for human glioma and breast

cancers.

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Footnotes

We thank all colleagues and members of our laboratory for the stimulating discussions, and F. Sulzmaier

for assisting with the apoptosis data analysis. Flow cytometry services were provided by the Molecular

and Cellular Immunology Core, which is supported in part by National Institute of General Medical

Sciences Centers of Biomedical Research Excellence Grant P20GM103516. This work was supported by

the National Cancer Institute Grants CA161931 (JT) and GM086171 (YC) and the University of Hawaii

start-up funds (JT).

The abbreviations used are: STAT, signal transducer and activator of transcription; MEF/ST3KO, Stat3

knockout mouse embryonic fibroblasts, EMSA, electrophoretic mobility shift assay; MAPK, mitogen-

activated protein kinase; ERK, extracellular signal-regulated kinase; GAPDH, Glyceraldehyde-3-

phosphate dehydrogenase; qPCR, quantitative polymerase chain reaction, ChIP, chromatin

immunoprecipitation.

Figure Legends

Figure 1. SH5-07 and SH4-54 and effects against STAT DNA-binding activities in vitro. (A) SH5-07

and SH4-54; and (B-D), Nuclear extract preparations from (B) NIH3T3/v-Src containing activated Stat3,




24

or (C, D) EGF-stimulated NIH3T3/hEGFR containing activated Stat1, Stat3 and Stat5 were pre-incubated

0-20 µM SH5-07 or SH4-54 for 30 min at room temperature prior to incubation with the radiolabeled (B,

C) hSIE probe that binds Stat3 and Stat1 or (D) MGFe probe that binds Stat1 and Stat5 and subjecting to

EMSA. Positions of STATs:DNA complexes are labeled; control lanes (0) represent extracts treated with

0.05% DMSO. Bands corresponding to STATs:DNA complexes were quantified using ImageQuant,

calculated as percent of control (cont), and are shown or plotted against concentration to derive IC50

values. Data are representative of 3-4 independent determinations.

Figure 2. Agents inhibit intracellular Stat3 activation. (A and B) EMSA analysis of Stat3 DNA-

binding activity in nuclear extracts from the designated malignant cells that were treated with (A) 0-10

µM SH5-07 (i) or SH4-54 (ii) for 1 h, or (B) 5 or 8 µM SH5-07 (i) or SH4-54 (ii) for 0-24 h; (C-E) Stat3

and pY705-Stat3 immunoblotting analysis of malignant cells treated with (C) 0-10 µM SH5-07 (i) or

SH4-54 (ii) for 1 h, (D) 5 or 8 µM SH5-07 (i) or SH4-54 (ii) for 0-24 h, or (E) 5 µM SH5-07 for 0-9 h,

with a repeat dosing at 5 h for additional 1 h (6#) or 4 h (9#); and (F) NIH3T3 fibroblasts were transiently

co-transfected with v-Src plasmid and the (i) Stat3-dependent (pLucTKS3) or (ii) Stat3-independent

(pLucSRE) luciferase reporter and treated with 0-8 µM SH5-07 for 24 h. Luciferase reporter activity in

cytosolic extracts is plotted as fold change over control. Positions of STATs:DNA complexes or proteins

in gel are labeled; control lanes (-, 0) represent nuclear extracts, cytosolic extracts, or whole-cell lysates

from 0.05% DMSO-treated cells. For each transfection, luciferase activity was normalized to transfection

efficiency with β-galactosidase activity as an internal control. Data are representative of 3-4 independent

determinations. Values, mean ± S.D., n=4, each performed in triplicate. * - <0.05.

Figure 3. Compounds inhibit Stat3 binding to EGF receptor and ligand-induced pY705Stat3.

Immunoblotting analysis of (A) EGFR immunecomplexes or (B, C) whole-cell lysates from

NIH3T3/hEGFR fibroblasts pre-treated or not with 10 μM SH5-07 or SH4-54 for 24 h prior to EGF




25

stimulation (100 ng/ml, 12 min), or (D) whole-cell lysates from MDA-MB-231 cells pre-treated for 1 h

prior to IL-6 stimulation (20 ng/ml, 10 min) and probing for EGFR, pY1068EGFR, pY705-Stat3, Stat3,

pERK1/2, ERK1/2, or β-actin. Positions of proteins in gel are labeled; control lane (-) represents whole-

cell lysates or EGFR immunecomplexes prepared from 0.05% DMSO-treated cells. Bands corresponding

to proteins were quantified using ImageQuant, calculated as percent of control (cont) and shown. Data are

representative of 2-3 independent determinations.

