ORIGINAL ARTICLE

Sequential combination of docetaxel with a SHP-1 agonistenhanced suppression of p-STAT3 signaling and apoptosisin triple negative breast cancer cells

Chun-Yu Liu1,2,3,4& Kuen-Feng Chen5,6

& Tzu-I Chao5 & Pei-Yi Chu7,8&

Chun-Teng Huang2,9 & Tzu-Ting Huang1,4 & Hsiu-Ping Yang1 & Wan-Lun Wang10 &

Chia-Han Lee1 & Ka-Yi Lau1& Wen-Chun Tsai1 & Jung-Chen Su11

& Chia-Yun Wu1,2,3&

Ming-Huang Chen1,2,3& Chung-Wai Shiau11

& Ling-Ming Tseng2,4,10

Received: 9 November 2016 /Revised: 7 May 2017 /Accepted: 19 May 2017# Springer-Verlag Berlin Heidelberg 2017

AbstractTriple negative breast cancer (TNBC) is an aggressive cancerfor which prognosis remains poor. Combination therapy is apromising strategy for enhancing treatment efficacy. Blockadeof STAT3 signaling may enhance the response of cancer cellsto conventional chemotherapeutic agents. Here we used aSHP-1 agonist SC-43 to dephosphorylate STAT3 thereby sup-pressing oncogenic STAT3 signaling and tested it in combi-nation with docetaxel in TNBC cells. We first analyzed mes-senger RNA (mRNA) expression of SHP-1 gene (PTPN6) in apublic TNBC dataset (TCGA) and found that higher SHP-1mRNA expression is associated with better overall survival inTNBC patients. Sequential combination of docetaxel and SC-

43 in vitro showed enhanced anti-proliferation and apoptosisassociated with decreased p-STAT3 and decreased STAT3-downstream effector cyclin D1 in the TNBC cell linesMDA-MB-231, MDA-MB-468, and HCC-1937. Ectopic ex-pression of STAT3 reduced the increased cytotoxicity inducedby the combination therapy. In addition, this sequential com-bination showed enhanced SHP-1 activity compared to SC-43alone. Furthermore, the combination treatment-induced apo-ptosis was attenuated by small interfering RNA (siRNA)against SHP-1 or by ectopic expression of SHP-1 mutants thatcaused SC-43 to lose its SHP-1 agonist capability. Moreover,combination of docetaxel and SC-43 showed enhanced tumorgrowth inhibition compared to single-agent therapy in mice

Chun-Yu Liu and Kuen-Feng Chen contributed equally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s00109-017-1549-x) contains supplementary material,which is available to authorized users.

* Ling-Ming Tsenglmtseng@vghtpe.gov.tw

1 Division of Medical Oncology, Department of Oncology, TaipeiVeterans General Hospital, No. 201, Sec. 2, Shih-Pai Road,Taipei 112, Taiwan

2 School of Medicine, National Yang-Ming University, No. 155, Sec.2, Li-Nong Street, Taipei 112, Taiwan

3 Division of Hematology and Oncology, Department of Medicine,Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road,Taipei, Taiwan

4 Comprehensive Breast Health Center, Taipei Veterans GeneralHospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 112, Taiwan

5 Department of Medical Research, National Taiwan UniversityHospital, No. 7, Chung-Shan South Road, Taipei 100, Taiwan

6 National Taiwan University College of Medicine, No. 1 Sec. 1,Jen-Ai Road, Taipei 100, Taiwan

7 Department of Pathology, ShowChwanMemorial Hospital, No. 542,Sec. 1, Chung-Shan Rd, Changhua City 500, Taiwan

8 School of Medicine, Fu Jen Catholic University, No. 510,Zhong-zheng Rd., Xin-zhuang Dist, New Taipei City 24205, Taiwan

9 Division of Hematology and Oncology, Department of Medicine,Yang-MingBranch of Taipei City Hospital, No. 145, Zhengzhou Rd.,Datong Dist, Taipei 10341, Taiwan

10 Department of Surgery, Taipei Veterans General Hospital, No. 201,Sec. 2, Shih-Pai Road, Taipei 112, Taiwan

11 Institute of Biopharmaceutical Sciences, National Yang-MingUniversity, No. 155, Sec. 2, Li-Nong Street, Taipei 112, Taiwan