Figure 4. Stat3 interacts with compounds and its activity is rescued by SH2 domain over-expression.

(A) Surface plasmon resonance analysis of SH4-54 interaction with Stat3; (B) NMR analysis of Stat3 in

solution with SH4-54. (i) Overlay of the 1H-13C HMQC spectra of Stat3, free (black) and SH4-54-bound

(red). Residues with significant changes in either resonance line-widths or NMR chemical shifts are

indicated; and (ii) the ribbon representation of Stat3:DNA complex showing the locations of the affected

amino acid residues; and (C) Stat3 DNA-binding assay/EMSA analysis of nuclear extracts from U251MG

cells transiently over-expressing Stat3 SH2 or DNA-binding (DB) domain and treated with 8 µM SH4-54

for 3 h. Positions of Stat3:DNA complex are labeled; control lanes (-) represent nuclear extracts treated

with 0.05% DMSO. Data are representative of 2-3 independent determinations

Figure 5. Antitumor cell effects in vitro of compounds against malignant cells harboring aberrantly-

active Stat3. (A) Cultured human tumor cells that harbor varying degrees of constitutively-active Stat3

and cells that do not (NIH3T3 (3T3), Stat3 null mouse embryonic fibroblasts (MEF/ST3KO), MCF7 and

A2780S) were treated once with 0-10 μM (i) SH5-07 or (ii) SH4-54 for 72 h and assayed for cell

proliferation and plotted as % cell viability against concentration, from which IC50 values (inserts) were

derived; (B and C) MDA-MB-231 cells were transiently-transfected with (B) FLAG-tagged Stat3 DNA-

binding (DBD) or SH2 domain expressing plasmid and untreated or treated with 3 or 5 µM SH4-54 for 72

h or (C) Stat3 siRNA or scrambled siRNA for 24 h and (B(i) and C) assayed for cell proliferation, or




26

(B(ii)) or immunoprobed for FLAG or β-actin; (D) Single-cell cultures of designated cells treated once

with 0-5 μM agents and cultured until large colonies were visible, which were stained with crystal violet

and imaged; (E) Plots of numbers of large visible colonies of tumor cells growing in soft-agar and treated

once with 0-8 µM SH5-07; (F and G) Flow cytometry analysis of cells in culture treated with 0-8 µM

SH5-07 for 24-48 h, stained with (F) propidium iodide (PI) for DNA content and cell cycle distribution,

which is plotted, or (G) PI and Annexin V for apoptosis, which is represented as percent Annexin V

positive cells. Control (-, 0) represents samples or whole-cell lysates prepared from scrambled siRNA-

transfected or 0.05% DMSO-treated cells. Values, mean ± S.D., n=3-4. Data are representative of 3

independent determinations.

Figure 6. Suppression of Bcl-2, Bcl-xL, Cyclin D1, c-Myc, Mcl-1 and Survivin expression and the

Stat3 occupancy of inducible nitric oxide synthase, bcl-2 and survivin promoters. (A and C)

Immunoblots of (A) Bcl-2, Bcl-xL, c-Myc, Survivin, Cyclin D1, Mcl-1, β-actin or GAPDH the

designated cells treated with 0 or 5 µM SH5-07 for 6-24 h, or (C) iNOS, Bcl-2 and Survivin from MDA-

MB-231 cells transiently-transfected with Stat3 siRNA or scrambled siRNA; and (B) percent enrichment,

as detected by qPCR, of the DNA fragments for the indicated gene promoters from Stat3 or Stat5

immunoprecipitated DNA complex relative to input. PCR amplifications were performed using specific

primers for the promoter regions. Positions of proteins in gel are shown; control (-, 0) lane represents

whole-cell lysates from scrambled siRNA-transfected or 0.05% DMSO-treated cells. Bands

corresponding to proteins were quantified using ImageQuant, calculated as percent of control (cont) and

shown. Values, mean ± S.D., n=3-4. Data are representative of 2-3 independent determinations. * - <0.05

Figure 7. In vivo antitumor efficacy response against subcutaneous human glioma and breast tumor

xenografts in mice and effect on Stat3-regulated genes. Growth curves for subcutaneous human (A)

glioma, U251MG or (B) breast, MDA-MB-231 tumor xenografts and the effects of (A and B, left panel)




27

intravenous administration (5 or 6 mg/kg, every 2 or 3 days), or (B, right panel) oral gavage delivery (3

mg/kg, every day) of agetns; and (C) Mcl-1, c-Myc or Cyclin D1 immunoblots in tumor tissue lysates

from 2 different control and 3 different treated mice for each inhibitor. Positions of proteins in gel are

labeled; control lanes represent tissue lysates from mice treated with 0.05% DMSO. Values, mean ± S.D.,

n=6. * - <0.05




SH5-07 SH4-54

Stat5:Stat5 Stat1:Stat1

B

D

Stat3:Stat3

SH5-07 (μM) 0 0.5 1 2 5 10 20

IC50 = 3.9 ± 0.6 μM

IC50 (μM)

>20 >20

SH5-07 (μM) 0 1 3 5 8 10 15 20

Fig. 1

i)

ii)

SH4-54 (μM) 0 1 3 5 8 10 15 20


IC50 (μM) >20 >20

SH4-54 (μM) 0 1 3 5 10 15 20 Stat3:Stat3

% of Cont 100 99 87 94 41 15 3

% of Cont 100 71 54 47 19 32 3

IC50 = 4.7 ± 0.5 μM

A

SH4-54 (μM) 0 1 3 5 10 20

Stat3:Stat3 Stat1:Stat3 Stat1:Stat1

SH5-07(μM) 0 1 3 5 10 20

Stat3:Stat3 Stat1:Stat3 Stat1:Stat1

C i)

ii)

i) ii)




0 1 3 5 8 10 Stat3:Stat3

Stat3:Stat3

Stat3:Stat3

MDA-MB-231

0 1 3 10 U251MG

A (ii)

Fig. 2

(i)

C

pY705-Stat3 Stat3

SH5-07 (µM, 1 h)

pY705-Stat3 Stat3

0 3 10 1 SH4-54 (µM, 1 h)

0 1 3 10

U251MG

U373MG

Stat3 pY705-Stat3 MDA-MB-231

Stat3:Stat3

Stat3:Stat3

(ii) (i)

DU145

SF295

U87MG

Stat3:Stat3

U373MG

SH5-07 (μM, 1 h) SH4-54 (μM, 1 h)

pY705-Stat3 Stat3

5 µM SH5-07 (h) 0 0.5 1 6 24 3

pY705-Stat3 Stat3

8 µM SH5-07 (h) 0 1 6 24 3

Stat3 pY705-Stat3

D

5 µM SH5-07 (h)

U373MG

8 µM SH4-54 (h) 0 0.5 1 6 24 3

U251MG

5 µM SH4-54 (h) 0 0.5 1 6 24 3

Stat3 pY705-Stat3

8 µM SH5-07 (h) 8 µM SH4-54 (h)

NIH3T3/vSrc

(ii) (i)

5 µM SH4-54 (h)

MDA-MB-231

0 0.5 1 6 24 3 0 0.5 1 6 24 3

0 0.5 1 6 24 3 0 0.5 1 6 24 3

0 1 3 10 0 1 3 10

0.5

MDA-MB-231

NIH3T3/v-Src U373MG

B


0 1 6 24


(i)

SH4-54 (8 μM, h) SH5-07 (8 μM, h) 0 1 6 24

0 1 6 24

(ii) SH4-54 (5 μM, h) SH5-07 (5 μM, h) 0 1 6 24

U251MG

E

Stat3 pY705-Stat3

0 1 3 6 6# 9#

U251MG

Lanes 1 2 3 5 6 4

SH5-07 (5 μM, h)