J Mol MedDOI 10.1007/s00109-017-1549-x

bearing MDA-MB-231 tumor xenografts. Our results suggestthat the novel SHP-1 agonist SC-43 enhanced docetaxel-induced cytotoxicity by SHP-1 dependent STAT3 inhibitionin human triple negative breast cancer cells. TNBC patientswith high SHP-1 expressions show better survival. Docetaxelcombined with SC-43 enhances cell apoptosis and reduces p-STAT3. SHP-1 inhibition reduces the enhanced effect ofdocetaxel-SC-43 combination. Docetaxel-SC-43 combinationsuppresses xenograft tumor growth and reduces p-STAT3.

Key messages& TNBC patients with high SHP-1 expressions show better

survival.& Docetaxel combined with SC-43 enhances cell apoptosis

and reduces p-STAT3.& Enhanced prolyl-hydroxylase inhibition increases mainly

CXCR4+/CD11b+ cells.& SHP-1 inhibition reduces the enhanced effect of docetaxel-

SC-43 combination.& Docetaxel-SC-43 combination suppresses xenograft

tumor growth and reduces p-STAT3.

Keywords STAT3 . Triple negative breast cancer . SHP-1 .

Docetaxel

Introduction

Breast cancer is the most common cause of cancer-associateddeath in women worldwide [1]. There are approximately onemillion new breast cancer cases globally per year of which15–20% are diagnosed as the triple negative (ER−, PR−,HER2−) subtype [2]. Recurrent or metastatic triple negativebreast cancers (TNBCs) are heterogeneous and remain a clin-ical challenge. Chemotherapy is the current mainstay system-atic treatment for TNBCs. The administration ofanthracyclines or taxanes for TNBCs is considered effectivein clinical settings [3–6]. Applying platinum-based therapy toimpair DNA repair and synthesis in tumor cells representsanother approach to treat TNBCs [7]. Combining platinumcompounds with standard chemotherapy for TNBC treatmentincreases the percentage of patients with pathological com-plete response (pCR) [5, 8, 9], but patients without pCR showworse response [10]. Part of the reason that TNBC patientshave unfavorable prognosis can be attributed to a lack of ef-fective targeted agents for TNBC treatments.

Dysregulated STAT3 signaling in breast cancer leads toaberrant expression of genes which control cell prolifera-tion, epithelial-mesenchymal transition, and angiogenesis.TNBC cells secrete large amounts of IL-6 in an autocrinemanner to activate STAT3 signaling and participate in theresistance to apoptosis [11–13]. STAT3 is highlyexpressed and activated in most breast cancers [14],

especially in TNBC [15]. Previous studies have alsoshown that high p-STAT3 levels are correlated with worseoutcomes in invasive breast cancers [16]. In addition, bio-informatics analysis has shown that STAT3-regulatedgene expression is common to basal-like breast cancersbut not to luminal A or luminal B cancers [17].Increased STAT3 activity has been correlated with thechemotherapy-resistant property of TNBCs [18].Constitutive activation of STAT3 in TNBCs may also beinvolved in metastasis [19]. These studies suggest thattargeting STAT3 pathway may be a promising approachfor patients with TNBC.

Despite growing recognition of the pivotal role ofSTAT3 in TNBCs and the development of blocking Absagainst IL-6 receptors or inhibitors against JAK kinases[15, 20, 21], strategic targeting of the negative regulationof STAT3 signals remains overlooked. Src homology re-gion 2 domain containing phosphatase 1 (SHP-1), a non-receptor protein tyrosine phosphatase and a negative mod-ulator of phosphorylated STAT3 (p-STAT3) [22], is com-posed of two N-terminal-located SH2 domains that inter-act with phosphotyrosine, a phosphatase domain, and a C-terminal end [22, 23]. We previously developed a potentSHP-1 agonist, SC-43, which abolishes p-STAT3 signals[24, 25] and exhibits strong anti-breast cancer activity[26]. Aiming to target STAT3 and its drug-resistant role[11, 27] while extending our previous work on the SHP-1agonist, in this study, we developed a novel regime thatconsists of a combination of docetaxel and SC-43 used insequence to treat TNBCs.

Our research on the RNA profile of the TNBC patientsrevealed that the messenger RNA (mRNA) expression ofSHP-1, a key element in the negative regulation of p-STAT3signals, correlates with better overall survival and suggeststhat SHP-1 has a pivotal role in TNBCs. We then applied asequential treatment by employing docetaxel followed by theSHP-1 agonist, SC-43, to TNBC cells. The combination index(CI) values were less than 1, showing that the sequential ac-tions of docetaxel and SC-43 synergistically contributed tocell death. The sequential setting also led to decreased p-STAT3, cyclin-D1, and increased apoptosis in all TNBC cellstested. Validation by STAT3 overexpression or by using smallinterfering RNA (siRNA) against SHP-1 confirmed that theincreased apoptosis was mediated by the BSHP-1-STAT3axis.^ Significantly reduced tumor size in the TNBC xeno-graft animal model was observed with the docetaxel and SC-43 regime compared to mice treated with either drug alone.The elevated SHP-1 activity and decreased p-STAT3 levelresulting from the docetaxel and SC-43 regime contributedto improved therapeutic efficacy. The novel approach devel-oped herein, by the sequential administration of docetaxel andSC-43, a SHP-1 phosphatase agonist, could provide a basis toadvance TNBC therapy.

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Materials and methods

Cell culture, reagents, and antibodies

The MDA-MB-231, MDA-MB-468, and HCC-1937 celllines were obtained from the American Type CultureCollection (Manassas, VA, USA). All cells were maintainedin DMEM with 10% FBS. All cell lines were incubated at37 °C in a 5% CO2 incubator. SC-43 was synthesized, andits quality was evaluated as described in a previous study [26].For cell-based studies, SC-43 at various concentrations wasdissolved in dimethyl sulfoxide and then added to the cells inDMEM with 1% FBS. Antibodies for immunoblotting, suchas p-STAT3 (Tyr705) (Cat No. 9145), STAT3 (Cat No. 4904),Caspase-3 (Cat No. 9662), PARP (Cat No. 9542), Cyclin-D1(Cat No. 2926), SHP-1 (Cat No. 3759), and SHP-2 (Cat No.3397), were purchased from Cell Signaling (Danvers, MA,USA). β-Actin (Cat No. ab6276) was purchased fromAbcam (Cambridge, MA, USA). Myc-tag (Cat No. LS-C154222) and PTPRD (Cat No. LS-B9625) were fromLifeSpan (Seattle, WA, USA).

Treatment protocols of drug combinations in vitroand in vivo

For in vitro drug treatment, different concentrations of drugswere added to the medium either separately or in combination.The combination used a fixed ratio of 1/10 for docetaxel andSC-43. In sequential treatment, cells were treated with doce-taxel for 24 h, then the docetaxel was removed and washedtwice with PBS, and cells were treated with SC-43 for 24 h.The effects of the combinations were estimated usingCompuSyn software [28]. We designed the in vivo experi-ments based on the results from in vitro combination testing,and we also took the clinical treatment schedule of docetaxelinto consideration. Clinically, docetaxel is often administeredevery 3 weeks to breast cancer patients [29]. Therefore, wefinalized the in vivo treatment schedule for docetaxel on day 1and day 22 (10 mg/kg i.p., every 3 weeks) and for SC-43 ondays 2, 4, 6, 9, 11, 13, 16, 18, 20, 23, 25, 27, 30, and 32 (5 mg/kg i.p., three times a week).

MTTand Western blot analyses

Cell viability and proliferation of breast cancer cells wereassessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) assay. MTT assay and Western blot as-say were performed as previously reported [26].

Apoptosis analysis

Drug-induced apoptotic cell death was assessed by Westernblot analysis of activated caspase-3 or cleaved poly (ADP-

ribose) polymerase (PARP) and measurement of apoptoticcells by flow cytometry (sub-G1 analysis).

Phosphatase and kinase activity assays

The RediPlate 96 EnzChek Tyrosine Phosphatase Assay Kit(R-22067) was used for SHP-1 activity assay (MolecularProbes, Carlsbad, CA, USA) as described previously [26].