0

5

10

15

20

25

0

10

20

30

40

Arb

itra

ry lu

cife

rase

act

ivit

y

Fold

of

chan

ge

F

v-Src (200 ng) pLucSRE (500 ng)

SH5-07 (μM, 16 h)

- +

-

+ + +

+ +

0 3 8 +

v-Src (200 ng) pLucTKS3 (500 ng) SH5-07 (μM, 16 h)

- +

-

+ + +

+ +

0 3 8 +

(i)

(ii)

NIH3T3

NIH3T3

Lane 1 2 3 4

Lane 1 2 3 4

* *

Fold

of

chan

ge

Arb

itra

ry lu

cife

rase

act

ivit

y




Fig. 3

C

A

Stat3

pY705-Stat3

Lane 1 2 3 4

β-actin

1 5 2 3 4

IP: EGFR Blot:

pY705-Stat3

Stat3 SH4-54 (10 µM, 1 h) SH5-07 (10 µM, 1 h)

IL-6 (20 ng/ml, 10 min)

D

Lane 1 2 3 4 5 6

MDA-MB-231

NIH3T3/hEGFR

NIH3T3/hEGFR

IgG

85 91 100 94

35 18 100 138

110 105 100 126

25 32 0 100

Lane EGF (100 ng/ml, 12 min)

SH5-07 (10 µM, 24 h)

SH4-54 (10 µM, 24 h)

EGF (100 ng/ml, 12 min) SH5-07 (10 µM, 24 h) SH4-54 (10 µM, 24 h)

% of cont

% of cont

% of cont

+

+ + +

+ -

-

- - - -

-

+

+ + +

+ -

-

- - - -

-

-

- -

-

+ - +

+ -

-

- + - -

-

+

- -

-

+ -

EGFR

SH4-54 (10 μM, 24 h) SH5-07 (10 μM, 24 h)

EGF (100 ng/ml, 12 min)

pY1068EGFR

ERK1/2 pERK1/2

- - + - - - - +

- + + +

NIH3T3/hEGFR B

Lane 1 2 3 4

Stat3

EGFR % of cont




Fig 4. 1

3C

Ch

em

ical

Sh

ift

(pp

m)

1H Chemical Shift (ppm)

Black : free state Red : inhibitor bound state

B

SH4-54

A

Time (s) 0 200 400 600 800

0

200

100

300

Re

spo

nse

(R

U)

- + - + - + Stat3:Stat3

SH4-54 (8 µM, 3 h) C

Mock SH2 DB

I364

1.5 1.1 0.7

27

19

23

11

15

0.3 -0.5 -0.1

(ii) (i)

I597

I439 I386

L411

I364




NIH3T3

MEF/ST3KO

A2780S

MCF-7

U251MG

U87MG

U373MG

SF295

U251MG

U87MG

U373MG

SF295

vSrc231DU145Panc-1

vSrc

231

DU145

Panc-1

Fig. 5

>10 >10

IC50, uM 1.9

1.3

3.7

7.4

IC50, uM

Ce

ll via

bili

ty (

% o

f co

ntr

ol)

0

20

100

80

60

40

SH4-54 (µM, 72 h) 0 2 8 6 4 0 2 10 8 6 4

A

ii)

6.4

5.2

2.7

1.1

IC50 (uM)

Ce

ll via

bili

ty (

% o

f co

ntr

ol)

SH5-07 (µM, 72 h)

IC50 (uM)

8.8

8.1

9.0

0

20

100

80

60

40

0 2 10 8 6 4 0 2 10 8 6 4 0 2 10 8 6 4

i)

10.8

0 2 10 8 6 4

9.5

9.2

4.5

IC50, uM

IC50 (uM)

3.8

10.3

5.8

4.1

5.3

9.6

NIH3T3

MEF/ST3KO

A2780S

B

C

Ce

ll via

bili

ty

(%

of co

ntr

ol)

+ 0

20

100

80

60

40

scrambled siRNA Stat3 siRNA + -

-

FLAG

FLAG

β-actin

MDA-MB-231 i)

ii)