Gene knockdown using siRNA

Smart-pool siRNA, including control (D-001810-10) andSHP-1, were all purchased from Dharmacon (Chicago, IL,USA). Briefly, cells were transfected with siRNA (final con-centration, 100 nM) in 6-cm plates using lipid-mediated trans-fection with DharmaFECT 1 Transfection Reagent(Dharmacon, Chicago, IL, USA) according to the manufac-turer’s instructions. After 48 h, the medium was replaced andthe breast cancer cells were incubated with docetaxel or SC-43or sequential treatment, harvested, and separated for Westernblot analysis and apoptosis analysis by flow cytometry.

Xenograft tumor growth

Female NCr athymic nude mice (5–7 weeks of age) wereobtained from the National Laboratory Animal Center(Taipei, Taiwan, ROC). The animal study was approved andall experimental procedures using these mice were performedin accordance with protocols approved by the InstitutionalAnimal Care and Use Committee of Taipei Veterans GeneralHospital. Each mouse was inoculated subcutaneously to themouse mammary fat pads with 5 × 106 breast cancer cellssuspended in 0.1 mL serum-free medium containing 50%Matrigel (BD Biosciences, Bedford, MA) under isofluraneanesthesia. Tumors were measured using calipers, and theirvolumes were calculated using a standard formula:width2 × length × 0.52. When tumors reached around100 mm3, mice were administered docetaxel (10 mg/kg i.p.)every 3 weeks, with or without SC-43 (5 mg/kg i.p.) threetimes a week. Controls received vehicle (1× PBS). To accu-rately quantify the drug effects on these xenograft tumors, wehave cut off any unnecessary fat tissue from the tumor ascleanly as possible to avoid an overestimation of the tumorweight or an incorrect interpretation of immunohistochemicalresults on markers between the groups.

Combination index calculation

Cells were treated with docetaxel (0.01–0.12 μM) and SC-43(0.1–1.2 μM). The combination index (CI) was determinedusing the Chou and Talalay method [30] and the softwarepackage CompuSyn (Biosoft, Ferguson, MO, USA). A CIvalue of less than 1 was defined as synergism [31].

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Immunohistochemical staining

Paraffin-embedded breast cancer xenograft tumor tissue sec-tions (4 μm) on poly-1-lysine-coated slides were prepared andstained according to the IHC staining procedure previouslydescribed [26]. The primary antibodies against p-STAT3(ab76315, 1:50 dilution, Abcam) and STAT3 (no. 9132L,1:100 dilution, Cell Signaling Technology) were used for in-cubation for 1 h at room temperature. Bound antibodies weredetected using the EnVision Detection Systems Peroxidase/DAB, Rabbit/Mouse kit (Dako, Carpinteria, CA, USA). Theslides were then counterstained with hematoxylin. Rabbit IgGwas used as a control for antibody specificity. To detect theapoptosis, tumor tissue samples were stained by the terminaldeoxynucleotidyl transferase-mediated deoxyuridine triphos-phate nick end labeling (TUNEL) method with S7100ApopTag peroxidase in situ Apoptosis Detection Kit (MerckMillipore Corporation, Darmstadt, Germany) according to themanufacturer’s instructions.

The cancer genome atlas data description

Level 3 data of mRNA expressions from triple negative breastcancer samples and normal tissue samples were downloadedfrom The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/) and the Broad GDAC Firehose data portal(https://gdac.broadinstitute.org/). The mRNA reads perkilobase of exon model per million (RPKM) of all sampleswere selected and analyzed to compare abundances [32]. Themedian value was used as the cutoff limit for the overall sur-vival and disease-free curves for patients with high SHP-1expression (n = 49) and patients with lower SHP-1 expression(n = 50).

Statistical analysis

Data are expressed as mean ± SD or SE. Statistical compari-sons were based on nonparametric tests, and statistical signif-icance was defined as a P value of less than 0.05. For survivalanalysis, disease-free and overall survival curves of patientswere generated by the Kaplan-Meier method and compared

by log rank test. All statistical analyses were performed usingSPSS for Windows software, version 12.0 (SPSS, Chicago,IL, USA).

Results

High levels of SHP-1 transcripts are associated with betteroverall survival in patients with TNBC

First, to assess the clinical relevance of SHP-1, the mRNAreads per kilobase of exon model per million (RPKM) ofTNBC tumor samples from a TCGA public database wereselected and analyzed to compare abundances by GraphPadPrism 5 software. As shown in Fig. 1, survival analysis fromthe TCGA database demonstrated that high levels of SHP-1transcripts were associated with better overall survival(P = 0.029) in patients with TNBC (Fig. 1). This suggests thatSHP-1 might be a tumor suppressor and potential predictor ofTNBC prognosis.