+ ST3 DBD-Flag

ST3 SH2-Flag + -

-

-

-

Ce

ll via

bili

ty (

% o

f co

ntr

ol)

SH4-54

Mock SH2 DBD 0

20

120

80

60

40

100

3 µM DMSO

5 µM

E

Co

lony n

um

ber

SH5-07

0

50

100

150

200

MDA-MB-231 U251MG

0 μM 2 μM 5 μM

MDA-MB-231 U251MG MEF/ST3KO

0 1 3 0 1 3 0 1 3

SH4-54 (μM) 0 1 3 0 1 3 5

MDA-MB-231 U251MG MEF/ST3KO

0 1 3 5

D

DMSO3 µM5 µM8 µM

F

MDA-MB-231 U251MG

Ce

ll cycle

(%

)

i) ii) MEF/ST3KO

(24 h)

0

20

40

60

80 DMSO

3 µM

5 µM

8 µM

0 h24 h48 h

iii)

G

% A

nnexin

V p

ositiv

e i) ii)

0

10

20

30

0

2

4

6

8MDA-MB-231 U251MG

0

2

4

6

8SH5-07 (24 h) SH5-07 (24 h) SH5-07 (24 h)

MEF/ST3KO iii)

(24 h) (5 µM)

SH5-07 (μM)

SH5-07 (µM, 72 h) SH5-07 (µM, 72 h)

SH4-54 (µM, 72 h) SH4-54 (µM, 72 h)

SH5-07




MDA-MB-231 U251MG

SH5-07 (5 μM, h)

Bcl-xL

β-actin

Bcl-2

Fig 6.

A

B Stat3 Promoter Occupancy

% E

nri

chm

ent

over

inp

ut

100 95 75 42 100 99 71 56

0 6 15 24 0 6 15 24

100 86 61 29

100 99 90 67 100 82 44 37

100 95 90 48 100 45 48 51

100 87 52 36

+ scrambled siRNA Stat3 siRNA + -

-

100 88 89 51

100 44 51 47 100 88 99 44

100 87 43 17

C

Mcl-1

GAPDH

Cyclin D1

Stat3

iNOS

β-actin

Survivin

Bcl-2

% of cont

% of cont

% of cont

% of cont c-Myc

% of cont

% of cont

Survivin

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

iNOS BCL2 Survivin

IgGDMSOSH454

0

0.1

0.2

0.3

0.4

0.5

0.6

β-Casein

IgG

DMSO

SH454

*

*

*

% of cont 100 5

% of cont 100 54

% of cont 100 75

% of cont 100 52

β-actin + scrambled siRNA

Stat3 siRNA + - -

SH4-54 SH4-54




Cont (1% DMSO)

SH5-07, 3 mg/kg

Duration time (day)

Fig 7.

Tum

or

siz

e x

10

2 (

mm

3 )

Duration time (day)

Tum

or

siz

e x

10

2 (

mm

3 )

MDA-MB231

Cont (1% DMSO)

SH5-07, 5 mg/kg

SH4-54, 6 mg/kg

Cont (1% DMSO)SH5-07, 5 mg/kgSH4-54, 6 mg/kg

Tum

or

siz

e x

10

2 (

mm

3 )

U251MG

Duration time (day)

* * * *

* *

0

10

15

5

0 10 40 60 30 20 50

0

10

5

0 5 20 30 15 10 25

30

0

20

10

0 5 20 30 15 10 25 35

*

*

A

B

c-Myc Cyclin D1

U251MG 1 2 3 4 5 6 7 8

Control SH4-54 SH5-07

MDA-MB-231

Control SH4-54 SH5-07

β-actin 1 2 3 4 5 6 7 8

C

Lane




Published OnlineFirst June 18, 2015.Cancer Res Peibin Yue, Francisco Lopez-Tapia, David Paladino, et al. and in vivosuppress human glioma and breast cancer phenotypes in vitro Hydroxamic acid and benzoic acid-based Stat3 inhibitors

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Hydroxamic acid and benzoic acid-based Stat3 inhibitors ... · inhibitors) (Roche, Indianapolis, IN) for lysis. To shear the DNA, the nuclear lysates were sonicated (Omni International,