Combination of SC-43 and docetaxel enhancesanti-proliferative effects and reduces p-STAT3 signalingin TNBC cells

SC-43, a potent SHP-1 agonist which dephosphorylatesSTAT3, has been shown to exhibit anticancer activity in he-patocellular carcinoma, colorectal carcinoma, and breast can-cer [24–26]. We performed MTTassay (Fig. 2a) and observedthat SC-43 displayed comparable anti-TNBC activity to doce-taxel (Fig. 2b), a common chemotherapy agent for TNBCtherapy. Building on the foundation laid by docetaxel, weincorporated SC-43 treatment after docetaxel dosing and eval-uated the cytotoxicity of this sequential setup (Fig. 2c). The CIvalues were less than 1 in all TNBC lines tested suggestingthat serial docetaxel and SC-43 treatment acted synergisticallywith a concentration ratio of docetaxel: SC-43 at 1:10(Fig. 2d).

The performance of the docetaxel-SC-43 regimenprompted us to explore its potency in induction of apoptosis.MDA-MB-468, MDA-MB-231, and HCC 1937 cells were

Fig. 1 Clinical relevance ofSHP-1 in triple negative breastcancer. Overall survival (left) anddisease-free curves (right) forpatients with high SHP-1expression (n = 49) and patientswith lower SHP-1 expression(n = 50) were generated using theKaplan-Meier method. Medianvalue was used as the cutoff limit

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subjected to docetaxel, followed by SC-43 exposure. Thepercentage of apoptotic TNBC cells was calculated by sub-G1 fraction analysis. Docetaxel-SC-43 treatment had syner-gistic effects on p-STAT3, cyclin D1, and cleaved PARP aswell as cell apoptosis in comparison with either treatmentused alone in these TNBC cells (Fig. 3a). The increasedTNBC cell apoptosis was positively correlated with in-creased SC-43 exposure, as 24 h treatment with SC-43 afterdocetaxel contributed to more programmed cell death than12 h of SC-43 exposure (Fig. 3b). Prolonged SC-43 treat-ment also abolished p-STAT3 signaling. However, thedocetaxel-SC-43 combination did not affect the expressionof SHP-1 and SHP-2 (Fig. 3c). Overexpression of STAT3alleviated the apoptotic effect and reduced the cleavagePARP triggered by the docetaxel-SC-43 treatment, suggest-ing that STAT3 has a pivotal role following the sequentialtreatment (Fig. 3d).

Inhibition of SHP-1 reduces the synergismof the docetaxel-SC-43 combination on p-STAT3and apoptosis

As the docetaxel-SC-43 combination had a synergistic effecton p-STAT3 and cell apoptosis, we next validated the role ofthe BSHP-1-STAT3 axis^ in the docetaxel and SC-43 formula.After the transfection of siRNA against SHP-1, the proteinlevels of p-STAT3 and the apoptotic population of MDA-MB-468 cells were restored (Fig. 4a). In addition, the activi-ties of SHP-1 were enhanced by treatment with SC-43 only orthe docetaxel-SC43 combination in MDA-MB-468 (Fig. 4b)and MDA-MB-231 (Supplementary Fig. 1) cells. To furtherconfirm that SHP-1 plays a crucial role in docetaxel-SC-43-mediated TNBC cell apoptosis, we also employed two typesof SHP-1 mutant constructs [24], in which the effects of SC-43 on SHP-1 were inhibited. As illustrated in Fig. 4c, the

Fig. 2 Anti-proliferative effects of docetaxel, SC-43, and combination inTNBC cells. aDocetaxel and b SC-43 both show anti-proliferative effectsin three triple negative breast cell lines. Cells were exposed to two drugsat the indicated doses for 48 h and cell viability was assessed by MTTassay. Points, mean; bars, SD (n = 3). c Sequential treatment schedule for

evaluating the cytotoxicity was treated in three TNBCs at the fixed ratioof 10:1 (0.1 μM docetaxel; 1 μM SC-43). d The combination index wascalculated as described in BMaterials and Methods.^ Experiments wereperformed in triplicate. CI values of less than one were considered assynergism

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ectopic expression of dN1 mutants, in which the N-SH2 do-main of SHP-1 was deleted, rescued the p-STAT3 signalingand reduced apoptosis in the cells subjected to the sequentialtreatment (Fig. 4d). Transfection of SHP-1 D61A mutants, inwhich the Asp at residue 61 of SHP1 was substitutedwith Ala,also desensitized MDA-MB-468 cells to docetaxel- SC-43-induced apoptosis (Fig. 4e).

Combination of docetaxel and SC-43 suppresses xenografttumor growth and reduces p-STAT3 signaling

To investigate the clinical therapeutic options, we tested thein vivo effect of the SC-43-docetaxel combination on tumorgrowth in a xenograft mouse model. According to the experi-ence of other groups [33] and our own experience, MDA-MB-

Fig. 3 Sequential combination of docetaxel with SC-43 enhancesapoptosis and reduces p-STAT3 signaling in TNBC cells. The treatmentstrategy followed Fig. 2c. a Cell apoptosis was analyzed by sub-G1fraction and PARP cleavage form. And the effects of combination on p-STAT3 and its downstream signal (cyclin-D1) were analyzed by Westernblot. b Time-dependent analysis of apoptosis and protein levels. c Effectof docetaxel-SC-43 combination on SHP-1 and SHP-2 expression. d

Overexpression of STAT3 reverses the apoptotic effect of the drugcombination. MDA-MB-468 cells stably expressing STAT3 with Myc-tag were treated with DMSO or the drug combination. The percentage ofapoptosis wasmeasured by sub-G1 analysis, and the effect on protein wasanalyzed by Western blot. Results are representative of at least threeindependent experiments. Columns, mean; bars, SD. *P < 0.05.**P < 0.01

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231 cells grow extremely well and give a 100% tumor takerate in the mammary fat pad. In addition, in this study, com-pared with the other two cell lines, MDA-MB-231 cells weremore sensitive to SC-43 or docetaxel treatment (Fig. 2).Therefore, we chose MDA-MB-231 for the in vivo experi-ment. Before we performed the animal experiments, wesearched the references regarding the doses of docetaxel inxenograft mouse models. We found that docetaxel is highlyactive and well-tolerated in a wide range of doses (2.5–20 mg/kg) in MDA-MB-231 xenograft mouse models [34,35]. On the other hand, the effective and well-tolerated dos-age range of SC-43 is 1–10 mg/kg [26, 36]. Based on thein vitro study, a low-dose docetaxel (0.1 μM) combined with

SC-43 (10 μM) is suitable to effectively induce TNBC cellapoptosis (Fig. 3). In order to balance the efficacy and toxic-ity, we used the median dosage of docetaxel (10 mg/kg) andSC-43 (5 mg/kg) in animal experiments. There were no ob-vious detrimental effects on phenotype but the tumor growth(Fig. 5a) as well as tumor weight (Fig. 5b) was significantlysuppressed in comparison with the control group. Moreover,compared to the control group, combined SC-43 and doce-taxel treatment effectively enhanced the activity of SHP-1(Fig. 5c) and reduced the levels of p-STAT3 proteins(Fig. 5d). Importantly, no significant weight change was ob-served (Fig. 5e) suggesting that this regime has a good safetyprofile. To further investigate the expression and localization

Fig. 4 Inhibition of SHP-1 reduced the synergism of the docetaxel-SC-43 combination on p-STAT3 and apoptosis. a Silencing SHP-1 by siRNAreduced the effect on p-STAT3 in MDA-MB-468. b SC-43 alone (left) orcombination with docetaxel (right) enhanced the activity of SHP-1 inMDA-MB-468. c Schematic representation of deletion and singlemutants of SHP-1. d, e Deletion of the N-terminal inhibitory domain

(dN1) and single mutant for SHP-1 (D61A) impair the drugcombination-induced STAT3 signaling and apoptosis effect. Apoptoticassay was performed by sub-G1 analysis. All results are representativeof at least three independent experiments. Columns, mean; bars, SD.*P < 0.05. **P < 0.01

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of p-STAT3 within the tissue section, the xenografted tumorswere subjected to immunohistochemistry analysis. The

docetaxel-SC-43 combination significantly downregulatedthe expression of p-STAT3 and promoted cell apoptosis

J Mol Med

compared with either docetaxel or SC-43 treatment alone inMDA-MB-231 tumors (Fig. 5f).

Discussion

Patients suffering from TNBCs have poor prognosis. As thereare no validated therapeutic targets, chemotherapy is widelyapplied in patients with TNBC. However, while initially re-sponsive to front-line chemoagents, this recalcitrant subtypeof breast tumor tends to recur and highlights the need forcontinuing therapy. Hence, strategically targeting moleculesinvolved in chemoresistance or aberrant cell proliferationmay provide a second-phase attack to this less-treatable tumorand bring clinical benefits.

Previous studies have revealed that SHP-1 protein was di-minished or abolished in several cancer cell lines and tumortissues [23, 37]. In addition, overexpression of SHP-1 sup-presses the growth of breast cancer cells [38], pancreatic can-cer [39], and prostate cancer [40], suggesting that SHP-1 mayfunction as a tumor suppressor. In our study, the data from apublic TCGA database indicated that higher expressions ofSHP-1 transcripts correlated with better overall survival inTNBC patients (Fig. 1). Tumorigenic STAT3 activation hasbeen observed in numerous malignant cancers, includinglung, breast, colon, liver, prostate, stomach, pancreas, kidney,and brain cancers [41]. Activated STAT3 is highly expressedin most breast cancers [14], especially in TNBC [15]. High p-STAT3 levels also correlated with poor outcomes in invasivebreast cancers [16] and contributed to chemotherapy resis-tance [42]. SHP-1 is a negative regulator of the cell cycle, aswell as inflammatory and JAK/STAT pathways in cancer pro-gression [43]. We previously generated a series of sorafenibderivatives that are devoid of angiokinase (VEGFR/PDGFR)inhibition and identified them as SHP-1 agonists [44, 45]. We

have shown that a direct SHP-1 agonist SC-43 is effective forcolorectal cancer [25], hepatocellular carcinoma [36], andbreast cancer [26]. These findings suggest that SHP-1-mediated STAT3 downregulation is a potential target of anti-cancer drugs that results in induction of apoptosis in cancercells.

Interestingly, the TNBC cell lines we used in this studyshowed a difference in molecular characteristics, MDA-MB-468 is so-called basal type [46], MDA-MB-231 is a claudin-low subtype [47], and HCC1937 is a BRCA-mutant cell line[48]. The results showed that these cell lines had differentialsensitivity to SC-43 and docetaxel (Fig. 3), but more studiesare necessary to elucidate the impact of these molecular char-acteristics (basal type, claudin-low, BRCA mutations, etc.) onthe effects of SC-43 in relation to SHP-1/STAT3 signaling.

Reports from breast cancer clinical trials have shown thatthe sequential application of single drugs, compared to simul-taneous co-administration of chemoagents, often resulted inless toxicity while maintaining similar survival benefits [49,50]. Lee et al. indicated that timing-specific inhibition ofEGFR by erlotinib, but not simultaneous co-administration,significantly sensitizes TNBC cancer cells to doxorubicin[51]. From a clinical point of view, patients with TNBCs ini-tially respond to chemotherapy, but a large portion of patientsexperience tumor relapse. Moreover, targeting another signal-ing pathway after chemotherapy, which universally attackscancer cells and normal cells, could potentially result in asynergistic effect. We thus adopted a sequential treatment,with an order-dependent administration of docetaxel followedby SC-43, to further advance TNBC therapy. The subsequentuse of SC-43 to abolish p-STAT3 after the first hit with doce-taxel, instead of co-administration of the two selected agents,significantly enhanced the percentage of apoptotic TNBCcells (Figs. 3 and 5d). Dosing with SC-43 after docetaxel notonly synergistically inhibited cell survival (Fig. 3) but also

Fig. 5 In vivo effect of docetaxel and SC-43 on MDA-MB-231xenograft nude mice. a The growth curves of tumors treated withvehicle, docetaxel (10 mg/kg i.p.), SC-43 (5 mg/kg i.p.), or docetaxel(10 mg/kg i.p.) combined with SC-43 (5 mg/kg i.p.). The treatmentschedule for docetaxel was day 1 and day 22 (every 3 weeks) and forSC-43 was days 2, 4, 6, 9, 11, 13, 16, 18, 20, 23, 25, 27, 30, and 32 (threetimes a week). Point, mean; bars, SE (n = 6). There were significantdifferences between the combined treatment group and the othergroups. b Average tumor weight measured at the end of experiments.Columns, mean; bars; SD (n = 6). *P < 0.05. c The activity of SHP-1in MDA-MB-231 tumors. Columns, mean; bars; SD (n = 6). *P < 0.05.**P < 0.01. d The expression of p-STAT3 and caspase-3 cleavage intumors was analyzed by Western blot. e Body weight measurement ofmice that received different treatments. Point, mean; bars, SD (n = 6). fImmunohistochemical staining (IHC) staining for p-STAT3, STAT3, andin situ apoptosis detection with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) in theMDA-MB-231 xenografts treated with vehicle, docetaxel, SC-43, orcombination therapy were performed as described in the BMaterials andMethods^ section. Representative staining results are shown in magneticfield power ×200

R

Fig. 6 Working model of the SC-43 and docetaxel combination in triplenegative breast cancer cells

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suppressed tumor growth (Fig. 5a). However, we did not eval-uate multiple dosing scheme for the in vivo experiments; wedesigned the in vivo experiments based on the results fromin vitro combination testing and also took the clinical treat-ment schedule of docetaxel into consideration. Clinically do-cetaxel is often administered every 3 weeks to breast cancerpatients [29]. Considering mice group with a single injectionof docetaxel might not be optimal for comparison with micegroups receiving either SC-43 monotherapy or combinationtreatment; therefore, we planned an every 3-week schedule forin vivo docetaxel injections for mice groups receiving doce-taxel treatment. In the future, more in vivo animal studies maybe needed for the optimal dosing scheme for docetaxel incombination with SC-43.

In conclusion, we found that high levels of SHP-1 tran-scripts correlate with better overall survival in patients withTNBC. Sequential application of docetaxel and a SHP-1 ago-nist SC-43 synergistically enhanced cytotoxicity through de-creasing p-STAT3 in TNBC cells (Fig. 6). This novel sequen-tial regime also triggers apoptosis with a positive correlationbetween the time of SC-43 exposure and the percentage ofapoptotic TNBC cells. Most importantly, the docetaxel-SC-43 treatment suppressed tumor growth in MDA-MB-231 xe-nograft model by elevating SHP-1 activity and reducing p-STAT3 expression. Taken together, the docetaxel-SC-43 com-bination might be another therapeutic strategy in patients withTNBC in the future.

Compliance with ethical standards

Conflict of interest The authors declare no conflicts of interest.

Research involving animals The animal study was approved and allexperimental procedures using these mice were performed in accordancewith protocols approved by the Institutional Animal Care and UseCommittee of Taipei Veterans General Hospital.

Funding information This work was supported by grants from theTaiwan Clinical Oncology Research Foundation; the Yen Tjing LingMedical Foundation (CI-104-07); the Ministry of Science andTechnology, Taiwan (MOST 103-2811-B-002-157, MOST 103-2325-B-075-002, MOST 104-2628-B-075-001-MY3, MOST 104-2811-B-002-030, MOST 104-2321-B-010-017, 105-2314-B-002-190-MY2);the National Health Research Institutes, Taiwan (NHRI-EX106-10608BI); Yang-Ming Branch of Taipei City Hospital (104 No. 35,M-1A00-B-B17-35); Taipei Veterans General Hospital (V103C-141,V104C-151, V105C-067); the TVGH-NTUH Joint Research Program(VN103-08, and VN105-09) from Taipei Veterans General Hospital andNational Taiwan University Hospital, and from the Ministry of Healthand Welfare, Executive Yuan, Taiwan (MOHW105-TDU-B-211-134-003, MOHW104-TDU-B-211-124-001 for the Center of Excellence,and MOHW103TDU-212-114-002, MOHW106-TDU-B-211-144-003for Cancer Research at Taipei Veterans General Hospital). This studywas also partially supported by the Chong Hin Loon Memorial Cancerand the Biotherapy Research Center of National Yang-Ming University,Taipei, Taiwan.

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